**Contents**


Reprinted from: *Biology* **2021**, *10*, 289, doi:10.3390/biology10040289 ................. **163**

## **Henrique Silva**


Reprinted from: *Biology* **2020**, *9*, 223, doi:10.3390/biology9080223 .................. **315**



## **Maria Fernanda Taviano, Natalizia Miceli, Rosaria Acquaviva, Giuseppe Antonio Malfa, Salvatore Ragusa and Deborah Giordano et al.**

Cytotoxic, Antioxidant, and Enzyme Inhibitory Properties of the Traditional Medicinal Plant *Matthiola incana* (L.) R. Br.


## **About the Editors**

## **Francisco Les**

Graduated in Veterinary Medicine from University of Zaragoza in 2012, Graduated in Pharmacy from San Jorge University (USJ) in 2015 and Doctor in Health Sciences also from USJ in 2017.

Professor of the degrees in Pharmacy and Nursing in subjects of Pharmacology, Plant Biology, Pharmacognosy and Phytotherapy.

His line of research is focused on investigating plant extracts and active ingredients of plant origin of pharmaceutical, cosmetic and agri-food interest, studying the bioactivity and toxicity of new compounds in in vitro or in vivo models.

He has published more than 40 articles on plant or food extracts and active ingredients with pharmacological and nutritional interest. He has participated in national and international conferences on pharmacy, herbal medicine, physiology, diabetes and obesity.

He defended a doctoral thesis with international mention entitled "Study of the bioactive properties of pomegranate juice and other compounds of polyphenolic origin", obtaining the qualification of cum laude and the Extraordinary Award of the Doctoral Program in Health Sciences. In addition, this has been awarded the first prize for doctoral theses in the areas of phytotherapy, medicinal plants, nutritherapy and natural ingredients of the "Arkopharma Institute Doctoral Thesis Awards 2021".

During his academic training, he has received six scholarships to support research and the subsequent completion of his doctorate, and has carried out international research stays at the "Dipartimento di Neuroscienze"in Florence (Universita di Firenze, Italy) in 2017, and at the "Institute ` of Cardiovascular and Metabolic Diseases"(Universite Toulouse III, France) in 2015. ´

#### **V´ıctor L ´opez**

Pharmacist and nutritionist with a PhD in Pharmaceutical Sciences currently working as Associate Professor and Vicedean at Universidad San Jorge in Spain. Involved in teaching and researching in the field of pharmacology, natural products and functional foods.

His line of research focuses on the pharmacology of natural products with particular emphasis on those bioactive compounds, natural antioxidants or plant extracts of pharmaceutical, cosmetic or agri-food interest. Head of Phyto-Pharm, a multidisciplinary and international research group with experience in the development of competitive projects for public calls but also through contracts with companies in the pharmaceutical sector.

Author of more than 75 articles in indexed international journals with numerous communications in specialized congresses, as well as several book chapters.

Other merits: collaborating researcher at the Aragon Agrifood Institute (IA2), member of the Board of Directors of the Spanish Society of Phytotherapy (SEFIT), member of the Spanish Society of Pharmacology, and member of the Kingdom of Aragon Academy of Pharmacy.

#### **Guillermo C´asedas**

Research and associate professor at San Jorge University (USJ). MSc in Pharmacy, Master in Business Administration (MBA) and Doctor in Health Sciences from the San Jorge University (USJ). He has carried out research stays at different laboratories such as the Department of Pharmacy and Pharmacology of the Faculty of Pharmacy of the Complutense University of Madrid, Department of Pharmaceutical Chemistry of the Univerza v Ljubljani (Slovenia), Department of Physiology of the University of Stellenbosch (South Africa), Research Unit in Santa Cristina University Hospital, Princess Health Research Institute (IIS-IP) of Madrid and Department of Genetics, Physical Anthropology and Animal Physiology of the University of the Basque Country (UPV/EHU).

His research line is focused on the study of active ingredients of plant origin and plant extracts of pharmaceutical and food interest. Mainly, in vitro pharmacological assays of enzyme inhibition (AChE, MAO-A, Tyrosinase) and antioxidant activity (DPPH radical, superoxide radical, ORAC), quantification of endogenous antioxidant systems (catalase, SOD, GPx, GR), oxidative stress (ROS) and protein expression in cellular models (glial and neuronal).

He is the author of the chapter "Engineering and Biomedical Effects of Commercial Juices of Berries, Cherries, and Pomegranates With High Polyphenol Content"of the book Non-alcoholic beverages Volume 6: The Science of Beverages (Elsevier).

He has published more than 20 articles on isolated bioactive compounds and polyphenols presented in plants in indexed scientific journals. He has participated and presented more than 20 communications in national and international conferences on phytotherapy, physiology, nutrition, pharmacology and pharmacy, oxidative stress and diabetes.

He is a peer reviewer for various scientific journals related to antioxidants and natural products.

## *Editorial* **Bioactivity of Medicinal Plants and Extracts**

**Francisco Les 1,2,\* , Guillermo Cásedas <sup>1</sup> and Víctor López 1,2**


Nature is an inexhaustible source of bioactive compounds and products with interesting medicinal properties and technological applications. Although natural products can be found in plants, animals, microorganisms and minerals, the vast majority of them come from plants [1].

Since the beginning of the ages, plants have produced a great variety of molecules through different biosynthetic routes. Some of them are considered essential for the normal performance and development of the plant, such as carbohydrates, lipids and proteins; this aggregate is called primary metabolites [2].

The biochemical pathways also lead to the production of relatively small molecules known as secondary metabolites. These secondary metabolites do not seem essential for plant development. However, science has demonstrated that secondary metabolites have important functions in plants, for instance, defence against ultraviolet radiation exposure; struggling against infections caused by viruses, fungi, bacteria and phytopathogens; or keeping herbivores away. These secondary metabolites are the most interesting in therapeutics and belong to three large groups known as polyphenols, terpenes and alkaloids [3].

Natural products have constituted the origin of pharmacology and therapeutics. Early on, they were used as medicinal plants or preparations, and later as isolated molecules or phytochemically characterized extracts. Plants are still a source in nature for obtaining and isolating molecules with pharmacological applications (drug discovery), but can also be used as herbal medicinal products in traditional or complementary medicine. In addition, the WHO has launched a Traditional Medicine Strategy (2014–2023), including herbal medicines as medicinal therapies, with the aim of ensuring the quality, safety, proper use and effectiveness of traditional medicines, among other objectives [4].

More recently, natural products have continued to enter clinical trials or to provide leads for compounds that have entered clinical trials, particularly as anticancer and antimicrobial agents. Further, research in natural products has shown many advantages [5,6]:


Throughout history, human have used plants for therapeutic purposes; the development of synthetic and organic chemistry allowed herbal medicines to be replaced by isolated molecules provided by the pharmaceutical industry, even though approximately 50% of them are not completely synthetic and have a natural origin [7–9]. It is also important to note that research on natural products has increased exponentially in recent years, but the percentage of new approved drugs that have a natural origin has decreased. This fact has been caused by different factors, such as aspects of intellectual property, respect for biodiversity, accessibility to living organisms or the amount of active substance available in nature [10,11].

**Citation:** Les, F.; Cásedas, G.; López, V. Bioactivity of Medicinal Plants and Extracts. *Biology* **2021**, *10*, 634. https://doi.org/10.3390/ biology10070634

Academic Editor: Zed Rengel

Received: 28 June 2021 Accepted: 7 July 2021 Published: 8 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In addition to their nutritional, industrial, ecological and environmental value, plants have played (and still continue to play) a crucial role in medicine and pharmacy [12].

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Article* **Essential Oil of** *Foeniculum vulgare* **Mill. as a Green Fungicide and Defense-Inducing Agent against Fusarium Root Rot Disease in** *Vicia faba* **L.**

**Mona M. Khaleil 1,2,\*, Maryam M. Alnoman 2, Elsayed S. Abd Elrazik 3, Hayat Zagloul <sup>4</sup> and Ahmed Mohamed Aly Khalil 2,5,\***


**Simple Summary:** Plant extracts, including essential oils, are a viable alternative method for controlling plant diseases. This work deals with the exploitation of fennel seed essential oil (FSEO) to inhibit *Fusarium solani* and control *Fusarium* root rot disease in *Vicai faba*. In vitro FSEO inhibited mycelium growth by up to 80% at 400 μL/mL of FSEO. In vivo, the protective effects against *Fusarium* root rot disease were recorded when FSEO was applied to *Vicia faba* seeds. The FSEO reduced the disease severity from 98% in plants grown in infested soil with *Fusarium solani* to 60.1% in plants that previously had their seeds treated with FSEO. GC-MS spectrometry analyses showed that the major chemical components in the essential oil were D-limonene, menthol, estragole and 2-decenal. Applications of the essential oil resulted in increased total phenolic and flavonoid contents in leaves compared with untreated inoculated (control) plants. The defense-related genes, such as defensin and chitinase, were differentially expressed. This study revealed that the essential oil of fennel seed was effective as a control agent against *Fusarium* root rot in broad beans.

**Abstract:** *Fusarium solani*, the causative agent of root rot disease is one of the major constraints of faba bean (*Vicia faba* L.) yield worldwide. Essential oils have become excellent plant growth stimulators besides their antifungal properties. *Foeniculum vulgare* Mill. (fennel) is a familiar medicinal plant that has inhibitory effects against phytopathogenic fungi. Herein, different concentrations of fennel seed essential oil (FSEO) (12.5, 25, 50, 100, 200 and 400 μL/mL) were examined against *F. solani* KHA10 (accession number MW444555) isolated from rotted roots of faba bean in vitro and in vivo. The chemical composition of FSEO, through gas chromatography/mass spectroscopy, revealed 10 major compounds. In vitro, FSEO inhibited *F. solani* with a minimum inhibitory concentration (MIC) of 25 μL/mL. In vivo, FSEO suppressed *Fusarium* root rot disease in *Vicia faba* L. by decreasing the disease severity (61.2%) and disease incidence (50%), and acted as protective agent (32.5%) of *Vicia faba* L. Improvements in morphological and biochemical parameters were recorded in FSEOtreated faba seeds. Moreover, the expression level of the defense-related genes defensin and chitinase was noticeably enhanced in treated plants. This study suggested using FSEO as a promising antifungal agent against *F. solani* not only to control root rot disease but also to enhance plant growth and activate plant defense.

**Keywords:** *Foeniculum vulgare*; chitinase; defensin; qRT-PCR; *Vicia faba*; plant disease; root rot; essential oil; plant promotion

**Citation:** Khaleil, M.M.; Alnoman, M.M.; Elrazik, E.S.A.; Zagloul, H.; Khalil, A.M.A. Essential Oil of *Foeniculum vulgare* Mill. as a Green Fungicide and Defense-Inducing Agent against Fusarium Root Rot Disease in *Vicia faba* L. *Biology* **2021**, *10*, 696. https://doi.org/10.3390/ biology10080696

Academic Editors: Francisco Les, Víctor López and Guillermo Cásedas

Received: 2 July 2021 Accepted: 13 July 2021 Published: 22 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

*Vicia faba* L., commonly known as faba bean or broad bean, is an economical legume grain that widely contributes to human consumption, animal fodder and silage making [1]. Faba beans have high nutritional value due to their high protein content, minerals, vitamins and considerable amounts of bioactive compounds [2]. This crop is usually planted at the end of summer. *Vicia faba* is susceptible to several diseases that reduce their yield, especially in moist conditions. Faba beans are attacked by a common disease known as root rot, which is caused by many fungal species including *Fusarium solani*, *Rhizoctonia solani* and *Sclorotium rolfsi* [2–4]. *Fusarium solani* is one of the most pathogenic fungi that deteriorate the quality and quantity of many crops' production [5]. *F. solani* is a common plant pathogen that invades a wide range of hosts including 111 plant species belonging to 87 genera [6]. Globally, *Vicia faba* L. is one of the most common plants suffering from root rot disease. *Fusarium* root rot takes place at the beginning of the growing season, resulting in the death of seedlings [4]. Otsyula et al. [7] reported that, the yield of common beans decreased by up to 84% due to root rot induced by *F. solani*. Management of *Fusarium* root rot is a complex task due to the soil-borne pathogens being located near the rhizosphere and their long-term survival by producing resistant spores [8]. Although there are fungicides that can control fungal diseases, they have negative impacts on human health, the ecosystem and evolving fungicide-resistant strains [9]. Therefore, the use of natural alternatives is the only reliable source for controlling plant diseases [10]. Essential oils (EOs) are among the most important alternatives that play a vital role in plant protection and food preservation, with a wide variety of applications. Several studies have confirmed the use of EOs to manage plant pathogenic fungi and improve the safety and quality of crops [11,12]. The chemical composition of EOs contains many bioactive molecules that have antifungal, antibacterial and antioxidant activity [13]. EOs have many antagonistic effects against bacteria and fungi, as they drive the plasma membrane to lose its ability to act as a barrier, followed by the release of intracellular components and suppression of cellular respiration with homeostasis failure [14]. Moreover, EOs can inhibit the formation of fungal cell walls and electron transport in the mitochondria [15]. Remarkably, one of the important actions of EOs on the plasma membrane is to suppress the secretion of toxins [16]. The usage of EOs effectively enhances the safety and quality of cereals and food products [11]. Regardless of antimicrobial activity, applying essential oils as biocides has many benefits beside antimicrobial activity, including pre-harvest restrictions, being non-toxic for human health, being reliable to use in any type of lands and their convenience for all types of cultivation, such as organic systems [11]. Herbs and aromatic plants are usually used for medicinal purposes and for inhibiting microbes, since they contain essential oils [17]. The fennel herb (*Foeniculum vulgare* Mill.), a member of the *Apiaceae* family, is an important medicinal plant found almost all over the world [18]. It is used as a carminative, antiseptic, diuretic, digestive and expectorant, aiding anticancer, anti-inflammatory, antimicrobial, and antioxidant activities [19]. The leaves and fruits of fennel are used in cosmetics and flavoring substances, while seed extracts exhibit antifungal effects against *Aspergillus* sp., *Candida* sp., *Sclerotinia sclerotiorum* and many other phytopathogens and dermatophytes [18,19]. The main components of fennel oil are trans-anethole (53.51%), carvacrol (11.93%), fenchone (8.32%) and thymol (8.11%) [20]. The essential oil of *F. vulgare* is enriched with phytochemicals such as polyphenols that give it antimicrobial and antioxidant properties [21]. The use of EOs can move from pathogen suppression to plant protection, presumably by different strategies; one such mechanism is defensin, a term used in the description of antimicrobial and antifungal proteins (AFPs), isolated from mammals, insects and plants, and serving as effectors molecules of innate immunity, providing an efficient initial defense against infectious pathogens [22]. Another such strategy is the synthesis of pathogenesis-related (PR) proteins such as chitinases, which hydrolyze chitin, a linear polymer of β-1,4-linked *N*-acetylglucosamine residues that is one of the primary cell wall components of many pathogenic fungi [23,24]. The level of expression of PR genes such as chitinase and defensin increases the defensive response

of plants against a wide range of pathogens [25]. Although FSEO has been used to inhibit some fungal species, several studies are still required to discover its efficiency against fungal plant pathogens and plant diseases, and its effect on plant quality and resistance. In addition, there is an increasing demand for this green, natural and safe product for future approaches to crop protection and organic farming. In this context, this study planned to investigate the antifungal potential of FSEO as a fungicide to overcome the economically damaging *Fusarium* root rot disease of *Vicia faba* L. in vitro and in vivo. Furthermore, the effect of FSEO was assessed on plant growth, antioxidant enzymes and the expression levels of defensin genes in *Vicia faba* L.

## **2. Materials and Methods**

## *2.1. Plant Material and Extraction of Fennel Seed Essential Oil*

*Foeniculum vulgare* (fennel) seeds were obtained from a local market (El-Hawag Company, Cairo, Egypt), and identified and authenticated by the Department of Botany, Faculty of Science, Mansoura University, Egypt. Fifty grams of fennel seeds was air dried and ground into a fine powder and then placed in a 2000 mL flask with 500 mL of distilled water and extracted by the hydro-distillation process using a Clevenger-type apparatus for 5 h to extract the essential oils. The oils were collected in a 250 mL conical flask, dried over anhydrous sodium sulphate and kept at 4 ◦C until use [26].

## *2.2. Gas Chromatography/Mass Spectral Analysis*

The chemical composition of the essential fennel oil was determined by gas chromatographymass spectrometry system (GC-MS-QP 2010, Shimadzu, Japan), equipped with a flame-ionization detector (FID) with a Rtx-5MS column (30 m × 0.25 mm, 0.25 μm thickness). The essential oil (10 μL) was dissolved in acetone (100 μL) and 1 μL of the solution was injected into the GC/MS system with the following properties: helium was the carrier gas, used at a flow rate of 1 mL/min; split mode (1:25), with 1 μL (1/10 in acetone, *v*/*v*) as the injected volume and 300 ◦C as the injection temperature. The mass spectra of the obtained compounds were matched with those in the NIST11 library (Gaithersburg, MD, USA) [27].

## *2.3. Isolation of Fusarium solani, Pathogenicity Test and Cultivation*

*Fusarium solani* was isolated in the laboratory from infected roots of faba bean plants (*Vicia faba* L.) displaying external signs of rot root disease. The plants were collected from agricultural areas in the production region of Behera, Egypt, in the winter of 2019. *F. solani* isolation was achieved by cutting the infected root into pieces (2 to 3 mm). The fragments were surface-sterilized with a 10% sodium hypochlorite solution for 2 min, then rinsed with sterile distilled water ternary. Pieces were cultured aseptically onto a *Fusarium* selective medium— Nash-Snyder agar (1 g/L KH2PO4, 0.5 g/L MgSO4-7H2O, 15 g/L peptone, 20 g/L agar, 1 g/L pentachloronitrobenzene, 0.3 g/L streptomycin sulfate, 0.12 g/L neomycin sulfate) [28]—and incubated at 25 ± 2 ◦C for 5–7 days. The fungal mycelium was sub-cultured on Czapek-Dox agar medium (CZA) (30 g/L sucrose, 3 g/L NaNO3, 0.5 g/L KCl, 100 mg/L FeSO4-7H2O, 0.5 g/L MgSO4-7H2O, 1 g/L K2HPO4). Morphological features as well as microscopic characteristics were investigated [29]. Moreover, molecular identification was also applied; the universal primers ITS1/ITS2 for the ribosomal internal transcribed spacer (ITS) were used. The sequence was compared with the suggested species using the BLAST sequence analysis tool and was registered into GenBank under the accession number MW444555. Koch's postulate was implemented to confirm that the symptoms of root rot belonged to *F. solani* KHA10 [30]. Eventually, cultures attained from single spores were maintained on CZA and kept at 4 ◦C for further use. The pure culture has been placed in the culture collection of the Botany and Microbiology Department, AUC (No. BMS0023).

## *2.4. In Vitro Evaluation of Antifungal Activity and Growth Inhibition*

## 2.4.1. Agar Well Diffusion Method

The antifungal activity of FSEO was tested by the well diffusion method with minor modifications. *F. solani* was inoculated on a Czapek-Dox (CZ) broth medium and then incubated at 25 ± 2 ◦C for 5–7 days [13]. Fungal inoculum of *F. solani* was spread on the surface of CZA plates. Next, 5 wells 8 mm in diameter were made using a sterile cork-borer on each agar plate (90 mm). The wells were filled with 100 μL of different concentrations of FSEO. Basically, 3 mL of Tween 80 was mixed with 97 mL of sterile distilled water. FSEO at 25, 50, 100, 200 and 400 μL/mL was prepared by adding 25, 50, 100, 200 and 400 mL of FSEO each to 1 L of sterile distilled water and Tween 80 (3%), respectively [31]. The culture plates were incubated at 25 ◦C for 7 days, and the zones of inhibition were observed and measured. All experiments were performed in triplicate.

## 2.4.2. Radial Growth Method

Radial growth of *F. solani* was evaluated at different concentrations of FSEO (25, 50, 100, 200 and 400 μL/mL) according to method used by Hashem et al. [1], with minor changes. The fennel essential oil was mixed well with the molten CZA medium at the desired final concentrations. Different concentrations of essential oil were prepared by dissolving the required amounts in sterile CZA amended with Tween 80 (0.1%, *v*/*v*) to obtain the desired concentrations (25, 50, 100, 200, 400 μL/mL). The medium was then poured into Petri dishes and kept until solidifying. The center of each plate was inoculated with a mycelium plug (6 mm diam.) from a 7-day-old culture, and the plates were then incubated at 25 ± 2 ◦C. Mycelium growth was assessed daily by measuring the diameters of the colony in each plate. Inhibition percentage of pathogen growth was calculated using the following equation:

Inhibition of pathogen growth (%) <sup>=</sup> Growth in the control <sup>−</sup> Growth in the treatment Growth in the control <sup>×</sup> <sup>100</sup>

## *2.5. Pot Experiment*

## 2.5.1. Preparation of Fungal Inoculum

The inoculum of *F. solani* KHA10 was prepared based on Büttner et al. [32] with slight modification as follows: a 500 mL sterilized Erlenmeyer flask containing 250 mL of the sterilized CZ medium was inoculated with 3 discs (5 mm in diameter) from the edge of 5-day-old *Fusarium* culture, and then incubated in the dark for 7 days at 25 ± 2 ◦C under shaking (125 rpm). Conidiospores were counted using a hemocytometer, and the inoculum suspension was adjusted to a final concentration of 10<sup>6</sup> spores/mL. The inoculum was kept chilled at 4 ◦C until use.

## 2.5.2. Fennel Seeds, Growth Conditions and Treatments

Seeds of *Vicia faba* L. (Nubaria1) were obtained from the Agriculture Research Center (ARC), Ministry of Agriculture, Egypt. The *Vicia faba* seeds were washed with distilled water then sterilized using 2% sodium hypochlorite for 2 min. *Vicia faba* seeds were grown in plastic pots (15 cm in diameter × 15 cm in depth), previously sterilized using a 5% formaldehyde solution and filled with 1 kg of sterile sandy clay soil (4:1). Two weeks before planting, the soil was infested with *F. solani* KHA10. Soil infestation was carried out by adding 90 mL of a 10<sup>6</sup> spore/mL suspension of *F. solani* KHA10/pot. The infested soil was kept moist for 7 days to stimulate fungal growth and ensure homogeneous distribution of the fungus. The control treatment was prepared by adding the same amount of the sterilized Czapek-Dox broth (CZ) to the sterilized soil of each pot. The pots were grown in greenhouse conditions at 25 ± 5 ◦C, with a 14 ± 2 h light regimen and humidity at 65 ± 10%, and irrigated as necessary. Treatments used in this study were as follows: (1) healthy control (C)—the sterilized *Vicia faba* seeds submerged in distilled water (D.W.) for 6 h and sowing in sterilized soil; (2) treated with FSEO (T)—the sterilized *Vicia faba* seeds

soaked in FSEO 400 μL/mL at the minimal fungal concentration (MFC) for 6 h and sown in sterilized soil; (3) control infected with *F. solani* (P)—sterilized *Vicia faba* seeds soaked in D.W. for 6 h and sown in soil previously inoculated with *F. solani*; (4) seeds treated with FSEO (T+P), sterilized *Vicia faba* seeds soaked in FSEO 400 μL/mL for 6 h and sown in soil previously inoculated with *F. solani*. The data were collected at 3 intervals (3 weeks, 6 weeks and 10 weeks). The results collected after 3 weeks of growth were much like the control because there was not much time for plant to be affected by both the pathogen and the treatments. After 10 weeks, it was very hard to collect data because the plant was old and affected too much by the pathogen to create a huge variation in the results (Supplementary Figure S1, Tables S1–S3). Five seeds/pot of *Vicia faba* for each treatment were applied. All experiments were arranged in a completely randomized split-plot design with 3 replicates per treatment. Six weeks after planting, all pots were evaluated for the incidence of *Fusarium* root rot. Percentages of seed rot, pre- and post-emergence damping off, and plant survival were also recorded [33].

## *2.6. Disease Assessments*

Disease severity (DS) and incidence (DI) of *Fusarium* root rot were assessed in *Vicia faba* L. 6 weeks after planting. Disease severity was evaluated using the 0–5 scale described by [34].

$$\text{Disease severity} \left( \% \right) = \sum \text{ab} / \text{AK} \times 100$$

where a = number of diseased plants with the same infection degree, b = infection degree, A = total number of the evaluated plants and K = the greatest infection degree.

Disease incidence (DI) of *Fusarium* root rot was assessed pre-emergence and at postemergence damping off after the treatments. Disease incidence was calculated for each treatment according to the following equation:

$$\text{Disease incidence (\%)} = \text{a/A} \times 100$$

where, a = number of diseased plants and A = total number of evaluated plants.

## *2.7. Analysis of Plant Growth Parameters*

Samples were assessed after 6 weeks of sowing. The morphological traits of treated and untreated faba bean plants were measured. Three plants with *Fusarium* root rot from each experiment were harvested and transferred to the laboratory, carefully uprooted and washed using tap water for measuring plant height, and shoot and root fresh and dry weight. For dry weight, samples were oven-dried at 40 ◦C for 48 h.

## *2.8. Biochemical Analyses*

For each treatment, 3 plants were collected 30 days after treatment and analyzed for total phenol content (TPC), total flavonoid content (TFC), phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and antioxidant enzymes.

#### 2.8.1. Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Activity

The scavenging activity of DPPH radicals was evaluated by adding a 1 mM solution of DPPH in ethanol to 1.5 mL (1 mg/L mL) of the EO extract solution. The freshly prepared DPPH solution was taken in test tubes and extracts were added, followed by serial dilutions (100–1000 μg) in every test tube such that the final volume was 2 mL, and the absorbance was evaluated at 517 nm against the corresponding blank solution, which was prepared by taking 3 mL ethanol, and the control O.D. was prepared by taking 3 mL of DPPH. The assay was repeated 3 times. DPPH percentage inhibition was estimated based on the control reading [1].

$$\text{DPPH szavænged (\%)} = \text{(A cont. } - \text{A test)} / \text{A cont. } \times 100^{\circ}$$

where A cont. is the absorbance of the control reaction and A test is the absorbance in the presence of the sample of the extracts.

## 2.8.2. Total Phenolic Content

Total phenols were measured in the uppermost leaves using the ethanol extraction method (80%, *v*/*v*); the supernatant was added to Folin and Ciocalteau's reagent as described [35].

## 2.8.3. Total Flavonoid Content

Total flavonoid content (mg·g−<sup>1</sup> fresh weight) was measured using aluminum chloride catechin equivalent (CAE) as the standard accordingly [35].

#### 2.8.4. Phenylalanine Lyase Assay

PAL activity was determined following the method described by Whetten and Sederoff [36]. The mixture of the assay, including 500 μL 50 mM Tris HCI and 100 μL plant extract, (pH 8.8), and 600 μL 1 mM L-phenylalanine, was incubated at room temperature for 1 h, and 2 N HCI was used to stop the reaction. Toluene (1.5 mL) was used to extract the assay mix by vertexing for 30 s. After centrifugation at 300 g, toluene was recovered for 5 min using a CRU-5000 centrifuge ITC. The toluene phase (containing trans-cinnamic acid) absorbance was measured at 290 nm. The enzyme activity was expressed as nmol trans-cinnamic acid released min−<sup>1</sup> g−<sup>1</sup> fresh weight.

## 2.8.5. Polyphenol Oxidase (PPO)

Extraction of PPO was performed as reported by [37]. Powdered samples (0.5 g) were homogenized with a buffer containing 20 mL of a 100 mM sodium phosphate buffer (pH 7.0) and 0.5 g polyvinyl pyrrolidone (PVP) (mol. wt 40,000) for the assay of the activity of PPO. The activity was measured in powder extracted with a 50 mM sodium phosphate buffer (pH 8.8) containing 5 mM β-mercaptoethanol. The extracts were filtered through 2 layers of Mira cloth, and the filtrates were centrifuged at 27,000× *g* at 4 ◦C for 30 min.

## 2.8.6. Antioxidant Enzyme Quantification

Samples (500 mg) of leaves were homogenized in a 50 mM KH2PO4 buffer (pH 7.8) with 0.1 mmol L−<sup>1</sup> EDTA, 0.1% (*v*/*v*) Triton X-100 and 2% PVP, and centrifuged at 4 ◦C for 10 min at 22,000× *g*. The supernatant obtained was reserved for the assays of the different antioxidants.

The activities of total superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) were recorded as follows: SOD activity was evaluated based on Kono (1978) [38] by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). The reduction of NBT was followed by an absorbance increase at 540 nm in a reaction mixture containing 1.3 mL Na-carbonate buffer (50 mM, pH 10.0), 500 μL NBT (96 μM) and 100 μL Triton X-100 (0.6%). The reaction was initiated by the addition of 100 μL hydroxylamine-HCl (20 mM, pH 6.0); 2 min later, 70 μL of the enzyme sample was added. The enzyme activity was calculated as the SOD concentration inhibiting the reduction of NBT by 50%. CAT activity was measured based on the method described by Aebi (1974) [39]. The rate of decomposition of H2O2 was superseded by a decrease in absorbance at 240 nm in a reaction mixture containing 1.5 mL K-phosphate buffer (100 mM, pH 7.0), 1.2 mL H2O2 (150 mM) and 300 μL of the enzyme extract. Enzyme activity was estimated by the extinction coefficient of 6.93 × 10−<sup>3</sup> mM−<sup>1</sup> cm<sup>−</sup>1. Moreover, APX activity was measured based on the method of Nakano and Asada (1981) [40] achieved by a decrease in absorbance at 290 nm in a reaction mixture containing 1.5 mL K-phosphate buffer (100 mM, pH 7.0), 300 μL ascorbate (5 mM), 600 μL H2O2 (0.5 mM) and 600 μL of the enzyme extract. Enzyme activity was determined using the extinction coefficient of 2.8 mM−<sup>1</sup> cm<sup>−</sup>1, and was calculated as the amount of enzyme required to oxidize 1 μmol of ascorbate min−<sup>1</sup> g−<sup>1</sup> tissue.

#### 2.8.7. Expression of Defense-Related Genes

Total RNA was extracted from 0.5 g fresh faba leaves at 1, 2 and 3 weeks after sowing from all treatments and the control using an RNA extraction kit (QIAGEN, Redwood, CA, USA). The obtained RNA was incubated with DNase for 1 h at 37 ◦C and quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). An RT-PCR kit (Omniscript RT; QIAGEN) was used for the synthesis of cDNA. Thermo QuantStudio 12K Flex Real-Time PCR System qRT-PCR was carried out in triplicate with 3 biological repeats using TOP real TM qPCR 2X Pre MIX SYBR Green (Enzynomics, Daejeon, Korea) according to the manufacturer's instructions using the given primers of the defense-related genes defensin and chitinase (Table 1) using β-actine as the reference gene. The PCR cycle was: 95 ◦C for 5 min (hot-start activation) followed by 40 cycles of 95 ◦C for 10 s (denaturation), 58 ◦C for 20 s (annealing) and 72 ◦C for 20 s (extension). The melting curve was generated after 40 cycles to test the specificity of each primer pair across the temperature range of 60–95 ◦C at a heating rate of 0.05 ◦C/s. Gene expression analyses were performed according to Rawat et al. [41].

**Table 1.** Primers used for qRT-PCR defense gene analysis.


## *2.9. Statistical Analysis*

Data Procession System (DPS) was used for analysis of variance (ANOVA). Two-way ANOVA was used to test the effect of E and P and their interactions on plant health, followed by the least significant difference (LSD). Correlation, PCA analysis and presentation were performed using R version 3.4.2.

## **3. Results**

## *3.1. Chemical Composition of Fennel Oil*

Since the researchers did not know the mechanism behind the antifungal activities of fennel seeds, this study attempted another experiment to check the chemical composition of fennel seeds and whether it could lead us to a significant result. Therefore, the chemical composition of fennel oil was inspected through gas chromatography/mass spectrometry (GC/MS) analysis, which revealed the presence of 10 major compounds in different percentages. The most abundant compound was cis-vaccenic acid (31.23%), followed by 9,12 octadecadienoic acid (29%), pentadecanoic acid (7.51%), estragole (4.39%), octadecadienoic acid (3.92%); 9-octadecadienoic acid (3.75%), D-limonene (2.93%), menthol (1.89%), 2,4-decadienal (1.68%) and 2-decenal (1.58%) (Figure 1 and Table 2).

**Figure 1.** Chromatogram: GC-MS chromatogram of *Foeniculum vulgare* essential oils.


**Table 2.** Chemical composition of *Foeniculum vulgare* oil by GC-MS analysis.

#### *3.2. In Vitro Control of F. Solani by Fennel Essential Oils*

## 3.2.1. Antifungal Activity and Minimum Inhibitory Concentration of FSEO

The antifungal activity of fennel seeds was investigated at different concentrations (25, 50, 100, 200 and 400 μL/mL) to inhibit *Fusarium solani* KHA10, the causative agent of *Fusarium* root rot disease in *Vicia faba* by the agar well diffusion method (Figure 2). The results revealed that all concentrations of FSEO showed antifungal activity against *F. solani* KHA10. However, 400 μL/mL presented the most antifungal activity, with a 38 mm inhibition zone, while 25 μL/mL exhibited the lowest antifungal activity, inhibiting the growth of *F. solani* with a 1 mm inhibition zone. According to the previous results, 25 μL/mL of FSEO was the MIC for controlling *F. solani*.

**Figure 2.** Antifungal activity of FSEO at different concentrations against *F. solani*. Data are expressed as means ± standard deviations in triplicate. Different alphabetic superscripts in the same column are significantly different (*p* < 0.05) based on Tukey's multiple comparison test.

3.2.2. Effect of FSEO on the Radial Growth of *F. solani* and Minimum Fungicidal Concentrations

In vitro, the antifungal activity of different concentrations of fennel seed essential oil extract was tested against the mycelial growth of *F. solani* KHA10 with different incubation periods from 1 to 7 days (Figure 3A–C). The radial growth was examined to measure the inhibition percentage of each FSEO concentration. The results demonstrated that the inhibition percentage increased with an increasing concentration of FSEO, while the radial growth ceased, as shown in Figure 3B. *Fusarium solani* could not grow at 400 μL/mL on the CZA surface, with an inhibition percentage of 100%, so this concentration had the minimum fungicidal activity (Figure 3). Moreover, FSEO at 25 μL/mL allowed the growth of *F. solani* with only 6% inhibition percentage.

ŚŖŖȱΐȦ ŘŖŖ ΐȦ ŗŖŖ ΐȦ śŖȱΐȦ ŘśȱΐȦ

#### *3.3. In Vivo Control of F. solani KHA10 by Fennel Essential Oil*

#### Efficacy of FSEO on *Fusarium* Root Rot Disease of *Vicia faba* L. under Pot Conditions

After applying the treatments to *Vicia faba*, morphological and disease progression were observed at the pre-emergence, post-emergence and 6 week stages (Table 2). Preand post-emergence, the damping off was decreased by 25% and 19%, respectively. The seeds exposed to the pathogen and FSEO (P+E) were much healthier compared were seeds treated with the pathogen (P) only. In addition, the P-only treated seeds clearly developed the disease.

The growth performance of FSEO-treated plants were much improved over the control (C) and P plants after 6 weeks of plantation. Broad bean plants treated with P+E were taller and healthier than P plants. The results showed that the disease resistance was higher in the 400 μL/mL FSEO treated plants.

After 400 μL/mL FSEO treatment of *Vicia faba* L. seeds, disease incidence (DI) and disease severity (DS) decreased significantly as compared with the pathogen-inoculated seeds only at 6 weeks after planting. The percentage of DI in faba seeds soaked with 400 μL/mL FSEO decreased to 33.5% as compared with pathogen-only infected plants. Plant survival and protection were clearly improved when FSEO was applied to infected seeds by approximately 44% and 50%, respectively (Table 3).


**Table 3.** Mean *Fusarium* root rot incidence and severity at pre- and post-emergence damping off after different treatments were applied to *Vicia faba* L.

## *3.4. Physiological Characterization of FSEO Treated Faba Bean Plants*

The physiological characterization data of greenhouse application treatments showed a significant increase in the growth parameters of *Vicia faba* plants, viz. plant height (P.h), shoot fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW) and root dry weight (RWD), by soaking seeds in 400 μL/mL FSEO, compared with *F. solani* KHA10 inoculation. Maximum P.h was recorded in the case of FSEO: 49.92 cm at 6 weeks after sowing. The control was second in rank, where a P.h of 43.20 cm was recorded, while T+P recorded a P.h. of 39.50 cm. As expected, the lowest P.h was observed in the case of the pathogen treatment: 36.70 cm at 6 weeks after sowing. Overall, SFW, RFW, SDW and RDW were significantly higher in the T+P treatment than in the pathogen-only treatment. Apart from that, we also observed a significant in of SFW, RFW, SDW, and RDW in the oil-treated plants as compared with the controls (Table 4).

**Table 4.** Effect of FSEO and *F. solani* KHA10 on morphological parameters of *Vicia faba* L. in pot conditions at 6 weeks of treatment.


Values are the means of 15 replicates ± standard errors. Values in each column followed by the same letter are not significantly different according to Duncan's multiple range test (*p* ≤ 0.05).

## *3.5. Influence of FSEO on Different Biochemical Parameters*

The second part of this study was the biochemical analysis of fennel seed extract and its influence on 2,2-diphenyl-1-picrylhydrazyl (DPPH), total phenolic contents (TPC), total flavonoid contents (TFC), phenylalanine ammonia lyase (PAL) and polyphenyl oxidase (PPO) (Figure 4). Significantly high DPPH was detected for *Vicia faba* L. treated with FSEO, which measured 57.05 μg g−<sup>1</sup> dry wt. 6 weeks after sowing, while the control recorded 49.97 μg·g−<sup>1</sup> dry wt. (Figure 4A). Pathogen-infected *Vicia faba* L. was strongly affected, where DPPH was 12.77 μg g−<sup>1</sup> dry wt., illustrating the strong influence of the pathogen on faba bean plants. A significant decrease in DPPH values was reported in the case of FSEO+P, namely 18.16 μg g−<sup>1</sup> dry wt., explaining the role of FSEO in plants defense against fungal diseases (Figure 4A).

**Figure 4.** Biochemical components and antioxidant enzymes of plants, after 6 weeks of treatment. 2,2-Diphenyl-1 picrylhydrazyl (DPPH) radical scavenging activity (**A**), total phenol content (TPC) (**B**), total flavonoid content (TFC) (**C**), phenylalanine ammonia lyase (PAL) (**D**), polyphenol oxidase (PPO) (**E**) and antioxidant enzymes (**F**). Error bars indicate ± standard error of the mean of three replicates. Different alphabetic superscripts in the same column are significantly different (*p* < 0.05) based on Tukey's multiple comparison test.

Total phenolic content is another biochemical contributor in plants that has redox properties, acting as an antioxidant (Figure 4B). The highest TPC was detected in control plants 6 weeks after sowing, recording 294.68 mg catechol 100 g−<sup>1</sup> dry wt., while in the case of the FSEO treatment, the recorded TPC was 284.34 mg catechol 100 g−<sup>1</sup> dry weight. In the case of FSEO+P, the recorded TPC was 235.13 mg catechol 100 g−<sup>1</sup> dry wt. Total

phenolic content dramatically decreased to 213.83 mg catechol 100 g−<sup>1</sup> dry wt. when plants were treated with *F. solani* (pathogen) (Figure 4B).

The level of total flavonoid content increased in *Vicia faba* L. treated with FSEO to 33.06 mg rutin 100 g−<sup>1</sup> dry wt. at 6 weeks after sowing, while the control was 27.29 mg rutin 100 g−<sup>1</sup> dry wt. The treatment of faba beans with FSEO +P recorded TFC at 15.09 mg rutin 100 g−<sup>1</sup> dry wt., while the level of TFC was significantly decreased at 11.84 mg rutin 100 g−<sup>1</sup> dry wt. in the case of the pathogen treatment (Figure 4C).

Additionally, the highest assay of PAL was detected when *Vicia faba* L. was treated with pathogen: 7.00 nM cinnamic g−<sup>1</sup> fresh wt. 6 weeks after sowing, while PAL was at 4.7 nM cinnamic g−<sup>1</sup> fresh wt. in the case of the control. PAL was at 5.81 nM cinnamic g−<sup>1</sup> fresh wt. when faba bean was treated with FSEO +P, whereas PAL was at 4.67 nM cinnamic g−<sup>1</sup> fresh wt. in the case of FSEO (Figure 4D).

Regarding polyphenol oxidase (PPO), the data showed a significantly increase PPO 12.50 μg g−<sup>1</sup> dry wt. when *Vicia faba* L. was treated with *F. solani*. Moreover, PPO was at 9.55 μg g−<sup>1</sup> dry wt. in case of FSEO +P, while FSEO only recorded 3.57 μg g−<sup>1</sup> dry wt. The smallest amount of PPO, 3.24 μg g−<sup>1</sup> dry wt., was recorded with the control (Figure 4E).

In addition, DI showed a strong negative (*p* ≤ 0.01) correlation with biochemicals such as DPPH, TPC and TFC, illustrating that an increase in disease incidence or severity will lead to a decrease in these biochemical or plant physiological characteristics and vice versa. However, there was a strong positive (*p* ≤ 0.01) correlation between DI with antioxidants, showing that a parallel increase or decrease in one will affect the other component positively (Figure 5).

**Figure 5.** Fold expression in the accumulation of the defense-related genes (**A**) defensin and (**B**) chitinase in *Vicia faba* L. samples, at different treatments relative to the control, and at different periods. Values are the means (±SD) of three repeated experiments. Different alphabetic superscripts in the same column are significantly different (*p* < 0.05) based on Tukey's multiple comparison test.

Concurrently, we also performed PCA analysis to identify the relationship of variables at 6 weeks in plants grown under different treatments. Correlations between variables were found via biplot analyses, where an acute angle means a positive correlation, an obtuse angle means a negative correlation and a right angle means no correlation between the measured parameters. The first principal component has the largest variance due to the orthogonal transformation. According to the PCA calculated for all the data, the first factor (PC1) explained 71.5% of the total variance of the variables, and the second factor (PC2) about 24.2%. In total, both PCs explained 95.7% of the total variance of all the analyzed variables (Figure 6).

**Figure 6.** Correlation coefficients (*r*) between different plant parameters. The *r* values from 0.50–0.70 have *p* ≤ 0.05 and those from 0.70–1.0 have *p* ≤ 0.01.

## *3.6. Expression Levels of Defense-Related Genes*

Pathogenesis-related genes are of great importance in plants that have greatly raised the level of their defense mechanisms against a wide range of pathogens. Therefore, in the third part of the current study, defensin and chitinase gene expression (GE) levels were evaluated. Not surprisingly, both genes showed highly significant levels of expression in the FSEO +P and P treatments, illustrating activation of the defense-related machinery in these treatments. In particular, defensin GE was significantly high (11.84, 11.65, 11.08 and 10.83) for the FSEO +P and P treatments on the second and third day, respectively. On the other hand, significantly low defensin GE levels were seen for other treatments. Accordingly, chitinase GE levels were significantly high (9.13, 9.07, 8.48, 8.26 and 6.29) in the FSEO +P and P treatments on the second and third day, while the other treatments showed relatively low chitinase GE levels (Figure 5).

#### **4. Discussion**

The resistance of many crops to fungicides continues to cause serious disease control problems. The practical research experience gathered over the past 50 years has highlighted the importance of using different strategies to control plant diseases. Moreover, scientific research is becoming alarmed not only due to losses from pathogen resistance but also environmental and health concerns. Therefore, there has been increasing interest in the serious pursuit of alternative biological phyto-therapeutic agents. Thus, this study is an attempt toward evaluating the applicability and the potential of essential oils derived from *Foeniculum vulgare* Mill. in the control of *Fusarium* root rot disease in *Vicia faba* L. under greenhouse conditions.

In the present study, we reported the antifungal activity of FSEO against *Fusarium solani* KHA10 in vitro and in vivo. Through GC-MS analysis of oil from *Foeniculum vulgare*, 10 different components were identified. In another study, gas chromatography of essential oils showed the presence of 18 main monoterpenoids in fennel oil. Limonene, transanethole, fenchone and estragole were common in fennel oil [21]. Another study reported

that the volatile oil of fennel contains different components: fenchone (1–20%), anethole (40–70%) and estragole (2–9%) [33]. Many other GC-MS screenings enumerated different components: fenchone (1–20%), estragole (2–9%) and anethole (40–70%) [42]. Similarly, in another experiment, fennel essential oil contained fenchone (less than 5%), but bitter types contained 20%. Sweet fennel oil contains 84–90% anethole, but bitter fennel contains 61–70% [18]. In this respect, the fennel seed essential oil, which contained a high amount of menthol, showed good antifungal activity against *F. solani*, as shown in previous results [33].

The results of the current study showed that fennel seed essential oils suppressed mycelium growth of *Fusarium solani* KHA10 in vitro at different concentrations from 25 to 400 μL/mL. Evidently, the inhibition of fungal growth increased with the increase in the concentration of the essential oil [33]. Plant-derived essential oils are compounds that have antibacterial and antifungal activity [15]. An illustration of this is a research study on *Botrytis cinerea*, in which different concentrations of essential oils promisingly and effectively suppressed the growth of *Botrytis cinerea* in a dose-dependent manner [26]. Our revealed data are in high accordance with the findings on another organism [43], which stated that the disease occurrence of powdery mildew on *Zinnia elegans* was significantly diminished through spraying with ginger, cinnamon, fennel and clove essential oils.

Another compelling piece of evidence was found from a research study investigating the high potential antifungal ability of essential oils of *Artemisia indica*, *Mentha spicata*, *Eucalyptus citriodora*, *Cymbopogon citratus* and *Cinnamomum tamala*, recording highly significant activity against *Fusarium oxysporum* and *Aspergillus niger* [44]. Among the tested essential oils, *Cymbopogon citratus* displayed the highest productive antifungal potential against *Fusarium oxysporum* (100% inhibition in mycelial growth) at 40 μL mL<sup>−</sup>1. *Mentha spicata* showed potent antifungal activity against *Aspergillus niger* (92.93% inhibition in mycelial growth) at 40 μL mL−<sup>1</sup> concentration [44]. The possible mechanism of action could be attributed to the disruption of the plasma membrane and disorganization of the mitochondrial structure caused by essential oils, as previously reported [45]. Therefore, a recent confirmative hypothesis reported that essential oils contain specific antifungal compounds and fungitoxic agents that inhibit the growth of certain microorganisms [45,46].

The current study reported a significant change in the physiological and morphological characteristics of *Vicia faba* L. plants treated with *F. solani* KHA10, confirming previously reported findings [30], which also stated that the morphological characteristics of asparagus were significantly reduced after inoculation with *Fusarium* species. In this study, the efficacy of fennel seed essential oil was examined in *Vicia faba* L. under greenhouse conditions. Both curative and preventive oil treatments were effective in reducing *F. solani* KHA10 infection. The disease incidence and severity were obviously decreased when plants were treated with FSEO.

Moreover, plant polyphenols are major compounds produced by plants for resistance to pathogens and many other functions [47]. In the present study, total phenolic and flavonoid contents were significantly increased when FSEO was applied on inoculated and non-inoculated faba bean plants when compared with seeds not treated with FSEO and sown in infested soil (pathogen). Similar results were described by [15,34,47], who stated that the phenolic compounds may prevent infection by the pathogen by increasing the mechanical strength of the host's cell walls and thus inhibiting pathogen infection.

Additionally, plant essential oils have unique antioxidant and antimicrobial properties, and are recommended to be a good alternative to synthetic antioxidants and chemical pesticides [48,49]. In this research, inhibition of the fungal activity using essential oil of fennel led to a significant increase in the fresh and dry weight of the shoot and root system of *Vicia faba* L. plants. There was a clear significantly negative relationship (Figure 6) between the degree of disease severity and fresh plant weight, indicating that infection with *F. solani* KHA10 was the main growth-limiting factor in all plants in the present and previous studies [1,25].

In order to evaluate the molecular mechanisms concerned in FSEO-induced resistance in faba bean, the expression of two defense-related genes, defensin and chitinase, was

assessed in treated faba bean at different times. It is worth mentioning here that the expression of defense-related genes can be induced by pathogen inoculation and environmental stresses [33]. The identification of such a broad mechanism involved in defense against pathogens and environmental stresses provides new opportunities for crop improvement. Plant defensins are a family of cysteine-rich peptides, many members of which have been shown to exhibit antimicrobial activity against various microbial attacks [50]. In the present study, we reported the high expression of defensin and chitinase GE treated with the pathogen and FSEO after the third day. These finding show that FSEO may also act as an inducer of the defense-related genes of plants when co-applied with pathogens.

Supporting such findings, it is worth mentioning that essential oils have two advantages: they are safe for use and have a low risk of the microorganisms developing resistance [46]. Essential oils are biodegradable and non-toxic. Since these bioactive compounds are extracted from plants, they are thought to be more acceptable and less risky to the environment than synthetic compounds [10]. In fact, different essential oils as antioxidants are naturally found in plants and have been considered as scavengers of active oxygen [13]. Due to the hydroxyl groups, phenolic compounds play an essential role in their scavenging ability [34,35,50]. Several reports highlighted the rapid advancement of essential oils as biodegradable, less toxic and eco-safe fungi-toxicants, showing the possibilities for their exploitation as natural fungicides [21].

#### **5. Conclusions**

Fennel seed essential oil, applied at a concentration of 400 μL/mL, has antifungal activities against *Fusarium solani* KHA10 in vitro and in vivo. The defensin and chitinase gene profile indicated that these genes may play vital roles in the resistance mechanism via reducing *Fusarium* root rot in faba beans. Although more studies are needed to fully verify and understand its mode of action, fennel seed oil is a promising fungicide against *F. solani* KHA10 as well as a plant growth promoter.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/biology10080696/s1, Figure S1: In vitro inhibitory effect of 400 μL/mL of FSEO against *F. solani*, Table S1: Mean Fusarium root rot incidence and severity in pre- and post-emergence damping off after different treatments were applied to *Vicia faba* L. after 3 weeks of treatments, Table S2: Effect of FSEO and F. solani KHA10 on morphological parameters of *Vicia faba* L. under pot conditions at 3 weeks of treatments, Table S3: Biochemical components and antioxidant enzymes of plants, after 6 weeks of treatment.

**Author Contributions:** Conceptualization, A.M.A.K. and M.M.K.; methodology, A.M.A.K., E.S.A.E., M.M.A. and H.Z.; software, A.M.A.K. and H.Z.; validation, M.M.K., M.M.A. and E.S.A.E.; formal analysis, H.Z., E.S.A.E. and A.M.A.K.; investigation, A.M.A.K. and M.M.K.; resources, H.Z., E.S.A.E. and A.M.A.K.; data curation, M.M.K. and E.S.A.E.; writing—original draft preparation, and A.M.A.K.; writing—review and editing, M.M.A., M.M.K., E.S.A.E. and A.M.A.K.; funding, M.M.A. and H.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The study did not report any data.

**Acknowledgments:** The authors express their sincere thanks to the Faculty of Science (Boyes), Al-Azhar University, Cairo, Egypt, for providing the necessary research facilities.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

1. Hashem, A.H.; Abdelaziz, A.M.; Askar, A.A.; Fouda, H.M.; Khalil, A.M.A.; Abd-elsalam, K.A.; Khaleil, M.M. Bacillus megaterium -Mediated Synthesis of Selenium Nanoparticles and Their Antifungal Activity against Rhizoctonia solani in Faba Bean Plants. *J. Fungi* **2021**, *7*, 195. [CrossRef] [PubMed]


## *Review* **Searching for Scientific Explanations for the Uses of Spanish Folk Medicine: A Review on the Case of Mullein (Verbascum, Scrophulariaceae)**

**José Blanco-Salas 1,\* , María P. Hortigón-Vinagre 2,\* , Diana Morales-Jadán <sup>3</sup> and Trinidad Ruiz-Téllez <sup>1</sup>**


**Simple Summary:** Mullein (*Verbascum* spp.) has been widely used in Spanish folk medicine to treat several pathologies, and these applications suggest the potential anti-inflammatory action of these plants. Based on the aforementioned, a deep bibliographic review of the chemical composition of the 10 species of Verbascum, catalogued by the Spanish Inventory of Traditional Knowledge related to Biodiversity, and virtual simulations using computer programs were used to demonstrate the molecular evidence supporting the use of these intuitive and traditional popular medicines.

**Abstract:** *Verbascum* species (common mullein) have been widely used in Spanish folk medicine to treat pathologies related to the musculature, skeleton, and circulatory, digestive, and respiratory systems, as well as to treat infectious diseases and organ-sense illnesses. These applications support the potential anti-inflammatory action of Verbascum phytochemicals. Based on the aforementioned facts, and following a deep bibliographic review of the chemical composition of the 10 species of Verbascum catalogued by the Spanish Inventory of Traditional Knowledge related to Biodiversity, we look for scientific evidences to correlate the traditional medical uses with the chemical components of these plants. To support these findings, in silico simulations were performed to investigate molecular interactions between Verbascum phytochemicals and cellular components. Most of common mullein traditional uses could rely on the anti-inflammatory action of phytochemicals, such as quercetin, and it could explain the employment of these plants to treat a wide range of diseases mediated by inflammatory processes such as respiratory diseases, otitis, arthrosis, and rheumatism among others.

**Keywords:** Verbascum; traditional knowledge; validation; flavonoid; terpene; inflammatory

## **1. Introduction**

The genus Verbascum (Scrophulariaceae, Lamiales) comprises more than 300 Eurasiatic species. It is the largest genus of the family, and its origin is the center of the Eastern Mediterranean Basin. In the Iberian Peninsula, it is represented by 26 species [1]. In Spain, they are popularly named "gordolobos" (in English, common mullein), and the Spanish Inventory of Traditional Knowledge related to Biodiversity [2] has catalogued 10 species which have been used to treat a wide range of pathologies. These are *Verbascum pulverulentum* Vill., *V. sinuatum* L., *V. thapsus* L., *V. boerhavii* L., *V. creticum* (L.) Cav., *V. dentifolium* Delile, *V. giganteum* Willk., *V. lychnitis* L., *V. rotundifolium* Ten., and *V. virgatum* Stokes in With.

In order to realize the potential pharmacological application of these species, we must perform a deep analysis of their chemical compositions as a starting point to understand

**Citation:** Blanco-Salas, J.; Hortigón-Vinagre, M.P.; Morales-Jadán, D.; Ruiz-Téllez, T. Searching for Scientific Explanations for the Uses of Spanish Folk Medicine: A Review on the Case of Mullein (Verbascum, Scrophulariaceae). *Biology* **2021**, *10*, 618. https:// doi.org/10.3390/biology10070618

Academic Editors: Francisco Les, Víctor López, Guillermo Cásedas and Zhongqi He

Received: 5 May 2021 Accepted: 29 June 2021 Published: 2 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

which phytochemicals could exert the medical actions described in the traditional knowledge. The chemical components of *Verbascum* spp., and the biological actions attributed to these phytochemicals, can be found in the literature [3–17], with the correlation between the phytochemicals' bioactivity and their traditional uses being a key point to validate their traditional ethnobotanical uses.

The aforementioned bibliographic prospection could be complemented by in silico approaches to demonstrate the phytochemicals' affinities using molecular targets. The combination of bibliographic research and computer programming could provide a strong tool to approach the botanical bioactive compounds existing in *Verbascum* spp. with the medical uses collected by folk knowledge.

The objective of this work is to analyze the affinities of phytochemicals from *Verbascum* spp. for mammalian molecular targets to perform a comprehensive scientific validation of its medical uses. This work could support further experimental studies on *Verbascum* spp. extracts and their phytochemicals as therapeutic agents, making the experimental approach easier and eventually contributing to reducing the number of animals employed in pre-clinical testing [18,19].

## **2. Materials and Methods**

#### *2.1. Ethnobotanical Uses and Chemical Composition of Verbascum Used in Spanish Folk Medicine*

We first carried out a bibliographic search, looking at the applications recorded by the Spanish Inventory of Traditional Knowledge related to Biodiversity [2], for the 10 *Verbascum* spp. catalogued in the Iberian Peninsula. We summarized them in a table, grouped by diseases and physiological systems.

Afterwards, we performed a bibliographic review of the chemical composition of the 10 Verbascum species. We used the databases Scopus, Dialnet, Medline, PubMed, ScienceDirect, Google Patents, Google Scholar, and Wiley Online. The employed keywords were: "*Verbascum sinuatum*", "*Verbascum thapsus*", "*Verbascum boerhavii*", "*Verbascum creticum*", "*Verbascum dentifolium*", "*Verbascum giganteum*", "*Verbascum lychnitis*", "*Verbascum rotundifolium*", "*Verbascum virgatum*" and/or "activity", "chemical composition", "pharmacology", and "medicin\*".

The bibliographic results were managed using a Prisma 2009 Flow Diagram Methodology [20]. A final summary was obtained. It contains the metabolites that had been identified in the aforementioned Verbascum species throughout the published literature and can be consulted in Appendix A (Table A1).

The chemical structures of these metabolites (83 molecules of Table A1) were retrieved from PubChem [21]. This is a database of chemical compounds maintained by the National Centre for Biotechnology Information (NCBI), a branch of the National Library of Medicine of the National Institute of Health (NIH). Structures were drawn and edited using ChemDraw Professional 17.0 (Perkin Elmer, Waltham, MA, USA) and/or Marvin Sketch 19.15 (ChemAxon, Budapets, Hungary). Finally, the respective SMILES codes were also compiled in the abovementioned Table A1 because they are essential to perform the in silico modelling planned for the next stage.

## *2.2. In Silico Modelling of Verbascum spp. Chemical Constituents' Affinities by Human Molecular Targets*

To obtain a virtual prediction of the probable molecular targets of the Verbascum metabolites listed in Table A1, we used the free Software SwissTargetPrediction (STP) [22]. This program allows one to estimate the most probable macromolecular targets of any small molecule assumed to be a bioactive metabolite. The prediction is founded on a combination of 2D and 3D similarity with a library of 370,000 known actives from more than 3000 proteins from 3 species. We focused our predictions on *Homo sapiens* targets. When a metabolite molecule SMILES code is uploaded to the SwissTargetPrediction Website, a document is obtained, which contains a list where proteins are ranked according to the probability of

the protein being a target of the query molecule (phytocompounds). Probabilities of ≥0.65 are considered to be significant in the metabolite–protein interaction [22].

We uploaded each of the Verbascum metabolites to the SwissTargetPrediction System; the significant results are summarized in a table available in Appendix B (Table A2). It corresponds to the list of 20 metabolites which showed a significant level of affinity for different targets. The results of Table A2 were analyzed and presented as a frequency histogram figure, structured from the perspective of the STP Target Classes.

The SwissTargetPrediction Program runs with a database system where the proteins included are linked to its own Class Target Classification System.

In summary, the total number of Verbascum metabolites tested in silico was 83, and the metabolites that showed target affinities (finally, 20) were then analyzed, studied, and discussed.

#### *2.3. Comparative Review of Ethnobotanical Uses and Physiopatological Molecular Targets*

The discussion consisted of making a qualitative comparison between the traditional use and biological activity of the components. The latter was considered in the published experimental results, which are accessible through bibliographic databases, and the in silico protein affinity tests performed using the aforementioned SwissTargetPrediction Program.

#### **3. Results**

### *3.1. Ethnobotanical Uses and Chemical Composition*

The use of *Verbascum* spp. in Spanish traditional medicine includes a wide range of formulations to treat disorders affecting a wide range of systems such as the circulatory, digestive, and respiratory systems, as well as skin diseases, sense organ illnesses, and infectious and parasitic diseases. The main applications collected by the Spanish Inventory of Traditional Knowledge related to Biodiversity [23] for the 10 *Verbascum* spp. catalogued in the Iberian Peninsula are summarized in Table 1, in which we have also included data on the method of administration.

#### 3.1.1. Circulatory System Diseases

Among the circulatory system applications, the anti-hemorrhoidal use of *Verbascum* spp. is the best established, as it has been reported for 7 out of 10 Iberian species. Topical application is the most common posology; it can be accomplished by sitz bath, with the liquid resulting from plant decoction [24–29], or by rubbing the mash or boiled plant onto the affected area [24,30–39]. Rubbing with hairy leaves has also been reported [40–42].

#### 3.1.2. Digestive Apparatus

Digestive system illnesses, in many cases, include conditions caused by an inflammatory process (tooth pain, gumboils, liver and gastric inflammation). Moreover, these species have also been used for their digestive properties and to treat gallstones, diarrhea, and constipation. Again, the liquid resulting after boiling to decoct the plant is the most common posology, together with plant infusions, which are commonly drunk to obtain healing benefits [28,31,36,38,42–49]. Nevertheless, these species can also be used in mouthwashes to treat teeth pain and gumboils [36,38,50,51], or as enemas for constipation, pediatric gut swelling, and indigestion [25]. The topic application of poultices or leaves (boiled or raw) is also used to treat abdominal pain, commonly attributed to liver or gut inflammation or diarrhea [25,28,33–35,52,53].

#### 3.1.3. Respiratory Diseases

The most common way to use *Verbascum* spp., to relieve respiratory system conditions, such as hoarseness, tonsillitis, cold, cough, asthma, or bronchitis, is through the ingestion of a wide variety of preparations (infusions, macerations, syrup) made with common mullein alone or mixed with other plants (mint, rosemary, mallow, hawthorn flower, coltsfoot, thymus and pine leaves, among others) or culinary ingredients (honey and

sugar) [24,25,28,30,31,33,35,36,38–40,43,45,46,49,51,54–68]. The ability of *V. thapsus* extracts to inhibit the growth of bacteria involved in respiratory infections has been proved using antibacterial assays, with the aqueous extracts being the most efficient [69].

#### 3.1.4. Musculature and Skeleton

Regarding the employment of *Verbascum* spp. to treat and relieve conditions affecting the musculature and skeleton, the healing properties attributed to common mullein could rely on its anti-inflammatory action, since most of the conditions treated share a strong inflammatory component (rheumatism, arthritis, swelling, contusions, and broken bones). The formulas employed include fresh, mashed, boiled, or infused plants, and the means of application is topical [25,26,29,30,33,35,43,52,67,70,71].

## 3.1.5. Skin and Sense Organs

A wide range of skin conditions are treated with *Verbascum* spp., including eczema, exanthema, cysts and zits, insect bites, and nail infections, as well as different types of wounds. The topical application of the liquid, resulting from boiling, infusing, or macerating the plant, is the most common posology [24–26,28,29,31,33,35,36,39,41,42,45–48,52,54,57,60,61,68,72–79]. The species' employment for chilblain relief is another common use (5 out of 10 *Verbascum* spp.). The most common means of application is rubbing the liquid, resulting from decoction [25,27,38,39,43,80–82], which, in Alicante, is carried out in milk instead of water [83]. In Caceres, a lead poultice is applied on the affected area [41].

A liniment made from mullein flowers, boiled or macerated in olive oil, is a common means for treating earache in different parts of Spain (Cataluña, Baleares, and Navarra) [24,25,35,66]. Conjunctivitis is another condition treated with common mullein [24].

## 3.1.6. Other Uses

Finally, another interesting application of *Verbascum* spp. is the treatment of infectious and parasitic diseases, such as diphtheria, helminthiasis, tuberculosis, typhus, and mange [25,28,35,62,68,77]. Despite the lack of experimental results showing the antimycobacterial action of Verbascum extracts, the British folk knowledge also point to the ability of common mullein to treat tuberculosis. Besides it, the nomenclature and local names of this genus are tightly connected with diseases caused by mycobacteria [84].

### 3.1.7. Chemical Composition

Spanish *Verbascum* spp. phytocompounds include two main classes: terpenes and flavonoids (see Table A1 and Figure 1). The best characterized species are *V. thapsus* [3,4,6,9,15–17], *V. sinuatum* [10–13], and *V. lychnitis* [5,7,14].

Monoterpene iridoids, sesquiterpenes, triterpene saponins, and phenyl propanoids are isoprene derivatives. Monoterpene iridoids are 10 C terpenes with a cyclopentanopyran cycle. Catalposide and specioside are metabolites belonging to this group. Their chemical structures are very similar, though differing in the way the phenol group is inserted, with specioside being more hydrophobic. Sesquiterpenes are 15 C terpenes, such as buddlindeterpene B. Triterpene saponins (vg. ursolic acid) are 30 C terpenes that reduce the surface tension, easing the mix of lipophilic and hydrophilic phases from liquid substances. Phenilpropanoid alcohols are glycosidic molecules, such as verbascoside and poliumoside.

Flavonoids share a flavonic nucleus (2-phenylbenzopyrane). They have been classified into three subgroups: flavonols, flavones, and O-methylated flavones. Flavones are pheny1-4 benzopyranones, flavonols are 3-hidroxyflavones, and O-metilated flavones have a methyl radical in the 3-hydroxilated part of the main pheny1-4-benzopyranone nucleus. The flavonoid components of Table A1 have a common structure of chromone (1-4 benzopyranone); are characterized by main functional groups such as hydroxyl, and carbonyl; have a conjugated double bond. They are soluble in water and ethanol, and they have oxygen bases varying from moderate to strong.

Some of these components have a powerful physiological activity, which has been shown in several experimental works [85–87]. This activity, usually with a narrow therapeutic margin (little difference between the minimum active concentration and the maximum tolerated concentration), has attracted interest in its associated biochemical processes.


**Table 1.** Traditional uses of Spanish Verbascum.

(*Vp: V. pulverulentum; Vs: V. sinuatum; Vt: V. tapsus; Vb: V. boerhavii; Vc: V. creticum; Vd: V. dentifolium; Vg: V. giganteum; Vl: V. lychnitis; Vr: V. rotundifolium; Vv: V. virgatum).* Administration T: Topic; I: Infusion; B: Boiled; M: Maceration; E: Enem; S: Steam.

#### *3.2. In Silico Modelling of Verbascum spp. Chemical Constituents' Affinities by Human Molecular Targets*

The review resulted in a library of 83 molecular structures identified in Verbascum. (Table A1). The application of the SwissTargetPrediction program yielded a final score of 20 molecules with ligand–target interactions with a probability of ≥0.65; thus, these were selected, and the rest were discarded. They are summarized in Table 2 and additional data are available in Table A2 (Appendix B).

The chemical structures of the 20 components are plotted in Figure 1, together with the probability values obtained by in silico modelling and target class, according to the SwissTargetPrediction classification.

Figure 2 shows the quantification of cases where the probability is greater than 0.65, in relation to the target class established by SwissTargetPrediction, and shown in Table A2. It

is necessary to emphasize the great affinity for the classes "enzymes" (44 cases), "kinases" (39 cases), and "lyases" (24 cases).

According to the data in Table A2, iridoids (catalposide, specioside) show affinity for the cytosolic protein HSP90AA1 (heat shock protein HSP90-α). The sesquiterpene, buddlindeterpene B, shows affinity for the transcription factors GLI1 and GLI2 (glioma-associated oncogen, which are zinc finger proteins). Ursolic acid, a triterpene saponin, mainly shows affinity for PTPN1 (protein-tyrosine phosphatase 1B) and other phosphatases (PTPN2 or T-cell protein-tyrosine phosphatase, P246666, or low molecular weight phosphotyrosine protein phosphatase), as well as the membrane receptor PTPRF (receptor-type tyrosineprotein phosphatase F), the nuclear receptor RORC (RAR-related orphan receptor γ), the DNA polymerase β (POLB), the aldo-ketoreductase 10 (AKR1B10), and the 11-beta hydroxysteroid dehydrogenase 1 (HSD11B1). The phenylpropanoid glycosides (verbascoside, poliumoside) show affinity for matrix metalloproteinases (MMP2, MMP12). The studied flavones (apigenin, apigenin-7-glucuronide, apigetrin, cynaroside, luteolin, luteolin-7 glucuronide, 6-hydroxyluteolin-7-glucoside, 7-methoxy-luteolin) show a wide profile of affinities, as summarized in Table A2. Among them are affinities for Cit P450, Glyoxalase 1 (GLO1), proinflammatory cytokine IL2, TNF-α secreted proteins, NADPH oxidase (NOX4), and arachinodate lipoxygenase (LOX), and the metalloproteinases (MMP 9 and 12) can be highlighted. The O-metilated flavones (acacetin, acacetin-7-O-α-D-glucoside) show affinity for cytochrome P450 (CYP1B1), interleukin-2 (IL2), and the Tumor Necrosis Factor (TNF-α).


**Table 2.** Chemical constituents of Spanish Verbascum, which is used in folk medicine, with a ligand–target interaction probability of ≥0.65 calculated by the SwissTargetPrediction software.

*Vl = V. lychnitis, Vt = V. thapsus; Vs = V. sinuatum; Vb = V. boerhavii.*

**Figure 1.** *Cont.*

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**Figure 1.** Chemical structures of Verbascum components with probability values and target class according to the SwissTargetPrediction classification. \* Probability—target class.

**Figure 2.** Quantification of cases where the Verbascum molecule–human target affinity is significant in the different classes of targets according to the SwissTargetPrediction classification system.

#### **4. Discussion**

#### *4.1. Anti-Inflammatory Action of Verbascum*

The role of biological molecules, such as inteleukins (ILs), lipooxygenase (LOX), cyclooxygenase (COX), nuclear factor κB (NF-κB), vascular endothelial growth factor (VEGF), matrix matalloproteinases (MMPs), and tumor necrosis factor (TNF), among others, with the onset of inflammation is well known as well as the link between inflammation and chronic diseases [88]. Therefore, the study of phytochemicals, able to block the action of the aforementioned molecules, is key in the search of new drug candidates to treat chronic diseases and other pathologies with a high inflammatory component.

Most of medicinal applications of *Verbascum* spp. collected from the folk knowledge, have in common an array of inflammatory processes; therefore, understanding the antiinflammatory molecular mechanisms displayed by Verbascum phytochemicals is essential in order to explain most of its healing properties.

The results generated by our affinities studies show the affinity of flavones (apigenin and luteolin) and flavonols (quercetin, 3 -methylquercetin and kaempferol) by arachinodate-lypoxygenases (LOX), a group of enzymes implicated in the synthesis of eicosanoids, such as leukotriens (LTs), which are molecules with an essential role in cell signaling, being also implicated in inflammation and disorders, such as asthma, skin diseases, rheumatoid arthritis, allergic rhinitis, inflammatory bowel, cardiovascular diseases, cancer, and osteoporosis [89–93]. It is well-known the anti-inflammatory role of polyphenolic compounds [94], in which flavones and flavonols are included. The ability of these compounds to interfere with enzymes implicated in the synthesis of eicosanoids, such as LOX, is one of the molecular mechanisms underlying their anti-inflammatory properties, and the ability of quercetin and lutein to suppress LOX product synthesis has been scientifically proven [90]. Despite our in silico approach cannot provide information about the molecular dynamic of phytochemical-target interaction, the affinity of flavones (apigenin and luteolin) and flavonols (quercetin, 3 -methylquercetin and kaempherol) for LOX, obtained by our in silico approach, is consistent with the scientific results found in the literature, in which the ability of quercetin and luteolin to suppress the formation of LOX products implicated in inflammation, such as LTs, is well demonstrated [90].

The polyphenolic compounds listed in Figure 1 shared a cathechol partial structure, which could be responsible for uncoupling the catalytic cycle of LOX, due to its iron chelating and antioxidant properties [90].

Another interesting result obtained from our in silico studies has shown the affinity of luteolin, quercetin, and kaempferol for interacting with NOX4 (NADPH oxidase-4), an enzyme implicated in the generation of superoxide anions and other downstream reactive oxygen species (ROS) [95]. For example, the protective role of luteolin against inflammation via the NOX4/ROS-NF-κB and MAPK pathways supports our findings and explains the anti-inflammatory action of mullein [96]. Compounds such as acacetin, apigetrin, and cynaroside have a high affinity to interact with the proinflammatory cytokines TNF-α and IL-2, which could also be related to their anti-inflammatory effects. In 2017, a paper from Hu et al. [97] demonstrated the anti-inflammatory effect of the flowers of Chuju (a medical cultivar of *Chrysanthemum morifolim* Ramat), which contain apigetrin and acacetin in their chemical composition [97]. A work of Zhao et al. (2014) [98] showed the ability of acacetin to block T-cell proliferation and IL-2 secretion, both essential to induce the inflammatory response underlying diseases such as rheumatoid arthritis and psoriasis [98]. The anti-inflammatory bioactivity of apigetrin has also been reported in an animal model of acute otitis media [99], which is a traditional use of Verbascum widely reported throughout the Iberian peninsula. Eventually, the anti-inflammatory effect of cynaroside has been demonstrated in a model of human periodontal ligament (hPDL) cells, a cell type essential in the maintenance of the periodontal tissues homeostasis, in which cynaroside has the ability to decrease the expression of pro-inflammatory cytokines, such as TNF-α, induced by LPS treatment [100].

Eventually, the in silico result, showing affinity between ursolic acid and the retinoic acid-related orphan receptor gamma (RORγ), a transcription factor essential for T helper cells differentiation, supported by experimental result showing an effective and selective inhibitory effect of this phytochemical over RORγ, could also explain the anti-inflammatory properties attributed to Verbascum spp. [101].

## *4.2. Circulatory System Diseases*

The most remarkable uses in this section are those related to circulation. The applications of these species against hemorrhoids and varicose veins are related to their local expansion processes in the peripheral circulation. This healing action can be explained by the presence of flavonoids, whose antioxidant and vasodilatory activities are associated with their protective cardiovascular action, widely referred to in the literature [102]. These compounds are common in aqueous extracts from the plants [103], so their presence is expected in many of the preparations recorded in Spanish traditional medicine and listed in Table 1. It has been reported that they are mainly used after being boiled and are then applied externally. The pathologies previously mentioned have also a local inflammatory component, therefore, the anti-inflammatory activity of common mullein, discussed in the previous section, could also underlie this group of healing remedies [93].

The antihypertensive use of *Verbascum* spp. reported in Table 1 could rely on the interaction of Verbascum phytocompounds with the α-adrenergic receptors implicated in peripheral vascular resistance walls. On the one hand, the α-adrenergic antagonist activity of flavonoids could explain Verbascum's antihypertensive action [104]. On the other hand, the affinity of rutin to interact with the α2-adrenoreceptors obtained in our in silico assays, and its anti-hypertensive action reported in the literature [105], could contribute to the antihypertensive action of Verbascum reported from folk knowledge [106].

#### *4.3. Digestive Apparatus*

The digestive process begins with activity in the oral cavity, chewing, salivation, and swallowing. Therefore, oral health is essential for proper digestion. The employment of infusions and decoctions, of these plants by Spanish folk medicine, to treat tooth pain and gumboil could be related to the anti-inflammatory activity discussed above. The anti-inflammatory effect of common mullein could rely on the anti-inflammatory action of its phytochemical cynaroside which has been demonstrated to confer protection against the inflammation underlying the periodontitis [100].

Other applications include for digestive problems, gastric ulcer, or inflammations in different parts of the digestive system (stomach, liver, gallbladder), for which there are treatments described in the traditional Spanish uses of the plant (Table 1). One study indicates the protective effect of ursolic acid against hepatotoxicity in mice [107].

In addition, some of these proteins are specifically related to the physiology of the gastro-intestinal tract. Salivary amylases help to break down food into its molecular components. Parietal cells in the stomach release various acids, pepsins, and enzymes, including gastric amylase, to achieve partial digestion and obtain chemo (semi-fluid and semidigested mass). Acids also neutralize salivary amylase, favoring gastric intervention. After about an hour, the chimo is pushed into the duodenum, where acidity acquired in the stomach stimulates the release of the hormone secretine. The pancreas then releases hormones, bicarbonate, bile, and numerous pancreatic enzymes, such as lipases (P04054), and those of the lipidic metabolism, such as aldoreductases and most of the ones consigned in the "Enzyme" file of Table A2. These are related to glucose conversion in NADPH-dependent sorbitol, the first step in the poliol pathway of glucose metabolism [108]. Afterwards, thanks to bicarbonate, the acidity of the chimo is changed into an alkaline form, allowing the better degradation of food and also creating a hostile environment for bacteria that survived the passage to the stomach. This process can be carried out effectively and smoothly if the enzyme system is healthy; otherwise, careful supplementation is required [109].

More difficult to validate, however, is the use related to defecation processes. These species have been used as both astringents and laxatives, and the only possible explanation for the traditional use of these plants is that in the first case, diarrhea (for which infusions are taken) has some infectious origin and causes inflammation. In the second case, where enemas are used because of the evacuating effect achieved by the mechanical action of water, this is favored by the presence of triterpene saponins, which have the ability to produce soapy solutions.

## *4.4. Respiratory Diseases*

Respiratory tract pathologies treated with mullein have different etiologies (hoarseness, tonsilitis, colds, coughs, asthma, bronchitis, and even hemoptysis) and treatments, but all have a common feature: the development of inflammatory processes. Besides this, in many cases, fever and cough are displayed. The relief properties of mullein could be explained by its antitussive and expectorant activities, which could be justified by the presence of mucilages in these species [110] which exert demulcent activity [111].

Ursolic acid is one of the most promising substances of biological origin for antimicrobial therapy. It has been identified as a phytochemical inhibitor of the main protease of COVID-19 using molecular modelling approaches [112–114]. Other potential phytochemicals of Verbascum spp., which could be useful to treat COVID-19, are the flavonoids apigenin, luteolin, and quercetin, which have been shown to be replication inhibitors of other coronaviruses [115].

Since, in severe COVID-19 patients, an elevation of pro-inflammatory cytokines occurs, also known as "cytokine storm", that is responsible of deteriorating their health conditions, the search of drugs able block target this "cytokine storm" and suppress the exacerbated inflammatory response is key in the treatment of the complications associated to the disease [116]. Our in silico results have evidenced affinity between mullein phytochemicals (Flavones and O-metilated flavones) and pro-inflammatory cytokines (IL-2 and TNF-α), molecules implicated in inflammatory processes related to the respiratory system and COVID-19 [117–119]. The previously validated anti-inflammatory activity of Verbascum components also supports the potential use of the extracts from the plants tackled in this review to achieve the desire anti-inflammatory action requested to prevent and treat COVID-19 acute clinical profile. The employment of natural compounds with

immunosuppressant properties could be useful as adjuvants to ameliorate the inflammatory process triggered by the out-of-control immune response which could be fatal for the patient, even causing death [120].

Our hypothesis suggesting the employment of Verbascum flavonoids as promising COVID-19 treatment is extensively supported by the existing literature which includes a large number of works using in silico and in vitro approaches which demonstrate the ability of flavonoids to interfere with the viral infection or to prevent/ameliorate the COVID-19 disease effects. Among SARS-CoV2 targets blocked by flavonoids 3CLpro (the protease responsible of processing the two polyproteins firstly translated after viral entry) can be highlighted due to its pivotal role in the initiation and progression of the viral cycle and the lack of its human homologue. Apigenin, luteolin, kaempferol, and quercetin are able to inhibit the proteolytic activity of 3CLpro, quercetin being the most effective. The ability of these phytochemicals to interact with 3Clpro could be due to the ability of the two phenyl groups of flavonoids to interact with the protease substrate binding pocket [121]. Another target is the RNA-dependent RNA polymerase (RdRp) responsible or virus genome replication. The RdRp activity, and therefore the viral replication, is affected by high Zn2+ levels and quercetin can act as Zn2+ ionophore facilitating the influx of Zn2+ into the cell [122]. The last molecular target to deal with SARS-CoV-2 infection is to block the interaction between the SARS-CoV-2 Viral Spike Protein (S) and its cellular receptor, the Angiotensin Converting Enzyme-2 (ACE2) protein, responsible of viral entry. In silico experiments have shown the capacity of two flavonoids (quercetin and luteolin) to block this process [123,124].

A recent review work has studied the potential action mechanisms of Chinese Traditional Medicines to treat COVID-19 by targeting key proteins for the initiation and progression of the disease (ACE 2 and 3CLpro) or inhibiting inflammatory mediators. The formulas tackled by this review shared components presented in Verbascum spp. such as luteolin, kaempferol and quercetin [125].

The main challenge found in the use of flavonoids, such as quercetin, with a widely supported antiviral action is the poor oral bioavailability due to its reduced absorption and biotransformation during digestion [126,127]. This issue can be tackled through alternative administration ways, such as nasal spray [128] or phytosomes [129].

#### *4.5. Musculature and Skeleton*

The use of analgesic, anti-inflammatory, and/or antipyretic drugs is very common in treating a wide range of medical conditions in current clinical pharmacology. Traditional medicine has also used many plants with identical purposes, such as the *Verbascum* spp. studied here. The applications listed in Table 1 extracted from the Spanish National Inventory include a wide spectrum of remedies to treat osteoarthritis, rheumatism, hand crack, kneeache, gout footache, contusions, and even broken bones, all of them characterized by the onset of inflammation and pain. The main aspects considered in the preceding paragraphs have already been discussed within inflammation section.

Pain has been defined by the IASP (International Association for the Study of Pain) as an unpleasant sensory and emotional experience associated with or resembling that associated with actual or potential tissue damage [130]. The phenomenon is a multidimensional entity and nuanced elements of pain are not easy to apprehend when pain is measured with the standard qualitative metrics [130]. From a biochemical and molecular biology point of view, the relationship of certain proteins with painful effects is well known [131], although the potential utility of proteomics to investigate pain management has just started to be considered. Cytochrome P450 [132], gyoxalase I [133], myeloperoxidase [134], and kinases [135] are proteins involved in the physiopathology of pain. Table A2 summarizes how the in silico study points to the great affinity of phytocompounds of these vegetables particularly quercetin, kaempferol, apigenin, and luteolin—with these proteins.

Osteoarthritis, one of the illnesses treated with common mullein by Spanish traditional medicine, is characterized by the degradation of cartilage, inflammation, and osteophyte formation in joints. Metalloproteinases are directly related to the onset of this medical condition due to their ability to proteolyze the extracellular matrix [136]. The affinity of some Verbascum phytochemicals (verbascoside, poliumoside, luteolin, quercetin, and kaempferol) for metalloproteinases could explain the traditional employment of mullein in osteoarthritis treatments. This notion is supported by a recent work which suggests the employment of verbascoside to treat osteoarthritis [136]. The employment of an ethanolic extract of Moussonia deppeana (high verbascoside content) shows an anti-edematous action in an experimental model of arthritis [137]. The ability of quercetin to reduce the severity of rheumatoid arthritis has also been demonstrated in vivo [138]. Another molecular mechanism, implicated in rheumatoid arthritis, is the invasion of fibroblast-like synoviocytes (FLS), which is responsible for cartilage destruction. Again, the metalloproteinases are involved in FLS invasion and kaempferol is able to reduce FLS migration and invasion both in vitro and in vivo [139].

A similar reasoning can be found regarding fever. Antithermic action is related to TNF-α secreted proteins [140] (P01375, Table A2), which have shown an in silico affinity with Verbascum flavones (6-hydroxyluteolin-7-glucoside, apigetrin, and cynaroside) and O-metilated flavones, such as acacetin-7-O-α-D-glucoside.

## *4.6. Skin and Sense Organs*

The topical dermatological use of various extracts (infusion, boiling, maceration) from these plants for the treatment of occasional or repetitive local eruptions (cysts, zits, eczemas, exanthemas), accidental or more serious conditions (wounds, ulcers, burns, bites), and even eye or ear inflammations are justified by their anti-inflammatory power reported throughout this manuscript.

The employment of common mullein to treat otitis could be explained by the presence of apigetrin in its chemical composition. We have shown the high affinity of apigetrin for TNF-α and IL-2 (P01375 and P60558, respectively), both belonging to the cytokine family and implicated in inflammatory processes. This hypothesis is supported by a recent work which demonstrates the healing effect of apigetrin in otitis media due to its ability to suppress inflammation and oxidative stress. Treatment with apigetrin reduces mucosa thickness, inhibits the inflammatory response by downregulating neutrophils and macrophages, and reduces ROS generation, eventually alleviating otitis [99].

#### *4.7. Other Uses*

Other popular uses, such as in the treatment of infectious diseases and parasitosis (diphtheria, helminthiasis, tuberculosis, typhus, and mange), require a direct validation that is difficult to explain with the data currently available. Indirectly, all the anti-inflammatory actions discussed throughout this work need to be taken into consideration.

#### **5. Conclusions**

The use of Spanish Verbascum spp. is in traditional medicine as a healing plant related to various pathologies, most of them involving inflammatory processes, can be justified from a scientific point of view, based on the chemical composition of these plants and the biological activities tested in vitro or in vivo employing the isolated phytochemicals or the plant extract itself, which can be found through the large bibliographic databases surveyed. The bibliographic prospection is supported by a simple in silico approach to look for specific protein affinities, in order to conduct the aforesaid bibliographic search.

The popular and most common use of Verbascum spp. is linked to its anti-inflammatory properties, which could be explained by the presence of flavonoids such as luteolin, quercetin, apigenin, and kaempferol within chemical composition. The anti-inflammatory properties of these molecules are well validated in the literature. Our in silico study's findings are in line with the experimental results found in the existing bibliography and have allowed us to select the phytochemicals with potential biological activities, among the preliminary list of compounds. This approach validates the employment of simple in silico

studies aimed to obtain the molecule-target affinities as a useful tool to be employed before starting bibliographic or experimental works aimed to validate the biological activities of phytochemicals. This kind of studies have a pivotal role to underlie the search of potential pharmacological compounds to be used as drug candidates to treat a wide range of pathologies. In the case of the species studied, the activity of molecules such as the flavonoids (apigenin, apigetrin, cynaroside, luteolin, quercetin, kaempferol, rutin, acacetin), iridoids (catalposide, specioside), phenylpropanoids (verbascoside, poliumoside), sesquiterpenes (buddlindeterpene), and saponins (ursolic acid) could serve as inspiration for the design of improved drugs to treat a wide range of pathologies, including respiratory pathologies, which are of particular interest at the moment, in the context of the COVID 19 pandemic.

**Author Contributions:** Conceptualization, T.R.-T. and J.B.-S.; methodology, T.R.-T.; software, T.R.-T., D.M.-J.; investigation, J.B.-S., T.R.-T., and M.P.H.-V.; data curation, J.B.-S., T.R.-T., and M.P.H.-V.; writing—original draft preparation, M.P.H.-V. and T.R.-T.; writing—review and editing, D.M.-J.; supervision, T.R.-T.; project administration, T.R.-T.; funding acquisition, T.R.-T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Consejería de Economía e Infraestructuras (Junta de Extremadura) Spain and Fondo Europeo de Desarrollo regional (FEDER) Spain, through the Grant (IB16003) Valorización de la Biodiversidad vegetal del espacio protegido, ZIR Sierra Grande de Hornachos como fuente de innovación para el desarrollo and Apoyos a los Planes de Actuación de los Grupos de Investigación Catalogados de la Junta de Extremadura: FEDER GR18169 and GR18116. M.P.H.-V. is supported by the Government of Extremadura (Grant No. TA18052). D.M.-J. is supported Universidad de las Américas, Ecuador (Grant One Health Research Group).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are grateful to Francisco Centeno Velazquez for his help and advice on how to use the SwissTargetPrediction Software.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

## **Abbreviations**


## **Appendix A**

**Table A1.** Metabolites of Spanish Verbascum (*Vp: V. pulverulentum; Vs: V. sinuatum; Vt: V. thapsus; Vb: V. boerhavii; Vc: V. creticum; Vd: V. dentifolium; Vg: V. giganteum; Vl: V. lychnitis; Vr: V. rotundifolium; Vv: V. virgatum*) and SMILES code.


#### **Metabolite Species Reference SMILES Code** <sup>21</sup> 6-O-(2"-O-feruloyl)-α-Lrhamnopyranosylcatalpol *Vt* [6,9,17] COc6cc(C=CC(=O)OC1C(O)[C@H](O)[C@H](C)O[C@@H]1O[C@H]4C3C= CO[C@@H](O[C@H]2OC(CO)[C@@H](O)C(O)[C@H]2O)C3[C@]5(CO)O [C@H]45)ccc6O <sup>22</sup> 6-O-(4"-O-feruloyl)-α-Lrhamnopyranosylcatalpol *Vt* [6,9,17] COc6cc(C=CC(=O)O[C@@H]5[C@H](C)O[C@H](O[C@H]3C2C=CO[C@@H] (O[C@H]1OC(CO)[C@@H](O)C(O)[C@H]1O)C2[C@]4(CO)O[C@H]34)C(O) C5O)ccc6O <sup>23</sup> 6-O-(2"-O-isoferuloyl)-α-Lrhamnopyranosylcatalpol *Vt* [6,9,17] COc6ccc(C=CC(=O)OC1C(O)[C@H](O)[C@H](C)O[C@@H]1O[C@H]4C3C= CO[C@@H](O[C@H]2OC(CO)[C@@H](O)C(O)[C@H]2O)C3[C@]5(CO)O [C@H]45)cc6O <sup>24</sup> 6-O-(3"-O-isoferuloyl)-α-Lrhamnopyranosylcatalpol *Vt* [6,9,17] COc6ccc(C=CC(=O)OC5[C@H](O)[C@H](C)O[C@H](O[C@H]3C2C=CO [C@@H](O[C@H]1OC(CO)[C@@H](O)C(O)[C@H]1O)C2[C@]4(CO)O[C@H] 34)C5O)cc6O <sup>25</sup> 6-O-(4"-O-isoferuloyl)-α-Lrhamnopyranosylcatalpol *Vt* [6,9,17] COc6ccc(C=CC(=O)O[C@@H]5[C@H](C)O[C@H](O[C@H]3C2C=CO [C@@H](O[C@H]1OC(CO)[C@@H](O)C(O)[C@H]1O)C2[C@]4(CO)O[C@H] 34)C(O)C5O)cc6O 26 Pulverulentoside I *Vp*, *Vs*, *Vt* [6,9,17] C=C(C=Cc1ccc(OC)cc1)OC6[C@@H](O[C@H]4C3C=CO[C@@H](O[C@H] 2OC(CO)[C@@H](O)C(O)[C@H]2O)C3[C@]5(CO)O[C@H]45)O[C@@H](C) [C@@H](O)C6OOC(C)=O 27 6-O-(2"-O-p-methoxy-transcinnamoyl-4"-O-asetyl)-α-Lrhamnopyranosylcatalpol *Vt* [6,9,17] COc6ccc(C=CC(=O)OC1C(O)[C@H](OC(C)=O)[C@H](C)O[C@@H]1O [C@H]4C3C=CO[C@@H](O[C@H]2OC(CO)[C@@H](O)C(O)[C@H]2O)C3 [C@]5(CO)O[C@H]45)cc6 <sup>28</sup> Pulverulentoside II *Vp* [9] COc6ccc(C=CC(=O)OC5C(O)C(C)OC(OC3C2C=COC(OC1OC(CO)C(O) C(O)C1O)C2C4(CO)OC34)C5OC(C)=O)cc6O <sup>29</sup> Catalposide *Vl* [5,9] C1=COC(C2C1C(C3C2(O3)CO)OC(=O)C4=CC=C(C=C4)O)OC5C(C(C (C(O5)CO)O)O)O 30 Specioside *Vl* [4,83] C1C(C(=CC(=O)OC2=C1C=CC(=C2)OC3C(C(C(C(O3)CO)O)O)O)C4= CC=C(C=C4)O)O.C1C(C(=CC(=O)OC2=C1C=CC(=C2)O)C3=CC=C(C=C3) O)OC4C(C(C(C(O4)CO)O)O)O 31 Ajugol *Vt*, *Vv* [6,9,17] CC1(CC(C2C1C(OC=C2)OC3C(C(C(C(O3)CO)O)O)O)O)O <sup>32</sup> 6-O-benzoyl ajugol *Vt* [6,9,17] [H][C@@]23C=CO[C@@H](O[C@@H]1O[C@H](CO)[C@@H](O) [C@H](O)[C@H]1O)[C@]2([H])[C@@](C)(O)C[C@H]3OC(=O)c4ccccc4 <sup>33</sup> 6-O-syringoyl ajugol *Vt* [6,9,17] CC1(CC(C2C1C(OC=C2)OC3C(C(C(C(O3)CO)O)O)O)OC(=O)C4=CC(=C (C(=C4)OC)O)OC)O <sup>34</sup> 6-O-vanilloyl ajugol *Vt* [6,9,17] CC1(CC(C2C1C(OC=C2)OC3C(C(C(C(O3)CO)O)O)O)OC(=O)C4=CC(=C (C=C4)O)OC)O 35 Harpagide *Vs*, *Vt* [6,9,17] CC1(CC(C2(C1C(OC=C2)OC3C(C(C(C(O3)CO)O)O)O)O)O)O <sup>36</sup> Harpagoside *Vp*, *Vs*, *Vt* [6,9,17] CC1(CC(C2(C1C(OC=C2)OC3C(C(C(C(O3)CO)O)O)O)O)O)OC(=O)C= CC4=CC=CC=C4 <sup>37</sup> Lychnitoside *Vl* [9] OCC2=CO[C@@H](O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H] 1O)C3C=CCC23 <sup>38</sup> Lateroside *Vt* [8] [H][C@@]24C=CO[C@@H](O[C@@H]1O[C@H](CO)[C@@H](O)[C@H] (O)[C@H]1O)[C@]2([H])[C@@](C)(OC(=O)C=Cc3ccccc3)C[C@H]4O 39 5-O-α-L-rhamnopyranosy (1α-3)-[α-D-glucuronopyranosyl (1α-6)]-α-D-glucopyranoside *Vt* [8] CC8OC(OC1C(O)C(O)C(O)OC1OC2C(O)C(O)C(C(=O)O)OC2OC7CC [C@]6(C)C5CC=C4C3CC(C)(C)CC(O)[C@]3(C)CC[C@@]4(C)[C@]5(C)CCC 6C7(C)CO)C(O)C(O)C8O 40 Ningpogenin *Vt* [8] [H][C@]12C=C(CO)[C@@H](CO)[C@@]1([H])CC(=C)O2 41 10-deoxyeucommiol *Vt* [8] CC1=C(CO)C(CCO)[C@@H](O)C1 42 Jioglutolide *Vt* [8] C[C@@]1(C[C@H]([C@H]2[C@@H]1COC(=O)C2)O)O <sup>43</sup> 6-β-hydroxy-2-oxabicyclo [4.3.0]Δ8-9-nonen-1-one *Vt* [8] [H][C@@]12CCOC(=O)C1=C(C)C[C@H]2O <sup>44</sup> 8-cinnamoylmyoporoside *Vt* [8] C[C@@]1(C[C@H](C2[C@@H]1[C@@H](OC=C2)O[C@H]3[C@@H]([C@H] ([C@@H]([C@H](O3)CO)O)O)O)O)OC(=O)/C=C/C4=CC=CC=C4

## **Table A1.** *Cont.*

45 Verbthasin A *Vt* [8] [H][C@@]12COC(=C)[C@]1([H])C[C@@H](O)C2=CCO



## **Appendix B**

**Table A2.** Targets and metabolites of *Verbascum* spp. Probability calculated by SwissTargetPrediction (http://www. swisstargetprediction.ch/ accessed date 10 June 2020).







## **References**


## *Article* **Development and Optimization of Supercritical Fluid Extraction Setup Leading to Quantification of 11 Cannabinoids Derived from Medicinal Cannabis**

**Sadia Qamar 1,\*, Yady J. Manrique 1,2, Harendra S. Parekh <sup>1</sup> and James R. Falconer 1,\***


**Simple Summary:** This study describes the design and development of setup for the extraction of cannabis strain 1 (Cannabidiol dominant) using supercritical carbon dioxide. For this purpose, two different supercritical fluid extraction instruments were used. The extraction conditions were maintained at 37 ◦C and 250 bar. Different carbon dioxide inlet and outlet positions were experimented to obtain the maximum yield. A separating chamber was also designed to reduce the throttling effect and dry ice formation during the depressurization process. After developing the supercritical fluid extraction setup, ultra-high performance liquid chromatography coupled with a diode array detection quantification method for 11 cannabinoids was developed.

**Abstract:** In this study, the optimal setup of supercritical fluid extraction (SFE) was designed and developed, leading to the quantitation of 11 distinct cannabinoids (cannabidivann (CBDV), tetrahydrocannabivann (THCV), cannabidiol (CBD), cannabigerol (CBG) cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabinol (CBN), delta 9-tetrahydrocannabinol (Δ9-THC), delta 8 tetrahydrocannabinol (Δ8-THC), cannabichomere (CBC) and delta 9-tetrahydrocannabinol acid (THCA-A)) extracted from the flowers of medicinal cannabis (sp. *Sativa*). Supercritical carbon dioxide (scCO2) extraction was performed at 37 ◦C, a pressure of 250 bar with the maximum theoretical density of CO2 (893.7 kg/m3), which generated the highest yield of cannabinoids from the flower-derived extract. Additionally, a cold separator (separating chamber) was used and positioned immediately after the sample containing chamber to maximize the yield. It was also found that successive washing of the extract with fresh scCO2 further increased yields. Ultra-high performance liquid chromatography coupled with DAD (uHPLC-DAD) was used to develop a method for the quantification of 11 cannabinoids. The C18 stationary phase was used in conjunction with a two solvent system gradient program resulting in the acquisition of the well-resolved chromatogram over a timespan of 32 min. The accuracy and precision of isolated cannabinoids across inter-and intra-day periods were within acceptable limits (<±15%). The assay was also fully validated and deemed sensitive from linearity, LOQ, and LOD perspective. The findings of this body of work are expected to facilitate improved conditions for the optimal extraction of select cannabinoids using scCO2, which holds promise in the development of well-characterized medicinal cannabis formulations. As to our best knowledge, this is the first study to report the uHPLC quantification method for the analysis of 11 cannabinoids from scCO2 extract in a single run with more than 1 min peak separation.

**Keywords:** cannabis flowers; neutral cannabinoids (sp. *Sativa*); supercritical extraction; supercritical carbon dioxide (scCO2); SFE Nottingham unit; SFE Helix unit

## **1. Introduction**

Cannabis is considered a highly promising medicinal plant due to its purported array of therapeutic properties, although according to a recent survey it is most commonly used

**Citation:** Qamar, S.; Manrique, Y.J.; Parekh, H.S.; Falconer, J.R. Development and Optimization of Supercritical Fluid Extraction Setup Leading to Quantification of 11 Cannabinoids Derived from Medicinal Cannabis. *Biology* **2021**, *10*, 481. https://doi.org/10.3390/ biology10060481

Academic Editors: Francisco Les, Víctor López and Guillermo Cásedas

Received: 29 March 2021 Accepted: 10 April 2021 Published: 28 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

as an illicit drug [1]. It contains a variety of phytochemicals (around 500 compounds) including sugars, cannabinoids, alkaloids, phenolic compounds, and terpenes [2]. However, the psychoactive and psychotropic property of cannabis is particularly related to the presence of cannabinoids. In medicinal cannabis, cannabidiol (CBD), and tetrahydrocannabinol (THC) are most commonly occurring cannabinoids. From an industrial point of view, CBD is currently considered the most valuable cannabinoid as it possesses a broad range of therapeutic properties, such as anticonvulsant, anxiolytic, neuroprotective, antibiotic, anti-inflammatory activity, and anti-oxidant [3,4]. That said cannabinol (CBN), cannabigerol (CBG), and cannabichromene (CBC) have also shown antifungal, antibacterial, anti-inflammatory, and analgesic properties [5].

The therapeutic properties in natural products are also due to the presence of various phytochemicals, such as glucosinolates, lignans, carotenoids, polyphenols, etc. [6]. Therefore, there is a growing interest to adopt the "natural" or alternative approaches to cure so-called lifestyle diseases, rather than using pharmacologic therapy. The use of natural products for the treatment or to prevent the diseases to gain the healthy lifestyle is progressing [7].

The supercritical fluid extraction (SFE) method has gained increasing interest as a means of extracting cannabinoids from cannabis due to its selective extraction, short processing time, low running cost, and low impact on the environment, compared to conventional solvent-based extraction methods. SFE is a process in which the supercritical fluid (SCF) separates or dissolves components from the plant matrix according to their solvating properties. The solvating property of extracting the component can be maintained by changing the temperature and pressure above the critical point. Therefore, due to the tunable nature of SCF it can only target the desired substance from the sample matrix [8]. Additionally, the design of the SCF extracting chamber also plays an important role in the interaction of SCF substance with a targeted analyte. Proper experimental design can also maximize the yield of the targeted component with high purity. Various theoretical and physical factors (such as inlet and outlet valves of SCF into the extracting chamber, and separating chamber) also participates simultaneously to obtain the high yield. Furthermore, managing the pressure and throttling effect of SCF during extraction collection can enhance the extractability of SCF [9,10].

After the extraction of cannabinoids, the fast and reliable quantification method is an essential step of the analysis. Gas chromatography (GC) is considered as the most useful quantifying and separating technique for the analysis of cannabinoids as it considers a simpler and faster technique compared to high-performance liquid chromatography (HPLC) [11]. However, during GC analysis, acidic cannabinoids convert into their neutral form due to the thermal effect. Therefore, the proper quantification of cannabinoids through GC derivatization step is necessary [12]. HPLC is also considered as the simplest method to analyze the cannabinoids from the cannabis plant and other matrixes, as it does not require high heating step for analysis of the cannabinoids. Therefore, previously a number of HPLC methods have been developed for the determination of cannabinoids [13].

Recent surveys have shown that cannabinoids quantification assays via HPLC focused on the analysis of main cannabinoids (THCA, THC, CBN, CBD, and CBDA) in a single run [14]. However, these methods were either not validated properly or unable to perform the efficient separation of cannabinoids [12,15,16]. Because of the complex nature of the plant extract, the major cannabinoids peaks overlap (such as, CBGA/CBN, and CBG/CBD), which affect the analysis [3].

Previously, a number of studies focused on the SCF conditions for the extraction of cannabinoids. However, the setup of SCF is equally important to gain a high yield of cannabinoids. Therefore, this study was aimed to develop a setup for the SCF extraction of cannabinoids with high yield at optimal operating conditions from cannabis plant material. In addition, reversed-phase uHPLC-DAD quantification assay was developed for the effective quantification of 11 main cannabinoids and their acids with good peak separation.

#### **2. Material and Methods**

#### *2.1. Chemical and Reagents*

Eleven cannabinoids, namely cannabidivann (CBDV), tetrahydrocannabivarin (THCV), cannabidiol (CBD) with 99.66% purity (Lot: FE08071702), cannabigerol (CBG) with 98.98% purity (Lot: FE06241604), cannabidiolic acid (CBDA) with 98.3% purity (Lot: FE12011601), cannabigerolic acid (CBGA), cannabinol (CBN) with 99.37% purity (Lot: FE06131701), delta 9-tetrahydrocannabinol (Δ9-THC) with 97.66% purity (Lot: FE1041701), delta 8 tetrahydrocannabinol (Δ8-THC), cannabichomene (CBC) with 97.60% purity (Lot: FE10011502), and delta 9-Tetrahydrocannabinol acid (THCA-A) with 99.18% purity (Lot: FE12121601) stock solution with the concentration of 1000 μg/mL in acetonitrile or methanol (acid forms) as reference standards were purchased from Cerilliant, a Sigma Aldrich company (Kinesis Australia Pty Ltd., Redland Bay, QLD, and Novachem Pty Lt, Victoria, Australia). All other solvents (methanol, acetonitrile, and phosphoric acid) were purchased from Merck.

## *2.2. Sample Collection*

Cannabis material has been obtained by the School of Pharmacy, The University of Queensland, under Queensland Health Approval license UNIR008335019; cannabis strain 1 (cannabidiol dominant; had <10% *w/w* of total cannabinoids and among them 90% *w/w* cannabinoids were CBD and CBDA, whereas, THC and THCA were around 5% *w/w*), and strain 2 flower material (had around 14% *w/w* total cannabinoids and among them ~50% *w/w* cannabinoids were cannabidiol and ~45% *w/w* cannabinoids were tetrahydrocannabinol, referred to as the 'balanced strain'). These cannabis samples with Sativa genotype were planted on 4th May, 2017 under the best-growing conditions (12 to 18 h light exposure at 23 ◦C). Cannabis sample (from plant flowers) collected at the fluorescence stage and dried for 5 to 8 days at 20 ◦C (with total moisture <10%). This sample was pulverized for 2 min in a coffee grinder (Breville, model BCG200) to obtain a particle size of <2.7 mm. The schematic representation of the cannabis sample preparation and analysis is shown in Figure 1.

**Figure 1.** The schematic representation of the cannabis sample preparation and analysis.

#### *2.3. The Supercritical Fluid Extraction (SFE) and Setup Optimization*

In this study, the supercritical fluid extraction (SFE) of cannabinoids was performed using scCO2. The density of CO2 plays an import role for the extraction of selective components from cannabis. Previously, Span and Wagner [15] studied the thermodynamic properties of CO2 at various states and found that at 250 bar and 37 ◦C it attained a high density (893.7 kg/m3) in a supercritical state. That is why this study was performed at 250 bar and 37 ◦C for 3 h to obtain a better extraction of cannabinoids from cannabis. Furthermore, two different SFE operating systems were used for studies to evaluate the optimal set-up for extraction.

## *2.4. Nottingham Unit*

The SFE Nottingham unit (Teledyne ISCO, D-series) was used for the experimental designs A to D. The assembly of Nottingham unit was based on the liquid CO2 cylinder and syringe compressor to convert into scCO2. However, one main upgrade of the experimental design was the sample holding chamber, and their inlet and outlet positions for CO2 were designs in the lab. This demonstrates the uniqueness of the designed experimental setup and has led to novel extraction results, which are not reported in literature in the authors' best knowledge. The maximum sample holding capacity of SFE extraction stainless steel vessel for Nottingham unit was 60.0 mL. The syringe pump attached to the liquid CO2 cylinder can hold up to 250 mL of liquid CO2 (60 bar) and regulate the desired pressure in extracting chamber. Glass wool and a stainless-steel filter was used on the top of sample holding reactor to separate the extract from grinded plant material.

## 2.4.1. Experimental Setup A

The 1.0 g sample of cannabis strain 1 was placed inside the extracting chamber and filled with CO2. The temperature of the extracting chamber was controlled by using a heating jacket to obtain the desired density of CO2 in sub or supercritical state. The liquid CO2 dissolved the matrix from the sample according to its density. An overhead stirrer was also used (200 rpm) to help the proper dissolution of a matrix (as represented in Figure 2A). After extraction, the extracting vessel was removed from the SFE extraction system and reverted into collecting vessel to acquire a maximum amount of extracting material with the help of gravity, as shown in Figure 2B.

**Figure 2.** Schematic representation of the SFE Nottingham unit for the experimental setup A (**A**); Sample containing chamber was inverted and placed on stirrer for the depressurization (**B**).

## 2.4.2. Experimental Setup B

To increase the yield of cannabinoids from the cannabis sample by using SFE, the extraction chamber was rewashed with fresh CO2. In this procedure, the sample containing chamber was firstly filled with CO2 (at 250 bar, 37 ◦C) for 3 h to complete the extraction. After extraction, it was depressurized from 250 bar to 100 bar in the extract collecting chamber, as shown in Figure 2, and then the pressure was maintained again at 250 bar with fresh CO2 through a pressure regulating syringe. After 30 min, the sample holding chamber was disconnected from a back pressure regulator and again depressurize to 100 bar. After collecting the extract, the sample holding chamber was refilled again with fresh CO2 at 250 bar to avoid the super-saturation of CO2 from cannabinoids. This extracting chamber was finally fully depressurized at 0 bar after 30 min of extraction. The safety valve was removed and washed with methanol (5 mL) to collect the extract stuck to it.

## 2.4.3. Experimental Setup C

In this experimental design, the extraction of cannabinoids was also performed at 250 bar and 37 ◦C for 3 h. However, the depressurization or washing of the sample after extraction was performed three times. In this process, after 3 h of extraction, the sample holding chamber was removed from the back pressure regulating syringe pump and inverted into a sample collecting chamber with the help of the stand, as presented in Figure 2B. The sample extracting chamber was fully depressurized at 0 bar. After depressurization, the safety valve was removed and washed with 5 mL methanol to obtain the extract if it stuck to the valve. This sample holding chamber was attached again to the pressure regulating syringe pump. The pressure and temperature were maintained again at 250 bar and 37 ◦C. After 30 min of extraction, depressurization was performed again. The safety valve was also removed and washed with methanol as there was no pressure inside the chamber. After refilling CO2, full depressurization of the sample holding chamber and washing of the rod with methanol was repeated similarly again to maximize the yield of the cannabinoids.

#### 2.4.4. Experimental Setup D

In this procedure the operating conditions of extraction were similar (250 bar, 37 ◦C, 3 h) as performed in earlier experiments. However, the glass wool was not used and depressurization of sample holding chamber was performed in an upward direction (as shown in Figure 2A). The full depressurization (at 250 bar to 0 bar) was acquired after 30 min of CO2 refilling. Overall, rewashing of the sample was performed three times (first after 3 h, second after 30 min and third after 30 min). After complete extraction, the safety valve was opened and washed with 5 mL methanol.

## *2.5. Helix Unit*

The SFE Helix unit (applied separations) was used for the experimental setup B. The maximum sample holding capacity of the helix stainless steel sample holding chamber was 100 mL. The desired internal temperature was monitored by the heating jacket. For the Helix unit, the maximum operating temperature and pressure were 60 ◦C and 700 bar. The back pressure was directly regulated by the preconditioning chamber from the liquid CO2 cylinder, as shown in Figure 3.

**Figure 3.** Schematic representation of the Helix unit for setup E extraction.

In this experiment, the SFE of the cannabis sample was performed at 250 bar and 37 ◦C for 3 h. The 1 gm grinded cannabis flowers sample strain 1 was placed on the bottom of the chamber. The CO2 stream was entered from the bottom inlet of the chamber and extraction was carried out for 3 h. In addition, there were two built-in filters on the bottom and top end of the sample holding cylinders to separate the plant material and CO2 from the chamber. After extraction, the CO2 with the dissolved matrix entered into the separating chamber, where the pressure was around 50 bar to avoid the throttling effect of dry ice (as represented in Figure 3). The extract was collected in the sample collecting vessel, attached to the bottom of the separating chamber. The weight of the extract collecting vessel was measured before and after extraction to estimate the yield of the extract. The sample was washed with the continuous flow of CO2 for 10 min and a dry sample was collected in the sample collecting vessel.

The SFE Helix unit (applied separations) was used for the experimental setup E to G. Similarly to the assembly of the Nottingham unit, the Helix unit was originally based on a liquid CO2 cylinder and preconditioning chamber to convert into scCO2. However, the sample holding chamber and their inlet and outlet positions for CO2 were designs in the lab. The maximum sample holding capacity of the helix stainless steel sample holding chamber was 100 mL. The desired internal temperature was monitored by the heating jacket. For the Helix unit, maximum operating temperature and pressure was 60 ◦C and 700 bar. The back pressure was directly regulated by the preconditioning chamber from the liquid CO2 cylinder, as shown in Figure 3.

#### 2.5.1. Experimental Setups E and F

The SCF extraction without using a separating chamber was performed in two different methods. In the first method, the CO2 stream was entered from the top of the sample holding chamber (as illustrated in Figure 2). However, all the other experimental conditions were similar, as performed in setup E experiment. After 3 h of extraction, the extract was collected from the bottom of the sample extracting chamber in a collection chamber. The weight of extract collecting vessel was measured before and after extraction to estimate the yield of extract.

## 2.5.2. Experimental Setup G

In this experiment, the SFE of the cannabis sample was performed at 250 bar and 37 ◦C for 3 h. The 1 gm ground cannabis sample was placed on the bottom of the chamber. The CO2 stream was entered from the bottom inlet of chamber and extraction was carried out for 3 h. In addition, there were two built-in filters on the bottom and top end of the sample holding cylinders to separate the plant material and CO2 from the chamber. After extraction, the CO2 with the dissolved matrix entered into the separating chamber (as shown in Figure 4), where the pressure was around 50 bar to avoid the throttling effect dry ice. The extract was collected in the sample collecting vessel, attached to the bottom of the separating chamber. The sample was washed with the continuous flow of CO2 for 10 min and the extract was collected in the sample collecting vessel.

**Figure 4.** Schematic representation of the Helix unit for setup B extraction.

### *2.6. uHPLC-DAD Quantification*

The uHPLC method was initially developed by Shimadzu Scientific Instruments and transferred to PACE with a loan of a Prominence-I LC-2030 C3D liquid chromatography unit, with a Shim-pack XR ODS-II (2.20 μm, 3.0 mm ID × 75 mm).

## 2.6.1. Standard Solution Preparation

The concentrated solution of each cannabinoid standard (1000 μg/mL) was diluted in methanol to make 250 μg/mL as a stock solution. The calibration curve of mixed standards with each cannabinoid at 1.0 to 25.0 μg/mL concentrations was prepared in methanol by using the cannabinoids stock solution. All standards solutions were stored at −80 ◦C.

#### 2.6.2. Instrumentation

Shim-pack XR-ODSII, spherical silica particles, 2.2 μm particle size (Shimadzu Scientific) reversed-phase C18 chromatographic column was used for the separation of cannabinoids. The quantitation method was standardized using Lab Solutions software or Cannabis Analyser (Shimadzu Scientific Instruments, Sydney, NSW, Australia).

#### 2.6.3. Mobile Phase Elution Program

The mobile phase A was the mixture of MilliQ water and phosphoric acid (millimolar; mM) (0.07% H3PO4/99.93% MilliQ H2O; adjusted to pH between 2.22 to 2.26). Mobile phase B was the mixture of methanol and phosphoric acid (mM; 0.07% H3PO4/99.92% methanol; adjusted to pH 2.43 to 2.48). The column oven temperature was maintained

at 50 ◦C and the flow rate was 1.0 mL/min to maintain the column pressure (~5400 to 5600 psi). The volume of injection was 10 μL and the total runtime was 45 min. Initially, mobile phase B (*v/v*) was adjusted at 65% for 1 min. Then, the percentage of mobile phase B was gradually increased from 65% to 72% over the 25 min time period. After that, it finally increases to 95% during a 5 min time period. After maintaining these conditions for 2 min the initial ratio of mobile phases was adjusted and re-equilibrated the column for 12 min.

## *2.7. uHPLC Method Validation*

## 2.7.1. Selectivity/Identification

To identify the specific cannabinoid in a mixture, the retention time of each standard cannabinoid was ensured separately. For this purpose, the complete UV-visible spectra of each cannabinoid were recorded and compared with the retention time of the mixture.

## 2.7.2. Precision and Accuracy

The accuracy, repeatability, and intermediate precession of the method were determined by preparing the three samples of each low medium and high concentration (2.5, 10, and 20 μg/mL) of 11 cannabinoids standard mixture on inter and intra days.

## 2.7.3. Linearity

The linearity of the method was demonstrated by preparing seven different concentrations of standard mixture solution in methanol (containing 11 cannabinoids) from 1.0 μg/mL to 25 μg/mL. The selected range of calibration curves was plotted in triplicates on three consecutive days. The regression of the coefficient (*r2*-value) of each calibration curve was calculated to determine the linearity of the method.

## 2.7.4. Limit of Detection (LOD) and Limit of Quantitation (LOQ)

LOD and LOQ were analyzed by plotting a calibration curve within the range of seven different non-zero detection limit (DL) and quantitation limit (QL) values. The DL and QL of all 11 cannabinoids were calculated by using the following formulas:

$$DL = \frac{\text{3.3 } \sigma}{\text{S}} \,\text{s} \tag{1}$$

$$QL = \frac{10\,\sigma}{\text{S}},\tag{2}$$

where σ = response of standard deviation and S = slope of the calibration curve.

## *2.8. Statistical Analysis*

All analyses were performed in triplicates. The statistical analysis was obtained by using Minitab 17 software.

## **3. Result and Discussion**

Due to the complexity and lack of knowledge of SFE factors interactions and indepth fluid dynamics, SFE is considered a black box design. However, by exploring different experimental parameters, extraction principles and detailed point-to-point process information can produce favorable results [8]. Optimization of the setup to obtain fruitful results is the first stage of every experimental design. Therefore, this study was conducted to design the best setup for the extraction of cannabinoids from cannabis by using SFE. For this study, two different units of SFE were used for the extraction, the Nottingham unit, and the Helix unit. The results are represented in Table 1.


**Table 1.** Conditions for SCF extraction of cannabinoids from cannabis.

#### *3.1. Nottingham Unit*

The Nottingham unit was used for experimental setup A and the performed conditions are presented in Table 1. The extraction of cannabinoids from the cannabis sample occurred at 37 ◦C and 250 bar. These conditions were selected to obtain the maximum density of CO2. Recent studies only focused on the temperature and pressure of CO2 for SFE. It was reported that the adoption of very high pressure (up to 500 bar) decreases the selectivity of the cannabis extract [16], because above 250 bar the vapor pressure of the solute also increases. That is why at high pressures, high temperature has a greater influence on the solubility than the density [17]. Therefore, in this study, a carefully low temperature was used to increase the mass transfer rate of cannabinoids from plant to CO2.In experimental setup A, the glass wool and stainless-steel filter was used for the proper separation of cannabinoids scCO2 extract from original plant material. However, during the depressurization of the scCO2 extract (from 250 bar to atmospheric pressure) it was found that all extracted material (oil) was soaked in glass wool and the yield was quite low (as represented in Figure 5A). To avoid the extract soaking in glass wool and to improve the extraction, experimental setup B was designed.

**Figure 5.** Schematic representation of glass wool after experimental setup A (**A**) and SFE Nottingham unit extraction (**B**).

In experimental setup B, a comparatively small amount of glass wool and 2 stainless steel filters were used to avoid the contamination of the extract from original ground material. Additionally, the depressurization was performed in triplicates (first; from 250 bar to 100 bar, second; after 30 min extraction with fresh CO2, from 250 bar to 100 bar, third; again, filled up with fresh CO2 for 30 min and fully depressurized from 250 bar to 0 bar). As a result, a sharp increase in the total yield of the extract was observed in experimental setup B (127.67 mg) as compared to experimental setup A (26.70 mg). Furthermore, the soaking of extract in glass wool also decreases markedly (Figure 5B).

After the inspiration of experimental set-up B results, experimental setup C was designed, in which full depressurization from 250 bar to 0 bar was performed in triplicates. From the results, it was shown that the total yield and the % of cannabinoids were increased two-fold as compared to experimental set-up B (as shown in Table 1). However, the two main issues were observed. Contamination with original material was not fully resolved and high leakage of cannabinoids on the safety valve and glass wool was found.

To resolve these main issues, the glass wool was fully removed in experimental set-up D and the experiment was performed only using stainless steel filters. However, from the results, it was cleared that the total yield decreased sharply as compared to experimental setups B and C. That is why a further study was performed on Helix unit of SFE as it has built-in filters and contamination chances with original plant material were almost zero or very low.

#### Helix Unit

After Nottingham, the Helix unit has been used to obtain the high yield of scCO2 extract with a high amount of cannabinoids from cannabis. For this purpose, experimental setups E to G were designed, as shown in Figures 3 and 4. Experimental setup E was very simple because the built-in stainless filter was placed on both ends of the sample holding the chamber. The CO2 was entered inside the sample holding chamber from the top inlet valve and the sample was placed in a stainless steel cone in the bottom of the sample holding chamber (Figure 6B). However, after the depressurization from the bottom inlet, it was found that the stainless steel filter gets blocked and the plant material stuck on the filter, as illustrated in Figure 6A. Therefore, the extract was not collected.

**Figure 6.** Schematic representation of bottom floor of the sample holding chamber in Helix unit. (**A**): bottom filter was blocked during depressurization of CO2 from bottom outlet. (**B**): cannabis sample placed at the top of cone.

To figure out this issue, experimental setup F was designed, in which the sample was also placed on the stainless steel cone in the bottom of the sample holding chamber. However, the stream of CO2 entered from the bottom inlet and depressurization from the top outlet (Figure 4). After the depressurization, it was observed that due to the sudden drop in pressure (250 bar to 0 bar), the throttling process occurred, as presented in Figure 7. As a result, the total yield of scCO2 extract was very low (14.20 mg, Table 1).

**Figure 7.** Throttling effect during the extract collection, (**A**): dry ice formation at sudden drop in pressure (250 bar to 0 bar), (**B**): Collection of extract at low pressure drop (50 bar to 0 bar).

Therefore, to improve the setup of SFE extraction with Helix unit, experimental setup F was designed, un which after the sample holding chamber (maximum size 100 mL), the low-pressure regulating chamber/separating chamber (maximum holding pressure 100 bar) was adjusted. As a result, the good yield (53.92 mg, Table 1) was obtained after single depressurization.

## *3.2. Ultra-High-Performance Liquid Chromatography Coupled with DAD (uHPLC-DAD)*

uHPLC with DAD is considered as the most accurate and simple method for the quantification of cannabinoids. Because it is easy to perform on a routine basis, it efficiently separates the analyte and does not degrade the sample during quantification [18]. Several studies developed methods for the quantification of cannabinoids. Such as, De Backer, Benjamin [19] designed and validated a method to analyze the eight cannabinoids by using three chemotypes (including, fiber-type, intermediate-type, and drug-type) extracts (chloroform: methanol; 1:9 *v/v*) of the cannabis plant. Additionally, this method was validated with a clearly separated HPLC profile. In another study Ciolino, Ranieri [20] developed a new HPLC-DAD quantitation method to determine the 11 cannabinoids in cannabis samples by using two different columns. The analytical column, ACE 5 C18-AR (250 mm × 4.6 mm ID, 5 μm) gives better separation than the conventional c-18 column. The isocratic mobile phase system for ACE 5 C18-AR and Luna C-18 was 34: 66 and

26:74 for 0.5% acetic acid: acetonitrile. The total run time was 50 min and the figures of chromatogram are shown in Figure 8.

**Figure 8.** HPLC profile of 11 cannabinoids using an analytical column, (**A**) ACE 5 C18-AR (250 mm × 4.6 mm ID, 5 μm) and (**B**) conventional c-18 column [20]. Elution order: 1-CBDV, 2-CBDA, 3-CBGA, 4-CBG, 5-CBD, 6-THCV, 7-CBN, 8-d9THC, 10-CBC, and 11-THCA. Reprinted with permission from Ref. [20]. Copyright 2021 Copyright Ciolino.

However, only five cannabinoid compounds were validated (CBN, THCA, CBDA, d9THCA, and CBD). These cannabinoids were also scrutinized in cannabis oils, extracts, plant, and their commercial products. In different states (free-flowing liquids or viscous compounds, semisolids, solids, emulsions, dispersions, aqueous and non-aqueous solutions) and polarities such as polar foodstuffs (beverages and sugary foods) nonpolar products (butter, balms/certain ointments), and substances with intermediate polarities (oral supplements and many topical foods).

HPLC analysis of 11 cannabinoids cannabis extract and biomass was also performed by Gul, Gul [21]. The mobile phase system was gradient (water and acetonitrile with 0.1% formic acid). The separation chromatogram was obtained by using Luna C-18 column at 220 nm in 22.2 min run time. The elution order is also represented in Figure 9. This is quite similar to Figure 8 but overall, the efficiency of the separated peaks was low. Whereas, this method was validated for all selected cannabinoids and their concentration was also measured in 13 various samples of cannabis.

**Figure 9.** HPLC profile of 11 cannabinoids by using Luna C-18 analytical column [21]. Elution order: 1-I.S, 2-CBDA, 3-CBGA, 4-CBG, 5-CBD, 6-THCV, 7-CBN, 8-d9THC, 9-d8THC, 10-CBL, 11-CBC, and 12-THCA.Reprinted with permission from Ref. [21]. Copyright 2021 Copyright Gul.

McPartland, MacDonald [22] used reversed-phase HPLC to investigate the binding affinity of THC and its acidic precursor THCA-A with CB1 and CB2 receptors in humans. In this method, the C18 column was used with a linear gradient mobile phase system and the chromatogram was obtained within 25 min. However, this study only focused on the stability of non-psychoactive cannabinoids (THCA-A) and their binding capability in human body receptors. However, the study revealed a greater binding affinity of THC CB1 (62-fold) and CB2 (125-fold) as compared THCA-A.

Various other studies developed methods to determine six to seven main cannabinoids and their acidic precursors by using HPLC. However, these methods are not validated. Such as Romano and Hazekamp [23] had been used preheated cannabis (with 19% THC) extracts in olive oil, olive oil with water, ethanol, petroleum ether naphtha for the quantification of cannabinoids through HPLC.

Therefore, this study aimed to develop a new method for the quantification of 11 main cannabinoids in cannabis and its derived products with good peak separation. In this study, psychoactive and non- psychoactive neutral cannabinoids and their acidic form were separated in 32 min (Figure 10).

## *3.3. Method Validation*

## 3.3.1. Selectivity/Identification

The peaks of all 11 cannabinoids were fully separated during 32 min of program run. The retention time of each cannabinoid is shown in Table 2 and presented in Figure 10. To identify each peak of cannabinoid in a standard mixture, all cannabinoids were analyzed separately. Their elution order, retention time, and sensitivity were also confirmed through system suitability. It was also noticed that the pH of both mobile phases plays a very important role in stable separation and in maintaining a good retention time.

**Figure 10.** *Cont*.

**Figure 10.** Peaks identification of eleven cannabinoids. (**a**) uHPLC profile of CBDV; (**b**) uHPLC profile of THCV; (**c**) uHPLC profile of CBD; (**d**) uHPLC profile of CBG; (**e**) uHPLC profile of CBDA; (**f**) uHPLC profile of CBGA; (**g**) uHPLC profile of CBN; (**h**) uHPLC profile of Δ9-THC; (**i**) uHPLC profile of Δ8-THC; (**j**) uHPLC profile of CBC; (**k**) uHPLC profile of THCA-A; (**l**) uHPLC profile of 11 cannabinoid mixture.

**Table 2.** System suitability for higher concentration (20 μg/mL) of 11 cannabinoid standard mixtures.


#### 3.3.2. Precision and Accuracy

The precision and accuracy of intra and inter days are represented in Table 3. The method for each cannabinoid was validated at three different levels of concentration, including low (2.5 μg/mL), medium (10 μg/mL), and high (20 μg/mL) as shown in Table 3. The %RSD of all selected 11 cannabinoids for the intra-day varied from 1.60% to 3.37% and for the inter-day from 0.20% to 1.75% respectively. Similarly, the variations in the accuracy level of each cannabinoid were also in an acceptable range. For the intra-day, the accuracy level for the low limit varied from 91.2 to 103.0 μg/mL, for the medium limit from 101.9 to 103.0 μg/mL, and the higher limit from 97.7 to 104.1 μg/mL. Whereas, for the inter-day, the accuracy level for the low limit varied from 88.26 to 99.6 μg/mL, for the medium limit from 101.12 to 103.04 μg/mL, and for the higher limit from 96.8 to 106.0 μg/mL. They are in the acceptable limit of 85.0 to 115.0% (±15%), except for LOQ 80.0 to 120.0% (±20%).

### 3.3.3. Linearity, the Limit of Detection (LOD), and Limit of Quantitation (LOQ)

The sensitivity of the method was obtained by determining the linearity, LOD, and LOQ (results are represented in Table 4). The obtained LOD of this analytical method ranged between 0.27 to 0.51 μg/mL, showing that a very low quantity of cannabinoids in extract can be measured by this method, without any guarantee of the imprecision or bias in the result of this assay.


**Table 3.** Precision and accuracy of 11 cannabinoids on intra-day and inter-day.

A calibration curve of 11 cannabinoid standard mixtures was performed to evaluate the concentration of cannabinoids in unknown samples or ground plant material. The calibration curve was conducted in triplicates on three consecutive days, with the stable, linear, and *r2*-value always >0.99 for each standard. Additionally, from the results of LOD (0.27 to 0.51 μg/mL) and LOQ (0.92 to 1.71 μg/mL), it was shown that the method was sensitive. The obtained LOD and LOQ values of cannabinoids were also comparable with previously developed methods [3,21].


**Table 4.** Linearity, the limit of detection (LOD), and limit of quantitation (LOQ).

Where, Δ9-THC is *Delta-9-tetrahydrocannabinol* and Δ8-THC is *Delta-8-tetrahydrocannabinol*.

#### *3.4. Analysis of Cannabinoids*

Two different cannabis strains were used for the SCF extraction (as shown in Table 5) and their chromatogram is also represented in Figure 11, in which strain 1 was CBD + CBDA dominant (around 90% *w/w*) and strain 2 had an almost equal amount of CBD + CBDA (50% *w/w*) and THC+THCA (45% *w/w*) as compared to other cannabinoids. The results of well cannabis strains from cannabis strain extracts obtained from the final SFE setup are also presented in the table.

**Figure 11.** uHPLC profile of cannabis strains: (**A**) with dominant CBD + CBDA (around 90% *w/w*); and (**B**) with 55% *w/w* CBD+CBDA and 35% THC + THCA in the cannabinoid mixture.


**Table 5.** Amount of cannabinoids in the SCF extract of two different cannabis strains.

## **4. Conclusions**

The optimal setup configuration of supercritical fluid extraction (SFE) was achieved by systematically changing the inlet and outlet valve position of SFE for CO2 entrance and depressurization. However, scCO2 extraction conditions were fixed at 250 bar, 37 ◦C, 180 min, and 1 g plant material, to measure the cannabinoids yield during setup optimization. Additionally, the purity of the extract was also increased by using stainless steel built-in filters and an additional separating chamber. Furthermore, for the quantification of neutral cannabinoids and their acids, a highly sensitive reverse-phase uHPLC-UV-DAD method was developed. All selected cannabinoids showed good separation over the 32 min runtime (45 min with re-equilibration). Their relative retention time was also strongly influenced on the pH of mobile phases and the operating pressure of the column. The method was validated by analyzing the linearity, LOQ, LOD, accuracy, and precision in triplicates on inter and intra-day according to US-FDA guidelines.

**Author Contributions:** Conceptualization, S.Q., J.R.F. and H.S.P.; methodology, S.Q., J.R.F. and H.S.P.; validation, S.Q., J.R.F., Y.J.M. and H.S.P.; formal analysis, S.Q., J.R.F., Y.J.M. and H.S.P.; writing original draft preparation, S.Q. and J.R.F.; visualization, S.Q. and J.R.F.; project administration, S.Q., J.R.F., Y.J.M. and H.S.P.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Sadia Qamar is a recipient of the Australian Government Research Training Program Scholarship from The University of Queensland, Brisbane, Australia. The authors also thank Andrew K. Whittaker of the Australian Institute for Bioengineering and Nanotechnology (AJBN) and Professor Kristofer Thurecht at the Centre for Advanced Imaging, The University of Queensland. Brisbane, QLD 4072 Australia, for their support and access to specialized equipment including the CO2 unit used in this search. AU the authors also acknowledge support from the School of Pharmacy, The University of Queensland.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article Jasonia glutinosa* **(L.) DC., a Traditional Herbal Tea, Exerts Antioxidant and Neuroprotective Properties in Different** *In Vitro* **and** *In Vivo* **Systems**

**Francisco Les 1,2,\*,† , Marta Sofía Valero 2,3,4,† , Cristina Moliner <sup>1</sup> , David Weinkove 5, Víctor López 1,2 and Carlota Gómez-Rincón <sup>1</sup>**


**Simple Summary:** *Jasonia glutinosa* (L.) DC or rock tea (RT) is a plant traditionally used to treat different pathologies. In this study the neuroprotective potential of an ethanolic extract of RT is analyzed. Caenorhabditis elegans model and *in vitro* assays with relevant central nervous system enzymes were used. The results showed antioxidant and neuroprotective potential of this plant.

**Abstract:** In traditional medicine, *Jasonia glutinosa* (L.) DC or rock tea (RT) has been mainly used to treat digestive and respiratory pathologies but also as an antimicrobial or an antidepressant herbal remedy. An ethanolic extract of RT has been demonstrated to have antioxidant and anti-inflammatory effects, which may be explained by its phytochemical profile, rich in polyphenols and pigments. The aim of this study is to investigate the neuroprotective potential of RT. For this purpose, the ethanolic extract of RT is assayed in *Caenorhabditis elegans (C. elegans)* as an *in vivo* model, and through *in vitro* assays using monoamine oxidase A, tyrosinase and acetylcholinesterase as enzymes. The RT extract reduces juglone-induced oxidative stress in worms and increases the lifespan and prevents paralysis of *C. elegans* CL4176, a model of Alzheimer's disease; the extract is also able to inhibit enzymes such as acetylcholinesterase, monoamine oxidase A and tyrosinase *in vitro*. Together these results demonstrate that *Jasonia glutinosa* is a good candidate with antioxidant and neuroprotective potential for the development of new products with pharmaceutical interests.

**Keywords:** medicinal plants; *C. elegans*; acetylcholinesterase; monoamine oxidase A; tyrosinase; herbal medicine; lifespan

## **1. Introduction**

*Jasonia glutinosa* (L.) DC. (Compositae), whose popular name is "té de roca" (rock tea, RT), is a medicinal plant distributed in Mediterranean countries, mainly Spain and France [1]. Ethnobotanical studies on this plant report that its main use, as an infusion, is for the treatment of diarrhea, dyspepsia or abdominal pain [2]. These digestive properties were demonstrated for the first time in a murine model of colitis [3,4]. In these studies, the oral administration of an ethanolic extract of RT (5, 25 and 50 mg/kg) ameliorated colitis symptomatology, prevented the macroscopic damage and histological changes induced by dextran sulfate sodium in mice and normalized the intestinal contractibility and the intestinal total transit disrupted by the colitis [3,4].

**Citation:** Les, F.; Valero, M.S.; Moliner, C.; Weinkove, D.; López, V.; Gómez-Rincón, C. *Jasonia glutinosa* (L.) DC., a Traditional Herbal Tea, Exerts Antioxidant and Neuroprotective Properties in Different *In Vitro* and *In Vivo* Systems. *Biology* **2021**, *10*, 443. https:// doi.org/10.3390/biology10050443

Academic Editor: Alessandra Durazzo

Received: 21 April 2021 Accepted: 14 May 2021 Published: 18 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In addition to its use as a digestive, *J. glutinosa* has been widely used for the treatment of respiratory or infective pathologies, hypertension, pain, emesis or even mood disorders [2,5–7]. Different studies reported an ethnopharmacological use of RT as a "stimulant", capable of improving mood and clearing the mind [6,7].

Some of these effects could be explained by anti-inflammatory [4,8] or antioxidant effects [4,5,9], which are related to its phytochemical composition. A recent *in vivo* study has shown that dietary supplementation with RT (10 or 30%) for 15 or 30 days in sea bream (*Sparus aurata L.*) improves short-term immunostimulatory capacity (15 days) and maintains antioxidant capacity in the long-term (30 days) [10].

Different studies have shown that *J. glutinosa* is rich in polyphenols, terpenes, esters, alkanes, lactones or flavonol glucopyranoside [1,4,9]. High-performance liquid chromatography with a diode-array detector (HPCL-DAD) analysis of the ethanolic extract of RT showed a rich content of phenolic compounds (134.4 mg/g, dry extract) and pigments (0.27 mg/g, dry extract) (Figure 1). Among the 15 phenolic compounds detected, 10 were phenolic acids and 5 flavones. 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid were the phenolic acids most represented (70% of the total phenolic content). The most abundant flavonoid is quercetin-3-Ogalactoside (50% of total flavonoid). In respect to pigments, two carotenoids, chlorophylls and xanthophylls were detected. Lutein represented 55% of the total pigments [4].

**Figure 1.** *J. glutinosa* photography taken by authors and the main phenolic compounds of the extract.

Oxidative stress and inflammation have been linked to the aging process as triggers of various diseases, including neurodegeneration. *J. glutinosa*, with a phytochemical composition rich in polyphenols, has previously demonstrated antioxidant and anti-inflammatory effects, and its use as an antidepressant has also been reported by certain inhabitants of Spain. However, there are no scientific reports showing the potential of RT as a neurotherapeutic agent. Therefore, the objective of this work is to study the antioxidant and neuroprotective activity of RT extract in a *Caenorhabditis elegans (C. elegans)* model, studying the effect of *J. glutinosa* on stress resistance, lifespan and amyloid toxicity and *in vitro* analyzing its ability to inhibit the enzymes of the nervous system such as acetylcholinesterase (AChE), monoamine oxidase A (MAO-A) and tyrosinase (TYR), involved in neurotransmitters' metabolism. These enzymes participate in the elimination of biogenic amines such as acetylcholine, serotonin and catecholamines (dopamine, epinephrine, norepinephrine), which are involved in the development of different pathologies of the nervous system such as Alzheimer's, Huntington's and Parkinson's. Therefore, the inhibitory substances of these enzymes could help the treatment of these diseases.

## **2. Materials and Methods**

#### *2.1. Reagents and Chemicals*

The following chemical reagents: gallic acid, xanthine, NBT (nitroblue tetrazolium), xanthine oxidase, DPPH (2,2 Diphenyl 1 picrylhydrazyl), galantamine, ATCI (acetylthiocholine iodide), DTNB (5,5 -dithiobis-(2-nitrobenzoic acid)), Tris, vanillic acid, 4-aminoantipyrine, horseradish peroxidase, acetylcholinesterase, tyramine, MAO-A, L-DOPA (levodopa) and tyrosinase were obtained through Sigma-Aldrich (Madrid, Spain). Clorgyline and α-Kojic acid were sourced from Cymit quimica (Barcelona, Spain). Na2CO3, HCl, NaCl, Methanol and potassium phosphate were acquired from Panreac (Barcelona, Spain). Juglone (5 hydroxy-1,4-naphthoquinone) and FUdR (5-fluoro-2 -deoxyuridine) were sourced from Alfa Aesar (Ward Hill, MA, USA). *C. elegans* strains and Escherichia coli OP50 were obtained from the Caenorhabditis Genetics Center (CGC, Minneapolis, MN, USA).

## *2.2. Plant Material and Extraction*

*Jasonia glutinosa* ethanolic extract was obtained as described in Valero et al., (2015) [11] and a plant voucher was kept in the Universidad San Jorge (ref. 001-2012). The ethanolic extract was analyzed phytochemically in a previous author's study using HPLC-DAD, determining its composition in phenolic compounds and pigments [4].

## *2.3. Caenorhabditis elegans Studies*

#### 2.3.1. *C. elegans* Strains and Maintenance Conditions

This study used the wild-type strain of *C. elegans* (N2) and a transgenic strain (CL4176 (smg-1 ts 131 (myo-3/Aβ1–42 long 3 -UTR)). The *C. elegans* CL4176 strain contains a temperature-sensitive transgene that expresses the human amyloid peptide β1–42 in muscle, which causes paralysis in worms.

The strains were maintained at 16 ◦C (CL4176) or at 20 ◦C (N2) on nematode growth media (NGM) agar plates seeded with *Escherichia coli* OP50 as a food source. Synchronized worms were obtained using an alkali-bleaching method [12] for the N2 strain and egglaying for the CL4176 strains.

#### 2.3.2. Assessment of Resistance to Lethal Oxidative Stress

Synchronized L1 worms were cultivated in NGM agar plates in the presence of different concentrations of RT extract (5, 10, 20 and 50 μg/mL) or in its absence (control). Under these conditions, the worms were incubated at 20 ◦C until the first day of adulthood. RTtreated and control adult worms were washed with sterile water and transferred to 96-well microplate with NGM agar containing 150 μM juglone (5-hydroxy-1,4-naphthoquinone), which produces lethal oxidative stress. After an incubation period of 24 h at 20 ◦C, survival was evaluated by the response to a mechanical stimulus [13]. The number of alive and dead worms was recorded and the survival rate % (% SR) was calculated:

$$\text{V\% SR} = \text{(N\% of workers alive} \times 100) / \text{Total number of workers} \tag{1}$$

Each concentration was tested in triplicate and 100 nematodes were used per assay.

#### 2.3.3. Lifespan Analysis

The lifespan of the wild-type *C. elegans* (N2) was tested using different concentrations of RT (5, 10, 20, 50 and 100 μg/mL) and compared with untreated controls following the method of Solis and Petrascheck [14]. Synchronized wild-type L1 larvae were transferred to 96-well plates (7–18 worms/well) and were cultured in an S-complete medium containing *E. coli* OP50 (1.2 × 109 bacteria/mL). On the first day of adulthood, FUdR (0.06 mM) was added to sterilize the adults (day 0). RT extracts were added 24 h later. Survival of nematodes was scored every two or three days. The scoring method was the same as used for the juglone oxidative stressed assays. Results are expressed as survival rate % (Equation (1)) and mean lifespan. Each assay was repeated three times and used 150 nematodes per condition.

## 2.3.4. Paralysis Assay

*In vivo* neuroprotective effects of RT extract were evaluated using the strain CL4176 according to previous research protocols [15]. The strain CL4176 was egg-synchronized onto the NGM plates seeded with *E. coli* containing 0, 5, 10 and 25 μg/mL of the RT extract and cultured at 16 ◦C. The temperature was changed to 25 ◦C to induce expression of the amyloid-β (Aβ) transgene 38 h later (day 0). Paralysis was scored twice a day for five days [16]. Worms that exhibited pharyngeal pumping, but did not move, or only moved the head after being touched with a platinum wire, were scored as paralyzed. The test was performed with 100 nematodes per condition.

#### *2.4. Bioassays Regarding CNS Enzymes*

#### 2.4.1. Acetylcholinesterase Inhibition

Ellman's method was performed in 96-well microplates using a microplate reader to measure absorbance as previously described [17]. Each assay well contained a mixture of 25 μL 15 mM ATCI in Milipore water, 125 μL 3 mM DTNB in buffer C (50 mM Tris-HCl, pH = 8, 0.1 M NaCl, 0.02 M MgCl2 6H2O), 50 μL buffer B (50 mM Tris-HCl, pH = 8, 0.1% bovine serum) and 25 μL RT extract at different concentrations (20.00, 10.00, 5.00, 2.50, 1.25 and 0.63 μg/mL) in buffer A (50 mM Tris-HCl, pH = 8). At last, 25 μL AChE enzyme (0.22 U/L) was added to begin the reaction. Controls and blanks were performed, containing buffer A instead of samples and a buffer instead of the enzyme, respectively. Absorbance was read 13 times every 13 s at 405 nm. Galantamine was used as a reference inhibitor.

## 2.4.2. Monoamine Oxidase A Inhibition

The MAO-A activity was performed in a 96-well microplate using a technique previously described [18]. The assay mixture contained 50 μL RT extract at different concentrations (0.0001, 0.0010, 0.0100, 0.1000, 1.0000 and 10.0000 mg/mL), 50 μL chromogenic solution (0.8 mM vanillic acid, 417 mM 4-aminoantipyrine and 4 U/mL horseradish peroxidase in potassium phosphate buffer pH = 7.6.), 100 μL 3 mM tyramine and 50 μL 8 U/mL MAO-A. Controls and blanks were also performed, with solvent instead of samples and a buffer instead of the enzyme, respectively. The absorbance was read at 490 nm every 5 min for 30 min. Clorgyline was used as a reference inhibitor.

## 2.4.3. Tyrosinase Inhibition

The TYR activity was assessed in 96-well microplates using a procedure previously described [19]. The reaction mixture contained 10 μL RT extract at different concentrations (0.001, 0.010, 0.100, 0.500, 1.000, 2.500 and 5.000 mg/mL), 40 μL of L-DOPA, 80 μL phosphate buffer, pH = 6.8 and 40 μL tyrosinase; these were mixed in each well. Controls and blanks were also performed with 50 μL solvent instead of samples and 50 μL buffer instead of the enzyme, respectively. Absorbance was read at 475 nm. α-Kojic acid was used as a reference inhibitor.

## *2.5. Statistical Analysis*

Data were expressed as mean ± SEM and for statistical analysis, GraphPad Prism version 6.0c (GraphPad Software, San Diego, CA, USA) was used. Extract concentration was required to inhibit 50% of the activity of the nervous system enzymes, IC50; this was estimated using non-linear regression. ANOVA following by Tukey's multiple comparisons test were used to evaluate resistance to oxidative stress. Lifespan and paralysis curves were tested using log-rank for significant fit to Kaplan–Meier survival curves. The significance level was set to *p* < 0.05.

#### **3. Results**

#### *3.1. Rock Tea Extract Improved the Stress Resistance of C. elegans*

To evaluate the antioxidant effect of *J. glutinosa*, wild-type *C. elegans* were pre-treated with RT extract (5, 10, 20 and 50 μg/mL) for 24 h and then exposed to a lethal dose of juglone, a natural pro-oxidant. As Figure 2 shows, this treatment resulted in 0.92% ± 0.52 survival of the control group. Pre-treatment with RT extract improved the survival rate of the worms in a dose-dependent manner. Groups pre-treated with the highest doses of RT extract, 20 and 50 μg/mL, significantly increased the survival rate compared to the control group, with rates of 9.15% ± 2.13 (*p* < 0.001) and 10.69% ± 2.51 (*p* < 0.0001), respectively. These results indicate that the RT extract improves oxidative stress resistance in *C. elegans*, protecting them from oxidative stress.

**Figure 2.** Rock tea (RT) extract increases the survival of wild-type *C. elegans* exposed to lethal oxidative stress. L1 worms were incubated on treatment plates with different doses of RT (5, 10, 20 and 50 μg/mL) until they reached adulthood and then exposed to juglone (150 μM) for 24 h. The results represent the mean ± SEM of the values from three independent experiments. The significance for the differences between the control and pre-treated worms is \*\*\* *p* < 0.001.

#### *3.2. Rock Tea Extract Increased C. elegans Lifespan*

With the object to study the effect of RT extract on lifespan, wild-type *C. elegans* grown at 20 ◦C in a liquid medium containing different doses of RT (5, 10, 20, 50 and 100 μg/mL) were used. *C. elegans* is a model widely used to study aging and age-related disorders due to the good conservation of the biochemical pathways and their short life cycle [20]. The lifespan curves showed that RT extract increases the lifespan of the worms in a dosedependent manner (Figure 3). Significant differences were found (*p* < 0.05) in survival curves between the control group and the worms treated with 100 μg/mL of RT extract (12.8 ± 0.56 days vs. 14.7 ± 0.58 days, respectively). The maximum lifespan, understood as the average lifespan of 10% of each population living longer, increased by 11.36% at doses of 20 and 50 μg/mL of RT and 13.81% at the dose of 100 μg/mL of RT with respect to the control. These results show that RT extract presents a positive effect on *C. elegans* lifespan.

**Figure 3.** Lifespan curves of wild-type *Caenorhabditis elegans (C. elegans)* in a liquid medium supplemented with different concentrations of RT extract (0–100 μg/mL) at 20 ◦C. Synchronized worms were exposed to the extract from the second day of adulthood (day one). Scoring of survival was carried out three times per week until all worms died. The results are representative of three independent biological replicates. The curves were analyzed using a long-rank test. Differences in the survival curves between the treatment and control groups were found at the dose of 100 μg/mL, with a *p*-value of 0.0153.

The means of lifespan were 12.8 ± 0.56 days in the control and 13 ± 0.57, 13 ± 0.56, 13.45 ± 0.58, 13.42 ± 0.57 and 14.7 ± 0.55 days in the RT 5, 10, 20, 50 and 100 μg/mL groups, respectively. The results of lifespan experiments were analyzed using the Kaplan-Meier survival model and for significance by means of a log-rank pairwise comparison test between the control and treatment groups. Differences in survival curves between the treatment and control groups were found for 100 μg/mL with a *p*-value of 0.0153.

## *3.3. Rock Tea Extract Delays the Onset of Paralysis Induced by Aβ Peptide*

Although there are multiple factors involved in Alzheimer's disease, numerous studies have shown that neuronal accumulation of the Aβ peptide plays a central role in the development of the disease [21]. To further investigate the *in vivo* neuroprotective effect of rock tea, an examination was carried out as to whether diet supplementation may affect the progression of paralysis induced by Aβ toxicity in the *C. elegans* transgenic strain CL4176. This strain has proven to be a good model for screening potential neuroprotective natural products [15,22,23]. Nematodes were exposed to different concentrations of RT extract (5, 10 and 25 μg/mL) from the egg stage. Then, human Aβ expression was induced by a temperature upshift that makes worms paralyze over time. The time to develop paralysis was analyzed using survival curves (Figure 4).

**Figure 4.** Effect of RT extracts on Aβ-induced paralysis in transgenic *C. elegans* CL4176. The statistical significance of the difference between the curves was analyzed using a log-rank (Kaplan–Meier) statistical test, which compares the survival distributions between the control and treatment groups. Differences in the survival curves between the treatment and control groups were found (*p* < 0.0001).

The time for 50% nematodes to become paralyzed (PT50) showed a significant increase (*p* < 0.0001) in the groups treated with RT extract compared to the untreated groups. However, between the different groups treated with RT extract, no significant differences were found in PT50 at 43 h. According to HR (hazard ratio) values obtained by log-rank analysis (0.76; 0.72 and 0.78), the RT extract significantly reduced the risk of paralysis by 23%, 28% and 21% in worms treated with 5, 10 and 25 μg/mL RT extract concentration, respectively.

#### *3.4. Bioassays Regarding CNS Enzymes*

The AChE, MAO-A and TYR enzymes are involved in processes that regulate neurotransmission in the CNS. RT extract was able to inhibit all the enzymes at high doses, although the dose-response curve was shifted to the right with respect to the reference substances for each enzyme (Figure 5). AChE inhibition of RT extract is achieved at high doses, with an IC50 of 4.5 mg/mL for RT (Figure 5B). The galantamine IC50 for AChE inhibition was 0.1 mg/mL. RT extract also revealed the potential to achieve a total inhibition of MAO-A (Figure 5B). RT extract IC50 was 76.34 μg/mL, a little far from that of the reference inhibitor, clorgyline, which was 0.12 μg/mL. Finally, RT extract also showed the inhibitory potential of TYR in a dose-dependent manner (Figure 5C). The extract reached 100% enzyme inhibition at a high dose, 5 mg/mL, and the IC50 of the assay were 1.05 and 0.004 mg/mL for RT extract and kojic acid, respectively.

**Figure 5.** Neuroprotective effect of RT extract on nervous system enzymes; (**A**) Acetylcholinesterase (AChE) inhibition, (**B**) Monoamine oxidase A (MAO-A) and (**C**) Tyrosinase (TYR) inhibition. Galantamine, clorgyline and kojic acid have been used as reference inhibitors, respectively.

#### **4. Discussion**

This study shows, for the first time, the neuroprotective effect of *Jasonia glutinosa* extract using different types of *in vitro* and *in vivo* bioassays. The results obtained demonstrate that RT extract prevented oxidative stress, increased the lifespan and delayed paralysis of

the transgenic amyloid *C. elegans*. In addition, the extract inhibited enzymes of the nervous system such as acetylcholinesterase, monoamine oxidase A and tyrosinase.

Various studies have associated reactive oxygen species with the pathogenesis of Alzheimer's disease (AD) by showing that reactive oxygen species promote the formation and accumulation of the β-amyloid peptide and hyperphosphorylation of Tau. On the other hand, oxidative stress is associated with aging. Many theories try to explain the connection, but it is complicated due to its multifactorial etiology. The so-called "Oxi-Inflamm-Aging" theory explains the events that occur during aging and the appearance of diseases related to it; during cellular aging, senescent cells produce pro-inflammatory cytokines that produce chronic systemic inflammation ("inflamm-aging"), leading to an increase in free radicals. Similarly, oxidative stress produces states of inflammation due to the impaired immune system, creating a vicious circle between oxidative stress, inflammation and aging. This process has been implicated in multiple disease states, such as cardiovascular disease, cancer, neurodegenerative diseases, diabetes or respiratory diseases [24,25]. Oxidative stress, β-amyloid peptide accumulation the lifespan have been correlated in the *C. elegans* model [26,27], making it an excellent model to study the bioactive properties of substances with neuroprotective potential.

Different RT extracts have shown anti-inflammatory effects [4,10] and a high capacity to eliminate superoxide and DPPH radicals [4,5,9]. Furthermore, one *in vivo* study showed these antioxidant properties by decreasing HSP70 levels, increasing peroxidase activity and upregulating the Nrf-2 transcription factor, producing an increase in the expression of antioxidant enzymes such as catalase and superoxide dismutase in fish [10]. These properties, which could be explained by its rich composition in phenolic acids, flavonoids and pigments, make RT an ideal candidate as a neuroprotective natural agent.

Thus, the RT extract has demonstrated antioxidant properties and lifespan extension in *C. elegans* in a similar manner to quercetin or kaempferol, two flavonoids present in the extract. The phytochemical analysis of the ethanolic extract showed a composition rich in phenolic compounds and pigments [4]. Of the phenolic compounds identified, the most representative were the hydroxycinnamic acids derived from caffeoylquinic acid, represented mainly by 3,4-di-O, 3,5-di-O (40.95 mg/g of dry extract), 1,5-di-O (24.73 mg/g of dry extract) and 4,5-di-O caffeoylquinic acids (23.14 mg/g of dry extract). Of the flavonoids, the most representative compound was quercetin-3-O-galactoside (15.16 mg/g of dry extract). In addition, pigments in the extract were determined for the first time, highlighting the presence of carotenoids and chlorophylls, lutein being the most representative, with content greater than 55% of the total.

The treatment of *C. elegans* with quercetin or its methyl derivates, isorhamnetin and tamarixetin (200 μM), increased worm survival rate under exposure to juglone (150 μM, 24 h exposure). Quercetin showed a greater capacity than methyl derivatives in decreasing oxidative proteins compared to untreated worms. Isorhamnetine showed greater protection (16%) compared to quercetin and tamarixetine (11%), prolonging the mean lifespan of the worms compared to the control worms [13]. These results were similar to those obtained by Kampkötter and colleagues (2008), who demonstrated that quercetin (100 μM) enhanced the percentage of survival against oxidative stress (juglone 150 μM, 72 h) by 19% and the mean lifespan by 15% of worms compared to the untreated worms [28]. Furthermore, worms pre-treated with kaempferol showed diminished oxidative stress and less accumulation of reactive oxygen species [29]. Kaempferol and quercetin showed scavenging capacity in mitochondrial reactive oxygen species levels, without affecting lifespan in *mev-1* mutant worms, a mutation that increases sensitivity to oxidative stress and reduces lifespan [30]. The effect on lifespan may be explained by the antioxidant effect of these compounds and/or by other complementary mechanisms, independent of antioxidant properties, such as stress-sensitive signaling pathways.

One of the main key regulatory pathways in *C. elegans* lifespan and the response to oxidative stress is the insulin/insulin-like growth factor (IGF-1) signaling (IIS) pathway, as well as the activation of different transcription factors such as DAF-16/FoxO, HSF- 1 and SKN-1/Nrf-2 that regulate stress-related genes as antioxidant enzymes. Several studies have shown that quercetin and kaempferol produced a translocation of DAF-16 from the cytosol to the nucleus in transgenic *C. elegans*, increasing the expression of defense proteins [28–30], the role of these DAF-16 target genes in longevity is unclear. Different studies have shown that transcription of this factor may not be responsible for the effect of flavonoids on worm lifespan, since quercetin-treated *daf-16* deletion mutants increased survival in comparison with the control [30–32]. It has recently been shown that resistance to oxidative stress by quercetin in worms would implicate genes involved in the IIS pathway such as *age-1*, *akt-1*, *akt-2*, *daf-18*, *sgk-1*, *daf-2* and *skn-1*, independent of transcription factors such as DAF-16 and HSF-1.

Other important compounds in RT extract are caffeoylquinic acids (CQAs), which are emerging as an interesting group of bioactive compounds with high potential as neuroprotective agents. These compounds have been shown to possess strong antioxidant and neuroprotective properties *in vitro* and *in vivo*. CQAs have also been shown to improve cognitive impairment in several rodent models of AD [33]. Recently, Chen et al. [34] showed that 1,5-O-dicaffeoyl-3-O-(4-malic acid methylester)–quinic acid (MQA), a derivative of caffeoylquinic acid, can protect against ischemic brain injury in rats. This neuroprotective effect may involve the inhibition of cell apoptosis due to p38 activation, the increase of the Bcl-2/Bax ratio and the modulation of NFk-B1 and caspase-3 expression. Furthermore, the observed decrease in lipid peroxidation suggests that the antioxidant activity of MQA could also contribute to the protective effect against cerebral ischemia. The association between antioxidant effects and neuroprotection has been previously demonstrated for other CQAs. Thus 3-O-caffeoylquinic acid and caffeic acid treatment improved memory in the mouse model of Aβ accumulation by the inhibition of oxidative stress, inflammation and apoptosis through the p38 mitogen-activated protein kinase (MAPK) signaling pathway [35,36]. Similarly, Chu et al. [37] observed that a flavone-rich extract of *Tetrastigma hemsleyanum* whose major components were 3-caffeoylquinic acid, 5-caffeoylquinic acid and quercetin-3- O-rutinoside, and kaempferol-3-O-rutinoside could attenuate glutamate-induced toxicity in PC12 cells and *C. elegans*. This extract lessened genotoxicity, relieved oxidative stress and recovered mitochondrial functions in PC12 cells via the MAPK pathway by suppressing the over-phosphorylation of ERK and p38. Furthermore, treatment suppressed O2 - generation, reduced GSH depletion and partially restored the normal motility lost after the glutamic acid exposition in *C. elegans*. Therefore, antioxidant properties could explain the positive effect of *J. glutinosa* extract in increasing the lifespan in N2 and transgenic *C. elegans* (CL4176 strain).

As mentioned above, oxidative stress and the malfunction of specific enzymes related to neurotransmitter effects, as AChE, are involved in the development of the pathogenesis of AD. The accumulation of Aβ peptide caused by free radicals would result in neuron dysfunction and death. Different works have shown that phenolic compounds present neuroprotective activity, decreasing the deleterious effects of oxidative stress, increasing ACh availability, reducing the anti-inflammatory effect and interacting directly with Aβ peptides inhibit their aggregation and oligomerization [15,23,38,39]. The reduction of the IIS pathway and expression of genes as DAF-16, HSF-1, HSP-16 and Nrf2/SKN-1 have also been related to a neuroprotective effect by decreasing the expression of Aβ peptides and improved the paralysis and lifespan in a *C. elegans* model of AD [40]. These results show that RT extract decreases the risk of paralysis in transgenic strain CL4176, and its neuroprotective effect can be explained by the synergic antioxidant and neuroprotective activities of phenolic acids and flavonoids.

On the other hand, *C. elegans* present numerous neurotransmitters, some of which are implicated in different pathologies of the nervous system such as ACh, dopamine, GABA or glutamate [41]. RT extract has demonstrated the ability to inhibit the AChE, MAO and TYR enzymes, which could explain its traditional uses as a stimulant, memory enhancer or antidepressant, reinforcing its neuroprotective role. Others isolated extracts of plants and flavonoids have demonstrated the capacity to inhibit CNS enzymes [15,18,23,42,43] and decrease the toxicity by Aβ accumulation [15,23,38,44]. Due to these mechanisms, many natural products have neurotherapeutic potential for the prevention and treatment of different CNS disorders.

## **5. Conclusions**

*Jasonia glutinosa* is a medicinal plant with a wide range of traditional uses. The neurotherapeutic potential of a polyphenolic extract has here been demonstrated in different models due to its protective activity against oxidative stress and the capacity to inhibit β-amyloid aggregation in *C. elegans*. This herbal extract has also shown the inhibitory capacity of nervous system enzymes, which might explain, from an *in vitro* perspective, possible mechanisms of its use as a stimulant and antidepressant. All in all, this plant species as well as its extracts and bioactive compounds are worthy of further investigation for the prevention of diseases associated with cellular aging and oxidative stress.

**Author Contributions:** Conceptualization, V.L., C.G.-R. and M.S.V.; methodology, F.L. and C.M.; analysis, F.L., C.M., M.S.V., V.L. and C.G.-R.; investigation, F.L. and C.M.; resources, V.L.; writing—original draft preparation, M.S.V. and F.L.; writing—review and editing, V.L., F.L. and D.W.; supervision, V.L. and D.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Government of Aragón (Phyto-Pharm, B44- 20D), Universidad San Jorge and Universidad de Zaragoza (ref. gJIUZ-2018-BIO-09).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in this article.

**Conflicts of Interest:** The authors declare that they do not have any conflict of interest.

**List of Abbreviations :** AChE: acetylcholinesterase, AD: Alzheimer's disease, Aβ: amyloid-β, CQAs: caffeoylquinic acids, NGM: nematode growth media, MAO-A: monoamine oxidase A, MQA: 1,5-Odicaffeoyl-3-O-(4-malic acid methylester)–quinic acid, RT: rock tea, TYR: tyrosinase, % SR: survival rate %.

#### **References**


## *Review* **Effects of Propolis on Infectious Diseases of Medical Relevance**

**Nelly Rivera-Yañez 1,2, C. Rebeca Rivera-Yañez <sup>3</sup> , Glustein Pozo-Molina 1,4, Claudia F. Méndez-Catalá 2,4, Julia Reyes-Reali 1,5, María I. Mendoza-Ramos 1,5, Adolfo R. Méndez-Cruz 1,5 and Oscar Nieto-Yañez 1,\***


**Simple Summary:** Propolis is a beekeeping product with a complex and highly variable chemical composition. Many beneficial health properties have been reported. In this review, we will be focusing on compiling the studies carried out with propolis on infectious diseases of greater medical relevance. Likewise, the promises and challenges that propolis has to consolidate itself as a complementary therapy for the treatment of these diseases are analyzed.

**Abstract:** Infectious diseases are a significant problem affecting the public health and economic stability of societies all over the world. Treatment is available for most of these diseases; however, many pathogens have developed resistance to drugs, necessitating the development of new therapies with chemical agents, which can have serious side effects and high toxicity. In addition, the severity and aggressiveness of emerging and re-emerging diseases, such as pandemics caused by viral agents, have led to the priority of investigating new therapies to complement the treatment of different infectious diseases. Alternative and complementary medicine is widely used throughout the world due to its low cost and easy access and has been shown to provide a wide repertoire of options for the treatment of various conditions. In this work, we address the relevance of the effects of propolis on the causal pathogens of the main infectious diseases with medical relevance; the existing compiled information shows that propolis has effects on Gram-positive and Gram-negative bacteria, fungi, protozoan parasites and helminths, and viruses; however, challenges remain, such as the assessment of their effects in clinical studies for adequate and safe use.

**Keywords:** propolis; antibacterial; antifungal; antiparasitic; antiviral; bioactive compounds

## **1. Introduction**

Currently, most health systems around the world are based mainly on the prevention of diseases. The world is constantly exposed to a large number of pathogens that cause emerging and re-emerging disease. These pathogens differ widely in terms of severity and probability and have varying consequences for morbidity and mortality, jeopardizing not only health but also social and economic well-being. It is absolutely necessary to have a global health system that is able to prevent and respond effectively to the expanding and evolving infectious diseases, as well as solving an increasingly widespread antimicrobial resistance [1]. The need to prevent, identify, and respond to any infectious disease that compromises global health stability remains a national, regional, and international priority [2].

**Citation:** Rivera-Yañez, N.; Rivera-Yañez, C.R.; Pozo-Molina, G.; Méndez-Catalá, C.F.; Reyes-Reali, J.; Mendoza-Ramos, M.I.; Méndez-Cruz, A.R.; Nieto-Yañez, O. Effects of Propolis on Infectious Diseases of Medical Relevance. *Biology* **2021**, *10*, 428. https://doi.org/10.3390/ biology10050428

Academic Editors: Francisco Les, Víctor López and Guillermo Cásedas

Received: 23 April 2021 Accepted: 10 May 2021 Published: 12 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Existing natural products could be potential resources to find different compounds for the development of new drugs and relevant medicine [3], creating an area of study of great importance, since the immense difference of natural molecules could contribute bioactive compounds that help in therapeutic improvement [4]. Propolis is a natural resinous product elaborated by bees from material obtained from various botanical sources; it is mixed with bees' wax and enzymes secreted by the bee's salivary glands [5]. Characteristically, its composition is 50% resin, 30% wax, 10% essential oils, 5% pollen, and 5% other substances [6]. The propolis was informed to present about 300 distinct compounds [7]. The characteristic chemical groups identified in propolis are phenolic acids or their esters, flavonoids, terpenes, aromatic aldehydes and alcohols, fatty acids, stilbenes, and β-steroids [7,8]. In addition, both the biomedical effect and composition of propolis have a very high variability according to the region of collection, the surrounding plant sources, and the seasons [9,10]. Many reports have shown that propolis possesses antibacterial, antifungal, antiparasitic, antiviral, antioxidant, anti-inflammatory, antitumor, antidiabetic, and immunomodulatory properties [11–19]. Propolis is a bee product that contains a great variety of biomedical properties and a great spectrum of components that could be promising candidates for drug discovery, which could be used to treat characteristic affections of distinct diseases. Notably, infectious diseases are a public health problem, since they do not have adequate treatment because many pathogens have developed resistance to the different drugs used against them. This is where propolis and many other alternative and complementary medicine products play an important role, since they are easily accessible, allowing a high percentage of the world population to use them, providing options to complement current treatments. As such, it is necessary to clinically analyze the effectiveness of propolis to evaluate its potential in human health promotion.

## **2. Antibacterial Activity of Propolis**

One of the main complications with diseases caused by bacteria is their resistance to the antibiotics commonly used against them. Antibiotics are chemical compounds that can act in two ways: inhibiting (bacteriostatic drugs) or killing (bactericidal drugs) bacteria. These drugs are characterized by a specific interaction with a defined target in the bacterial cell, and they are arguably the most important medical intervention introduced by humans [20]. Currently, the figures related to this problem are alarming: according to conservative numbers mentioned by the Centers for Disease Control (CDC), approximately 23,000 people are estimated to die annually only in the USA as a result of an infection with an antibiotic-resistant organism [21]. According to a report, antibiotic resistance is predicted to cause around 300 million premature deaths by 2050, with a loss of up to USD 100 trillion to the global economy [22]. Next, we address the main research describing the use and activities of propolis from different countries on some bacterial agents of greater medical relevance today.

#### *2.1. Staphylococcus Infections*

The genus *Staphylococcus* causes different infections in the human population like impetigo, scalded skin syndrome, toxic shock syndrome, pneumonia, endocarditis, and urinary tract infections, among others [23]. Some species of this genus are resistant to antibiotics, such as methicillin-resistant *Staphylococcus aureus* (MRSA) [24]. In this genus, we can highlight to *S. aureus* and *Staphylococcus epidermidis*.

Records exist that demonstrate the use of propolis since ancient civilizations, as it possesses a large number of biological properties, one of which is its antibacterial effect [9,25–27]. Currently, various investigations around the world have demonstrated the antibacterial capacity of different types of propolis; hence, various studies report that all the distinct varieties of propolis have different antibacterial activities [28]. On the American continent, propolis varies widely, each having different characteristics. In this context, the antibacterial activity of Canadian propolis was evaluated, which showed activity on *S. aureus* [29]. Likewise, the antibacterial effect of Brazilian propolis (red, green, and brown)

from distinct areas was studied, with the authors finding that the red extracts demonstrated activity against different bacterial species, including *S. aureus*; however, the green and brown extracts showed less activity than red extracts [30]. Similarly, in Europe, French propolis demonstrated significant antibacterial activity against both methicillin-susceptible *Staphylococcus aureus* (MSSA) and MRSA [31]. Likewise, Polish propolis showed variability in its activity on twelve MSSA and MRSA clinical isolates [32].

In 2017, Al-Ani et al. mentioned the antibacterial activity of propolis of various geographic origins such as Germany, Ireland, and the Czech Republic. The three propolis samples showed moderate antibacterial effect on *S. aureus*, MRSA, and *S. epidermidis* [28]. Similarly, Italian propolis showed antibacterial activity on clinically isolated *S. aureus* and *S. epidermidis*. The propolis demonstrated an inhibition on lipase activity of 18 *Staphylococcus* spp. and an inhibition on the coagulase of 11 tested *S. aureus*. Propolis showed an inhibitory activity of the adhesion and consequent biofilm growth of *S. aureus* [33]. In another study, 53 propolis were obtained from various areas in Serbia, which revealed one type of blue propolis and one orange, depending on floral and geographical origin. Propolis samples showed an effect against different bacteria, including *S. aureus*, with the orangetype propolis samples showing higher antibacterial activity compared with the blue-type propolis samples (Table 1) [34]. The variety of climates and flora in Africa results in propolis with very particular characteristics; however, as with samples from the Americas and Europe, they showed an effect on strains of *S. aureus* and *S. epidermidis* [35]. Another study reported that Kenyan propolis showed differences in the antibacterial activity against *S. aureus* in three studied geographical areas [36]. In this context, we agree with the different authors who found a great variety of propolis that present a diversity of activity against Staphylococci in distinct regions around the world; these investigations have been important for the study of infections caused by this bacterial genus. However, in these studies, the chemical components of the propolis were not described, so the adequate and standardized use of propolis cannot yet be achieved [6].


**Table 1.** Effect of propolis from several parts of the world on various *Staphylococcus* species.

MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration.

As propolis functions to support the sterility and health of the beehive, the protective properties of the bioactive compounds in propolis can provide significant benefits for human health [37,38]. In this context, the flavonoids and esters of phenolic acids present in propolis are habitually the active components related to antibacterial effect [39]. Samples of distinct types of propolis from diverse regions in Brazil were studied (red, green, and brown), showing distinct antibacterial activities against different microorganisms, including *S. aureus*. Ferulic acid, gallic acid, caffeic acid, coumaric acid, *p*-coumaric acid, catechin, drupanin, kaempferide, artepillin C, luteolin, and pinocembrin have been identified in propolis, the researchers concluding that Brazilian propolis have various compounds, which present antibacterial activities that could be used for the elaborate of new medicines [40–42]. Similarly, the antibacterial activity of 20 Polish propolis obtained from distinct areas and 5 propolis from agricultural localities were studied. These samples showed distinct antibacterial activity toward *S. aureus* and *S. epidermidis*. For the 20 clinical isolates of *S. aureus* (16 MSSA and four MRSA), the propolis samples presented different activities, two of which showed higher antistaphylococcal activity, probably because these two samples contain more flavonoids that the other samples of propolis studied. The propolis originating from agricultural areas in Southern Poland presented a higher content of bioactive components (different flavonoids and phenolic acids). The samples of Polish propolis effectively eradicated staphylococcal biofilm, suggesting that the identified components are essential for the antibacterial effect of propolis [27,43]. Pinocembrin, galangin, and chrysin identified in South African propolis are known to possess antibacterial activity; combinations of these three flavonoids presented higher inhibition than flavonoids alone against different bacterial strains, including *S. aureus*. These flavonoids showed a synergistic effect to obtain a better antibacterial activity (Table 2) [44]. Propolis from Northern Morocco showed inhibitory effects against *S. aureus*, with the authors identifying different phenolic compounds such as caffeic acid, *p*-coumaric acid, ferulic acid, naringenin, pinocembrin, chrysin, galangin, pinobanksin, and quercetin [45]. We also agree with the studies that have reported the different propolis around the world presenting antistaphylococcal activity; these investigations have described the main active components of propolis and their activity against staphylococci, alone or in combination, which is of relevance for future research [44], since the search for more compounds and better combinations is necessary to treat infections occasioned by the *Staphylococcus* genus.

In another study, synergistic interactions were reported regarding combinations of Irish propolis and antibiotics (two-drug combinations: vancomycin, oxacillin, and levofloxacin) against different microbial pathogens, including MRSA. The authors concluded that the propolis from Ireland increased the synergistic effect and the effectiveness of antibiotics, mainly of vancomycin and oxacillin, that interact on cell-wall synthesis on drugresistant bacteria [28]. In 2019, Grecka et al. observed the synergistic antistaphylococcal effect against *S. aureus* of one sample of Polish propolis combined with different drugs and fusidic acid; notably, all these drugs present an inhibitory action on protein synthesis [27]. Similarly, the activity of the combination of propolis from Poland with 10 antibiotics against staphylococci on *S. aureus* clinical isolates was proven, suggesting that the combinations of Polish propolis with different drugs potentiated the antibacterial effect on the various strains; however, no synergism was observed in the case of ciprofloxacin and chloramphenicol [32]. Likewise, the synergetic effect of propolis from Italy with some antibiotics on different bacterial strains was assessed, including *S. aureus* and *S. epidermidis*, reporting that Italian propolis enhanced the antibacterial activity of six different antibiotics [33].

Recently, Malaysian propolis and propolis nanoparticles (prepared with Malaysian propolis) exhibited antibacterial and antibiofilm properties against *S. epidermidis.* Propolis nanoparticles drastically inhibited biofilm growth by *S. epidermidis* and reduced the viability of biofilm bacteria compared with propolis extract. Propolis nanoparticles treatment showed significant disruption of biofilm and partial disruption by Malaysian propolis extract, decreasing bacteria in the biofilm. The gene expression in the tested bacteria described that genes related in intercellular adhesion (*IcaABCD, embp*) were downregulated by

propolis nanoparticles. Propolis nanoparticles presented a synergistic effect with different drugs, suggesting efficient treatment. The authors concluded that propolis nanoparticles are more efficient than propolis extract alone in inhibiting bacterial biofilms by produce membrane alteration and reducing biofilm growth (Table 3) [46]. Notably, drug-resistant bacteria significantly affect health systems at present; in this context, studies with propolis and its potential antibacterial activity are promising. We agree with the different studies that have demonstrated the effectiveness of propolis, its bioactive components, and the combinations with various antibiotics against the *Staphylococcus* genus. These new sources of natural products are not aimed to replace antibiotic treatment but could be a complement in the treatment of these pathogens that now have resistance to antibiotics [47].


**Table 2.** Antibacterial effect of diverse propolis and its chemical composition.

**Table 3.** Effects of propolis in combination with various drugs on different bacterial species.



#### **Table 3.** *Conts.*

MIC: minimum inhibitory concentration.

## *2.2. Streptococcus Infections*

The genus *Streptococcus* is classified as Gram-positive and catalase-negative, appearing as cocci in pairs and chains on Gram stains. When grown on blood agar, they appear as small colorless colonies that cause beta or complete hemolysis [48]. The species of this genus are the cause of a large number of diseases in the human population, from acute to chronic infections with a wide array of manifestations in both adults and children [49]. In this genus, we can highlight *S. pyogenes, S. pneumoniae*, and *S. mutans*.

Three propolis samples of different geographic origins (Germany, Ireland, and Czech Republic) present moderate antibacterial effect on *S. pyogenes* and *S. pneumoniae* [28]. Similarly, Italian propolis showed antibacterial activity on different clinically isolated Grampositive strains, including *S. pneumoniae* [33]. Similarly, a sample of Mexican propolis presented antibacterial activity against different microorganisms, including *S. mutans*; compounds such as pinocembrin, chrysin, galangin, alpinetin, dillenetin, isorhamnetin, ferulic acid, syringic acid, and caffeic acid were identified in the propolis. Several compounds (galangin, ferulic acid, syringic acid, and caffeic acid) also showed antibacterial activity against this oral pathogen [50]. In another study, the antibacterial effect of various samples of South Brazilian propolis was assessed: all showed activity against different bacterial strains, including *S. mutans*. All samples of propolis have an inhibitory action on *S. mutans* biofilm growth. In all these samples, diverse compounds were described, concluding that South Brazilian propolis could be an important resource of active components with properties for use in the pharmaceutical sector [42]. In similar research, the antibacterial and antibiofilm activities of propolis from Iran and its main compound, quercetin, were described on different bacterial strains, including *S. mutans* and *S. pneumoniae*, suggesting that Iranian propolis and quercetin were effective on the different bacteria studied and showed an inhibitory activity *S. mutans* biofilm adherence (Table 4) [51]. Several investigations have studied the activity of propolis and some of its bioactive compounds against the genus *Streptococcus*; although the results are encouraging, a limitation of these studies is that the possible mechanisms of action must be studied in vitro and in models [52], which would help to better understand this type of infection and its possible complementary treatments.

Interactions were reported regarding combinations of Irish propolis and distinct drugs (two-drug combinations: vancomycin, oxacillin, and levofloxacin) against different bacterial strains, including *S. pneumoniae* and *S. pyogenes*. The propolis from Ireland increased synergistic effect and the effectiveness of drugs that interact on cell-wall synthesis (vancomycin and oxacillin) [28].


**Table 4.** Antistreptococcal activity of diverse propolis and its chemical composition.

N.I., none identified; MIC: minimum inhibitory concentration.

#### *2.3. Gastrointestinal Infections*

Gastrointestinal infections constitute a great proportion of the acute and chronic disease burden in all the world. Some bacterial, viral, and parasitic microorganisms infect through contaminated food and water or from human to human. The WHO mentions that diarrhea causes 2.2 million deaths each year worldwide (about 4%) [53]. For this reason, the studies examining the effects of propolis on the main bacterial pathogens that cause gastrointestinal diseases are analyzed below.

The propolis of different geographic origins presented a moderate antibacterial effect on *Escherichia coli*, *Salmonella choleraesuis*, and *Shigella flexneri* [28]. In another study, 53 propolis were obtained from various areas of Serbia; the orange-type propolis samples showed higher antibacterial activity against *E. coli, Salmonella enteritidis, S. flexneri,* and *Listeria monocytogenes* [34]. Another study reported that Kenyan propolis showed differences in the antibacterial activity from three different geographical areas against different bacterial strains, including *E. coli* [36].

Similarly, Brazilian propolis (red, green, and brown; collected in diverse regions) as well as Southern Poland propolis showed distinct antibacterial activities against different microorganisms, including *E. coli* and *L. monocytogenes*; bioactive components such as ferulic acid, *p*-coumaric acid, caffeic acid, catechin, luteolin, drupanin, kaempferide, artepillin C, pinocembrin, chrysin, pinobanksin, apigenin, and kaempferol were identified [40,41,43]. In another study, pinocembrin, galangin, and chrysin (principal components South African propolis) were found to possess antibacterial activity against different microorganisms, including *L. monocytogenes* and *E. coli*, and combinations of these three flavonoids presented higher inhibition activity than components alone. The authors observed that these compounds worked synergistically to achieve the best antibacterial effect (Table 5) [44]. Propolis from Northern Morocco showed inhibitory effects against different Gram-negative strains, including *E. coli*; the researchers identified caffeic acid, *p*-coumaric acid, ferulic acid, naringenin, pinocembrin, chrysin, galangin, pinobanksin, and quercetin [45]. As mentioned by different authors who have studied the activity of propolis and some of its bioactive compounds on the effect against pathogens that cause gastrointestinal infections, we agree that the propolis present a great antibacterial diversity; nevertheless, these investigations contribute limited conclusions; therefore, it is necessary to carry out more studies focusing on understanding the antibacterial activity of propolis and trying to find its possible mechanism of action [54]. In addition, it is important to conduct in vivo and clinical trials in

propolis of various areas to consider the differences in the chemical components of each one and, therefore, the different antibacterial activities that it may present [55].

**Table 5.** Antibacterial activity of different propolis on various microorganism species.


N.I., none identified; MIC: minimum inhibitory concentration.

Another research showed that Brazilian propolis presents a bacteriostatic effect on *Salmonella typhi*, and Bulgarian propolis presented a bactericidal effect and a synergism with chloramphenicol, tetracycline, and neomycin (act on the ribosome) on this same pathogen [56].

#### *2.4. Nosocomial Infections*

Nosocomial infections are not commonly found when admitted to hospital or are probably incubating. These infections are typically contracted in the hospitalization and generally manifest 48 h after [57]. The numbers of these infections are worrying: according to estimated figures from the CDC, in 2014, 11,282 patients suffered from healthcareassociated infections in USA hospitals alone. The main infections encompass primary bloodstream infection, surgical site infections, pneumonia, and urinary tract infections [58]. In this area, *Haemophilus influenza, Pseudomonas aeruginosa*, and *Klebsiella pneumoniae* are the cause of most of respiratory and renal nosocomial infections, respectively.

The antibacterial effect of Brazilian propolis (red, green, and brown) of various areas was studied; the red extracts demonstrated higher activity than green and brown extracts against different bacterial species, including *Klebsiella* sp. [30]. Similarly, a study reported the moderate antibacterial effect of propolis of distinct geographic regions (Germany, Ireland, and Czech Republic) on *P. aeruginosa, H. influenzae, K. pneumoniae*, and two clinical isolates of *K. pneumoniae* [28]. Another studies reported that Cameroonian, Congolese, and Kenyan propolis showed differences in antibacterial activity against various microorganisms, including *K. pneumoniae* and *P. aeruginosa* (Table 6) [35,36]. The greatest limitation of these investigations is that they remained at a qualitative level, only describing whether or not propolis presented activity; they did not mention any possible mechanism of action of propolis against these pathogens, which is essential to better understanding nosocomial infections and how to combat them [59,60].

Another study mentioned that various South Brazilian propolis showed activity against different bacterial strains, including *P. aeruginosa*. In all samples, gallic acid, caffeic acid, coumaric acid, artepillin C, and pinocembrin were identified [42]. Propolis originating from Southern Poland showed stronger antibacterial activity against different microorganisms, including *K. pneumoniae* and *P. aeruginosa*. Additionally, pinocembrin, chrysin, pinobanksin, apigenin, kaempferol, *p*-coumaric acid, ferulic acid, and caffeic acid were identified [43]. In other research, the effects of Albanian propolis were evaluated in various virulence factors of *P. aeruginosa*. Propolis inhibited the microbial development and biofilm

growth; also, propolis decreased extracellular DNA release and phenazine production. Compounds were identified in the propolis, such as caffeic acid, *p*-coumaric acid, ferulic acid, isoferulic acid, quercetin, apigenin, pinobanksin, chrysin, pinocembrin, galangin, and caffeic acid phenethyl ester (CAPE), with the authors including that Albanian propolis contains different components with activity on biofilm-related infections [61]. In another investigation, combinations of pinocembrin, galangin, and chrysin (principal components of South African propolis) showed a better inhibitory effect than single compounds against different bacterial strains, including *P. aeruginosa* and *K. pneumoniae*, suggesting that these compounds present a synergistic interaction favoring antibacterial activity (Table 7) [44]. Likewise, propolis from Northern Morocco showed inhibitory effects against different Gram-negative strains, including *P. aeruginosa*. Different phenolic compounds, such as caffeic acid, *p*-coumaric acid, ferulic acid, naringenin, pinocembrin, chrysin, galangin, pinobanksin, and quercetin, were identified from the propolis [45]. We consider the studies on propolis and some of its bioactive compounds against pathogens that cause nosocomial infections to be of relevance, since they mentioned a possible mechanism of action [62], although more studies are needed related to this area. It is also necessary to carry out research using in vivo models and then to clinical trials to help knowledge possible via action of propolis and be able to combat this type of infection [63,64].

**Table 6.** Antibacterial activity of diverse propolis on different microorganisms that cause nosocomial infections.


**Table 7.** Effect of diverse propolis and its main identified components on *P. aeruginosa* and *K. pneumoniae*.


Another study reported that two-antibiotic combinations (vancomycin, oxacillin, and levofloxacin) and Irish propolis showed synergism on *H. influenzae*, concluding that the propolis from Ireland increases the synergism and effectiveness of vancomycin and oxacillin, which act on cell wall synthesis [28].

As we already mentioned, resistance to antibiotics is a serious health problem, since it makes it difficult to properly treat several diseases of bacterial origin. The documented effects of propolis and its derivatives on bacteria such as MRSA make them ideal candidates for clinical studies in order to evaluate their effectiveness on antbiotic-resistant bacterial diseases. The clinical application of propolis should not focus on the substitution of antibiotics, but on complementing and improving the efficacy of these when co-administered.

### **3. Antifungal Activity of Propolis**

Fungal infections are responsible for over one million human deaths annually and are an increasingly important cause of mortality and morbidity [65]. In recent years, fungal infections have increased significantly, being considerably high in immunosuppressed patients [66]. Unfortunately, the low number of available treatments and the misuse of the antifungal medications have led to the selection of resistant microorganisms [67], which is why the search for new, effective, and inexpensive antifungal agents is crucial to overcoming existing resistance mechanisms [66]. Different natural products from distinct places and latitudes come to constitute a little-explored group of agents with antifungal capacity; of all these, propolis has special relevance [66], as recent studies have evaluated it as a natural product with potential for the development of antifungal drugs without toxicity [68,69].

## *3.1. Candidiasis*

The genus *Candida* is a group of fungi known for their dimorphic capacity and is commonly isolated from the microbiome of healthy individuals (intestinal tract, oral cavity, skin, and vaginal cavity) [70–72]. However, when the host's immunity becomes compromised by diseases such as HIV, AIDS, cytotoxic therapies, uncontrolled diabetes mellitus, or people of very young or very old age, *Candida* can behave like a pathogenic fungus. The progressive increase in the number of infections caused by *Candida* worldwide has increased in recent decades; this may be due to the significant increase in the population at risk, particularly the spread of HIV, immunosuppressive therapies, and the increase in the use of permanent devices [72–74]. The different species of *Candida* were classified as the fourth main agent that generate highly relevant infections worldwide; the magnitude of these diseases worldwide is alarming [72,75], and it has been recorded that infections caused by Candida species have a high crude mortality rate, exceeding the number caused by *S. aureus* and *P. aeruginosa* in nosocomial infections of the bloodstream [76]. It is important to highlight that the incidence in the annual rates of nosocomial infections of the bloodstream caused by Candida at the beginning of the century presented a variability of 6.0 to 13.3 and from 1.9 to 4.8 cases per 100,000 inhabitants in the United States and Europe, respectively [77–79]. Hence, *Candida* is a healthcare priority, and new antifungal therapeutic approaches are urgently needed. The propolis from different geographical regions has demonstrated anti-*Candida* activity, as described below.

The distinct clinical isolates of different species of the genus *Candida* extracted from vaginal exudates of patients with vulvovaginal candidiasis were completely suppressed by Brazilian propolis with a very small variation independent of the yeast species [80]. Likewise, Brazilian green propolis showed the ability to suppress the growth and biofilm formation of vaginal isolates of *C. albicans* [81]. In another research, the fungicidal effect of Brazilian propolis was demonstrated on three morphogenetic types of *C. albicans*, and the induced cell death was mediated by metacaspase and Ras signaling. This was corroborated by propolis inhibiting yeast transformation to hyphal growth. Moreover, a topically applied pharmaceutical formula based on propolis can partially control *C. albicans* infections in a vulvovaginal candidiasis infection in a mouse model [82].

Within Europe, the antifungal effect of different propolis has been investigated. The effect of four different Polish propolis samples on azole-resistant *Candida* clinical isolates was studied, with only one of the four propolis samples revealing high antifungal activity [83]. Similarly, Portuguese and French propolis presented distinct antifungal activities against *C. albicans* and *C. glabrata* [31,84]. The antifungal effect of propolis obtained in distinct geographical areas of the European continent was investigated. All propolis used reported an antifungal effect both in reference strains and different species of the *Candida* genus from clinical isolates. Propolis from Ireland and Czechia showed very good fungicidal effects, while propolis from Germany showed mostly fungistatic activity; *C. glabrata*, *C. parapsilosis*, and *C. tropicalis* were the most sensitive *Candida* [28].

In Asia in 2020, Alsayed et al. reported a fungicidal effect of propolis from Saudi Arabia against *C. zeylanoides*, *C. famata*, *C. sphaerica*, *C. guilliermondii*, *C. magnoliae*, and *C. colliculosa* and a fungistatic effect against *C. krusei*, *C. pelliculosa*, and *C. parapsilosis* [85]. In other study, propolis from Turkey showed antifungal activity against different clinical isolates of *Candida* [86]. Similarly, the antifungal effect of aqueous and ethanolic extracts of Iranian propolis was described against *Candida* samples that were collected from 23 oral cavities of patients presenting candidiasis in the oral cavity (isolating 22 samples of *C. albicans* and one *C. glabrata*). Both extracts of Iranian propolis demonstrated inhibitory effects on *Candida*, but the extract that presented greater effectiveness even above the aqueous one was the ethanolic [87]. Other researchers evaluated the antifungal activity of propolis and propolisloaded nanoparticles (EEP-NPs) from Thailand, observing the impact they have on specific factors that contribute to the pathogenesis of *C. albicans*, where EEP-NPs were mostly active compared to propolis in its free form, inhibiting virulence factors such as adhesion, hyphal germination, biofilm formation, and invasion. It should be noted that the EEP-NPs showed a decrease in the expression of genes related to adhesion processes linked to the hyphae of *C. albicans*, demonstrating that the EEP-NPs have the ability to mediate a great anti-*Candida* activity, attacking key factors of virulence, such as the inhibition of the expression of genes related to adhesion-related proteins, which mediate the morphological change of *C. albicans*, attenuating the virulence of the yeast (Table 8) [88]. In Africa, few studies were found for this review. One was conducted by Papachroni et al. in 2015, who analyzed four propolis of distinct areas of Africa, which showed an effect on *C. albicans*, *C. tropicalis*, and *C. glabrata* [35]. We think that the aforementioned studies that have demonstrated the anti-*Candida* activity of propolis have a significant impact on this issue, since most of them reported fungicidal or fungistatic activity exhibited by propolis from different regions on different strains and clinical isolates of *Candida* [89], as well as some possible mechanisms of action through which propolis inhibits this yeast, such as virulence factors that favor the pathogenicity of *C. albicans*. Some studies even reported the activity of propolis being very promising in in vivo models of candidiasis infections; however, the limitation of all these investigations is that they did not mention the composition of the different propolis, since, as we mentioned earlier, it is important to know and determine the active components present in propolis to identify which molecules are responsible for this antifungal effect [90].

Various studies around the world have focused on the search for components in the propolis that have antifungal effect; below, we describe some of the investigations that revealed the antifungal potential of this natural product. A fraction of the Brazilian red propolis rich in benzophenones was analyzed, which showed activity against different clinical isolates of *C. parapsilosis* and *C. glabrata* resistant to antifungal agents, like fluconazole [91]. Equally, other propolis from Brazil analyzed by different researchers showed fungicide action on different strains, with *C. albicans* being more sensitive and *C. parapsilosis* being the most resistant strain studied. An in vivo study described that gels based on propolis had an antifungal effect similar to clotrimazole cream. In this propolis, different compounds were identified [92]. In Europe, one of the most extensive studies of propolis extracts was conducted, analyzing the effects of 50 different propolis extracts from Polish hives against 89 *Candida* spp. clinical isolates. Most of the samples of propolis produced satisfactory activity, showing high activity in the inhibition of biofilm formation generated by *C. glabrata* and *C. krusei* on the surfaces of polyvinyl chloride and silicone catheters. The propolis inhibited the yeast-to-mycelia morphological change and mycelial growth of *C. albicans*. In addition, the propolis combined with fluconazole or voriconazole on *C. albicans* was shown to have a clear synergism. The chemical composition of three propolis with high and one with low antifungal effect was determined (finding different flavonoids and phenolic compounds), providing evidence that the fungal cell membrane could be the target of propolis [66]. Similarly, in 2019, Pobiega et al. analyzed different Polish propolis (agricultural regions and Southern Poland), the latter being noted by greater antifungal

activity against different microorganisms, including *C. albicans* and *C. krusei*, also showing a higher content of bioactive components (Table 9) [43]. Within Africa, samples providing satisfactory results were reported: one of them was Egyptian propolis, which presented antifungal activity against *C. albicans*. In addition, the authors identified compounds such as ferulic acid, cis- and trans-caffeic acids, pinostrobin, and galangine, among others [93]. We agree that the identification of the composition of propolis from distinct areas of the world is crucial, which provides an approach to elucidating some bioactive compounds with antifungal activity and thus paving the way for future research, for example, to complement antifungal drugs with propolis or with its bioactive compounds. However, in the aforementioned studies, the mechanism of action by which propolis or any of its identified molecules exerts their antifungal effect on the different strains of *Candida* must be further investigated [94]. In addition, this pathogen is the cause of many infections in which alternative treatment is needed; propolis could be a promising option.



MFC: medium fungicidal concentration.


**Table 9.** Antifungal activity of diverse propolis and its chemical composition on different *Candida* strains.

## *3.2. Trichophyton Infections*

The diseases known as dermatophytoses are mycoses generated by fungi that commonly cause different infections in the superficial epithelia in animals and principally in humans. Various filamentous fungi are the cause of these diseases that can invade and acquire nutrients from keratinized epithelia (skin, hair, and nails) [95,96]. Dermatophytoses have the ability to affect people all over the planet, having a higher incidence level in hot tropical countries with high humidity. Approximately 10% to 15% of people are infected by dermatophytes at some time in their life [97]. It is known that dermatophytoses have the ability to affect approximately 25% of the world population according to data from WHO, as well as to generate adults carrying the disease with completely asymptomatic characteristics in a percentage ranging from 30% to 70% [98]. In developed countries, dermatophytes are the main causes of onychomycosis identified with a frequency ranging from 80% to 90%. Around the world, the prevalence of tinea pedis is has been reported approximately at 5.5%, representing 50% of all cases of nail disease [99]. The main cause of these diseases is the genus *Trichophyton*.

The Brazilian Amazon rainforest is a huge source of plant biodiversity, which is why the propolis derived from this area has many biological properties, one of which is the antifungal activity described by Silva et al. in 2015, who stated that red and green propolis are active against strains of *T. rubrum*, *T. tonsurans*, and *T. mentagrophytes*, with red propolis being more efficient than green [100]. Similarly, Brazilian green propolis showed antifungal activity against the preformed biofilms of two clinical isolates of *Trichophyton* from onychomycosis cases, the authors observing that the total biomass and the percentage of living cells of the biofilms that were subjected to the treatment with propolis were lower than in the control for both isolates; therefore, Brazilian propolis had the ability to decrease the number of cells in the preformed *Trichophyton* biofilm. Sixteen patients infected of onychomycosis were treated with topical propolis twice a day, with a 6-month follow-up period. After treatment, the data obtained were encouraging, observing a mycological and clinically total resolution in the nails and showing a complete improvement of the natural morphology of the nail and the disappearance of the fungus of up to 56.25% of patients.

Brazilian propolis is a therapy drug with great potential to be used to topically treat onychomycosis caused by *Trichophyton* [101]. Likewise, the Portuguese propolis presented distinct antifungal activity against *T. rubrum* (Table 10) [84]. The different previous studies showed that propolis has variable antifungal activity on different *Trichophyton* strains, inhibits the biofilm of clinical isolates, and a topical treatment based on propolis improved onychomycosis in patients; however, there are several limiting factors in the research of the activities of propolis against this fungus. One of the recurring omissions in this type of research is to omit to description of the components of propolis, since this is transcendental for identifying the bioactive components. Future studies must search for a possible mechanism of action against this pathogen, as it causes very common infections; therefore, it is important for there to be accessible options or traditional medicine to treat them, since a large part of the population uses this type of treatment. In addition, it is vitally relevant to carry out clinical studies that help to validate the distinct doses of propolis that help the treatments, because variety in active principles and the biomedical effects of the several propolis have to be taken into account [102].


**Table 10.** Effect of propolis on different species of *Trichophyton*.

## *3.3. Aspergillus and Penicillium Infections*

Some species of the *Aspergillus* genus are responsible for chronic pulmonary Aspergillosis (CPA) disease, which can range from nonprogressive to severe effects, such as chronic necrotizing pulmonary Aspergillosis [103]. The number of people around the world who have CPA is estimated at three million, and it is believed that Asia has the highest number of disease cases in comparison to other continents [104]. For the year 2019, it was calculated that after pulmonary tuberculosis, 12 million patients developed CPA [105]. In addition, the species of the genera *Aspergillus* and *Penicillium* produce various secondary metabolites known as mycotoxins [106]. In this set of toxins, we find the aflatoxins, fumonisins, deoxynivalenol, ochratoxin A, and zearalenone are agriculturally important and dietary mycotoxins exposure is associated with many chronic health risks, such as cancer, immune suppression, digestive, blood, and nerve defects [107,108]. Next, studies performed with propolis and these species give these fungal gears are described.

Portuguese propolis presented varying antifungal activity against *Aspergillus fumigatus* [84]. Propolis from different latitudes can present very similar biological activities, as in the case of United States and Chinese propolis against *Penicillium notatum*. With both propolis, the structure and morphology of hyphae were damaged, inhibiting the development of mycelium. Propolis treatment raised extracellular conductivities, showing that propolis probably affects the cell membrane. In addition, a decrease in the activity of enzymes related to the functioning of cellular respiration of *P. notatum* (succinate dehydrogenase and malate dehydrogenase) was observed. Additionally, quantitative proteomic analysis (iTRAQ-based) related to energy metabolism and sterols biosynthetic pathway of *P. notatum* in presence the propolis was described, which showed that 88 proteins (25.8%) were upregulated and 253 (74.2%) were downregulated, of a total of 341 proteins. The major compounds

in both propolis were pinocembrin, pinobanksin-3-O-acetate, galanin, chrysin, pinobanksin, and pinobanksin-methyl ether. The authors suggest that all these different properties that propolis has on *P. notatum* can interfere with its development [109]. Similarly, Southern Poland propolis showed antifungal activity against different microorganisms, including *A. niger* and *A. ochraceus*, and were found to contain pinocembrin, chrysin, pinobanksin, apigenin, kaempferol, *p*-coumaric acid, ferulic acid, and caffeic acid [43]. Few studies have been conducted on the effects of propolis against the genus *Aspergillus*, indicating a large gap in the literature. However, the previous studies only focused on observing whether propolis has antifungal activity; only the identified compounds were mentioned, and no correlation was mentioned between the activity and the propolis components, so research is lacking on this topic [110]. We note that study by Xu et al. provides a clear example of how to study natural products on different microorganisms to determine if they present activity or not, to later identify the chemical composition, and then try to find a possible mechanism of action by which natural products could inhibit the pathogen. Finally, the next step in Xu et al.'s research is to conduct studies on in vivo models and clinical trials to provide an alternative and complementary treatment for fungal infections.

Currently, some antifungal drugs are available; however, the problem in treating these diseases is the toxicity toward the host or the emergence of drug resistance in pathogen populations. Here, we describe the efficacy of propolis against different fungal pathogens, where the effect of propolis has been demonstrated in vitro and in vivo [111], as well as in different pathogenicity mechanisms; in some cases, an activity similar to that of the drugs used to treat mycoses has been reported. One of the most remarkable aspects is the use of propolis in clinical studies, where it was shown that propolis can be an alternative that complements the treatment of some of these diseases [112], so it is important to realize more clinical trials to support the effectiveness of propolis in addition to implementing trials focused on evaluating toxicity to determine a standardized dose of propolis that is safe for consumption and application in humans, as well as study the components of each propolis used. These studies could give scientific support to natural products widely used as a therapeutic alternative in rural communities and in developing nations.

## **4. Antiparasitic Activity**

Parasitic diseases continue to take an enormous toll on human health globally, particularly in tropical regions [113,114]. Intestinal and protozoan infections are the most common parasitic diseases. Protozoan parasites are unicellular eukaryotes responsible for 1.3 million deaths worldwide annually [114,115]. In several countries, these diseases are unfortunately not a priority with respect to their surveillance, prevention, and treatment. Among these diseases are malaria, Chagas disease, leishmaniasis, trichomoniasis, amebiasis, and giardiasis [116–118]. Below, we discuss the studies that have been conducted with propolis on the pathogens that cause these diseases.

## *4.1. Malaria*

Malaria is a disease that can cause death generated by a protozoan parasite of the Plasmodium genus. This disease is transmitted by female Anopheles mosquito bites. It is estimated to impair about 219 million people each year in 87 countries, mainly affecting pregnant women and children aged between 0 and 5 years [119]. Propolis has been used from some countries to study its antimalarial effects on species of the genus Plasmodium. In an effort to find alternatives for this disease, 20 propolis from different provinces in Cuba were evaluated in vitro, with three showing significant activity on *Plasmodium falciparum*. Chemical composition analyzes were carried out for propolis, where compounds of phenolic origin and triterpenes such as linquiritigenin and lupeol were found; these compounds were already reported to have activity against *P. falciparum* [120]. Similarly, twelve propolis from Libya were evaluated in vitro and demonstrated antiprotozoal activity, including against *P. falciparum* [121]. Propolis also showed antimalarial properties in vitro, for example, in the case of four Iranian propolis that showed in vitro and in vivo activity at different

concentrations against *P. falciparum*. The chemical composition of the two extracts with higher activity was determined, and molecules such as palmitic acid, stearic acid, pinocembrin, tectochrysin, and 4 ,5-dihydroxy-7-methoxyflavanone with an antiplasmodial effect were identified [122]. Saudi propolis considerably suppressed parasitemia and demonstrated an important effect on decreasing anemia in *Plasmodium chabaudi*-infected mice, reducing oxidative damage by enhancing the catalase function and the glutathione concentrations, and enhancing the quantities of pro-inflammatory cytokines. It is reported that these cytokines promote phagocytosis, chemotaxis, and antibody-dependent cytotoxicity. Furthermore, they are responsible for the activation of neutrophils as well as protection against this parasite (Table 11) [123]. These works demonstrate the promise of using propolis against this complex disease; however, some of these works lacked a chemical analysis of propolis. As mentioned previously, it is necessary that future studies with propolis include the origin and description of the chemical composition [6]. Some of the compounds such as lupeol and liquiritigenin have been reported to have antiplasmodial activity [124,125] and could be related to the effects of Cuba's propolis against this parasite. However, it is still necessary to realize different studies to better understand the effects of propolis in the treatment of this disease.


**Table 11.** Antimalarial effect of propolis and its chemical composition.

N.I. = none identified.

### *4.2. Chagas Disease*

*Trypanosoma cruzi* is a protozoan parasite responsible for Chagas disease, which is transmitted mainly by Hematophagous triatomine insects, and to lesser extent by oral, congenital, blood transfusion and organ transplantation [126]. This disease is a problem for most of Latin America and especially affects marginalized zones; it is estimated that 8–10 million people are infected each year [126,127]. Extensive research has been conducted on propolis as an anti-trypanosome agent using in vitro models. We highlight the thorough study on various propolis from Brazil, of which the activity of propolis extract on *T. cruzi* trypomastigotes was reported [30]. Some propolis were effective on the three forms of the parasite, and treatment with propolis strongly inhibited infection levels by promoting lysis of bloodstream trypomastigotes and diminished the number of parasites in peritoneal

macrophages and infected heart muscle cells [128]. Some propolis from Brazil showed an in vitro effect against *T. cruzi*; their chemical composition was determined, and caffeic acid, cinnamic acid, pentenoic acid, ferulic acid, linoleic acid, amyrin, and pinostrobin, amongst others, were identified; however, in these studies, the anti-trypanosomal activity of these compounds was not evaluated [129,130]. Interestingly, other authors reported that the application of natural products obtained from propolis produced anti-trypanosome effects; for example, four components were isolated from Brazilian propolis and two were effective on *T. cruzi* [131]. Similarly, Brazilian and Bulgarian propolis were shown to have activity against this parasite, diminishing replication of the parasite without damaging the membrane of the host cell. Microscopic analysis showed that the main organelles damaged by the extracts were mitochondrion and reservosomes [132]; two Bulgarian propolis share many bioactive compounds, mainly flavonoids and a remarkable antitrypanosomal effect; epimastigotes were more sensitive than trypomastigotes. The efficacy of either of the two Bulgarian propolis on trypomastigotes was similar to that of the reference drug [133].

Despite the encouraging results from the in vitro tests, in vivo studies are scarce. For example, treatment with Bulgarian propolis in *T. cruzi*-infected mice led to a reduction in parasitemia and showed no toxic hepatic or renal effect, the spleen mass decreased, and the initial inflammatory reaction was modulated, favoring a greater number of CD8+ and partially inhibiting the increase in CD4 [134]. Studies conducted with propolis from Brazil in infected mice recorded a decrease in the number of parasites and mortality of the animals without generating toxicity or injury on other tissues, so it could be assayed in combination with other drugs as a potential metacyclogenesis blocker (Table 12) [135].


**Table 12.** Anti-trypanosome activity of different propolis and its chemical composition.


N.I. = none identified.

#### *4.3. Leishmaniasis*

Leishmaniasis is a neglected disease group, occasioned by 20 species of protozoan parasites belonging to the genus *Leishmania* and spread by female sand flies of the genus Phlebotomus or Lutzomyia. Present in nearly 100 countries and endemic in Asia, Africa, the Americas, and the Mediterranean region, more than 12 million people, about 25,000 deaths, and 1 million new cases are reported annually; according to the WHO, it is a Category I (emerging or uncontrolled) disease [136]. In humans, four clinical forms of this disease can develop: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), or post-Kala-azar dermal leishmaniasis (PKDL). For more than six decades, pentavalent antimonials (SbV) were the first-line drugs against leishmaniasis; however, the toxicity and resistance of the parasites are the main limitation of these drugs [137]. Other treatments such as pentamidine, paromomycin, or amphotericin B has been employed, but its high costs and side effects make it difficult to use [137,138]. Therefore, alternatives are urgently required that complement and help with the adequate treatment of leishmaniasis. Propolis has been studied as an alternative to various protozoa, including parasites of the genus *Leishmania*. The main investigations on the leishmanicidal effect of propolis have mainly been conducted in vitro, focusing on determining the effect of propolis on the mortality of this parasite, e.g., the brown, green, and red propolis from Brazil and that from Portugal, which showed significant growth inhibition of *L. braziliensis* Vianna, *L. infantum*, and *L. amazonensis* promastigotes, and decreased the number of internalized amastigotes in infected murine macrophages [84,139–141]. Some of the countries with the highest incidence of Leishmaniasis are in the Middle East; the administration of propolis as an antileishmania agent has also been reported in this region. The composition of propolis from three regions of Turkey (Adana, Hatay, and Bursa) was analyzed, and differences were found in the type of compounds and in their quantities; the main component of Adana propolis was found to be cembrene, that of Hatay was chrysin, and Bursa's was cinnamyl cinnamate. All three registered a good antileishmanial activity against *L. tropica* or *L. infantum*, but the propolis from Bursa was the most effective [142,143]. The effects of propolis on Leishmaniasis have been studied more: as mentioned earlier, various in vitro studies have shown the benefits of this apiculture product. Fortunately, in several of these studies, the chemical compounds present in each propolis were identified. Several of these pure compounds have been tried individually against different species of *Leishmania*, and these compounds are likely related to the antileishmanial effect of propolis [144–146]. Although these studies are limited, since they only involved in vitro tests, they provide support for the use and application of propolis in animal models.

Many have evaluated the effect of propolis from Latin American countries on various species of the genus *Leishmania* and have identified a large part of the chemical composition of these propolis. For example, 20 propolis from Cuba presented in vitro antimicrobial properties; the major effect was found on *L. infantum*. The results demonstrated an association between the biological effect and compounds identified. The propolis that contain acetyl

triterpenes as amyrin, lupeol, and cycloartenol as the main constituents are the best options for future studies [120]. Similarly, three propolis obtained from distinct areas in Ecuador (Quito, Guayaquil, and Cotacach) avoided *L. amazonensis* growth, highlighting the activity of sample rich in flavonoids as naringenin, sakuranetin, eupatolitin, and rhamnazin [147]. Brazil is one of the countries with the most studies of the biological and chemical properties of its propolis. Brazilian propolis (Ribeirao Petro and Minas Gerais) were proven on *Leishmania* species associated with different clinical forms of leishmaniasis, and the chemical composition was determined. Propolis from Minas Gerais showed great antileishmanial effect on *L. amazonensis*, *L. braziliensis*, *L. chagasi*, and *Leishmania major*, with the last species being the most susceptible. Ribeirao Petro propolis was only evaluated against *L. amazonensis*; it recorded a dose-dependent activity against promastigotes, also the number of parasites decreased inside macrophages. Although a leishmanicidal effect on *L. amazonensis* was reported in the two studies, the effects were not the same, because the extracts from Minas Gerais and Ribeirao Petro had different chemical compositions: the main compounds of the first were diethyl 2-methylsuccinate, cinnamic acid, pentanedioc acid, and hydrocinnamic acid; for the second, they were artepillin C, 4,5-dicaffeoylquinic acid, *p*-coumaric acid, and drupanin [148,149]. Brazilian propolis also showed strong in vivo effects: in an experimental infection model with *L. braziliensis* using BALB/c mice treated previusly with propolis, it reduced growth and promoted morphologic alterations on promastigotes and also favored the TNF-α levels in supernatants from liver cells and peritoneal exudate [150]. Green propolis decreased more than 75% in lesion development caused by L. brazilienzis, while the glucantime treatment showed a 57.7% decrease (Table 13) [151].

(50–1000 μg/mL) showed


**Table 13.** Antileishmanial activity of various propolis and its chemical composition.



**Table 13.** *Conts.*

When used in combination with nitric oxide (NO) during infection with *L. amazonensis* at the lesion site, the levels of NO, healing, collagen synthesis, the function of macrophages and fibroblasts were favored, in addition to decreased parasitized cells, pro-inflammatory factors, and tissue damage [152]. Propolis from Brazil was also used in combination with first line antileishmaniasis medications. Green propolis was administered in combination with liposomal meglumine antimoniate, decreasing the parasitic burden in the liver without damaging or altering the functions of the kidney, liver, spleen, and heart (Table 14) [153]. The two works mentioned in this section are noteworthy as they used propolis as a complementary or combination treatment with another substance. This type of study demonstrates the path that can be followed in the examination of propolis and its bioactive compounds, since propolis is not intended to replace existing treatments but to supplement them with new alternatives [154,155]. Finally, clinical trials are still needed to demonstrate the effectiveness of propolis in humans.


**Table 14.** Activity of Brazilian propolis on different *Leishmania* infection models.

## *4.4. Giardiasis*

Giardiasis is a parasitic intestinal disease, the etiological agent of which is *Giardia duodenalis*, also known as *G. intestinalis* or *G. lamblia*. This parasite is transmitted mainly by consuming water or food that contains Giardia cysts. Symptoms of infection are usually diarrhea, nausea, epigastric pain, and weight loss. Giardiasis annually affects about 200 million people worldwide. Since 2004, it is listed by the WHO as a neglected disease by the World Health Organization. The prevalence of *Giardia* infection is higher in developing countries [156,157]. Several remedies of traditional medicine have been administered as a complement to treat this disease [158], among which is propolis.

There are reports of the in vitro effect of three samples from Sonoran Desert propolis in Mexico (Caborca, Pueblo de Alamos, and Ures) and some of its bioactive compounds. The Ures propolis presented a remarkable activity on *G. lamblia* in a dose-dependent manner, as well as one of its components (CAPE), which registered the highest antigiardia effect [159]. Similarly, propolis from Brazil showed efficacy in eliminating trophozoites of *G. lamblia*. The effect on the proteolytic activity of excretory/secretory products (ESPs) from trophozoites treated with propolis was studied; however, no significant differences were found between hydrolysis patterns and inhibition on the protease activity of propolis-treated and untreated trophozoites [160,161]. Propolis from Egypt was reported to be effective in an in vivo model of giardiasis with immunodeficient mice. The propolis produced a diminution in intensity of infection, as well an augment in the IFN-γ serum level and in the CD4+:CD8+ T cell ratio. Combination propolis and metronidazole presented a great effect in reducing the number of parasites than that produced by each drug alone. Futhermore, this combination induced an immunological regulation, mainly in T lymphocytes, which favor intestinal homeostasis and histological integrity (Table 15) [162]. While several drugs against this parasitosis are available, its incidence continues to be higher in developing countries [163]. In these regions, it is common for people to use traditional or alternative medicine to address health problems [164]. For this reason, it is necessary to determine the effectiveness of these treatments against this parasite. The works included in this review on this disease demonstrate the in vitro and in vivo effect of propolis and its effect on the immunological response against *G. lamblia*. Further research is important to support the utility of propolis as an alternative against Giardiasis; the work conducted to date and the information available are limited.

In this work, we addressed the properties of propolis in the defense against these parasitic diseases; in most cases, propolis showed interesting and promising antiprotozoal activities [165]. However, several challenges remain, such as the wide variety in the components of propolis in each geographical area, isolating the components responsible for the activities, and describing their mechanisms of action and synergy. It is also necessary to promote and conduct research using animal models, since very few studies have been published. It should be noted that the implementation of clinical studies is necessary to support the antiparasitic activity of propolis, as well as conducting research focused on the combined treatment of propolis with different drugs used to treat parasitic infections in humans, and to find more effective complementary treatments in order to be able to reduce the dose and toxicity of the drugs currently implemented.

**Table 15.** Effect of different propolis on *G. lamblia* in in vitro and in vivo models.


NS: not specified.

## *4.5. Helminths*

Humans are exposed to a remarkable number of parasites, including protozoans (over 70 species), helminths (about 300 species), and arthropod parasites. There are two major phyla of helminths: the nematodes (also known as roundworms) and the Platyhelminthes (Trematoda and Cestoda) [166,167]. According to the WHO, more than 2 billion cases of intestinal worms were registered in 2018, mainly affecting disadvantaged communities [168]. However, this number could be higher, since many of these diseases are not reported [169]. Another relevant aspect regarding helminths is that they not only parasitize humans, but also affect many domestic animals and the livestock industry, resulting in large economic losses [168]. In addition, in the absence of therapeutic options in developing regions, this population resorts to the use of traditional remedies or alternative medicine to treat these parasites.

The anthelmintic activity of some propolis from Egypt on adult flukes of *Fasciola gigantica* was reported. Alteration of the architecture was found as lifting base of the spines and large blisters in the apical cone, several of which seemed to have burst, generating injuries. The inhibitory activity on the viability and hatchability of immature *F. gigantica* eggs was also found, showing the highest inhibitory effect compared with other treatments. The chemical composition of these propolis was determined. Compounds such as diprenyl-dihydrocoumaric acids, coumarate esters, ferulate esters, hydroxy acetophenones, furanon derivative, furofuran lignans, benzofuran lignans, and valeric acids derivatives could be related to anthelmintic activity [170,171]. Other propolis from Egypt was evaluated against *Schistosoma mansoni* in mice: propolis alone or in combination with praziquantel were administrated. Propolis administration did not eliminate the worms of infected mice but significantly reduced the hepatic granuloma number, hepatic, splenic, and plasma myeloperoxidase (MPO) activity, as well the liver and thymus NO levels, and also regulation of plasma antioxidant proteins evidenced by decrease in malondialdehyde (MDA) and normalization of glutathione (GSH) [172]. The antihelmintic activity of propolis from Turkey on *Echinococcus granulosus* was reported: 1 μg/mL of propolis killed all the protoscoleces in the in vivo part of the study, without causing side effects when administered intraperitoneally. However, the mechanism of action and chemical composition were not reported [173]. There are also reports of the anthelmintic effect of the essential oil of Brazilian red propolis, larvae of *Toxocara cati*, were incubated during 48 h with the essential oil, and then later inoculated in mice. The authors informed 100% effectivity to disable the infective capacity of the larvae [174]. Five propolis from distinct parts of Libya were studied, and they presented moderate activity against *Trichinella spiralis*. The components of the propolis was analyzed, and fourteen compounds were identified, of which cycloartanol, mangiferolic

acid, agathadiol, isocupressic acid, and isoagatholal were highlighted (Table 16) [175]. These compounds may play interesting roles in the effects of propolis on helminths in each of the studies reviewed. As each propolis has a complex and changing chemical composition, it is a priority to determine whether the antihelmintic activity is due to a specific compound or a synergism phenomenon to identify new pharmacological alternatives [176]. Since the results in these works show the favorable effects of propolis against various helminths, these propolis could be tested against parasites such as *Tenia*, *Enterobius*, and *Ascaris*, which have high incidence in several countries and are a public health problem [177,178].


**Table 16.** Anthelmintic activity of propolis and its chemical composition.

N.I., none identified; MPO, myeloperoxidase; MDA, malondialdehyde; GSH, glutathione.

Helminth parasites mainly continue to be a public health problem in countries with disadvantaged and low-resource communities. Currently available anthelmintic drugs include the benzimidazoles (albendazole and mebendazole), pyrantel pamoate, and ivermectin [179]. Although these drugs are usually well-tolerated and efficient for the treatment of helminth parasites, they are limited in number, and the susceptibility among helminth species has been shown to vary greatly in different populations [180]. Another concern is the emergence of resistance, which have mainly been observed in veterinary medicine over the past decade [181]. Traditional and alternative medicine offers a wide repertoire of compounds that could complement the treatment of these diseases. Research in animal models must be increased, and clinical trials are needed to confirm the safe utilization of propolis in these diseases.

## **5. Antiviral Activity of Propolis**

Viruses need the host cells' biosynthetic machinery to replicate [182,183]. Viral infections are responsible for some diseases in humans and cause serious public health problems in populations worldwide [184]. Therefore, recent research on new antiviral medications is increasing due to the development of resistance to antiviral drugs [185]. As such, the study of natural products that present antiviral activity, such as propolis and some of its identified compounds, is vital [186,187].

Two propolis from Czech Republic (aqueous and ethanolic extracts) were studied, and both showed great antiviral effect on herpes simplex virus type 2 (HSV-2). Both propolis decreased the infection and exhibited a concentration- and time-dependent antiviral effect. Additionally, the two propolis showed a high antiviral effect when viruses were pretreated prior to infection; thus, both propolis could be used to treat recurrent herpetic infection topically [188].

In other research, the antiviral effect of different propolis from several geographic regions, such as the United States, Brazil, and China, was against human immunodeficiency virus type 1 (HIV-1). All propolis inhibited viral expression in CD4+ lymphocytes and microglial cell in a concentration-dependent manner. In another study, propolis from the United States suppressed cell fusion HIV-1 in cultures of CD4+ lymphocytes, suggesting that the possible mechanism of propolis' antiviral property in CD4+ lymphocytes is produced in part by inhibition viral entry into cells [189].

The antiviral activity of four Brazilian propolis on influenza virus was examined, and all propolis presented anti-influenza virus effect in vitro. In this same study, the four propolis were studied in a murine influenza virus infection model (propolis was orally administered three times daily for seven days), and only one propolis sample effectively prolonged the lifetime of infected mice. The authors concluded that the Brazilian propolis possessed antiviral effect and amelioration influenza symptoms in mice [190]. In another study, three different extracts of Brazilian propolis were administered orally three times daily for six days to cutaneous herpes simplex virus type 1 (HSV-1)-infected mice to study their effect on HSV-1 infection. The three propolis presented anti-HSV-1 activity and favored immunological effect on intradermal HSV-1 infection in mice (Table 17) [191]. At present, diseases caused by viruses are a priority in any health system due to the severity of their symptomatology, their high infective capacity, and mortality. Viral diseases can affect the economy of a country or the globe [192,193]. The previously mentioned propolis studies are notable and highly relevant, since they reported antiviral properties capable of inhibiting viral replication, cell fusion in cultures of CD4+ lymphocytes, and stimulation of immunological activity [189]. However, these works lack a chemical analysis, which limits the explanation of some possible action mechanisms related to the secondary metabolites present in each propolis.

Some flavonoids and phenolic acids, also described in propolis, presented antiviral activity [187]. One antiviral study showed that Canadian propolis had a pronounced viricidal effect against HSV-1 and HSV-2 and interfered with virus adsorption. Different compounds were identified in this propolis. The interaction with propolis indicates damage to the HSV and suggests that propolis could damage protein components of envelopes essential for adsorption and penetration of the virus into the cells (Table 18) [194].


**Table 17.** Antiviral activity of different samples of propolis.

**Table 18.** Anti-HSV-1 and HSV-2 activity of several propolis and its chemical composition.


Similarly, the antiviral effect of aqueous and ethanolic extract of Czech Republic and some compounds identified in propolis against HSV-1 in cell culture was analyzed. Both samples presented high anti-HSV-1 effect in cells effect in prior to viral infection; of the compounds tested, galangin and chrysin were the most active components. However, the propolis with various compounds presented higher antiviral activities than the isolated constituents alone. The authors concluded that the antiherpetic activity of propolis is due to a combination of several components; therefore, the propolis from Czech Republic was found to be more effective on herpes infection than the individual compounds [187].

In another study, the replication of HSV-1 and HSV-2 was inhibited with the propolis from the south of Turkey. Propolis started to suppress HSV-1 replication after 24 h of incubation and effect on HSV-2 started at 48 h after incubation. This activity of propolis on HSV-1 and HSV-2 was checked by a lower in the number of viral copies. They found that propolis showed activity similar to that of acyclovir, since both started to suppress HSV-1 replication following 24 h of incubation. They also found a synergistic effect of combined propolis and acyclovir on HSV-1 and HSV-2 replication compared with acyclovir alone. Some compounds in the propolis were identified. The propolis from the south of Turkey was found to present relevant antiherpetic activities in comparation with acyclovir; particularly, the synergism generated by the antiherpetic effect of propolis and acyclovir in combination has a stronger activity on HSV-1 and HSV-2 than acyclovir alone. The authors mentioned that the possible mechanism of synergism between acyclovir and propolis may be attributed to some of the components of propolis [185]. As described in each work, most of the molecules identified in each propolis are compounds of phenolic origin. Some of these have been reported as having the ability to stimulate antiviral responses in in vitro and in vivo models, promoting the production of interferons as well as the activation of cytotoxic T lymphocytes and natural killer cells [195–199]. This supports the use of propolis as a source of new molecules with antiviral effects and an alternative to complement the treatment of these diseases.

Although little literature about the antiviral activity of propolis in clinical studies exists, in 2019, Jautová et al. reported that one lip cream with propolis extract from Central Europe produced a better effect than acyclovir to treat patients with herpes labialis in the vesicular phase, confirming the clinical efficacy of lip cream composed of European propolis in the early and late start of treatment during an episode of herpes labialis [200]. Similarly, a clinical study reported the contribution of propolis extract from Central Europe as a constituent in a lotion for complementary treatment of Herpes zoster. A total of 33 patients with a diagnosis of Herpes zoster applied a treatment with a propolis-based lotion for 28 days as a complementary treatment to oral antiviral treatment with acyclovir. The healing of lesions was improved and faster with the propolis treatment; approximately 50% of propolis-treated patients had no injuries on day 14 and the growth of new vesicles was inhibited, clinically confirming the antiviral effects of European propolis and demonstrating the properties of complementary therapy on the systemic antiviral treatment of Herpes zoster (Table 19) [201].


**Table 19.** Antiherpetic activity of Central European propolis in patient studies.

The current situation related to COVID, which has compromised all health systems, makes it necessary to search for therapies that prevent or mitigate the complications of this disease. Natural products such as propolis are an interesting option in the search of complete therapies. Some recent research mentions the potential benefits of using propolis against this disease. These studies are based on previously reported activities against other viruses and on in silico models that allow predictions of activities against this virus. These studies focus mainly on reported bioactive compounds in the different propolis; they include antiviral activities that could be applied against SARS-CoV-2 or immunomodulatory effects that would reduce the symptoms of the disease. One of the clearest examples is quercetin, one of the most abundant and consumed flavonoids in the diet. Quercetin has been shown to inhibit the replication cycle of the virus, since it reduces the functioning of the main protease (Mpro) and S protein of SARS-CoV-2. CAPE, one of the main components of many propolis, is able to inhibit the transmembrane protease serine 2 (TMPRSS2), angiotensin-converting enzyme-related carboxypeptidase (ACE-II), and Mpro; these molecules are crucial for access and replication viral of SARS-CoV-2 in cells. Another interesting compound is the rutin that reduced the function of S protein, ACE-II, and others non-structural proteins of SARS-CoV-2. These flavonoids are also able to regulate JAK/STAT-mediated signaling and the production of ROS, NO, pro- and anti-inflammatory cytokines, avoiding a cytokine storm. They even reduce the risk of comorbidities that complicate the betterment of patients with COVID-19 [202–204]. Propolis and its bioactive compounds open new means for future works that describe in detail their effects on SARS-Cov-2 and are able to be applied as a complementary therapy in clinical studies.

Considering all the research mentioned above, the search for new strategies for the control and complementary treatments of infections caused by viruses has become a global public health priority. However, more in vitro and clinical studies with propolis are needed to elucidate its mechanisms of action and identify the molecules responsible for the antiviral effects of this natural product.

### **6. Conclusions**

We collect the main studies of the effect of propolis on pathogens related to infectious diseases of medical relevance. The reports of the efficacy of the different propolis are encouraging: this bee product showed effectiveness on bacteria, fungi, protozoa, helminths, and viruses. Propolis presents a great spectrum of components that could be used to treat characteristic affections of distinct diseases. Not all propolis present the same activities; depending on the flora of the geographical area, each propolis has a different chemical composition with unique biological activities, making propolis a promising source of discovering molecules, which can be used in different clinical situations. Propolis offers potential for research into the treatment of infectious diseases that lack adequate therapies due to the resistance of pathogens to drugs, either isolating active components to be studied alone or combined with different current drugs. Despite the in vitro and in vivo evidence suggesting that propolis can be a reliable alternative to existing drugs, the effect of propolis must be investigated in the clinic to improve tour comprehension of the mechanisms of action of the different propolis, attain the synergism of their compounds, and generate a standardized and safe consumption protocol.

Another relevant aspect is that clinical tests with propolis, bee products, or other natural products are scarce but necessary. From products used in traditional medicine, modern medicine has obtained compounds such as taxol, valproic acid, polycarpine, ephedrine, digoxin, and acetylsalicylic acid, just to name a few. The therapeutic uses and applications of natural products and their derivatives are promising in the search for new treatments, so clinical studies against diseases caused by microbes resistant to drugs or treated with toxic agents should be a priority in future clinical research.

Finally, a new perspective to consider in future research is to investigate the presence and function of microRNAs (miRNAs) in propolis. Recent studies have proposed that the miRNAs present in honey from plants visited by bees during their collection could play a determining role in the development of larvae. The finding of these molecules

could be surprising related to the beneficial effects on the health of consumers of this bee product. The identification of miRNAs in propolis would be crucial to understanding and explaining many of its biological and medicinal activities, and these activities are currently attributed mainly to compounds such as flavonoids and terpenes. miRNAs in bee products can be the subject of various investigations, and their clinical applications could generate new treatments based on nutritional supplements with various specific benefits for health [205,206].

**Author Contributions:** O.N.-Y. and N.R.-Y. performed the conceptualization; N.R.-Y., O.N.-Y., and C.R.R.-Y. collected the data; C.R.R.-Y., N.R.-Y., and O.N.-Y. wrote the first draft with contributions from G.P.-M., C.F.M.-C., J.R.-R., M.I.M.-R., and A.R.M.-C.; review and editing were performed by G.P.-M., C.F.M.-C., J.R.-R., M.I.M.-R., and A.R.M.-C. All authors reviewed and worked in the final version. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was financially supported, in part, by the institutional Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) de la UNAM IA206819 and PAPIIT de la UNAM IA207921.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Article* **Effects on Steroid 5-Alpha Reductase Gene Expression of Thai Rice Bran Extracts and Molecular Dynamics Study on SRD5A2**

**Chiranan Khantham <sup>1</sup> , Wipawadee Yooin 1,2 , Korawan Sringarm 2,3 , Sarana Rose Sommano 2,4 , Supat Jiranusornkul 1, Francisco David Carmona 5,6 , Wutigri Nimlamool <sup>7</sup> , Pensak Jantrawut 1,2, Pornchai Rachtanapun 8,9 and Warintorn Ruksiriwanich 1,2,8,\***


**Simple Summary:** Dihydrotestosterone (DHT), the most potent androgen hormone, is an important aetiologic factor of androgenetic alopecia (AGA), or hair loss. Steroid 5-alpha reductases (SRD5As) increase DHT production in the scalp hair follicles, resulting in hair thinning and hair loss. Even though synthetic SRD5A inhibitors (finasteride and dutasteride) are effective in treating AGA, they cause adverse effects. This has led to an increased interest in alternative treatments from natural sources. The value of Thai rice bran has increased because several of its components may have use in AGA treatment. This study aimed to compare the suppression of the expression of *SRD5A* genes (type 1–3) exerted by several Thai rice bran extracts and investigate the interactional mechanism of their components towards SRD5A type 2. Tubtim Chumphae rice bran (TRB) had the highest sum of overall bioactive compounds. Among all extracts, the expression of *SRD5A* genes was suppressed by TRB as well as finasteride. In silico simulation showed that α-tocopherol had the greatest interaction with SRD5A type 2. Our findings identified α-tocopherol as the key bioactive in TRB; it could be developed as an anti-hair loss product.

**Abstract:** Steroid 5-alpha reductases (SRD5As) are responsible for the conversion of testosterone to dihydrotestosterone, a potent androgen, which is the aetiologic factor of androgenetic alopecia. This study aimed to compare the *SRD5A* gene expression suppression activity exerted by Thai rice bran extracts and their components and investigate the interactional mechanism between bioactive compounds and SRD5A2 using molecular dynamics (MD) simulation. Bran of *Oryza sativa* cv. Tubtim Chumphae (TRB), Yamuechaebia Morchor (YRB), Riceberry (RRB), and Malinil Surin (MRB), all rice milling by-products, was solvent-extracted. The ethanolic extract of TRB had the highest sum of overall bioactive compounds (γ-oryzanol; α-, β-, and γ-tocopherol; phenolics; and flavonoids). Among all extracts, TRB greatly downregulated the expression of *SRD5A1*, *SRD5A2*, and *SRD5A3*; there were no significant differences between TRB and finasteride regarding *SRD5A* suppression. The linear relationship and principal component analysis supported that the α-tocopherol content was correlated with the *SRD5A* suppression exerted by TRB. Furthermore, MD simulation demonstrated

**Citation:** Khantham, C.; Yooin, W.; Sringarm, K.; Sommano, S.R.; Jiranusornkul, S.; Carmona, F.D.; Nimlamool, W.; Jantrawut, P.; Rachtanapun, P.; Ruksiriwanich, W. Effects on Steroid 5-Alpha Reductase Gene Expression of Thai Rice Bran Extracts and Molecular Dynamics Study on SRD5A2. *Biology* **2021**, *10*, 319. https://doi.org/10.3390/ biology10040319

Academic Editors: Francisco Les, Víctor López and Guillermo Cásedas

Received: 10 March 2021 Accepted: 9 April 2021 Published: 11 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

that α-tocopherol had the highest binding affinity towards SRD5A2 by interacting with residues Phe118 and Trp201. Our findings indicate that α-tocopherol effectively downregulates the expression of *SRD5A* genes and inhibits SRD5A2 activity, actions that are comparable to standard finasteride. TRB, a source of α-tocopherol, could be developed as an anti-hair loss product.

**Keywords:** 5α-reductase; androgenetic alopecia; rice bran; tocopherol; SRD5A2; molecular dynamics; RD69; Tubtim chumphae

#### **1. Introduction**

Androgenetic alopecia (AGA), characterised by the progressive replacement of terminal hair into small vellus hair, is generally known as a hereditary androgen-dependent disorder [1]. Although AGA is not a serious threat to health, it may impact the social and psychological well-being of an individual [2]. Different tissues, including hair follicles, require an optimal androgen concentration. High androgen levels can have deleterious effects on health [3]. Overexpression and intensive activity of steroid 5-alpha reductase (SRD5A) in scalp follicles have been shown to be involved in AGA development [4].

SRD5A comprises five members, SRD5A1, SRD5A2, SRD5A3, and the little characterised glycoprotein synaptic 2 (GSPN2) and GSPN2-like [5]. SRD5As are dihydronicotinamide adenine dinucleotide phosphate (NADPH)-dependent [6] and play a significant role in steroidogenesis by catalysing 4-ene-3-keto steroids into more active 5α-reduced derivatives, including the reduction of testosterone (T) to dihydrotestosterone (DHT) [7]. SRD5A1, SRD5A2, and SRD5A3 are encoded by separate genes: *SRD5A1*, *SRD5A2*, and *SRD5A3*, respectively. While these isozymes share sequence homology and show similar substrate preferences, they vary in biochemical properties, sensitivity to SRD5A inhibitors, physiological functions, and also tissue distribution [8]. DHT has a 5-fold higher affinity for the androgen receptor and a 10-fold greater potency for provoking androgen-sensitive genes compared with its precursor [9]. These androgen-sensitive gene products, transforming growth factor beta 1, interleukin 6, and dickkopf 1, have been identified as androgen-inducible negative mediators for AGA development [10].

*SRD5A* genes are expressed differently in androgen-responsive tissues, which include the adrenal glands, the testis, the placenta, and the skin. When translated, the proteins are located mainly in the endoplasmic reticulum membrane [11]. Beyond sexual functions, they have also been implicated in and influence the diverse biological activities of the skin [12]. Both SRD5A1 and SRD5A2 are well-characterised enzymes involved in AGA; they are expressed principally in skin and annexes, including hair follicles, sweat glands, and sebaceous glands [4]. The expression levels of *SRD5A1* and *SRD5A2* are higher in the frontal hair follicles in both men and women with AGA, a pattern that indicates they play a major role in AGA [13]. The level of *SRD5A2* expression is higher in dermal papilla cells (DPCs) from an AGA scalp than in DPC from other sites [1]. In contrast, *SRD5A3* is involved in protein *N*-glycosylation and shows slight or no potential to catalyse steroid substrates [8,14]. Interestingly, a previous study identified that the ratio of the total amounts of DHT to T is significantly depleted in *SRD5A3*-knockdown cells [15]. Moreover, tissue distribution analysis demonstrated that the expression level of *SRD5A3* is higher in peripheral tissues, including the skin, compared with *SRD5A1* and *SRD5A2* [16]. *SRD5A3* is overexpressed in prostate cancer [17], and several studies have proposed an association between prostate cancer and AGA [18,19]. A subsequent study also reported that a higher expression level of *SRD5A3* in plucked hair derived from AGA, suggesting its possible role in the pathogenesis of AGA [20].

To date, oral finasteride and dutasteride, SRD5A competitive inhibitors, have been approved to treat AGA. Finasteride specifically inhibits SRD5A2 as well as SRD5A3 but restrains less effectively SRD5A1. On the other hand, dutasteride exhibits great inhibitory effects against the three distinct types of SRD5A [5,16]. Despite their promising efficacy, these drugs are associated with several side effects, especially erectile dysfunction and loss of libido [21]. In recent years, several herbal extracts and their bioactive constituents have been implemented as an alternative treatment to promote hair growth or prevent hair loss [22].

Rice (*Oryza Sativa* Linn.) is used as a staple food by more than half of the globe [23]. Thailand is recognised as the biggest rice exporter and the fifth-biggest producer in the world [24]. Rice bran is an important by-product that is largely generated during the milling process [25]. In addition, it is a rich source of biologically active compounds, including γ-oryzanol, phytic acid, vitamin E isoforms (α-, β-, and γ-tocopherol), unsaturated fatty acids (such as oleic acid, linoleic acid, and γ-linolenic acid), and phenolic compounds (such as ferulic acid, gallic acid, and caffeic acid) [26]. The geographical origin and genetic diversity among rice varieties also influence the types, appearances, and bioactive content in rice bran [25].

Rice bran and its biomolecules have been shown to possess the potential for application as a treatment for hair loss and androgen-dependent disorders such as benign prostatic hyperplasia, hirsutism, and hypertrichosis [22]. Researchers suggest that specific aliphatic unsaturated fatty acids, especially oleic acid, linoleic acid, and γ-linolenic acid, inhibit the activity of SRD5A in androgen-responsive tissue [27]. In addition, various extracts from *Serenoa repensits*, *Thujae occidentalis*, *Cucurbita pepo*, and *Panax ginseng* inhibit SRD5A activity; these findings suggest that free fatty acids contribute to SRD5A inhibition [22]. Rice bran supercritical CO2 extracts and linoleic acid have been reported to suppress the messenger RNA (mRNA) expression of *SRD5A1* in cell lines [28]. Several studies have focused on the effect of plant extracts or their bioactive constituents, especially unsaturated fatty acids, on the inhibition of SRD5A activities, but only a few have focused on the gene expression levels [22].

Previous studies have investigated the inhibitory effects of rice bran extracts and their constituents on SRD5A1 and SRD5A2, but the effect on SRD5A3 is still unknown. Furthermore, the effect of other major components (vitamin E isoforms, phytic acid, and phenolic compounds) of rice bran extracts on the expression of *SRD5A* genes has not been thoroughly determined [29]. SRD5A2 is an important causative factor of AGA, and the aforementioned study was limited to SRD5A2. Moreover, the atomic-level mechanism between SRD5A2 and the bioactive compounds of rice bran extracts remains unclear. Molecular dynamics (MD) simulation can help to understand the binding mode of ligands towards SRD5A2, screen potential ligands, and accelerate candidate identification instead of the experimental method. With regards to these rationales, it is necessary to understand the biochemical actions of rice bran extracts and their bioactive compounds before converting this agricultural waste to a high-value-added anti-hair loss product. Hence, this study aimed to compare the *SRD5A* suppression exerted by four Thai rice bran varieties and their bioactive compounds and also to investigate the interactional mechanism at an atomic level of bioactive compounds in rice bran towards SRD5A2.

## **2. Materials and Methods**

## *2.1. Reagents and Chemicals*

Sulphorhodamine B (SRB); the Folin–Ciocalteu reagent; (−)-epigallocatechin gallate (EGCG); ferulic acid, phytic acid, gallic acid, oleic acid, linoleic acid, and γ-linolenic acid; γ-oryzanol; and α-, β-, and γ-tocopherol were obtained from Sigma Chemical (St. Louis, MO, USA). Finasteride and dutasteride were obtained from Wuhan W&Z Biotech (Wuhan, China). Agarose gel, Tris base, and 50X Tris/acetic acid/EDTA (TAE) were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Foetal bovine serum (FBS; cat no. 16000044) and Roswell Park Memorial Institute medium (RPMI-1640; cat no. 31800022) were obtained from Gibco Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). Penicillin/streptomycin solution (100X) was purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Ethanol, dimethyl sulphoxide (DMSO), acetic

acid, trichloroacetic acid, and other chemical substances were obtained from RCI Labscan (Bangkok, Thailand). All other chemicals were of analytical grade.

## *2.2. Plant Material and Extraction*

Rice bran of *Oryza sativa* Linn. cv. Tubtim Chumphae (RD69; TRB), Riceberry (RRB), and Mali Nil Surin (SRNC05053-6-2; MRB) was provided by Phrao Green Valley Co., Ltd. (Chiang Mai, Thailand). Rice bran of Yamuechaebia Morchor (YMCB 3 CMU; YRB) was obtained from Lanna Rice Research Center, Chiang Mai University, Thailand. Herbarium voucher specimens of TRB (PNPRDU63021), YRB (PNPRDU63022), RRB (PNPRDU63023), and MRB (PNPRDU63024) were deposited in the Pharmaceutical and Natural Products Research and Development Unit, Faculty of Pharmacy, Chiang Mai University. Rice bran (2 kg) was macerated in 95% (*v*/*v*) ethanol (ratio of solid/solvent: 1:3) for 24 h. The extract solutions were filtered through Whatman filter paper no. 4 and then no. 1 and concentrated by a vacuum evaporator (Hei-VAP value, Heidolph, Schwabach, Germany) at 50 ◦C. All extracts were stored in a sealed vial in the dark at 4 ◦C before further analysis.

#### *2.3. Determination of Bioactive Compounds*

## 2.3.1. γ-Oryzanol and Tocopherols

The high-performance liquid chromatography (HPLC) conditions were adapted from a previous study [30]. The analytical system consisted of an Agilent 1220 Infinity DAD LC module (Agilent Technology, Palo Alto, CA, USA) and a fluorescent detector (Agilent 1260 FLD Spectra, Agilent Technology). The separation was performed on an Ultra C-18 column (250 mm × 4.6 mm, 5 μm particle size; Restek, Bellefonte, PA, USA). Briefly, 10 mg of the sample was diluted with 1 mL of isopropanol, mixed and then filtered through a 0.45 μm syringe filter into a 1.5 mL vial. Twenty microliter aliquots were injected. A mixture of acetonitrile/methanol/isopropanol in different ratios served as the mobile phase (solvent A: 50:40:10, *v*/*v*/*v*; solvent B: 30:65:5, *v*/*v*/*v*). The following procedure was used for the separation of both γ-oryzanol and tocopherols: isocratic elution with phase A for 5 min, followed by a 10 min linear gradient from phase A to 100% phase B, and a final 5 min isocratic elution with phase B. The column temperature was 25 ◦C with the flow at 1 mL/min. γ-Oryzanol was detected by a UV–VIS spectrophotometric detector at 325 nm. Tocopherols were detected by using a fluorescence detector with excitation and emission wavelengths at 290 and 330 nm, respectively. OpenLAB software (Agilent Technology) was used to acquire and process the data. The standard compounds were γ-oryzanol and the mixture of α-, β-, and γ-tocopherol.

### 2.3.2. Total Phenolic Content

The total phenolic content (TPC) of all samples was determined using the Folin– Ciocalteu colourimetric method, as described in a previous study [31]. The calibration curve was plotted using the absorbance of standard gallic acid against its concentration in the range between 0.2 and 0.0016 mg/mL. The TPC was calculated according to the standard curve equation of gallic acid (*y* = 13.463*x* + 0.0406, *R*<sup>2</sup> = 0.9991) and is expressed as mg gallic acid equivalents per 100 g dried sample (mg GAE/100 g). All the samples were prepared in triplicate.

## 2.3.3. Total Flavonoid Content

An aluminium chloride colourimetric assay was used to estimate the total flavonoid content (TFC) of all extracts following the method described by Zeng et al. [32]. The calibration curve was created using different concentrations of EGCG (0.01–0.32 mg/mL) and its absorbance was measured at 515 nm. The standard curve equation of EGCG was y = 0.3587x + 0.0041 (*R*<sup>2</sup> = 0.9993). The results are represented as mg EGCG equivalents per 100 g dried sample (mg EGCGE/100 g). All the samples were prepared in triplicate.

### *2.4. Cell Culture*

DU-145 human prostate cancer cells were obtained from the American Type Culture Collection (Rockville, MD, USA). DU-145 was grown in RPMI-1640 containing 10% FBS and 1% antibiotics (100 μg/mL of streptomycin and 100 unit/mL of penicillin). DU-145 cells were maintained at 37 ◦C in a humidified incubator containing 5% CO2. The cells in passages 3–6 were used for all experiments.

## *2.5. Cell Viability Assay*

The samples were tested to determine the non-cytotoxic concentration and cell viability of DU-145 cells by using SRB assay, as previously described [33]. Briefly, cells were seeded at a density of 1 × 104 cells/well in a 96-well plate and incubated overnight for cell attachment in a 5% CO2 atmosphere at 37 ◦C. Cells were then exposed to five serial concentrations (0.0001–1 mg/mL) of the ethanolic rice bran extracts (TRB, YRB, RRB, and MRB), their bioactive compounds (ferulic acid, phytic acid, gallic acid, oleic acid, linoleic acid, and γ-linolenic acid; γ-oryzanol; and α-, β-, and, γ-tocopherol), and standard controls (dutasteride and finasteride). Control cells were treated with 10% (*v*/*v*) DMSO in incomplete RPMI-1640, whereas incomplete RPMI-1640 served as a blank. After 24 h treatment, the adherent cells were fixed with 50% (*w*/*v*) trichloroacetic acid for 30 min, and cells were washed with water and then air-dried. Cells were stained with 0.04% (*w*/*v*) SRB for 30 min. The unbound dye was removed by washing with 1% (*v*/*v*) acetic acid. The bound stain was solubilised with 10 mM Tris base, and absorbance was detected at 515 nm using a 96-well plate reader (EZ Read 400 Flexi, Biochrom, Cambridge, UK). The experiments were performed in triplicate. The highest non-toxic concentration that gave more than 80% cell viability was selected for further studies. The percentage of cell viability was calculated by Equation (1), where Abs denotes absorbance:

$$\text{Cell viability} \left( \% \right) = \left( \frac{\text{Abs}\_{\text{sample}} - \text{Abs}\_{\text{blank}}}{\text{Abs}\_{\text{control}} - \text{Abs}\_{\text{blank}}} \right) \times 100. \tag{1}$$

## *2.6. RNA Extraction and Semiquantitative RT-PCR Analysis*

#### 2.6.1. RNA Extraction

Total RNA was extracted from DU-145 cells treated with 0.10 mg/mL of the ethanolic rice bran extracts (TRB, YRB, RRB, and MRB), 0.01 mg/mL of bioactive compounds, 0.10 mg/mL of standard controls (finasteride and dutasteride), or untreated cells using the NucleoSpin® RNA isolation kit (cat no. 740955.50; Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions. The concentration of isolated RNA was quantified using a Qubit 4 fluorometer (Invitrogen, Carlsbad, USA) and Qubit™ RNA HS Assay Kit (Invitrogen). The total RNA solution was kept at −20 ◦C until use.

#### 2.6.2. Semi-Quantitative RT-PCR

Complementary DNA (cDNA) was synthesised by using the RT-PCR Quick Master Mix (Toyobo, Osaka, Japan) according to the manufacturer's instructions. Briefly, the 20 μL reaction mixture contained 1 μg of total RNA, 10 μL of the RT-PCR Quick Master Mix, 1 μL of manganese acetate, 1.2 μL of oligo dT primers (Integrated DNA Technologies, Coralville, IA, USA), and nuclease-free water. The transcript levels of the genes of interest (*SRD5A1*, *SRD5A2*, and *SRD5A3*) and the reference gene (glyceraldehyde 3-phosphate dehydrogenase (*GAPDH*)) were measured in triplicate. The sequences of primers are listed in Table 1. The cycle consisted of denaturation at 94 ◦C for 30 s, annealing at 50–55 ◦C for 30 s, and extension at 72 ◦C for 1 min; there were 40 amplification cycles.

The RT-PCR products were analysed by electrophoresis on 1% (*w*/*v*) agarose gels in a chamber with 1X TAE buffer at 100 V for 60 min. The gel was imaged with a Gel Doc™ EZ System (Version 3.0; Bio-Rad) to obtain images of the bands. Image Lab™ software (Bio-Rad) was used to analyse quantitatively the intensity of the bands. The expression of target genes was normalised by the GAPDH expression value and is expressed as the

relative expression (*RE*) value. The percentage of SRD5A suppression was calculated according to Equation (2):

$$SRD5A \text{ suppression } (\%) = \left(\frac{RE\_{control} - RE\_{sample}}{RE\_{control}}\right) \times 100\tag{2}$$


**Table 1.** The sequences of the primers used for RT-PCR.

## *2.7. Computational Method Details*

#### 2.7.1. Protein and Ligand Preparation

The structure of human SRD5A2 comprises seven transmembrane domains and six loops that construct a cavity inside SRD5A2 (Figure 1). A carboxyl-terminal side (Cterminal side) faces the cytosol, whereas an amino-terminal side (N-terminal) faces the endoplasmic reticulum lumen. The space between transmembrane (TM)1 and TM4 is the entry port that opens for the ligand to enter the binding site. Loop 1 (L1) has been suggested to be a gate domain that controls the NADPH/NADP<sup>+</sup> exchange from the cytosol. A previous study reported that SRD5A2 catalyses the hydride transfer from NADPH to finasteride, resulting in the formation of a stable intermediate adduct, namely NADP–dihydrofinasteride (NADP–DHF), via a covalent bond [34]. Consequently, SRD5A2 is inhibited irreversibly. The key residues E57 (Glu57), R114 (Arg114), and F118 (Phe118) inside the SRD5A2 pocket have been suggested to interact with the intermediate adduct of finasteride and steroid substrate [34].

The crystal structure of human SRD5A2 in complex with NADPH and finasteride was retrieved from the Protein Data Bank (PDB) with the PDB ID 7BW1 [34]. SRD5A2 was prepared in Discovery Studio version 2.5 software. In the SRD5A2 binding pocket, finasteride is already fused with NADP+. The finasteride structure was removed by breaking the chemical bond, while the reduced form of NADP+ (NADPH) only remained in the substrate-binding cavity. SRD5A2 with NADPH in the binding pocket (Figure 1) is the state before ligand entry and formation of the intermediate adduct. This model was used to evaluate and compare the binding mode of ligands. The structure of finasteride retrieved from the crystal structure was used as a positive control.

The three-dimensional structure of nine bioactive compounds (ferulic acid, phytic acid, oleic acid, linoleic acid, γ-linolenic acid, γ-oryzanol, and α-, β-, and γ-tocopherol), which can be purified from rice bran extracts and dutasteride, another positive control, were retrieved from the PubChem compound database and optimised with the Gaussian 09 program (Gaussian Inc., Wallingford, CT, USA) using the B3LYP model with a 6-31G (d, p) basis set [35]. Figure 2 illustrates the chemical structures of selected ligands.

**Figure 1.** Structure of steroid human 5-alpha reductase 2 (SRD5A2) comprises seven transmembranes (7 TMs) and six loops. Dihydronicotinamide adenine dinucleotide phosphate (NADPH) (orange) is located inside the binding site. Crucial residues involving ligand interaction are coloured in yellow.

**Figure 2.** Chemical structure of selected ligands. (**a**) α-tocopherol, (**b**) β-tocopherol, (**c**) γ-tocopherol, (**d**) oleic acid, (**e**) linoleic acid, (**f**) γ-linolenic acid, (**g**) ferulic acid, (**h**) γ-oryzanol, (**i**) finasteride, and (**j**) dutasteride.

#### 2.7.2. Molecular Docking Study for 5-Alpha Reductase 2

Nine compounds and two positive controls were docked into the binding site of SRD5A2 using AutoDock 4.2.6 and AutoDockTools, the graphical user interface [36]. In all dockings, a grid box was generated with a centre over the native finasteride position. Grid box dimensions were set to 34, 30, and 30 in x, y, and z dimensions, respectively. The grid spacing was kept at 0.375 Å. One hundred conformations were generated by Lamarckian genetic algorithm searches with an initial population size of 300 random positions and conformation. Each run had two stop criteria: a maximum of 2,500,000 energy evaluations and a maximum of 27,000 generations. The reference root-mean-square deviation (RMSD) was kept as the default, with a mutation rate of 0.02 and a crossover rate of 0.8. The RMSD tolerance of 2 Å was kept for clustering of docked poses and ranked according to their binding energy. The docked conformation with the lowest binding energy of the most populated cluster was selected.

## 2.7.3. Molecular Dynamics Simulation

The best-docked complexes of native finasteride (positive control); α-, β-, and γtocopherol; the best top-three compounds; and SRD5A2 were selected for the MD simulation using the AMBER 14 program. The Amber ff14SB force field was used for the conformational analysis of protein systems [37,38]. For the non-standard unit in AMBER, antechamber was used to determine the GAFF atom type for structures of the four ligands and NADPH, and the restrained electrostatic potential (RESP) charges were employed for these ligands [39]. The systems were neutralised with 14 chloride counterions and centred in a 10 Å truncated octahedral box of pre-equilibrated TIP3P water molecules using the tLEaP program [40]. All MD simulations were carried out with the GPU-capable PMEMD.CUDA in AMBER14. The solvated structures were first energy-minimised to remove possible steric stress using the steepest descent (SD) method and the conjugate gradient techniques (CONJ) with a different part of the system [41]. They were then heated gradually from 0 to 310 K for 500 ps and equilibrated at 310 K at 1 atm pressure to obtain a stable density for 1000 ps. The unconstrained production simulations were run in an NPT ensemble at 310 K and 1 atm for 50 ns. The Langevin thermostat was used to maintain the temperature of the system [42], the SHAKE algorithm was used to constrain all of the chemical bond lengths involving hydrogen atoms [43], and the time step was set at 2 fs for all MD simulations.

## 2.7.4. Trajectory Analysis

Visual molecular dynamics (VMD) [44] and PyMOL [45] were used to visualise and analyse MD trajectories. The structural analysis of the conformational ensemble was performed by evaluating the RMSD with the CPPTRAJ module implemented in AMBER 14 [46]. The binding free energy and decomposed binding free energy of SRD5A2/ligand complexes were evaluated by the molecular mechanics–generalised Born surface area (*MM*/*GBSA*) protocol using the MMPBSA.py module, as embedded in AMBER 14 [47,48]. The snapshots were extracted from 50 ns of MD trajectories for the analysis of the binding free energy; all water molecules and chloride counterions were removed prior to calculations. In *MM*/*GBSA*, binding free energy (Δ*Gbind*) was estimated through Equation (3) [49]:

$$\begin{aligned} \Delta \mathbf{G}\_{\text{bind}} &= \mathbf{G}\_{\text{complex}} - \mathbf{G}\_{\text{protein}} - \mathbf{G}\_{\text{ligand}} \\ &= \Delta H + \Delta \mathbf{G}\_{\text{solution}} + T \Delta \mathbf{S} \\ &= \Delta E\_{MM} + \Delta \mathbf{G}\_{GB} + \Delta \mathbf{G}\_{SA} - T \Delta \mathbf{S} \\ &= \Delta E\_{vdw} + \Delta E\_{ele} + \Delta \mathbf{G}\_{GB} + \Delta \mathbf{G}\_{SA} + T \Delta \mathbf{S} \end{aligned} \tag{3}$$

where Δ*EMM* is the gas-phase interaction energy between protein and ligand, containing van der Waals interaction energy (Δ*Evdw*) and electrostatic energy (Δ*Eele*); Δ*GGB* and Δ*G* denote the polar and nonpolar desolvation free energy, respectively; and -*T*Δ*S* indicates the conformational entropy contribution at temperature *T*, where *T* is the absolute temperature

and *S* the entropy of the molecule. Here, the generalised Born (*GB*) approximation model was used to estimate the polar desolvation term (Δ*GGB*) [50,51], while the solvent-accessible surface area (*SASA*) model with the LCPO model used to estimate the nonpolar desolvation term (Δ*GSA*): Δ*GSA* = 0.0072 × Δ*SASA* [52].

## *2.8. Statistical Analysis*

Data are expressed as the mean of three independent experiments ± standard deviation. Principal component analysis (PCA) of bioactive contents and *SRD5A* suppression was performed using a free trial version of XLSTAT (Addinsoft, New York, NY, USA). Linear correlation between variables is expressed by Pearson's correlation coefficient (*r*). Analysis of variance (ANOVA) followed by post hoc analysis (Tukey's test) was used to compare means and evaluate statistical differences. The null hypothesis was rejected at the calculated probability value of less than 5%. Both linear correlation and ANOVA were carried out in SPSS Statistics for Windows, Version 17.0 (SPSS Inc., Chicago, IL, USA).

## **3. Results and Discussion**

#### *3.1. Extraction Yield and Bioactive Compounds*

The extraction yields, relative to 100 g of dried material, were 7.49% ± 0.89% (TRB), 5.68% ± 0.27% (YRB), 4.54% ± 0.92% (MRB), and 3.08% ± 1.67% (RRB); TRB had the highest yield among the four varieties of rice bran extracts. The physical appearances of all extracts were viscous semisolid and greasy with different colours due to pigment deposition in the pericarp or the bran of the rice kernel [26,53], specifically dark reddish-brown (TRB and YRB), black-purple (MRB), and dark purple (RRB).

The active constituents of rice extracts are presented in Table 2. Tocopherols are the major group of natural antioxidants in rice bran and have a similar chemical structure based on a 6-chromanol amphiphilic ring with one to three methyl groups and a phytyl tail with three chiral centres, resulting in α-, β-, γ-, and δ-tocopherol [26]. The highest content of α-tocopherol was found in TRB (20.76 ± 0.13 mg/kg extract), followed by YRB (12.52 ± 0.01 mg/kg extract), RRB (11.95 ± 0.04 mg/kg extract), and MRB (7.61 ± 0.01 mg/kg extract). Pigmented rice bran contains about 9.67–116.60 mg/kg of α-tocopherol, which is higher than that in non-pigmented rice bran [54]. β-Tocopherol is present at only minor concentrations in rice bran extracts [55], and the pair of β- and γ-tocopherol isomers is not completely separated using reversephase HPLC [30]. Hence, the pair of β- and γ-tocopherol was quantified and interpreted jointly as (β+γ)-tocopherol. The content of (β+γ)-tocopherol in YRB (50.98 ± 0.02 mg/kg extract) was much higher than in the other rice bran extracts, while δ-tocopherol was not detected in the extracts. These results are in agreement with several studies, where α- and β-tocopherol are predominantly present in rice bran and rice whole grain [55,56].

Rice bran is a major source of γ-oryzanol, a mixture of steryl ferulates, and speculated to be the primary constituent of rice bran oil [26]. The content of γ-oryzanol was enriched in TRB (8600.45 ± 0.13 mg/kg extract) and RRB (9174.01 ± 0.09 mg/kg extract). It has been reported that the mean value of γ-oryzanol in non-pigmented rice bran is approximately 3067.10 mg/kg [54]. Overall, pigmented TRB and RRB contained a high content of γoryzanol, and black-purple rice (MRB) had the lowest amount of tocopherols and γoryzanol. It has been suggested that environmental factors as well as the origin and genotype of rice varieties influence the composition of steryl ferulates, resulting in the variation of the γ-oryzanol profiles in black-purple rice varieties [57].

Phenolic and flavonoid contents were measured by colourimetric methods. Highest levels of TPC and TFC were found in red rice bran (YRB): 254.97 ± 5.20 mg GAE/100 g dried sample and 880.16 ± 22.86 mg EGCGE/100 g dried sample, respectively. Phenolic acids can be classified as free, conjugated, and bound. The free form is suggested to be easily extracted from rice bran [53]. Phenolic compounds, including phenolic acids and flavonoids, are mainly present in pigmented rice and act as metal ion chelators, free radical scavengers, and reducing agents [53,58]. The foremost phenolic acids found in bran

include ferulic acid (56–77% of total phenolic acids), followed by *p*-coumaric acid, sinapic acid, gallic acid, protocatechuic acid, *p*-hydroxybenzoic acid, vanillic acid, and syringic acid [46]. A previous study reported that Thai red rice bran contains a higher TPC than black and white rice bran extracts, resulting in better antioxidant activity [59]. The main flavonoids in non-pigmented rice varieties are flavones, whereas proanthocyanidins and anthocyanins are primarily found in pigmented rice varieties [54,60]. Anthocyanins and proanthocyanidins are responsible for purple-to-blue pigmentation and red pigmentation, respectively [26,61]. Moreover, a study reported that red and purple bran rice has greater TPC and TFC than light-coloured bran rice and other cereals due to higher concentrations of proanthocyanidins and anthocyanins, respectively [62].


**Table 2.** Bioactive compounds of four types of rice bran extracts.

Rice bran extracts of Tubtim Chumphae (TRB), Yamuechaebia Morchor (YRB), Mali Nil Surin (MRB), and Riceberry (RRB). Total phenolic content (TPC) presented as mg gallic acid equivalents per 100 g of dried sample (mg GAE/100 g). Total flavonoid content (TFC) expressed as mg (−)-epigallocatechin gallate equivalents per 100 g of dried sample (mg EGCGE/100 g). Not detected (ND).

> Several studies have reported the use of topical α-tocopherol in cosmetic applications and treatments of cutaneous diseases such as wounds, sunburn, atopic dermatitis, and hair loss [63–65]. A recent study reported that α-tocopherol-loaded hydrogel promoted the healing of the dorsal skin injury in a rat model. Based on the histopathological results, the α-tocopherol-treated group showed epidermal proliferation and the generation of new hair follicles [63]. In addition, α-tocopherol and α-tocopheryl acetate lotions showed the acceleration of the hair growth rate in a rabbit model within two weeks [66]. Nevertheless, whether tocopherols affect an androgen-dependent pathway involving AGA has not been directly observed. Therefore, tocopherols were selected for further study to determine their effect on *SRD5A* isozyme expression and for a molecular docking study. In addition, other bioactive compounds in rice bran extracts, such as ferulic acid [67], phytic acid [68], oleic acid [27,69], linoleic acid [27,69,70], γ-linolenic acid [69,71], and γ-oryzanol [28], which are involved in androgen metabolism pathways, were compared to tocopherols and standard controls (finasteride and dutasteride).

## *3.2. Effect on the Expression of 5-Alpha Reductase Isoenzymes*

The activity of SRD5A in hair follicles was first identified by Takayasu et al. [72]. *SRD5A1* and *SRD5A2* gene expression levels in men were about threefold higher than in women, resulting in the higher prevalence of AGA among men compared with women [4]. The expression of *SRD5A3* has been suggested to be a predisposing factor to AGA development [18–20]. Moreover, the absence of temporal regression and baldness in cases of *SRD5A* deficiency supports the crucial role of *SRD5A* in the pathogenesis of AGA [73]. Downregulation of *SRD5A* gene expression could lead to a reduction in their protein translation in the downstream pathways involving the pathology of AGA. Several studies have indicated that the DU-145 human androgen-insensitive prostate adenocarcinoma cell line expresses the three types of *SRD5A*; these cells have been used in the present study to observe the regulation of *SRD5A* gene expression [16,28,74,75]. With regards to these, the effects of the selected bioactive compounds and rice bran extracts on *SRD5A1*, *SRD5A2*, and *SRD5A3* expression were investigated. The percentage of *SRD5A* suppression and the relative expression of each *SRD5A* gene are given in Supplementary Materials (Table S1 and Figure S1).

The four rice bran extracts significantly decreased the mRNA expression levels of *SRD5A1*, *SRD5A2*, and *SRD5A3* compared with the negative control groups in the follow-

ing order: TRB > RRB > YRB > MMB (Figure 3). Interestingly, treatment with TRB greatly decreased the expression levels of *SRD5A1*, *SRD5A2*, and *SRD5A3* by 35.79% ± 6.94%, 22.26% ± 3.73%, and 21.97% ± 0.01%, respectively (Table S1). Finasteride, a dual inhibitor of SRD5A2 and SRD5A3, suppressed *SRD5A1*, *SRD5A2*, and *SRD5A3* by 54.21% ± 3.05%, 6.48% ± 8.22%, and 7.70% ± 3.58%, respectively. However, there were no significant differences between TRB and finasteride regarding *SRD5A* gene suppression. Dutasteride, a triple inhibitor of SRD5As, downregulated *SRD5A1*, *SRD5A2*, and *SRD5A3* by 43.50% ± 0.01%, 20.84% ± 0.54%, and 45.57% ± 0.03%, respectively (Table S1). Among the selected bioactive compounds, the mRNA levels of *SRD5A1*, *SRD5A2*, and *SRD5A3* were downregulated markedly by γ-tocopherol (0.10 mg/mL), specifically 48.32% ± 4.29%, 42.57% ± 1.91%, and 61.04% ± 9.10%, respectively (Table S1). The overall effect of γtocopherol on *SRD5A* gene suppression was significantly greater than dutasteride. However, complete downregulation of *SRD5A* genes may not be the best choice for AGA patients, considering the required androgen balance for normal health and tissue homeostasis [3,4]. In skin, androgens also regulate sebum production and secretion, wound healing, cutaneous barrier formation, and hair growth [4]. Undesirable side effects of long-term use of SRD5A inhibitors on skin changes have been reported, including dry skin, thinning skin, changes in skin texture and tone, and penile and scrotal shrinkage [76,77]. Moreover, excessive suppression may lead to ejaculation problems, erectile dysfunction, sexual anhedonia, decreased sperm count, gynaecomastia, and loss of libido in patients [78].

**Figure 3.** Effects of selected bioactive compounds and rice bran extracts on 5α-reductase isoenzyme (*SRD5A*) expression in DU-145 cells treated with 0.10 mg/mL of ethanolic rice bran extracts (TRB, YRB, RRB, and MRB), 0.01 mg/mL of selected bioactive compounds, and 0.10 mg/mL of standard controls (finasteride and dutasteride). A statistical significance in comparison to dutasteride and finasteride is indicated as a and b (*p* < 0.05), respectively. A significant difference between TRB and other extracts is expressed as c (*p* < 0.05).

The *SRD5A1* mRNA level was significantly lower after treatment with most of the bioactive compounds, including tocopherols, ferulic acid, linoleic acid, γ-linolenic acid, γ-oryzanol, and standard control groups (finasteride and dutasteride), compared with the negative control group (Figure S1a). This result is similar to previous studies [28,79]. The extracts (0.50 mg/mL), including *Nelumbo nucifera*, *Sesamum indicum*, and bran of *O. sativa*, greatly diminished *SRD5A1* expression, suggesting that a high content of linoleic acid is responsible for the activity with a synergist of ferulic acid, vanillic acid, phytic acid, and γ-oryzanol [79]. Nevertheless, in this study, the *SRD5A1* mRNA level slightly decreased in the groups treated with phytic acid, gallic acid, and linoleic acid. The concentration of standard bioactive compounds (0.01 mg/mL) and rice bran extracts (0.10 mg/mL) used in this study was lower than the concentration used in the previous study, suggesting that the *SRD5A* genes may be suppressed in a concentration-dependent manner.

The expression of *SRD5A2* was not remarkably changed with the treatments of YRB, MRB, RRB, and the major compounds (Figure S1b). *SRD5A2* was only downregulated in the cells treated with TRB, β- and γ-tocopherol, oleic acid, γ-linolenic acid, and dutasteride. The *SRD5A2* suppression exerted by the four extracts was in the following order: TRB > RRB > YRB > MMB. TRB suppressed *SRD5A2* by 22.26% ± 3.73%, which was not significantly different compared with standard dutasteride (20.84% ± 0.54%). This suppression might be from the synergistic effect of β- and γ-tocopherol, oleic acid, and γ-linolenic acid in the extract. The *SRD5A3* mRNA level was downregulated significantly in cells treated with all forms of tocopherol, phytic acid, phenolic acids, and γ-oryzanol compared with the control group, but it was not altered in groups treated with unsaturated fatty acids or finasteride (Figure S1c).

The sum of overall bioactive compounds (α-, β- and γ-tocopherol; γ-oryzanol; TPC; and TFC) was ranked in descending order: TRB > RRB > YRB > MMB. In addition, the overall results of *SRD5A* mRNA expression levels from all four rice bran extracts were arranged in the order of decreasing suppression: TRB > RRB > YRB > MMB. Taken together, these bioactive compounds provided potential synergy that enhances the downregulation of *SRD5A* genes.

## *3.3. Correlation Analysis*

## 3.3.1. Pearson's Correlation

Pearson's correlation coefficients (*r*) between the potential suppressive effect on *SRD5A* expression and the content of bioactive compounds in four rice bran extracts (α-tocopherol, (β+γ)-tocopherol, γ-oryzanol, TPC, and TFC) were determined to estimate the relationship between variables. The correlation coefficients are classified into four levels: particularly high (*r* > 0.9), high (0.9 > *r* > 0.7), moderate (0.7 > *r* > 0.5), and poor (*r* < 0.5) [80]. There were strong and significant linear relationships between the content of α-tocopherol and the suppressive effect on *SRD5A1* (*r* = 0.814, *p* < 0.01), *SRD5A2* (*r* = 0.917, *p* < 0.01), and *SRD5A3* (*r* = 0.943, *p* < 0.01). In contrast, a previous study reported a significant positive linear correlation between *SRD5A1* suppression and the unsaturated fatty acid content (*r* = 1.00, *p* < 0.01) and the linoleic acid content (*r* = 1.00, *p* < 0.01) [28]. Because the rice bran extracts in the previous study were obtained by supercritical CO2 extraction, the bioactive compounds in extracts were different from our study, especially the unsaturated fatty acid content [28].

Regarding HPLC results, YRB contained the highest content of (β+γ)-tocopherol among rice bran extracts, followed by TRB, RRB, and MRB. Furthermore, treatment with γtocopherol exhibited the greatest suppression of *SRD5A1*, *SRD5A2*, and *SRD5A3*. However, there was not a significant relationship between the content of (β+γ)-tocopherol in rice bran extracts and their suppression of *SRD5A* mRNA levels. Instead, there was a strong relationship between the α-tocopherol content and the suppressive effects on these genes. The most abundant form of tocopherols in pigmented rice bran was α-tocopherol, followed by γ-, β-, and δ-tocopherol [54]. In addition, a study reported that α-tocopherol is the major tocol in two Taiwanese rice varieties [26]. TRB showed the highest α-tocopherol

content, followed by YRB, RRB, and MRB. There was a clear concordance between αtocopherol and the suppressive effects on *SRD5A* genes. The content of γ-tocopherol in YRB may be lower than the content of α-tocopherol, resulting in the minor effect on gene regulation. These findings indicate that the expression levels of all *SRD5A* genes are greatly downregulated by TRB, suggesting that the higher α-tocopherol content contributes to the more pronounced effect.

#### 3.3.2. Correlation by Principal Component Analysis

PCA was performed to classify the rice bran extracts and cluster the samples. Correlation between rice bran extracts (TRB, YRB, MRB, and RRB) and their biological activity (*SRD5A* suppression) and biological contents (α-tocopherol, (β+γ)-tocopherol, γ-oryzanol, TPC, and TFC) is shown as a PCA biplot in Figure 4. The PCA space distributed 52.64% in PC1 and 34.63% in PC2. The data were separated into four clusters of rice extracts across the PCA space. YRB was separated from other extracts due to the TPC, the TFC, and the content of (β+γ)-tocopherol. TRB showed a high correlation between *SRD5A* gene suppression and the contents of α-tocopherol and γ-oryzanol. RRB was slightly correlated with the content of γ-oryzanol, whereas MRB was not correlated with any variables. Regarding the groups and clustering seen in PCA, the biological activities and biological contents of samples were speculated to be similar. However, there was a strong correlation between the suppression of *SRD5A* genes and the content of α-tocopherol in TRB.

**Figure 4.** Principal component analysis (PCA) biplot of rice bran extracts and their biological and phytochemical properties. Rice bran extracts of Tubtim Chumphae (TRB), Yamuechaebia Morchor (YRB), Mali Nil Surin (MRB), and Riceberry (RRB). Total phenolic content (TPC). Total flavonoid content (TFC).

#### *3.4. Molecular Docking Study for 5-Alpha Reductase 2*

Both *SRD5A1* and *SRD5A2* are expressed and active in scalp hair follicles. Specialised fibroblasts in hair follicles or DPC, known as androgenic targets, induce surrounding epidermal cells to form hair follicles and regulate the hair growth cycle [81]. *SRD5A2* is expressed mainly in DPC obtained from scalp hairs, and its activity is 14-fold higher than in the remaining hair follicles [4]. In addition, the absence of AGA in males with congenital SRD5A2 deficiency provides strong evidence that SRD5A2 activity is the most importance factor in AGA development [82]. Even though it is more favourable for SRD5A1 to catalyse androstenedione as a substrate to generate 5α-androstenedione, SRD5A2 has been implicated mostly in the reduction of T to DHT [6,83,84]. Furthermore, finasteride, a selective SRD5A2 inhibitor, is a Food and Drug Administration (FDA)-approved drug to treat AGA and has proven favourable efficacy in increasing hair density and hair diameter [85]. Thus, SRD5A2 was selected to perform the further molecular docking with selected bioactive compounds of rice bran extracts.

Molecular docking was performed to predict the potential target/ligand interaction. The binding free energy, which indicates the ligand-binding possibilities with target protein SRD5A2, was calculated. The predicted binding free energies of finasteride, dutasteride, and nine bioactive compounds are ranked and presented in Table 3. The ranking is based on the binding affinity of the SRD5A2/ligand complex; the lowest binding energy (highest negative value) is projected to specify the best-possible interaction. The results indicated that the binding energy of finasteride (−10.13 kcal/mol) was lower than that of dutasteride (−8.75 kcal/mol). These results are in agreement with a previous study that indicated finasteride more specifically inhibits SRD5A2 (IC50 = 14.3 ± 2.7 nM) compared with dutasteride (IC50 = 57.0 ± 6.8 nM) [5,16]. Several studies have shown that both synthetic and plant-derived unsaturated fatty acids inhibit SRD5A and, consequently, block the conversion of T to DHT [27,33,86,87]. Ferulic acid, γ-oryzanol, and phytic acid appeared to have unfavourable interactions. Similarly to a previous study, there was no correlation between SRD5A inhibitory activity and the TPC [88]. Strikingly, tocopherols displayed a higher affinity towards SRD5A2 compared with dutasteride and other active constituents. The binding free energies of tocopherol are in ascending order: β-tocopherol (−9.83 kcal/mol), γ-tocopherol (−9.53 kcal/mol), and α-tocopherol (−9.47 kcal/mol). Among all considered compounds, finasteride and tocopherols were subjected to MD simulation.

**Compounds AutoDock Binding Free Energy, ΔG (kcal/mol)** Native finasteride −10.13 β-Tocopherol −9.83 γ-Tocopherol −9.53 α-Tocopherol −9.47 Dutasteride −8.75 γ-Linolenic acid −7.03 Linoleic acid −6.62 Oleic acid −6.49 Ferulic acid −5.22 γ-Oryzanol −4.26 Phytic acid −2.02

**Table 3.** Binding free energy of finasteride, dutasteride, and nine bioactive compounds with 5αreductase 2.

#### *3.5. Molecular Dynamics Simulation*

3.5.1. Stability of the Molecular Dynamics Trajectories

The structural dynamics at the atomistic level of SRD5A2 upon interacting with all ligands were carried out by MD trajectories over a 50 ns simulation time. The RMSD values of the protein backbone atoms were calculated to determine the stability of each SRD5A2/ligand complex and were plotted relative to the first frame of the original structure (Figure 5a). The RMSD values of the SRD5A2 backbone for all ligands were in the range of 2 Å, establishing their overall stability over the explored timescale [89]. There were large fluctuations of SRD5A2/α-tocopherol and SRD5A2/β-tocopherol at 10–20 ns and 20–30 ns, respectively. All systems reached the same state after 40 ns.

**Figure 5.** Root-mean-square deviation (RMSD) analysis over 50 ns MD stimulation time: (**a**) RMSD plot of the steroid 5α-reductase 2 (SRD5A2) in complex with each ligand (finasteride and α-, β-, and γ-tocopherol). (**b**) RMSD plot of each ligand.

In the case of the ligand RMSD (Figure 5b), finasteride was stable throughout the 50 ns MD simulation. RMSD plots of α- and β-tocopherol showed almost the same pattern. The RMSD values of three ligands (finasteride and α- and β-tocopherol) were less than ∼2 Å and remained stable to the end of the 50 ns simulation, indicating that these ligands have a stable conformation in the binding site. However, the RMSD of γ-tocopherol increased from the beginning of the MD simulation until approximately 5 ns. Hence, γ-tocopherol dramatically underwent a conformational change at the starting period and then maintained a constant trend until 50 ns. Overall, finasteride and α- and β-tocopherol exhibited a reciprocal stabilisation in the SRD5A2 pocket, contributing to an increase in the binding affinity of the ligands.

#### 3.5.2. Binding Free Energy Analysis

οοο The binding free energy of all ligands towards SRD5A2 was estimated using the snapshots obtained from a 50 ns MD simulation [90]. The different individual energies including van der Waals forces (VDW), electrostatic energy (EEL), nonpolar contribution to the solvation free energy (ESURF), ΔGgas, and ΔGsolv are summarised into the total binding free energy, or ΔGTotal (Table 4). α-Tocopherol possessed the highest binding affinity against SRD5A2, with ΔGTotal of −54.55 ± 3.67 kcal/mol. VDW, a major support of ΔGgas, was more favourable in all ligands compared with electrostatic interaction.

**Table 4.** Estimated binding free energy of the complex of 5α-reductase 2 with the ligands (finasteride and α-, β-, and γ-tocopherol) using molecular mechanics–generalised Born surface area (MM/GBSA).


The energy of each compound comprised individual energy terms, including van der Waals forces (VDW), electrostatic energy (EEL), the electrostatic contribution to the solvation free energy (EGB), and nonpolar contribution to the solvation free energy (ESURF). The binding free energy terms are given by ΔGgas (gas-phase free energy) = VDW + EEL, ΔGsolv (solvation free energy) = EGB + ESRUF, and ΔGTotal (total binding free energy) = ΔGgas + ΔGsolv.

#### 3.5.3. Decomposition of Binding Free Energy

The free energy contribution of each amino acid residue of the SRD5A2 pocket for ligand interaction was elucidated by energy decomposition analysis using MM/GBSA with the configurations based on a 50 ns MD simulation. A residue is considered a

favourable contributor to ligand binding when the total energy decomposition is more negative than −1 kcal/mol [71]. All hotspot residues and NADPH as a donor cofactor are represented in Figure 6. The most crucial residue in all SRD5A2/ligand complexes is Phe118, which contributed favourably to the ligand binding in the following order: α-tocopherol (−9 kcal/mol), β-tocopherol (−6 kcal/mol), finasteride (−4.5 kcal/mol), and γ-tocopherol (−4.2 kcal/mol). It is obvious that finasteride was more likely to interact with NADPH via VDW and *pi*-alkyl interaction. Nevertheless, a covalent bond has been suggested to connect the nicotinamide C-4 atom of NADPH and the C-2 atom at the pyridone ring of finasteride and then create an intermediate adduct between NADPH and finasteride (NADP–dihydrofinasteride) [34,91]. In addition, the key residues in the SRD5A2 binding cavity, such as Ser31, Gly32, Trp53, Glu57, Try91, and Arg94, were prone to form interactions with finasteride. Conversely, tocopherols were mainly involved with the hotspot residues Lue20, Leu11, Arg114, Gly115, Phe219, and Phe233. These findings indicate that finasteride and tocopherols interact with crucial residues in the SRD5A2 pocket at different levels and with distinct binding patterns.

**Figure 6.** Energy decomposition of amino acid residues in the binding site towards the ligands (finasteride and α-, β-, and γ-tocopherol).

## *3.6. Post-Molecular Dynamics Simulation Binding Mode Analysis*

The dynamic data at atomic spatial resolution revealed that α-tocopherol, which can be found in rice bran extract, is considered a promising SRD5A2 inhibitor. Our preliminary molecular docking study indicated that α-tocopherol, with a binding free energy of −9.47 kcal/mol, had less interaction with SRD5A2 compared with other tocopherols and finasteride. According to the stability of trajectories, all systems of the SRD5A2/ligand complex reached the same state after 40 ns (Figure 5a). The ligand RMSD values of αand β-tocopherol were quite low, within the acceptable limit of less than 2 Å, whereas the RMSD value of γ-tocopherol was higher (Figure 5b). These data suggest that α- and β-tocopherol undergo a slightly conformational change. However, the binding free energy analysis indicates that α-tocopherol possesses the most favourable interaction by the energy of −54.55 kcal/mol.

Molecular interaction and binding mode conformations of key residues were executed using the final snapshot from the MD simulation of each complex and constructed into 3D and 2D plots (Figure 7). The study demonstrated that tocopherols occupy the SRD5A2

binding pocket similarly to finasteride but display distinct binding patterns. SRD5A2 contains seven transmembrane α-helices with NADPH buried in cytosolic loops [7]. The post-MD binding pose showed that all ligands enter the port and mainly interact with residues in TM2 and TM4.

α-Tocopherol formed different intermolecular interactions between its benzene ring and residues in the binding pocket, including Phe118 (*pi-pi* interaction) and Trp201 (hydrogen bonding). The energy decomposition analysis indicates that NADPH is likely to interact with a chromanol head of *α*-tocopherol via hydrophobic interactions. The binding environment and the formation of an intermediate adduct between NADPH and α-tocopherol may provide better interaction inside the binding pocket. Moreover, the total energy decomposition of the residue Phe118 is the most negative in the SRD5A2/α–tocopherol complex (−9 kcal/mol). A previous study reported that a substitution mutation at the residue Phe118 in the SRD5A2 binding cavity could dramatically restrain the binding of testosterone [92]. Together with our findings, the residue Phe118 is the most crucial amino acid that is prone to interact with steroid substrates and ligands, including α-tocopherol.

By contrast, finasteride had strong hydrogen bond interaction with residues Arg94 and Glu57. The residue Phe118 interacted with finasteride via hydrophobic interaction (−4.5 kcal/mol) but less compared to its interaction with α-tocopherol. In accordance with our results, a previous study suggested that residues Glu57 and Arg114 interact with an intermediate adduct of finasteride and NADPH in the binding pocket [34]. In addition, residues Tyr235, Asp241, and Lys244 interact with finasteride inside the SRD5A2 binding cavity [91], but these residues were not significantly observed in this study.

Although β- and γ-tocopherol form a number of notable interactions with residues in the SRD5A2 binding pocket, the complexes were much less stable than α-tocopherol and finasteride. This may be due to fact that the chromanol ring of α-tocopherol contains three methyl groups at the C5-, C7-, and C8-positions, while β- and γ-tocopherol have two methyl groups. Consequently, the residues in the pocket are likely to form a hydrophobic interaction with these methyl groups of α-tocopherol, resulting in the stabilisation of ligands [93]. The rigid moieties at the phytyl tail of β- and γ-tocopherol might contribute to the steric hindrance effect and affect the binding pocket insertion [94].

We performed MD simulations to screen for promising SRD5A2 inhibitors and gain insight into the stability and overall dynamics of each ligand in the SRD5A2 pocket before the intermediate adduct is formed. However, the present study only provides a proofof-concept by focusing on the molecular interactions of the ligand in the binding site without a membrane environment. Because SRD5A2 is a membrane-embedded steroid reductase, the influence of the membrane environment is an important factor for the protein conformational dynamics [95]. A previous advanced MD study has demonstrated that steroid substrates presumably access the SRD5A2 binding pocket from the lipid bilayer through the opening between TM1 and TM4. Furthermore, high conformation dynamics of the cytosolic region were observed during the NADP+/NADPH exchange [34]. Membrane– protein interactions may affect ligand entry, ligand binding modes, and SRD5A2 structural conformations. Thus, future MD studies of SRD5A2 should include the lipid membrane to evaluate its influence on the system. The experimental study of promising ligands towards SRD5A2 needs further investigation to confirm the inhibitory activity.

**Figure 7.** Molecular interaction patterns of 3D and 2D interactions of the ligands (finasteride and α-, β-, and γ-tocopherol) in the pocket of 5α-reductase type 2 after molecular dynamics simulation; the interacting residues are highlighted with different colours: (**a**,**e**) binding pose of finasteride, (**b**,**f**) binding pose of α-tochopherol, (**c**,**g**) binding pose of β-tocopherol, and (**d**,**h**) binding pose of γ-tocopherol.

#### **4. Conclusions**

Ethanolic TRB rice bran extract, a dark-brown semisolid extract, contained a decent amount of α-tocopherol and had the largest sum of overall bioactive compounds (γ-oryzanol; α-, β-, and γ-tocopherol; TPC and TFC). TRB also showed the highest antiandrogenic suppression of *SRD5A1*, *SRD5A2*, and *SRD5A3* mRNA. There was no statistically significant difference in the suppression of the mRNA expression of the three types of *SRD5A* between TRB and finasteride (FDA-approved for anti-hair loss treatment). The linear relationship and PCA indicated that the content of α-tocopherol shows a strong contribution to the suppression of all *SRD5A* genes. Strong evidence from previous studies indicates that SRD5A2 activity is implicated mostly in the reduction of T to DHT and expressed mainly in DPC obtained from hair follicles, highlighting the vital role of SRD5A2 in AGA development. SRD5A2 was subsequently docked with selected bioactive compounds based on the prior results of the studies indicating the involvement in androgen pathways. MD simulation demonstrated that α-tocopherol forms a stable interaction with SRD5A2 similarly to finasteride; it forms hydrophobic and hydrogen bond interactions with crucial amino acid residues in the SRD5A2 binding pocket, supporting the potential inhibition of this bioactive compound in TRB. Our findings bring new insights that TRB suppresses *SRD5A* gene expression as well as SRD5A2 activity. However, additional studies of enzymatic inhibition need to be evaluated. This will help to progress the uses of this rice by-product for future pharmaceutical and cosmeceutical applications as anti-hair loss products.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/biology10040319/s1: Figure S1. Effects of selected bioactive compounds and rice bran extracts on steroid 5α-reductase isoenzyme (*SRD5A*) expression in DU-145 cells treated with 0.10 mg/mL of ethanolic rice bran extracts (TRB, YRB, RRB, and MRB), 0.01 mg/mL of selected bioactive compounds, and 0.10 mg/mL of standard controls (finasteride and dutasteride). (a) Suppression of *SRD5A1*. (b) Suppression of *SRD5A2*. (c) Suppression of *SRD5A3*. A statistical significance in comparison to controls is indicated as \* *p* < 0.05; Table S1. The percentage of *SRD5A* suppression of four rice bran extracts and their bioactive constituents.

**Author Contributions:** Conceptualisation, C.K. and W.R.; methodology, W.Y., W.R. and C.K.; software, W.Y. and S.R.S.; validation, K.S.; formal analysis, S.J.; investigation, C.K.; resources, W.R.; data curation, C.K.; writing—original draft preparation, C.K. and W.R.; writing—review and editing, C.K., F.D.C., W.N., P.J., P.R. and W.R.; supervision, W.R.; project administration, W.R.; funding acquisition, W.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research project is supported by National Research Council of Thailand (NRCT): NRCT5-RRI63004-P05 and partially supported by Chiang Mai University.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in this article.

**Acknowledgments:** The authors are grateful to the NRCT for supporting research facilities (grant no. NRCT5-RRI63004-P05). We would like to thank Phrao Green Valley Co., Ltd., and the Lanna Rice Research Center, Chiang Mai University, Thailand, for providing the rice bran samples. Special thanks to Ricky Kabir and Yasir Nazir for assistance with proofreading this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

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## *Review* **Beneficial Role of** *Carica papaya* **Extracts and Phytochemicals on Oxidative Stress and Related Diseases: A Mini Review**

**Yew Rong Kong 1,†, Yong Xin Jong 1,†, Manisha Balakrishnan 1,†, Zhui Ken Bok 1,†, Janice Kwan Kah Weng 1,†, Kai Ching Tay 1,†, Bey Hing Goh 1,2 , Yong Sze Ong 1, Kok Gan Chan 3,4,\* , Learn Han Lee 5,\* and Kooi Yeong Khaw 1,\***


**Simple Summary:** This review highlights the medicinal benefits of a natural remedy, the *Carica papaya* extracts and its phytochemicals. In this review, the potential of *Carica papaya* against various conditions, including cancer, inflammation, aging, healing of the skin, and lifelong diseases has been summarized and discussed. In short, more research and development should focus on this natural remedy that can potentially act as a prophylaxis against chronic diseases.

**Abstract:** Oxidative stress is a result of disruption in the balance between antioxidants and prooxidants in which subsequently impacting on redox signaling, causing cell and tissue damages. It leads to a range of medical conditions including inflammation, skin aging, impaired wound healing, chronic diseases and cancers but these conditions can be managed properly with the aid of antioxidants. This review features various studies to provide an overview on how *Carica papaya* help counteract oxidative stress via various mechanisms of action closely related to its antioxidant properties and eventually improving the management of various oxidative stress-related health conditions. *Carica papaya* is a topical plant species discovered to contain high amounts of natural antioxidants that can usually be found in their leaves, fruits and seeds. It contains various chemical compounds demonstrate significant antioxidant properties including caffeic acid, myricetin, rutin, quercetin, α-tocopherol, papain, benzyl isothiocyanate (BiTC), and kaempferol. Therefore, it can counteract pro-oxidants via a number of signaling pathways that either promote the expression of antioxidant enzymes or reduce ROS production. These signaling pathways activate the antioxidant defense mechanisms that protect the body against both intrinsic and extrinsic oxidative stress. To conclude, *Carica papaya* can be incorporated into medications or supplements to help manage the health conditions driven by oxidative stress and further studies are needed to investigate the potential of its chemical components to manage various chronic diseases.

**Keywords:** oxidative stress; antioxidant; *Carica papaya*; inflammation; diabetes; cancer; aging; wound healing; periodontal disease; Alzheimer's disease

**Citation:** Kong, Y.R.; Jong, Y.X.; Balakrishnan, M.; Bok, Z.K.; Weng, J.K.K.; Tay, K.C.; Goh, B.H.; Ong, Y.S.; Chan, K.G.; Lee, L.H.; et al. Beneficial Role of *Carica papaya* Extracts and Phytochemicals on Oxidative Stress and Related Diseases: A Mini Review. *Biology* **2021**, *10*, 287. https:// doi.org/10.3390/biology10040287

Academic Editor: Francisco Les

Received: 8 March 2021 Accepted: 30 March 2021 Published: 1 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Oxidative stress is a natural phenomenon, resulting from the disruption of the redox equilibrium due to the amount of pro-oxidants outweighing antioxidants, which can eventually result in cell or tissue damage. As the name suggests, either oxidative stress can be induced by the presence of a high amount of pro-oxidants or the incompetence of antioxidant defense mechanism in the human body. In normal circumstances, human body is capable of scavenging free radicals, inhibiting the generation of oxidative stress with the help of several antioxidant enzymes, including glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). Among various pro-oxidants, those free radicals that contain oxygen are known as reactive oxygen species (ROS), and ROS are considered as the secondary products of aerobic metabolism. Examples of ROS include singlet oxygen, hydroxyl radicals, superoxide radicals, and hydrogen peroxide. Sources of free radicals include diet, environment, and sunlight exposure and their accumulation can lead to oxidative stress and tissue injury, consequently leading to aging of the skin and medical conditions, including Alzheimer's disease (AD), rheumatoid arthritis, asthma, atherosclerosis, and cancers [1,2]. Various researches have been conducted to investigate the pathophysiology of diseases related to oxidative stress and the benefits of antioxidants in treating those diseases. Antioxidants can be found abundantly in plants. An example of antioxidant-rich plant is the *Carica papaya* L., which is a flowering and dicotyledonous plant, classified as violales order, *Caricaceae* family, *Carica* L. genus, and papaya species [3]. The *Carica papaya* L. has a single hollow light greenish to brownish stem with scarring, bearing big leaves and big oval fruits. Besides, this plant is cultivated in countries, such as Malaysia, Brazil, South America, Australia, and Indonesia, which are located near to the equator. The *Carica papaya* L. plant is known as many different names, such as kepaya, paw paw, or tapaya, based on its geographical distribution. In fact, this plant is acclaimed for an array of medicinal values from each part of the plant including fruit, roots, leaves, and seeds of the plant. Therefore it has been used as a traditional treatment regimen for various diseases [4]. Some of the medicinal properties of the plant can be explained by its antioxidative property, which confer protection on the cells from being harmed by oxidative stress [5]. Papain is the most widely exploited proteolytic enzyme from the *Carica papaya* L. and it has been used to help with meat tenderization and digestion. It is worth to note that papain exhibited great potential as a medication [4], as it is suggested to exhibit drug-like properties for atherosclerosis and associated conditions, which involve monocyte-platelet aggregate (MPA)-regulated inflammation [6]. Relevant and significant studies have been conducted to evaluate the benefits of the *Carica papaya* extracts and chemical constituents. This review aims to gather and summarize the research findings linking the *Carica papaya* to its antioxidant properties and the utilization of this natural resource as a pharmaceutical, cosmeceutical, and nutraceutical products.

#### **2. Methods**

All literature was retrieved from databases (PubMed, Semantic Scholar, Web of Science, WorldWideScience, and Embase) using search terms, including "*Carica papaya*", "inflammation", "cancer", "Alzheimer's Disease", "diabetes", "aging", "wound healing" and "oxidative stress". Literatures published from 2000 to 2020, investigating the benefits of the *Carica papaya* plant towards various conditions, were included. Literatures that were not related to oxidative stress mechanisms were excluded. Literatures selected were categorized based on related conditions including inflammation, cancer, skin aging, wound healing, diabetes, periodontal diseases, and Alzheimer's disease (AD). The mechanisms of action of the *Carica papaya* towards each condition were also presented in tables in the respective sections.

## **3.** *Carica papaya* **Counteracts Oxidative Stress in Inflammation, Skin Aging, and Healing, Chronic Diseases, and Cancers**

Oxidative stress occurs due to excessive ROS production, which will cause oxidative damage to tissues. Consequence effects of oxidative stress has known to cause inflammation, leading to the development of various health conditions, including AD, rheumatoid disease, cardiovascular diseases (CVDs), cancers, cataracts, as well as cosmetic issues, such as the formation of wrinkles and loss of elasticity of the skin [7,8]. Figure 1 provides an overview of the role of oxidative stress in these conditions.

**Figure 1.** Role of oxidative stress in different medical conditions.

## *3.1. Inflammation*

Inflammation is a complicated pathway of the body's own protective mechanism against pathogens, which is associated with symptoms such as pain, swelling, and redness due to the release of a mediator "prostaglandin" [9]. This defensive action can be divided into innate and adaptive responses [10]. In short, the pathogenesis of inflammation starts with tissue injury, which causes infiltration and activation of macrophages and relevant antigen-presenting cells (APCs). This causes the release of proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) and interleukins (ILs). Cytokines stimulate the release of chemokines, which further recruit and activate lymphocytes and leukocytes. ROS are produced to eliminate invaders whereby activates Nuclear factor kappa-B (NF-*κ*B). NF-*κ*B is a transcription factor and plays a role in inducing inducible nitric oxide synthase (iNOS) activity and, thus, nitric oxide (NO) production. Excessive ROS upregulated prostaglandin E2 (PGE2) synthesis and, hence, cyclooxygenase-2 (COX-2) expression, which eventually leads to oxidative stress that causes tissue damage and worsens inflammation [11–13].

Another study further suggested that oxidative stress and inflammation are interrelated as oxidative stress resulting from high ROS can precipitate the formation of inflammation by increasing the gene expression coding for inflammatory proteins, including NF-*κ*B, peroxisome proliferator activator receptor gamma (PPAR-γ), and activator protein 1 (AP-1). Consequently, inflammatory chemokines and cytokines are produced to induce inflammation. On the other hand, inflammation can increase ROS production via several signaling cascades. Polymorphonuclear neutrophils (PMN) is an immune cell that is largely involved in inflammatory processes. During inflammation, they congregate the gp91-phox, which is a catalytic subunit of NADPH oxidase 2 (NOX) and generate more ROS, including hydroxyl radical, superoxide anion, and hypochlorous acid, thereby enhance inflammation through mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and c-Jun-N-terminal kinase (JNK) pathways [14]. Activation of these signaling cascades lead to production

of more inflammatory chemokines and cytokines. Therefore, this forms a vicious cycle leading to chronic inflammation and eventually a range of medical conditions, including cardiovascular diseases, neurodegenerative diseases, and cancers [12,14].

Table 1 shows anti-inflammatory activities of *Carica papaya*. Different parts of *Carica papaya* possess anti-inflammatory effects. Aqueous extract of *Carica papaya* seeds significantly reduced NO radical by 69.4% in a cell free assay in vitro. Meanwhile, the aqueous extract at a concentration of 150 μg/mL inhibited the release of lysosomal enzymes and stabilized human red blood cell membrane by 22.7%. On the contrary, the extract exhibited least potent hydroxyl radical scavenging action (69.1%) at a concentration 95 mg/mL and reducing power at a concentration of 20 mg/mL [15]. Meanwhile, Aruoma and colleagues demonstrated that fermented papaya preparation (FPP) inhibited H2O2-induced phosphorylation of Akt and p38, as well as downregulating MAPK pathway [16]. An in vivo study showed that *Carica papaya* leaf extract at a dose of 1.32 μg/mL demonstrated immune modulation properties [17].


**Table 1.** Anti-inflammation activities of *Carica papaya.*


**Table 1.** *Cont.*

Abbreviation: NO, nitric oxide; HRBC, human red blood cell; TLR, toll-like receptors, MDA, serum malondialdehyde.

Somanah and co-workers revealed that papaya extracts at a dose of 2 mg/mL showed protective effects through attenuated ROS production and pro-inflammatory cytokines secretion of interleukin-6 (IL-6) and TNF-α as well as upregulating antioxidant enzymes activities [18]. Another in vivo study showed that papaya juice demonstrated anti-obesity properties by reducing obesity markers, inflammation and oxidative stress in high-fat diet rats by upregulating SOD levels, attenuated serum malondialdehyde (MDA), PPAR-γ, lipid peroxidation, and ROS production at a treatment dose of 1 mL per 100 g of body weight [19].

The anti-inflammatory effect of *Carica papaya* was further investigated on various in vivo experimental studies. For instance, ethanolic extract of *Carica papaya* leaves was found to reduce paw edema induced by carrageenan, granuloma formation, as well as inflammation in formaldehyde-induced arthritis rats at doses of 25–200 mg/kg [20]. Methanolic extract of *Carica papaya* seeds at dosage range of 50 to 200 mg/kg exhibited antiinflammation activities in egg albumin induced inflammation on Wistar albino rats [21]. Similarly, the aqueous extract of *Carica papaya* seeds at a dose of 400 mg/kg showed anti-inflammatory in carrageenan and formalin induced pedal edema rats [22].

High phenolic and flavonoid content in papaya seed extracts were proposed to act as free radical scavengers and metal ion chelators [15]. Phytochemicals including tocopherols and quercetin are showed to enhance AMP-activated protein kinase (AMPK) activation, as well as the inhibition of COX-2 expression [17]. In addition, a range of phytochemicals with great strength of anti-inflammatory effect, such as benzyl isothiocyanate (BiTC), β-carotene, lycopene, and vitamin C could be found in various parts of papaya fruits, in either pulp or seeds. These phytochemicals were proven to inhibit pro-inflammatory cytokines including TNF-α, IL-6 and monocyte chemoattractant protein-1 (MCP-1) [18,19]. In addition, polyphenols within *Carica papaya* could act as free radical scavenger and at the same time exerting its effects in upregulating the antioxidant enzymes activities [18].

## *3.2. Diabetes*

Diabetes is a chronic disease, predominantly due to the insulin resistance or insulin insufficiency phenomenon, which leads to elevation of blood glucose level, a condition known as hyperglycemia [23]. Uncontrolled diabetes can lead to various macro and microvascular complications in which ultimately affect the quality of life of diabetes patients. It has been shown that oxidative stress plays an important role in diabetes and its progression [24]. There are tremendous amount of evidences revealed that uncontrolled hyperglycemia might induce oxidative stress by promoting ROS production and weakening antioxidant defenses via several mechanisms, including inducing lipid peroxidation of lowdensity lipoprotein (LDL), glycation of proteins, and glucose oxidation. Non-enzymatic interaction of glucose with proteins generates advanced glycation end products (AGEs), and increases nitric oxide (NO). Excessive free radicals can cause dysfunction of β-cells of the islets of Langerhans of pancreas and lead to complications. Thus, these findings support the role of antioxidants in diabetic control [25,26]. Table 2 shows anti-diabetic activities

of *Carica papaya*. Agada and co-workers demonstrated that the ethyl acetate extract of *Carica papaya* seeds significantly reduced postprandial glucose levels in streptozotocininduced diabetic rats. Along with in vivo study, ethyl acetate showed α-glucosidase and α-amylase enzyme inhibitory effect and antioxidant activities in vitro, whereas hexane extract exhibited slightly more potent enzyme inhibitory activities [27].


**Table 2.** Anti-diabetes activities of *Carica papaya.*


Abbreviation: DPPH, 2,2-diphenyl-1-picryl-hydrazyl-hydrate; TBA, thiobarbituric acid; FPP, fermented papaya preparation; TAC, total antioxidant capacity; SOD, superoxide dismutase; AST, aspartate transaminase; ALT, alanine aminotransferase; NO, nitric oxide. Footnote: IC50 = concentration needed for 50% inhibition; AA50 = concentration needed to achieve 50% antioxidant activity.

> *Carica papaya* FPP extract showed protective effect against diabetic complications such as atherosclerotic plaque formation, upregulated SOD level and ameliorated lipid peroxidation at a concentration of 50 μg/mL. In addition, increasing platelet membrane fluidity of diabetic patients and preventing chronic hyperglycemia-induced platelet malfunction [28]. Likewise, Somanah and colleagues conducted a study on the impact of short-term supplementation of fermented papaya preparation (FPP) on the biomarkers of diabetes mellitus. The randomized controlled trial showed that daily consumption of 6 g of FPP for a period

of 14 weeks enhanced the antioxidant status of the subjects and improved general health status of several organs that were potentially at risk of damage from diabetes. In addition, the FPP extract reduced the aspartate transaminase (AST) and alanine aminotransferase (ALT) levels to enhance insulin sensitivity of the liver and stabilize blood glucose level in diabetic patients [29]. Furthermore, a continuous human trial conducted by Somanah and co-workers showed that the supplementation of FPP successfully reduced erythrocyte hemolysis rate in pre-diabetics. FPP possessed hydroxyl-quenching properties that could possibly prevent DNA damage and boost the total phenolic content that exhibited antioxidant activities [30].

Unripe papaya has been used as a folk medicine, e.g., to relieve menstrual pain, improve ingestion, wound healing, and heart disease. An in vitro study showed that unripe *Carica papaya* fruit inhibited α-amylase and α-glucosidase enzymes. In addition, the fruit extract protected β-cell against oxidative stress in streptozotocin induced diabetes rats. The phytochemical analysis of *Carica papaya* fruits revealed the presence of phytochemicals, including kaempferol, quercetin, and caffeic acid [31].

An in vivo study suggested that chloroform extract of *Carica papaya* leaves protected β-cells of islet of Langerhans from oxidative stress-induced damage and promoted pancreatic β-cells regeneration at a dose of 31 mg/kg, leading to an increase in insulin production [32]. In addition, Juarez-Rojop and colleagues reported that *Carica papaya* leaf extract stimulated the healthy β-cells to release more insulin in vivo. At concentrations of 0.75 and 1.5 g/100 mL, *Carica papaya* leaf aqueous extract also demonstrated antioxidant properties via increasing NO production, consequently lowering ROS production, and diminishing diabetes-induced oxidative stress. As a result, this mechanism delayed or prevented the progression to diabetic complications, such as neuropathy and nephropathy [33].

## *3.3. Alzheimer's Disease (AD)*

Oxidative stress is correlated with the induction and progression of Alzheimer's disease (AD). AD is manifested by generation of neurofibrillary tangles and an aggregation of β-amyloid peptides in the brain [34]. As the amount of β-amyloid accumulates, it generates ROS that causes lipid and protein peroxidation in the brain, and resultant in neurotoxicity. In an AD brain, there is an impairment of the defense mechanism against oxidative stress due to a reduction in the concentration of glutathione. Furthermore, generation of ROS in the brain inhibits the activity of α-secretase whilst promoting the activity of γ- and β-secretase via generation of neurotoxic β-amyloid 40 and 42 [35]. These two mechanisms form a vicious cycle in the AD pathology. Another mechanism notably suggested that β-amyloid impairs mitochondrial function of neuronal cells in AD patients; therefore, promoting neuronal cell death by inducing oxidative injury in isolated mitochondria. β-amyloid impairs the antioxidative stress mechanism by lowering the expression of uncoupling proteins (UCPs) that act to promote mitochondrial uncoupling and reduce ROS generation [35]. A limited number of studies proposed that oxidative stress susceptibility is increased by overexpression of tau protein in neuronal cells. In addition, presence of transition metals including iron, zinc, and copper can react with the β-amyloid to produce hydrogen peroxide (H2O2) in the brain [34–36].

Fermented papaya preparation (FPP) is a popular health-promoting product which owns protective properties against free radicals to improve general health. Meanwhile, fermented papaya preparation (FPP) exerted neuroprotective properties against copper induced neurotoxicity in Swedish mutant human APP (APPsw) cells at a dose of 2.4 mg/mL by reducing 64% of ROS generation. In addition, FPP significantly reduced scavenging of superoxide anion and hydroxyl radicals and upregulation of SOD-1 enzyme. FPP exerted anti-apoptotic effect and attenuated pro-apoptotic Bax gene expression, upregulated BCL level, and maintained calcium homeostasis, leading to improvement of neuronal cell survival and AD condition. Administration of 2.4 mg/mL FPP inhibited up-regulation of expression of iNOS, nNOS, and NO by about 43%, 71%, and 40%. Moreover, treatment with 2.4 mg/mL FPP lowered the secretion of Aβ peptide by 30.6% [37].

FPP significantly reduced the 8-hydroxy2 -deoxyguanosine (8-OHdG) level in AD patients treated with FPP at a dose of 4.5 g/day for 6 months. During the study period, no neurotrophic drugs were administered to the study participants; therefore, proving the value of the *Carica papaya* plant in improving AD. The proposed mechanisms of action by FPP include decreased peroxidation of lipids, aluminum and iron induced neuronal toxicity and free radicals' production [38]. Overall, FPP showed promising anti-Alzheimer's disease in cell-based model and human trial. Studies including discovery of novel phytochemicals, safety profile, and efficacy warrants future investigation.

## *3.4. Periodontal Disease*

Periodontal disease is an infection on the tissue that supports the tooth, which is closely related to oxidative stress. Several examples of periodontal diseases include gingival inflammation, chronic periodontitis, aggressive periodontitis, necrotizing periodontal disease and periodontal associated lesion. These conditions can happen to anyone ranging from a juvenile to an adult [39]. When the integrity of tissues supporting the tooth is compromised, the immune response of the host is triggered secondary to pathogen invasion and eventually led to inflammation. Periodontal inflammation can be augmented by excessive ROS and leukocytes and damaging the periodontal tissues. Likewise, periodontal tissue injury also occurs when there is a disruption in the redox equilibrium, due to either over-generation of ROS or diminished antioxidant enzymes, including GPx, CAT, and SOD, which defend against oxidative stress. ROS plays a role in activating signaling pathways, such as NLRP3 inflammasomes, NF-*κ*B, and JNK, which eventually lead to inflammation and cell death [40]. Human trial has shown that *Carica papaya* leaf extract significantly alleviated gingival bleeding and inflammation [41].

Kharaeva and colleagues reported that the standardized fermented papaya gel (SFPG) application at 7 g/day for 10 consecutive days significantly reduced gingival inflammation and bleeding in participants by decreasing nitrate (NO3 <sup>−</sup>) and nitrite level (NO2 <sup>−</sup>), and regulating the level of inflammatory cytokines. Reduced NO3 <sup>−</sup> and NO2 <sup>−</sup> attenuated production of peroxynitrite and oxidative stress generation. Furthermore, the antioxidant effect was reported to last as long as 35 days after stopping SFPG application. Interestingly, SFPG was able to augment bacterial killing by impeding activation of bacterial catalase and eventually prevent infection at the periodontal sites [42].

Studies have also shown the protective effects of *Carica papaya* leaf extract dentifrice on interdental gingival bleeding. Participants who used dentifrice containing *Carica papaya* leaf extract demonstrated a significant decrease in the gingival bleeding and inflammation especially in advanced (>70%) gingival bleeding cases. This result could be attributed to the high phenolic content of *Carica papaya* leaf extract that possess antioxidant properties. A study revealed that *Carica papaya* leaf extract exerted anti-inflammatory action by decreasing TNF-α [41].

#### *3.5. Skin Aging*

Skin aging is characterised by extracellular matrix (ECM) degradation in which human skin naturally becomes drier, thinner, unevenly pigmented, and wrinkled, as a human being ages, due to the inevitable intrinsic aging factors. Extrinsic aging factors are avoidable, in which both factors may synergize and lead to premature skin aging. ROS is known to be the culprit of skin aging by contributing to oxidative stress and inflammation. Photoaging is a process that produces ROS, which eventually leads to augmented ECM turnover and degradation. Although not fully deleterious to the cells, excessive ROS can oxidise skin proteins and lipids leading to roughen the skin by altering the function of the skin barrier and further stimulate wrinkle formation [43].

In addition, ultraviolet (UV) and UV-generated ROS hasten aging via activation of mitogen-activated protein kinase (MAPK), p38, Jun N-terminal kinase (JNK), extracellularsignal-regulated kinase (ERK), recruitment of c-Fos, and c-Jun, as well as increased expression of activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). AP-1 is known to

lower transforming growth factor-beta (TGF-β), which is responsible for collagen production and induce expression of matrix metalloproteinase (MMP) 1, 3, and 9 in keratinocytes, and fibroblast leading to the disruption and loss of ECM components (collagen and elastic fibers) [43]. UV and ROS causes the skin to be in the state of "sunburn" (erythema). This further stimulates production of advanced glycation end products (AGEs). Activation of receptor for AGEs (RAGE) increases NF-κB activation, thereby upregulates pro-inflammatory gene transcriptions and RAGE leading to a vicious inflammatory state cycle characterised by elevated PGE2 synthesis [43,44].

Furthermore, ROS induces melanogenesis by increasing the number of tyrosinaserelated protein 1 (TYRP-1) and tyrosinase, which are both known as melanogenic factors resulting in skin pigmentation [44]. In addition, UV radiation induced greater amounts of oxidised lipids, triglyceride hydroperoxides, and cholesterol hydroperoxides generation, leading to increased sebum secretion. This condition in turn promotes the formation of acne vulgaris by *Propionibacterium acnes* (*P. acnes*). *P. acnes* infects skin cells and will further induce the production of free oxygen radicals that eventually lead to the formation of inflammatory lesions [44].

In past decades, research on strategies against skin aging attracted a great attention of researchers. For instance, *Carica papaya* is a potential candidate to be exploited for its anti-skin aging specialty, owing to its antioxidant and anti-inflammatory activities. Table 3 shows anti-skin aging activities of *Carica papaya* extracts. An in vitro study by Jarisarapurin and colleagues focused on unripe *Carica papaya* fruit extract against skin aging related endothelial oxidative stress [45]. It was proposed that activated endothelial cells contributed to a low-grade inflammatory state and the generation of oxidative stress. As a result of this unfavorable microenvironment, MMP-1 expression in dermal fibroblasts was induced leading to a significant loss of type I collagen, and accelerated ECM degradation [46]. The study demonstrated the ability of unripe *Carica papaya* fruit extract to inhibit H2O2-induced endothelial cell death at concentrations ranging from 100 to 1000 μg/mL. It was found to exert its effect via modulating intracellular stress and antioxidant defenses in endothelial cells. The mechanisms were consisted of a dose-dependent ROS scavenging effect and NFκB attenuation, upregulation of SOD and CAT activities, and prevention of H2O2-induced Nrf2 over activation. The study further explained that, although activation of antioxidant defenses was prompted by uncoupling of the Nrf2/Keap1 complex, followed by translocation of Nrf2 into the nucleus, the early (or over activation) of Nrf2 induced by oxidative stress can lead to depletion of endogenous antioxidants. The consequence of depletion of natural antioxidants produced by skin cells may promote skin aging. Therefore, the restraining properties of unripe *Carica papaya* on NF-kB elevation and Nrf2 dysregulation were proposed to be beneficial in maintaining redox homeostasis, thereby delaying skin aging [45].

A recent study by Seo and colleagues investigated the anti-aging mechanisms of *Carica papaya* leaf ethanol extract on UVB-irradiated human dermal fibroblast cells in vitro. At concentrations ranging from 10 to 250 μg/mL, the extract demonstrated radical scavenging and ROS suppressing action in a dose-dependent manner. At concentrations of 1 to 50 μg/mL, the extract was shown to enhance synthesis and attenuate degradation of type I procollagen in UVB-irradiated fibroblasts, increment in TGF-β1, and reduction in MMPs (MMP-1 and MMP-3) generation. Interestingly, Seo and colleagues further evaluated that the leaf extract possessed reversal action on UVB-induced AP activation at mRNA level via downregulating MAPK activation and protein phosphorylation of c-Fos and c-Jun. The effect of *Carica papaya* leaf extract on MAPK was proposed to act mainly on p38, showing 82% inhibition against p38 phosphorylation, followed by ERK and JNK. The extract demonstrated to acquire anti-inflammatory action by depleting production of cytokines, such as IL-6. Wrinkles formation induced by sun exposure as a result of erythema and diminished Type I collagen in skin. The ROS-conquering mechanisms and collagen synthesis promoting effects were described by Seo and colleagues, lending support on the potential use of *Carica papaya* leaf extract against skin aging [47].


**Table 3.** Anti-skin aging of *Carica papaya.*

Abbreviation: DPPH, 2,2-diphenyl-1-picryl-hydrazyl-hydrate; DCFH-DA, dichloro-dihydro-fluorescein diacetate; SOD, superoxide dismutase; MMP, matrix metalloproteinase; RCT, randomized controlled trial, MDA, serum malondialdehyde.

> A human trial by Bertuccelli and colleagues revealed that sublingual FPP 4.5 g sachet twice daily lowered biomarkers of skin aging. While both treatments attenuated skin MDA level, FPP showed superior anti-aging effects than antioxidant cocktails [45]. In addition, FPP elevated levels of SOD, NO, aquaporin-3 (AQP-3), and down-modulation of pro-aging cyclophilin-A (CyPA) and CD147 genes. The study proposed that the regulating effects of FPP on AQP-3 and pro-aging factors were crucial for significant improvement in skin health [48].

> The potentiality of *Carica papaya* being formulated as cosmetic products was demonstrated by Saini and colleagues, as the ideal oil-in-water *Carica papaya* fruit cream prepared was uniform, stable, and had a shiny and smooth texture. This study further proved ROS suppression as the main mechanism of *Carica papaya* fruit against anti-aging, in which the 5% cream was potent, owing reducing power against H2O2 free radicals [49].

> Flavonoids and phenolic acids were found in *Carica papaya* leaf and fruit extracts [50,51]. Flavonoids in *Carica papaya* are mainly kaempferol, myricetin, quercetin, and their glycosides, phenolic acids, such as caffeic acid and ferulic acid, are the key ROS suppressors

and antioxidant that displayed radical scavenging and metal chelating potential [50,52]. Caffeic acid and rutin were detected and proposed to be the main anti-skin aging components. Both phytochemicals were reported to downregulate MMPs expression and photoprotective against collagen degradation. Caffeic acid mitigated skin erythema via inhibitory action towards NF-κB and AP-1 signaling [48]. The ability of caffeic acid in film formulation to permeate and retentate in epidermis (stratum corneum) and dermis layer enhance its efficacy [50,53]. Besides, the anti-skin aging role of rutin was supported by a human trial, which showed enhanced skin elasticity and less wrinkles in individuals treated with rutin-containing cream. The findings of elevated type I collagen via lowering MMP expression and potent ROS scavenging in human dermal fibroblast cells further supported the anti-skin aging of *Carica papaya* chemical constituents [48,54].

Albeit several mechanisms were compiled and proposed, however, studies regarding the *Carica papaya* anti-skin aging effect were scarce. More evidence regarding various parts, therapeutic range, and the relevant phytochemicals of *Carica papaya* on skin aging are needed to ensure their efficacy.

## *3.6. Wound Healing*

Wound healing is rather complex and well-coordinated with involvement of several stages of cellular responses, including inflammation, proliferation, and remodeling. The duration of each phase usually ranges from 1 to 4 days, 5 to 10 days, and 11 days onwards. Characterisation for each phase includes presence of leukocytes, angiogenesis, protein synthesis and deposition, epithelialization, wound contraction, and scar formation [55]. These processes can be altered by the presence of oxidative stress [56]. The efficiency of the wound healing process decreases with advancing age [57]. Oxidative stress can alter the speed of wound recovery as it depends on the amount of ROS present at the wound site. Although minimal ROS prevents infection, excessive ROS is known cytotoxic to fibroblasts and reduce flexibility of skin lipids. In addition, it also causes impairment to lipids, DNA, proteins, and cellular membranes, and subsequently, severely damages the tissue and promotes inflammation [56].

In the case of chronic wounds or impaired wounds, ROS production is excessive in response to NADPH oxidase (NOX) activation in macrophages and neutrophils during the inflammatory phase of the wound healing process, contributing to high oxidative stress that leads to the wound remaining not healed. Thereby, extracts and phytochemicals with great strength of antioxidative properties are beneficial in wound healing [58,59]. Another key factor for wound healing is the extent of inflammation level at different stages of healing. Inflammation is essential to prevent infection, stimulate angiogenesis, and matrix deposition via secretion of cytokines and angiogenic factors at the early stage of wound healing. However, excessive or prolonged pathological inflammation causes delayed wound healing and fibrosis. Hence, inflammation at certain phases is deemed crucial for wound healing, but not throughout the entire healing process [55,60].

It is known that oxidative stress plays a vital role in wound healing. Table 4 shows wound healing activities of *Carica papaya* extracts. The protective action of aqueous extract *Carica papaya* seeds against oxidative stress-induced apoptosis in human skin fibroblast further supported its role in wound healing. An extensive mechanistic study conducted found potent antioxidant action of the papaya extract against H2O2-induced oxidative stress specifically on fibroblast cells was activated via radical scavenging, reduction of calcium ions influx into cytoplasm, reversal of oxidative stress-induced mitochondrial dysfunction, and maintaining oxidative balance inside the cells [61]. Mikhal and colleagues showed that FPP possesses antioxidative stress and anti-inflammation activities. FPP inhibited superoxide (IC50 = 5 mg/mL), hydroxyl radicals (IC50 = 1.1 mg/mL), and total ROS (IC50 =2 mg/mL) in blood, as well as reduction in myeloperoxidase (MPO) and radical generation at wound sites in vivo [62].

In addition, topical application of 5 mg/mL *Carica papaya* fruit extract enhanced wound healing by exerting effect on regulation antioxidant enzymes, inflammation, and

arginine metabolism. The addition of an antioxidant, selenium to the regimen, further shortened the time for wound healing significantly and, hence, confirmed the mechanisms. Antioxidative stress related mechanisms include inhibition of lipid peroxidation, lower MDA level and enhanced expression of SOD, CAT, and GPx. *Carica papaya* fruit extract reduces inflammation associated with oxidative damage through upregulation of antioxidant enzymes, arginine metabolism, and cyclooxygenase specific inhibition in an excision wound model. The extract demonstrated an attenuated inflammatory state, increased collagen synthesis and vascularization at wound site. Transforming growth factor-beta (TGF-β), a cytokine that generates fibroblast recruitment was high at the inflammatory phase and reduced at the repairing phase. While expression of vascular endothelial growth factor A (VEGFA), an angiogenic factor was increased throughout the wound healing process. The further study showed that addition of selenium to the papaya fruit extract synergistically upregulated TGF-β and VEGFA resulting in a significant acceleration in the wound healing process [63,64].

An oral FPP supplementation at a dose of 0.2 g/kg body weight for 8 weeks was found to enhance diabetic wound closure via improved macrophages respiratory-burst function and iNOS production. Diabetic wounds are hard-to-heal due to being prone to infections as a result of compromised NO at the wound site. Another reason was the antibacterial effect of macrophages via NOX was downregulated by hyperglycemia, consequently, respiratory burst dysfunction was seen in diabetic patients. FPP was shown to reverse these conditions. Similar to previous report by Nafiu and colleagues, FPP supplement showed an increase in VEGFA expression, deemed as a crucial regulator in current scenario [65].

Dickerson and colleagues further examined the diabetic wound healing effect of FPP on type II diabetes mellitus patients. The participants were given oral FPP (9 g/day for 6 weeks), and showed that NADPH and cellular ATP level increased in human monocytic THP-1 cells treated with FPP. Besides, FPP also exhibited higher oxygen usage and mitochondrial membrane potential on monocytic cells, which further revealed its capability to correct the respiratory burst function, enhancing the defense mechanisms against pathogens in diabetics. FPP upregulated the mRNA expression of Rac2, which was essential for NOX activation and eventually enhancing respiratory burst in macrophages [66,67].

Meanwhile, Indran and colleagues investigated the protective effect of *Carica papaya* leaf aqueous extract against alcohol-induced hemorrhagic lesions. Pretreatment with 500 mg/kg leaf extract significantly reduced gastric ulcer index via reducing lipid peroxidation, MDA levels, and improving GPx activity at gastric mucosa. The study showed the radical scavenging activity, which might be contributed by polyphenols within leaf extract. It was suggesting that the alkaline content of the extract and its ability to neutralize stomach acidity, thereby protecting stomach against gastric ulcer [68]. The concepts were further supported by in vivo studies evaluating different parts of *Carica papaya* on incised, burned, and diabetic wounds respectively. The recent findings show that *Carica papaya* fruit and seed extracts demonstrated dose-dependent increment in hydroxyproline, fibrillation, epithelial thickness, shortened wound contraction, and epithelialization time [69–72].

Cysteine endopeptidases including papain and chymopapain showed wound healing activity that can be attributed to their proteolytic wound debridement and antibacterial effects [63,64,70,72]. This was established by an in vivo study using papain-based wound cleanser. The cleanser was formulated with 5 g of papain and α-tocopherol. The results showed superior collagen deposition and least fluid exudates compared to betadine cleanser leading to eschar reduction and quicker epithelialization [73]. Safety of *Carica papaya* extracts and dressings is of less concern as it is traditionally used to treat wounds and certain skin conditions [74]. Several studies further assured its safety to be used [61,67,71]. In addition, papaya dressing was safe to be used and compatible in hydrogel formulation [70,75].


**Table 4.** Wound healing activities of *Carica papaya.*


**Table 4.** *Cont.*

Abbreviation: MMP, matrix metalloproteinase; HRBC, human red blood cell; HSP-70, heat shock protein 70; NO, nitric oxide; ROS, reactive oxygen species; COX-2, cyclooxygenase-2; MPO, myeloperoxidase; VEGFA, vascular endothelial growth factor A; TGF, transforming growth factor; FPP, fermented papaya preparation; MDA, serum malondialdehyde.

#### *3.7. Cancers*

Cancer is a prevailing topic and there is no absolute cure to date for various types of cancers. ROS generation as a result of metabolic reactions in the mitochondria plays a role in both initiation and potentially elimination of cancers. With a low amount of ROS that is tolerable by the body cells, the progression of cancer could occur through either promoting genomic DNA alterations or DNA damage that alters the normal physiological signaling pathways. For instance, mitogen-activated protein kinase (MAPK) activation, c-Jun Nterminal kinase (JNK), extracellular signal-regulated kinase (ERK) phosphorylation, and cyclin D1 expression are correlated to cancer progression and survival [76]. In the normal healthy cells, a significantly high level of ROS can lead to cellular damage and eventually cell death [77]. However, cancer cells generally have a higher resistance to oxidative stress than normal cells to allow for uncontrolled proliferation and to compensate for the survival

of cancer cells during metastasis from their site of origin [76]. However, increasing ROS to a specific threshold level, specifically for cancer cells is proven to attenuate cancer cell growth and progression.

Several studies showed the correlation of microRNAs and oxidative stress in the progression of cancer. The recent advancement of genomic studies has showcased the presence of certain groups of microRNAs may promote cancer cell proliferation and progression. For example, there is an overexpression of miR-210 detected in hepatocellular and breast carcinoma under hypoxia. miR-210 acts to regulate the ROS production and mitochondrial function by promoting cancer cell adaptation, survival, and proliferation [77]. It also suggested that ROS is able to induce carcinogenesis by induced mutations in the tumour-suppressor gene in the normal skin cells leading to a transformation of normal cell into cancerous cells by halting the initiation of programmed cell death. An example of this mutation is seen in the alteration of a guanine in the p53 gene through oxidative mechanisms in basal cell and squamous cell carcinoma [43].

DNA damage is pivotal in cancer formation. A study proposed that FPP was capable of impeding DNA fragmentation induced by free radicals and H2O2-induced DNA damage at a dose of 100 μg/mL [16]. In addition, the aqueous extract of *Carica papaya* fruit suppressed proliferation of human breast epithelial cancer (MCF-7) cells in vitro. The aqueous extract of *Carica papaya* showed significant anti-proliferation activity (~53%) in MCF-7 cells at a dose of 4% *v*/*v* after 72 h treatment. The anti-cancer activity of FPP might be attributed to the mechanisms including triggering cell signaling to induce apoptosis [78]. An in vitro study showed that the aqueous extract of *Carica papaya* leaves showed antiproliferation activity of MCF-7 at a IC50 of 1.31 mg/mL and induced apoptosis of MCF-7 cells at 22.5% with a dose of 0.65 mg/mL [79].

*Carica papaya* enriched with phytochemicals, including flavonoids, has been found to possess chemopreventive properties. The mechanisms of action underlying the chemoprevention effects include activating tumour-suppressor genes, deactivating oncogene products transcriptionally, decreasing oxidative damage via acting as free radical scavengers and impeding the commencement of lipoxygenase reaction by chelating with ROS-generating agents. For example, the benzene fraction of aqueous extract of *Carica papaya* showed chemoprotective effects in benzo(a)pyrene and 7,12-dimethyl benz(a)anthracene -induced carcinogenic animal models. It was reported that a significant reduction of lung adenomas (>50%) at a treatment dose of 1 g/kg body weight. In addition, a significant reduction in skin papillomagenesis incidence at 64.20% was compared with tumour incidence in the control group. It was suggested that those flavonoids contained within different parts of the *Carica papaya* plant act via multi-signaling networks as the viable chemoprevention agents [80].

An in vivo study reported that FPP at a concentration of 500 mg/kg significantly elevated antioxidant enzymes, including GPx (66.1%), SOD (20%), and CAT (81%). Furthermore, FPP was also capable of preventing DNA structural damage possibly induced by free radicals and genotoxins [81]. This was supported by another study suggesting that *Carica papaya* peel extract significantly increased glutathione (GSH), while decreasing MDA and ROS production. Thus, preventing DNA damage and induction of colonic carcinogenesis azoxymethane treated group [82]. Another study by Mukami and colleagues revealed that orally administration of FPP at 450 mg/kg showed complete disappearance of the tumours in a radiation-induced leukemia mice model [83]. Overall, several research groups revealed the anticancer properties of papaya extracts. Further studies are needed to standardize the extract for quality control of the efficacy, and discover novel compounds, owing to the anticancer activities.

### **4. Conclusions**

To summarize, the *Carica papaya* counteracts oxidative stress via its potent antioxidant properties. Therefore, it can be incorporated into nutraceuticals or conventional medications to be used as a potential preventative or treatment option for various health

conditions. The antioxidant properties of the *Carica papaya* plant might be attributed to the various chemical constituents that the plant contains, including caffeic acid, myricetin, quercetin, rutin, α-tocopherol, papain, BiTC, kaempferol steroids, alkaloids, and saponins.

There is no doubt that emerging evidence has proven the potential of *Carica papaya* as a natural resource that can be exploited as a medicinal product. However, more safety data are needed to justify its use in different medical conditions. Many plants—although exerting therapeutic benefits, having been used traditionally for diseases since the ancient times—are potentially cytotoxic [7,84]. The acute toxicity study of the *Carica papaya* leaf extract revealed that there were no significant toxic effects of *Carica papaya* leaf extract at the concentration up to 2 g/kg of body weight, which corresponded to 14 times the dose incorporated in traditional medication. Moreover, it was also suggested that any concentration of *Carica papaya* leaf extract below 2 g/kg of body weight posed no significant toxicity and adverse effects when administered orally for a 14-day interval [85,86]. In terms of the *Carica papaya* extract with different methods of extraction, namely ethanol and aqueous extract, it might possess different safety profiles, owing to the extractive chemical constituents. For example, the ethanol extract might be more nephrotoxic and hepatotoxic than the aqueous extract in the Wistar rats model at 1 g/kg of body weight concentration [87]. However, all these studies suggest that more extensive evaluation studies pertaining the cytotoxicity profile of oral administration of the *Carica papaya* extract are needed to further validate the safety for consumption.

It was also suggested that the medicinal properties of the *Carica papaya* plant can be attributed to other mechanisms of action. Several studies have suggested that *Carica papaya* extract exerted antimicrobial properties that aided in wound recovery [69,71]. Therefore, more studies should be done in order to unravel the benefits of the *Carica papaya*.

**Author Contributions:** Conceptualization, K.Y.K. Writing–original draft preparation, Y.R.K., Y.X.J., M.B., Z.K.B., J.K.K.W. and K.C.T. Writing—review and editing, Y.R.K., Y.X.J., M.B., Z.K.B., J.K.K.W., K.C.T., K.G.C., L.H.L., B.H.G., K.Y.K. and Y.S.O.; supervision, K.Y.K. and Y.S.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the University of Malaya (to Kok-Gan Chan, FRGS grant number: FP022-2018A) and The SEED Funding from Microbiome and Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine and Health Sciences, (To Learn-Han Lee, Vote Number: MBRS/JCSMHS/02/2020).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We would like to acknowledge School of Pharmacy, Monash University Malaysia for administrative and technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


## *Article Curcuma amarissima* **Extract Activates Growth and Survival Signal Transduction Networks to Stimulate Proliferation of Human Keratinocyte**

**Wutigri Nimlamool 1,2,\* , Saranyapin Potikanond 1,2 , Jirapak Ruttanapattanakul <sup>1</sup> , Nitwara Wikan 3, Siriporn Okonogi 2,4, Salinee Jantrapirom <sup>1</sup> , Pornsiri Pitchakarn <sup>5</sup> and Jirarat Karinchai <sup>5</sup>**


**Simple Summary:** Like many plants in the family of Zingiberaceae, *Curcuma amarissima* has been traditionally used to induce healing and tissue regeneration. However, there is no scientific evidence to explain how *Curcuma amarissima* works to accelerate wound healing. Our data clearly proved that *Curcuma amarissima* extract could potentially accelerate the closure of scratch wounds of human keratinocytes by stimulating cell proliferation. The potential mechanisms underlying these effects were defined to be associated with the activated signal transduction pathways relevant to cell proliferation and survival. This strongly suggests the ability of *Curcuma amarissima* to enhance the process of keratinocyte reepithelization during wound healing. Our current study provides convincing evidence that supports the possibility to develop an effective wound-healing promoting agent from this plant.

**Abstract:** Many medicinal plants have been used to treat wounds. Here, we revealed the potential wound healing effects of *Curcuma amarissima* (CA). Our cell viability assay showed that CA extract increased the viability of HaCaT cells that were cultured in the absence of serum. This increase in cell viability was proved to be associated with the pharmacological activities of CA extract in inducing cell proliferation. To further define possible molecular mechanisms of action, we performed Western blot analysis and immunofluorescence study, and our data demonstrated that CA extract rapidly induced ERK1/2 and Akt activation. Consistently, CA extract accelerated cell migration, resulting in rapid healing of wounded human keratinocyte monolayer. Specifically, the CA-induced increase of cell monolayer wound healing was blocked by the MEK inhibitor (U0126) or the PI3K inhibitor (LY294002). Moreover, CA extract induced the expression of Mcl-1, which is an anti-apoptotic protein, supporting that CA extract enhances human keratinocyte survival. Taken together, our study provided convincing evidence that *Curcuma amarissima* can promote proliferation and survival of human keratinocyte through stimulating the MAPK and PI3K/Akt signaling cascades. These promising data emphasize the possibility to develop this plant as a wound healing agent for the potential application in regenerative medicine.

**Keywords:** human epidermal keratinocytes; HaCaT; *Curcuma amarissima*; PI3K/AKT; RAS/ERK; wound healing; proliferation; survival

**Citation:** Nimlamool, W.;

Potikanond, S.; Ruttanapattanakul, J.; Wikan, N.; Okonogi, S.; Jantrapirom, S.; Pitchakarn, P.; Karinchai, J. *Curcuma amarissima* Extract Activates Growth and Survival Signal Transduction Networks to Stimulate Proliferation of Human Keratinocyte. *Biology* **2021**, *10*, 289. https:// doi.org/10.3390/biology10040289

Academic Editors: Francisco Les, Victor López and Guillermo Cásedas

Received: 4 March 2021 Accepted: 29 March 2021 Published: 1 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Ineffective skin wound healing is becoming a big problem in the public health sector. Several factors including aging, diabetes, infection, immunodeficiency, and cancers can lead to unsuccessful wound treatment and eventually cause morbidity and mortality [1–5]. Although the human body has a great protective skin barrier, sometimes an unexpected injury is unavoidable. A process of organisms, namely "wound healing", is responsible for the reconstruction of injured skin. This physiological repairing system requires four stages, which include hemostasis (blood clotting), inflammation, proliferation (growth of new tissue), and maturation (remodeling) [6,7]. A successful step of re-epithelialization is considered to be an essential indicator of wound closure to prevent further infection and chronic wound development [8]. In response to skin damage, the process called reepithelialization is triggered to restore the damaged epidermis. The most important cell types responsible for re-epithelialization are keratinocytes, which proliferate, differentiate, and migrate to heal the open wound [9]. Failure of keratinocytes to maintain skin integrity and/or wound closure could cause reoccurrence of affected lesions and further complicated skin reaction [10]. At the cellular and molecular level, growth and survival signaling cascades are very crucial for enhancing certain stages of re-epithelialization. Specifically, the signaling pathway of mitogen-activated protein kinase (MAPK) is involved in regulating cell proliferation and migration [11,12]. In particular, ERK1/2 kinase phosphorylation is induced by various groups of growth factors [13–16]. Therefore, certain growth factors such as EGF or PDGF are approved to be used to stimulate the skin healing process [17–19]. Nevertheless, growth factors (used at high concentrations) can cause adverse effects, including uncontrolled cell growth, chronic inflammatory skin disease such as psoriasis, or aberrant skin functions [20,21]. Therefore, discovering novel wound healing agents that have lesser side effects would be beneficial for patients with certain conditions.

Since ancient times, people have used herbal medicines as one of the components in wound management to accelerate wound healing. In particular, for treatments derived from plants, both topical and systemic herbal agents have been widely used in wound healing. Several properties, including anti-inflammatory, antioxidant, and antimicrobial activities, are required for being effective wound healing agents [22,23]. Thus, any herbs with these properties will be worth investigating and using for the development of effective wound healing agents. Generally, extracts or the isolated phytochemical compounds derived from plants promote the tissue regeneration process via certain mechanisms, which ultimately provide a synergistic effect on healing efficiency [24]. Currently, some natural products that include herbs [25] have been included as active components for wound treatments. For instance, it has been demonstrated that *Poincianella pluviosa* extract could potently accelerate keratinocyte migration and proliferation [26]. Recently, *Boesenbergia rotunda* or fingerroot, belonging to the Zingiberaceae family (same family with *Curcuma amarissima*) has been revealed to promote human keratinocyte proliferation via stimulating the phosphorylation of ERK1/2 and Akt [27]. Although many plant-derived medicines are believed to be effective, affordable, and cause minimal unwanted side effects [28], it is necessary to carefully define the exact mechanisms of action to be certain how medicinal plants function.

*Curcuma amarissima* Roscoe (CA) or "black turmeric" or "Kamin-dum" is in the family Zingiberaceae and is usually used to treat amoebic dysentery, enteritis, and vermicide [14]. Lectin isolated from the rhizomes of this plant showed anti-fungal activity against several different species, including *Colectrotricum cassiicola, Exserohilum turicicum,* and *Fusarium oxysporum*. Moreover, the purified lectin could also reduce cell proliferation in a breast cancer cell line, BT474 [29]. Accumulating evidence has suggested that plants in the Zingiberaceae family may be one of the great candidates for wound healing [27,30,31]. Specifically, several activities of herbs in this family contain essential combinations that are necessary for the healing process. Those activities include antioxidant, antimicrobial, anti-inflammation, promoting collagen production, promoting dermal cell proliferation, promoting new blood vessels, and central/peripheral antinociceptive effects [32,33]. Previous studies disclosed that the rhizomes of CA contain several different active constituents similar to those found in *Curcuma longa*. Those compounds include curcumenol, curdione, curzerenone, germacene, isofrtungermacrene, and zedoarone [34].

Although many plants in the family of Zingiberaceae have been revealed to have promising effects suitable for wound treatment, there is no evidence showing that *Curcuma amarissima* Roscoe has regenerative effects on skin. Therefore, it is of our interest to investigate whether the *Curcuma amarissima* Roscoe extract has specific pharmacological activities that help enhance wound healing processes.

Here, we discovered that CA can enhance human keratinocyte, HaCaT, cell proliferation and migration via inducing ERK1/2, and Akt phosphorylation, which are important molecular pathways involved in re-epithelialization. Our current study provided information that CA can be developed as an agent for accelerating skin wound repair.

## **2. Materials and Methods**

#### *2.1. Preparation of Ethanolic Extract from the Rhizomes of Curcuma aeruginosa (CA)*

The rhizomes of *Curcuma amarissima* Roscoe were obtained from the cultivating areas in Mae Taeng District, Chiang Mai, Thailand, and were identified by a botanist at the Faculty of Pharmacy, Chiang Mai University. The samples of authenticated *Curcuma amarissima* Roscoe were deposited in the Herbarium of the Faculty of Pharmacy, Chiang Mai University, with the voucher specimen number 0023261. For preparing the ethanolic extract, the fresh rhizomes of *Curcuma amarissima* Roscoe were washed, cut into small pieces, dried, and ground. Next, the ground powder was mixed with ethanol (95%) at room temperature (RT) for 24 h. The mixture was filtered through Whatman No.1 filter paper (Sigma-Aldrich, Saint Louis, MO, USA), and the filtered solution was subjected to a rotary evaporator at 40 ◦C to eliminate the solvent. Next, one gram (g) of the obtained CA extract was diluted in 1 milliliter (mL) 100% dimethyl sulfoxide (DMSO) and used as a stock solution. For each treatment, the CA extract stock solution (1 g/mL in DMSO) was further pre-diluted in medium to obtain the final working concentrations. However, the final concentration of DMSO was not allowed to exceed 0.5% *v*/*v* in the diluted media throughout the experiment.

## *2.2. HPLC Fingerprint of CA Extract*

HP1100 system (HPLC LC-10, Shimadzu, Kyoto, Japan) with an Agilent C-18 column (150 × 4.6 mm, 5 μm) was applied for visualizing the HPLC fingerprint of the extract. The system was performed with a thermostatically controlled column oven and a UV detector set at 254 and 360 nm. The mobile phase was methanol–water system with gradient elution as follows: 40–70% methanol for 0–30 min, 100% methanol for 80–100 min, 40% methanol for 105–115 min. The extract was diluted with methanol to 50 mg/mL before injection (10 μL of sample volume) into the column, with 1.0 mL/min of flow rate.

Additionally, quantitative analysis of curcuminoids and polyphenolic contents in CA extract by HPLC was performed. CA extract was determined for the existence of curcuminoid and polyphenolic contents by HPLC using a C18 column (250 × 4.6 mm, 5 μm) (Agilent Technologies, Santa Clara, CA, USA). The detection of curcuminoids, including bis-demethoxycurcumin, demethoxycurcumin, and curcumin, was carried out using isocratic mode of mobile phase (2% acetic acid in water and acetonitrile 50:50 *v*/*v*) with a flow rate of 1 mL/min. Ten microliters of the extract (20 mg/mL dissolved in 1 mL of MeOH) was injected into the column, and detection was done at 425 nm. The polyphenolic content in the extract was determined by using a gradient system of mobile phase A (1% acetic acid in water) and mobile phase B (100% acetonitrile) with a total run time of 50 min for the detection with a flow rate of 0.7 mL/min and detection wavelength at 280 nm. The gradient system used was 90% A in 0 min–60% in 28 min, followed by 40% in the next 39 min and 10% in the next 50 min.

## *2.3. Culture of Human Keratinocyte Cell Line, HaCaT*

HaCaT cell line was purchased from CLS Cell Lines Service GmbH (CLS Cell Lines Service GmbH, Eppelheim, Baden-Wurttemberg, Germany) and maintained in DMEM (Gibco, New York, NY, USA), supplemented with 10% (*v*/*v*) fetal bovine serum (FBS) (Gibco, New York, NY, USA), 100 U/mL penicillin and 100 μg/mL streptomycin) (Gibco, New York, NY, USA) at 37 ◦C in a humidified atmosphere of 5% CO2. For experiments that required serum deprivation, cells were cultured in serum-free media.

## *2.4. Cell Viability Determination of HaCaT Cells Treated with CA Extract*

MTT assay was conducted in CA-extract-treated HaCaT cells, which were cultured in either media containing fetal bovine serum (FBS) or FBS-free media. The viability characteristics of the cell were observed. Moreover, toxic and non-toxic concentrations of CA were obtained. Briefly, we cultured (5 × 104 cells/well in 96-well plates) in complete media overnight. For CA extract treatment, the CA extract stock (1 g/mL) was pre-diluted in FBS-free media to make a final concentration of 160 μg/mL (containing DMSO at 0.16%). DMSO (as a vehicle control) was diluted in the same manner. Next, we prepared the treating media containing various concentration of CA extract (ranging from 160 down to 0.625 μg/mL) and DMSO (0.000625–0.16%) by making a two-fold dilution of CA extract or DMSO in either FBS-rich or FBS-free media. Cells were treated with individual treating media (200 μL/well in 96-well plates) for 48 h and subjected to a working solution (0.4 mg/mL) of MTT reagent for 1 h in an incubator. After MTT was discarded, 100 μL of DMSO was added to each well, and the developed color was detected (at 570 nm) by an absorbance reader plate spectrophotometer (BioTek Instruments, Winooski, VT, USA). From this experiment, we chose three concentrations (2.5, 5, and 10 μg/mL) of CA extract for all experiments. Moreover, the proliferative effect of CA extract at specific non-toxic concentrations on HaCaT cells was determined by phase-contrast microscopy. For this experiment, cells were treated with CA extract (2.5, 5, and 10 μg/mL) in FBS-rich media or FBS-free media, and the changes in the size of cell colonies were captured at various time points (0, 24, 48, and 72 h). In some experiments, HaCaT cells were fixed and permeabilized with absolute methanol and stained with crystal violet to clearly verify the density and the size of the colonies.

## *2.5. Determination of HaCaT Cell Monolayer Healing by Scratch Wounding Assay*

Wound healing assay of HaCaT cell monolayer was performed. Briefly, confluent HaCaT cells cultured in 24-well plate were scratched by a P200 pipette tip to make a thin wound. Scratch wounds were created in confluent monolayers using a P200 disposable micropipette tip. Cells were then treated with CA extract at all three non-toxic concentrations diluted in serum-deprived media, with or without 10 μM U0126 (CST, Boston, MA, USA) or 50 μM of LY294002 (CST, Boston, MA, USA). The rate of keratinocyte migration was monitored over time, and representative pictures were taken at different time points (0, 20, 30, 40, and 50 h). The measured wound areas were analyzed by the ImageJ program.

## *2.6. Effects of CA Extract on Increasing Number of HaCaT Cells*

We directly counted total number of CA extract-treated cells to confirm the effects of CA extract on inducing keratinocyte proliferation. Low density of cells (2.5 × 104 cells/well) was seeded in 24-well plates overnight. Then, media were changed to serum-free media before treating cells with 10 μg/mL. At each incubation time point (0, 24, and 48 h), cells were detached from the plate and counted. The differences in the number of cells between control and experimental groups were calculated.

#### *2.7. Effects of CA Extract on Stimulating Molecular Signaling Pathways*

To investigate the responsible molecular signaling in which CA extract can be activated, Western blot analysis was conducted to detect certain molecular players responsible for conveying signal transduction for cell proliferation and survival. Specifically, cells were

seeded in complete media in 3-cm dishes for 24 h. Cells were then cultured in media without serum for 24 h. For a positive control, HaCaT cells were treated with 100 ng/mL of EGF for 15 min before harvesting. For determining whether CA extract stimulates the early signaling, we treated HaCaT cells with 10 μg/mL of CA extract and harvested them at a certain time (0 min to 24 h). For evaluating the concentration-dependent effects of CA extract, we treated HaCaT cells with 3 different non-toxic concentrations of CA extract for 15 min (with or without U0126 or LY294002). For any experiment involving inhibitors, cells were pre-treated with inhibitors (10 μM of U0126 or 50 μM of LY294002) for 2 h prior to CA extract addition. For the experiment that aimed to detect the level of Mcl-1 protein, cells were treated with CA extract at 2.5, 5, 10 μg/mL, or DMSO as a vehicle control, for 24 h before harvesting cells. The effects of CA on inducing the expression and activating the phosphorylation of ERK1/2 (pERK1/2), and Akt (pAkt), cells were treated with CA extract (10 μg/mL) for 30 min. After preparing cell lysates, all samples were subjected to SDS-PAGE and Western blot analysis. Membranes were then incubated with 5% skim milk dissolved in TBST (Sigma-Aldrich, Saint Louis, MO, USA) at room temperature (RT) for 1 h. After washing trice, membranes were incubated with primary antibodies for 24 h at 4 ◦C. Primary antibodies (Cell Signaling Technology (CST), Boston, MA, USA) included (1) an anti-pErk1/2 antibody, (2) an anti-total Erk1/2 antibody, (3) an anti-pP38 antibody, (4) an anti-total p38 antibody, (5) an anti-pSAPK/JNK antibody, (6) an anti-total APK/JNK antibody, (7) an anti-pAkt antibody, (8) an anti-total Akt antibody, (9) an anti-pPRAS40 antibody, (10) an anti-pEGFR antibody, (11) an anti-total EGFR antibody, (12) an anti-Mcl-1 antibody, and a an anti-β-actin antibody. Next, membranes were incubated with appropriate secondary antibodies (Li-COR Biosciences, Lincoln, NE, USA), which were an anti-mouse IgG (conjugated with IRDye®800CW) or an anti-rabbit IgG (conjugated with IRDye®680RT) for 2 h, at RT. The Western blot signal was detected with Odyssey® CLx Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and the intensity of each immunoreactive band was analyzed by Image J.

## *2.8. Detection of Early Signaling Pathway Induced by CA Extract in Individual Cells by Immunofluorescence Study*

The phosphorylation of key kinases including ERK1/2 (pERK1/2), Akt (pAkt), EGFR (pEGFR at tyrosine 1068), and the expression of total EGFR and Mcl-1 proteins upon CA extract stimulation were determined in individual cells by immunostaining technique. Sample cover slips were prepared by seeding HaCaT cells on glass cover slips to confluence, and cells were then treated with CA extract (10 μg/mL) for 30 min. Next, fixation was performed, 15 min at RT, using 4% paraformaldehyde (Sigma-Aldrich, Saint Louis, MO, USA). After washing three times with PBS, 0.3% Triton X-100 was added to permeabilize cells for 5 min. Sample cover slips were then blocked with 1% BSA for 1 h at RT and incubated with primary antibodies overnight at 4 ◦C. Primary antibodies were an antipErk1/2 antibody, an anti-pAkt antibody, an anti-pEGFR receptor antibody, an anti-total EGFR antibody, and an anti-Mcl-1 antibody. Sample coverslips were then probed with antirabbit IgG (Alexa488-conjugated) or goat anti-rabbit IgG (Alexa594-conjugated) (Thermo Fisher Scientific, Waltham (HQ), MA, USA) for 2 h at RT. The nuclei of HaCaT cells were stained with DAPI (1 μg/mL) (Sigma-Aldrich, Saint Louis, MO, USA). Sample coverslips were washed three times with PBS, and one time with distilled water before being subjected to mounting with Fluoromount-G (SouthernBiotech, Bermingham, AL, USA) as a mounting medium. Positive fluorescent signals were detected and recorded by a fluorescence microscope, Axio Vert.A1 (Carl Zeiss Suzhou Co. Ltd., Suzhou, China) equipped with Colibri 7 illumination system (Carl Zeiss Microscopy GmbH, Gottingen, Germany) and Axiocam 506 color digital camera (Carl Zeiss Microscopy GmbH, Gottingen, Germany) using immersion objective (100×/1.3 Oil M27). Signal of the nuclei stained with DAPI was excited by the UV mode (385/30 nm), signal of Alexa488 was excited by the blue light mode (469/38 nm), and signal of Alexa594 was excited by the green mode (555/30 nm). Representative pictures were taken with the Zen 2.6 (blue edition) Software.

## *2.9. Data and Statistical Analysis*

Results were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed, followed by Tukey's post hoc multiple comparisons (SPSS Inc., Chicago, IL, USA). *p* values less than 0.05 were considered statistically significant.

#### **3. Results**

#### *3.1. Curcuma Amarissima (CA) Extract Enhances Cell Viability of HaCaT Cells*

After obtaining the extract, we performed chromatographic fingerprint analysis of the ethanolic extract from *Curcuma amarissima* (CA) by high-performance liquid chromatography (HPLC). The results (both detected at 254 and 360 nm) showed the unique characteristics of compound fingerprint in CA extract (Figure S1). Like many plants in the genus *Curcuma* such as *Curcuma longa, Curcuma amarissima* may contain some active compounds including curcumin that has been shown to have significant wound healing properties. Therefore, quantitative analysis of curcuminoids in *Curcuma amarissima* extract was examined by HPLC. As expected, the results revealed the existence of two curcuminoids in the extract which were desmethoxycurcumin (5.87 ± 0.001 μg/g extract) and curcumin (10.81 ± 0.001 μg/g extract) (Figure 1). We also found that the extract contained ferulic acid (Figure S2).

**Figure 1.** The HPLC profile of (**A**) curcuminoids (50 μg/mL) including bis-demethoxycurcumin, desmethoxycurcumin, and curcumin with their chemical structures. (**B**) The HPLC profile of *Curcuma amarissima* extract (20 mg/mL) indicating the existence of desmethoxycurcumin and curcumin.

We performed MTT cell viability assay to monitor changes in cellular metabolic activity and viability of HaCaT cells in media containing CA extract (CA extract in DMSO as a solvent from the stock solution) with the presence of 10% Fetal Bovine Serum, FBS. Cells were also treated with the FBS-rich media containing DMSO (as a vehicle control) with varied concentrations corresponding to those present in the CA extract-treated group. Results demonstrated that CA extract at concentrations lower than 40 μg/mL had no significant effect on the viability of HaCaT cells cultured in FBS-rich media (Figure 2A).

However, the extract at 80 and 160 μg/mL caused significant reduction in cell viability to approximately 30% and 10%, respectively. However, DMSO at all concentrations used did not cause any change in HaCaT cell viability (Figure 2A). We also performed similar experiments where treatment with CA extract was done in FBS-free media. Results showed that HaCaT cell viability in serum-free media was more sensitive to CA treatment. In particular, when cells were treated with CA at 20 μg/mL, cell viability was observed to be significantly reduced, and the cell viability was maximally decreased to approximately 20% in cells treated with CA at 40 μg/mL or more (Figure 2B). Interestingly, we observed that HaCaT cell metabolic activity dramatically increased in response to treatment with CA extract at 2.5, 5, and 10 μg/mL (Figure 2B). The results indicate that CA extract induced HaCaT cell metabolic activity, and this activity may be caused by an increasing number of cells upon CA extract treatment.

**Figure 2.** Cell viability HaCaT cells upon treatment with *Curcuma amarissima* (CA) extract. Viability of HaCaT cells treated with CA extract (0.625 to 160 μg/mL) or DMSO (0.000625–0.16%) for 48 h in FBS-rich media (**A**) and in FBS-free media (**B**). Data from three experiments were analyzed and presented as mean ± standard deviation (SD); \* *p* < 0.05 in comparison to the untreated group.

## *3.2. CA Extract Stimulates Colony Formation and Proliferation of HaCaT Cells*

From the previous experiment, data clearly showed that CA extract at non-toxic concentrations (2.5, 5, and 10 μg/mL) could significantly induce HaCaT cell metabolic activity in a concentration-dependent fashion, which could be seen when serum was deprived. We thought that an increase in cell viability of HaCaT cells may have resulted from the proliferative effects of CA extract. Therefore, we observed whether CA extract can accelerate the growth of the HaCaT colonies over time by a phase-contrast microscope. Our

data demonstrated that at 0 h of CA treatment or DMSO vehicle control (in both complete and serum-free media), cells were detected to be single cells with equal distribution and similar cell density on the surface of the dish (Figure 3A,B). Over the course of 72 h, we noticed that the colony size of HaCaT cells incubated with CA extract in FBS-rich media gradually increased from single cells at 0 h to 100% confluence at 72 h (Figure 3A). We also observed that the colonies of CA-extract-treated cells (at 10 μg/mL) were slightly larger than those of the untreated or DMSO-treated cells (Figure 3A). These observations were clearly verified by the results obtained from CA treatment in serum-free media, where HaCaT cells were suppressed to undergo slow cell proliferation due to the lack of growth factors in FBS that generally stimulate cell proliferation. Specifically, CA extract at 10 μg/mL showed strong effects on accelerating the growth of the HaCaT colonies at 24, 48, and 72 h of incubation (Figure 3B).

**Figure 3.** CA extract induced the growth of HaCaT colonies. HaCaT cells were left untreated or treated with CA extract at 2.5, 5, and 10 g/mL in media containing 10% FBS (**A**) or in media without the presence of FBS (**B**). The sizes of HaCaT colonies were monitored, and the pictures were captured at 0, 24, 48, and 72 h by a phase-contrast microscope (10× magnification, scale bar = 200 μm). Cells were also treated with DMSO which served as a vehicle control. The pictures were representative of three individual experiments.

We attempted to clarify whether the observed phenomenon is an effect of CA extract on stimulating cell proliferation. Therefore, we treated HaCaT cells with CA extract (5 and 10 μg/mL) in media containing FBS or FBS-free media for 2 days, and then cells were stained with crystal violet and we observed clear phenotypic changes of the HaCaT colonies (Figure 4A). Consistent with these results, our cell counting assay revealed that CA extract exhibited the ability to stimulate an increase in cell number over 48 h (Figure 4B).

**Figure 4.** (**A**) Crystal violet staining of CA-extract-treated cells (5 and 10 μg/mL) for 24 h in FBS-rich media or FBS-free media detected by a phase-contrast microscope (10× magnification, scale bar = 200 μm). (**B**) Analysis of total number of cells treated with CA extract at 5 and 10 μg/mL over the course of 24 and 48 h in FBS-free media. Data, from three experiments, present mean ± SD; \* *p* < 0.05 (in comparison to the untreated (UT) group).

## *3.3. CA Extract Induces Migration of HaCaT Cell Monolayer into The Wounded Area*

Besides proliferation, migration of human keratinocytes is an additional crucial step that contributes to efficient healing. To test that CA extract can stimulate HaCaT cells migration, we monitored the rate of migration into the wounded area of keratinocytes treated with CA extract at varied concentrations and time points. Our quantitative analysis clearly demonstrated that CA extract significantly promoted percent cell migration into the wounded areas in a concentration-dependent manner and in all time points (20, 30, 40, and 50 h), compared to those of the untreated and DMSO-treated groups (Figure 5A,B).

**Figure 5.** CA extract stimulated HaCaT cell monolayer healing. (**A**) HaCaT cell monolayer treated with CA extract at various concentrations in FBS-free media was monitored for the ability to heal over the course of 50 h by a phase-contrast microscope (10× magnification, scale bar = 200 μm). The vehicle control group was treated with DMSO. (**B**) The analysis of percent migration of CA extract-treated HaCaT cells in FBS-free media at each time point (0, 20, 30, 40, or 50 h). Regions confined by the red lines indicate the space with no cell occupation. Data present mean ± SD (\* *p* < 0.05, compared to the untreated).

## *3.4. Proliferation and Survival Signal Transductions Are Induced in HaCaT Cells upon Treatment with CA Extract*

Since we observed that CA induced HaCaT cell proliferation and migration, we hypothesized that CA extract stimulates growth and survival signaling in HaCaT cells. Therefore, we tested our hypothesis by focusing on relevant molecular signaling cascades. It is well-defined that the MAPK signaling pathway is important for activating cell proliferation and migration. In particular, this signal transduction pathway is active in response to damage of epidermis, and blockade of ERK activation suppresses keratinocyte migration. Therefore, we investigated the effects of CA extract on stimulating ERK phosphorylation by Western blot analysis. Our data (Figure 6A) revealed that when compared to the untreated group, CA extract rapidly stimulated ERK1/2 phosphorylation (pERK1/2) approximately 3 fold, starting at 5 min after treatment and reaching the maximum activation at 1 h post-treatment. Phosphorylation of ERK1/2 was determined to be approximately 10 fold (Figure 6B). The phosphorylation of ERK1/2 upon CA extract treatment exhibited the unique pattern of a bell-shaped curve where ERK1/2 phosphorylation gradually increased over time and then decreased after 1 h of CA stimulation (Figure 6A,B). Observing ERK1/2 activation led us to the belief that CA may also stimulate the survival signal transduction pathway. Therefore, we detected the activation of Akt by examining phosphorylation of serine 473 of Akt, which is normally responsible for promoting cell survival by inhibiting apoptosis and regulating cell cycle. Data from Western blot analysis showed that CA could rapidly activate Akt phosphorylation, and the phosphorylation pattern was similar to that of pERK1/2. Consistently, PRAS40 which is a downstream substrate of active Akt was also phosphorylated, and the maximal phosphorylation was detected to be between 1 to 3 h post-CA extract treatment (Figure 6A). The rapid activation of ERK1/2 and Akt by CA extract may have occurred as a result of the activation of the upstream molecules of the signaling cascade. Therefore, we detected the activation of epidermal growth factor receptor (EGFR) by targeting tyrosine 1068 (Y1068) phosphorylation and found that CA extract did not activate EGFR receptor, in comparison to the results obtained from HaCaT cells activated with EGF, where EFGR was strongly phosphorylated (Figure 6A).

**Figure 6.** CA extract strongly stimulated ERK1/2 and Akt phosphorylation. (**A**) Time-dependent detection by Western blot analysis for the expression and phosphorylation of crucial signaling molecules (ERK1/2, Akt, PRAS40, and EGFR) in HaCaT cells treated with 10 μg/mL of CA extract over 24 h. (**B**) Quantification of the phosphorylation of ERK1/2 in CA extract-incubated HaCaT cells. (**C**) Quantification of the phosphorylation of Akt in CA extract-treated HaCaT cells. Actin was used as a loading control. Data from three individual experiments present mean ± SD; *p* < 0.05.

Besides Western blot analysis, an immunofluorescence study was done to clearly verify that CA extract activates ERK1/2 and Akt in individual cells. Our studies confirmed that CA extract at 10 μg/mL could strongly induce phosphorylation of ERK1/2 (Figure 7A) and Akt (Figure 7B) in human keratinocytes, but this activation was not seen in the untreated group. Nevertheless, CA extract had no effect on inducing EGFR phosphorylation (Figure 7C) or the receptor's expression pattern (Figure 7D).

**Figure 7.** CA extract activated ERK1/2 and Akt phosphorylation in single cells. Immunofluorescence study showing the signal of phosphorylated form of ERK1/2 (green) (**A**) and Akt (green) (**B**) in HaCaT cell treated with 10 μg/mL of CA extract for 15 min. Additionally, phosphorylation status of EGFR at tyrosine 1068 (**C**) and the expression of EGFR protein (red) (**D**) were examined. The nucleus of HaCaT cells were stained with DAPI (blue). Representative pictures were taken by a fluorescent microscope at 100× magnification (scale bar = 200 μm).

Additionally, when we detected the expression of Mcl-1, an anti-apoptotic protein in which its expression is regulated by the PI3K/Akt pathways, we found that CA extract induced the expression of this protein in a concentration-dependent manner (Figure 8A). Results from immunofluorescence study verified the findings from Western blot analysis and provided more information on the intracellular location of Mcl-1, which was likely to cluster in the mitochondria where this anti-apoptotic protein normally functions (Figure 8B).

## *3.5. Suppression of ERK1/2 and Akt Activation by Specific Inhibitors Attenuates CA Extract-Induced HaCaT Cell Monolayer Wound Healing*

To further confirm the possible mechanism of action of CA extract, we designed additional experiments by using a MEK1 inhibitor (U0126) and a PI3K inhibitor (LY294002) to verify that ERK1/2 and Akt kinases are responsible molecular players in stimulating HaCaT cell proliferation and survival in response to CA extract. Data from Western blot analysis revealed that CA extract could strongly activate ERK1/2 and Akt, but not other MAPKs, including p38 and JNK (Figure 9A,B). As expected, U0126 could specifically inhibit CA extract-induced ERK1/2 phosphorylation (Figure 9C), whereas LY294002 could specifically block Akt phosphorylation (Figure 9D). Moreover, when U0126 and LY294002 were combined, the activation of ERK1/2 and Akt in HaCaT cells stimulated with CA extract was completely inhibited (Figure 9E).

**Figure 8.** Effects of CA extract on Mcl-1 expression in HaCaT cells detected by Western blot analysis (**A**); and immunofluorescence study (100× magnification, scale bar = 200 μm) (**B**). Data were obtained from three individual experiments.

**Figure 9.** U0126 and LY294002 inhibited CA extract-induced ERK1/2 and Akt phosphorylation. (**A**) Western blot detecting phosphorylated ERK1/2 and Akt in CA-extract-treated HaCaT cells. (**B**) Quantitative analysis of the fold change of ERK1/2 and Akt kinase phosphorylation. (**C**) Western blot analysis for phosphorylated ERK1/2 and Akt in CA-extract-treated HaCaT cells with U0126. (**D**) Western blot detecting phosphorylated ERK1/2 and Akt in CA-extract-treated HaCaT cells with LY294002. (**E**) Western blot detecting phosphorylated ERK1/2 and Akt in CA-extract-treated HaCaT cells with U0126 plus LY294002. Data from three individual experiments present mean ± SD; \* *p* < 0.05.

We next performed a functional test to evaluate whether suppression of growth and survival signaling by U0126 and LY294002 can attenuate healing-enhancing effects of CA extract. Scratch wound healing assay revealed that the effects of CA extract on stimulating HaCaT cell migration were remarkably blocked when U0126 alone, LY294002 alone, or the combination of these two inhibitors were present (Figure 10).

**Figure 10.** U0126 and LY294002S strongly inhibited the healing-promoting activities of CA extract. Phase-contrast microscopy at 10× magnification (scale bar = 200 μm) was performed to monitor the rate of migration into the wounded site of HaCaT cells treated with CA extract in media containing U0126, LY294002, or the combination of U0126 and LY294002 over the course of 50 h. Regions confined by the red lines indicate the space with no cell occupation. Data are from 3 different experiments.

## **4. Discussion**

Here we studied *Curcuma amarissima* (CA) ethanolic extract by focusing on its wound healing activities by using HaCaT cell monolayer as a study model, since this cell type is derived from human skin. In particular, we attempted to investigate it to gain concrete evidence for its molecular mechanisms. We first performed a cell viability test by MTT assay to select a range of concentrations that were not toxic to a human epithelial keratinocytes (HaCaT) cell line. Interestingly, results from this experiment where we treated cells with CA extract in FBS-free media clearly showed that CA extract may be able to promote the viability of HaCaT cells. However, this positive effect of CA extract on cell viability was not observed when the treatment system was in complete media where it contained 10% fetal bovine serum (FBS). This phenomenon may be caused by high amount of growth factors in culture media that helps maintain a degree of cell viability so high that it conceals the effects of CA extract. Serum-free condition lacks growth factors, thus allowing its effects on cell viability enhancement to stand out. Data from MTT assay suggest the possibility that CA extract may be able to induce specific cellular events that increase the metabolic activity of the cell, or it may have resulted from increased cell proliferation. As expected, our results from colony-forming assay demonstrated that CA extract could dramatically stimulate the growth of HaCaT colony, suggesting that CA extract may contain potential active constituents that can regulate cell proliferation. Data from crystal violet staining and cell counting clearly confirmed that CA extract promotes cell proliferation to

increase a significant number of keratinocytes over time. The possible mechanisms of CA may be similar to those of several growth factors that are critical factors to stimulate the proliferation of human keratinocytes and eventually contribute to efficient healing [35,36]. We then performed a functional test by using a scratch wound model and discovered that CA extract induced a drastic increase in the rate of cell monolayer wound healing. According to our findings, it provides a close correlation between the closure of scratch wound and an increase in HaCaT cell number, suggesting that a contribution to wound healing of CA extract mainly derives from increased cell proliferation.

On the basis that the mitogen-activated protein kinase (MAPK) signaling normally plays crucial roles in cell migration and proliferation regulation [11,12,37,38], we therefore examined whether CA extract contributes to HaCaT cell proliferation through activation of this signal transduction pathway. Interestingly, we disclosed that CA extract stimulated ERK1/2 phosphorylation, and when the phosphorylation of this kinase was inhibited with U0126, the migration rate of HaCaT monolayer was dramatically suppressed. These data strongly suggest that CA extract may possess wound-repairing effects by enhancing cell proliferation and migration, at least in part through ERK1/2 activation. To support our statement about the involvement of ERK1/2 in keratinocyte, many studies previously demonstrated that ERK phosphorylation promoted the migration of this cell type [39,40]. Although ERK1/2 was activated by CA extract, the phosphorylation of p38 kinase and JNK kinase was not affected. These results suggest that not all MAPK signals are involved in CA extract-induced HaCaT cell monolayer healing and indicate that ERK1/2 is the primary signal pathway activated by CA extract. Besides ERK1/2, the phosphatidylinositol 3-kinase (PI3K/Akt) signaling transduction pathway is well characterized to be responsible for migration of many cell types [41–43]. Therefore, we determined the effect of CA extract on Akt phosphorylation status in human keratinocytes. Like ERK1/2, we observed a strong increase in phosphorylated Akt level in HaCaT cells incubated with CA extract. Consistent with results from Western blot, an increase in phosphorylation of Akt in individual cells was also confirmed by immunofluorescence study. Additionally, the involvement of this signaling pathway in HaCaT cells induced by CA extract was verified by wound closure assay where a PI3K inhibitor, LY294002, was present. When both U0126 and LY294002 were combined, no monolayer wound healing was evident, indicating that the wound healing activities of CA extract in human keratinocytes are regulated through the activation of the MAPK and PI3K/Akt signal transduction pathways. In addition to the effects on cell proliferation, CA extract has an influence on cell survival by increasing the production of Mcl-1, which is a key anti-apoptotic protein. Mcl-1 production is under the influence of the PI3K/Akt pathway [44]. Moreover, Mcl-1 protein can be upregulated by some cytokines such as IL-6 [45] and growth factors such as EGF, which conveys the cellular signal to control Mcl-1 translation via the MAPK pathway [46,47]. We observed that Mcl-1 was upregulated in individual cells treated with CA extract, and the localization pattern of Mcl-1 suggests that this protein resides in the mitochondria of the cells. Induction of strong expression of Mcl-1 anti-apoptotic protein suggests that CA extract helps increase cell survival of keratinocytes. However, transactivation of epidermal growth factor (EGFR) was not affected by CA extract, indicating that CA extract does not stimulate ERK1/2 and Akt phosphorylation via the EGFR signaling cascade. The activity of CA extract in inducing an increase in cell proliferation and cell survival may be caused by its curcuminoids, including curcumin and demethoxycurcumin. It is evident that curcumin (predominantly found in *Curcuma longa*) stimulates fibroblast proliferation, enhances the formation of granulation tissue, and promotes the contraction and epithelialization of wounds [48,49]. Moreover, the finding that CA extract contains ferulic acid strengthens the possibility to utilize this plant for wound healing since ferulic acid has been documented to be beneficial for skin repair, and this compound has recently been incorporated into the formulation of wound healing [50]. Therefore, our study suggests that CA extract possesses its wound-healing effects, at least in part, through the action of curcuminoids and ferulic acid.

However, our study, using HaCaT cells as a model, has some limitations that require future investigations by other models since HaCaT contains certain genetic alterations that may cause the cell to respond to certain stimuli in a different manner compared to human keratinocyte in actual physiologic conditions. For this reason, human primary keratinocyte should be used to verify the effects of CA extract and its active compound on proliferation and survival. Nevertheless, the use of normal primary human keratinocytes can be limited by the complexities involved in their recovery from donors, cultivation, and limited number of passages. Moreover, primary keratinocytes in vitro have little in common with ordinary naive keratinocytes in vivo under homeostatic conditions. Considering that wounding is a stress that requires several different complex series of communicating processes to respond to various stimuli and to heal the wound [51,52], it needs specific models that can be able to address the promoting effects of a certain agent on wound healing. To overcome these limitations, animal models and clinical trials in humans should be performed in the future to achieve our complete understanding of physiologic and pathologic processes as well as translational efficiency. Altogether, our study revealed that CA extract can potentially trigger and strengthen the molecular healing cascades in human keratinocytes. These events are of great interest for the development of CA as an alternative option for wound healing occurring in some diseases, such as complicated diabetes mellitus, which is less sensitive to treatment by growth factors [53].

## **5. Conclusions**

Our present study provides evidence that *Curcuma amarissima* (CA) possesses pharmacological properties in activating human keratinocyte proliferation and survival through its ability to strongly stimulate the phosphorylation of ERK1/2 and Akt kinases. These properties are generally required for promoting wound healing. We demonstrate that this plant contains curcumin, demethoxycurcumin, and ferulic acid, which are potential active compounds reported to be able to promote skin regeneration. Our discovery provides information beneficial for potential uses of CA in regenerative medicine. When mechanisms of action of CA in wound healing is completely defined, and further investigation in animal and human models are done, this plant may be an excellent candidate for the development of a wound-healing agent.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/biology10040289/s1, Figure S1: Chromatographic fingerprint analysis of the ethanolic extract from *Curcuma amarissima* (CA) by high-performance liquid chromatography (HPLC), Figure S2: The HPLC profile of standard ferulic acid and the extract from *Curcuma amarissima*.

**Author Contributions:** Conceptualization, investigation, funding acquisition, writing—original draft preparation, review and editing, W.N.; investigation, resources, S.P.; investigation, J.R., P.P. and J.K.; resources, data curation, formal analysis, N.W.; resources, S.O.; writing—original draft preparation, S.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research project was funded by the Faculty of Medicine, Chiang Mai University (Grant number 008-2564). Partial support was provided by the Research Center of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Chiang Mai University, Thailand.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in this article.

**Acknowledgments:** The authors would like to thank Sathit Monkaew for facilitating laboratory work in Molecular Pharmacology Laboratory Unit, Department of Pharmacology, Faculty of Medicine, Chiang Mai University.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Review* **A Descriptive Overview of the Medical Uses Given to** *Mentha* **Aromatic Herbs throughout History**

## **Henrique Silva 1,2**


Received: 20 November 2020; Accepted: 8 December 2020; Published: 21 December 2020

**Simple Summary:** Mints are aromatic herbs with a millenary tradition of use for diverse medicinal purposes since ancient civilizations, and they are still presently used in different clinical practices. Mints have been used since ancient Babylon, but it was in Classical Antiquity that their medical uses flourished, with major contributions from Pliny the Elder. In the Middle Ages, the increased knowledge surrounding mints came from Byzantine physicians, while, in the Modern Age, technological developments allowed the production of mint-based products, such as extracts and essential oils, which have become part of elaborate galenic formulas employed by an increasing number of physicians, and have also stimulated both scientific and artistic interests alike. In present-day medicine, several mints and mint-based products are being researched as potential therapeutic alternatives for many diseases, while also being vastly employed in food and cosmetic industries.

**Abstract:** Mints have been among the most widely used herbs for medicinal purposes since ancient civilizations. They are still presently used for numerous purposes, including non-medicinal, which makes them economically relevant herbs. Information regarding the medical and scientific uses given to mints throughout history are vastly scattered and/or incomplete. The aim of this paper is to provide an extensive descriptive overview of the medical uses given to these herbs, highlighting both the authors in medical culture responsible for their dissemination, as well as their major galenic formulations. Databases on medical science, reference textbooks on medical history, botanics (aromatic herbs), and pharmacognosy were consulted. The use of mints remotes to Classical Antiquity, with major contributions from Pliny the Elder. In the Middle Ages, the increased knowledge surrounding mints came from Byzantine physicians, while, in the Modern Age, technological developments allowed the production of mint-based products which have become part of elaborate galenic formulas employed by an increasing number of physicians, as well as have also stimulated both scientific and artistic interests alike. In present-day medicine, several mints and mint-based products are being researched as potential therapeutic alternatives for many diseases, while also being vastly employed in food and cosmetic industries.

**Keywords:** *Mentha* L. genus; aromatic herb; history; pharmacognosy; therapeutics

## **1. Introduction**

Medicinal plants have been at the forefront of most medical therapies for many centuries and have been used in many societies worldwide. A large number of medical treatises have established several plant-based medicinal products as the most important components of the available therapeutic arsenal. Members of aromatic mint herbs have been among the most widely used plants throughout history for a multitude of medicinal purposes. This paper provides a thorough review of the medical uses given to mint herbs in western medicine from ancient civilizations to modern day medicine and provides recent scientific data to support the reasons behind the longevity of their use. Mint herbs consist of perennial aromatic members of the Lamiaceae family, the *Mentha* L. genus, classified into 42 species, 15 hybrids, and hundreds of subspecies, varieties and cultivars. Among the best-known mint species, we find watermint (*Mentha aquatic* L.), spearmint (*Mentha spicata* L.), wildmint (*Mentha arvensis* L.), horsemint (*Mentha longifolia* (L.) L.), pennyroyal (*Mentha pulegium* L.), and peppermint (*Mentha x piperita* L.), the last of which is a natural sterile hybrid of watermint and spearmint [1,2]. While most mints have been known to man since ancient times, it was not until 1696 that peppermint was described by English Botanist John Ray (1627–1705) in his *Synopsis Methodica Stirpium Britannicarum* [3].

Most mints invariably grow in moist environments near ponds, lakes, and rivers and require partial shade, although some species grow well in warm environments [4]. Interestingly, one of the first written references to mints, the poem *Theriaca* by Nicander of Colophon (197 BC–170 BC), Greek poet and physician, alludes to their habitat and describes them as 'delighting in gleaming rivers' [5]. Sterile mint hybrids, such as peppermint, only display vegetative reproduction, while the majority display sexual reproduction and contain both male and female organs [4]. It is likely that mints' sexual dimorphism justified the comparison made between pennyroyal and the fictitious magical plant *Pantagruelion*, as featured in François Rabelais' *Gargantua and Pantagruel* [6]. The first attempt to classify them from a botanical perspective came from Pedanius Dioscorides (c. 40–90), the renowned Greek physician and herbalist, whose teachings became a reference from classical antiquity well into the Renaissance period. A more formal classification was proposed several centuries later by Carl Nilsson Linnæus (1707–1778) in his *Species Plantarum* (The Species of Plants) published in 1753 [7], which has been further perfected in recent years based on the genetic relations between these species [8]. To contribute to the complexity of the mint taxonomy, all the different species are referred to in many medical treatises as simply mint, which prevents a more precise appreciation of the uses given to each particular species. From a pharmacognosy perspective, the therapeutic value of herbs lies in aerial parts, which can be dried and ground into powder or used fresh. If used fresh, aerial parts can be subjected to water or steam distillation to produce an essential oil that is extracted [4].

The name of the mints' genus, *Mentha*, was probably coined by the Greek philosopher Theophrastus (371 BC–287 BC), who described several species from botanical and agricultural standpoints in his *Enquiry into Plants* [9]. The name *Mentha* probably alludes to Minthe, a figure of Greek mythology, although different ancient texts describe different versions of her myth. According to the myth told in the didactic epic *Halieutika* (On Fishing) by Oppianus of Corycus (fl. 2nd century), the Greek-Roman poet contemporary of emperor Marcus Aurelius and of Galen, Minthe was a nymph of the river Cocythus and wife of Aidoneus. But when Aidoneus raped Persephone, of the Aetnaean hill, Minthe, overtaken by jealousy, claimed to be nobler of form and more excellent in beauty than Persephone, and, for this, she was trampled upon and destroyed by Demeter, Persephone's mother, and from the earth sprang the herb that carries her name [10]. In the versions told by Strabo (c. 64 BC–24), the Roman geographer, and by the Roman poet Ovid (43 BC–17), Minthe was beloved by Hades, and it was Persephone, the god's jealous wife that destroyed the nymph [11,12].

The oldest written records of mint herbs are attributed to King Hammurabi of ancient Babylon (1800 BC), who prescribed them for medicinal purposes, namely gastrointestinal [13]. Mints are also referred to in the Ebers papyrus and on the walls of the temple of Horus in Edfu [14,15]. A great deal of knowledge on the mints' usage was acquired during classical antiquity by Greek and Roman philosophers, especially Gaius Plinius Secundus (Pliny the Elder, 23–79), who described most of the medical uses that would be given to these herbs throughout the history of western medical practice in his *Naturalis Historia* (Natural History) [16], an important work that would be the reference for several posterior medical texts. Knowledge of these herbs spread throughout the Middle East, partly due to the conquests of Alexander the Great (356 BC–333 BC) and later to the Crusades [17]. Given their many uses, mints became valuable herbs and were probably used as currency by the Pharisees, as can be inferred from two passages from the Bible: '( ... ) Woe to you, scribes and Pharisees, hypocrites! For you tithe mint, dill, and cumin, and have left undone the weightier matters of the Law: justice,

mercy, and faith ( ... )' (Matthew 23:23) and '( ... ) But woe to you Pharisees! For you tithe mint and rue and every herb, but you bypass justice and the love of God ( ... )' (Luke 11:42) [18]. Several references are also found in the Babylonian Talmud, a compilation of Jewish teachings on several knowledge areas that contains many pieces of practical advice of a medical nature [19]. In the Middle Ages, medical and scientific learning shifted to Constantinople, the capital of the Byzantine Empire, with the majority of important references to mints coming from Islamic physicians, such as Ibn Sinna (Avicenna, 980–1037), author of *Canon of Medicine* [20]. In Christian Europe, medical knowledge was kept mostly in universities and monasteries by physician-monks, where the teachings of classical philosophers were still followed [17]. Charlemagne (c. 747–814), leader of the Holy Roman Empire and founder of the first medical school in Salerno in the 9th century, ordered medicinal plants, in which mints were already included, to be cultivated inside state-owned land, as stated in his *Capitulare de villis* [21]. The Renaissance period allowed a change to medico-scientific paradigms and the progressive abandonment of empiric frameworks as regards medical plants to embrace more experimentally ascertained facts. This was also a period of artistic flourishment, when the printing press was invented which facilitated the dissemination of literary works [22]. Incidentally, one of the first *incunabula* (i.e., earliest printed books, between 1450 and 1500), the famous and obscure *Hypnerotomachia Poliphili* (Poliphilo's Strife of Love in a Dream) attributed to the Italian Dominican priest Francesco Colonna (c. 1433–1527), features several mint species among the numerous botanical varieties described in the story's oneiric landscapes [23]. The 16th century was marked by the rupture with the orthodox medical doctrine based on the theory of humors, and a new doctrine emerged called *nova medicina*, which was rooted in alchemy, astrology, magic, and natural philosophy. The advent of iatrochemistry made possible for new plant extracts and essential oils to be prepared, some from the recently discovered New World, which increased the number and variety of available medicine products [24]. The development of pharmaceutical technology from the 19th century onward has largely contributed to the diversification of mint-based products [25]. Peppermint essential oil, for example, became such a popular medicine in the 19th century that it found its way into the post-impressionistic painting *Still Life with Peppermint Bottle* by French painter Paul Cézanne [26]. Apart from being a source of inspiration to artistic expression, mints also aroused curiosity in the emerging scientific fields. For example, one mint species was used by Joseph Priestley (1733–1804) in his pioneering studies on photosynthesis [27], while Charles Darwin (1809–1882) used peppermint essential oil for his studies on botanics [28]. Mint active compounds concentrate mainly in the leaves of herbs, which can be used by dry grounding them into powder or fresh and being subjected to solvent extraction or steam distillation, the latter of which produces mint essential oils. These oils contain a variety of volatile compounds and are widely used in food, cosmetic, and perfume industries, largely for their flavoring, fragrance, and preservative properties, which make them very economically valuable. The cultivation and processing of mint herbs is a very large business and one that is responsible for a considerable part of the economy of many countries [4].

Most of the data regarding the medical and scientific uses given to mints throughout history are vastly scattered and/or incomplete. The aim of this paper was to provide an extensive descriptive overview of the medical uses given to these herbs, highlighting both the authors in medical culture responsible for their dissemination, as well as their major galenic formulations. The next section references these authors and respective written sources in chronological order (Table 1), together with a concise and up-to-date appreciation of these medical uses in light of ongoing scientific research (Table 2). A comprehensive review of the composition of the mint herbs and a correlation with their ancient/actual medical properties is beyond the scope of this paper. Databases on medical science (Pubmed, Springer Link, Google Scholar, Internet Archive, U.S. National Library of Medicine) were searched using combinations of the following keywords: "mentha", "mint", and its variations to include the several species' names, "medical" and "history". Reference textbooks on medical history, botanics (aromatic herbs), and pharmacognosy were also consulted. The literary works of the most relevant authors were accessed and analyzed as regarding the previous keywords. From the numerous

medical/science research papers accessed, the most relevant ones for the historical discussion comprised in this paper were selected. Given that some authors of the many analyzed written sources do not clearly identify the mint species used, the term "mint" will be used throughout this paper with the meaning "unknown species". Nevertheless, whenever possible, the names of the concerned species will be provided.

## **2. Medical Applications**

## *2.1. For Scenting and Perfuming*

Probably the most recognizable quality of mints is their appealing fragrant scent, which is why they have been used since early times in embalming funerary rites. The embalming practice continued until the Middle Ages in Christian Europe but was reserved to the elite. In fact, mint was recently found among the embalmed remains of King Richard the Lionheart (1157–1199) [29] and of John Plantagenet of Lancaster, first Duke of Bedford (1389–1435) [30]. The purpose of using aromatic herbs, such as mints, in these practices was not only to create conditions for long-term body conservation but to also confer it a good odor, similarly to the body of Christ (i.e., the sanctity odor) [29].

Mints were also employed to manufacture perfumes and to mask distasteful substances in medicines. For hygienic purposes, Paulus Aegineta (625–690), a Byzantine Greek physician and compiler of Greek–Roman medicine, best known for his encyclopedia *Medical Compendium*, mentioned the use of ground pennyroyal in a mint decoction, among several ingredients, to mask the displeasing taste of medicinal draughts [31]. Peppermint essential oil is featured in several formulations containing either laudanum (i.e., an opium tincture) or morphine (i.e., the main component of opium). Albert Ethelbert Ebert (1840–1906), a prominent pharmacist of the late 19th century, featured peppermint essence in *The Standard Formulary* as an ingredient of a complex formulation in which morphine was included [32]. Similar uses were later carried out to mask the odor of fish oil [33], cod liver oil [34,35], and laudanum. An example of the last case was the inclusion in the infamous nostrum *Shiloh's consumption cure* to be used for colds, coughs, bronchitis, asthma, and irritation of the throat [36]. Unquestionably, the most explored mint compound for scenting and perfuming is menthol. This is easily explained by its capacity to induce the perception of coolness, which can strongly affect cognition, emotion and behavior [37]. It is legitimate to interpret that, given the inflammatory component of many diseases, manifested topically with redness and warmth, a "cooling" or "fresh" sensation would be logically sought for to calm the effect. Furthermore, the known local menthol anesthetic seems to have been also an important reason to include these herbs in medical recipes.

## *2.2. For Gastrointestinal Disorders*

The medical use of mints for gastrointestinal affections is present in the works of most philosophers and physicians who came across them, from classical antiquity to present-day medicine. Mints have been consistently referred to as possessing anti-emetic and carminative properties, and being used to facilitate digestion and assist in the treatment of gastrointestinal disorders. Pliny was among the first to document these properties and, quoting Democritus, wrote that mints were a suitable treatment for vomiting [17]. Aëtios of Amida (502–575), a Byzantine physician and writer, credits Kyrillos, the archbishop, with a recipe of a digestive composed of a mixture of plants, including pennyroyal macerated in vinegar [38]. Paulus Aegineta left many references to the digestive benefits of mints. For stomach problems, he advised drinking 'a draught of juice of endive sprinkled with mint' or 'a mixture of juice of kernel, pomegranate and mint' [31]. Centuries later, Hildegard von Bingen (1098–1179), a German Benedictine abbess and polymath, left many texts about the use of medical plants in her *Physica*. To adding to her theoretical knowledge, she gained practical experience in the use of the plants that she grew in her monastery's garden. To ease digestion, she advised consuming watermint and spearmint for they 'warm the stomach' [39]. Trota, a prominent figure of the medical school of Salerno in the 12th century, was another female healer and medical writer to have contributed

to extend knowledge on female healthcare. In her *Trotula*, a famous compendium of texts on women's medicine, she detailed several recipes involving mints [40]. For constipation one recipe consisted on cooking mint in honey and water, a beverage that was to be drunk by after bloodletting. Another recipe consisted in a mixture of wild celery, mints, cowbane, mastic, cloves, watercress, madder root, sugar, castoreum, zedoary, and gladden. This mixture was to be made into a very fine powder and be given with wine to relieve abdominal distension caused by trapped gas during pregnancy and the consequent risk of miscarriage. Another mention in the *Trotula, Potio Sancti Pauli* (Saint Paul's Potion), a potion including horsemint, was made to diverse gastric ailments. With the extended use of mints throughout Europe, several recipes began to appear among the royal apothecaries' arsenals. For example, a recipe for a plaster made of wheat bread, cumin, wormwood, mint, and rose leaves figures in the long list of medicines to Katherine Neville, Duchess of Norfolk by John Clerk (15th century), king's apothecary to Edward IV, which represents both the fear of epidemics and the need to correct the excesses of the aristocratic lifestyle [41]. Mint medicines also appear in the apothecary's list for Anne of Bohemia, first wife of King Richard II. Although the use given to mints in these lists is not specified, the intended treatment of gastrointestinal disorders is probable [42].

Theophrastus von Hohenheim (Paracelsus, 1493–1541), the German-Swiss physician and alchemist, is credited with bridging medical practice and chemistry fundaments. For difficult digestions, Paracelsus suggested consuming a mixture of mint water and syrup of gillyglower [43]. Garcia de Orta (1501–1568), a Portuguese physician and herbalist who pioneered tropical medicine while working in the empire's eastern colonies, wrote an important medical treatise on pharmacognosy entitled *Colóquios dos simples e drogas da India* (Conversations on the simples, drugs and medicinal substances of India). In this work, Garcia de Orta advised mixing mint water and mastic powder for 'vomiting and weakness of the stomach' [44], which would be later referenced by the also celebrated Portuguese physician and naturalist Cristóvão da Costa (1515–1594) in his *Tractado de las drogas y medicinas de la Indias Orientales* (Treatise of the drugs and medicines of the East Indies), based on Garcia de Orta's own work [45]. Prosper Alpinus (1553–1617), a Venetian physician and botanist, referenced a treatment for bile vomiting consisting in the administration of 'sub-acid fruits, juice of wormwood or of mint in wine' [31]. Herman Boerhaave (1668–1738), the prominent Dutch physician, was also interested in mints for their usefulness in digestive ailments. In his *Materia medica* (On Medical Material), he wrote about watermint and peppermint as anti-emetics [46]. Thomas Sydenham (1624–1689), the prominent English physician, used mint water as a solvent for several anti-emetic agents [47].

The carminative (i.e., reliever of flatulence) and spasmolytic properties of mints were also much appreciated. Paulus Aegineta provided a recipe for constipation in children, for which he advised rubbing the abdomen with a mixture of mint and honey [31]. Robert Burton (Democritus Junior, 1577–1640), Oxford scholar and author of the celebrated *The anatomy of melancholy*, described watermint as an effective carminative [48]. The prominent English physician and founding member of the Royal Society, Thomas Willis (1621–1675), wrote recipes using mint as an anti-emetic and antispasmodic in his *Dr. Willis's Receipts for the Cure of All Distempers* [49], while Sydenham used watermint to relieve the so-called 'iliac passion' (i.e., intestinal volvulus) [47]. Reverend Joseph Townsend (1739–1816), English physician and vicar, listed peppermint as an effective spasmolytic in his *Elements of Therapeutics: or, a Guide to Health; being Cautions and Directions in the Treatment of Diseases* [50]. In the late 19th century, a nursing book by Isabel Robb (1860–1910), a nurse theorist, mentions the internal application of peppermint water to ameliorate colic in infants [51].

The gastrointestinal usefulness attributed to mints has been uncovered in recent scientific publications. Mint oils possess substances that increase gastric emptying to improve digestion [52] and relax the bowel [53,54]. Its antiemetic properties are of considerable magnitude as they can reduce postoperative, chemotherapy-induced nausea and vomiting [55,56]. Their spasmolytic properties are known to relieve symptoms of irritable bowel syndrome [57–59]. They are safe to be used in endoscopic procedures, as a suitable alternative to conventional spasmolytics, and increase the diagnostic sensitivity of the procedure itself [60].

References to intestinal infections, especially of parasitic etiology, can be found in numerous medical treatises, and mints appear as useful therapeutic herbs. Pliny advised taking dry powdered mint in water to expel intestinal worms [17], a reference which was imported into one of the most read works in the Middle Ages, *Macer Floridus*, a hexametric poem on medicine and botanics, presumably authored by Odo de Magdunensis (1070?–1112?) [61,62]. Dioscorides recognized the ability of spearmint to kill roundworms [63]. The Babylonian Talmud advises eating pennyroyal with seven white dates to kill intestinal worms caused by eating raw meat [64]. Paulus Aegineta advised the external application of 'mint or gith in rose-oil' to the navel for worm infection and to also give 'sebesten plums and mint' to aid low fevers brought up by worm infections [31]. Alexander of Tralles (525–605), a prominent physician from the early Byzantine period and an apparently accomplished helminthologist, also listed watermint among several effective antihelminths [65]. Centuries later, the *Regimen Sanitatis Salernitanum* (The Salernitan Rule of Health), a medieval didactic poem from the Salerno medical school, briefly refers to mints as antihelmintic herbs [66].

The use of mints to treat cholera is described in several medical texts, with the first being once again attributed to Pliny [17]. For cholera, Paulus Aegineta advised drinking 'juice of pomegranate sprinkled with mint' [31]. Sydenham used mint as a component of a nourishing drink for cholera patients [47]. Physician William Currie (1754–1828) who, in the 19th century described the disease in his work *Of the Cholera*, also used mints as therapeutic herbs. He also recommended using peppermint water as a palliative treatment for digestive and spasmodic problems and mentioned the activity of mints' oils against a multitude of pathogenic microorganisms, including *Vibrio cholera*, the causative agent of cholera [67]. Recent studies have shown that the essential oils of several mint species have shown in vitro activity against *Echinococcus*, *Trichostrongylidae*, and roundworm parasites [68–70], as well as in vivo activity against *Giardia* and *Entamoeba* species, to name just a few [71].

## *2.3. For Reproductive Purposes*

Mints have had vast reproduction applications for not only female hygiene and contraceptive purposes but also for their abortifacient properties. The first records on female health date back to ancient Greece, where contraception was almost limited to the so-called "barrier methods". Mints were added to pessaries, especially balls of wool, inserted in the female reproductive tract, probably for the cooling/calming sensation they evoked [31,72]. As for hygienic measures, mints were used for washing female genitalia after coition. Soranus of Ephesus (98–138), a Greek physician best known for his gynecological and obstetric work *Gynecology*, prescribed pennyroyal for sitz baths [73]. Similarly, John of Gaddesden (c. 1280–1361), a famous practitioner in 14th century England and the author of *Rosa Medicinae*, advised using watermint for the hygienic washing of female genitalia [74].

The best-known use of mint herbs for female health undoubtedly comes from their recognized effect of increasing uterine contractile strength (i.e., oxytocic). They were used as emmenagogues, especially in the clinical context of dysmenorrhea. Galen (129–c. 210), in his *On the Mixtures and Powers of Simple Drugs*, suggested using watermint and pennyroyal as complementary treatments to bloodletting, a popular treatment for plethoric ailments, and mentioned that they 'bring on an abundant menstrual flow' [75]. Centuries later, Luis de Mercado (1525–1611), physician to Phillip II of Spain, also listed pennyroyal as an herb to relieve menstrual retention [76]. The most popular mint for its abortifacient properties was pennyroyal, which has been featured in numeral medical treatises, as well in mythological texts. In the Homeric *Hymn to Demeter*, there is a reference to a recipe for *kykeon*, a porridge containing honey-sweet wine, barley, water, and pennyroyal that was drunk by the goddess Demeter herself. This reference is probably a connotation between pennyroyal and female health and sexuality, but whether it precedes the medical use of the herb or was responsible for it is still a matter of discussion [77]. Pliny described pennyroyal and other mints as being capable of increasing uterine contractions and to 'help expel the placenta and a dead fetus' [17], a belief later repeated by Dioscorides [78]. Quintus Serenus Sammonicus (d. 212), the Roman author of *Liber Medicinalis*, a didactic poem on medicine, advised administering pennyroyal in tepid water to induce

abortion in women with pregnancies less than 1 month old and whose 'embryo was weak' [79], a practice also mentioned in Odo de Magnudensis' work [61]. Pennyroyal is referenced in *Acharnians*, *Peace*, and *Lysistrata*, three plays by the celebrated dramaturge Aristophanes (c. 450 BC–c. 388 BC). In *Peace*, pennyroyal is suggested to have contraceptive properties [79], and in *Acharnians* and *Lysistrata*, the blossoming of pennyroyal is mentioned as a metaphor for pubic hair [80–82]. Trota advised the anointment of pennyroyal oil on a cloth to be placed in the vagina to induce receding of prolapsed uterus caused by traumatic coitus due to the 'excessive size of the male member'. Pennyroyal also appears in Trota's work as an herb to be applied in a bath, among several others, which 'flegmatic and emaciated' women who could not conceive should take to increase the chances of fertilization. Trota also described several recipes for stimulating menses. In a simple recipe, honey was to be cooked in mint water; mint also appeared in the herb mixture *Tyriaca diathessaron*. A mixture of mint, pennyroyal, rue, red cabbage, leek, and salt would be cooked together in a plain pot, and be drunk in the bath. In addition, she advised the fumigation of cumin, fennel, dill, calamint, mint, and nettle, either individually or mixed. Furthermore, a mixture of ground castoreum, white pepper, costmary, mint, and wild celery in white or sweet wine to be drunk in the evening also appeared. To help women having difficulties to give birth, mint and wormwood powder would also be given, besides mint and other odoriferous herbs being applied to the cervix during the prepartum period. The beverage *Tyriaca magna Galeni*, taken with mint water, was also given to stimulate menses or parturition [40,83]. Centuries later, Hildegard von Bingen wrote in her *Physica* that eating pennyroyal 'acts to expel the afterbirth' that remained inside the uterus after delivering the fetus [39]. In the 17th century, James Primerose (d. 1659), English physician and notable opponent of William Harvey's Theory of Circulation, who dedicated a large portion of his career to gynecology and obstetrics, had also mentioned pennyroyal to be an abortifacient [76]. Nicholas Culpepper (1616–1654), physician, astrologer, and herbalist warned in his *A Directory for Midwives* 'give not [pennyroyal] to any that is with Child, lest you turn Murderess' [84]. The almanacs of two female authors of the 17th century England, Sarah Jinner of London (fl. 1658–1664) and Mary Holden of Sudbury (c. 1648–1726), transmitted classically-based medical cures for women, challenging the existing medical hierarchy [85]. Sarah Jinner, a student of astrology and a contemporary of Primerose, wrote the first series of almanacs that focused on female health and destined to transmit updated gynecological knowledge from the medical elite to common literate persons and rural physicians [86]. These almanacs contained herbal useful remedies for managing and treating gynecological disorders, among which a list of 'pills to expel a dead child' is included, several containing pennyroyal as an ingredient, which she also noted as being able to regulate menses [87]. Thus, pennyroyal became a household herb for inducing abortion and is reported to have been taken with gin during menstruation as recently as the 1950s [88]. The strong effect of pennyroyal is attributed to the oxytocic terpene pulegone, which has, nonetheless, an important hepatotoxic profile [89].

Regarding male health and fertility, several considerations regarding the possible effect of mints on sperm generation existed. Pliny wrote that adding watermint to milk prevented it from curdling and thickening or turning sour. Assuming that a similar effect would occur with semen, Pliny wrote that ingesting watermint could change the consistency of semen and, therefore, affect fertility [17]. Dioscorides also believed that mints, if taken in large quantities, changed sperm quality and affected erection [31]. Aëtius of Antioch (d. 367), Syrian bishop and physician, wrote that watermint consumption 'generates much semen, but of a feeble nature' [31]. Avicenna wrote about watermint as a spermicide, used as a female suppository before coition [74]. In contrast, Trota wrote that men should apply pennyroyal (presumably externally), among others, to increase fertility [40]. Thus, pennyroyal was a valued herb through the ages for female health, but, in the 20th century, its use has steadily declined mainly due to the creation of abortifacient drugs and to the increase in the awareness against this herb's toxicity profile, with only scarce records of its use being found today.

## *2.4. For Modulating Libido*

Reports of mints' effects on libido are diverse and often controversial. This may be partly explained by the differences in used species and also in the quality and quantity consumed. An obvious consensus was reached by classical philosophers that watermint was aphrodisiac. This notion was shared by Aristotle, Dioscorides, Galen, and, centuries later, by the Persian polymath Muhammad ibn Zakariya al-Razi (Rhases, 854–925) [31] and Nicholas Culpeper in his *The English Physician Enlarged* [90]. Contrary records exist but, strangely enough, appeared only in the Middle Ages. Avicenna recommended taking watermint as a treatment to reduce sexual desire [91], and a similar description is present in Hildegard von Bingen's *Physica* [39]. Similarly, Trota wrote that mints could be given to placate the repressed sexual desire [40]. Later, the Italian physician Paolo Zacchia (1584–1659), considered one of the fathers of forensic medicine, believed, in accordance with Hippocrates' beliefs, that eating watermint was responsible for erectile dysfunction [92]. However, the mints' effect on libido have generated only a modest interest thereafter. Recent animal and clinical studies agree that ingestion of mints have an anti-androgenic effect in males, which might affect erection and may also decrease libido [93].

## *2.5. For Repelling Insects and for Animal Bites*

Providing protection against the different elements of the natural world, including animals, was always a concern for human beings, and mints have been employed as insect repellants and adjuncts in animal bite treatments since ancient times. By quoting Xenocrates (396 BC–314 BC), Pliny advised smelling pennyroyal and placing it near patients with tertian fevers (probably referring to malaria) that were prevalent in Hellenistic Greece and Egypt [17]. Trota also mentioned the utility of medicinal drinks *Tyriaca diatesereon* and Saint Paul's potion for patients with quartan fever (once again, probably malaria) [40]. These practices would likely have been intended to prevent *Plasmodium* protozoa from spreading, the causative agents of malaria from mosquito bites based on herbs' insect repellant activity. Paulus Aegineta noted that spreading herbs, including pennyroyal, was useful for repelling reptiles [31]. It has already been established that *Mentha* herbs, especially pennyroyal, have repellant, larvicidal and growth/reproduction regulatory activities against a wide variety of insects, including the mosquitoes responsible for spreading malaria (*Anopheles sp.*), yellow fever, dengue (*Aedes aegypti*), and zika (*Culex quinquefasciatus*) [94–98].

Mints were also valued for their scent in the Black Plague pandemic of the Middle Ages, which decimated at least one third of the European population. During this period, a vinegar-based formulation against the Black Plague called the "Four Thieves Vinegar" is thought to have originated in Medieval France, created by thieves that plundered the dead and dying plague victims. It is thought that, because they smeared this formulation on their skin, they were able to come in contact with their victims without being themselves affected [99]. Mints do not appear in the original formulation but were added to later variations of this formulation [100,101]. In this time period, it was still believed that (infectious) diseases were spread by miasmas, disease carrying vapors that emanated from corpses and decaying matter or from the breath of infected persons, a theory introduced by Hippocrates and Galen [102]. The discovery that plague was transmitted by fleas carrying the causative microbe *Yersinia pestis* was made centuries later. For these reasons, it is more logical that mints were added for their ability to offset pestilent odors rather than for their flea-repellant property; in fact, recent studies suggest that mint oils show only a weak flea repellant activity [103].

For the purpose of dealing with animal bites, several records are noteworthy. Nicander of Colophon used mints to create a 'repellant stench' to chase off snakes [5], while Pliny wrote of watermint and pennyroyal's usefulness for snake, scolopendra, and scorpion bites [17]. Trota also suggests people drinking *Tyriaca diatesereon* who had been bitten by poisonous animals and rabid dogs, as well as applying it to the wound itself. She also advised using *Tyriaca magna Galeni* with added mint water to help with poisoned wounds [40]. It is noteworthy that later references to mints are lacking, and recent scientific publications that have addressed the usefulness of mint-derived products to counteract animal venoms are scarce and inconclusive [104].

## *2.6. For Respiratory Disorders*

Mints were also used to control respiratory ailments, although literary sources are not abundant. Plutarch (46–119) in his *Moralia* wrote of the habit of Heraclitus, the Greek philosopher of Ephesus, of drinking cold water with spigs of pennyroyal before giving a speech [105]. Pliny made a similar reference and advised taking watermint juice 'when a person is about to engage in a contest of eloquence, but only when taken just before' [17]. For lower respiratory tract problems, Theodorus Priscianus (b. 300), a physician from the 5th century Byzantine empire, used pennyroyal in an herbal mixture for chestpain [38]. Aëtios of Amida wrote of a cough medicine containing pennyroyal in a mixture of pepper, hyssop, terebinth, fresh butter, and honey [38]. Gil Rodrigues de Valadares (Giles of Santarém, 1185–1265), a Portuguese Dominican friar and physician, wrote mint-based medicinal preparations, which can be found in *Códice Eborense CXXI*/*2-19*. For aphonia, Giles of Santarém prescribed a mixture of mint juice, ground pepper, malva seeds, egg yolk powder, and honey to be applied to the tongue [106]. Another interesting reference found centuries later is made to Jean-Paul Marat (1743–1793), the famous French physician, scientist, and politician from the French Revolution. Marat prescribed a mixture of mint and tolu balms and vegetable extracts, as well as a secret nostrum, to treat the Marquise de Laubespine from tuberculosis, a case that increased his popularity among French elites [107]. Research has shown that mints' compounds create the perception of nasal decongestion but also display bronchodilator and antitussive properties [108–110]. Indeed, the menthol-based Vicks® chestrub is extremely popular in the United States for treating common colds [111].

## *2.7. For Cardiovascular and Urinary Disorders*

There are only a few references made to using mint herbs for cardiovascular and urinary systems. Pliny wrote of pennyroyal as a diuretic and as one capable of removing bladder stones [17], an idea also defended by Aulus Cornelius Celsus (Celsus, c. 25 BC–c. 50) in regard to spearmint [112]. Later, Trota also advised using horsemint and pennyroyal in baths or fumigations to help with strangury (i.e., slow and painful discharge of small volumes of urine), likely due to their diuretic effect [38]. Interestingly, recent studies have shown that menthol does indeed have beneficial effects on the urinary system.

For example, menthol administration is associated with an improved inflammatory profile and clinical manifestations in female patients with interstitial cystitis [113]. As for their cardiac effect, Avicenna wrote of watermint in his opus *Canon of Medicine* as a valuable herb for heart conditions, especially palpitations [114]. Recent research suggests that the *Mentha x villosa* species has a hypotensive effect through bradycardia and vasodilation [115]. Although several mint substances may display this vasoactive effect, it has definitely been detected in menthol [116].

## *2.8. For Pain and Inflammation*

Although the pathophysiological mechanisms of inflammation were not uncovered until the 20th century, its central concept was understood many centuries before by Celsus [117]. Accordingly, mints were used either topically or systemically to control the inflammatory manifestations of several diseases, including pain, erythema, and fever, since classical antiquity. Pliny used mints in formulas for cases of ophthalmic and oral inflammation [17]. The Talmud mentions using pennyroyal in a mixture of boiled herbs to be applied to the scalp to 'soften the skull' prior to cranial surgery, which would suggest that pennyroyal may have been used as a local anesthetic in ancient Jewish medical practice [118]. Giles of Santarém advised applying a complex mixture of vegetable products, including mint juice, for abscess pain and otalgia. One of these preparations was used as a rubefacient to be applied to persons with leg atrophy, probably as a result of poliomyelitis, demyelinating diseases or even trauma [106]. Thomas Sydenham used liniments containing multiple herbs, including mints, for local applications to the abdomen and armpits in patients with 'scrophular diseases', which probably refer to cervical tuberculous lymphadenitis and rickets [47].

Odo de Magdunensis mentions a mixture of mint, strong rue, tansy, and milk cooked in olive oil with virgin wax, made into a plaster and applied to the kidney area of women who could not deliver their child. This could be to relieve pain [83]. Still on the subject of ophthalmic afflictions, William Mackenzie (1791–1868), author of the *Practical Treatise of the Diseases of the Eye*, one of the first British textbooks of ophthalmology, used a formulation of peppermint water, camphorated spirits of opium tincture and borax as a local anesthetic to be applied to the lacrimal puncta prior to mechanical unblocking with a hair pencil [119].

Gout is an inflammatory arthropathy characterized by the painful swelling of joints resulting from the precipitation of uric acid crystals. Mints are among the several types of treatment tried for this disease throughout history. Pliny exalted the usefulness of pennyroyal for treating gout, presumably by covering the affected body region with the herb [17]. In the work of Hildegard von Bingen also mentioned the usefulness of mints for treating gout [39]. Sydenham, having himself been afflicted with gout in the last years of his life, made very accurate descriptions of the disease and included mints in several electuaries (i.e., medicines, generally in powder, mixed in with a palatable medium) form to be taken by patients, as well as for rheumatism [47]. The anti-gout effect of mints may have a scientific basis as recent studies have shown that mint extracts inhibit xanthine oxidase *in vitro*, an enzyme involved in the formation of uric acid, as well as in generating oxidative stress that contributes to the pathophysiology of the disease [120]. The local anesthetic effect of mints is attributed mainly to volatile monoterpene menthol which, at low concentrations, activates receptors on cold nervous fibers by creating a perception of coolness, whereas it is irritating at high concentrations [121,122].

## *2.9. For Oral Health*

The first mention of mint herbs for oral health dates back to a 4th century Egyptian papyrus, probably written by a Christian monk, on which a recipe for toothpaste appeared. This toothpaste was a mixture of 'one drachma of rock salt, two drachmas of mint, one drachma of dried iris flower and 20 grains of pepper' [123]. Abu Al Qasim Al Zahrawi (Albucasis, 936–1013) recommended washing or gargling with mint decoctions to help with the 'swelling and erosion of the mouth, tongue and throat' brought up by the toxic effects of topically applied mercury, a metal already used in Islamic medicine for its therapeutic value [124]. He also advised inhaling the vapors of pennyroyal fumigation in absinthe and vinegar for a swollen uvula [125]. He added mint to borax for oral hygiene purpose [126]. Nikolaos Myrepsos (fl. 13th century), a physician at the court of John III Doukas Vatatzes at Nicaea wrote in his *Dynameron*, one of the richest treatises of the late Byzantine era, about the virtues of mints for the inflammation of teeth, mouth, and palate [127].

Gilbertus Anglicus (Gilbert of England, 1180–1250) wrote *Compendium Medicinae* (Compendium of Medicine), an important medical treatise of the 13th century, which mentions mints as promoters of oral health. For halitosis caused by teeth or gum decay, Gilbertus advised a mouthwash made from birch and mint soaked in wine, after which a linen cloth would be rubbed against gums until they hemorrhaged. Finally, the patient should chew marjoram, oregano, mint, and pellitory leaves, as well as rub the mixture into gums [128]. Paulus Aegineta wrote that 'Pseudo-Dioscorides recommends mint triturated with honey, red sumach, and rose oil with honey, or by itself for cleaning the tongue' [31]. Guy de Chauliac (1300–1368), the famous surgeon and pioneer in odontology, advised in his magnum opus *Chirurgia Magna* (Great Surgery) using wine and mint mouthwashes for discolored teeth. For carious teeth, Chauliac advocated an antiseptic gargle made of wine mixed with mint, sage, and pepper or pellitory [129]. For odontalgia, Rev. Townsend references one of Boerhaave's own prescriptions for a pill to be applied to the decaying tooth composed of opium, camphor, oil of cloves, and peppermint essential oil [50]. Despite the many recipes found for oral health that included mints, obviously the primary therapeutic intention was to create a local anesthetic/anti-inflammatory effect on the affected site.

## *2.10. For Cutaneous Disorders*

A few references are made about using these herbs for dermatological purposes. One famous effect, apparently to have been discovered by chance, refers to the treatment of elephantiasis. According to Pliny, elephantiasis was prevalent while Gnaeus Pompeius Magnus (Pompey the Great, 106 BC–48 BC) governed and had been imported from Egypt. He wrote that a person, ashamed of being affected with a facial form of the disease, smeared watermint on his face with shame and was cured [17]. A similar account comes from Paulus Aegineta, who mentions a mixture of 'watermint, juniper and mezereon', previously used by Marcellus Empiricus (fl. 385) and Quintus Serenus Sammonicus (d. 212), both medical writers from the late Roman empire [31]. The latter described remedies for different skin problems, such as 'juice of the bark of the juniper, the ashes and blood of the weasel, mint' [31]. Paulus Aegineta advised 'washing the head frequently with a lotion made from marjoram, mint or centaury' for porrago favosa (i.e., favus, a dermatophytosis) [31]. Theophanes Chryssobalantes (fl.c. 950), chief physician of the educated Emperor Constantine VII the Porphirogenitus, in his book *Epitome*, quotes a preparation of the ancient physician Archigenes (1st century AD) that consisted of laudanum and mint in equal quantities for alopecia [130]. Theophanes also indicates a concoction made of celandine (swallow-wort), Egyptian rose, and mint to dye the hair blonde [130].

In *Trotula*, Trota advises anointing *Theriaca magna Galeni* or 'juice of mint' to treat the thickness of lips [40]. William Augustus Hardaway (1850–1923) in his *Manual of Skin Diseases* mentions using peppermint essential oil in a formula for ameliorating generalized pruritus in urticaria and papular eczema, which included carbolic acid, glycerin and water, which would then be sprayed onto skin with an atomizer [131]. Recent research shows that several mint-based products appear promising to treat dermatological conditions. Peppermint essential oil is effective for controlling pruritus and for relieving irritation and inflammation [132,133].

## *2.11. For Nervous Disorders*

Since ancient times, mint-based preparations have also been known to affect cognition and emotion, and have been used as restorative agents to enable to regain vigor. The oldest record on the effect that mints have on the central nervous system is attributed to Aretaeus of Cappadocia (fl. 2nd century), who advised that epileptics should take walks among acrid and aromatic herbs, such as mint and pennyroyal [134]. Pliny also wrote about the use of watermint to control epileptic seizure, besides it helping with hangovers [17]. For the latter, Marcus Terentius Varro (116 BC–27 BC), a roman scholar and writer, advised hanging garlands of pennyroyal in one's chambers [17]. In contrast, in the *Hippocratic Corpus* it is written that mint should be avoided by epileptics because of its 'pungent nature' [135] There are also several records about using mints to treat headaches. Paulus Aegineta treated throbbing headaches and those resulting from heat exposure with pennyroyal and watermint [33]. Galen wrote about a head compress, previously recorded by Asklepiades, but probably attributed to Nikomedes IV of Bithunia, which contained 'sulfurwort (hog-fennel), rue, mint and other herbs in rose oil' [31]. Finally, some attempts have been made to use mints for psychiatric disorders. Galen also wrote about hysteria, and, among his treatments, he administered a mixture of plants, such as mint, hellebore, laudanum, belladonna extract, and valerian [136]. In Trota's work, *Potio Sancti Pauli*, which included horsemint, was given to treat several nervous states, including 'epileptics, cataleptics and analeptics' [40]. Centuries later, Rev. Townsend tried peppermint essential oil, without much success, to treat hysterical fits [50], as did John Quincy (d. 1722), the English apothecary and medical writer, with pennyroyal water [137].




**Table 1.** *Cont.*


**Table 1.** *Cont.*

**Table 2.** Description and main results of relevant studies assessing the health-promoting effects of mint-based products.


## **3. Conclusions**

This paper makes a thorough descriptive review of the many medical uses given to mints throughout history, from ancient civilizations to modern day medicine, beyond previously published material, highlighting both the authors in medical culture responsible for their dissemination, as well as their major galenic formulations. Most medical knowledge came from Ancient Greek and Roman philosophers and medical authors, while later contributions consisted mainly of progressive technological improvements. From the immediately perceived qualities of these herbs, it can be inferred that the primary intention of their topical application was to probably cause local anesthesia and to control irritation and inflammation, being especially used in gastrointestinal tract affections. Their particular scent and flavor also came of use for the purpose of masking the unpleasant taste of many medicinal formulae long before the advent of pharmaceutical technology. The longevity and diversity of the use of mints in medicine are a testament of their importance, receiving still noble place in herbal medicine.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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*Biology* **2020**, *9*, 484

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## *Article*
