**Analysis of Natural Bioactive Compounds in Plant, Food, and Pharmaceutical Products Using Chromatographic Techniques**

Editor

**Faiyaz Shakeel**

Basel • Beijing • Wuhan • Barcelona • Belgrade • Novi Sad • Cluj • Manchester

*Editor* Faiyaz Shakeel King Saud University Riyadh, Saudi Arabia

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Separations* (ISSN 2297-8739) (available at: https://www.mdpi.com/journal/separations/special issues/189U457VRA#Editorial).

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## **Contents**

with UPLC-MS/MS


## **El Mustapha El Adnany, Najat Elhadiri, Ayoub Mourjane, Mourad Ouhammou, Nadia Hidar, Abderrahim Jaouad, et al.**

## **Mayra Beatriz G ´omez-Patino, ˜ Juan Pablo Leyva P ´erez, Marcia Marisol Alcibar Munoz, ˜ Israel Arzate-V ´azquez and Daniel Arrieta-Baez**


## **About the Editor**

## **Faiyaz Shakeel**

Prof. Faiyaz Shakeel received his Masters and Ph.D. in Pharmaceutics from Jamia Hamdard (Hamdard University, New Delhi, India). At Jamia Hamdard, he worked on nanoemulsion-based drug delivery systems for some poorly soluble drugs. Then, he became a lecturer at the University of Benghazi (Libya), where he worked on nanoemulsion and self-nanoemulsifying drug delivery systems of some biologically active molecules. In 2011, he was awarded the Young Scientist Award from the Association of Pharmacy Professionals (APP). One of his group's research articles was awarded with the most cited paper award from the European Journal of Pharmaceutics and Biopharmaceutics in March 2012. Currently, he is working as a professor at the Department of Pharmaceutics, College of Pharmacy, King Saud University. At King Saud University, he developed several nanocarrier-based formulations of various drugs. He also developed a double nanoemulsion for a self-nanoemulsifying drug delivery system of 5-fluorouracil. He has very good expertise in the solubilization of drug molecules using cosolvency models. He developed various analytical methods for the determination of various drugs in a variety of sample matrices. His research interests lie in the general area of pharmaceutics and novel drug delivery systems. He is the author of more than 430 journal articles and several book chapters. He also has a US patent. He is Editor/Editorial Board Member of several journals such as Pharmaceutics, Molecules, Separations, Current Drug Delivery, and Pharmaceutical Sciences, among others. He has 11,114 total citations with an H-index of 49 and an i10 index of 237. In 2020, 2021, and 2022, he was named to the Stanford/Elsevier list of the top 2% scientists in the world for both his career (coveted) and a single year.

## *Editorial* **Analysis of Natural Bioactive Compounds in Plant, Food, and Pharmaceutical Products Using Chromatographic Techniques**

**Faiyaz Shakeel**

Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; fsahmad@ksu.edu.sa

## **1. Introduction**

A growing tendency toward the discovery and use of natural bioactive compounds that are the least harmful, have the fewest side effects, and are the most natural for the human body has been noticed during the past few decades [1]. As evidenced by the rise in recent studies on the therapeutic properties of plants, this trend has caused a return of healthcare professionals to nature and plants, but with a modern approach that specifically questions how plants help to heal humans and what their exact effects on the human body are [2]. The medicinal properties of plants are related to their phytochemical makeup, which is a complex matrix with a large number of naturally occurring bioactive molecules that must be distinguished between in order to be identified [3,4]. The separation of natural bioactive chemicals from plants can be accomplished utilizing cutting-edge, high-tech, hyphenated chromatographic approaches, which also provide us with lots of information to be able to identify compounds [5–7]. In order to explain a plant's mechanism of action and therapeutic effect, modern healthcare providers need to be able to link the phytochemical profile of a plant employed in therapy to a biological activity [8]. In addition to plants, natural bioactive substances can be found in a variety of foods and pharmaceuticals [8,9]. As a result, it is crucial to analyze these chemicals in plant, food, and pharmaceutical products [8–10].

In order to identify and analyze natural bioactive compounds in plant, food, and pharmaceutical products, this Special Issue has attempted to compile the latest improvements, advancements, and analytical innovations in chromatographic techniques. Additionally, this Special Issue seeks to enable researchers to link the phytochemical profiles of plants, foods, and pharmaceuticals with proven therapeutic effects, which may later substantiate the health-related claims made for these products. This Special Issue includes 10 research articles focused on the analysis of natural bioactive compounds and phytochemicals in plant, food, and pharmaceutical products using innovative chromatographic techniques.

## **2. Overview of Published Articles**

This Special Issue begins with an article by Mohiuddin et al. [11], who studied the chemical composition and antibacterial effects of *Cerana indica* propolis from the Kashmir region. GC-MS analysis was performed to identify the chemical compounds of Kashmiri propolis, and showed the presence of 68 different phytochemicals in Kashmiri propolis. The ethanolic extract of Kashmiri propolis showed the maximum zone of inhibition against *Staphylococcus aureus*. The findings of this research indicate the presence of various secondary metabolites with distinct pharmacological activities.

Abdel-Baki et al. [12] next assessed the chemical constituents, in vitro cytotoxicity, and scolicidal, acaricidal, and insecticidal activities of *Lavandula steochas* essential oil. The phytoconstituents of *L. steochas* essential oils were detected using spectrometry and gas chromatography techniques. The analyses of *L. steochas* oil showed camphor as being the major compound (58.38%). The oil presented significant cytotoxicity and scolicidal activities. The essential oil also showed 100% adulticidal activity against *R. annulatus* at a

**Citation:** Shakeel, F. Analysis of Natural Bioactive Compounds in Plant, Food, and Pharmaceutical Products Using Chromatographic Techniques. *Separations* **2023**, *10*, 541. https://doi.org/10.3390/ separations10100541

Received: 14 August 2023 Accepted: 12 September 2023 Published: 13 October 2023

**Copyright:** © 2023 by the author. 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

10% concentration, whereas the larvicidal activity was 86.67%. However, the oil showed no insecticidal activity. In addition, *L. steochas* oil demonstrated 100% larvicidal and pupicidal effects. The findings of this work suggest that *L. steochas* essential oil could serve as a potential source of scolicidal, acaricidal, insecticidal, and anticancer agents.

Haq et al. [13], in the next article, developed and validated a greener, stabilityindicating, high-performance liquid chromatography (HPLC) approach to determine curcumin (CCM) in an in-house developed nanoemulsion, *Curcuma longa* L. extract, and marketed tablets. The greener HPLC approach was found to be linear, rapid, accurate, precise, and sensitive for measuring CCM. The AGREE approach showed an AGREE score of 0.81 for the proposed HPLC method, which indicated an outstanding greenness profile. The proposed HPLC method successfully determined the CCM in the in-house developed nanoemulsion, *Curcuma longa* extract, and commercial tablets. Furthermore, the greener HPLC method was found to be stability-indicating. The results of this work indicate that CCM can be routinely measured in all studied sample matrices using the greener HPLC method.

The capillary electrophoresis (CE) technique with light-emitting diode-induced fluorescence detection was used for the analysis of sugars in honey samples by Andrasi et al. [14]. The optimized CE technique was applied in the measurement of fructose and glucose via the direct injection of honey samples. The proposed CE technique provides high separation efficiency and sensitivity within a short analysis time. Furthermore, it enables the injection of honeys without sample pretreatment. The findings of this study showed the rapid and sensitive analysis of sugars in honey samples using the CE technique with fluorescence detection.

Altharawi et al. [15] developed and validated an UPLC-MS/MS method for the simultaneous determination of neratinib and naringenin in rat plasma using imatinib as the internal standard (IS). The mass spectra of studied compounds were recorded via the multiple reaction monitoring of the precursor-to-product ion transitions. The proposed UPLC-MS/MS method was found to be linear, selective, precise, accurate, and stable. The proposed method was also found to be eco-friendly for the measurement of neratinib, naringenin, and IS. The analytical results of this work showed that the developed method has implications for its applicability in pharmacokinetic studies in humans to support the therapeutic drug monitoring of combination drugs.

Haq et al. [16] developed a rapid and sensitive HPLC approach for the determination of a natural bioactive compound, pterostilbene (PTT), in commercial capsule dosage form, solubility, and stability samples. The developed HPLC approach was linear, rapid, accurate, precise, and sensitive. The proposed HPLC approach was successfully applied in the measurement of PTT in commercial capsule dosage form, solubility, and stability samples. The results indicated that PTT in commercial products, solubility, and stability samples may be routinely determined using the proposed HPLC approach.

In another article, Suleman et al. [17] used two different spices, Chinese prickly ash and cinnamon, to mitigate the formation of heterocyclic aromatic amines (HAAs) in superheated steam-roasted patties. The findings demonstrated significant differences (*p* < 0.05) in the content of both polar and non-polar HAAs in comparison to the control patties. In cinnamon-roasted and Chinese prickly ash patties, both polar and non-polar HAAs were considerably reduced. The results of this study showed that both spices and superheated steam controlled HAAs to a significant level in lamb meat patties.

El Adnany et al. [18] improved the extraction efficiency of phenolic compounds from olive leaves (*Moroccan picholine*) while minimizing the use of harmful chemicals. Ultrasonic extraction using ethanol was found to be the most effective and environmentally friendly approach. The antioxidant activity of the phenolic compounds of olive leaves was also evaluated. Various parameters, such as the extraction time, solid/solvent ratio, and ethanol concentration (independent variables), were evaluated using a response surface methodology (RSM) based on the Box–Behnken design (BBD) to optimize the extraction conditions. The phenolic compounds of olive leaves were identified using the HPLC-MS

technique. Various phenolic compounds, such as hydroxytyrosol, catechin, caffeic acid, vanillin, naringin, oleuropein, quercetin, and kaempferol, were found in high concentrations. The findings of this work showed the efficient extraction of phenolic compounds with great antioxidant activity.

Zasheva et al. [19] studied the effects of *Haberlea rhodopensis* methanol extract fractions on the cell viability and proliferation of two model breast cancer cell lines (MCF7 and MDA-MB231 cells) with different characteristics. In addition to the strong reduction in cell viability, two of the fractions showed a significant influence on the proliferation rate of the hormone receptor expressing MCF7 and the triple-negative MDA-MB231 breast cancer cell lines. The results of this study presented a good background for future studies on the use of myconoside (an active constituent of *Haberlea rhodopensis*) for targeted breast cancer therapy.

In the final article, Gomez-Patino et al. [20] developed a rapid protocol for the extraction and separation of the components of the aerial parts of *Gymnosperma glutinosum*. The chemical compounds of chloroformic and methanolic extracts of *G. glutinosum* were identified using the GC-MS technique. The findings revealed the presence of (−)-α-bisabolol (BIS) as the main component in the chloroformic extract, which was isolated and analyzed via 1H NMR to confirm its presence in *G. glutinosum*. The evaluation of methanolic extracts using the UPLC-MS technique demonstrated the presence of six methoxylated flavones and a group of C20-, C18-hydroxy-fatty acids. The findings of this study concluded that the presence of BIS, an important sesquiterpene with therapeutic skin effects, as well as some antioxidant compounds such as methoxylated flavones and their oils, could play an important role in cosmetology and dermatology formulations.

## **3. Conclusions and Future Perspectives**

In the last few decades, a tremendous amount of research on the analysis of natural bioactive compounds in plants, foods, and pharmaceutical products, using a wide range of chromatography techniques, has been performed. This Special Issue has brought together prominent researchers who have explored the diverse application range of chromatographic techniques in the extraction, separation, identification, and analysis of natural bioactive compounds. This Special Issue provides sufficient information on the analysis of natural bioactive compounds in plant, food, and pharmaceutical products using chromatographic techniques. However, one article reported the pharmacokinetic profile of natural bioactive compounds using the highly sensitive UPLC-MS/MS technique. I believe that further applications of these techniques on the biological samples, pharmacokinetic evaluation, and therapeutic drug monitoring of natural bioactive compounds are still required to explore the clinical applications of these techniques. Furthermore, the correlation of identified bioactive compounds and phytochemicals with their biological activity is required and will add the advantages for future studies.

**Acknowledgments:** The authors who contributed to this Special Issue are highly appreciated. The reviewers who reviewed the articles of this Special Issue are also thankful for their significant efforts to enhance the quality of this Special Issue.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


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## *Article* **GC-MS Analysis, Phytochemical Screening, and Antibacterial Activity of** *Cerana indica* **Propolis from Kashmir Region**

**Ishfaq Mohiuddin 1,\*, T. Ramesh Kumar 1, Mohammed Iqbal Zargar 2, Shahid Ud Din Wani 2, Wael A. Mahdi 3, Sultan Alshehri 3, Prawez Alam <sup>4</sup> and Faiyaz Shakeel <sup>3</sup>**


**Abstract:** Propolis is a resinous compound produced by honey bees. It contains bioactive molecules that possess a wide range of biological functions. The chemical composition of propolis is affected by various variables, including the vegetation, the season, and the area from which the sample was collected. The aim of this study was to analyze the chemical composition and assess *Cerana indica* propoli's antibacterial efficacy from the Kashmir region. Gas chromatography-mass spectrometry (GC-MS) analysis was used to determine the chemical composition of Kashmiri propolis. A range of bacterial strains was tested for antimicrobial activity using different extracts of propolis by agar well diffusion technique. Propolis was found to be rich in alkaloids, saponins, tannins, and resins. The chemical characterization revealed the presence of 68 distinct phytocompounds using GC-MS, and the most predominant compounds were alpha-D-mannopyranoside, methyl, cyclic 2,3:4,6-bis-ethyl boronate (21.17%), followed by hexadecanoic acid, methyl ester (9.91%), and bacteriochlorophyll-c-stearyl (4.41%). The different extracts of propolis showed specific antibacterial efficacy against multidrug-resistant (MDR) strains viz., *Pseudomonas aeruginosa* (MTCC1688), *Escherichiacoli* (MTCC443), *Klebsiella pneumonia* (MTCC19), *Cutibacterium acnes* (MTCC843), and *Staphylococcus aureus* (MTCC96). The EEKP showed the highest zone of inhibition against *S. aureus* (17.33) at 400 μg mL<sup>−</sup>1. According to the findings of this study, bee propolis contains a variety of secondary metabolites with various pharmacological activities. Furthermore, because of its broad spectrum of positive pharmacological actions and the fact that it is a promising antibacterial agent, more research on propolis is warranted.

**Keywords:** *C. indica*; propolis; GC-MS; antibacterial; bioactive compounds; chemical composition

## **1. Introduction**

Propolis (also known as bee glue) derives its name from the Greek word pro, which means "in front of" or "at the entrance to," and polis, which means "community" or "city". This natural substance is a unique mixture of various natural components with distinct properties and aids in the protection of the beehive [1,2]. It is made up of a resinous substance that honeybees collect from the buds and exudates of certain trees and plants, which they then combine with beeswax and the enzyme-rich secretion of bee salivary glands, including β-glycosidase [3–5].

Honeybee colonies frequently employ propolis for hive repairs as a sealant to plug the cracks and restrict the openings, limiting the entry of intruders and maintaining the temperature inside the hive at the ideal level for bees, about 35 ◦C [6]. In addition, they embalm dead invaders with bee glue to prevent decomposition, protect the bee larvae, store honey, and remove potential sources of microbial infestations [7]. Propolis becomes

**Citation:** Mohiuddin, I.; Kumar, T.R.; Zargar, M.I.; Wani, S.U.D.; Mahdi, W.A.; Alshehri, S.; Alam, P.; Shakeel, F. GC-MS Analysis, Phytochemical Screening, and Antibacterial Activity of *Cerana indica* Propolis from Kashmir Region. *Separations* **2022**, *9*, 363. https://doi.org/10.3390/ separations9110363

Academic Editor: Rosario Rodil

Received: 25 October 2022 Accepted: 7 November 2022 Published: 9 November 2022

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

**Copyright:** © 2022 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/).

5

sticky when above room temperature, but it is hard and brittle at low temperatures [8]. It has a very distinctive and pleasant aroma. Depending on its origin and age, its coloration varies from yellow to red to dark brown. Even the existence of transparent propolis has been reported [9,10]. Propolis has been revealed to have antimicrobial properties [11,12], and it has been used for centuries in traditional medicine and remedies.

The complex chemical content of propolis is constantly changing due to various geographical conditions; generally, raw propolis is composed of plant resin (45–55%), wax (25–35%), essential (5–10%), and aromatic oils (5%), pollen, and other natural components (5%). [13]. Depending on the species of bee, the plant's origin, the habitat, and the storage conditions, thechemical composition of propolis varies [14,15]. So far, over 300 chemical compounds have been isolated and identified from the propolis, including sugars, polyols, hydroxy acids, fatty acids, cardanols, flavan derivatives, triterpenes, prenylated flavanones, anacardic acids, aromatic acids, and their esters, and chalcones, [6,16]. Additionally, due to its complex chemical composition, it has been shown that propolis possesses antimicrobial [17,18], antioxidant [19–21], anti-inflammatory [22,23], and antifungal properties [24]. The bioactivity of propolis has been implicated in its flavonoid, phenolic, diterpenic acid, and aromatic acid contents [25]. Globally, propolis formulations are effective antibacterial agents [26–28]. Propolis is effective against a wide range of bacterial strains, though it is most effective against Gram-positive bacteria and less effective against Gram-negative bacteria [4,29]. The findings of numerous studies have revealed that the antibacterial action is based on obstruction of bacterial movement and the activity of various bacterial enzymes, as well as a weakening of the cytoplasmic membrane [29]. Propolis from several countries, including Spain, Romania, and China, was found to contain fructose, glucose, galactose, stachyose, and sucrose [30]. Propolis contains essential amino acids, such as proline and arginine, which are needed for cell regeneration and can vary depending on the flora and environment around bee colonies [31]. Terpenoids are the volatile components of propolis that are responsible for the odor or scent that propolis emits. It also significantly contributes to propolis essential oil extract's biological effects, such as antibacterial and anti-inflammatory properties [32]. According to Sturm and Ulrih (2020), terpenoids were isolated in propolis for the first time in 2011, and 133 terpenes have been reported in propolis so far. Only 16 alkaloids have been isolated from Brazil and Algeria to date [33]. Hexadecanoic acid, methyl ester, is a fatty acid with antioxidant, hepatoprotective, anticancer, anti-inflammatory, anticoronary, anti-arthritic, antieczemic, antihistaminic, and antiandrogenic properties [34].

Therefore, the current study aims to evaluate the antibacterial activities, physicochemical characterization, phytochemical screening, and gas chromatography–mass spectrometry (GC-MS) analysis of various propolis extracts from the chosen area.

## **2. Materials and Methods**

## *2.1. Chemicals and Apparatus*

All of the chemicals and reagents used were of analytical grade purity. Methanol, ethanol, nutrient agar, Mueller–Hinton agar (MHA), and dimethyl sulfoxide (DMSO) were purchased from Himedia (Mumbai, India). Other chemicals used in this experiment were of the highest quality and commercially available on the market. Streptomycin was also procured from Himedia (Mumbai, India). Throughout the media preparations and measurements, ultrapure water was used.

#### *2.2. Collection of Propolis Sample*

Crude propolis samples produced by Ceranaindica bees were obtained from honeybee colonies in Chunt-Waliwar, Ganderbal, Jammu, and Kashmir, India (Figure 1a,b). By scraping the surfaces of the walls, frames, entrances, and coverings, propolis was recovered from the beehive [35]. Prior to analysis, the propolis sample was stored at −20 ◦C.

**Figure 1.** (**a**) The image of map showing the location from where the *C. indica* propolis was collected. (**b**) The image of crude propolis sample produced by *C. indica* honeybees.

## *2.3. Extraction Procedure*

## 2.3.1. Ethanolic Extract (EEKP)

The 30 g of powdered propolis was extracted using a maceration process with 100 mL of ethanol for at least 3 days. The extract was filtered by using Whatman No.1 filter paper. The extract was dried under pressure using a rotatory evaporator and kept at −20 ◦C prior to further analysis.

## 2.3.2. Methanolic Extract (MEKP)

The 30 g of powdered propolis was extracted using a maceration process with 100 mL of methanol for at least 3 days. The extract was filtered using Whatman No. 1 filter paper. The extract was dried under pressure using a rotatory evaporator and kept at −20 ◦C prior to further analysis.

## 2.3.3. Aqueous Extract (AqEKP)

About 30 g of Propolis was chopped into pieces and extracted with 100 mL of distilled water. The samples were heated for 5 min on a hot plate with constant agitation at 60 ◦C before being left at room temperature for a night before being filtered. The resulting mixture was filtered through Whatman filter paper No.1 and concentrated at low temperatures using the freeze-drying method. The extracted samples were kept at −20 ◦C before the analysis.

## *2.4. Qualitative Phytochemical Analysis*

The different extracts of propolis were subjected to qualitative analysis to identify its constituents, including alkaloids, saponins, tannins, flavonoids, terpenoids, phlobatanins, proteins, and carbohydrates, by following standard protocols [36–39].

#### *2.5. Identification and Quantification of Bioactive Compounds Using GC-MS*

GC-MS analysis was performed by using GC-MS/MS-7000D Agilent (Agilent technologies, Santa Clara, CA, USA), equipped with Agilent J&W, GC-MS column HP-5Ms (15 m × 250 mm × 0.25 μm). The carrier gas was helium at a flow rate of 1 mL/min, and the injector temperature was 280 ◦C in split-less mode. The oven temperature was initially held at 60 ◦C for 4 min before being increased to 150 ◦C at a rate of 10 ◦C/min for 15 min. The following parameters were used to optimize the mass spectra: the source temperature is 280 ◦C, and the transfer temperature is 150 ◦C. The solvent delay time was 2 min, and the scan range was 35–500 Da. The temperature was finally raised to 310 ◦C. The GC's total run time was 40.5 min. By comparing their mass spectra to data from the National Institute of Standards and Technology (NIST) library, the compounds were identified.

## *2.6. Antibacterial Screening*

The agar well diffusion method was used to assess the antibacterial activity of various propolis extracts [40]. After being sterilized at 121 ◦C for 25 min, all the selected bacterial colonies were first sub-cultured in Nutrient Agar Media 2 (NAM) with a pH of 7. About 4 mL of the NAM was added to test tubes to produce the agar slants, which were then incubated at 37 ◦C overnight. Tubes containing 15 mL of MHA were inoculated with freshly prepared bacterial inoculums using a sterile loop to ensure uniform distribution of inoculums. The pre-inoculated medium was poured into Petri plates and allowed to solidify before cutting 8 mm wells with a sterile cork borer. The wells were filled with 100 μL of extract at various concentrations (100, 200, and 400 μg mL<sup>−</sup>1) and an equivalent volume of DMSO, which served as a negative control. After standing for 30 min to allow for pre-diffusion of the extract into the medium, the plates were incubated at 37 ◦C for 16–20 h. The inhibition zones on the plates were ascertained, and the findings were compared to the positive control containing streptomycin (10 μg mL−1). All assays were carried out in triplicate, and mean values were obtained.

## *2.7. Bacterial Strains*

The five bacterial strains used in this study, viz. *Pseudomonas aeruginosa* (MTCC1688), *Escherichia coli* (MTCC443), *Klebsiella pneumoniae* (MTCC19), *Cutibacterium acnes* (MTCC843), and *Staphylococcus aureus* (MTCC96) were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India. As a positive control, streptomycin (10 μg mL<sup>−</sup>1) was used.

## **3. Results**

#### *3.1. Phytochemical Analysis*

The propolis extracts were phytochemically analyzed and found to contain bioactive components, such as terpenoids, flavonoids, alkaloids, phenols, tannins, and saponins. The efficacy of the three solvents in extracting various components of propolis is compared in Table 1.

#### *3.2. GC-MS Analysis*

Since EEKP produced the maximum number of phytochemicals when compared to MEKP and AqEKP, GC-MS analysis was performed to determine EEKP's chemical profile. The GC-MS chromatogram of *C. indica* propolis extract shown in Figure 2 shows a total of 68 peaks corresponding to bioactive compounds identified by comparing their mass spectral fragmentation patterns to those of known compounds described in the NIST library. The chemical constituents identified in Table 2 are listed by retention time, peak area (%), and molecular weight. The most common phyto-constituents found in propolis extract are alpha-D-Mannopyranoside, methyl, cyclic 2,3:4,6-bis-ethyl boronate (21.17%), hexadecanoic acid, methyl ester (9.91%), nona-2,3-dienoic acid, ethyl ester (4.75%), bacteriochlorophyllc-stearyl (4.41%), 10-methyldodecan-4-olide (3.89%), and nickel, cyclopentadienyl-1,2,3 trimethylallyl (3.12%).


**Table 1.** Phytochemical screening of propolis extracts.

EEKP: ethanolic extract of Kashmiri propolis; MEKP: methanolic extract of Kashmiri propolis; AqEKP: aqueous extract of Kashmiri propolis; ++: abundant, +: moderate, -: absent.

**Figure 2.** Gas chromatography–mass spectrometry (GC-MS) chromatogram of significant propolis compounds.



#### **Table 2.** *Cont.*



#### **Table 2.** *Cont.*

## *3.3. Antibacterial Activity*

Table 3 and Figure 3 show the antibacterial properties of different extracts of Kashmiri propolis. EEKP, MEKP, and AqEKP had nearly identical antimicrobial activities. Small differences were observed in the majority of cases (1–3 mm). Only in the cases of *E. coli* and *C. acnes* did MEKP and AqEKP show no activity at 100 μg mL<sup>−</sup>1.

**Table 3.** Antimicrobial capacity of various extracts of propolis against tested bacterial strains using agar well diffusion method.


Results in the table are expressed in millimeters (mm) and each value is in triplicate represented as mean ± S.E.M.; - represents no zone of inhibition. EEKP: ethanolic extract of Kashmiri propolis; MEKP: methanolic extract of Kashmiri propolis; AqEKP: Aqueous extract of Kashmiri propolis.

**Figure 3.** The zone of inhibition (mm) of different extracts of *C. indica* propolis against the selected bacterial strains. (**A**) EEKP vs. *P. aeruginosa*. (**B**) EEKP vs. *S. aureus.* (**C**) EEKP vs. *K. pneumoniae*. (**D**) MEKP vs. *K. pneumoniae*. (**E**) MEKP vs. *P. aeruginosa.* (**F**) AqEKP vs. *S. aureus.* EEKP: ethanolic extract of Kashmiri propolis; MEKP: methanolic extract of Kashmiri propolis; AqEKP: Aqueous extract of Kashmiri propolis.

#### **4. Discussion**

The propolis extracts used in the current study showed the existence of a range of bioactive components, including carbohydrates, aldehydes, flavonoids, alkaloids, terpenoids, alcohols, cardiac glycosides, tannins, coumarins, amino acids, phytobatanins, saponins, etc. These phytochemicals could be responsible for the pharmacological properties of the propolis extracts. Propolis is commercialized globally and is considered a significant source of phytochemicals that have pharmacological effects [41]. The presence of 68 compounds from various groups was revealed by the GC-MS analysis, including flavonoids, flavonoid derivatives, terpenes, aromatic acids, and their related esters.

The extracts of propolis have a strong effect against bacteria, such as *Enterococcus* spp., *Escherichia coli*, and *Staphylococcus aureus* [42,43]. In the present studies, the antibacterial activity of various extracts of propolis was tested against an array of bacterial strains. The EEKP was found to possess comparatively stronger antimicrobial activity than MEKP and AqEKP. The highest microbial activity in the EEKP may be due to the highest concentration of flavonoid and phenolic compounds, which may inhibit microorganisms [44]. Another factor is that the majority of naturally occurring secondary metabolites are highly soluble in organic solvents; as a result, the ethanolic extract has the highest microbial activity, followed by MEKP and AqEKP [45]. The propolis extracts had significant antibacterial activity against Staphylococcus aureus but no activity against *E. coli,* according to studies from China and Canada [46,47]. The propolis ethanolic extracts have shown the highest levels of antimicrobial activity against a variety of microorganisms in Brazil [48]. In Vietnam, propolis crude extract demonstrated significant antibacterial activity against S. aureus and inhibited E. coli at lower doses [49]. This is because propolis contains flavones and flavonols that have been isolated from it [50], as well as large levels of terphenyl esters and hydroxybenzoic acid, both of which have antibacterial and antifungal properties [51]. Components of propolis, such as Pinocembrin, exhibit antibacterial action against *Streptococcus* species, whereas p-Coumaric acid, artepillin,3-phenyl-4-di hydrocinnamylocinnamic acid inhibits *H. pylori*, and Apigenin substantially inhibits bacterial glycosyltransferase [52].

#### **5. Conclusions**

The present work is the first approach to identify numerous bioactive components by GC-MS analysis and to assess the antibacterial potency of various extracts of *C. indica* propolis from the Kashmir region. The GC-MS analysis of propolis has revealed the presence of 68 bioactive compounds that have a wide range of pharmacological potential, including antibacterial, antifungal, anti-protozoal, antioxidant, hepatoprotective, antiinflammatory, anticancer, and so on. The presence of these bioactive compounds in propolis is responsible for various therapeutic and pharmacologic properties in traditional medicine. More research is needed to isolate a specific substance that results in a positive result in a biological assay, and appropriate methodologies for in-depth studies should be developed.

**Author Contributions:** Conceptualization, I.M. and F.S.; methodology, I.M., T.R.K., M.I.Z. and P.A.; software, S.A. and F.S.; validation, S.U.D.W., F.S., S.A. and W.A.M.; formal analysis, S.U.D.W.; investigation, T.R.K. and M.I.Z.; resources, W.A.M. and S.A.; data curation, P.A.; writing—original draft preparation, I.M.; writing—review and editing, F.S., S.A. and W.A.M.; visualization, W.A.M.; supervision, I.M.; project administration, I.M.; funding acquisition, W.A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research project was supported by Researchers Supporting Project number (RSP2022R516), King Saud University, Riyadh, Saudi Arabia, and APC was supported by the RSP.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Authors are thankful to the Researchers Supporting Project number (RSP2022R516), King Saud University, Riyadh, Saudi Arabia, for supporting this work.

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

## **References**


## *Article* **Cytotoxic, Scolicidal, and Insecticidal Activities of** *Lavandula stoechas* **Essential Oil**

**Abdel-Azeem S. Abdel-Baki 1, Shawky M. Aboelhadid 2,\*, Saleh Al-Quraishy 3, Ahmed O. Hassan 4, Dimitra Daferera 5, Atalay Sokmen <sup>6</sup> and Asmaa A. Kamel <sup>2</sup>**


**Abstract:** Essential oils (EOs) have recently attracted more interest due to their insecticidal activities, low harmfulness, and rapid degradation in the environment. Therefore, *Lavandula steochas* (*L. steochas*) essential oil was assessed for its chemical constituents, in vitro cytotoxicity, and scolicidal, acaricidal, and insecticidal activities. Using spectrometry and gas chromatography, the components of L. steochas EOs were detected. Additionally, different oil concentrations were tested for their anticancer activities when applied to human embryonic kidney cells (HEK-293 cells) and the human breast cancer cell line MCF-7. The oil's scolicidal activity against protoscolices of hydatid cysts was evaluated at various concentrations and exposure times. The oil's adulticidal, larvicidal, and repelling effects on *R. annulatus* ticks were also investigated at various concentrations, ranging from 0.625 to 10%. Likewise, the larvicidal and pupicidal activities of *L. steochas* against *Musca domestica* were estimated at different concentrations. The analyses of *L. steochas* oil identified camphor as the predominant compound (58.38%). *L. steochas* oil showed significant cytotoxicity against cancer cells. All of the tested oil concentrations demonstrated significant scolicidal activities against the protoscoleces of hydatid cysts. *L. steochas* EO (essential oil) showed 100% adulticidal activity against *R. annulatus* at a 10% concentration with an LC50 of 2.34%, whereas the larvicidal activity was 86.67% and the LC50 was 9.11%. On the other hand, the oil showed no repellent activity against this tick's larva. Furthermore, *L. steochas* EO achieved 100% larvicidal and pupicidal effects against *M. domestica* at a 10% concentration with LC50 values of 1.79% and 1.51%, respectively. In conclusion, the current work suggests that *L. steochas* EO could serve as a potential source of scolicidal, acaricidal, insecticidal, and anticancer agents.

**Keywords:** lavender oil; cytotoxic; scolicidal; Musca domestica; acaricide

## **1. Introduction**

In developing countries, parasitic diseases, associated with both ectoparasites and endoparasites, represent a severe hazard to both human and animal health [1]. Cystic echinococcosis (CE) is caused by the larval stage (hydatid cyst) of *Echinococcus granulosus*, which can develop in the liver, heart, lungs, brain, spleen, bone, and kidneys of the host, and can be fatal [2,3]. In several endemic areas, the incidence rate of CE may vary from 1 to 200 per 100,000 persons annually [4]. Currently, a range of chemical scolicidal compounds, such as benzimidazole derivatives, are used to deactivate hydatid cyst protoscolices [5,6]. However, these chemicals cause a variety of negative side effects, including impairments in liver function, leucopenia, and abdominal pain [6,7]. A perfect scolicidal agent is one that

**Citation:** Abdel-Baki, A.-A.S.; Aboelhadid, S.M.; Al-Quraishy, S.; Hassan, A.O.; Daferera, D.; Sokmen, A.; Kamel, A.A. Cytotoxic, Scolicidal, and Insecticidal Activities of *Lavandula stoechas* Essential Oil. *Separations* **2023**, *10*, 100. https:// doi.org/10.3390/separations10020100

Academic Editor: Faiyaz Shakeel

Received: 3 January 2023 Revised: 24 January 2023 Accepted: 26 January 2023 Published: 1 February 2023

**Copyright:** © 2023 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/).

remains stable after being diluted with cyst fluid, eliminates cyst protoscolices, is non-toxic, causes no harm to the tissue of the host, is inexpensive, and is easily accessible [3,7–9].

The use of scolicidal compounds is essential to the therapeutic treatment of hydatid cysts and helps prevent the spread of the protoscoleces through surgery [2]. Due to the adverse effects often associated with the use of protoscolicidal agents during hydatid cyst surgery, more emphasis is now placed on the toxicity of these agents and the search for safe alternatives [10]. Significant amounts of research have recently been directed towards examining herbal extracts as a source of novel, powerful, and nontoxic anti-scolicidal compounds [11]. Numerous plant extracts and their essential oils, including *Mentha pulegium, Curcuma longa, Allium sativum*, *Nigella sativa*, *Zataria multiflora, Salvadora persica*, *Origanum minutiflorum*, and *Zingiber officinale* have been shown to carry significant scolicidal effects [2,8,11–13]

Globally, ectoparasites pose a significant risk to both the economy and animal health [14]. Ectoparasitic infestation has been linked to a variety of health issues, including anemia, weight loss, abscesses, and tissue damage. It can also serve as a vector for several deadly diseases of great concern to livestock [14,15]. Among the varieties of ectoparasites, ticks are considered a main threat due to the severe irritation, anemia, paralysis, and toxicosis they can cause, as well as the fact that they can transmit diseases such as anaplasmosis, theileriosis, and babesiosis [16–19]. Similarly, one of the most globally prevalent arthropods with medicinal and veterinary significance is the house fly (*Musca domestica* L.) [20,21]. This species is known to harbor more than 100 types of microorganisms, including bacteria, viruses, parasites, worms, and protozoa, which can lead to serious and potentially fatal diseases in both humans and domestic animals [22,23].

Chemically derived drugs are the primary approach used globally to manage ectoparasites and endoparasites affecting different kinds of animals. This has resulted in several serious problems, including the development of resistance [24,25], toxic damage to nonspecified organisms, and environmental pollution [26]. Consequently, new eco-friendly alternatives are being introduced into strategic parasite-monitoring programs [27]. The usage of essential oils and plant extracts as insect control agents has become the subject of intensive investigation in a number of countries because of the efficiency of their insecticidal and acaricidal effects, which have negligible environmental impacts [28,29]. These oils comprise combinations of chemical substances that are toxic to insects, and toxicity operates via a number of mechanisms including enzyme inhibition and protein denaturation [30]. It is known that several plants in the Mediterranean region possess insecticidal and acaricidal properties [31]. The genus "*Lavandula*" (Lamiaceae) is a wild plant found in the Mediterranean basin that comprises over 34 species and is well-known for having insecticidal effects against different species [32,33]. Along with being employed in conventional treatment, different species of genus "*Lavandula*" are also utilized in the pharmaceutical and cosmetic sectors [34,35]. One of the most widely studied and used lavender species in the world is *Lavandula stoechas* (L. stoechas) [36]. Some studies have focused on the antibacterial [37,38], antifungal [39,40], and antioxidative [40,41] characteristics of *L. stoechas.* Correspondingly, the objectives of the current study were to determine the following: (i) the total chemical constituents; (ii) the in vitro cytotoxic activities of *L. steochas*; (iii) the in vitro scolicidal activity of *L. steochas* against the protoscoleces of hydatid cysts; (iv) the in vitro adulticidal, larvicidal, and repellent activities of *L. steochas* against *R. annulatus* ticks; and (v) the in vitro larvicidal and pupicidal activities of *L. steochas* against *M. domestica.*

### **2. Materials and Methods**

#### *2.1. Plant Material*

In August 2017, the aerial components of *Lavandula stoechas* subsp. Stoechas were gathered from the city of Elmal-Antalya, and identification was performed by the plant taxonomist Dr. A¸skn Akpulat from the Faculty of Education, Cumhuriyet University, Sivas, Turkey. A voucher specimen was deposited at the Herbarium of the Department of Biology, Cumhuriyet University (CUFH). The leaves of the gathered plants were removed from the

stems and flowers, dried in the shade, and then crushed until they could pass through a 2 mm mesh.

#### *2.2. Essential Oil Extraction*

Dried and finely crushed leaves (100 g) were hydrodistilled for 3 h in a Clevenger-type distillation apparatus with 2 L of double-distilled water [41,42]. The produced EOs were filtered, dried over anhydrous sodium sulfate, and kept at 4 ◦C until use.

GC–MS analysis of Lavandula stoechas essential oil was performed using a Trace Ultra gas chromatograph, (GC) coupled with a DSQ II mass spectrometer (MS; Thermo Scientific). The compounds were separated on a TR-5MS (30 m × 0.25 mm × 0.25 μm) capillary column (Thermo Scientific), operating on a temperature program of 60 to 250 ◦C with an elevation speed of 3 ◦C/min and a helium flow rate of 1 mL/min. The injector and MS transfer line temperatures were set at 220 and 250 ◦C, respectively. The samples were prepared via the dilution of 1 mg of EO in 1 mL of acetone. In total, 1 μL of the diluted sample was injected manually in the splitless mode. The MS was operated in the EI mode at 70 eV. The ion source temperature was 240 ◦C, and the mass spectra were acquired in the scan mode based on a mass range of 35–400. We tentatively identified the compounds based on comparisons of their relative retention indexes and mass spectra with corresponding data found in the literature and different databases [42]. A series of n-alkanes (C8–C24) was used in the determination of the relative retention index (RRI). Relative percentages of the compounds were obtained electronically from area percentage data.

#### *2.3. Cytotoxic Activity of L. stoechas*

#### Cell Culture

Human embryonic kidney cells (HEK-293 cells) and the human breast cancer cell line MCF-7 were cultured in a DMEM culture medium accompanied with 10% fetal bovine serum (FBS), 0.2% sodium bicarbonate, and an antibiotic/antimycotic solution. The cells were grown in a CO2 incubator (5% CO2–95% atmosphere) at ±37 ◦C and a high humidity [43]. The trypan blue dye exclusion assay was used to determine the vitalities of all cell lines, and batches of cells with over 98% cell viability were utilized.

## *2.4. Cytotoxicity Assessment by MTT Assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide Tetrazolium)*

The cytotoxicity assessment was performed according to the protocol set out by Siddiqui et al. [43]. In brief, the cells were plated in 96-well culture plates and adhered for 24 h in a CO2 incubator at ±37 ◦C. Then, the cells were exposed to *L. steochas* oil in several concentrations (0.0156–1%) for 24 h. Following exposure, 10 mL of MTT (5 mg/mL of stock) was added to each well, and the plates were then incubated for a further 4 h in a CO2 incubator. After the supernatant was discarded, 200 mL of DMSO was added to each well and thoroughly mixed. The plates were read at a wavelength of 550 nm. The cytotoxic activity of *L. steochas* oil against the cancer cell line was inferred from the estimated LC50 value.

## *2.5. Scolicidal Activity*

#### Collection of Protoscoleces from Hydatid Cysts and the Viability Test

Using sheep livers that were naturally infected with hydatid cysts, the careful excision of the hydatid was performed. The hydatid fluid samples were aspirated with protoscoleces and kept in glass cylinders for 20 min to allow the protoscoleces to settle. The supernatant fluid was then discarded and the reaming protoscoleces were washed three times in saline solution. An eosin stain at 0.1% was used to confirm the viability of the protoscoleces. Staining was performed for five minutes and the protoscoleces that did not take on the dye were considered alive, while those that were stained were considered dead [44]. Protoscoleces with a viability of 95% were selected for further study.

## *2.6. Determination of In Vitro Scolicidal Activity*

To assess the in vitro activity, the protoscoleces treated with three concentrations of essential oil (0.025, 0.05, and 0.1%) were evaluated. A total of 2 mL of each concentration was placed into three tubes to which ~5 × 103 protoscolices were added, which were gently mixed. Then, the three tubes of each concentration (nine in total) were incubated at ±37 ◦C for 1, 3, and 5 min, respectively. After incubation, the supernatant was carefully removed to avoid unsettling the remaining protoscoleces. Then, the settled protoscoleces were gently mixed with 1 mL of 0.1% eosin. Stained protoscoleces were then examined under a microscope five minutes later to determine their vitality. A group of at least <sup>5</sup> × 103 protoscoleces in 2 mL of distilled water with no oil treatment was used as the control.

## *2.7. Acaricidal Activity of L. steochas against Larvae and Adult of R. annulatus*

In various villages in the Beni-Suef province of central Egypt, and further south towards Cairo (29◦04 N, 31◦05 E), adult engorged females of *R. annulatus* were collected from naturally infected cattle. The collected ticks were taken to the Parasitology Lab at the Faculty of Veterinary Medicine, Beni-Suef University. The ticks were identified according to the process of Estrada-Pena et al. [45]. A portion of the collected ticks were employed in the adult immersion test, while the remaining were incubated at 27 ± 1.5 ◦C and 70–80% relative humidity (RH) to produce eggs, which were then allowed to develop into larvae for use in subsequent bioassays.

## *2.8. Adult Immersion Test of R. annulatus*

*L. stoechas* essential oil was evaluated for adulticidal activities against adult female *R. annulatus* ticks taken from naturally infected cattle. This assay was performed following the method of Drummond et al. [46]. This test was performed over five replicates (ten ticks/replicate) for each concentration. The ticks of the control group were treated with ethanol 70% and deltamethrin 5%. Briefly, the ticks were immersed in 10 mL of each concentration in a Petri dish with a diameter of 7 cm at room temperature with occasional gentle agitation. After 2 min, the solution was discarded, and the female ticks were removed and gently dried on a paper towel. For ovipositioning, the treated ticks were maintained in a BOD incubator at a temperature of 27 ± 2 ◦C and a relative humidity of 80 ± 10%. On day 14 post-application (PA), the eggs deposited by the treated ticks were collected and weighed. After 14 days, the number of dead adult ticks was determined, and the egg production index (EPI) was calculated for the ticks that were still alive [47,48]. Egg production index (EPI) = weight of egg mass/initial weigh of engorged female × 100.

#### *2.9. Larvicidal Activity against R. annulatus*

The larvicidal activity of *L. stoechas* essential oil was assessed via application against the larvae of *R. annulatus* ticks using the larval packet technique (LPT) with the modifications suggested by Matos et al. [49]. In brief, about one hundred ten-day-old larvae were placed on the center of 7 × 7 cm filter paper, which was impregnated with 100 μL of each concentration and then folded into a pocket shape. After 24 h, the packets were checked to assess the mortality rates—motionless larvae were considered dead. The filter paper of the control groups was impregnated with ethanol (70%) and deltamethrin 5%. This test was conducted three times for each concentration.

## *2.10. Repellent Activity against R. annulatus*

This bioassay was based on the vertical migration behavior of tick larvae and was modeled after that elucidated by Wanzala et al. [50] with some modifications. In this assay, we used a device consisting of two aluminum rods (0.7 × 15 cm) and filter paper (7 × 7 cm) impregnated with 200 <sup>μ</sup>L (covering approximately 28 cm2) of the different concentrations. The treated filter paper was clipped to one rod, while on the other rode, filter paper impregnated with 70% ethanol was clipped, and this acted as a negative control. Nearly 30 ten-day-old *R*. *annulatus* larvae were placed at the base of each rod; the rods were observed after 15 min and after 1 h, and then followed up 4 h post-application. Larvae that were found on the tops of the impregnated filter paper were not considered repelled, while those at the base of the impregnated filter paper (the uncovered part of the rod) were considered repelled. This test was performed five times for each concentration.

$$\text{The repellerce } (\%) = \frac{\text{NC} - \text{NT}}{\text{NC}} \times 100$$

NC = number of larvae on the negative control; NT = number of larvae on the treated paper

## *2.11. Insecticidal Activity against Musca domestica* Rearing of Housefly Colony

Adult house flies were collected from a farm in Beni-Suef province, Egypt. The collected house flies were taken to the Parasitology Lab at the Faculty of Veterinary Medicine, Beni-Suef University. The flies were kept in plastic jars (35 × 15 cm) at 28 ± 2 ◦C and 60–70% relative humidity (RH), covered with muslin cloth. A cotton swab soaked in milk (10% *w*/*v*) was introduced as food to the adult flies, and this also served as a substratum for oviposition. For hatching and larval development, the eggs were transferred to a different set of jars containing animal feed or cotton swabs soaked in milk. Similarly, pupae were collected and maintained in a separate container until they emerged as adults. Larvae and pupae were used in the bioassays, as recommended by Jesikha [51] and Abdel-Baki et al. [52].

### *2.12. Larvicidal Bioassay against Musca domestica*

The residual film method, as set out by Busvine [53] and modified by Palacios et al. [54], was used to evaluate the larvicidal activity of *L. stoechas* essential oil. Briefly, 1 mL of each test solution was applied to filter paper discs placed in Petri dishes (90 mm diameter) in such a way as to generate a homogenous film. The treated Petri dishes were first air-dried for a short time to let the solvent evaporate, then the larvae (*n* = 10) were released, and finally the Petri dishes were kept under observation in the laboratory for 24 h. The positive control group was treated with deltamethrin at a concentration of 2 L/mL, while the negative control group was treated with acetone. Three replicates of each test were performed.

$$\text{Percentage mortality} = \frac{\text{Number of dead larvae}}{\text{Number of larvae introduced}} \times 100$$

## *2.13. Pupicidal Bioassay against Musca domestica*

In this bioassay, ten 2- to 3-day-old pupae were placed in a glass Petri dish. They then received a single application of 10 μL of each test solution [55]. Acetone was given to the negative control group, while the positive control group received deltamethrin at a concentration of 2 μL/mL. The Petri dishes were put in an incubator set to 28 ± 2 ◦C and 75–85% relative humidity. The treated pupae were monitored for six days to evaluate the emergence of adults. Three replicates of each test were performed. The adult emergence rate was evaluated following the method of Kumar et al. [56,57] and the percentage of inhibition rate (PIR) was calculated using the following equation:

$$\text{PIR} = \frac{\text{Number of newly emerged in Sect.} \times \text{Number of newly emerged in Sect.} + \text{Number of new ways.}}{\text{Number of newly employed insect in control}} \times 100$$

#### *2.14. Statistics*

For each treatment, three to five replicates were carried out and mean ± SE values were calculated. ANOVA was used to analyze larval mortality, followed by Duncan's multiple range test (*p* < 0.05). To determine the LC50 and LC90 values, as well as their 95% confidence limits, probit analyses were used [58]. SPSS for Windows (version 22.0) was used to conduct all statistical analyses.

#### **3. Results**

#### *3.1. Chemical Composition of the Essential Oil*

Hydrodistillation yielded a pale-yellow essential oil. The yield was 1.9% (*v*/*w*). Thirty-six compounds were identified, accounting for 95.56% of the EO's total volatile fraction. The GC–MS analyses of the *L. stoechas* essential oil showed that the predominant compound was camphor (58.38%), followed by fenchone (18.15%) and eucalyptol (6.93%). The fraction of oxygenated monoterpenes constituted almost 87% of the oil, while hydrocarbon monoterpenes and sesquiterpenes constituted about 4.8% and 3.4%, respectively (Table 1 and Figure 1).


**Table 1.** Chemical composition of the essential oil of *Lavandula stoechas*.

RT: Retention time. R.I.ex: Experimental retention index calculated on Rt-5MS column. R.I.lt: Retention index from the literature for relative columns. \* Components representing less than 0.1%. \*\* Correct isomer not identified. Unknown &: main m/z is given (% relative intensity). Comp No 5: 91 (100), 119 (58), 77 (40), 134 (32). Comp No 30: 143 (100), 157 (80), 200 (55), 128 (40), 185 (34). MH: Monoterpene hydrocarbon. OM: Oxygenated monoterpene. HS: Hydrocarbon sesquiterpene.

#### *3.2. Cytotoxicity Assessment*

Cell viability was affected by the highest concentrations of lavender, whereby the concentrations of 0.25, 0.5, and 1.0% showed toxicity against 80% of HEK-293 (Figure 2). Additionally, this compound was shown to be safe for normal cells up to a concentration of 0.0625%, at which point a strong cytotoxic effect appeared at around 0.125%. Lavender oil showed clear cytotoxic effects on the MCF-7 cell line, even at a low concentration of 0.0625%, causing cell death at a rate of 30%, while high concentrations caused significant cell death (Figure 3).

**Figure 1.** GC–MS chromatogram of *Lavandula stoechas* essential oil.

**Figure 2.** Cytotoxic activity of lavender oil against HEK-293 cells determined by MTT assay. Cells were exposed to different concentrations of the oil for 24 h. All values are presented as mean ± SD.

**Figure 3.** Cytotoxic activity of *L. steochas* EO against MCF-7 cells determined by MTT assay. Cells were exposed to different concentrations of EO for 24 h. All values are presented as mean ± SD.

#### *3.3. In Vitro Scolicidal Activity*

Table 2 displays the scolicidal effects of the *L. stoechas* essential oil at various concentrations and with different exposure times. At a concentration of 0.025%, the scolicidal effectivity values of *L. stoechas* oil were 33.66, 50.4, and 88.07% after 1, 3, and 5 min, respectively. The values at a concentration of 0.05% were 41.07, 72.47, and 98.17% after 1, 3, and 5 min respectively. After 3 and 5 min of exposure, an oil concentration of 0.1% caused 95.5% and 100% mortality, respectively (Figure 4). Overall, the scolicidal activity of the oil was clearly concentration- and time-dependent.


**Table 2.** Scolicidal effect of *Lavandula stoechas* essential oil on the viability of *E. granulosus* protoscolices.

Means within the same column followed by different superscripts are significantly different (Duncan's multiple range test: *<sup>p</sup>* ≤ 0.05). LC = lethal concentration, CL = confidence limit, X2 = chi square, df = degree of freedom.

**Figure 4.** Live non-stained protoscolices (**A**), live protoscolices after staining with 0.1% eosin (**B**), dead protoscolices after treatment with *L. steochas* EO and staining with 0.1% eosin (**C**). Scale-bar = 100 mm.

#### *3.4. Acaricidal Activity L. stoechas EO against Adult and Larvae of R. annulatus Ticks*

*L. stoechas* EO showed significant adulticidal activity against *R*. *annulatus* ticks, especially at concentrations of 5 and 10%, showing tick mortality rates of 86.66 and 100%, respectively, and the LC50 was 2.34%. Moreover, the egg production index of the treated groups showed lower values compared to those of the control, i.e., the untreated ticks (Table 3).

**Table 3.** Adulticidal and lethal concentrations (LC50, LC90) of *Lavandula stoechas* against *R. annulatus* adult ticks.


Means within the same column followed by different superscripts are significantly different (Duncan's multiple range test: *<sup>p</sup>* ≤ 0.05). LC = lethal concentration, CL = confidence limit, X2 = chi square, df = degree of freedom.

Regarding larval toxicity, *L. stoechas* oil achieved a larvicidal activity of 86.7% at the highest concentration (10%), with an LC50 of 9.11% (Table 4).

**Table 4.** Larvicidal activity and lethal concentrations (LC50, LC90) of *Lavandula stoechas*, against larvae of *R. annulatus*.


Means within the same column followed by different superscripts are significantly different (Duncan's multiple range test: *<sup>p</sup>* ≤ 0.05). LC = lethal concentration, CL = confidence limit, X2 = chi square, df=degree of freedom.

Moreover, regarding the repellence activity of *L. stoechas*, we found a weak repellency in the first hour, even at the highest concentration of 10%, and no repellent effect was seen in the succeeding hours (S Table 1). There were no significant differences in results between the low concentrations and the control, treated with 70% ethyl alcohol.

## *3.5. Insecticidal Effect of L. stoechas against Larvae and Pupae of Musca domestica*

The larval toxicity of *L. stoechas* oil, assessed via the residual film method, increased significantly with increasing concentrations. Within 24 h after application, the lavendertreated groups showed significant mortality at 5 and 10% concentrations, with rates of 93.33 and 100%, respectively, and an LC50 value of 1.79%. The treated larvae died within 24 h, with clear blackening of the cuticles. Moreover, in the deltamethrin-treated and negative control groups, no larvicidal activities were observed whatsoever (Table 5).

Regarding pupal toxicity, *L. stoechas* oil achieved a percentage inhibition rate (PIR), ranging from 13.33% to 100% at various concentrations after six days of application with an LC50 value of 1.51%. Moreover, a concentration of 10% *L. steochas* essential oil led to the complete inhibition of adult emergence, and the dead pupae displayed darker colors (Table 6).


**Table 5.** Larvicidal activity, and lethal concentrations (LC50, LC90) of *Lavandula stoechas*, against larvae of *Musca domestics*.

Means within the same column followed by different superscripts are significantly different (Duncan's multiple range test: *<sup>p</sup>* ≤ 0.05). LC = lethal concentration, CL = confidence limit, X2 = chi square, df = degree of freedom.

**Table 6.** Percentage inhibition rate (PIR) of *Lavandula stoechas* against housefly pupae in contact toxicity assay.


Means within the same column followed by different superscripts are significantly different (Duncan's multiple range test: *<sup>p</sup>* ≤ 0.05). LC = lethal concentration, CL = confidence limit, X2 = chi square, df = degree of freedom.

#### **4. Discussion**

Synthetic chemicals have been widely used to control parasitic infections, but their indiscriminate and excessive usage has resulted in drug resistance, as well as detrimental effects to the environment and food supply [25,26,57]. Plant essential oils (and/or active components) can be used as natural alternatives or adjuncts to current therapies used against a variety of ectoparasites and endoparasites of medical/veterinary significance [52,58–61]. The use of essential oils as therapeutic agents is more affordable, effective, and safe [14,62]. As such, the present work was designed to investigate the in vitro acaricidal, insecticidal, and scolicidal activities of *L. steochas* essential oil against certain cell lines, as well as its safety.

Our GC–MS analyses revealed camphor and fenchone as the main components of *L. steochas* essential oil, accounting for 58.38% and 18.15%, respectively, followed by 6.93% eucalyptol and 2.04% camphene. These outcomes agree with those of several previous studies, which showed that the predominant components of *L. steochas* oil are camphor and fenchone [63–67]. The timing of plant collection, the duration of hydrodistillation, the selection of plant parts to be used, and environmental factors were all found to have a significant impact on the essential oil's yield and composition [68].

The in vitro cytotoxic activity of *L. steochas* EO against human embryonic kidney cells (HEK-293 cells) and human breast cancer cell line (MCF-7) MCF-7 cells was assessed in the present investigation. The results demonstrate that *L. steochas* oil was extremely cytotoxic to HEK-293 and MCF-7 cells, even at low concentrations. Our results corroborate those found in [69], who noted that *L. stoechas* flowers were cytotoxic to Allium cepa root-tip meristem cells. According to Siddiqui et al. [70], the cytotoxic effect of *L. stoechas* EO, perhaps resulting from apoptosis, apparently induces deformations in the nuclei and cell membrane. The ethanolic fraction of *L. stoechas* has an anticancer effect, which may be attributed to the presence of phytosterols [70]. Furthermore, it strongly inhibits the growth of human gastric adenocarcinoma (AGS), melanoma MV3, and breast cancer MDA-MB-231 cells, with median inhibitory concentrations (IC50) of 0.035 ± 0.018, 0.06 ± 0.022, and 0.259 ± 0.089 μL/mL, respectively [71].

We assessed the in vitro scolicidal efficacy of *L. steochas* EO at a variety of concentrations and exposure times on the protoscoleces of hydatidosis. The findings show that all tested concentrations of *L. steochas* EO had significant scolicidal activities, with 98.17% and 100% mortality achieved at doses of 0.05% and 0.1%, respectively, at 5 min post-treatment. The results of the current investigation suggest that *L. steochas* is a potential natural source of novel protoscolicidal agents that could be used in hydatid cyst surgery.

The current study revealed that *L. steochas* EO induced 100% mortality in *R. annulatus* adult ticks at concentration of 10%, with an LC50 value of 2.34%. Regarding the larvicidal activity, 86.67% larval death was achieved at a concentration of 10% with an LC50 of 9.11%. Similarly, *L. stoechas* EO showed significant acaricidal activities against adults and larvae of the *Hyalomma suspense* tick [10]. Other species of lavender have acaricidal activities. *Lavandula angustifolia* is effective against *Rhipicephalus* (*Boophilus*) *annulatus*) [11], and *Lavandula luisieri* has larvicidal effects against *Hyalomma lusitanicum* [12]. Additionally, Sertkaya et al. [13] found that *L. steochas* EO had an acaricidal effect against the red spider mite *Tetranychus cinnabarinus*. It was surprising, however, to see that *L. steochas* essential oil has no repellent effect against larvae of *R. annulatus*. Other studies did identify a repellent effect of *L. steochas* and other types of lavender. *Lavandula angustifolia* shows repellency against *Hyalomma marginatum* adults [72] and nymphs of *Ixodes ricinus* (L.) (Acari: Ixodidae) [73]. This discrepancy may be due to the tick species and tick stages used in our tests.

In terms of the insecticidal activity against *M. domestica*, our results indicate that *L. steochas* EO exhibits maximum efficacy (100%) against house fly larvae at a concentration of 10% with an LC50 of 1.79% at 24 h post-application. Additionally, a 10% concentration of *L. steochas* EO showed the highest level of toxicity against house fly pupae, and completely inhibited adult emergence (100% PIR). Essential oils from the genus *Lavandula* have also shown insecticidal efficacy against several insect species. The essential oil of *L. stoechas*, a member of this genus, showed significant toxic effects against the adults and/or larvae of *Anopheles labranchiae*, *Culex pipiens molestus*, and *Orgyia trigotephras* [33,67,74]. The essential oils of *L. dentate* and *L. angustifolia*, also belonging to the same genus, exhibited significant insecticidal actions against larvae of *M. domestica* and *Chrysoma albiceps* and have thus been suggested for use as a safe and effective natural means to control these dipterans [75–80]. Bosly [79] observed morphological abnormalities in *M. domestica* larvae after treatment with *Lavandula* spp. EOs and attributed these deformities to hormonal imbalances that interrupt insect metamorphosis. Meanwhile, Khater and Khater [81] found that the oil may limit larval motility and prevent the larvae from constricting during the pupal stage, thus contributing to the observed deformities. According to Conti et al. [31] and Sajfrtova et al. [82], the concentration of volatile compounds in the oil is directly related to the toxic effects found. It is difficult to compare the efficacy of essential oils across different studies because the methods used for oil extraction vary greatly, and this impacts the subsequent essential oils' efficacy [79].

Generally, the presence of volatile components in essential oils is largely responsible for the oils' acaricidal, insecticidal, and cytotoxic actions [80–82]. For instance, the main component of *L. stoechas* EO, camphor, has been proven to have insecticidal effects [83,84] and creates a fragrant vapor that repels mosquitoes [66,67]. Another key ingredient in *L. stoechas* EO, camphene, also has insecticidal activities resulting from its ability to repel insects, including flies and moths [63,64,83,84]. All of this can thus explain *L. stoechas*' effectiveness as an acaricidal, insecticidal, scolicidal, and anticancer agent.

In conclusion, *L. stoechas* EO has potential applicability as a potent protoscolicidal agent with high effectiveness at low doses and in shorter times. However, more research is required to fully assess the potential use of this oil in the prevention and treatment of cystic echinococcosis, including via in vivo assays and its main components. Additionally, this oil shows significant activity against *M. domestica* and *R. annulatus* ticks, which pose a substantial risk to public health; camphor is the first known substance to exhibit this activity. The mechanisms of action of these essential oils are poorly understood. One of the theories is that the monoterpenes operate on other sensitive locations, such as the neurological system, but further research is required to verify and expand on this.

**Author Contributions:** Conceptualization, A.-A.S.A.-B., S.M.A. and A.A.K.; methodology, A.A.K., A.S. and S.M.A.; software, D.D. and A.O.H.; validation, A.S., D.D. and A.A.K.; formal analysis, A.A.K. and A.S.; investigation, A.-A.S.A.-B., A.A.K. and S.M.A.; resources, S.A.-Q.; data curation, D.D. and A.S.; writing—original draft preparation, A.A.K. and S.M.A.; writing—review and editing, A.-A.S.A.-B., S.A.-Q. and S.M.A.; visualization, A.O.H. and S.A.-Q.; supervision, A.-A.S.A.-B. and S.M.A.; project administration, S.A.-Q.; funding acquisition, S.A.-Q. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Researcher supporting Project [RSP-2021/3], King Saud University.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All relevant data are within the paper and its supporting information.

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

## **References**


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## *Article* **Greener Stability-Indicating HPLC Approach for the Determination of Curcumin in In-House Developed Nanoemulsion and** *Curcuma longa* **L. Extract**

**Nazrul Haq 1, Faiyaz Shakeel 1, Mohammed M. Ghoneim 2, Syed Mohammed Basheeruddin Asdaq 2, Prawez Alam 3, Saleh A. Alanazi 4,5 and Sultan Alshehri 6,\***

	- <sup>4</sup> King Abdullah International Medical Research Center, College of Pharmacy, King Saud Bin Abdulaziz University for Health Sciences, Riyadh 11481, Saudi Arabia
	- <sup>5</sup> Pharmaceutical Care Department, King Abdulaziz Medical City, Ministry of National Guard Health Affairs, Riyadh 11426, Saudi Arabia

**Abstract:** Despite the fact that several analytical methodologies have been reported for the determination of curcumin (CCM) in a wide range of sample matrices, the greener liquid chromatographic approaches to determine CCM are scarce in the literature. Therefore, this research is designed to develop and validate a greener stability-indicating "high-performance liquid chromatography (HPLC)" methodology to determine CCM in an in-house developed nanoemulsion, *Curcuma longa* L. extract, and commercial tablets. CCM was measured on a Nucleodur (150 mm × 4.6 mm) RP C18 column with 5 μm-sized particles. Ethanol and ethyl acetate (83:17 *v/v*) made up the greener eluent system, which was pumped at a flow speed of 1.0 mL/min. At a wavelength of 425 nm, CCM was detected. The greener HPLC methodology was linear in the 1–100 μg/mL range, with a determination coefficient of 0.9983. The greener HPLC methodology for CCM estimation was also rapid (Rt = 3.57 min), accurate (%recoveries = 98.90–101.85), precise (%CV = 0.90–1.11), and sensitive (LOD = 0.39 μg/mL and LOQ = 1.17 μg/mL). The AGREE approach predicted the AGREE score of 0.81 for the established HPLC technique, indicating an outstanding greenness profile. The utility of the greener HPLC methodology was demonstrated by determining CCM in the in-house developed nanoemulsion, *Curcuma longa* extract, and commercial tablets. The % amount of CCM in the in-house developed nanoemulsion, *Curcuma longa* extract, and commercial tablets was found to be 101.24%, 81.15%, and 78.41%, respectively. The greener HPLC methodology was able to detect its degradation product under various stress conditions, suggesting its stability-indication characteristics. These results suggested that CCM in developed nanoemulsion, plant extract samples, and commercial tablets may be routinely determined using the greener HPLC methodology.

**Keywords:** AGREE; *Curcuma longa*; curcumin; nanoemulsion; greener HPLC; validation

## **1. Introduction**

Turmeric is a spice, which is obtained from *Curcuma longa* L [1]. It is a rich source of phenolic compounds, namely curcuminoids [1,2]. Three main curcuminoids have been reported in *C. longa* extract: curcumin (CCM) (Figure 1), demethoxycurcumin (DCCM), and bisdemethoxycurcumin (BDCCM) [1,3]. Marketed CCM contains CCM, DCCM, and BDCCM, but its main constituent is CCM [4]. Various therapeutic activities have been reported for *C. longa* extract, which are mainly due to the presence of curcuminoids [1,4].

**Citation:** Haq, N.; Shakeel, F.; Ghoneim, M.M.; Asdaq, S.M.B.; Alam, P.; Alanazi, S.A.; Alshehri, S. Greener Stability-Indicating HPLC Approach for the Determination of Curcumin in In-House Developed Nanoemulsion and *Curcuma longa* L. Extract. *Separations* **2023**, *10*, 98. https://doi.org/10.3390/ separations10020098

Academic Editor: Josef Cvaˇcka

Received: 11 December 2022 Revised: 24 January 2023 Accepted: 28 January 2023 Published: 1 February 2023

**Copyright:** © 2023 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/).

CCM is a yellow-colored pigment, which is used for the coloring of various food products [4]. In the literature, the variety of therapeutic activities of CCM are reported, including antioxidant [1,2], anti-inflammatory [5], antimicrobial [6], antibacterial [7], antiviral [8], antiparasitic [9], antimutagenic [10], and antiproliferative activities [11–13] etc. As a consequence, the quality control and standardization of CCM in its pharmaceutical products, food products, and herbal extracts are significant due to its variety of therapeutic activities.

**Figure 1.** Molecular structure of curcumin (CCM).

Different analytical techniques have been utilized for the qualitative and quantitative detection of CCM in herbal extracts, commercial pharmaceutical formulations, commercial food products, and biological materials. For the determination of CCM in food products, spices, and herbal extracts, various ultra-violet (UV) spectrometry methods have been reported [14–16]. A spectrofluorometric assay has also been reported to determine CCM in a nanoliposomal formulation and mice plasma samples [17]. Fluorescence detection of CCM has also been carried out in the literature [18,19]. Reported spectrometry and fluorescence methods of CCM measurement were less accurate and sensitive than the current method [14–16,18,19]. The variety of high-performance liquid chromatographic methods (HPLC) have also been used to determine CCM in food products, pharmaceutical products, and herbal extracts [4,20–27]. However, most of the reported HPLC methods were environmentally toxic and less sensitive than the current HPLC method [4,20,25–27]. CCM in food products and herbal extracts has also been identified using liquid chromatography tandem mass-spectrometry (LC-MS) and ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS) approaches [28–31]. Various high-performance thin-layer chromatography (HPTLC) approaches are also reported to determine CCM in herbal extracts and CCM dosage forms [32–37]. Some LC-MS and UPLC-MS approaches have also been used to determine CCM in the plasma samples of rat, equine, and human [38–41]. Carbon nanotube-based composites have also been used to determine CCM in food products [42,43]. Reported LC-MS, UPLC-MS, and HPTLC approaches were also found to be more environmentally toxic than the current HPLC method [28–43]. Some voltammetry approaches have also been reported to determine CCM in natural food supplements and food spices [44–46]. Some electrochemical approaches were also used to determine CCM in its pure form and human blood serum sample [47,48]. Some other techniques, such as the Fourier transform near infrared spectroscopy approach, nanosensor, and solvatochromic approach have also been reported to determine CCM [49–51].

The detailed literature survey revealed the wide range of analytical techniques for the determination of CCM in distinct sample matrices. However, the greener/sustainable HPLC approaches of CCM detection are scarce in the literature. Furthermore, no greenness index of any reported HPLC method of CCM analysis has been reported. Several qualitative and quantitative approaches are reported to evaluate the greener characteristics of analytical procedures [52–56]. However, the "analytical GREEnness (AGREE)" metric methodology exclusively considers all twelve green analytical chemistry (GAC) principles for the determination of the greenness profile [54]. As a result, the "AGREE metric methodology" was used for the determination of the greener profile of the present HPLC assay of CCM analysis [54]. Based on all these assumptions, the objective of this research was to design and validate a simple, rapid, and greener HPLC methodology for the determination of CCM in an in-house developed CCM nanoemulsion, *C. longa* extract, and commercial

tablets. The greener HPLC methodology for determining CCM was validated, following the "International Council for Harmonization (ICH)-Q2-R1" procedures [57].

#### **2. Materials and Methods**

### *2.1. Materials*

The working standard of CCM was obtained from "E-Merck (Darmstadt, Germany)". HPLC-grade solvents, such as methanol, ethyl acetate, acetone, and ethanol were provided by "Sigma Aldrich (St. Louis, MO, USA)". High-pure water was collected from "Milli-Q® water purifier (Millipore, Lyon, France)". All other chemicals and reagents used were of analytical grades. The fresh rhizomes of *Curcuma longa* were purchased from the hypermarket in Riyadh, Saudi Arabia. The commercial tablets (having 1000 mg of standardized *C. longa* extract) were obtained from a pharmacy shop in Riyadh, Saudi Arabia. The nanoemulsion formulation of CCM was developed in the laboratory by the aqueous phase titration method using clove oil (oil phase), Tween-20 (surfactant), Transcutol-HP (cosurfactant), and purified water (aqueous phase).

## *2.2. Instrumentation and Analytical Conditions*

Waters HPLC system, composed of a 1515 isocratic pump, a 717 automatic sampler, quad LC-10A VP pumps, a programmable UV-visible variable wavelength detector, a column oven, an SCL 10AVP system controller, and an inline vacuum degasser, was used to measure CCM at a temperature of 25 ± 1 ◦C. The Millennium program (Version 32, Waters, Milford, MA, USA) was used to process and evaluate the data. CCM was determined using a Nucleodur (150 mm × 4.6 mm) RP C18 column with 5 μm-sized particles. The mixture of ethanol and ethyl acetate (83:17% *v/v*) was used as the greener eluent system. The eluent system flowed with a flow speed of 1.0 mL/min. At a wavelength of 425 nm, CCM was detected. The samples (20 μL) were injected into the system using a waters autosampler.

### *2.3. CCM Calibration Curve*

In triplicates (*n* = 3), the appropriate amount of CCM was dispensed in the eco-friendly eluent system to produce a stock solution with a 200 μg/mL concentration. To obtain the serial dilutions in the required range (1–100 μg/mL) in triplicates (*n* = 3), the requisite aliquots from the stock solution of CCM (200 μg/mL) were diluted with the greener eluent system. Using the greener HPLC methodology, the chromatographic response for each concentration of CCM was identified. To produce the CCM calibration curve, CCM concentrations were plotted against the measured chromatographic response in triplicates (*n* =3).

## *2.4. Sample Preparation for the Determination of CCM in Curcuma Longa Extract*

Approximately 10.0 g of fresh rhizomes of *C. longa* were taken and powdered finely. The fine powder was soaked in 100 mL of ethanol-water mixture (50:50 *v/v*) and ultrasonicated for about 60 min. The temperature was kept constant at 25 ◦C. The supernatant was recovered and filtered using nylon filter paper. The solvents were evaporated using a rotary vacuum evaporator at 40 ◦C. The obtained sample was used to determine CCM in *C. longa* extract using the greener HPLC approach.

## *2.5. Analytical Method Development*

As the eluent systems, different combinations of greener solvents were examined to develop a trustworthy stability-indicating greener HPLC assay for the detection of CCM in an in-house developed nanoemulsion, *C. longa* extract, and commercial tablets. The greener solvent compositions of methanol-water, ethanol-water, acetone-water, methanol-ethanol, ethyl acetate-methanol, ethyl acetate-ethanol, ethyl acetate-acetone, acetone-ethanol, and acetone-methanol were among the numerous greener solvents that were examined. Various aspects were taken into account when determining the best greener eluent system, including the affordability, greenness/toxicity profile, the assay's sensitivity, the analysis duration, the chromatographic parameters, and the solvents' compatibilities with one another. As a consequence, various greener solvent compositions were examined as the eluent system in combined forms. Finally, the most trustworthy eluent system for future investigation was discovered to be a 83:17 volume-to-volume blend of ethanol and ethyl acetate.

## *2.6. Validation Parameters*

Following ICH-Q2-R1 procedures, the greener HPLC technique for the measurement of CCM was verified for various parameters [57]. By drawing the linearity graphs, the linearity of the greener HPLC methodology was examined in the 1–100 μg/mL range. CCM solutions that had just been prepared were added to the HPLC system in triplicates (*n* = 3), and the chromatographic response was recorded. The CCM calibration curve was derived by plotting the CCM concentration vs. chromatographic response.

The system appropriateness parameters for the greener HPLC technology were derived using a number of chromatographic characteristics, including resolution (Rs), selectivity factor (α), tailing factor (As), capacity factor (k), and theoretical plates number (N) [58,59].

The intra-day and inter-day accuracy of the greener HPLC technology was assessed using a standard addition/spiking method in terms of % recovery [57]. To investigate intra-day accuracy at three different quality control (QC) levels, the pre-analyzed QC level of CCM (10 μg/mL) was spiked with an extra 50, 100, 150% of CCM solution to obtain low QC (LQC = 15 μg/mL), middle QC (MQC = 20 μg/mL), and high QC (HQC = 25 μg/mL). The obtained LQC, MQC, and HQC of CCM were analyzed on the same day in three replicates (*n* = 3) to measure intra-day accuracy. On three distinct days, three replicates (*n* = 3) of CCM's LQC, MQC, and HQC levels were obtained by the spiking method and used to examine inter-day accuracy. The percentage recovery, percentage coefficient of variance (%CV), and standard error were computed at each QC level.

The greener HPLC methodology's precision was examined using intra-day and interday variations. On the same day, three replicates (*n* = 3) of the LQC, MQC, and HQC levels of CCM were used to assess the intra-day precision. At the LQC, MQC, and HQC of the CCM on three distinct days, inter-day precision was assessed in three replicates (*n* = 3).

To examine the influence of intentional chromatographic alterations on CCM measurement, the robustness of the greener HPLC methodology was evaluated. The CCM MQC (20 μg/mL) was chosen for the robustness assessment. By altering the greener eluent's composition, flow speed, and detecting wavelength, robustness was assessed. The initial ethanol: ethyl acetate (83:17 *v/v*) eluent system was altered to ethanol: ethyl acetate (85:15 *v/v*) and ethanol: ethyl acetate (81:19 *v/v*) for the robustness assessment, and the differences in chromatographic response were recorded. For the purpose of evaluating robustness, the original flow speed of 1 mL/min was changed to flow rates of 1.15 mL/min and 0.85 mL/min, and the changes in chromatographic response were noted. For the robustness assessment, the initial detection wavelength (425 nm) was changed to detection wavelengths of 430 nm and 420 nm, and the differences in chromatographic response were recorded.

The standard deviation approach was used to evaluate the sensitivity of the greener HPLC methodology in terms of "limit of detection (LOD) and limit of quantitation (LOQ)" [57]. The standard deviation of the blank sample (without CCM) was computed following the injection of the blank sample into the HPLC apparatus three times (*n* = 3). After that, established techniques that have been documented in the literature were used to calculate the LOD and LOQ values for CCM [57,58].

The solution stability of CCM in stock solution and nanoemulsion was performed at the MQC level (20 μg/mL) at two distinct temperatures, namely, the bench temperature (25 ± 0.5 ◦C) and refrigeration temperature (4 ± 0.5 ◦C). These studies were carried out for the period of 72 h. The MQC concentration of CCM was freshly produced in a greener eluent system. The freshly prepared nanoemulsion was also diluted with the greener eluent system to obtain the MQC level of CCM. Both solutions were stored at 25 ± 0.5 ◦C and

4 ± 0.5 ◦C for 72 h and the decomposition of CCM was evaluated by measuring the rest of CCM after storage.

#### *2.7. Forced Degradation/Selectivity Studies*

Forced degradation studies under a variety of stress conditions, including acidic (HCl) stress, basic (NaOH) stress, oxidative (H2O2) stress, thermal stress, and photolytic stress conditions, were conducted in order to evaluate the selectivity and stability-indicating property of the greener HPLC methodology [56,60]. The degradation studies were performed in mild conditions, as recommended by ICH [57]. The 40 μg/mL of CCM was freshly prepared into the eluent system for acid and base-induced degradation. By mixing 4 mL of 1 M HCl and 4 mL of 1 M NaOH into an aliquot (1 mL) of this solution, acid and base hydrolysis were applied. For the determination of CCM in the presence of its acidand base-degradation products, respectively, these mixtures were refluxed for 48 h at 60 ◦C before being evaluated using the greener HPLC approach [60].

The 40 μg/mL of CCM was freshly produced and introduced into the eluent system for oxidative degradation testing. This solution was oxidatively degraded by adding 4 mL of 30% H2O2 to an aliquot (1 mL) of it. For the detection of CCM in the presence of its oxidative-degradation products, this mixture was refluxed for 48 h at 60 ◦C before being evaluated using the greener HPLC approach [60].

The 40 μg/mL concentration of CCM (1.0 mL) was diluted with eluent system to produce a total volume of 5.0 mL. This solution was then subjected to a hot air oven for 48 h at 60 ◦C for thermal degradation tests. It was then assessed utilizing the ecofriendly HPLC technology for the detection of CCM in the presence of its thermal-degradation products [60].

For photolytic degradation investigations, a 1.0 mL aliquot of 40 μg/mL concentration was diluted with the eluent system to obtain the total volume of 5.0 mL. This solution was then subjected to a UV chamber at 254 nm for 48 h. Then, CCM was determined using the ecofriendly HPLC methodology while the photolytic-degradation products were present [60].

## *2.8. Greenness Measurement*

The greenness profile for the ecofriendly HPLC methodology was determined using the "AGREE metric approach" [54]. The AGREE scores (0.0–1.0) were derived using the "AGREE: The Analytical Greenness Calculator (version 0.5, Gdansk University of Technology, Gdansk, Poland, 2020)".

## *2.9. Application of Greener HPLC Methodology in Determination of CCM in In-House Developed Nanoemulsion*

In the lab, a CCM nanoemulsion was created and evaluated. One mL of an in-house developed nanoemulsion containing 20 mg/mL of CCM was appropriately diluted with the eluent system to produce 100 mL of stock solution in order to determine the CCM content. Following a suitable dilution with the eluent system and a 15-min sonication of this solution, the CCM content was determined using the ecofriendly HPLC approach. The potential for interference from components of the formulation that are nanoemulsions was also investigated.

#### *2.10. Application of Greener HPLC Methodology in Determination of CCM in Curcuma Longa Extract*

An amount of 1 mL of freshly prepared *Curcuma longa* extract was appropriately diluted with the eluent system to create 50 mL of stock solution in order to measure the CCM content. Following a suitable dilution with the eluent system and a 15-min sonication of this solution, the CCM content was determined using the ecofriendly HPLC approach.

#### *2.11. Application of Greener HPLC Methodology in Determination of CCM in Commercial Tablets*

The average mass of ten marketed tablets—each having 1000 mg of *C. longa* standardized extract—was determined. Ten tablets were crushed using a glass pestle and mortar

to obtain fine powder. An amount of 100 mL of the eluent system was combined with a portion of the powder containing an average weight of the tablet. Then, 1 mL of this solution was added to 50 mL of the eluent system. To eliminate any insoluble excipient, the obtained solution of the commercial tablet was filtered using Whatman filter paper (No. 41) and sonicated at 25 ◦C for 25 min. Using the eco-friendly HPLC approach, the amount of CCM in commercial tablets was determined.

#### **3. Results and Discussion**

#### *3.1. Analytical Method Development*

Table 1 provides a summary of the measured chromatographic characteristics and the composition of distinct greener eluent systems. The use of methanol-water, ethanol-water, and acetone-water in different compositions during the analytical method development step resulted in a subpar chromatographic response of CCM, which was exhibiting higher As values (As > 2.0) with low N values (<2000). Furthermore, the use of methanol-ethanol, methanol-acetone, and methanol-ethyl acetate in distinct compositions caused CCM to have a poor chromatographic response, as well as increased As values (As > 1.20) and low N values (<3000). Further, the use of ethanol-acetone and ethyl acetate-acetone was also examined as the greener eluent systems. With high As values (As > 1.80) and low N values (<2500), the chromatographic response of CCM was once more subpar. However, a well-resolved and intact CCM chromatographic peak with good As values and greater N values was shown by the binary mixture of ethanol and ethyl acetate in distinct composition. The binary mixture of ethanol and ethyl acetate (83:17 *v/v*) gave the best chromatographic response and reliable retention time (Rt), as well as As and N values, among the various ethanol and ethyl acetate mixes examined (Figure 2). As a consequence, the binary combination of ethanol and ethyl acetate (83:17 *v/v*) was selected as the final greener eluent system for measuring CCM with an appropriate As (1.09) and N (5081), quick analysis (Rt = 3.57 min), and a good analysis period (5 min). The finally used solvents, such as ethanol and ethyl acetate, are non-toxic and environmentally safe [61,62]. Both solvents have been extensively studied as green solvents in the literature [58–62]. As a result, these solvents were used in this study to create a greener HPLC approach to determine CCM.

**Figure 2.** A greener high-performance liquid chromatography (HPLC) chromatogram of CCM in solution derived using ethanol: ethyl acetate (83:17 *v/v*) greener eluent system.


**Table 1.** The optimization of greener eluent systems and measured analytical responses for standard curcumin (CCM) (mean ± SD, *n* = 3).

As: tailing factor; N: number of theoretical plates; Rt: retention time.

#### *3.2. Validation Parameters*

Numerous validation parameters for the greener HPLC methodology were assessed following ICH-Q2-R1 procedures [57]. The linearity graphs were produced using freshly prepared CCM samples (1–100 μg/mL). The outcomes of a linear regression analysis of the CCM calibration curve are summarized in Table 2. The linear calibration curve for CCM was between 1 and 100 μg/mL. According to estimates, the calibration curve's determination coefficient (R2) and regression coefficient (R) values are 0.9983 and 0.9991, respectively, suggesting a good relation between CCM concentrations vs. the measured response. These data demonstrated the efficiency of the greener HPLC methodology for determining CCM.

**Table 2.** Linear regression data for the calibration curve of CCM for the greener high-performance liquid chromatography (HPLC) methodology (mean ± SD, *n* = 3).


R2: determination coefficient; R: regression coefficient; SE: standard error; CI: confidence interval; LOD: limit of detection; LOQ: limit of quantification.

The system suitability parameters for the greener HPLC methodology were estimated using the Rs, α, As, k, and N and results are summarized in Table 3. The greener HPLC methodology's values for Rs, peak symmetry, α, As, k, and N were found to be satisfactory and trustworthy for determining CCM.

**Table 3.** Optimized chromatographic peak parameters to determine CCM for the greener HPLC methodology (mean ± SD, *n* = 3).


Rs: resolution; α: selectivity; As: tailing factor; k: capacity factor; N: number of theoretical plates.

The percent recovery at three distinct QC levels was used to evaluate the intra-day and inter-day accuracy of the greener HPLC methodology. The results are summarized in Table 4. At three distinct QC levels, the intra-day and inter-day percent recoveries of CCM were determined to be 99.84–101.85 and 98.90–101.44 percent, respectively. High percent recoveries for the greener HPLC methodology for determining CCM point to its accuracy.

**Table 4.** Intra-day and inter-day accuracy data of CCM for the greener HPLC methodology (mean ± SD; *n* = 3).


The results of the intra-day and inter-day precisions are included in Table 5 and are expressed in %CV. For CCM, the intraday precision percent CVs were observed to range from 0.86 to 0.94%. Contrarily, the %CVs for inter-day precision ranged between 0.96 and 1.17%. Low %CVs in the greener HPLC methodology for determining CCM indicated its precision.

**Table 5.** Intra-day and inter-day precision data of CCM for the greener HPLC approach (mean ± SD; *n* = 3).


Table 6 summarizes the outcomes of the robustness evaluation for the MQC level of CCM. When evaluating robustness by altering the composition of the eluent system, the %CV and Rt were found to be 1.02–1.28% and 3.55–3.59 min, respectively. The %CV and Rt were calculated to be 1.16–1.18% and 3.23–3.81 min, respectively, in the scenario of a robustness assessment when the flow speed was altered. The %CV and Rt were found to be 1.14–1.22% and 3.54–3.60 min, respectively, in the scenario of the robustness assessment by altering the detecting wavelength. Low CVs and minimal Rt value variation in the greener HPLC methodology for detecting CCM point to its robustness.

The results of analyzing the sensitivity of the ecofriendly HPLC methodology in terms of "LOD and LOQ" are presented in Table 2. According to calculations, the "LOD and LOQ" using the ecofriendly HPLC approach are 0.39 ± 0.03 μg/mL and 1.17 ± 0.09 μg/mL, respectively. These results indicated that the ecofriendly HPLC technology would be sensitive enough to identify and measure CCM in a wide range of concentrations.

The stability of CCM in stock solution and nanoemulsion formulation at two distinct temperatures was also determined. The results of stability determination at two distinct temperatures are presented in Table 7. The CCM degradation was determined by measuring the rest of CCM concentration after storage. The CCM decomposition was very low when stored for 72 h at 25 ± 0.5 ◦C and at 4 ± 0.5 ◦C when the peak areas of the stored CCM solution and nanoemulsion were compared to those derived using a freshly prepared CCM solution and CCM nanoemulsion. The precision of the CCM stock solution and nanoemulsion in terms of %CV was measured to be 0.71–0.80% and 0.74–0.79%, respectively, at two distinct temperatures. In addition, the % recovery of the CCM stock solution and nanoemulsion was determined to be 98.45–99.85% and 100.70–100.85%, respectively, at two distinct temperatures. CCM was found to be sufficiently stable in stock solution and nanoemulsion formulation at 25 and 4 ◦C based on these results.

**Table 6.** Robustness data of CCM at MQC (20 μg/mL) for the greener HPLC methodology (mean ± SD; *n* = 3).


**Table 7.** Stability data of CCM in stock solution and nanoemulsion formulation at MCQ level at two different temperatures (mean ± SD; *n* = 3).


## *3.3. Forced Degradation and Selectivity Studies*

By subjecting the 40 μg/mL concentration of CCM to various stress conditions, the selectivity and stability-indicating properties for the greener HPLC methodology were assessed. Figure 3 and Table 8 provide an overview of the outcomes of selectivity under various stress circumstances using the ecofriendly HPLC approach.

**Table 8.** Results of forced-degradation studies of CCM at 40 μg/mL concentration under various stress tests for the greener HPLC assay (mean ± SD; *n* = 3).


**Figure 3.** Greener HPLC chromatograms of CCM recorded under (**A**) acid-induced degradation, (**B**) base-induced degradation, (**C**) oxidative degradation, (**D**) thermal degradation, and (**E**) photolytic degradation of CCM.

The forced degradation investigations' chromatograms showed well-separated CCM peaks, along with a few other peaks of degradation products (Figure 3). In total, 87.85% of CCM was preserved under acid stress degradations, while 13.15 percent was degraded (Table 8). As a result, it was discovered that CCM was adequately stable under acidic degradations. The Rt value for CCM breakdown under acid stress was somewhat off (Rt = 3.53 min) (Figure 3A). In total, 84.02% of CCM was still present at base-stress degradations, while 15.98% was degraded (Table 8). As a result, it was discovered that CCM was adequately stable under alkali degradations. Additionally, a small shift was made to the Rt value of CCM during base-stress degradation (Rt = 3.56 min) (Figure 3B). Only 3.15% of CCM was found to be destroyed under oxidative-stress degradations, leaving 96.85% of the original material intact. As a result, it was discovered that CCM was adequately stable under oxidative stress degradation. The Rt value of CCM during oxidative stress degradation was not altered (Rt = 3.57 min) (Figure 3C). Under thermal degradation, 99.92% of CCM remained and only a negligible amount (0.08%) was degraded. Hence, CCM was found to be highly stable under thermal degradation. The Rt value of CCM under thermal degradation was not shifted (Rt = 3.57 min) (Figure 3D). Under photolytic degradation, 45.60% of CCM remained and 54.40% was degraded. As a result, CCM was found to be highly unstable under photolytic degradation. The Rt value of CCM under photolytic degradation was not shifted (Rt = 3.57 min) (Figure 3E). Overall, the maximum degradation of CCM was found under photolytic degradation. The degradation patterns of CCM were found to be identical with those reported previously in the literature [23]. Since the greener HPLC methodology was able to detect CCM in the presence of its degradation products, it can be considered as a stability-indicating one. Overall, these results indicated the selectivity and stability-indicating characteristics of the greener HPLC methodology.

#### *3.4. Greenness Measurement*

A number of analytical techniques are used to assess the greenness characteristics of analytical procedures [52–56]. Only the AGREE approach takes into account all twelve GAC principles while evaluating the analytical approaches' greenness [54]. As a result, the new HPLC methodology's greenness properties were identified utilizing the AGREE approach. The predicted overall AGREE score employing the twelve distinct GAC principles is summarized in Figure 4. Figure 5 includes the AGREE report sheet and AGREE score for each GAC concept. The different AGREE scores for each criteria of GAC were assigned by the AGREE calculator. The assigned scores ranged from 0.0 to 1.0. The established HPLC methodology's overall AGREE score was 0.81, indicating that it possesses exceptional greenness properties for the measurement of CCM. The AGREE score for the reported analytical methods of CCM measurement has not been determined in the literature. However, the AGREE score for the HPLC assay of some other drugs has been reported recently [58,59]. The AGREE score for the HPLC assay of the emtricitabine measurement has been reported to be 0.72 using the AGREE calculator [58]. Similarly, the AGREE score for the HPLC assay of the doxorubicin measurement has been reported to be 0.79 using the AGREE calculator [59]. The recorded AGREE score for the HPLC assay of the CCM measurement was better than those reported for the measurement of emtricitabine and doxorubicin [58,59]. Overall, the greenness profile of the current HPLC assay was outstanding.

**Figure 4.** Analytical GREEnness (AGREE) score for the established HPLC assay of CCM determination predicted using AGREE calculator.

#### *3.5. Determination of CCM in In-House Developed Nanoemulsion, Curcuma Longa Extract, and Commercial Tablets*

The stability-indicating greener HPLC methodology for CCM determination was shown to be efficient, rapid, and sensitive. This technique was therefore applied to ascertain CCM in an in-house developed nanoemulsion, *Curcuma longa* extract, and commercial tablets. The CCM percentage assay was 101.24 ± 0.72% in the in-house developed nanoemulsion. The amount of CCM was determined to be 81.15 ± 0.64% in *Curcuma longa* extract. The amount of CCM in commercial tablets (containing 1000 mg of standardized *C. longa* extract) was found to be 78.41 ± 0.58%. The assay of CCM in ordinary emulsion formulation using the HPLC method has been reported to be 99.45% [23], which was similar to the present method of CCM assay in nanoemulsion formulation. The amount of CCM in six different commercial *C. longa* extracts using the HPLC method has been reported in the range of 69.82 to 86.79% [26]. The amount of CCM in *C. longa* extract using the present HPLC method was superior to most of the reported extracts [26]. Using another

HPLC method, the amount of CCM in *C. longa* extract has been reported as 30.76 mg/g (equivalent to 3.06%) [27], which was inferior to the present HPLC method. These findings indicated that the greener HPLC methodology would work well for figuring out CCM in its laboratory developed formulations, commercially available products, and distinct plant extracts.


**Figure 5.** AGREE scale sheet presenting AGREE scale for 12 distinct components of GAC for the established HPLC methodology of CCM determination recorded using AGREE calculator.

## **4. Conclusions**

The CCM in an in-house developed nanoemulsion, *Curcuma longa* extract, and commercial tablets has been identified and verified using a quick, sensitive, stability-indicating, and greener HPLC approach. The stability-indicating greener HPLC methodology was validated according to ICH-Q2-R1 procedures. For detecting CCM, the greener HPLC approach is more efficient, accurate, precise, stability-indicating, robust, sensitive, and selective. The greener HPLC methodology was found to be suitable for the determination of CCM in the in-house developed nanoemulsion, *Curcuma longa* extract, and commercial tablets. The AGREE evaluation suggested outstanding greenness characteristics of the established HPLC methodology. Because of its selectivity and stability-indicating properties, the greener HPLC method was able to identify CCM, even in the presence of its degradation products. Based on these findings, it is possible to effectively determine CCM in a variety of sample matrices using the stability-indicating greener HPLC methodology. In future, further studies can be carried out to determine CCM in the complex matrices of biological samples and CCM pharmacokinetic evaluation.

**Author Contributions:** Conceptualization, S.A. and F.S.; methodology, N.H., M.M.G. and S.M.B.A.; software, N.H., S.A.A. and F.S.; validation, S.A. and P.A.; formal analysis, S.A.A. and P.A.; investigation, F.S. and N.H.; resources, S.A.; data curation, P.A.; writing—original draft preparation, F.S.; writing—review and editing, N.H., S.A. and P.A.; visualization, S.A.; supervision, F.S. and S.A.; project administration, F.S. and S.A.; funding acquisition, S.A. and M.M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study is supported via funding from Prince Sattam Bin Abdulaziz University project number (PSAU/2023/R/1444). The APC was funded by PSAU.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to Prince Sattam Bin Abdulaziz University for supporting this work via project number (PSAU/2023/R/1444). The authors are also grateful to AlMaarefa University for their generous support.

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

## **References**


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## *Article* **Analysis of Sugars in Honey Samples by Capillary Zone Electrophoresis Using Fluorescence Detection**

**Melinda Andrasi \*, Gyongyi Gyemant, Zsofi Sajtos and Cynthia Nagy**

Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem ter 1., H-4032 Debrecen, Hungary

**\*** Correspondence: andrasi.melinda@science.unideb.hu

**Abstract:** The applicability of capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF) for the separation of sugars in honey samples was studied. An amount of 25 mM ammonium acetate (pH 4.5) with 0.3% polyethylene oxide (PEO) was found to be optimal for the efficient separation of carbohydrates. 8-aminopyrene-1,3,6-trisulfonic acid (APTS) was used for the labeling of the carbohydrate standards and honey sugars for fluorescence detection. The optimized method was applied in the quantitative analysis of fructose and glucose by direct injection of honey samples. Apart from the labeling reaction, no other sample preparation was performed. The mean values of the fructose/glucose ratio for phacelia honey, acacia honey and honeydew honey were 0.86, 1.61 and 1.42, respectively. The proposed method provides high separation efficiency and sensitive detection within a short analysis time. Apart from the labeling reaction, it enables the injection of honeys without sample pretreatment. This is the first time that fluorescence detection has been applied for the CE analysis of sugars in honeys.

**Keywords:** capillary electrophoresis; fluorescence detection; honey; sugars

## **1. Introduction**

Honey is a natural, aqueous, supersaturated sugar substance produced by honeybees. It also contains other minor substances, such as minerals, enzymes, vitamins, organic acids, amino acids, flavonoids and phenolic acids. Honey is used as a nutritional product, but it can also exert several health-benefitting effects. The main components of honey are carbohydrates, representing around 85–97% of its weight. A significant part is made up of mainly fructose and glucose. Small amounts of other monosaccharides (galactose, mannose), disaccharides (maltose, sucrose, beta-trehalose) and oligosaccharides (melezitose, maltotriose, panose, erlose, isomaltotriose, theanderose) are present in honey [1,2].

The determination of sugars is a common approach for describing the quality of honey. The principle of classical chemical methods for the analysis of carbohydrates is based on the fact that reducing sugars (non-reducing carbohydrates can become reducing via hydrolyzation) react with other compounds to form precipitates or colored complexes, which can be determined by titration, gravimetric and spectrophotometric techniques [3,4]. The main disadvantages of the classical chemical methods are that carefully controlled, time consuming reaction parameters must be provided.

Separation techniques are the most powerful methods for the identification and quantification of carbohydrates, of which chromatographic methods, especially normal phase [5], anion-exchange [6], and hydrophilic interaction [7] high-performance liquid chromatography have been employed. A refractive index detector is commonly used in chromatographic analysis of sugars [8–10].

Capillary electrophoresis (CE) has emerged as an alternative tool for the analysis of carbohydrates. It provides high separation efficiency within a short analysis time, and low sample volume consumption. CE allows for the direct injection of real samples without

**Citation:** Andrasi, M.; Gyemant, G.; Sajtos, Z.; Nagy, C. Analysis of Sugars in Honey Samples by Capillary Zone Electrophoresis Using Fluorescence Detection. *Separations* **2023**, *10*, 150. https://doi.org/10.3390/ separations10030150

Academic Editor: Faiyaz Shakeel

Received: 7 February 2023 Revised: 15 February 2023 Accepted: 19 February 2023 Published: 23 February 2023

**Copyright:** © 2023 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/).

any pretreatment. On the other hand, carbohydrate analysis by CE is quite challenging in terms of separation and detection. Most carbohydrates have no charge or absorbing chromophores in the UV-VIS regions. In recent years, different strategies have been developed to overcome these limitations [11–13]. The use of borate buffers with direct UV detection is the simplest way to analyze carbohydrates. Borate forms complexes with the vicinal hydroxyl groups of sugars, converting them into anions. This complex shows increased UV sensitivity at 191–195 nm [14]. The detection of carbohydrate-borate complexes can also be carried out using electrochemical methods (amperometric, contactless conductivity) [15]. Another possibility for making carbohydrates amenable to CE analysis is the use of strong alkaline background electrolytes (BGEs). Using a BGE with a pH above the pKa of the sugars ensures that their hydroxyl groups are dissociated; hence, sugars can migrate as anions in capillary zone electrophoresis (CZE) [16]. Different detection modes can be applied, such as indirect UV [17], fluorescence detection [18], electrochemical detection [19], and mass spectrometry (MS) [20]. The main merit of the indirect UV mode is that no time-consuming derivatization procedure is required; however, the limit of detection (LOD) is weaker than that of fluorescence detection. The laser-induced fluorescence (LIF) or LEDIF detection mode can ensure high sensitivity. The most frequent fluorescent labeling reaction is reductive amination [21]. There are several types of labeling reagents (8-aminopyrene-1,3,6-trisulfonic acid (APTS), 8 aminonaphthalene-1,3,6-trisulfonic acid (ANTS), aminonaphthalene-2-sulfonic acid (ANA), and 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid (AHNS)) that can be used depending on the excitation and emission wavelengths [22]. APTS is a popular derivatizing agent because it can be excited with the commonly used argon-ion laser set at 488 nm and emitting at 520 nm. Furthermore, it adds three negative charges to the molecule, allowing for the possibility of CZE separation [23–25].

There are many examples of CE-based honey analysis in the literature, but very few of them deal with the determination of sugars. Indirect CE–UV was used by Rizelio et al. for the determination of fructose, glucose and sucrose in seven multifloral honey samples using 20 mM sorbic acid, 0.2 mM cetyltrimethylammonium bromide (CTAB) and 40 mM sodium hydroxide (NaOH) at pH 12.2. The detection limits for the three analytes were in the range of 0.022 to 0.029 g/L [26]. The drawbacks of indirect CE-UV are that the baseline is often not stable, and the sensitivity is worse compared to the fluorescence detection mode. A BGE consisting of 10 mM sodium benzoate and 1.5 mM CTAB, with a pH of 12.4, was applied for the simultaneous determination of fructose, glucose and sucrose in honey, using the indirect UV detection mode. The LOD for fructose, glucose and sucrose were 0.58 g/L, 0.67 g/L and 0.12 g/L, respectively [27]. The molar ratios of carbohydrates in 10 kinds of honey were determined by CZE after reductive, UV-active derivatization reactions with 1-phenyl-3-methyl-5-pyrazolone (PMP). Eleven PMP-labeled aldoses were separated in 200 mM borate-4% methanol at pH 11.0 [28]. A graphene–cobalt microsphere (CoMS) hybrid paste electrode was used for the detection of carbohydrates in three honey samples, for which the separation medium was 75 mM NaOH [29].

To the best of our knowledge, fluorescence detection has not yet been applied for the analysis of sugars in honeys. In this work, we applied APTS for the labeling of honey sugars. We optimized a CZE method for the separation of labeled sugars. The aim of this work was to demonstrate the applicability of CE for the determination of APTS-labeled sugars in honey samples.

## **2. Materials and Methods**

#### *2.1. Chemicals*

All chemicals were of analytical grade. Ammonium acetate, hydrochloric acid (HCl), NaOH, PEO (average Mv~90,000 g/mol), 6-aminocaproic acid (EACA), hydroxypropyl methylcellulose (HPMC), APTS, sodium cyanoborohydride (NaBH3CN), tetrahydrofuran (THF), acetic acid, fructose, mannose, glucose, galactose, maltose, arabinose, xylose, ribose, and galactose were obtained from Sigma Aldrich (St. Louis, MO, USA). Different types of honey samples (phacelia honey, acacia honey, honeydew honey) were a kind offer from

Hungarian producers. The ladder standard, malto-oligosaccharides composed of 1 to 7 glucose units, was made from β-cyclodextrin by acetolysis.

#### *2.2. Instrumentation*

CE separations were carried out using a 7100 CE System (Agilent, Waldbronn, Germany) coupled to UV and LEDIF (Zetalif Picometrics) detectors. Separations were performed using a fused-silica capillary (Polymicro, Phoenix, AZ, USA) of 65 cm, 50 cm × 50 μm i.d. The precondition procedure was 1 M HCl for 5 min, BGE for 5 min. Hydrodynamic sample injection (50 mbar × 2 s) was carried out at the anodic end of the fused silica capillary. For the electrophoretic separation, −30 kV was used. Fluorescence detection was performed by an LED-induced fluorescence detector; the excitation and emission wavelengths were 480 nm and 520 nm, respectively. The photomultiplier's high voltage was set to 700 V. For UV, on-capillary detection at a wavelength of 240 nm was chosen. Chemstation version B.04.02 software (Agilent) was used for operating the CE instrument and for processing the results.

## *2.3. Sample Preparation for Fluorescence Detection*

The derivatization by APTS was carried out according to the recipe of Evangelista [30]; only minor modifications were made, mainly regarding the incubation and the amount of reagents. For the labeling reaction, 0.5 mg of the carbohydrate standard and 1 mg of the honey sample were measured and were dissolved in a mixture of 6 μL of 20 mM APTS (in 15% acetic acid) and 2 μL of 1 M NaBH3CN (in THF) solution. In the first step, a Schiff base was formed, which was reduced to a secondary amine by NaBH3CN. The samples were homogenized using a vortex and then incubated for 1 h at 50 ◦C. The reaction was stopped by adding 92 μL of distilled water into the labeling reaction. The reaction mixtures were further diluted before direct sample injection.

### **3. Results and Discussion**

## *3.1. CZE Separations of APTS-Labeled Carbohydrates*

Fluorescence detection provides extremely sensitive detection for the analysis of sample injection plugs in the nanoliter range. As depicted in Figure 1, for the carbohydrate components, the LEDIF detection resulted in a high analytical response signal with very little baseline noise (Figure 1A), while detection at 240 nm yielded a small signal with high baseline noise (Figure 1B). The signal-to-noise ratio was much lower (S/N = 30) compared to fluorescence detection, where S/N was 81,000. Although UV detection is made possible by derivatization, it allows three orders of magnitude of lower sensitivity based on the calculated S/N values.

When labeling carbohydrates, derivatization should not simply be done for detectability; it is crucial to select a fluorophore that renders the labeled sugar component charged. The tagged sugar molecule gains three negative charges, since APTS has three sulfonate groups, enabling their migration in the electric field. Good resolution was achieved in a short migration window using a simple acetate buffer (pH 4.5) in the case of the analysis of the carbohydrate ladder containing oligomers of 1 to 7 glucose units (Figure 1). The electric field is applied under reversed polarity to drive anions toward the detection window. The separation relies on differences in charge-to-size ratio. All labeled sugar components possess a triple negative charge, but their molecular masses are different, so their electrophoretic mobilities differ, as well. The characteristic electrophoretic peak pattern is due to the method of sample production. The intensity decreases as the number of glucose units decreases. After the ring opening of β-CD, with 7 glucose units, an oligosaccharide composed of 7 glucose units was formed in the largest amount. During acidic decomposition, a decreasing amount of shorter oligosaccharides was formed, and glucose was present in high concentration in the sample due to the cleavage of glucose units (Figure 1B).

**Figure 1.** The CZE electropherograms of an APTS-labeled malto-oligosaccharide ladder standard using LEDIF (**A**) and UV (**B**) detection. The GX numbers indicate the numbers of glucose residues of the ladder. Conditions: fused silica capillary, ltot: 50 cm × 50 μm id, leff: 30 cm (**A**), 42 cm (**B**), BGE: 25 mM ammonium acetate (pH 4.5), separation voltage: −30 kV, injection: 50 mbar × 2 s, preconditioning: 1 M HCl for 5 min, BGE for 5 min washing. LEDIF detection was performed at 480 nm excitation/520 nm emission, UV detection was performed at 240 nm.

A four-component carbohydrate mixture (glucose, fructose, mannose and maltose) most often found in honey was investigated using the same BGE that proved optimal in separating the members of the carbohydrate ladder (Figure 2A). The disaccharide maltose appeared as a peak well separated from the monosaccharides upon application of a simple ammonium acetate buffer, but no adequate resolution was obtained in the case of the three monosaccharides (Figure 2A). All three monosaccharides are hexoses (C6H12O6), and their molar masses are the same (M = 180 g/mol); hence, there is no big difference in their electrophoretic mobility, which explains the merging of the peaks. The separation of fructose from mannose and glucose can be attributed to the fact that electrophoretic mobility is also determined by the shape and hydrodynamic radius of the particle. The use of simple, additive-free buffers does not always provide adequate resolution for sugars with the same number of carbon atoms [31].

In order to increase the selectivity, PEO (average Mv~90,000) was added into the BGE, as a result of which a suitable resolution was achieved for the three hexoses (Figure 2B). The effect of PEO on the enhancement of the separation is complex [32]. Due to its neutral polymer nature, it is connected to the inner surface of the fused silica capillary by secondary bonds, so it has a surface modification effect. The neutral coating suppresses the adsorption of the negatively charged components to protonated silanol groups. PEO also has a sieving effect by increasing the viscosity of the BGE. The increase in migration times is due to these effects (Figure 2B).

6-aminocaproic acid (EACA) as a buffer in CE has a selectivity-enhancing effect, which is related to its ion-pair-forming property [33,34]. A significant improvement in resolution was observed compared to the analysis in acetate, although it did not provide baseline resolution (Figure 2C). To improve the selectivity for aldoses, an HPMC linear polymer was added to the 40 mM EACA buffer at a concentration of 0.02%. The cellulose derivative improved the resolution between glucose and mannose and extended the analysis time compared to the PEO-containing buffer (Figure 2D). The effect of HPMC in increasing the separation efficiency is similar to that of PEO. From among the BGEs investigated, 25 mM ammonium acetate-0.3% PEO 90,000 (pH 4.5) provided the best separation and the best

resolution values (Supplementary Material Table S1); therefore, this BGE was applied for further analyses.

**Figure 2.** The CZE separations of a mixture of four carbohydrates using LEDIF detection. Conditions: fused silica capillary, ltot: 65 cm × 50 μm id, leff: 45 cm, BGE: (**A**) 25 mM ammonium acetate (pH 4.5), (**B**) 25 mM ammonium acetate-0.3% PEO (pH 4.5), (**C**) 40 mM EACA (pH 4.5), (**D**) 40 mM EACA-0.02% HPMC (pH 4.5), separation voltage: −30 kV, injection: 50 mbar × 2 s, preconditioning: 1 M HCl for 5 min, BGE for 5 min washing. LEDIF detection was performed at 480 nm excitation/520 nm emission. Sample: 1: fructose, 2: glucose, 3: mannose, 4: maltose.

The use of PEO enabled the separation of positional isomers of oligosaccharides, as well. Two samples of maltotetraose (G4)- isomaltotetraose (isoG4) and maltotriose (G3)-isomaltotriose (isoG3) were analyzed (Figure 3A,B). In the case of G3 and G4, the monosaccharide units are connected by an α(1-4) bond, whereas in the isoG3 and isoG4, the position of the bond is different; the glucose units are linked by an α(1-6) bond. Since the electrophoretic mobility is also influenced by the shape of the particle, it was possible to separate the G3 and G4 from their positional isomers possessing the same mass and charge but different form. The α(1-4) linkage affords an elongated, thinner molecular shape; therefore the friction coefficients of G3 and G4 are higher, so their electrophoretic mobility is lower than that of their isomers. The spherical shape of the isomers led to lower friction coefficients and, as a result, higher migration speed (Figure 3A,B).

During method development, sample injection was also examined, since the honey samples were introduced into the capillary without any sample preparation procedure, except for the labeling reaction. Electrokinetic sample introduction of a standard carbohydrate solution with 5 kV × 5 s delivered the same amount of each carbohydrate into the capillary as hydrodynamic injection by 50 mbar × 2 s (Supplementary Material Figure S1). The ratio of individual carbohydrates did not vary when electrokinetic injection was applied because there is no difference in the charge of APTS-labeled sugars. It is possible to examine APTS-marked sugar components in samples of honey via electrokinetic injection.

The calibration curves and analytical performance data are given for two sugars (fructose and glucose) that are present in honey in the largest amounts (Table 1). The calculated LOD and limit of quantitation (LOQ) in the range of ng/mL enable a more sensitive determination than what is usually available with UV-VIS spectrophotometric detection in the case of CE (μg/mL). The relative standard deviation (RSD%) values of migration time were around 0.5, whereas RSD% values of area were between 2.5 and 4.4. The theoretical plate number data correspond to the separation efficiency expected from CE (Table 1).

**Figure 3.** The analysis of isomaltotetraose (**A**) and isomaltotriose (**B**) by CZE-LEDIF. The analysis conditions were the same as stated at Figure 2B.

**Table 1.** Analytical performance data of fructose and glucose. The analysis conditions were the same as stated at Figure 2B.


### *3.2. Analysis of Sugars in Honey Samples*

The sugar content of three different honey samples (phacelia honey, honeydew honey, and acacia honey) was analyzed by CE-LEDIF using ammonium acetate with a PEO additive as a BGE (Figure 4C–E). The sugar content of honeys was characterized using a carbohydrate ladder (Figure 4A) and a standard mixture containing five carbohydrates (arabinose, xylose, ribose, galactose and maltose) (Figure 4B). All three honeys contain two monosaccharides in higher concentrations, and other sugars appeared in smaller concentrations in the monosaccharide region (G1), according to the ladder (Figure 4C–E). This corresponds well with the literature data showing that honey consists of about 90% sugars, mainly glucose and fructose. Identification of glucose and fructose peaks in honeys was performed by standard addition. This is illustrated through the example of phacelia honey, wherein the addition of fructose resulted in an enhancement in the peak height of fructose, which is proportional to the added amount (Supplementary Material Figure S2). Apart from glucose and fructose, other sugars were not identified by spiking. The fructose and glucose content of honey in mass percentages and their relative proportion data were given using the prepared calibration curves (Table 2). The fructose-to-glucose ratio is one of the most important data for describing the quality of honeys [2,35]. Honey is crystallized when this ratio is less than 1.2. This data reveals which honey contains more glucose and indicates

how likely it is to crystallize. Phacelia honey had the highest glucose content and the lowest fructose-to-glucose ratio. Honey is in a liquid state if the fructose-to-glucose ratio exceeds 1.2, so phacelia honey crystallized, whereas acacia and honeydew honeys retained their liquid characters due to the lower glucose content. Honeydew honey differed from phacelia and acacia honey in terms of monosaccharide composition. It had a lower carbohydrate content (Table 2). Honeydew honey is not of flower nectar origin, and, actually, this difference in origin is what causes the diversity in composition. Honeydew is excreted by aphids and other insects, and this excreted, sticky substance is collected by bees.

**Figure 4.** CZE-LEDIF determination of APTS-labeled sugars in model solutions and in honeys. (**A**) G7 malto-oligosaccharide ladder, (**B**) mixture solution of arabinose (1), xylose (2), ribose (3), galactose (4), maltose (5), (**C**) phacelia honey, (**D**) acacia honey, (**E**) honeydew honey. The analysis conditions were the same as stated at Figure 2B.

**Table 2.** The comparison of fructose and glucose content and their proportion in phacelia honey, acacia honey, honeydew honey, n = 3 (each of them). The analysis conditions were the same as stated at Figure 2B.


In addition to monosaccharides, the presence of small amounts of disaccharides was confirmed in the honey samples using the carbohydrate ladder (Figure 4C–E). According to the literature, maltose, sucrose, maltulose, nigerose, kojibose, trehalose and turanose are the disaccharides that are most commonly found in honey [1,2]. None of the disaccharide peaks visible on the electropherogram come from sucrose; because sucrose is not a reducing disaccharide, it cannot be labeled by APTS. According to the carbohydrate ladder, the electropherograms between G1 and G2 showed the positional isomers of the disaccharides. The honeys analyzed contained very trace amounts of trisaccharides. Less than 0.1% of the total monosaccharide amount was made up by the peaks migrating at the G3 location.

The evaluation of the greenness of analytical methods was performed by the Analytical Greenness calculator (AGREE). The obtained score was 0.74, which is in the middle of the AGREE pictogram with values close to 1; the green color indicates that the applied CE-LEDIF analytical procedure has little environmental impact [36–38].

## **4. Conclusions**

In this work, we studied the possibilities of determining the sugar composition of honey samples by capillary electrophoresis with light-emitting diode-induced fluorescence detection (CE-LEDIF). We applied 8-aminopyrene-1,3,6-trisulfonic acid (APTS) for the labeling of honey sugars, which made possible not only the sensitive detection but also the electrophoretic separation. LEDIF detection allowed for the determination of sugars with high sensitivity (~30 ng/mL limit of quantitation (LOQ) values). The effect of background electrolyte (BGE) additives on the separation efficiency was investigated, and the addition of polyethylene oxide (PEO) improved the resolution of labeled sugars having the same carbon number. We determined the glucose and fructose content of three different types of honey samples. The CE-LEDIF method has the potential to be a suitable platform for the routine analysis of honey samples in food and biology laboratories. Although the developed method is well suited for the determination of sugar components which are presented in honey, the method does not allow for the direct determination of sucrose, which plays a major role in the adulteration of honey. Further developments are needed for the determination of non-reducing disaccharides, for instance, sucrose (after enzymatic hydrolysis). We plan to analyze honey samples of different ages and different geographical origin.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/separations10030150/s1. Table S1: The comparison of resolutions using different BGEs. The analysis conditions were the same as stated in Figure 2. Figure S1: The CZE-LEDIF determinations of a mixture of four carbohydrates using hydrodynamic (A) and electrokinetic injection (B). The analysis conditions were the same as stated in Figure 2B. Injection: +5 kV × 5 s (S1B), samples: 1: fructose, 2: glucose, 3: mannose, 4: maltose, (\*): APTS. Figure S2: Identification of fructose peak in honey using standard addition. The analysis conditions were the same as stated in Figure 2B. Sample: 1: fructose, 2: glucose, (\*): APTS, (S2A) phacelia honey, (S2B) phacelia honey spiked with fructose, (S2A', S2B') narrow scale of electropherograms of (A, B).

**Author Contributions:** Conceptualization, M.A., G.G. and Z.S.; methodology, M.A.; investigation, M.A.; writing—original draft preparation, M.A.; writing—review and editing, C.N.; supervision, G.G., Z.S. and C.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research presented in the article was carried out within the framework of the National Research, Development and Innovation Office (K127931 and K142134), Hungary.

**Data Availability Statement:** Data available upon request from the authors.

**Acknowledgments:** The authors acknowledge the financial support provided to this project by the National Research, Development and Innovation Office (K127931 and K142134), Hungary.

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

## **Abbreviations**

capillary electrophoresis (CE); light-emitting diode-induced fluorescence detection (LEDIF); polyethylene oxide (PEO); 8-aminopyrene-1,3,6-trisulfonic acid (APTS); ultraviolet-visible (UV-VIS); background electrolyte (BGE); acid dissociation constant (pKa); capillary zone electrophoresis (CZE); mass spectrometry (MS); limit of detection (LOD); laser-induced fluorescence detection (LIF); 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS); aminonaphthalene-2-sulfonic acid (ANA); 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid (AHNS); cetyltrimethylammonium bromide (CTAB); sodium hydroxide (NaOH); 1-phenyl-3-methyl-5-pyrazolone (PMP); graphene–cobalt microsphere (CoMS); hydrochloric acid (HCl); 6-aminocaproic acid (EACA); hydroxypropyl methylcellulose (HPMC); sodium cyanoborohydride (NaBH3CN); tetrahydrofuran (THF); signal-to-noise ratio (S/N); numbers of glucose residues of the malto-oligosaccharide ladder standard (GX); maltotetraose (G4); isomaltotetraose (isoG4); maltotriose (G3); isomaltotriose (isoG3); limit of quantitation (LOQ); relative standard deviation (RSD%).

## **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

## *Article* **UPLC-MS/MS Method for Simultaneous Estimation of Neratinib and Naringenin in Rat Plasma: Greenness Assessment and Application to Therapeutic Drug Monitoring**

**Ali Altharawi 1,\*, Safar M. Alqahtani 1, Sagar Suman Panda 2, Majed Alrobaian 3, Alhumaidi B. Alabbas 1, Waleed Hassan Almalki 4, Manal A. Alossaimi 1, Md. Abul Barkat 5, Rehan Abdur Rub 6, Shehla Nasar Mir Najib Ullah 7, Mahfoozur Rahman <sup>8</sup> and Sarwar Beg <sup>6</sup>**


**Abstract:** Tyrosine kinase inhibitors have often been reported to treat early-stage hormone-receptorpositive breast cancers. In particular, neratinib has shown positive responses in stage I and II cases in women with HER2-positive breast cancers with trastuzumab. In order to augment the biopharmaceutical attributes of the drug, the work designed endeavors to explore the therapeutic benefits of neratinib in combination with naringenin, a phytoconstituent with reported uses in breast cancer. A UPLC-MS/MS method was developed for the simultaneous estimation of neratinib and naringenin in rat plasma, while imatinib was selected as the internal standard (IS). Acetonitrile was used as the liquid extractant. The reversed-phase separation was achieved on a C18 column (100 mm × 2.1 mm, 1.7 μm) with the isocratic flow of mobile phase-containing acetonitrile (0.1% formic acid) and 0.002 M ammonium acetate (50:50, % v/v) at flow rate 0.5 mL·min<sup>−</sup>1. The mass spectra were recorded by multiple reaction monitoring of the precursor-to-product ion transitions for neratinib (m/z 557.138→111.927), naringenin (m/z 273.115→152.954), and the IS (m/z 494.24→394.11). The method was validated for selectivity, trueness, precision, matrix effect, recovery, and stability over a concentration range of 10–1280 ng·mL−<sup>1</sup> for both targets and was acceptable. The method was also assessed for greenness profile by an integrative qualitative and quantitative approach; the results corroborated the eco-friendly nature of the method. Therefore, the developed method has implications for its applicability in clinical sample analysis from pharmacokinetic studies in human studies to support the therapeutic drug monitoring (TDM) of combination drugs.

**Keywords:** breast cancer; liquid chromatography; bioanalytical methods; neratinib; naringenin

## **1. Introduction**

Breast cancer is considered one of the leading causes of mortality in the female population and the second most commonly reported cancer globally [1]. Over recent years,

**Citation:** Altharawi, A.; Alqahtani, S.M.; Panda, S.S.; Alrobaian, M.; Alabbas, A.B.; Almalki, W.H.; Alossaimi, M.A.; Barkat, M.A.; Rub, R.A.; Mir Najib Ullah, S.N.; et al. UPLC-MS/MS Method for Simultaneous Estimation of Neratinib and Naringenin in Rat Plasma: Greenness Assessment and Application to Therapeutic Drug Monitoring. *Separations* **2023**, *10*, 167. https://doi.org/10.3390/ separations10030167

Academic Editor: Léon Reubsaet

Received: 4 January 2023 Revised: 15 February 2023 Accepted: 20 February 2023 Published: 1 March 2023

**Copyright:** © 2023 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/).

there has been continuous drug approval for the treatment and management of breast cancer. As per the WHO statistics of 2020, around 2.5 million deaths are estimated between 2020 and 2040. The early diagnosis of breast cancer, with confirmation of its genetic basis and heterogeneity, helps in selecting the right chemotherapeutic drugs for breast cancer treatment. In the past few years, the United States Food Drug Administration (USFDA) has approved several tyrosine kinase inhibitors for breast cancer treatment, which are very effective in HER2-overexpressed/amplified breast cancers [2,3]. Tyrosine kinase inhibitors specifically block the abnormal signal transduction, which is necessary for the proliferation of the cancer cells, and also show anti-epithelial growth factor receptor (EGFR) activity.

Neratinib (NER), a tyrosine kinase inhibitor, was approved by the USFDA in July 2017 as an extended adjuvant therapy for adult patients prescribed with trastuzumab for early-stage breast cancers [4,5]. Moreover, it is available in the form of tablets for oral administration at a dose of 40 mg. In contrast, the oral pharmacokinetics of neratinib exhibit a nonlinear absorption profile along with a food effect [6]. To understand the pharmacokinetic and biodistribution profile of the drug, a fast, sensitive, and efficient analytical method is always very useful for preclinical and clinical sample analysis. Very few bioanalytical methods of neratinib alone have been reported in the literature for estimation in rat and human plasma using high-performance liquid chromatography (HPLC) with ultraviolet (UV) and diode/photodiode array detectors (DAD/PDA) [7] and ultra-performance liquid chromatography (UPLC) with mass spectrometer (MS/MS) detectors [8–10]. In contrast to these existing methods, the present work aimed to establish a rapid and sensitive bioanalytical method for the quantification of multiple compounds in human plasma samples. This new method also endeavors to support the therapeutic drug monitoring (TDM) program. LC-MS/MS methods have been quite frequently used in TDM owing to their high sensitivity and specificity compared to other analytical techniques, as they quantify drugs irrespective of their natural chromophores or fluorophores [11,12]. Upon the optimization of sample preparation and mass spectrometry conditions, column and internal standard selection, LC-MS/MS methods greatly help in avoiding interference due to the matrix effect and the presence of other analytes and metabolites [13,14].

For better pharmacotherapeutic action, chemotherapeutic drug combinations with phytopharmaceuticals, especially antioxidants, have been extensively explored in the past few decades [15,16]. In this regard, we found naringenin (NRN) to be one of the potential phytopharmaceutical agents for various cancers, including breast cancer [17,18]. NRN potentially exhibits a dose-dependent increase in caspase-3 and caspase-9 activity-mediated apoptosis in breast cancer cells [17]. Additionally, it reduces cell proliferation and inhibits the migration of breast cancer cells via inflammatory and apoptosis cell signaling pathways. For the quantification of NRN in preclinical and clinical samples, a suitable bioanalytical method is required to understand the pharmacokinetic and pharmacodynamic behaviors of the drug. A score of research studies have documented the analytical estimation of NRN in biological matrices employing HPLC [19–21], LC-MS/MS [22], and related techniques. However, the sensitivity or lower limit of quantification (LLOQ) reported in the HPLC methods was in the range of 0.1–15 <sup>μ</sup>g·mL−<sup>1</sup> [19–21], while the LC-MS/MS method by Ma et al. (2006) reported double peaks for NRN in rat plasma despite a good LLOQ of 5 ng·mL−<sup>1</sup> [22]. On the contrary, the current method showed a single peak for NRN with high resolution, although LLOQ was found to be 10 ng·mL−1, which is still good for the routine analysis of the drugs in preclinical and clinical samples.

Green analytical chemistry (GAC) is a recent approach growing in popularity amongst analysts. However, any analytical procedure's green or eco-friendly nature dramatically affects environmental sustainability and the overall economy of the method development. With many screening and identification processes, chemists have recommended solvents and reagents that promise to fulfill the above intent. With scientists presenting newer approaches now and then, it has been a critical task for the analyst to choose between the various greenness assessment tools available. The National Environmental Methods Index (NEMI), Green Analytical Procedure Index (GAPI), and eco-scale (etcetera) are some

of the most widely utilized tools for assessing a method's greenness. Several scientists have reported the benefits and justifications of using such approaches for examining the eco-friendly nature of different analytical procedures [23,24]. A detailed discussion of their goals and rationale for assessment can be found elsewhere. In the current study, two assessment approaches, namely NEMI and AES, were adopted to qualitatively and semi-quantitatively determine the method greenness score.

In analytical science, method development for simultaneous estimation of two or more compounds provides cost and time economy in the analysis and colossal resource savings. However, method development for the simultaneous estimation of compounds is quite tedious, as many factors tend to influence the retention capacity of the drugs due to variations in their physicochemical characteristics. Therefore, in the present work, the researchers focused on developing a UPLC-MS/MS method to estimate NER and NRN in rat plasma simultaneously. Furthermore, the developed analytical method was validated according to current regulatory guidelines and employed to assess the stability of both analytes [25]. Additionally, the bioanalytical method's greenness was assessed considering some of the latest approaches discussed by various analysts [23,24].

## **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

NER was purchased from Weihua Pharma (Hangzhou, China), and NRN (Purity > 99%) was purchased from TCI Chemicals (India) Pvt. Ltd. (Chennai, India), while the internal standard (IS), imatinib (Purity > 98%), was generously provided by Dr. Reddy's Laboratories Ltd. (Hyderabad, India). The LC-MS grade acetonitrile (ACN) and distilled water were purchased from J.T. Baker Chemicals (Mumbai, India), and ammonium acetate, ethyl acetate, formic acid, and ammonium formate were obtained from Fluka Analytical (Seelze, Germany). The other chemicals and reagents obtained were of analytical reagent grade.

## *2.2. UPLC-MS/MS Instrument and Conditions*

The instrumental analysis was performed on the ACQUITY® UPLC-MS/MS system (Waters Corp., Milford, MA, USA) fitted with a binary pump system, autosampler unit, and column compartment. A ZsprayTM Xevo TQD (Waters Corp., Milford, MA, USA) mass spectrometer working with positive mode electrospray ionization (ESI) detected and quantified the analytes of NER and NRN. The detailed instrumental setting preferences for different test parameters are displayed in the Supplementary Information, Table S1.

#### *2.3. Animal Ethical Approval*

The animal study protocol was approved by the Institutional Animal Ethic Committee (IAEC) of Roland Institute of Pharmaceutical Sciences (RIPS) (Berhampur, Odisha 760010, India), with the protocol number 146/Chairman IAEC, RIPS, Berhampur (Approval Date: 13 November 2021). The animal care and maintenance were carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Healthy male Wistar rats (weighing between 180 and 220 g) were housed in polypropylene cages with free access to a standard diet and water *ad libitum*. Moreover, the animals were exposed to regular day and night cycles to maintain their circadian rhythms.

## *2.4. Preparation of Standards and Sample*

Separate stock solutions (1 mg·mL<sup>−</sup>1) of NER, NRN, and IS were prepared using ACN as a diluent. Precisely measured, 0.05 mL and 0.1 mL of analytes and IS were pipetted from the stocks and spiked into rat plasma samples (0.5 mL). After liquid-liquid extraction (LLE), the final dilution of eight concentrations of 10, 20, 40, 80, 160, 320, 640, and 1280 ng·mL−<sup>1</sup> of NER and NRN and 10 ng·mL−<sup>1</sup> of IS were obtained. Then, three specified concentrations viz. 100, 500, and 1000 ng·mL−<sup>1</sup> were defined as QC solutions for the analytes, while

10 ng·mL−<sup>1</sup> was taken as the IS for the validation results. The solutions were stored at −20 ◦C until the final analysis.

#### *2.5. Bioanalytical Extraction from the Rat Plasma*

Blood was collected from the retro-orbital plexus of anesthetized Wistar rats. The heparinized blood produced plasma when centrifuged at 10,000 rpm for 10 min. The recovered plasma was kept at −20 ◦C after the plasma harvesting operation. Aliquots of 0.05 mL of the analytes and 0.1 mL of 200 ng·mL−<sup>1</sup> IS were accurately measured and spiked in Eppendorf tubes, which were well mixed for 5 min. The extraction medium, ACN (1 mL), was added to all samples, followed by 2 min of vortex mixing and 5 min centrifugation at 3000 rpm (4 ± 2 ◦C). Precisely measured 0.5 mL of the organic fraction was pipetted and dried. The final dilution was prepared to 1 mL using mobile the phase, and the syringe was filtered before injection into the instrument. The prepared samples were always stored at −20 ◦C until analysis.

## *2.6. Validation Protocol*

Multiple bioanalytical method validation guidelines, such as the USFDA, China Food and Drug Administration (CFDA), etc., were referred to and employed to establish the validity of the current UPLC-MS/MS method.

## 2.6.1. Selectivity

Selectivity plays a critical role in establishing the uninterefered simultaneous quantification of analytes in a biological matrix, such as plasma. The absence of interference at analyte retention was used to establish method selectivity by comparing the chromatograms of the blank plasma from six animals with spiked samples.

## 2.6.2. Linearity and Sensitivity

Over the course of three days, eight-point analyte linearity curves were created utilizing the spiked plasma samples with concentrations ranging from 10 to 1280 ng·mL<sup>−</sup>1. The linearity plots were created using the drug-to-IS peak area ratio (*y*-axis) versus the spiked NER and NRN concentrations (*x*-axis). Further, the lower limit of quantification (LLOQ) was determined using six replicate samples that qualify for a minimum level of trueness and precision. For establishing trueness and precision at LLOQ, a maximum allowable deviation of 20% was permitted.

## 2.6.3. Trueness and Precision

We conducted hexaplicate analysis at LLOQ (10 ng·mL<sup>−</sup>1) and low, mid, and high QC samples with 100, 500, and 1000 ng·mL−<sup>1</sup> of NER and NRN, respectively, over three days to examine the trueness and precision of the method. The authors used percent relative standard deviation (% RSD) to define the method's trueness and precision, in addition to the amount of analyte recovered. The maximum variance allowed was 15% of the specified nominal level.

#### 2.6.4. Carryover and Dilution Integrity

A blank sample was injected after the immediate analysis of the highest calibration standard to test carry-over. The absence of a significant carry-over can be confirmed by obtaining a peak area not more (<20%) than that of the LLOQ.

Subsequently, the dilution integrity was tested by spiking the blank rat plasma with analytes and diluting it ten times in blank plasma within the studied range, with a maximum allowed deviation of ±15% for trueness and precision.

## 2.6.5. Matrix Effect and Extraction

The extracted peak regions of NER, NRN, and IS were directly compared to the spiked plasmatic blanks, revealing the extraction recovery at three QC levels. Additionally, a comparison of the peak areas of the analytes and IS in the plasma of rats to that of the equi-concentration standard solutions leads to inferring the extent of the influence of the matrix.

## 2.6.6. Stability Study

NRB and NRN were studied for their stability in bench top and short-term (room temperature) for 6 h and 24 h, respectively. At a temperature of −20 ◦C, long-term (14 days) and freeze–thaw (3 cycles) stability was assessed. The post-preparative stability study of the analytes was performed at 8 ◦C. All of the above stability studies were performed using analytes at their LQC and HQC concentration levels (n = 6).

## *2.7. Integrative Application of Multiple Green Metrics Tools*

The integrated approach of combining the National Environmental Methods Index (NEMI) procedure and the Analytical Eco-scale (AES) method was followed to ensure the greenness of the developed bioanalytical method [24,25]. NEMI deals with the identification of chemicals and reagents that are harmful to the environment and assigns green shades to eco-friendly ones in a typical pictogram highlighting each of them. Such reagents and chemicals are broadly classified as PBT (persistent, bioaccumulative, and toxic), H (hazardous), C (corrosive), and W (waste generation capability). Contrary to NEMI, a purely semi-quantitative approach of AES allocates penalty points (PPs) to an analytical method according to a corresponding amount of reagent depleted and subsequent possible hazards per single analysis. Additionally, it considers overall power demand, waste produced, and any operational risks for assigning PPs. Final greenness scores of 75 and above identify a method as being eco-friendly [26–28].

## **3. Results**

## *3.1. Preparation of the Plasma Samples*

A single-step extraction procedure was used to extract NER, NRN, and the IS from the rat plasma. The procedure employed acetonitrile as the most suitable extraction medium with higher recoveries. The addition of acetonitrile helped in protein precipitation and the partition of the analytes and IS during the liquid–liquid extraction process. The extraction recovery was observed to be quite satisfactory for the investigated analytes and the IS. There were no other peaks observed for the plasma components except for the analytes and IS, which ratified a lack of interference with the analytes.

## *3.2. Mobile Phase Selection and Optimization: A Greenness Perspective*

The mobile phase of selection for a UPLC-MS/MS relies solely on the volatility properties and compatibility with the MS/MS system. Only a few restricted options befit these criteria and are used accordingly for vivid applications. However, if a green UPLC-MS/MS method is intended for development, one must adhere to the principles of green analytical chemistry (GAC) and prioritize the use of ethanol as an organic proportion. However, the greenest solvent, ethanol, is less preferred in reversed-phase chromatography because of higher viscosity and UV cutoff than methanol or acetonitrile. With lower viscosity and UV cutoff values than methanol, acetonitrile is the most preferred organic phase for UPLC-MS/MS. Additionally, it has better volatility properties than methanol, which support its use as a mobile phase component. Adding 0.1% formic acid into the mobile phase supports investigations under the positive ionization mode. Ammonium acetate in LC-MS grade water with a pH adjusted to 3.5 using acetic acid served as the aqueous phase. Finally, the method's greenness was ensured by executing the NEMI and AES work strategies, which are described below in detail.

## *3.3. Investigation of Method Greenness*

For a preliminary qualitative method greenness evaluation, the NEMI pictogram procedure was executed. In this procedure, the persistent, bioaccumulative, toxic, hazardous,

and corrosive chemicals and reagents and their probable waste production capabilities were assumed to be alarming if they crossed 50 g. In the present evaluation, three quadrants indicated greenness, except for the quadrant denoted for hazard (H). Figure 1 portrays the NEMI multi-quadrant plot with notions for associated hazards. This helped us to be cautious for minimal possible hazards and to proceed with the penalty scoring system of AES. An overall score of 90 (Table 1) categorized the present method as green. This integrated approach ensured method greenness. From the above investigations, we inferred the exceptionally green nature of the method and its suitability for routine use, as it supports the current thinking to establish an AES framework.

**Figure 1.** NEMI-oriented method greenness assessment pictogram.



(a) Penalty points. (b) Total PP = Amount PP × Hazard PP.

#### *3.4. Optimized LC and MS Conditions*

ACQUITY UPLC® ethylene bridged hybrid (BEH) columns (Waters Corp., Milford, MA, USA) made of 1.7 μm particles of C8 and C18 were tested. Upon testing with the isocratic mode of separation, desired chromatographic results were obtained within 4 min. The flow of the mobile phase was 0.5 mL·min−<sup>1</sup> (Supporting Information, Table S1). Inbuilt Intellistart functionality automated the MS scanning and ESI optimization for NER,

NRN, and the IS. This function selected the ion transitions (Figure 2A–C) for NER (m/z of 557.138→111.927), NRN (m/z of 273.115→152.954), and the IS (m/z 494.5→394) out of the three available peaks. The Intellistart supported automated optimized MS/MS conditions for the valid quantification of the analytes, which are listed in the Supporting Information, Table S2. The typical MS/MS fragmentation products for NER, NRN, and the IS have also been provided in Figure 2.

**Figure 2.** Representative parent to daughter ion MRM transitions and the fragmentation products formed for (**A**) NER, (**B**) IS, and (**C**) NRN at LLOQ.

## *3.5. Validation of Results*

The results for the method validation parameters and their acceptance criteria are described in the below sections.

#### 3.5.1. Method Selectivity

The analyzed MRM chromatograms recorded for the blank plasma of six separate animals (Supporting Information, Figure S1A–C) showed an absence of any interference at the identified retention times for NER and NRN (Supporting Information, Figure S2A–C). This ensured adequate method selectivity to support the quantification of the analytes.

## 3.5.2. Method Linearity and Quantitation Limits

A concentration range over 10–1280 ng·mL−<sup>1</sup> of both NER (y = 0.082x + 0.071) and NRN (y = 0.060x + 0.058) was found to be linear (R<sup>2</sup> = 0.998). Figure 3 depicts the linear

calibration plot for NER and NRN between concentrations (ng·mL<sup>−</sup>1) versus the analyte/IS peak area ratio. At the lowest concentration of 10 ng·mL<sup>−</sup>1, both the analytes (Table 2)were adequately quantified (Figure 4A–C)and established as the method's LLOQ values.

**Figure 3.** Calibration curve of NER and NRN.



RE: Relative error; RSD: Relative standard deviation.

#### 3.5.3. Trueness and Precision

The results obtained for trueness (% recovery) and precision (% RSD) at LLOQ, LQC, MQC, and HQC levels are listed in Table 2. In addition, the intraday and interday recoveries for NER were found to be between −8.7 and −10.5% (% RE), while for NRN, the values were found to be between −8.76 and −10.6% (% RE), respectively. Moreover, the method's preciseness was found to be between 1.1 and 2.8% (% RSD) for both the analytes, indicating its acceptability for bioanalytical applications.

**Figure 4.** Representative MRM chromatogram of blank plasma samples of (**A**) NER, (**B**) IS, and (**C**) NRN at LLOQ.

## 3.5.4. Carryover and Dilution Integrity

Carryover was not observed by analyzing the blank plasma samples instantly after analyzing the highest calibration concentration of the analytes. In the dilution integrity studies for NER and NRN, the actual samples' concentrations were above the upper limit of the calibration range. The mean diluted concentrations were acceptable within ±6.8 and 7.5% of the nominal concentration for NER and NRN, respectively.

#### 3.5.5. Extraction Recovery and Matrix Effect

Analyte recovery was found to range between 85.81 and 88.21% for NER and 87.09 to 89.44% for NRN, respectively (supporting information, Table S3), at different studied concentrations. The matrix effect was found to be 10.12–14.13% and 9.61–13.07% for NER and NRN, respectively. In addition, the %RSD values of NER, NRN, and IS were found to be within 2%. These results implied the good extraction of NER and NRN in the plasma of rats and negligible influence of the studied matrix.

#### 3.5.6. Stability of the Analytes

The stability test conditions such as bench-top, short-term, long-term, freeze–thaw, and post-processing were tested for NER, NRN, and IS in the rat plasma. The data in Table 3 indicate the stable nature of the analytes in the plasma of rats under the investigated stress conditions, where good extraction recovery and a lack of interference of any plasma components with analytes were observed.


**Table 3.** Stability data of the method.

RE: Relative error, RSD: Relative standard deviation.

## **4. Discussion**

NER and NRN are a promising combination of modern and natural constituents for the effective management of breast cancer. The former controls breast cancer by its irreversible binding to epidermal growth factor receptors. In contrast, the latter is a derivative aglycone of hydrogenated flavanone origin that inhibits the migration of cancer cells to other body regions, alongside other health benefits that control disease progression. However, to efficiently monitor the therapeutic output of such a combination of drugs, sound bioanalytical LC-MS/MS methods are necessary. The qualitative identification and accurate quantitative determination of analytes in complex biological samples are key benefits of using such techniques. The current UPLC-MS/MS method was newly developed and validated per the regulatory guidelines governing the bioanalysis of drugs in biological fluids. The uncomplicated recovery of analytes from the rat plasma signifies the aptness of the current hyphenated method for the proposed purpose. Combined greenness assessment using contemporary techniques and a high greenness score (score = 90) for the optimized chromatographic condition aptly separated and quantified the analytes and promised environmental sustainability for future use. The method's validation results were satisfactory, with a sensitive linear range of analyte concentrations that engulfed the trueness, precision, selectivity, and good LLOQ values. The final results of all such studies were beneficial to the guidance and reference values that the USFDA and other regulatory bodies have established. No carryover was observed, and the dilution integrity was acceptable. The recovery of analyte greater than 85%, supported with the least matrix effect, vouches for the method's suitability. Finally, the results of the stability study under different stress conditions suggest the acceptable nature of the analytes in the studied matrix.

## **5. Conclusions**

A UPLC-MS/MS method was developed to simultaneously quantify NER and NRN from rat plasma. During the pre-development and optimization phase, the investigations constituted the use of greenness assessment tools such as NEMI and AES, which construed the greenness of the present method. In addition, sensitive LLOQ values were found to befit the method linearity. Further, the validation results from trueness and precision, selectivity, carryover, dilution integrity, recovery and matrix effect, and stability studies matched method intent and were satisfactory. Hence, after assessing the overall study results, the present method conforms to the requirement of simultaneous bioanalytical quantification of the cited analytes for therapeutic drug monitoring in plasmatic samples. The excellent outcome of this work can potentially be linked to evaluating the pharmacokinetic parameters of the drugs in animals and humans.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/separations10030167/s1, Table S1: Current UPLC-MS/MS method parameter details, Table S2: Details of MS/MS parameters for studied analytes, Table S3: Data showing extraction recovery and matrix effect; Figure S1: Representative MRM chromatograms of blank plasma samples of (A) NER, (B) I.S. and (C) NRN at 80 ng·mL−<sup>1</sup> concentration; Figure S2: Representative MRM chromatograms of plasma samples spiked with (A) NER, (B) IS, and (C) NRN at 80 ng·mL−<sup>1</sup> concentration

**Author Contributions:** A.A. and S.M.A.—study design and funding support; S.S.P.—method validation, data analysis, manuscript writing; M.A., A.B.A. and W.H.A.—data analysis, funding support; M.A.B., R.A.R. and S.N.M.N.U.—experimental support; M.A.A. and M.R.—manuscript writing; S.B.—software support, data analysis, manuscript writing, and language corrections. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors extend their appreciation to the Deanship for Research & Innovation and the Ministry of Education in Saudi Arabia for funding this research work through project number: IFP22UQU4310387DSR180.

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Animal Ethic Committee (IAEC) of Roland Institute of Pharmaceutical Sciences (RIPS) (Berhampur, Odisha 760010, India), with the protocol number 146/Chairman IAEC, RIPS, Berhampur (Approval Date: 13 November 2021).

**Data Availability Statement:** The data are contained within the article or Supplementary Material. The data presented in this study are available in its tables, figures, and Supplementary Materials.

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

## **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

## *Article* **Determination of Pterostilbene in Pharmaceutical Products Using a New HPLC Method and Its Application to Solubility and Stability Samples**

**Nazrul Haq 1, Faiyaz Shakeel 1, Mohammed M. Ghoneim 2, Syed Mohammed Basheeruddin Asdaq 2, Prawez Alam 3, Fahad Obaid Aloatibi <sup>4</sup> and Sultan Alshehri 1,\***


**Abstract:** The quantification of a natural bioactive compound, pterostilbene (PTT), in commercial capsule dosage form, solubility, and stability samples was carried out using a rapid and sensitive high-performance liquid chromatography (HPLC) approach. PTT was quantified on a Nucleodur (150 mm × 4.6 mm) RP C18 column with a particle size of 5 μm. Acetonitrile and water (90:10 *v/v*) made up the mobile phase, which was pumped at a flow speed of 1.0 mL/min. At a wavelength of 254 nm, PTT was detected. The developed HPLC approach was linear in 1–75 μg/g range, with a determination coefficient of 0.9995. The developed HPLC approach for PTT estimation was also rapid (Rt = 2.54 min), accurate (%recoveries = 98.10–101.93), precise (%CV = 0.59–1.25), and sensitive (LOD = 2.65 ng/g and LOQ = 7.95 ng/g). The applicability of developed HPLC approach was revealed by determining PTT in commercial capsule dosage form, solubility, and stability samples. The % assay of PTT in marketed capsules was determined to be 99.31%. The solubility of PTT in five different green solvents, including water, propylene glycol, ethanol, polyethylene glycol-400, and Carbitol was found to be 0.0180 mg/g, 1127 mg/g, 710.0 mg/g, 340.0 mg/g, and 571.0 mg/g, respectively. In addition, the precision and accuracy of stability samples were within the acceptable limit, hence PTT was found to be stable in solution. These results suggested that PTT in commercial products, solubility, and stability samples may be routinely determined using the established HPLC method.

**Keywords:** dosage form; HPLC; pterostilbene; solubility; stability; validation

## **1. Introduction**

As strong antioxidants, natural polyphenols have a key role in regulating a variety of physiological diseases [1,2]. Pterostilbene (PTT) is one of the polyphenolic antioxidants with the chemical structure shown in Figure 1 [3]. Although it can be found in a wide range of plants and fruits, PTT is primarily derived from *Pterocarpus marsupium* [4–6]. It is used to manage diabetes and hypertension in conventional medical care [7]. In the literature, it has also demonstrated for a number of therapeutic efficacies, including antioxidant [8,9], anti-inflammatory [9], anticancer [9,10], antidiabetic [11], cardioprotective [12], and neuroprotective [13] effects, among others.

Due to its wide spectrum of medicinal efficacies, PTT's quality control and standardization in its commercial polyherbal products are crucial. For the qualitative and quantitative detection of PTT in plant extracts, commercial products, commercial polyherbal products, and biological samples, numerous analytical approaches have been reported. These

**Citation:** Haq, N.; Shakeel, F.; Ghoneim, M.M.; Asdaq, S.M.B.; Alam, P.; Aloatibi, F.O.; Alshehri, S. Determination of Pterostilbene in Pharmaceutical Products Using a New HPLC Method and Its Application to Solubility and Stability Samples. *Separations* **2023**, *10*, 178. https://doi.org/10.3390/ separations10030178

Academic Editor: Kenichiro Todoroki

Received: 2 February 2023 Revised: 24 February 2023 Accepted: 1 March 2023 Published: 7 March 2023

**Copyright:** © 2023 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/).

71

analytical approaches include ultraviolet (UV) spectrometry, high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography (UHPLC) for the determination of PTT [14–20]. PTT has also been identified in biological samples using several HPLC and UHPLC techniques, either alone or in conjunction with other bioactive chemicals [21–24]. To determine PTT in plant extracts, a high-performance thin-layer chromatography (HPTLC) method has also been used [25]. Recently, a greener and sustainable HPTLC approach has also been used by our research group to determine PTT in commercial capsule dosage forms [26].

**Figure 1.** Chemical structure of pterostilbene (PTT).

A thorough literature evaluation suggested a variety of analytical approaches to determine PTT in different kinds of sample matrices. However, the methods in the literature have not been utilized for the measurement of PTT solubility. The solubility of bioactive compounds such as PTT is an important characteristic, and therefore its measurement in a variety of green solvents is important. As a result, simple and cost-effective analytical approaches are still required for its analysis and for further applications. Therefore, the aim of this research was to design and validate a simple, rapid, and cost-effective HPLC approach to determine PTT in commercial capsule dosage forms, solubility, and stability samples. The developed HPLC approach for determining PTT was validated according to the "International Council for Harmonization (ICH)-Q2-R1" protocols [27].

#### **2. Materials and Methods**

## *2.1. Materials*

The working standard of PTT was provided by Sigma-Aldrich (St. Louis, MO, USA). Chromatography-grade solvents, including, methanol, acetonitrile, and ethanol were provided by E-Merck (Darmstadt, Germany). High pure water was obtained from Milli-Q® water purifier (Millipore, Lyon, France). All other materials used were of analytical grade. Commercial PTT capsules were bought from the neighborhood pharmacy in Riyadh, Saudi Arabia.

## *2.2. Instrumentation and Analytical Conditions*

PTT was measured at 25 ± 1 ◦C using a Waters HPLC system 1515 isocratic pump, a 717 automatic sampler, a programmed UV-visible variable wavelength detector, a column oven, a SCL 10AVP system controller, and an inline vacuum degasser. The data were processed and analyzed using Millennium software (version 32, Waters, Milford, MA, USA). PTT was determined using a Nucleodur (150 mm × 4.6 mm) RP C18 column with 5 μm-sized particles. The mixture of acetonitrile and water (90:10% *v/v*) was used as the mobile phase. The mobile phase was flowed with a flow speed of 1.0 mL/min. At a wavelength of 254 nm, PTT was detected. The samples (20 μL) were injected into the system using a Waters autosampler.

## *2.3. PTT Calibration Curve*

An appropriate quantity of PTT was dispensed in a mobile phase to create a PTT stock solution with a 100 μg/g concentration. The required aliquots from the stock solution of

PTT (100 μg/g) were diluted with the mobile phase to create the serial dilutions in the necessary range (1–75 μg/g). Instead of volume/volume, all of the dilutions were prepared on a mass/mass basis. Using the established HPLC method, the chromatographic area for each concentration of PTT was identified. To create the PTT calibration curve, eight different PTT concentrations (1, 5, 10, 15, 20, 25, 50, and 75 μg/g) were plotted against the measured chromatographic area. All the sample preparation and experiments were performed in three replicates (n = 3).

## *2.4. Analytical Method Development*

As the eluent system/mobile phase, various combinations of organic/hydro-organic solvents were investigated for the development of a trustworthy HPLC approach for the detection of PTT in commercial capsules, solubility, and stability samples. The mixtures of methanol–water, ethanol–water, acetonitrile–water, methanol–ethanol, acetonitrile– methanol, acetonitrile–ethanol, methanol–formic acid, ethanol–formic acid, and acetonitrile– formic acid were among the numerous solvents that were investigated. Numerous aspects were taken into account when determining the best solvent or combination of solvents, including the solvents' affordability, the assay's sensitivity, the length of the analysis, the chromatographic parameters and the solvents' compatibility with one another. As a result, different solvents including methanol, ethanol, and acetonitrile were investigated as the mobile phase in both their individual and combined forms with water and formic acid.

## *2.5. Validation Parameters*

Following ICH-Q2-R1 procedures, the developed analytical approach for the measurement of PTT was verified for several parameters [27]. By drawing the linearity plots, the linearity of the developed analytical approach could be investigated in the 1–75 μg/g range. PTT solutions that had just been produced were added to the HPLC apparatus in triplicates (n = 3), and the peak area was estimated. A PTT calibration curve was obtained by plotting PTT concentration vs. peak area.

The peak symmetry, tailing factor (As), capacity factor (k), and theoretical plates number (N) were obtained to examine the system suitability parameters for the developed analytical approach [28,29].

The developed analytical approach's intra-day and inter-day accuracy was estimated using the percent recovery technique. Three replicates (n = 3) were performed on the same day to test intra-day accuracy at three different quality control (QC) levels: low QC (LQC = 10 μg/g), middle QC (MQC = 50 μg/g), and high QC (HQC = 75 μg/g). On three separate days, three replicates (n = 3) of the PTT's LQC, MQC, and HQC levels were used to test inter-day accuracy. The percentage recovery, percentage coefficient of variance (%CV), and standard error were computed for each QC level.

The developed analytical approach's precision was evaluated using intra-day and inter-day variations. On the same day, the same QC levels of PTT (as those used for accuracy) were used to determine the intraday precision. At the same QC levels of the PTT on three consecutive days, inter-day precision was assessed. Both precisions were measured in three replicates (n = 3).

To investigate the impact of intentional chromatographic alterations on PTT analysis, the robustness of the developed analytical approach was evaluated. The PTT MQC (50 μg/g) was selected for the robustness analysis. By adjusting the mobile phase's composition, flow speed, and detecting wavelength, robustness was examined. The initial acetonitrile: water (90:10 *v/v*) mobile phase was adjusted to acetonitrile: water (92:8 *v/v*) and acetonitrile: water (88:12 *v/v*) for the robustness investigation, and the differences in chromatographic response were recorded for each combination of mobile phase. The original flow speed (1 mL/min) was changed to flow rates of 1.1 mL/min and 0.9 mL/min for robustness evaluation by adjusting flow speed, and the variations in chromatographic response were recorded for each set of flow rates. The initial detection wavelength (254 nm) was changed to detection wavelengths of 256 nm and 252 nm for the robustness evaluation

by altering the detection wavelength, and the variations in chromatographic response were recorded at each wavelength.

The developed analytical technique's sensitivity was evaluated in terms of the limit of detection (LOD) and limit of quantitation (LOQ), utilizing the standard deviation approach [27]. After the sample was injected into the HPLC system three times (n = 3), the standard deviation of the response was calculated. The LOD and LOQ for PTT were determined using the following equations [27,28]:

$$\text{LOD} = 3.3 \times \frac{\sigma}{\text{S}} \tag{1}$$

$$\text{LOQ} = 10 \times \frac{\sigma}{\text{S}} \tag{2}$$

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

#### *2.6. Application of Developed HPLC Approach in the Assay of PTT in Commercial Capsules*

Ten capsules (each containing an equivalent of 200 mg of PTT) were consumed at random for the test of PTT in commercial capsules, and the average weight was determined. The capsule contents were taken out from the capsule shell and mixed well to obtain the fine powder. The fine powder, with an equivalent to 200 mg of PTT, was dispersed in 100 g of methanol and sonicated for about 15 min. Then, 1 g of this solution was further diluted with methanol to obtain the stock of 100 g. The obtained mixtures of capsules were filtered [26]. The obtained solutions were used for the pharmaceutical assay of PTT in commercial capsules using the developed HPLC approach.

## *2.7. Application of the Developed HPLC Approach in the Determination of PTT in Solutions*

The main purpose of measuring PTT solubility was to enhance the application of the developed method. The solubility of PTT in five different green solvents including water, propylene glycol (PG), ethanol, polyethylene glycol-400 (PEG-400), and Carbitol was determined at 25 ◦C using a previously reported shake flask method [30]. The excess of PTT was placed into known amounts (10 g) of each green solvent and examined in three replicates (n = 3). The obtained concentrated suspensions were vortexed for about 5 and transferred to a biological shaker for continuous shaking at 100 rpm speed for 72 h [31,32]. The samples were cautiously removed from the shaker once equilibrium had been reached. All the samples were centrifuged at 500 rpm for 30 min. The supernatants from each sample were taken, diluted with mobile phase (wherever required), and subjected to determination of PTT using the developed HPLC approach at a wavelength of 254 nm.

## *2.8. Application of the Developed HPLC Approach in the Determination of the Stability of PTT in Solutions*

The main purpose of determining PTT stability was to enhance the application of developed method. The stability of PTT solution was performed at MQC level (50 μg/g) at two different temperatures, i.e., bench temperature (25 ± 1 ◦C) and refrigeration temperature (4 ± 0.5 ◦C). In this work, solution studies were performed; these studies were performed for a short period of time (72 h). The MQC of PTT solution was prepared in mobile phase and stored at 25 ± 1 ◦C and 4 ± 0.5 ◦C for about 72 h, and the decomposition of PTT was determined by measuring the rest of PTT after storage.

## **3. Results and Discussion**

#### *3.1. Analytical Method Development*

Table 1 provides a summary of the measured chromatographic characteristics and the composition of various eluent systems. The application of methanol and water in various ratios during the analytical method development step led to a subpar chromatographic response of PTT, which exhibited higher As values (As > 1.30) with low N values (<3000). Additionally, the use of ethanol and water in various ratios caused PTT to have a poor chromatographic response as well as increased As values (As > 1.45) and low N values (<2000). The combination of organic solvents, including acetonitrile and ethanol, acetonitrile and methanol, and methanol and ethanol, was also looked at as an eluent system. With high As values (As > 1.35) and low N values (<3000), the chromatographic response of PTT was once more subpar. We also looked at the binary combinations of organic solvents with formic acid, including methanol: formic acid and ethanol: formic acid. Additionally, the PTT chromatographic response of these binary combinations was subpar, with bigger As values (As > 1.35), and lower N values (<2000).

**Table 1.** Summary of the eluent systems and measured analytical responses for pterostilbene (PTT) (mean ± SD, n = 3).


As: tailing factor; N: number of theoretical plates; Rt: retention time.

However, a well-resolved and intact PTT chromatographic peak with good As values and greater N values was shown by the binary mixture of acetonitrile and water in various ratios. The binary mixture of acetonitrile and water (90:10 *v/v*) gave the best chromatographic response (Figure 2). As a consequence, this mixture was chosen as the final eluent system for measuring PTT, with an acceptable As (1.07) and N (5125), rapid analysis (Rt = 2.54 ± 0.02 min), and a suitable analysis duration (5 min). Therefore, the most trustworthy eluent system for future investigation was a 90:10, volume-to-volume blend of acetonitrile and water.

**Figure 2.** High-performance liquid chromatography (HPLC) chromatogram of PTT (10 μg/g concentration) in solution, produced using a binary eluent system that consisted of acetonitrile and water (90:10 *v/v*).

#### *3.2. Validation Studies*

Several validation parameters for the developed HPLC approach were determined following ICH-Q2-R1 protocols [27]. The linearity graphs were constructed using freshly produced PTT samples (1–75 μg/g). The outcomes of a linear regression analysis of the PTT calibration curve are shown in Table 2. The linear calibration curve for PTT was between 1 and 75 μg/g. According to estimates, the calibration curve's determination coefficient (R2) and regression coefficient (R) values are 0.9995 and 0.9997, respectively. These outcomes revealed the efficiency of the developed analytical approach for determining PTT.

**Table 2.** Linear regression analysis for the calibration curve of PTT for the "high-performance liquid chromatography (HPLC)" approach (mean ± SD, n = 3).


R2: determination coefficient; R: regression coefficient; SD: standard deviation; SE: standard error; CI: confidence interval; LOD: limit of detection; LOQ: limit of quantification.

The system's appropriateness parameters for the developed analytical approach were determined using the peak symmetry, As, k, and N. The results are shown in Table 3. The developed analytical approach's values for peak symmetry, As, k, and N were found to be satisfactory and acceptable for determining PTT.

**Table 3.** Optimized chromatographic peak parameters for the resolution of PTT for HPLC approach (mean ± SD, n = 3).


As: tailing factor; k: capacity factor; N: number of theoretical plates.

The percent recovery at LQC, MQC, and HQC was used to determine the intra-day and inter-day accuracy of the established HPLC technique. The results are shown in Table 4. At three different QC levels, the intra-day and inter-day percent recoveries of PTT were found to be 98–102 and 98–101 percent, respectively. According to ICH guidelines, the percent recoveries of analytical method should be within the limit of 100 ± 2% [27]. The percent recoveries of two literature HPLC methods have been reported as 98–99 and 96–100 percent, respectively [17,18]. The percent recoveries of current HPLC method were similar to first reported method [17] and superior to second reported method [18], as per ICH guidelines. High percent recoveries for the established HPLC method for determining PTT point to its accuracy.

The results of the intra-day and inter-day precisions are summarized in Table 5 and are indicated in %CV. For PTT, the intraday variation percent CVs were observed to range from 0.59 to 1.15%. On the contrary, the %CVs for inter-day precision ranged between 0.60 and 1.25 percent. The %CVs of current HPLC method were similar to reported methods [17,18]. Low %CVs in the devised HPLC method for calculating PTT indicated its precision.


**Table 4.** Intra-day and inter-day accuracy results of PTT for HPLC approach (mean ± SD; n = 3).

**Table 5.** Intra-day and inter-day precision of PTT for HPLC approach (mean ± SD; n = 3).


Table 6 contains the results of the robustness assessment for the MQC level of PTT. When evaluating robustness by altering the composition of the mobile phase, the %CV and Rt were discovered to be 0.78–1.18% and 2.53–2.55 min, respectively. The %CV and Rt were found to be 0.43–1.45% and 2.28–2.75 min, respectively, in the scenario of a robustness assessment when the flow speed was changed. The %CV and Rt were calculated to be 1.17–1.55% and 2.55–2.57 min, respectively, in the scenario of a robustness assessment by shifting detecting wavelength. Low CVs and minimal Rt value swings in the devised HPLC method for detecting PTT indicate its robustness.

**Table 6.** Robustness results of PTT at MQC (50 μg/g) for the HPLC approach (mean ± SD; n = 3).


Table 2 lists the findings from evaluating the developed analytical approach's sensitivity in terms of LOD and LOQ. The LOD and LOQ for the developed analytical approach were discovered to be 2.65 ± 0.09 ng/g and 7.95 ± 0.27 ng/g, respectively. These results suggested that the developed analytical approach would have sufficient sensitivity to determine PTT.

The developed HPLC approach for the determination of PTT was compared with reported analytical assays used to determine PTT in solution form. The validation parameters of present HPLC approach compared with reported analytical methods are listed in Table 7. Most of the validation parameters of reported HPLC assays were within the limits of ICH protocol, and hence were similar to the present HPLC approach [17,18]. However, the linearity range, accuracy, precision, LOD, and LOQ values of the HPTLC approaches of PTT analysis in the literature were also found to be inferior to the present HPLC approach [25,26]. Furthermore, the LOD and LOQ values of PTT for the present method were lower than the reported HPLC and HPTLC methods, and were hence found to be more sensitive than the reported HPLC and HPTLC methods. Overall, the newly developed and validated HPTLC approach has been found to be reliable for the determination of PTT.


**Table 7.** Comparative summary of validation parameters of the present HPLC method with reported methods for the determination of PTT.

#### *3.3. Assay of PTT in Marketed Capsules*

The developed analytical approach for the PTT assay was shown to be efficient, quick, and sensitive. This approach was therefore used to ascertain PTT in its commercial capsule dosage form. The PTT percentage assay was 99.31% in the commercial capsule dosage form. The PTT percentage in different brands of commercially available capsule dosage forms has been reported as 98.75–98.94% using an HPLC method from the literature [17]. The PTT percentage in the marketed capsule dosage form has been reported as 92.59 and 100.84%, respectively, using routine and sustainable HPTLC methods [26]. The current HPLC method in terms of PTT assay was identical to the reported HPLC and sustainable HPTLC methods [17,26]. However, it was much superior to the reported routine HPTLC method [26]. These findings suggest that the HPLC method would work well for determining PTT in commercially available dosage forms.

## *3.4. Determination of PTT in Solubility Samples*

The potential of the developed HPLC approach was demonstrated by determining the solubility of PTT in five different green solvents, including water, PG, ethanol, PEG-400, and Carbitol, at 25 ◦C. At 25 ◦C, the solubility of PTT in water, PG, ethanol, PEG-400, and Carbitol was found to be 0.0180 mg/g, 1127 mg/g, 710.0 mg/g, 340.0 mg/g, and 571.0 mg/g, respectively. Based on these results, PTT was found to be poorly soluble in water, freely soluble in ethanol, PEG-400, and Carbitol, and very soluble in PG [33,34]. Similar solubility characteristics of PTT in water, PG, ethanol, PEG-400, and Carbitol at 25 ◦C have also been reported in the literature [31]. Hence, the obtained solubility results of PTT were in accordance with those reported in the literature [31]. These results suggested that the developed HPLC approach would be suitable for determining PTT in solubility samples.

## *3.5. Stability Studies of PTT in Solution*

The potential of the developed HPLC approach was also demonstrated by determining the stability of PTT in solution at two different temperatures. The solution of PTT was prepared in mobile phase (acetonitrile: water, 90:10 *v/v*). The findings of stability evaluations at two different temperatures are included in Table 8. The PTT degradation was measured by determining the rest of PTT concentration after storage. The PTT degradation was very low when held for 72 h at 25 ± 1 ◦C, and at 4 ± 0.5 ◦C, when the peak areas of the stored PTT solution were compared to those obtained from a freshly made PTT solution. The precision of PTT in terms of %CV was found to be 1.04–1.07% at two different temperatures. Furthermore, the percent recovery of PTT was found to be 99.84–100.42 percent at two different temperatures. PTT was discovered to be sufficiently stable in solution form

at 25 and 4 ◦C as a result. These findings indicated that PTT stability in solution could be determined using the HPLC method that was established.


**Table 8.** Stability data of PTT at MCQ level at two different temperatures (mean ± SD; n = 3).

#### **4. Conclusions**

A rapid, sensitive, and economical HPLC approach has been designed and validated for the quantification of PTT in its marketed products, solubility, and stability samples. The developed HPLC approach was validated per ICH-Q2-R1 protocols. The developed analytical approach is rapid, accurate, precise, robust, sensitive, and economical for estimating PTT. The developed HPLC approach was found to be reliable for the determination of PTT in commercial capsule dosage forms, solubility, and stability samples. Based on these findings, it is possible to effectively estimate PTT in a variety of sample matrices using the established HPLC approach. In future, further studies can be carried out to determine PTT in the complex matrices of biological samples, and to accomplish pharmacokinetic assessment of PTT.

**Author Contributions:** Conceptualization, S.A. and F.S.; methodology, N.H., M.M.G. and S.M.B.A.; software, N.H., F.O.A. and F.S.; validation, S.A. and P.A.; formal analysis, F.O.A. and P.A.; investigation, F.S. and N.H.; resources, S.A.; data curation, P.A.; writing—original draft preparation, F.S.; writing—review and editing, N.H., S.A. and P.A.; visualization, S.A.; supervision, F.S. and S.A.; project administration, F.S. and S.A.; funding acquisition, S.A. and M.M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Researchers Supporting Project (number RSP2023R146) at King Saud University, Riyadh, Saudi Arabia. This study was also supported via funding from Prince Sattam bin Abdulaziz University project number (PSAU/2023/R/1444). The APC was funded by RSP.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are thankful to the Researchers Supporting Project (number RSP2023R146) at King Saud University, Riyadh, Saudi Arabia for supporting this research. The authors are also thankful to Prince Sattam bin Abdulaziz University for supporting this work via project number (PSAU/2023/R/1444). The authors are also thankful to AlMaarefa University for their generous support.

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

#### **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

## **Evaluation of Chinese Prickly Ash and Cinnamon to Mitigate Heterocyclic Aromatic Amines in Superheated Steam-Light Wave Roasted Lamb Meat Patties Using QuEChERS Method Coupled with UPLC-MS/MS**

**Raheel Suleman 1,2, Muawuz Ijaz 3, Huan Liu 1, Alma D. Alarcon-Rojo 4, Zhenyu Wang <sup>1</sup> and Dequan Zhang 1,\***


**Abstract:** Chinese prickly ash and cinnamon contain many antioxidants, which scavenge free radicals and can reduce many harmful compounds, such as heterocyclic aromatic amines (HAAs). Modern technologies used for cooking, such as the use of superheated steam roasting, are beneficial in decreasing the development of HAAs. The current study was based on the use of these two spices in roasted lamb patties to mitigate the formation of HAAs in superheated steam roasted patties. Results exhibited significant differences (*p* < 0.05) in the content of both polar and non-polar HAAs as compared to control patties. In cinnamon roasted patties, polar HAAs were reduced from 23.76 to 10.56 ng g−1, and non-polar HAAs were reduced from 21.34 to 15.47 ng g−1. In Chinese prickly ash patties, polar and non-polar HAAs were 43.60 ng g−<sup>1</sup> and 35.74 ng g<sup>−</sup>1, respectively. Similarly, cinnamon-treated patties showed a significantly higher (*p* < 0.05) reduction in polar HAAs (23.52 to 12.41 ng g−1) than non-polar (16.08 to 9.51 ng g−1) at concentrations of 0.5–1.5%, respectively, as compared to the control, with 45.81 ng g−<sup>1</sup> polar and 35.09 ng g−<sup>1</sup> non-polar HAAs. The polar HAAs tested were PhIP, DMIP, IQx, and 8-MeIQx, while the non-polar were harman and norharman. Both spices and superheated steam controlled HAAs to a significant level in lamb meat patties.

**Keywords:** lamb meat; heterocyclic aromatic amines; roasted; spices

## **1. Introduction**

Heterocyclic aromatic amine (HAA) formation depends on heat transfer, lipid degradation, oxidation, and cooking methods, such as barbecuing and grilling [1]. Heterocyclic amines can be reduced by the application or induction of antioxidants, as well as the modification of cooking methods [2]. The addition of antioxidants to meat has been considered to be an effective strategy to reduce HAA exposure because of the hypothetical free radical pathway leading to HAA formation [3].

According to Adeyeye [4], antioxidants may trap free radicals, such as intermediates of HAAs, to prevent the formation of HAAs. Among spices, cinnamon and Chinese prickly ash are good sources of antioxidants in meat and meat products in Asian countries [5]. In a lipid peroxidation assay test by Thaipong [6], it was estimated that cinnamon showed more significant activity than anise, ginger, licorice, nutmeg, or vanilla. Chinese prickly ash is also consumed in Central Asian countries, such as China, and is used as an important spice [7]. Antioxidant compounds, such as sanshools and sanshoamides, are very beneficial

**Citation:** Suleman, R.; Ijaz, M.; Liu, H.; Alarcon-Rojo, A.D.; Wang, Z.; Zhang, D. Evaluation of Chinese Prickly Ash and Cinnamon to Mitigate Heterocyclic Aromatic Amines in Superheated Steam-Light Wave Roasted Lamb Meat Patties Using QuEChERS Method Coupled with UPLC-MS/MS. *Separations* **2023**, *10*, 323. https://doi.org/10.3390/ separations10060323

Academic Editor: Faiyaz Shakeel

Received: 13 May 2023 Revised: 21 May 2023 Accepted: 22 May 2023 Published: 25 May 2023

**Copyright:** © 2023 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/).

compounds in this spice, and are responsible for its antioxidant activity [8]. The spices can be a beneficial strategy to control HAAs in cooked meat products [9].

Modern cooking methods can be more helpful in the reduction of HAAs than conventional barbecuing or grilling, which include direct contact of the meat with flame [10]. Modern cooking methods, such as infrared grilling and superheated steam-light wave roasting, offer great potential to reduce HAAs in cooked lamb meat products [11]. One new technology introduced superheated steam-light wave roasting, which is applied by using water vapor to form steam in the oven, and has a higher temperature, which can be helpful in controlling HAAs at a very significant level [12]. The study aimed to analyze the effect of spices and superheated steam-light wave roasting to inhibit HAAs in roasted lamb meat patties.

#### **2. Materials and Methods**

### *2.1. Chemicals and Reagents*

The HAA standards 2-amino-9H-pyrido [2,3-b]indole (AαC), 2-amino-3-methyl-9Hpyrido[2,3-b]indole (MeAαC), 1-methyl-9Hpyrido[3,4-b]indole (Harman), 9Hpyrido[3,4-b]indole (Norharman), 2-amino-6-methyldipyrido[1,2-a:3 ,2 -d]imidazole (Glu-P-1), 2-amino-1,6-dimethylfuro[3,2-e]imidazo[4,5-b]pyridine (IFP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (TrP-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b] indole (TrP-P-2), 2 amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-1-methylimidazo[4,5-b]quinoline (IQ[4,5-b]), 2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-1-methylimidazo[4,5-f] quinoline (ISO-IQ), 2-amino-1,6-dimethylimidazo[4,5-b]pyridine (DMIP), 2-amino-5-phenylpyridine (Phe-P-1), 2-amino-3-methyl-3H-imidazo [4,5-f]quinoxaline (IQx), 2-amino-3,8-dimethylimidazo[4,5 f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx) were purchased from Toronto Research Chemicals (Canada). The purity of all standards was greater than 99.9%. The other reagents included for HAA extraction and purification included QuEChERS extraction packets, containing 4 g of magnesium sulfate and 1 g of ammonium acetate. The primary and secondary amine (PSA), endcapped C-18EC extraction column, and MgSO4, together in 15 mL centrifuge tubes, were procured from Agilent Technologies (Santa Clara, CA, USA). DPPH, BHT, ammonium acetate, and acetonitrile were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Chemicals and reagents were obtained in the packaged form and kept at a suitable required temperature until used for analysis.

## *2.2. Lamb Meat and Spices*

A total of 12 fresh lamb shoulder oyster cut muscles of 8 month old sheep were obtained from the Hongbao sheep meat industry of Bayannur, Inner Mongolia, China. The spices, cinnamon powder and Chinese prickly ash powder, were bought from the local spice market in Beijing, China [5].

## *2.3. Determination of DPPH Activity of Spices*

The antioxidant activity of spices was determined using the DPPH scavenging activity of spices, following the method of Thaipong [13]. Three different concentrations (10, 20, and 30 uL/mL) of Chinese prickly ash and cinnamon were prepared. From each concentration of spices, 1 mL of extract solution was added to 2 mL of freshly prepared DPPH solution (0.1 mM in 95% methanol). This mixture was vortexed for 10 s and then placed in a dark place for 30 min. The absorbance was measured at 517 nm wavelength using the UV-vis spectrophotometer (Persee TU-1810 UV-vis; Persee Instruments Co., Ltd., Beijing, China) at room temperature. Lower absorbance indicated higher radical-scavenging activity. Radicalscavenging activity was calculated as the percentage of DPPH discoloration using the following equation:

DPPH-radical-scavenging activity % = 100 × [1 − *AE*/*AD*],

where *AE* represents the solution absorbance at 517 nm when 1 mL of each spice solution was mixed with 2 mL of 0.1 mmol·L−<sup>1</sup> DPPH solution after incubation (30 min) at room temperature, and *AD* represents the absorbance of 2 mL of 0.1 mmol·L−<sup>1</sup> DPPH solution with 1 mL Milli-Q H2O. The final unit is % DPPH activity of the sample that has decreased the DPPH content by scavenging the radicals.

## *2.4. Preparation of Lamb Patties with Spices*

After the sheep were slaughtered, their carcasses were cooled at 4 ◦C for 24 h. Later, the shoulder oyster muscles were removed from the carcasses and kept at −80 ◦C until they were employed in further experiments. To make the patties, slices of the meat from the lamb oyster muscles were used. It was ensured that visible fat from muscles was properly removed. The 12 muscles were individually ground using a grinder with 5 mm blades to produce ground meat and ensure proper grinding of each muscle. Each muscle weighed 10 g after being ground. Fifty grams of fresh ground lamb meat was taken, and spices (cinnamon and Chinese prickly ash) were added in concentrations of 0.5%, 1%, and 1.5% to the ground meat paste to create each patty, using a 6 cm × 1.5 cm mold to make patties of the same size. The patties with two spices were separately prepared in triplicates. Three patty treatments contained spices, and one without spice was used as control. There were 12 patties in each spice-treated group; therefor, a total of 24 patties were made for each spice (cinnamon and Chinese prickly ash) [5].

## *2.5. Cooking of Patties*

The spiced lamb patties were cooked using a superheated steam-light wave roasting method at 240 ◦C. The patties were roasted for 17 min, which was needed to reach a core temperature of 72 ◦C in the oven. The patties were cooked in two batches; one batch for Chinese prickly ash and the other for cinnamon. A digital data logger (Hangzhou Co., Ltd., Hangzhou, China) with a digital thermometer probe was used to observe the internal temperature of each patty. The lamb patties were further cooled at room temperature for one to two hours, and then packed in zip-lock bags and stored at −20 ◦C until used [11].

## *2.6. Determination of HAAs in Roasted Patties*

HAAs in roasted patties were determined using the method developed by Hsiao, Chen, and Kao [14]. Two grams of ground patty sample were obtained and places in a centrifuge tube. A total of 10 mL of deionized water and one ceramic stone were placed inside the centrifuge tube containing the sample. The tubes were then mixed for 10 min. After adding 10 mL of acetonitrile containing 1% acetic acid, the tube was shaken once more for 10 min. After mixing, 4 g of anhydrous MgSO4 and 1 g of anhydrous C2H3NaO2 were dispersed. After 1 min, the centrifuge tube was spun at a speed of 3200× *g* of relative centrifugal force for 10 min at a temperature of 4 ◦C (RCF). A tube containing 900 mg of anhydrous MgSO4, 300 mg of propylsulfonic acid modified silica (PSA), and 300 mg of C18 was used to purify the supernatant after 10 min of centrifugation. The tube was oscillated for 1 min and centrifuged at 3200× *g*, at 4 ◦C, for 5 min.

One milliliter of the supernatant was removed after 1 min of centrifugation and nitrogen was used to freeze-dry it. The freeze-dried sample received 0.2 mL of methanol, which was then vortexed. The material was then filtered using a polyvinylidene difluoride (PVDF) membrane filter with a pore size of 0.22 μm. The samples were then analyzed using UPLC-MS/MS (Agilent model 1290). Mass spectrometric analyses were performed on an AB Sciex API 4000™ triple quadrupole mass spectrometer equipped with an electrospray ionization source to look for heterocyclic aromatic amines [15]. Separation was achieved on a Shim-pack GIST C18 (2.1 × 100 mm, 3 μm, 100 Å) at 37 ◦C. For further dilution, standard stock solutions containing 10 mg in 5 mL methanol were prepared. To establish calibration curves, LODs, and LOQs, stock solutions of standards mixed solutions with final concentrations of 5, 10, 25, 50, 100, 300, and 500 ppb in methanol were created. The mobile phase for analysis at UPLC-MS/MS was composed of (A) 100% HPLC/UPLC-grade

acetonitrile and (B) 10 mM ammonium acetate solution (pH 2.9) [16]. For the purpose of achieving equilibrium in the column, a linear gradient profile with 85% A and 15% B was maintained for the first 8 min, changing to 45% A and 55% B after 13 min, and then to 91% A and 9% B after 16 min. For the analysis, a column temperature of 25 ◦C was maintained with a flow rate of 0.4 mL/min. The injection volume for each sample's analysis was 2 μL. By using UPLC-MS/MS, each sample was examined for 30 min. Table 1 shows the limits of detection and limits of quantification for all HAAs compounds detected through UPLC-MS/MS.


**Table 1.** Values of limit of detection and limit of quantification of all HAA compounds.

The linear range of 0.1–5.00 ng g−<sup>1</sup> and recovery was from 54.86% to 108.32% for all HAA standards.

Figure 1 shows the peaks of the standards obtained by UPLC-MS/MS. The internal standard was a mixture of 20 HAA standards, and was used for the analysis of HAAs.

**Figure 1.** UPLC-MS/MS chromatograms of 20 HAA standards and one internal standard (4,7,8−TriMeIQx) detected by SRM mode. The standard mixture contained 500 ppb of each HA and 4,7,8−TriMeIQx. (1: DMIP; 2: Glu-P-2; 3: Iso-IQ; 4: IQ; 5: IQx; 6: MeIQ; 7: Glu-P−1; 8: 8−MeIQx; 9: IQ[4,5−b]; 10: IFP; 11: 7, 8−DiMeIQx; 12: 4,8−DiMeIQx; 13: Norharman; I.S. (internal standard): 4,7,8−TriMeIQx; 14: Harman; 15: Phe−P−1; 16: Trp-P−2; 17: PhIP; 18: Trp-P−1; 19: AαC; 20: MeAαC.).

#### **3. Results**

#### *3.1. DPPH Activity of Cinnamon and Chinese Prickly Ash*

Figure 2 shows the DPPH activity of cinnamon and Chinese prickly ash. Both spices showed significant antioxidant activity. Cinnamon showed inhibition rates of 91.96%, 92%, and 91.66% at concentrations of 10, 20, and 30 μL/mL, respectively, while Chinese prickly ash showed inhibition rates of 86.23%, 87.96%, and 89.4% at concentrations of 10, 20, and 30 μL/mL, respectively, which was lower than cinnamon.

**Figure 2.** DPPH activity of cinnamon and Chinese prickly ash. Different small (a, b) and capital (A, B) letters show significant differences (*p* < 0.05) among the concentrations of the two spices.

#### *3.2. Quantity of Polar and Non-Polar HAAs in Superheated Steam-Light Wave Roasted Patties and Chinese Prickly Ash*

Table 2 lists the findings of the polar HAAs found in the patties treated with Chinese prickly ash. Due to the high antioxidant capacity of Chinese prickly ash, it was shown that its inclusion reduced the production of several polar HAAs [16]. Some polar HAAs, such IQ (4,5-b) and 7,8-DiMeIQx, were not found in detectable amounts. Other polar HAAs, such as IQx, IQ, and 4,8-DiMeIQx, were found in low concentrations; their respective concentrations ranged from 3.25 to 1.91 ng g−1, 0.16 to 0.12 ng g−<sup>1</sup> (at 1.5% there was no detection of IQ), and 0.78 to 0.38 ng g<sup>−</sup>1. Results are given as means ± standard errors with superscripts (a, b, c) in columns showing significant differences (*p* < 0.05) within treatments of lamb patties. In beef patties which had Chinese prickly ash also showed a reduction in the polar and the non-polar HAAs [17].

According to the Table 3 findings, the level of non-polar HAAs, such as harman and norharman, which ranged from 8.89 to 6.66 ng g−<sup>1</sup> and 9.28 to 6.08 ng g<sup>−</sup>1, respectively, was greater in lamb patties [18]. The concentration of Glu-P-1 ranged from 2.10 to 1.66 ng g<sup>−</sup>1, and that of Glu-P-2 from 1.29 to 1.12 ng g−1. The lowering of both types of HAA content was observed to be positively impacted by Chinese prickly ash [19]. The Chinese prickly ash-treated patties had lower levels of HAAs than the control samples. According to Figure 3, control patties contained 35.74 ng g−<sup>1</sup> of non-polar HAAs and 43.60 ng g−<sup>1</sup> of polar HAAs.


**Table2.**PolarHAAs(ngg−1)intheChinesepricklyashpowder-treatedpattieswithsuperheatedsteam-lightwaveroasting.

Results are given as means ± standard errors with superscripts (a, b, c.,d) in columns showing significant differences (*<sup>p</sup>* < 0.05) within treatments of lamb patties.


**Table 3.** Non-polar HAAs (ng/g) in the Chinese prickly ash powder-treated patties with superheated steam-light wave roasting.

Results are given as means ± standard errors with superscripts (a, b, c) in columns showing significant differences (*p* < 0.05) within treatments of lamb patties.

**Figure 3.** Total polar and non-polar HAAs in Chinese prickly ash lamb patties. Means with different letters (a, b, c, d) denote significant difference (*p* < 0.05) within treatments of lamb patties.

In comparison to the control, the Chinese prickly ash-treated patties had lower concentrations of polar (23.76 ng g−1) and non-polar (21.34 ng g−1) HAAs at 0.5%. At 1%, polar HAA concentrations were marginally lower (20.69 ng g−1), while non-polar HAA concentrations were found to be 17.73 ng g<sup>−</sup>1. Chinese prickly ash patties contained 10.56 ng g−<sup>1</sup> of polar HAAs at a concentration of 1.5%, while 15.47 ng g−<sup>1</sup> of non-polar HAAs were present. The findings demonstrated that the content of the polar HAAs varied significantly between concentrations, as well as when compared to control patties. With the exception of 1.5% concentration, where total non-polar HAAs decreased relative to total polar HAAs, the overall content of polar HAAs was somewhat higher than non-polar HAAs [5].

## *3.3. Quantity of Polar and Non-Polar HAA Content in Cinnamon and Superheated Steam-Light Wave Roasted Lamb Patties*

Table 4 displays the results of the polar HAAs in lamb patties with cinnamon added in various amounts. The polar HAAs were found and contrasted with the no-spice control. The outcomes revealed that all polar HAA levels were greater in the control group [20]. Although there was a significant difference (*p* < 0.05) among the contents seen in the results, they were consistent with the other two spices studied in [21]. The highest values among polar HAAs were observed in 8-MeIQx, DMIP, and PhIP, which ranged from 11.80 to 4.05 ng g<sup>−</sup>1, 2.93 to 2.24 ng g−1, and 4.47 to 3.16 ng g−1, respectively, and corresponded to treatments 0.5%, 1%, and 1.5% cinnamon powder, respectively. IQ (4,5-b), IQ, ISO-IQ, and 7,8 DiMeIQx content was too low at 1% and 1.5% to be recognized by suppression of cinnamon antioxidants in comparison to the control, while the content was still visible in roasted patties [22].



**Table 4.** Polar HAAs (ng/g) in the cinnamon powder-treated patties with superheated steam-light wave roasting.

Results are given as means ± standard errors with superscripts (a, b, c) in columns showing significant differences (*<sup>p</sup>* < 0.05) within treatments of lamb patties.

IQx and 4, 8-DiMeIQx levels were also very low among polar HAAs in the patties with the addition of cinnamon powder, with content reduced from 2.91 to 2.26 ng g−<sup>1</sup> and 0.98 to 0.68 ng g<sup>−</sup>1, or from 0.5% to 1.5% concentration, in cinnamon patties. The results of non-polar HAAs, reported in Table 5 below, show that harman and norharman were higher in content than Glu-P-1 and Glu-P-2; harman and norharman levels were 7.65–3.56 ng g−<sup>1</sup> and 5.95–3.89 ng g−1, respectively, from 0.5% to 1.5%, while the content of Glu-P-1 and Glu-P-2 was detected to be 1.37–1.03 ng g−<sup>1</sup> and 1.28–1.02 ng g<sup>−</sup>1, respectively. The results showed that the amount of the polar HAAs was higher generally, and, individually, harman and norharman showed higher content at each concentration, but there was a reduction at all concentrations as compared to content observed in the control.

**Table 5.** Non-polar HAAs (ng/g) in the cinnamon powder-treated patties with superheated steamlight wave roasting.


Results are given as means ± standard deviation with different superscripts (a, b, c) in columns showing significant differences (*p* < 0.05) in the treatments and within treatments of lamb patties.

Cinnamon powder has shown a very potent activity towards the reduction of HAAs after addition in different concentrations [23]. At all concentrations, cinnamon powder had a diminishing effect on the content of HAAs, while the control patties, without cinnamon, had higher contents; the content of polar HAAs was 45.81 ng g<sup>−</sup>1, while the non-polar HAA content was 35.09 ng g<sup>−</sup>1, as shown in Figure 4. At 0.5% concentration of cinnamon powder added to the lamb patties, the content of polar HAAs was 23.52 ng g<sup>−</sup>1, while for non-polar HAAs content was 16.08 ng g−1. At 1%, the content of the polar HAAs was 19.78 ng g<sup>−</sup>1, while for non-polar HAAs it was 14.04 ng g<sup>−</sup>1. At 1.5% concentration of cinnamon powder, the content of polar HAAs was 12.41 ng g−1, while for non-polar HAAs it was 9.51 ng g<sup>−</sup>1. Overall, the results showed that polar HAAs were higher in content both in control as well as in the treated patties with cinnamon powder.

**Figure 4.** Total polar and non-polar HAAs in the cinnamon lamb patties. Means with different letters (a, b, c, d) denote significant difference (*p* < 0.05) within treatments of lamb patties.

## **4. Discussion**

#### *4.1. DPPH Activity of Cinnamon and Chinese Prickly Ash*

Antioxidant compounds present in foodstuffs play a vital role in human life, acting as health-protecting agents. In addition to this role, antioxidants are one of the key additives used in fats and oils [24]. Cinnamon is a very popular spice used in many foods as a flavor additive. It has many other beneficial properties as well, such as anti-inflammatory and antioxidant characteristics [25]. China uses various traditional spices for aroma and taste. One of the most popular spices is Chinese prickly ash, which is added to meat dishes for taste and aroma [7,26]. Based on our results, cinnamon and Chinese prickly ash are both good sources of natural antioxidants, as both showed good or higher levels of inhabitation of DPPH; levels were almost as high as BHT, which is an artificial antioxidant source. Processed meat is cooked at high temperatures, due to which some harmful compounds are produced, such as d-heterocyclic aromatic amines (HAAs) which can cause cancer. The use of cinnamon and Chinese prickly ash in meat products can prevent and block the oxidation of HAAs because of their high antioxidant activity [5].

## *4.2. Effect of Chinese Prickly Ash Powder and Superheated Steam-Light Wave Roasting on HAAs in Roasted Patties*

In China, various traditional spices have become popular around the globe which provide particular taste and aroma in food. Most meat products cooked with these spices are symbolic of Chinese cuisine around the world. One of the most popular spices is Chinese prickly ash (*Zanthoxylum bungeanum*), which is added to meat dishes for taste and aroma [7,26]. Moreover, it is considered to have many important antioxidants and exhibit anti-inflammatory and anti-cancer activity. It contains mainly sanshools and sanshoamides as the main antioxidant compounds [27]. We found that, due to this property, Chinese prickly ash can very efficiently reduce HAAs in roasted lamb meat. The results of the present study are consistent with another study [18], which showed that Chinese prickly ash decreased the content of all polar HAAs. At concentrations of 0.5% to 1% of Chinese prickly ash in roasted beef patties, the contents of PhIP, DMIP, MeIQx, and 4,8-DiMeIQx decreased from 11 to 6.06 ng g−1, 0.42 to 0 ng g−1, 0.69 to 0.32 ng g−1, and 0.25 to 0.24 ng g<sup>−</sup>1, while in non-polar HAAs, harman content was seen to increase from 0.73 ng g−<sup>1</sup> to 0.96 ng g<sup>−</sup>1, and norharman content from 5.12 ng g−<sup>1</sup> to 6.51 ng g<sup>−</sup>1, which was opposite to the trend of our results, where we observed a decline of non-polar HAAs at each concentration.

In another study [10], the role of this spice was observed to be very effective in the inhibition of many HAAs in grilled beef. The content of polar HAAs, such as PhIP, was reduced from 1.28 to 0.80 ng g−<sup>1</sup> by the addition of 0.5–1.5% concentration of Chinese prickly ash, while IQx was only found at 0.5%, with 0.12 ng g<sup>−</sup>1. MeIQx was reduced from 1.13 to 0.33 ng g−<sup>1</sup> with a 0.5% to 1.5% concentration of Chinese prickly ash. The content of 4,8-DiMeIQx was reduced from 0.04 ng g−<sup>1</sup> to 0.02 ng g−<sup>1</sup> at concentrations of 0.5% to 1.5%. We detected that content of PhIP, IQx, IQ, and 4,8-DiMeIQx was slightly higher than in this study, but the decrease we observed was similar to that observed in this previous study. For non-polar HAAs, the content of harman was reduced from 2.94 to 2.41 ng g−1, while norharman was slightly increased from 16.91 to 17.96 ng g−<sup>1</sup> at three concentrations, which is quite a contradictory trend compared to our results. In our study, the non-polar HAAs harman and norharman decreased in lamb patties with increasing concentrations of Chinese prickly ash. These results are comparable with those reported by other authors who studied the presence of antioxidant compounds in lamb meat [28].

## *4.3. Effect of Cinnamon Powder and Superheated Steam-Light Wave Roasting on HAAs in Roasted Patties*

Cinnamon (*Cinnamomum cassia* or *Cinnamonum zeylanicum*), which is a popular spice in South Asian and Central Asian countries, is an ordinary spice used on meat products as a flavoring and aromatic agent [29]. Cinnamon has many other beneficial properties, such as anti-inflammatory and antioxidant characteristics [30]. The beneficial bioactive compounds (cinnamaldehyde, cinnamic acid, cinnamyl alcohol, coumarin, and eugenol) in cinnamon make it an important plant spice for medicinal purposes [31]. It is also used to treat many illnesses, such as cancer and diabetes [32]. [33] stated that cinnamon has the potential to inhibit HAAs in beef. Our findings are consistent with a prior study, which found that cinnamon decreased PhIP in descending order, starting with 0.58 ng g−<sup>1</sup> in beef [33]. There are few publications on DMIP in the literature. Here, we discovered that cinnamon powder in lamb meat considerably (*p* < 0.05) decreased DMIP. In another study [34], it was shown that the addition of cinnamon, at 0.5% concentration, to cooked beef reduced the content of IQ to a lower content than all tested spices. The content of IQ was reduced to the very low level of 0.85 ng g<sup>−</sup>1, which is very similar to our results.

Similar results were observed in all concentrations, and the contents were reduced to a significant level in lamb meat by the addition of cinnamon. However, in this study PhIP content was increased to 0.43–1.94 ng g−<sup>1</sup> which is surprising, because we observed a significant decline in our results of PhIP and all polar HAAs. However, the contents of MeIQx and IQx were lower than our values of HAAs at 0.5% cinnamon powder. The MeIQx values were consistent with our results, but there was variation in samples, including kind of meat, cooking method, and temperature variation, as well as cooking method used, between the studies.

#### **5. Conclusions**

The spices Chinese prickly ash and cinnamon have great potential as antioxidants, and this property of these spices has an impact on HAA formation. Cinnamon, especially, has great potential to inhibit HAAs, as the antioxidant compounds in it hinders the formation of HAAs. Both kinds of HAAs were reduced by the addition of these spices. Moreover, superheated steam-light wave roasting proved to be a cooking method that was very beneficial in the reduction of HAAs at a significant level as compared to other traditional methods, observed in our previous studies. Therefore, we conclude that the use of cinnamon in meat products can be an important natural source of antioxidants; furthermore, the use of superheated steam-light wave roasting can be a beneficial technology to reduce harmful compounds, which needs further exploration in this area of meat science.

**Author Contributions:** Conceptualization, R.S.; methodology, R.S.; software, M.I.; validation, R.S. and Z.W.; formal analysis, M.I. and R.S.; investigation, R.S.; resources, R.S.; data curation, M.I.; writing—original draft preparation, R.S.; writing—review and editing, H.L., D.Z. and A.D.A.-R.; visualization, H.L. and D.Z.; supervision, R.S.; project administration, R.S.; funding acquisition, R.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded from the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2022-IFST-SN2022) and the National Key R&D Program of China (2019YFC1606204).

**Data Availability Statement:** The datasets generated for this study are available on request to the corresponding author.

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

## **References**


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## *Article* **Impact and Optimization of the Conditions of Extraction of Phenolic Compounds and Antioxidant Activity of Olive Leaves (***Moroccan picholine***) Using Response Surface Methodology**

**El Mustapha El Adnany 1, Najat Elhadiri 1, Ayoub Mourjane 2, Mourad Ouhammou 1,\*, Nadia Hidar 1, Abderrahim Jaouad 1, Khalid Bitar <sup>3</sup> and Mostafa Mahrouz <sup>1</sup>**


**Abstract:** The *Moroccan picholine* tree's leaves contain phenolic compounds that benefit human health. However, the amount and type of these compounds can vary based on factors such as the extraction method and conditions. This study aimed to improve phenolic compounds' extraction while minimising harmful chemicals' use. It has been found that using ethanol as a solvent with ultrasonic extraction is the most effective and environmentally friendly technique. Several parameters, such as the extraction time, solid/solvent ratio, and ethanol concentration as independent variables, were evaluated using a surface response method (RSM) based on the Box–Behnken design (BBD) to optimize the extraction conditions. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis and also examined using the appropriate statistical methods. In optimal conditions, the ultrasonic time, the ratio (solvent/solid) and the concentration (ethanol/water), the content of total polyphenols (TPC), total flavonoids (TFC), and antioxidant activity (by DPPH, ABTS, FRAP) were, respectively, 74.45 ± 1.22 mg EAG/g DM, 17.08 ± 1.85 mg EC/g DM, 83.45 ± 0.89% 82.85 ± 1.52%, and 85.01 ± 2.35%. The identification of phenolic compounds by chromatography coupled with mass spectrum (HPLC-MS) under optimal conditions with two successive extractions showed the presence of hydroxytyrosol, catechin, caffeic acid, vanillin, naringin, oleuropein, quercetin, and kaempferol at high concentrations.

**Keywords:** olive leaves; extraction; optimization; ultrasound; polyphenols; flavonoids; antioxidant

## **1. Introduction**

The food and pharmaceutical industries are interested in agricultural wastes due to their high content of phenolic bioactive compounds, carbohydrates, oils, and other biochemical molecules [1].

The olive tree is commonly found in the Mediterranean region and is widely spread throughout Morocco, covering 65% of the national tree area. The regions of Fez-Meknes and Marrakech-Safi have the highest concentration of olive-growing areas, covering 54% of the total area and meeting 19% of the demand for edible oils. The olive transformation by-products, including the skin, pulp, pits, and leaves, have caught the attention of the food and pharmaceutical industries due to the presence of phenolic compounds.

Studies have shown that phenolic compounds found in olive tree leaves have beneficial properties, such as antioxidants [2], anticancer, antimicrobial [3], and hypolipidemic activities [4]. However, the amount of phenolic compounds present can vary based on

**Citation:** El Adnany, E.M.; Elhadiri, N.; Mourjane, A.; Ouhammou, M.; Hidar, N.; Jaouad, A.; Bitar, K.; Mahrouz, M. Impact and Optimization of the Conditions of Extraction of Phenolic Compounds and Antioxidant Activity of Olive Leaves (*Moroccan picholine*) Using Response Surface Methodology. *Separations* **2023**, *10*, 326. https:// doi.org/10.3390/separations10060326

Academic Editor: Faiyaz Shakeel

Received: 15 April 2023 Revised: 17 May 2023 Accepted: 19 May 2023 Published: 25 May 2023

**Copyright:** © 2023 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/).

97

climate, moisture, plant age and variety [5], and extraction methods [6]. Traditional extraction methods, such as maceration and Soxhlet extraction, are slow and yield low amounts of bioactive products [1]. The ultrasonic method, a newer extraction technique, has been developed to efficiently extract organic bioactive compounds from plants [7]. This step is critical in the production of bioactive.

Ultrasonic-assisted extraction (UAE) is considered the greenest extraction process compared to microwave-assisted extraction (MAE), meeting the requirements of the green extraction method [8] as it reduces the temperature, time, and solvent usage [9–11]. This method has been widely used to extract valuable bioactive compounds from various plant materials. One of the food and pharmaceutical industries' dilemmas is improving extraction efficiency while reducing costs, which can be achieved by optimizing extraction conditions [12]. In addition, this technique is usually performed to study some independent factors, requiring more experiments, leading to increased cost and time [13].

Response surface methodology (RSM) is a powerful statistical tool that optimizes complex processes. It has gained popularity for its effectiveness in extracting methods, identifying optimal variable combinations, and simplifying experiment interpretation. This tool has been widely used in various fields [14]. The objective of this study was to examine the effect of certain independent factors of extraction (time, solid/solvent ratio, ethanol (%)) of bioactive compounds (TPC, TFC) and antioxidant activity (DPPH, ABTS, FRAP) with the ultrasonic-assisted extraction (UAE) method using response surface methodology (RSM).

## **2. Materials and Methods**

## *2.1. Preparation of the Powder*

Olive leaves of the Moroccan picholine variety were harvested in Marrakech, Morocco. The leaves were rinsed with water and dried in a ventilated oven (OVEN 19L DRYING AND STERILIZATION DIGITHEAT J.P.SELECTA) with a thickness of 1 cm at 80 ◦C (according to previous studies [15]) for 5 h (stable weight), then ground using a propeller mill (Mill Grinder For Spices And Professional Coffee, 1 kg). The leaf powder obtained was sieved (digital vibrating laboratory analysis sieve/GKM Siebtechnik GmbH) into four fractions (>125 μm, (125 μm; 50 μm), (50 μm, 25 μm), and <25 μm). The particle size was set at 25–50 μm. The leaf powder was stored at 4 ◦C in plastic bags.

#### *2.2. Chemicals*

The reagents used were pure ethanol, methanol (HPLC grade), Folin–Ciocalteu's, Sodium carbonate (Na2CO3), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), aluminum trichloride (AlCl3), potassium persulfate (K2S2O8), tripyridyltriazine complex (TPTZ), sodium acetate buffer (C2H3 NaO2 3H2O and C2H4O2), and hydrochloric acid (HCl).

## *2.3. Experimental Design and Statistical Analysis*

The Box–Behnken design was used to determine the best combination of extraction variables for organic bioactive compounds based on the results of the preliminary singlefactor test. Different variables such as extraction time and temperature, sample/solvent ratio, solvent percentage, and pH influence the determination of the content of phytochemicals [16]. Extraction time (min, X1), sample/solvent ratio (g/mL, X2), and ethanol concentration (*v/v*, X3) were chosen as independent variables, and their coded and uncoded (real) levels of independent variables are shown in Table 1.


**Table 1.** Coded and actual values for Box–Behnken design (BBD).

The variation of the response values (Y), with respect to the three variables was fitted into a response surface model and presented in the form of the second-order polynomial equation, is as follows:

$$\forall i = \beta 0 + \sum\_{i=1}^{k} \beta iXi + \sum\_{i=1}^{k} \beta iiXi^2 + \sum\_{j=1} \sum\_{\substack{\ell=2 \ \ell \neq i}}^{k} \beta ijXiXj + \varepsilon\_{\ell}$$

where Yi are the experiment responses; β0 represents the theoretical mean value of the response; βi, βj are the coefficients of the linear terms; βii, are the coefficients of the quadratic terms; βij are the coefficients of the interaction terms, and ε the error term.

## *2.4. Ultrasound-Assisted Extraction of Bioactive Compounds*

Extraction with organic solvents has economic and environmental drawbacks. The "green chemistry" concept encourages developing and using less hazardous processes and materials without reducing efficiency [17]. Consequently, solvent extraction of bioactive compounds must be optimized for maximum response using fewer organic solvents. Thus, water was chosen to be studied in combination with ethanol. Ethanol was chosen instead of methanol as the extraction solvent due to the high toxicity of methanol in the human body [18]. Ethanol has the highest affinity for phenolic compounds; therefore, it the first choice for extracting phenolic compounds from fruit and vegetable wastes [19]. We mixed 1 g of olive leaf powder with ethanol/water. The extraction process was performed using a typical ultrasonic apparatus (Heating cleaning bath "Ultrasound HD"-Model 3000866), and the extract was filtered to collect the supernatant. A UV-T80 spectrophotometer was used to analyse the polyphenols, flavonoids, and total antioxidant activity in the samples.

#### *2.5. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)*

The determination of total polyphenols by the method using the Folin–Ciocalteu reagent is described by Singleton et al. [20]. A total of 0.25 mL of leaf extract was mixed with 0.25 mL of Folin–Ciocalteu and 2 mL of distilled water; the mixture was vortexed. After 3 min, 0.25 mL of sodium carbonate (20%) was added; the mixture was stirred and then incubated for 30 min in the dark at room temperature. The absorbance was measured at 750 nm using a UV/VIS spectrophotometer "T80-PG Instruments. The calibration curve for gallic acid was performed, and the results are expressed as mg gallic acid per g dry matter (mg GAE/g DW).

Flavonoid content was determined based on the formation of a flavonoid–aluminum complex that absorbs at 430 nm. The flavonoid assay was performed according to the protocol described by Djeridane et al. (2006) [21]. A total of 1.5 mL of the olive leaf extract was added with 1.5 mL of aluminum trichloride (AlCl3: 2%). After 30 min incubation at room temperature, the absorbance of the reaction mixture was read at 430 nm using a UV/VIS spectrophotometer (T80-PG Instruments). The flavonoid content in the extracts was calculated by reference to a calibration curve established with catechin. Results are expressed as mg catechin equivalent per 1 g dry matter (mg EC/g DW).

#### *2.6. In Vitro Antioxidant Activity*

#### 2.6.1. DPPH Radical Reduction Test

For the anti-radical activity of the different extracts of the leaves dried at different temperatures, we used the method based on DPPH (1,1-diphenyl-2-picrylhydrazyl) as a relatively stable radical, according to the protocol described by Abdel Hameed et al. [22]. Briefly, 1 mL of leaf extract was added to 1 mL of DPPH solution (prepared by solubilizing 4 mg of DPPH in 100 mL of ethanol). The mixtures were incubated in the dark for 30 min at room temperature. The decolorization compared to the negative control containing only DPPH solution measured at 517 nm using a UV/visible spectrophotometer type T80. The radical-scavenging activity of DPPH was calculated as follows: %(AA) = ((A517 control − A517 sample)/A517 control) × 100. A517 control is the absorbance of DPPH solution (without sample extract), and A517 sample is the absorbance of the sample with DPPH solution.

## 2.6.2. ABTS Radical Test

The ABTS•+ radical cation decolorization test also evaluated the anti-radical activity according to the method used by Aadesariya et al. [23]. The ABTS+- radical was generated by the reaction of 7 mM ABTS+ and 2.45 mM potassium persulfate. An equal mixture volume was incubated in the dark for 12–16 h. The ABTS+-solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm before use. Then, 1 mL of ABTS+ solution was mixed with 10 μL of leaf extract. The mixture was incubated for 30 min at 30 ◦C, and the absorbance was measured at 734 nm. The radical scavenging activity was expressed as the percentage of free radical inhibition by the sample and was calculated by the following formula:

ABTS scavenged (%) = [(A734 control − A734 sample)/A517 control] × 100.

A734 control is the absorbance of the control reaction, and A 734 test is the absorbance in the presence of the sample extracts.

#### 2.6.3. Ferric Reducing Antioxidant Power (FRAP) Test

The FRAP (ferric reducing antioxidant power) method is based on the reduction of ferric ions (Fe3+) to ferrous ions (Fe2+). This method evaluates the declining power of compounds at low pH [24]. The ferrous tripyridyltriazine (TPTZ) complex has an intense blue color measured by a spectrophotometer at 593 nm. The FRAP assay was performed according to the protocol of [25]. The FRAP reagent was prepared by mixing 300 mM sodium acetate buffer (3.1 g C2H3NaO2 3H2O and 16 mL C2H4O2), pH: 3.6; 10 mM solution of TPTZ in 40 mM HCl; and 20 mM FeCl3 at a ratio of 10:01:01 (*v*/*v*/*v*). One hundred microliters of each extract were added to 3 mL of FRAP reagent and 300 μL of H2O. After incubation at 37 ◦C for 30 min; the absorbance was measured at 593 nm against the blank [26].

#### *2.7. Model Verification*

The extraction conditions were numerically optimized for maximum TPC and TFC content with high antioxidant activities based on regression analysis and 3D surface curves of independent variables. Responses were determined according to the recommended extraction conditions.

## *2.8. Qualitative and Quantitative Analysis by HPLC-MS*

Identification and quantification of phenolic compounds by HPLC-MS were performed according to the method used by Puigventos et al. (2015) [27]. The injection volume of each sample was 10 μL with separation between solvent A (0.1% aqueous formic acid solution) and solvent B (methanol) as follows: 0–3 min, linear gradient from 5 to 25% B; 3–6 min, at 25% B; 6–9 min, from 25 to 37% B; 9–13 min, at 37% B; 13–18 min, from 37 to 54% B; 18–22 min, at 54% B; 22–26 min, from 54 to 95% B; 26–29 min, at 95% B; 29–29. 15 min, back to initial conditions at 5% B; and 29.15 to 36 min, at 5% B. The mobile phase flow rate was 1 mL/min. Ion transfer tube temperature was set at 350◦ and the full scan MS acquisition mode to *m/z* 50–1000. The polyphenolic compounds were obtained at 31 min.

## *2.9. Statistical Analysis*

Analysis of variance (ANOVA) and multiple regression analysis were performed to fit the mathematical model using Design Expert 13 software. Significant terms (*p* < 0.05) in the model for each response were found by analysis of variance, and significance was judged by the F statistic calculated from the data. The experimental data were evaluated with various descriptive statistical analyses such as *p*-value, F-value, sum of squares (SS), degrees of freedom (DF), coefficient variation (CV), the mean sum of squares (MSS), coefficient of determination (R2), and adjusted coefficient determination (Radj2); this is to obtain the statistical significance of the developed quadratic mathematical model.

## **3. Results and Discussion**

## *3.1. Evaluation and Optimization of Extraction Conditions*

This study determined the relationship between response functions and process variables using a three-factor based on the Box–Behnken (BBD) design. The goal was to optimize the extraction conditions for bioactive compounds such as total polyphenols (TPC), total flavonoids (TFC), their corresponding antioxidant activities. Similar scientific studies were used as a reference. [28].

The outcomes of the conducted responses are reported in Table 2. The total polyphenol (TPC) content ranged from 48.69 to 72.98 mg EAG/g DM. The total flavonoid (TFC) content ranged from 10.45 to 16.36 mg EC/g DM. The entire content of TPC and TFC was obtained for trials 13, 14, and 15 under the experimental conditions of X1 = 45 min; X2 = 12.5 mL/g; and X3 = 60%. Regarding DPPH radical scavenging capacity, ABTS and FRAP ranged from 70.95% to 85.69%, 74.67 to 85.41%, and 71.92 to 86.95%, respectively. The highest antioxidant activity was obtained for tests 2 and 13 under X1 = 60 min; X2 = 5 mL/g; X3 = 60% and X1 = 45 min; X2 = 5 mL/g; and X3 = 60%, respectively. Based on these data, the extraction process was optimized to achieve the maximum desirable response.


**Table 2.** The experimental run from Box–Behnken design (BBD).

Pearson's test showed a strong positive correlation, r = 0.8- 0.85, between TPC and TFC and between TFC and FRAP. This implies that these answers evolve proportionally. The positive average correlations r = 0.5–0.75 appear between the other answers.

#### *3.2. Fitting the Model and Analysis of Variance*

The extraction process was optimised by applying the second-order polynomial model fit. The results are presented in Table 3. The model shows a high significance level and a good fit, with the experimental data of TPC and TFC contents showing less variation around the mean (R2 values 0.969 and 0.991), respectively.


**Table 3.** Experimental design of the surface response and statistical table of results.

#### **Table 3.** *Cont.*


\* Significant (*p* < 0.05).

The antioxidant activity (DPPH, ABTS, and FRAP) shows that the model is significant, and the polynomial equation fit using the coefficient of determination (R2) 0.90, 0.93, and 0.90, respectively. The regression coefficients for the dependent variables were obtained by multiple linear regressions, as shown in Table 3.


The ANOVA result for each variable response indicates that at least one of the model parameters can explain the experimental variation of the response variables (Table 3).

The corresponding variables would be more significant if the F-value becomes larger and the *p*-value becomes smaller [29]. The *p*-value < 0.05 showed that the model terms were significant. In terms of coefficients of variation (CV), the models recorded a CV for CPT, CFT, DPPH, ABTS, and FRAP of 4.24%, 3.69%, 2.42%, 1.9%, and 3.59%, respectively.

Generally, the acceptable coefficient of variation (CV) value should be less than 20%. The diagnostic diagram as the predicted versus actual values (Figure 1) evaluates the relationship and model satisfaction between the experimental and predicted values obtained from the developed models. From Figure 1, it is observed that the data points are located near the straight line, which means a high correlation between experimental and predicted data obtained for TFC and a medium correlation between experimental and predicted data of TPC and antioxidant activity (DPPH, ABTS, and FRAP) from the models.

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**Figure 1.** *Cont*.

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**Figure 1.** Diagnosis between experimental and predicted values for TPC, TFC, DPPH, ABTS, and FRAP.

## *3.3. Development of Second Order Polynomial Models*

A statistical analysis was conducted using a second-order polynomial equation with interaction terms to model the connection between three process variables and the efficiency of ultrasonic extraction. This model can be utilized to anticipate the efficiency of ultrasonic extraction for varying combinations of process variables.

Five models were developed from this study to have the ultrasound extraction efficiency of TPC, TFC, DPPH, ABTS, and FRAP from olive leaves. The second-order equations of the responses in terms of coded factors are given below:

$$\rm Y\_{\rm TFC} = 72.41 - 3.18 \mathbf{X}\_2 - 3.66 \mathbf{X}\_3 - 8.23 \mathbf{X}\_{11}^2 - 6.21 \mathbf{X}\_2^2 - 11.62 \mathbf{X}\_{33}^2$$

$$\rm Y\_{\rm TFC} = 16.34 + 0.77 \mathbf{X}\_1 - 0.68 \mathbf{X}\_1 \mathbf{X}\_2 - 1.84 \mathbf{X}\_{11}^2 - 2.28 \mathbf{X}\_{22}^2 - 2.81 \mathbf{X}\_{33}^2$$

$$\rm Y\_{\rm DPPH} = 81.65 + 2.2 \mathbf{X}\_1 - 2.61 \mathbf{X}\_1 \mathbf{X}\_2 - 3.8 \mathbf{X}\_3^2$$

$$\rm Y\_{\rm ABTS} = 82.65 - 3.13 \mathbf{X}\_1 \mathbf{X}\_2 - 2.60 \mathbf{X}\_1 \mathbf{X}\_3 - 3.23 \mathbf{X}\_2^2 - 3.59 \mathbf{X}\_{33}^2$$

$$\rm Y\_{\rm FRAP} = 86.28 - 3.95 \mathbf{X}\_1^2 - 5.68 \mathbf{X}\_2^2 - 6.55 \mathbf{X}\_3^2$$

## *3.4. Effect of Process Variables*

This study analyzed the impact of process variables on the extraction of bioactive compounds (TPC, TFC, and free radical antioxidants) from olive leaves using a two-level Box–Behnken design with three factors: extraction time, solid/solvent ratio, and ethanol concentration. The results were presented using a three-dimensional response surface, demonstrating the relationship between the independent and dependent variables. By holding two factors constant and varying the third, the response surface curves display the main effects and interactions of the independent variables on/with the dependent variables [1]. These graphs provide insight into how the different variables affect the extraction process.

## 3.4.1. Effect of Extraction Time

Based on the results obtained, a longer contact time between the sample and solvent leads to a higher transfer rate of bioactive compounds, ultimately resulting in better extraction efficiency. Figure 2 illustrates that the extraction time of 30–45 min was particularly impactful for flavonoids and DPPH. The linear effect of extraction time was significant (*p* < 0.05) for these two variables but not for the other independent variables. This effect is likely due to the extended time the plant matrix is exposed to the solvent, improving the solubility of the leaves' constituents. Essentially, the duration of the solvent exposure facilitates the migration of chemical compounds into the solution [30].

**Figure 2.** Response surface plot showing the variation of responses as a function of extraction time.

Studies have shown that the longer the extraction time, the higher the content of polyphenols and flavonoids. However, it is essential to note that excessively long extraction times can lead to the degradation of the bioactive compounds, as indicated in [31]. On the other hand, some studies found that extraction time was not a significant factor in the ultrasound-assisted extraction of phenolic compounds [32,33]. In other studies, such as those on Genipap berry pulp, blueberries, and carob pulp, the extraction time for bioactive compounds using ultrasonic-assisted extraction (UAE) was around 49, 50, and 57 min, respectively [34–36].

## 3.4.2. Effect of Solid–Liquid Ratio

The amount of solvent used in organic bioactive compounds and antioxidant extraction is a crucial factor. When evaluating its impact on the extraction of phenolic compounds, the results, presented in Figure 3, indicate that increasing the solid-liquid ratio up to 12.5 mL/g resulted in an increase in polyphenol and flavonoid content and antioxidant activity. This indicates that the volume of solvent used plays a significant role in

achieving good infusion and easy release of bioactive compounds into the surrounding environment [37]. However, according to Zakaria Fazila (2021) [38], a high solvent-to-solid ratio (between 10–30 mL/g) can lead to a decrease in phenolic compound content. The most effective ratio for maximum phenolic compound extraction was found to be 10 mL/g.

**Figure 3.** Response surface plot showing the variation of responses as a function of solid/ solvent ratio.

This research found that once the solid–liquid ratio surpasses 12.5 mL/g, the solution becomes oversaturated with solute. This can lead to a reduction in the rate of mass transfer and a hindrance in the penetration of organic bioactive compounds into the solution, ultimately resulting in a decrease in the yield of the extraction process.

#### 3.4.3. Effect of Solvent Concentration

The solubility of organic bioactive compounds can be enhanced by varying the concentration of the solvent [39]. Olive leaves were extracted using different ethanol concentrations at no more than 50 ◦C for 60 min under ultrasonic conditions. The results, shown in Figure 4, indicate that the samples containing 60% ethanol had the highest TPC, TFC, DPPH, ABTS, and FRAP values. This is because the solubility of polyphenol compounds increases with increasing ethanol concentrations. This may explain why the presence of water in ethanol improves the swelling of the plant material, while ethanol disrupts the binding between the solute and the plant. Other studies [40,41] have found that the recovery of organic bioactive compounds increased and peaked at an ethanol concentration of around 70% before slightly decreasing. Studies have shown that increasing the ethanol concentration can enhance the yield of phenolic compounds up to an average concentration of 40% ethanol [42]; however, Caldas et al. [43] observed that the highest phenolic compound content was achieved at an average concentration of 60% ethanol, possibly due to the different polarities of organic bioactive compounds in grape skin. For *Triticum aestivum*, the maximum yield of phenolic compounds was obtained using an ethanol concentration of 56% [44]. Similarly, studies on various plants such as *Jaboticaba* bark, blueberry, and apple pulp have found that the concentration of ethanol required to extsract the maximum amount of phenolic compounds ranges from 40–80% (*V/V*) [35–46].

**Figure 4.** Response surface plot showing the variation of responses with ethanol concentration.

## *3.5. Determination and Validation of Optimal Conditions*

This study aimed to determine the best experimental conditions for extracting phenolic compounds that benefit human health, such as antioxidant activity. This involves determining the ideal sonication time, solid/liquid ratio, and ethanol concentration for maximum extraction yield. The software Design Expert Version 13 was used to find the composite optimum for achieving the highest bioactive compounds extraction yield and the most significant antioxidant activity.

The optimization process involved assigning the response's desirability values between 0 and 1. Figure 5 displays a desirability value of 0.8735 and the predicted optimal values. Experiments were conducted under optimal triplicate conditions to compare the experimental and predicted values of the responses. The mean values are presented in Table 4. The optimal conditions for the experiments were 53.52 min, a solid/liquid ratio of 9.83 mL/g, and an ethanol concentration of 59.7% (*V/V*). The experimental values for TPC and TFC bioactive compounds were 74.45 ± 1.22 mg EAG/g DM and 17.08 ± 1.85 mg EC/g DM, respectively, while the antioxidant activity DPPH, ABTS, and FRAP were 83.45 ± 0.89%, 82.85 ± 1.52%, and 87.01 ± 2.35%, respectively. These experimental results were found to be similar to the predicted model for TPC (72.40 mg EAG/g DM), TFC (16.42 mg EC/g DM), DPPH (82.58%), ABTS (83.06%), and FRAP (85.79%). Therefore, there is a synergy between the results found and the Box–Behnken design.

**Table 4.** Optimal conditions and predicted and actual response values of olive leaf extract.


**Figure 5.** The predicted optimal values and desirability of olive leaf optimization.

## *3.6. HPLC-MS Analysis*

Figure 6 displays the phenolic compounds identified in olive leaf extracts through ultrasound in the first and second extractions. The peaks on the graph correspond to these compounds. The results indicate that the first extraction in optimal conditions (Figure 6a) resulted in a high release of phenolic compounds. In contrast, the second extraction (Figure 6b) had fewer bioactive compounds. This suggests that the ultrasonic extraction method is effective. Eight phenolic compounds were identified: hydroxytyrosol, catechin, caffeic acid, vanillin, naringin, oleuropein, quercetin, and kaempferol. As shown in Table 5, oleuropein was the most abundant compound, with a concentration of 114.10 mg/g DM, followed by hydroxytyrosol, caffeic acid, and kaempferol. The optimized ultrasound-assisted extraction method was likely responsible for the high amount of phenolic compounds in the first extraction.

**Figure 6.** HPLC chromatograms of polyphenols of olive leaf extracts from the First UEA under the optimal conditions (**a**) and the Second UEA under the same optimal conditions (**b**).


**Table 5.** Concentration of phenolic compounds identified in olive leaf extracts under the optimal conditions of the first and second ultrasound extraction (mg/g DM).

## *3.7. Scanning Electron Microscopy*

After being dried and ground, the leaf powder was examined under a scanning electron microscope. There was a visible difference between the untreated sample and the one treated with EWM and UAE (as shown in Figure 7). The untreated sample was densely compacted, while the treated sample showed structural changes. UAE caused more damage and formation of cracks than maceration, possibly due to the cavitation effects of the ultrasound [47]. During extraction, high ultrasound intensities can enhance solvent penetration and destroy cell membranes [48], releasing more bioactive compounds from the sample matrix.

**Figure 7.** SEM images of olive leaf powder before extraction (RM), leaf powder after ultrasoundassisted extraction (UAE) under optimal conditions, and leaf powder treated by ethanol water maceration (EWM).

## **4. Conclusions**

The Box–Behnken design (BBD) method, along with the surface response design approach (RSM), was used to study the impact of ultrasonic-assisted extraction (UAE) process parameters on the content of polyphenolic compounds in *Moroccan picholine* olive leaves. The results showed that this eco-friendly technique is beneficial in optimizing the conditions for extracting phenolic compounds (TPC, TFC). To achieve a high yield, it is recommended to use an extraction time of 53.5 min, a solvent/solid ratio of 9.95 mL/g, and an ethanol concentration of 59.7%. The content of TPC and TFC are 74.45 ± 1.22 mg EAG/g DM and 17.08 ± 1.85 mg EC/g DM, respectively, while the antioxidant activity of DPPH, ABTS, and FRAP are 83.45 ± 0.89%, 82.85 ± 1.52%, and 87.01 ± 2.35% respectively. The presence of certain phenolic bioactive compounds in high concentrations, specifically oleuropein and hydroxytyrosol, was confirmed through analysis by HPLC-MS.

To better understand the extraction and optimization phenomena, it would be beneficial to study additional parameters such as sonication temperature, frequency, and solvent nature.

**Author Contributions:** E.M.E.A.: doctoral student, practical work, and main leader of the operative manipulations; A.M.: doctoral assistant of operations and preparation of raw material; N.E. (FSSM/UCA), A.J. (FSSM/UCA) and M.M. (Emeritus FSSM/UCA): professors who initiated the follow-up and planning of the thesis work; M.O. and N.H.: assistants doctors of analysis; K.B., doctor, pharmacist, and CEO of the company, Iris Cosmétologie (IRCOS) Laboratoires, our industrial partner: availability of raw materials, logistics and production machines. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Center for Scientific and Technical Research (CNRST) and the Ministry of Higher Education, Scientific Research and Vocational Training of Morocco, "PPR-BR2BINOV-Mahrouz-FS-UCA-Marrakesh".

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank the City of Innovation of Marrakech and the company, Iris Cosmétologie (IRCOS) Laboratoires.

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

## **References**


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## *Article* **Fractions of Methanol Extracts from the Resurrection Plant** *Haberlea rhodopensis* **Have Anti-Breast Cancer Effects in Model Cell Systems**

**Diana Zasheva 1,\*, Petko Mladenov 2, Krasimir Rusanov 2, Svetlana Simova 3, Silvina Zapryanova 1, Lyudmila Simova-Stoilova 4, Daniela Moyankova <sup>2</sup> and Dimitar Djilianov <sup>2</sup>**


**Abstract:** Breast cancer is among the most problematic diseases and a leading cause of death in women. The methods of therapy widely used, so far, are often with many side effects, seriously hampering patients' quality of life. To overcome these constraints, new cancer treatment alternatives are constantly tested, including bioactive compounds of plant origin. Our aim was to study the effects of *Haberlea rhodopensis* methanol extract fractions on cell viability and proliferation of two model breast cancer cell lines with different characteristics. In addition to the strong reduction in cell viability, two of the fractions showed significant influence on the proliferation rate of the hormone receptor expressing MCF7 and the triple negative MDA-MB231 breast cancer cell lines. No significant effects on the benign MCF10A cell line were observed. We applied a large scale non-targeted approach to purify and identify highly abundant compounds from the active fractions of *H. rhodopensis* extracts. By the combined NMR/MS approach, myconoside was identified in the fractions and hispidulin 8-C-(6-O-acetyl-2-O-syringoyl-β-glucopyranoside) was found in one of them. We further performed molecular docking analysis of possible myconoside interactions with several proteins, important for breast cancer proliferation. High probability of binding was established for GLUT1 transporter, estrogen receptor and MYST acetyltransferase. Our results are a good background for future studies on the use of myconoside for targeted breast cancer therapy.

**Keywords:** breast cancer; *Haberlea rhodopensis*; myconoside; hispidulin 8-C-(6-O-acetyl-2- O-syringoyl-β-glucopyranoside); GLUT1 transporter; estrogen receptor and MYST acetyltransferase

## **1. Introduction**

One of the most problematic diseases related to women's health is breast cancer. Cases of breast cancer diagnosed in 2008 were 1.38 million [1] and their number increased to 2.3 million in 2020 [2], thus reaching 12% of all cancer cases [3]. At the same time, breast cancer is the second leading cause of death in women [4]. The first step in finding new anticancer substances is to test them on model cell lines that have features common to different types of cancer. Cell cultures remain indispensable tools in cancer research, despite some limitations due to phenotypic drifts, some heterogeneity and existence of clonal variants. The cellular characteristics of breast cancer and the changes in their cell signal pathways complicate the therapeutic methods used so far. Invasive cancer types, named basal, are characterized with low expression of HER (Human Epidermal Growth Factor Receptor) and a loss of estrogen and progesterone receptors [5]. They are also known

**Citation:** Zasheva, D.; Mladenov, P.; Rusanov, K.; Simova, S.; Zapryanova, S.; Simova-Stoilova, L.; Moyankova, D.; Djilianov, D. Fractions of Methanol Extracts from the Resurrection Plant *Haberlea rhodopensis* Have Anti-Breast Cancer Effects in Model Cell Systems. *Separations* **2023**, *10*, 388. https:// doi.org/10.3390/separations10070388

Academic Editor: Faiyaz Shakeel

Received: 6 June 2023 Revised: 27 June 2023 Accepted: 28 June 2023 Published: 1 July 2023

**Copyright:** © 2023 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 triple negative and are unresponsive of hormonal replacement therapy. A good model system to study new anticancer agents suitable for this very aggressive type of cancer is the cell line MDA-MB231 [6]. The expression of HER2 receptor and two hormonal receptors (estrogen and progesterone ones) characterizes luminal B types of breast cancer, known also as triple positive. A widely used model system of the hormone receptor expressing cancer is the cell line MCF7 [4]. This cell line is also used to study epigenetic regulation of cancer growth, mediated by higher expression and activity of MYST acetyltransferases in estrogen dependent breast tumors [7]. Several breast cancer cell lines, including MDA-MB231 and MCF7, are characterized with high rates of glucose uptake and high expression of glucose transporters of the GLUT family [6], which is not typical for noncancerous epithelial cell lines such as MCF-10A.

Conventionally used therapeutic methods are invasive and with many side effects. Often, the standardly used chemotherapeutic drugs lose effectiveness because of multidrug resistance developed by cancer cells. Side effects of chemotherapy and radiotherapy seriously hamper patients' quality of life. To overcome these constraints, new cancer treatment alternatives are constantly tested, including bioactive compounds of plant origin [8–10]. Polyphenol substances like e.g., coumarins [11], flavones like genistein [12], phenols like thymol [13], monotherpenes like thymoquinone [14] have been found to reduce the viability of breast cancer cell lines of various origins by mechanisms related to switching on apoptotic pathways, by blocking cell proliferation or different kinase pathways. The development of analytics with high resolution complementary instruments allows identification and determination of the molecular structure of many new active compounds from various plant species. In this respect, the complementary data obtained by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) could provide reliable information for compound discovery in natural products research [15,16]. In addition, identification of bioactive compounds from various plant species is a good perspective for drug discovery, with the valuable contribution of Artificial Intelligence (AI) and synthetic chemistry, for therapy or even prevention of cancer. In the search for new therapeutic agents, there has been recent attention on the resurrection plant species. They are a group of higher plants that are able to withstand a drastic decrease in the water content of their vegetative tissues and after long periods of dryness, they are able to recover fast and fully when water is available again [17]. These plant species belong to various botanical families and live under differing environments but share high desiccation tolerance as a common characteristic. This feature makes resurrection plants a suitable model for intensive studies for stress tolerance at molecular, physiological, biochemical and metabolomics levels. The antioxidative component of resurrection plants' desiccation tolerance is well recognized [18]. Additional attention is paid to the specific secondary metabolites, constitutive or accumulated during stress, with potential application as food additives, cosmetic agents or medicinal components.

The Balkan endemite *Haberlea rhodopensis* is among the only few resurrection plants growing in Europe and as all other species on the continent, belongs to the Gesneriaceae botanical family [19]. It is among the most studied desiccation tolerant model systems, at whole plant level or as detached leaves assays [20–25].

In addition, following long established strategy [26], knowledge on the specific metabolome of *H. rhodopensis* was gradually generated and the potential application of extracts or isolated compounds in various areas has been studied, including human diseases [27–31].

The present study is the first attempt to follow the behavior of two breast cancer cell lines with different characteristics in comparison to a normal breast epithelial cell line after treatment with *H. rhodopenis* extracts and their fractions. The latter are resulting from a non-targeted approach to purify and identify highly abundant compounds involved in the significant reduction in cell viability and proliferation of the cancer cell lines. Molecular docking analysis has been performed on one of the identified compounds—myconoside to propose a model for its interaction with several cancer proteins in an attempt to explain

the potential mechanisms of its penetration in cells and the reduction in their viability and proliferation. The results are discussed as a background for further studies and potential applications.

## **2. Materials and Methods**

## *2.1. Chemicals and Reagents*

Acetonitril and methanol of HPLC grade from Macron Fine Chemicals™ (Avantor, Gliwice, Poland) and Acetonitril of LC/MS grade from Sigma-Aldrich (St. Louis, MO, USA) were used. Dimethyl sulfoxide (DMSO) and formic acid were sourced in analytical grade from Sigma-Aldrich (St. Louis, MO, USA). Sephadex LH-20 was purchased from Cytiva (Marlborough, MA, USA). Antibiotic/antimycotic solution was purchased from GE Healthcare (Boston, MA, USA). CD3OD was purchased from Euriso-top (Saint-Aubin, France). All reagents for cell cultures treatments and cell culture assay tests were purchased from the Sigma-Aldrich (St. Louis, MO, USA).

## *2.2. Plant Material and Leaf Extract Preparation and Fractionation by Size Exclusion Chromatography*

*H. rhodopensis* plants were propagated in vitro and adapted in pots under controlled greenhouse conditions at 22–24 ◦C, a 16-h photoperiod, 60% relative humidity and a photon flux density of 36 μmolm−<sup>2</sup> s−<sup>1</sup> [32]. Fully developed leaves from well-hydrated pot plants were detached and air-dried for methanol extract preparation. Homogenised leaves (6 g) were macerated in 60 mL methanol and extracted for 30 min at 70 ◦C on water bath. After centrifugation at 10,000× *g* for 10 min, the supernatants were collected, dried at 40 ◦C by SpeedVac Labconco (Kansas City, MI, USA) and stored at −20 ◦C for further use.

1.1276 g of crude extract were dissolved in 14 mL methanol and subjected to size exclusion chromatography (SEC) on a Cytiva XK 50/100 column filled with Sephadex LH-20 with the help of an AKTA Pure FPLC system (Cytiva) using a constant flow of 3 mL/min of methanol. Monitoring of the elution was carried out at 220 nm. SEC fractions were collected in 50 mL tubes based on the observed elution of UV absorbing compounds.

## *2.3. Cell Cultures Treatments and Cell Culture Assay Tests*

## 2.3.1. Cell Lines

In this study adherent breast cancer cell line MCF7 (ATCC cell culture collection N NTB-22TM) Manassas, Virginia, USA and MDA-MB231 (ATCC cell culture collection N HTB-26™) and normal adherent breast epithelial cell line MCF 10A (ATCC cell culture collection N CRL-10317™) Manassas, Virginia, USA) were used. The normal cell line was grown on DMEM/F12 medium and MDA-MB231 cancer cell line was grown on DMEM medium with high glucose (4.5 g/L) supplemented with 5% FBS and 10% FBS, respectively, and antibiotic/antimycotic solution Amino acid mix solution was added to the cancer cell line MDA-MB231 medium. The normal cell line needed insulin in concentration 10 μg/mL, endothelial growth factor in concentration 20 ng/mL and hydrocortisone in concentration 500 ng/mL. The cancer cell line MCF7 was incubated in DMEM with low glucose (1 g/L) supplemented with 10% FBS and antibiotic/antimycotic solution and insulin in concentration 0.01 mg/mL. Cells were incubated in 5% CO2 incubator at 37 ◦C. The cells were cultivated to 80–90% confluence and were trypsinized with trypsin/EDTA solution. The cells were centrifuged, suspended in in FBS with 5–10% DMSO and stored in freezer at −150 ◦C.

## 2.3.2. MTT Cell Viability Assay

The studied cell lines were grown in 25 cm<sup>2</sup> well plates to confluence 80–85%. 104 cells were seeded per 96 well plate. The cells were grown 24 h and then treated with *H*. *rhodopensis* total extract (fractions). The viability was determined 48 h after the treatment with total extract and fractions at 48th, 72nd hour. MTT test followed standard procedure [33]—20 μL of MTT stock solution (5 mg/mL) were added to the well and the cells were incubated

in CO2 incubator for 4 h. The formazan crystals were solubilized in 150 μL dimethyl sulphoxide. The color intensity was measured at wave length 600 nm (ELISA Reader Fluostar Optima/BMG Tech) ThermoFisher Scientific Corporation, Waltham, MA, USA. The untreated samples were used as control with 100% viability. The percent of viable cells in experimental conditions were scored as a percent of control sample.

% of viable cells = E treated sample/E control sample × 100)

Each condition in experiment was performed in triplicate. Three biological experiments were made for each of the cell lines. The standard error bars in percent are presented in the graphs. The total extract was applied in concentrations 10, 100, 200 and 300 μg/mL, respectively, and the fractions were applied in concentration 300 μg/mL.

## 2.3.3. Proliferation Assay

The trypan blue-excluding proliferation assay test was made following the next procedure [33]. The cells were grown in 24-well plate to 4.5 × <sup>10</sup><sup>4</sup> cell for MCF10-A cell line, to <sup>6</sup> × <sup>10</sup><sup>4</sup> cells for MCF7 cell line and to 4 × <sup>10</sup><sup>4</sup> cells for MDA-MB231 cell line. For treatment, the medium was changed with fresh medium containing *H*. *rhodopensis* fractions numbers 14 and 18 at concentration 300 μg/mL. Total number of cells was counted for 96 h at 24 h interval. The cells were washed with PBS and then they were suspended in PBS, 10–20 μL of their suspension was trypan blue stained. Three biological replicates were made. The living cells presented in the graphs are averages of total number of cells in well minus averages of number of dead cells in well, stained by trypan blue dye.

## 2.3.4. Statistical Methods

The results are presented with standard error bars [34]. The absolute values of MTT assay of breast cancer cell lines data for total extract treatment concentration 300 μg/ml and fractions with effect on cancer cell lines are processed in ANOVA for multiple comparison plot and the data are presented extrapolated in absolute values to log2 scale. For each of the groups was applied one-way ANOVA analysis of fractions treatment versus control at each time point of the studied normal and two cancer cell lines in proliferation assay to estimate variations within the group [35].

## *2.4. Compound Identification*

## 2.4.1. Semi-Preparative HPLC

Semipreparative LC analysis of SEC fractions FR14 and FR18 was performed on an Agilent 1260 Infinity II LC System (Santa Clara, CA, USA) equipped with a quaternary pump, multicolumn thermostat, autosampler and multiple wavelength detector. Separations were performed on an Agilent ZORBAX StableBond C18 (5 μm, 9.4 × 150 mm) at room temperature. SEC fraction FR14 was dissolved in 3 mL 30% methanol to a final concentration of 42 mg/mL. SEC fraction FR18 was dissolved in 3 mL 30% methanol to a final concentration of 15 mg/mL. The mobile phase consisted of water (A) and Acetonitrile (B). The flow rate was 4 mL/min. Signal was detected at 220 nm. Increasing amount of SEC fractions were injected starting from 40 μL, 60 μL, 80 μL, 100 μL, 120 μL, 140 μL and 160 μL for FR14 and 24 μL, 48 μL, 100 μL and 250 μL for FR 18 until all the amount was injected. Individual compounds were manually collected in 50 mL tubes, evaporated to dryness using Labconco CentiVap vacuum concentrator (Kansas city, USA) and used for 1H NMR and LC-MS/MS analysis.

## 2.4.2. 1H NMR

Dried semi-preparative HPLC fractions were dissolved in 600 μL of CD3OD with deuterated 25 mM 196 potassium phosphate buffer at pH 5.91 in ratio 1:1 (*v*/*v*). Proton NMR spectra were acquired on a Bruker NEO 600 spectrometer (600.18 MHz, Biospin GmbH, Rheinstetten, Germany) at 293.0 ± 0.1 K using a Prodigy probehead. Standard parameters have been used—pulse programs zg30 and noesypr1d for experiments with water presaturation, pulse width 30◦/90◦, spectral width 13.66 ppm, 64 K data points, 1/64 scans, acquisition time 4.0 s, and relaxation delay 4.0 s. The signal of the rest proton signal of the solvent CD3OD at 3.3 ppm was used as an internal reference.

## 2.4.3. LC-MS/MS

After acquisition of 1H NMR spectra, LC-MS/MS analyses was performed on an Agilent 1260 Infinity II LC System equipped with a quaternary pump, autosampler, multicolumn thermostat and Agilent 6546 QTOF detector. Analytical separations were performed on an Agilent InfinityLab Poroshell 120 SB-C18 (2.7 μm, 3 × 150 mm) (Santa Clara, CA, USA) at room temperature. ESI-MS spectra were recorded in negative ion mode between *m*/*z* 20–3200. The fragmentor energy of ESI was set to 120 V. The injection volume was 10 μL (1 mg/mL dry weight). The mobile phase consisted of 0.1% aqueous formic acid (A) and 0.1% formic acid in Acetonitrile (B). The flow rate was 0.6 mL/min. The following gradient profile was used for qualitative analysis of SEC fraction FR14: 0 min 15% B, gradient 0–20 min 18% B, 20–22 min 100% B, 22–30 min 100% B isocratic, 30–32 min to 15% B. The following gradient profile was used for qualitative analysis of SEC fraction FR18: 0 min 25% B, 0–6 min isocratic 25% B, gradient 6–12 min 42% B, 12–14 100% B, 14–24 min 100% B isocratic, 24–26 min to 25% B. Three different collision-induced dissociation (CID) energies including 10, 20 and 40 eV were used for MS/MS verification of the myconoside structure. Hyspiduline hispidulin 8-C-(6-O-acetyl-2-O-syringoyl-β-glucopyranoside) was identified using a CID energy of 10 eV during MS/MS analysis.

## *2.5. Molecular Docking*

For the molecular modeling we used MOL structure file of myconoside (J796.651B, Japan Chemical Substance Dictionary (Nikkaji)) as ligand and three receptor macromolecules: human glucose transporter pdb ID 4PYP [36], MYST acetyltransferase pdb ID 6OIO [37] and Estrogen Receptor pdb ID 3OS8 [38]. Complex X-ray structures, including a receptor protein bound to a low-molecular-weight ligand, were used to determine the macromolecular complexes. Prior to docking, the primary ligands were removed. 3D macromolecular docking was performed with the Seamdock [39]. For human glucose transporter Charmm-Gui membrane builder [40] has been used to orientate the transmembrane spans through lipid bilayer and the resulting supramolecular structure was used to bind to the myconoside. All structures were optimized by free energy minimum and visualized with molecular dynamics programs Chimera 1.15 [41] and protein modeling—RasTop.

#### **3. Results**

## *3.1. Cell Viability and Cell Proliferation*

## 3.1.1. Cell Viability after Treatment with *Haberlea rhodopensis* Extracts and Fractions

Our preliminary experiments showed that the application of total extracts to control and breast cancer cell lines for 24 h were not informative enough. Longer exposure (for 48 h) of the cell lines to concentrations of extracts up to 300 μg/mL brought no differences in reaction to the treatment (Table S1). This triggered the application of nontarget fractionation of the extracts with liquid chromatography. We obtained 21 fractions and based on the availability of a sufficient amount of dry substance, 11 were selected for further analyses, and performed with 300 μg/mL for 48 h (Table S1). The results obtained were subjected to an ANOVA (Figure 1). A significant reduction in cancer cells viability was achieved after treatment with several of the fractions, among which 14 and 18 were most effective.

To further evaluate the effect of the selected fractions 14 and 18, the treatments have been prolonged for 72 h (Figure 2). The viability of the MCF-10A normal cell line was only slightly reduced to about 80% for both fractions. The viability of MCF7 cell line was significantly reduced below 50% for both fractions, while MDA-MB231 cell line was slightly less affected to 55% ± 10 after a treatment with fraction 14 and to 68% ± 2.2 for fraction 18

(Figure 2). The results obtained gave a good background for further proliferation assays with both fractions.

**Figure 1.** ANOVA multiple comparison plots of two breast cancer cell lines for statistical significance of difference between control conditions and treatment with total extract and different fractions for 48 h. (**A**) MCF7 and (**B**) MDA-MB231. Bars represent the viability of cells shown on x axis as log2 values of measured extinction for each treatment (shown on y axis); 1untreated; 2—total extract; 3—fr 14; 4—fr 15; 5—fr 16; 6—fr 17; 7—fr 18. The group tested for significance is represented with blue, with red are assigned groups with significant difference from the tested group, and with grey are shown the groups without significant changes.

**Figure 2.** Cell viability of normal breast cell line MCF-10A and breast cancer cell lines MCF7 and MDA-MB231 after treatment with fractions 14 and 18 in concentration 300 μg/mL for 72 h. Untreated samples are used as controls. The standard error bars are shown in percent. \*—the means are statistically significant at *p* ≤ 0.05; \*\*\*—the means are statistically significant at *p* ≤ 0.001.

#### 3.1.2. Cell Proliferation Assay after Treatment with Two Selected Fractions

The proliferation assay was performed with the studied cell lines—normal MCF-10A and two cancer cell lines MCF7 and MDA-MB231 (Figure 3). The cells were treated with two *H. rhodopensis* fractions—No. 14 and 18 with a concentration of 300 μg/mL dry substance. The viable cells were scored in absolute numbers at different time points (from 24 to 96 h). The cell line MCF-10A (panel A) had a normal growth and the treated cells showed slightly reduced numbers—reaching about 82.5% of the untreated cells for fraction 14 and 75% for fraction 18 at the end of the assay. The proliferation curves of control and treated cells of line MCF7 were very different (panel B). The numbers of treated cells were reduced at each time point. This was particularly true at the end of the assay where the absolute numbers of proliferation were reduced to 37.2% (fraction 14) and 36.3% (fraction 18). The cell proliferation of triple negative cell line MDA-MB231 was also significantly reduced after treatment with the fractions (Panel C). The reduction started after 24 h treatment and continued till the end of the assay. On the other hand, the proliferation rate of the cells at the last stages of treatment—72 and 96 h formed a plateau. Nevertheless, at the end of the assay, the reduction was 40–45% in comparison with control untreated cells. It should be underlined that in both cancer lines the reduction in proliferation rate showed no differences between both fractions after 48 h of treatment. The proliferation of MCF7 cell line was slightly more reduced than that of the MDA-MB231.

#### *3.2. Identification of Phytoactive Compounds in Plant Extract*

To identify the most abundant compounds from the fractions with the strongest effects on cancer lines—Fr 14 and Fr 18 (Figure 4A), we used semi-preparative HPLC to collect the most abundant peaks for each fraction followed by 1H NMR and mass spectrometry for identification (Figures 4B and 5). According to results from MS, the abundances of compounds **1** and **2** represent 24% and 14% from TIC of fractions 18 and 14, respectively (Tables S2 and S3, Figure 4A).

Semi-preparative fraction of the most abundant compound (**2**) from fraction 14 consists exclusively of myconoside as indicated by the NMR spectra (Table S4, Figure 5A). The MS and MS/MS spectra confirmed the mass of the pseudomolecular ion of myconoside (743.2399 (M – H)) as well as the presence of characteristic products of its fragmentation (Figures 4B and S1). This compound consists 14% from the total metabolite content in Fr14 followed by another unidentified compound with 6% of TIC. All other detected compounds showed very low abundances in fraction (Table S2, Figure 4A). The yield of the purified myconoside by semi-preparative fractionation was 34 mg (Figure 5A). Fr 18 shows several compounds above 6% of TIC including myconoside with 8.5% of TIC (Table S3, Figure 4B). The NMR spectra of semi-preparative fraction corresponding to the most abundant peak (23.5% of TIC) showed two main components in ratio 1.7:1, the higher concentrated one corresponding to hispidulin 8-C-(6-O-acetyl-2-O-syringoyl-β-glucopyranoside) (Table S3, Figure 5B). All signal assignments are in line with published data [42,43]. This identification was further confirmed by the corresponding pseudomolecular ion (683.1635 (M – H)) and fragmentation products from the MS<sup>2</sup> spectra corresponding to the loss of syringoyl and sugar moiety (Figures 5B and S2). However, further purification steps and analyses are needed for better evaluation of the active compound in this fraction.

**Figure 3.** Proliferation curves of normal epithelial breast cell line (**A**) and breast cancer cell lines MCF7 (**B**) and MDA-MB231 (**C**) treated with *H. rhodopensis* fractions N14 and N18. The standard error bars are shown. All means at each time point are statistically significant at *p* ≤ 0.05 (\*), *p* ≤ 0.01 (\*\*), and *p* < 0.001 (\*\*\*).

**Figure 4.** Mass spectrometry analysis of SEC fractions with significant effect on cancer lines and semi preparative fractionation of most abundant compounds. (**A**) Chromatograms of MS spectra of fractions 14 and 18. The most abundant peaks in each fraction are designated with numbers. (**B**) HPLC chromatograms of semi-preparative purification of the most abundant compounds from fraction 14 and 18.

**Figure 5.** Identification of the most abundant compounds in fractions 14 and 18 by combined NMR/MS2 analysis on same sample. The 1H NMR spectra for each compound is represented according to chemical shift (ppm) (upper panel), while ions of fragmentation from MS2 are represented according to their *m*/*z* (lower panel). (**A**) Identification of purified myconoside (compound 2) from fraction 14. (**B**) Identification of purified hispidulin 8-C-(6-O-acetyl-2-O-syringoyl-b-glucopyranoside) from fraction 18.

#### *3.3. Docking Analysis of Myconoside with Breast Cancer Proteins*

Considering our ability to purify and identify myconoside as the main compound in Fr 14 which significantly reduced cancer cell viability and proliferation and the available 2D and 3D deposited structures, we performed flexible docking analysis of this glycoside with several breast cancer proteins involved in cellular transport, signaling and DNA modification (Figure 6A).

**Figure 6.** Molecular docking of myconoside with several protein targets from breast cancer lines. (**A**) Ribbons of three-dimensional structure of binding of myconoside with GLUT 1, MYST acetyltransferase and estrogen receptor. The protein backbone is represented as a cartoon with different colors for each protein. The docked myconoside is represented with 3D stick model of chemical formula. GLUT 1 transporter (green) is integrated and oriented in membrane phospholipid bilayer; myconoside is given in blue. (**B**) Docking of myconoside into the binding cavity of the proteins with the corresponding intermolecular interactions and amino acid residues.

Our results showed that myconoside can interact with the three tested proteins. However, the interaction with GLUT 1 displayed more binding affinity according to ΔG (−19.8 kcal/mol) than other two proteins—MYST acetyltransferase (−12.3 kcal/mol) and Estrogen Receptor (−4.2 kcal/mol). Residues of the amino acids E380, F379, F287, F291, W412, T137 and W388 from GLUT 1 transporter are involved in interactions with myconoside. The amino acid residues E353, I386, L387, L391, W393, R394, F445 and K449 of estrogen receptor are involved in the represented receptor-ligand complex; while R655, R656, R660, F663, S690, Y691, S684, L683, F600, L601, W525 and Q654 were assigned in binding pocket of MYST acetyltransferase (Figure 6B). The docking model of myconoside with GLUT 1 is mainly determined by hydrogen bonds and hydrophobic interactions of Phenylalanine and Tryptophan residues. Most interactions with estrogen receptor are in a hydrophobic manner namely by Phenylalanine and Tryptophan residues as well by Leucine and Isoleucine residues. The binding of myconoside with MYST acetyltransferase is due to hydrophobic interactions of Leucine and Isoleucine residues as well as hydrogen bonds with Arginine and Serine.

#### **4. Discussion**

Breast cancer is among the most challenging human diseases. Despite the significant progress achieved in cancer treatment, the search for new natural products continues to be very intensive. Plant metabolites are tested for possible anticancer effects. Some of them can be used as food additives for cancer prevention or as therapeutics of side effects relief after radiotherapy. Others are used to enhance the effect of conventional drugs [11,12,44,45]. In this respect, promising results were reported for the proliferation rate reduction and cytostatic effects of some plant-derived alkaloids [10,33,46]; however, their application was limited by the multi-drug resistance developed by the cancer cells. The additional burden of the side effects of chemo- or radio-therapy paves the way for further studies on new potential sources of useful compounds.

Resurrection plants are a rich source of secondary metabolites with high antioxidant potential. Here, we describe, we believe for the first time, promising results of breast cancer cell lines treatment with extracts and fractions derived from leaves of the Balkan resurrection plant species *Haberlea rhodopensis*. The application of total leaf extracts, obtained with various extraction agents led to encouraging results in studies with several human diseases, including some types of cancer [27–30]. Viability reduction was described in two prostate cancer cell lines after methanol extracts treatment [27]. The same types of extracts were reported to have unique synergetic inhibitory effects against the herpes virus [29]. In addition, they could be a good candidate to be involved in complex treatments of pathological dermatological conditions [28]. Recently [30] extensive study with six human cancer cell lines—A549 (non-small cell lung adenocarcinoma, HepG2 (hepatocellular carcinoma)), HT29 and Caco-2 (colorectal adenocarcinomas), and PC3 and DU145 (prostate adenocarcinomas) showed that alcohol extraction appeared to be more effective than the aqueous. Significant antimigratory concentration-dependent effects were achieved for non-small cell lung adenocarcinoma and hepatocellular carcinoma (HepG2) cell lines [30].

Our study showed that the total extract was unable to significantly reduce the viability of the MCF7 and MDA-MB231 breast cancer cell lines (Supplementary Table S1). This triggered our further efforts to fractionate the extracts to achieve enrichment of active substances. Two of the fractions (14 and 18) showed significant effects on breast cancer cell viability (Figure 1) and a negligible effect on normal breast epithelia cell line MCF10-A (Supplementary Table S1, Figure 2). These fractions had a high impact on cell proliferation of the studied breast cancer cell lines and an insignificant effect on the proliferation of the normal breast epithelial cell line. One of the fractions (Fr 14) is enriched of myconoside and another one (Fr 18)—of hispidulin 8-C-(6-O-acetyl-2-O-syringoyl-β-glu-copyranoside) (Figure 4).

## *4.1. Potential Role of Myconoside*

Myconoside has been isolated earlier from the three European members of Gesnereiacea, including *H. rhodopensis* [42,47–51]. The myconoside molecule has a phenyl glycoside structure with 12 hydroxyl groups (Supplementary Figure S1) which determined its chemical activity and possibility to form a 3D structure which was deposited in the Japan Chemical Substance Dictionary (Nikkaji) database. The structure file was used to propose models of its binding to three proteins with an essential role for breast cancer and breast cancer cell lines development—estrogen receptor, glucose transporter GLUT1 and MYST acetyltrasferase (Figure 6). All of these three proteins are expressed in the MCF7 cell line whereas two of them—GLUT1 and MYST acetyltrasferase are expressed in MB-MDA231. We presumed a possibility for myconoside binding to glucose transporter GLUT1. The GLUT family of transporters are localized on the cell membrane and are connected by hydrophilic loops [52]. They are expressed in high levels in different types of tumors, including breast tumors [53–55] and in particular in the two cancer cell lines under study—MCF7 and MDA-MB231 [6]. The chemically synthesized transporter inhibitors WZB117 and STF-31 block cell proliferation of MCF7 and MDA-MB231 cell lines by an increase in extracellular glucose and a decrease in extracellular lactose [55]. WZB27 and

WZB115 are two transporter inhibitors which reduce glucose uptake and block cell proliferation in MCF7 cell line [56]. Various polyphenol substances of plant origin—gossypol, genistein, resveratrol, quercetin have been described to influence glucose metabolism in breast cancer cell lines [7]. We have two hypotheses about myconoside binding to the glucose transporter. It could be a glucose transporter blocker, thus reducing glucose uptake in cells, or this could be the way for myconoside penetration in the cell. The prediction of molecular binding of myconoside with GLUT1 transporter makes possible the penetration of myconoside in cancer cells by binding to this membrane localized transporter. These hypotheses should be a subject of future studies.

The estrogen receptor has an essential role for estrogen-dependent growth in estrogen expressing tumors and cancer cell lines. We presume a possible binding of myconoside to the estrogen receptor of the MCF7 cell line—a good model to search possibilities for estrogen receptor agonists/antagonists in breast cancer therapy [57]. Drugs with polyphenyl structures block the DNA binding receptor domain and are competitive antagonists of estradiol. This is a mechanism for hormone dependent growth blockage of breast cancer cells [57,58]. Such estrogen antagonists of plant origin are the coumarins with antiproliferative effects on the breast cancer cell line MDA-MB435 [57]. We propose a role of myconoside as an estrogen receptor modulator. Plant substances were virtually screened and 162 of them have been validated by docking with estrogen receptor α. Eight of them have ER α competition effects. Genistein, daidzein, phloretin, ellagic acid, ursolic acid, (−)-epigallocatechin-3-gallate, kaempferol are with different antagonistic activities against estrogen receptor α [59].

Our docking analyses allowed the presumption that another target of myconoside could be MYST acetyltransferase. This could be a mechanism, affecting cell proliferation in studied breast cell lines related to epigenetic DNA regulation. We found that interactions of MYST acetyltransferase with myconoside are docked by amino acids residues ARG655, LEU686, GLN760, ARG660, LEU689 and LYS763, which are previously reported to interact with different compounds of the medicinal plant *Withania somnifera* (L.) [60]. The MYST acetiytransferases are related to the activation of estrogen receptor α by acetylation of the hystone acetyltransferase domain in the estrogen receptor promotor. This mechanism of epigenetic regulation activates the estrogen receptor expression in estrogen receptor positive breast cancer cell lines like MCF7 [7]. The blockage of acetyltransferase enzyme suppresses the activation of estrogen receptor α promotor. Studies of acetyltransferase mRNA and protein expression showed different levels of their expression in a panel of breast cancer cell lines [7]. This is a pathway to limit estrogen dependent growth in estrogen receptor positive cells and could be a possible explanation for the higher inhibitory effect on the triple positive cell line MCF7. Purified myconoside was shown to have antimygratory effects and cell proliferation blockage on the A549 lung adenocarcinoma cell line with an IC50 of 20 μg/mL [61]. Our results showed that fractions containing myconoside did not significantly affect cell viability or proliferation of the normal cell line MCF10A (Figures 2 and 3). This is in agreement with the report that *H. rhodopensis* extracts fractions with identified myconoside and calceolarioside E has effects on protein expression of neutrophil essential transcription factor regulating redox potential Nrf2 in bone marrow neutrophils [42]. In addition, when applied in low concentrations, the natural phenyl glycoside induces hormetic-like response in MDCKII cell line [62] or has photoprotective effects on UVA/UVB irradiated immortalized keratinocytes [53].

## *4.2. Potential Role of Hispidulin 8-C-(6-O-acetyl-2-O-syringoyl-β-glu-copyranoside)*

The proliferation rate was significantly reduced in both breast cancer cell lines after treatment with Fraction 18. This fraction is more complex, containing several compounds, including myconoside and hispidulin 8-C-(6-O-acetyl-2-O-syringoyl-β-glu-copyranoside). We identified the most abundant compound as hispidulin 8-C-(6-O-acetyl-2-O-syringoylβ-glu-copyranoside) (Figure 5B). It was reported earlier in a mixture of flavone glycosides of the same plant species [43,63]. While myconoside is found exceptionally in some

Gesmneriaceae resurrection plant species, hispidulin is common for many plant species widely applicable in traditional medicine, such as *Grindelia argentina*, *Arrabidaea chica*, *Saussurea involucrate*, *Crossostephium chinense*, *Artemisia* and *Salvia* species. It was shown to possess various activities—antioxidant, antifungal, anti-inflammatory, antimutagenic, and antitumor [64]. The potential therapeutic usefulness of hispidulin has been studied in a variety of tumors [65–68]. The molecular mechanisms mediating its effects on cancer cell lines of different origin have been analyzed. Effects on cell viability and proliferation have been established for prostate cancer cell lines [65], GLUT1-HEK293T transformed cells and Hep2G hepatocellular carcinoma cells [66], as well as for human melanoma A253 cancer cells [67]. Its mechanism of influence on cell signal pathways depends on the type of cancer. In the case of prostate cancer cell lines it is related to limitation of cell migration, invasion, proliferation and apoptosis initiation mediated via PPARγ activation and autophagy induction [65]. Hispidulin modulates epithelial-mesenchymal transition in breast cancer cells—a process associated with the disruption of cell junctions, increase in cell mobility and metastasis by suppressing the TGF-β1-induced Smad2/3 signaling pathway [68]. The effects on human melanoma cells are mediated by activation of apoptosis rather than autophagy, inhibiting kinase signaling pathways AKT and ERK [4]. Recently, the inhibitory effect of various flavonoids on GLUT1 transporter in HEK293T and HepG2 has been reported [66]. Several flavonoids, including hispidulin inhibit hepatocellular carcinoma cell line Hep2G and GLUT1 expressing HEK2893T cell line by binding to glucose transporter1, which was shown by docking analysis and validated [66]. The suppression of GLUT1 transporter activity by hispidulin was established to 40% of control untreated sample at the concentration range 100–150 μM. This fact could explain our results on cell viability reduction and suppression of proliferation in the studied cell line MCF7 and MDA-MB231, which both express GLUT1 in high levels and are characterized with intensive glucose metabolism. The combination of identified hispidulin 8-C-(6-O-acetyl-2- O-syringoyl-β-glu- copyranoside) and the second highly abundant unidentified substance in the fraction should be a subject of future investigations related to its identification and the clarification of molecular mechanisms of influence on breast cancer cell proliferation.

## **5. Conclusions**

This study described for the first time the effect of *H. rhodopensis* methanol extract fractions on the viability and proliferation of two breast cancer cell lines with different characteristics and a normal cell line. The inhibitory effects are specific for cancer cell lines and are better for the hormone responsive one. Myconoside appears to be a suitable agent for cancer therapy and a possible model for its action was proposed, targeting three specific cancer hallmark proteins.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/separations10070388/s1. Table S1. cell viability, Table S2. Metabolic content of fraction 14, Table S3. Metabolic content of fraction 18, Table S4. signals of 1H and 13C of myconoside, Table S5. 1H signals of hispidulin, Figure S1. Molecular structure of myconoside (MS/MS identification), Figure S2. Molecular structure of hispidulin (MS/MS identification), Figure S3. Structure of myconoside (NMR), Figure S4. Structure of hispidulin (NMR).

**Author Contributions:** Conceptualization, D.Z. and P.M.; methodology, D.Z. and P.M.; validation, D.Z., P.M., K.R. and S.S.; formal analysis, D.Z., P.M., S.S. and K.R.; investigation, D.Z., P.M., S.S., K.R., S.Z., L.S.-S., D.M. and D.D.; resources, D.Z., L.S.-S., D.D., S.S. and K.R.; writing—original draft preparation, D.Z., P.M., D.M., L.S.-S. and D.D.; writing—review and editing, D.Z., P.M., S.S., K.R., S.Z., L.S.-S., D.M. and D.D.; visualization, D.Z., P.M., S.S. and K.R.; supervision, D.Z., P.M., S.S. and D.D.; project administration, D.Z., P.M., D.D. and L.S.-S.; funding acquisition, D.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was funded by NSF of Bulgaria, Grant number КΠ-06-Н41/6, Operational Program Science and Education for Smart Growth 2014–2020, co-financed by the European Union through the European Structural and Investment Funds, Grant BG05M2OP001-1.002-0012.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors highly appreciate the help of Zlatina Gospodinova (Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences) by kindly providing cell line MCF-10A and of Radostina Alexandrova (Institute of Experimental Morphology and Anthropology Bulgarian Academy of Sciences, Bulgarian Academy of Sciences) for kindly providing MCF7 cell line and of Milena Mourdjeva (Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences) for kindly providing cell line MDA-MB231. We highly appreciate the help of Svetlana Hristova, Ph.D (Department of Medical Physics and Biophysics, Medical Faculty, Medical University–Sofia, Zdrave Str. 2, 1431 Sofia, Bulgaria) for kindly contribute with software for analyses of molecular docking.

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

## **References**


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## *Article* **Rapid and Simultaneous Extraction of Bisabolol and Flavonoids from** *Gymnosperma glutinosum* **and Their Potential Use as Cosmetic Ingredients**

**Mayra Beatriz Gómez-Patiño 1, Juan Pablo Leyva Pérez 2, Marcia Marisol Alcibar Muñoz 3, Israel Arzate-Vázquez <sup>1</sup> and Daniel Arrieta-Baez 1,\***


**Abstract:** *Gymnosperma glutinosum* is a plant popularly known as "popote", "tatalencho", "tezozotla" or "pegajosa", and it is used in traditional medicine in the region of Tehuacán, Puebla (Mexico), for the treatment of jiotes and acne and to cure diarrhea using the aerial parts in infusions. To analyze the phytochemical composition, we have developed a rapid protocol for the extraction and separation of the components of the aerial parts of *G. glutinosum*. After a maceration process, chloroformic and methanolic extracts were obtained and analyzed. Extracts were evaluated by GC-MS (gas chromatography-mass spectrometry), and their composition revealed the presence of (−)-α-bisabolol (BIS) as the main component in the chloroformic extract, which was isolated and analyzed by 1H NMR to confirm its presence in the plant. The analysis of methanolic extracts by UPLC-MS (ultraperformance liquid chromatography-mass spectrometry) revealed the occurrence of six methoxylated flavones with *m*/*z* 405.08 (C19H18O10), *m*/*z* 419.09 (C20H20O10) and *m*/*z* 433.11 (C21H22O10), and a group of C20-, C18-hydroxy-fatty acids, which give the plant its sticky characteristic. The presence of BIS, an important sesquiterpene with therapeutic skin effects, as well as some antioxidant compounds such as methoxylated flavones and their oils, could play an important role in cosmetology and dermatology formulations.

**Keywords:** *Gymnosperma glutinosum*; cosmetology; skin care; antioxidants; flavonoids; bisabolol

## **1. Introduction**

The human skin is an organ that covers 15% of the total weight of the human body, which is not only important for aesthetic reasons, but also because it is responsible for many vital functions, among them the protection against external factors, regulation of fluid balance, metabolism, elimination of toxins and body shape maintenance [1,2]. Most people care about maintaining healthy skin, which promotes mental health by increasing people's self-confidence [3–5]. The use of cosmetics has become essential in our society, and although many plant products have been replaced by synthetic chemical compounds, a replacement has not been found for the safety and efficacy of natural products, which is why in recent years, the preference for natural products has resurfaced [6–10]. Since the awareness of the long-term benefits of natural ingredients in cosmetic products is increasing, they are being considered more, and recent studies indicate that plant components, such as phenolics, flavonoids and polysaccharides, have a high potential for cosmetic applications [11–15]. Besides the presence of compounds that demonstrated certain benefits, it is important

**Citation:** Gómez-Patiño, M.B.; Leyva Pérez, J.P.; Alcibar Muñoz, M.M.; Arzate-Vázquez, I.; Arrieta-Baez, D. Rapid and Simultaneous Extraction of Bisabolol and Flavonoids from *Gymnosperma glutinosum* and Their Potential Use as Cosmetic Ingredients. *Separations* **2023**, *10*, 406. https://doi.org/10.3390/ separations10070406

Academic Editor: Faiyaz Shakeel

Received: 5 June 2023 Revised: 4 July 2023 Accepted: 9 July 2023 Published: 14 July 2023

**Copyright:** © 2023 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/).

to consider their synergy. Extracts from medicinal plants are rich in compounds that act synergistically, and it is important to study these compounds in their natural percentage to understand their optimal biological activity [15]. Thus, in mild skin disorders, the topical application of certain preparations based on natural products, such as infusions, creams, balms and tinctures, having in mind these concepts, can be effective in preventing the development of more severe diseases.

Ethnobotanical studies have helped us to understand the use of plants in traditional medicine [16]. These studies have not only helped us to understand the historical relevance of plants but also the importance they play in human health. Based on this type of study, it was determined that *G. glutinosum*, a plant that is popularly known as "popote", "tatalencho", "pegajosa" or "tezozotla", is used in traditional medicine in the region of Tehuacán, Puebla (Mexico) [17–21], for the treatment of jiotes and acne and in infusions to cure diarrhea. In recent years, ethnobotanical studies showed that *G. glutinousm* is one of the most important plants used in traditional medicine from the Tehuacan-Cuicatlan Biosphere Reserve, San Rafael, Coxcatlan, and Zapotitlan Salinas, Puebla (Mexico), for the treatment of diarrhea. Phytochemical studies of *G. glutinosum* have demonstrated the presence of essential oils, flavonoids and diterpenes. Most of the compounds isolated from *G. glutinosum* are methoxylated flavones such as 5,7-dihydroxy 3,6,8-trimethoxyflavone and 5,7-dihydroxy 3,6,8,2 ,4 ,5 -hexamethoxyflavone, and some ent-labdane-type diterpene [20–23]. More recent studies showed the isolation of a diterpene ent-labdene-type: ent-dihydrotumanoic acid (DTA) with antidiarrheal and antinociceptive effects [24–26]. However, even when this plant is used in the treatment of some skin problems, there are no studies about the relation between the isolated compounds and these diseases.

(−)-α-bisabolol (BIS), a sesquiterpene alcohol, has been mostly isolated from chamomile [27], and there are no reports of the presence in the genus *Gymnosperma*. BIS has different biological activities, including antioxidant, anticancer, anti-inflammatory, anti-infection, and skin-shoothing and -moisturizing properties [28–31]. At present, BIS is mainly manufactured through the steam-distillation of chamomile essential oils. Some products are produced by synthesized BIS; however, the process requires an additional economically unviable purification step due to the presence of the diastereomer (+)-α-bisabolol [32]. Therefore, finding new natural sources of bisabolol is essential to specialty pharmacological and cosmetologically industries.

In this work, based on ethnobotanical studies, we studied *G. glutinosum* to find compounds as ingredients for cosmetic purposes, and three groups of compounds were identified under the bases of GC-MS and UPLC-MS analyses. (−)-α-bisabolol and 6-epishyobunol, six methoxylated flavones, and a group of C20-, C18-hydroxy-fatty acids were identified in this extract and represent interesting mixtures for further investigations in the cosmetic industry.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Chloroform (CAS 110-54-3, 99%), acetonitrile (CAS 75-05-8, 99%), methanol (CAS 64-578-6, 99%), water (CAS 7732-18-5) and the ESI-TOF (electrospray ionization-time of flight) tuning mix calibrant were obtained from Supelco (Toluca, Edo. de México, México). The deuterated CDCl3, MeOD and TMS were acquired from Sigma Aldrich (Toluca, Edo. de Mexico, Mexico).

#### *2.2. UPLC and GC Coupled to Mass Spectrometry Analysis*

Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis was conducted in a Ultimate3000 UPLC system (Dionexcorp., Sunnyvale, CA, USA) with photodiode array detection (PAD), coupled to a Bruker MicrOTOF-QII system by an electrospray ionization (ESI) interface (Bruker Daltonics, Billerica, USA). Mobile phase used in the system consisted of 0.1% formic acid in water (A) and acetonitrile (B) using a gradient program of 5–35% (B) in 0–10 min, 35–80% (B) in 10–10.1 min, 80–80% (B) in 10.1–11, 80–45% (B) in 11–11.1, 45–5% (B) in 11.1–12 min and 5% (B) in 12–15 min. The chromatographic column used was a Hypersil C18 column (3.0 μm, 125 × 4.0 mm) (Varian). The solvent flow rate used was 0.5 mL/min, and the column temperature was set to 30 ◦C. For the mass spectrometer, conditions in the negative mode were as follows: drying gas (nitrogen), flow rate, 8 L/min; gas temperature, 180 ◦C; scan range, 50–3000 *m*/*z*; end plate off-set voltage, −500 V; capillary voltage, 4500 V; and nebulizer pressure, 2.5 bar.

Direct injection electrospray ionization-mass spectrometry (DI-ESI-MS) analyses were conducted on Bruker MicrOTOF-QII system by an electrospray ionization interface (Bruker Daltonics, Billerica, MA, USA) operating in the negative ion mode (ESI-).

A total of 1 mg of the sample was resuspended in 1 mL of methanol, filtered through a 0.25 μm polytetrafluoroethylene (PTFE), and diluted 1:100 with methanol to avoid saturation of the capillary and cone soiling. To inject the sample directly into the spectrometer, a 74900-00-05 Cole Palmer syringe pump (Billerica, MA, USA) was used and set at 8 μL/min to obtain a constant flow rate. The capillary voltage was set to 4500 V, and nitrogen was used as a drying and nebulizing gas, using a flow rate of 4 L/min (0.4 Bar) with a gas temperature of 180 ◦C. The spectrometer was calibrated with an ESI-TOF tuning mix calibrant (Sigma-Aldrich, Toluca, Estado de Mexico, Mexico).

To analyze compounds' structure, a tandem mass spectrometry (MS/MS) analysis was performed using negative electrospray ionization with the appropriate mass set. According to the obtained pattern, suitable fragments were analyzed by a Bruker Compass Data Analysis 4.0 (Bruker Daltonics), which provided a list of possible elemental formulas using Generate Molecular Formula Editor, as well as a sophisticated comparison of the theoretical with the measured isotope pattern (σ value) for increased confidence in the suggested molecular formula (Bruker Daltonics Technical Note 008, 2004). The accuracy threshold for confirmation of elemental compositions was established at 5 ppm.

Gas chromatography analysis was performed using a gas chromatograph 456-GC SCION TQ (Bruker, Biellerica MA, USA). The injector port was set at 220 ◦C with split 10. Separation was achieved using an RXI-5SIL (Fused silica, 30 m × 0.32 mm (ResteK) and helium at a flow rate of 1 mL/min. Column oven was programmed in the following conditions: 55 ◦C for 1.0 min, 55 ◦C to 155 ◦C at 20 ◦C/min, 155 ◦C for 2 min, 155 ◦C to 255 ◦C at 10 ◦C/min, 255 ◦C for 5.0 min, and finally from 255 ◦C to 280 ◦C at 10 ◦C/min and 280 ◦C for 5 min. The MS was set in TIC mode with an EI (electron ionization) of 70 eV.

## *2.3. NMR Spectroscopy*

1H nuclear magnetic resonance (1H NMR) experiments of the soluble products were conducted on a Bruker Instruments ASCEND 750 spectrometer (Billerica, MA, USA). The resonance frequency was 750.12 MHz, with a typical acquisition time of 2.1845 s and a delay time of 1.0 s between successive acquisitions. The 1H and 13C chemical shifts are given in units of δ (ppm) relative to tetramethylsilane (TMS), where δ (TMS) belongs to 0 ppm.

#### **3. Results**

Aerial parts of *G. glutinosum* were collected in Santa Maria La Alta, a village in the municipality of Tehuacan, Puebla (Mexico) (18.60001:−97.65754) (Figure 1). A total of 800 g of the plant were collected in two different months to standardize the extraction process and determine if there could be some changes in the production of the phytochemical compounds. The first collection was completed in June and the second one in December.

**Figure 1.** Presence of *G. glutinosum* ("popote, pegajosa") in the Tehuacan Valley.

## *3.1. Isolation and Standardization of the Method of Extraction*

With plants collected in June and December 2022, the next procedure was applied: 250 g of the aerial parts of the fresh plant material were ground and extracted two times with 500 mL of chloroform at room temperature. After filtration, the solvent was evaporated to obtain the chloroform extract. This procedure was completed twice for each plant material collected. The chloroform extract was partitioned with chloroform and methanol. Two partitioned fractions were obtained: a chloroform fraction 1 (CHCl3 Fc1) and methanolic fraction 1 (MeOH Fc1). The yields are shown in Table 1.

**Table 1.** Yields of the fractions obtained from the *G. glutinosum* chloroform extraction.


<sup>1</sup> Extraction yield is the average of two processes.

To analyze if the extraction with chloroform had been exhausted, the residue of the plant material was subject to another extraction with methanol and the extract was subject to the same partition as those applied to the chloroform extract. Two fractions were obtained: a chloroform fraction 2 (CHCl3 Fc2) and a methanolic fraction 2 (MeOH Fc2). The yields are shown in Table 2.

**Table 2.** Yields of the fractions obtained from the *G. glutinosum* methanolic extraction.


<sup>1</sup> Extraction yield is the average of two processes.

## *3.2. Chemical Composition Assay by GC-MS of the CHCl3 Fc1*

The fraction partitioned with chloroform was analyzed through GC-MS. Samples were analyzed by triplicate, injecting 1 mL, and carried out at 1 mL-min−<sup>1</sup> by ultrapure helium. Two peaks were detected as the main compounds, and the mass spectra of each molecule were compared with those in the NIST (National Institute of Standards and Technology) database software. Under these conditions, 6-epi-shyobunol and (−)-α-bisabolol (BIS) were detected at 2.5 and 97.5%, respectively (Figure 2).

**Figure 2.** Chromatogram of the GC-MS analysis and compounds detected in the CHCl3 Fc1 from *G. glutinosum*.

From the methanol extraction, chloroform partitioned extract (CHCl3 Fc2) was analyzed under the same conditions. However, no compounds were detected indicating that BIS was completely extracted with chloroform.

## *3.3. Chemical Composition by UPLC-MS Assay of MeOH Fc1*

MeOH Fc1 was subject to a UPLC-MS analysis and according to Figure 3, different peaks were detected and analyzed by the extracted ion chromatogram (EIC) from the total ion chromatograph (TIC) to generate two main groups of compounds detected in this extract. In the first group, two types of long-chain fatty acids were present for the *m*/*z* 321.23 and 319.21. For the detected molecular ion at *m*/*z* 321.23 [M-H]−1, a molecular formula C20H34O3 was assigned. According to Figure 3(1), at least six peaks correspond to this molecular weight indicating the presence of isomers. Under the bases of *ms/ms* analysis, only the main peak was identified as 20-hydroxyicosa-(5,8,11)-trienoic acid (Figure 3(1)). In the same regard, the molecular ion at *m*/*z* 319.21 [M-H]−<sup>1</sup> was consisting of a molecular formula C18H32O3. In this case, four peaks were detected, indicating the presence of the same number of isomers that could be assigned to 20-hydroxyicosa-(5,8,11,14)-tetraenoic acid derivatives (Figure 3(2)).

For the second group, a methoxylated flavones group was detected and analyzed. Two peaks at Rt of 11.9 and 13.5 min (Figure 3(3)) showed the same molecular ion at *m*/*z* 405.08, which consisted of the molecular formula C19H18O10 (5,7,2 ,4 -Tetrahydroxy-3,6,8,5 -tetramethoxyflavone and 5,7,4 ,5 -Tetrahydroxy-3,6,8,2 -tetramethoxyflavone). *m*/*z* 419.09 detected an Rt of 13.1 and 13.9 min (Figure 3(4)) and consisted of the molecular formula of C20H20O10 for other two methoxylated flavones (5,7,2 -Trihydroxy-3,6,8,4 ,5 pentamethoxyflavone and 5,7,4 -Trihydroxy-3,6,8,2 ,5 -pentamethoxyflavone). Finally, another two peaks at Rt of 14.3 and 15.4 min (Figure 3(5)) showed the same molecular ion at *m*/*z* 433.11, assigned to the molecular formula C21H22O10 (5,7-Dihydroxy-3,6,8,3 ,4 ,5 hexamethoxyflavone and 2-(5-Hydroxy-2,3-dimethoxyphenyl)-5-hydroxy-3,6,7,8-tetramethoxy-4H-1-benzopyran-4-one).

**Figure 3.** UPLC-MS chromatogram (upper chromatogram, TIC) and EIC-chromatogram analysis of the compounds detected in MeOH Fc1 from *G. glutinosum*. (1. EIC *m*/*z* 321, 2. EIC *m*/*z* 319, 3. *m*/*z* 405, 4. EIC *m*/*z* 419 and 5. EIC *m*/*z* 433).

The retention time (Rt), formula, name and relative percentage of the compounds detected are given in Table 3.

**Table 3.** Retention time and relative percentage of the compounds detected in the chromatogram of the UPLC-MS analysis of the MeOH Fc1 of *G. glutinosum*.


Under these conditions, BIS was not detected in the methanolic fraction.

The methanol extract, which was partitioned into a chloroform fraction (CHCl3 Fc2) and a methanolic fraction (MeOH Fc2), was analyzed by means of DIESI-MS. As we can see in Figure 4, small peaks corresponding to the methoxylated flavones were detected, especially peaks with molecular ions at *m*/*z* 321, 405, and 419.

**Figure 4.** DIESI-MS analysis of the MeOH Fc2 from *G. glutinosum* compared with that obtained from MeOH Fc1. Upper spectrum: MeOH Fc1; middle spectrum: MeOH Fc2 and lower spectrum: CHCl3 Fc2.

## *3.4. NMR Analysis of the Chloroform Extract of G. glutinosum*

CHCl3 Fc1 was analyzed through 1H NMR. In Figure 5, a comparison of simulated and obtained spectra is shown. Vinylic protons at δ 5.2 and 5.4 ppm, as well as the methyl groups at δ 1.3 and 1.7 ppm confirm the presence of BIS, which was detected before by means of GC-MS.

**Figure 5.** 1H NMR spectra of CHCl3 Fc1 from *G. glutinosum* (lower spectra), compared with a predicted spectrum of BIS (software MestReNova ver 6.0.2).

### **4. Discussion**

In addition to the use of *G. glutinosum* in traditional medicine in the Tehuacan Valley for stomach problems, it has been used to help with some skin problems. Under these ethnobotanical studies, this plant that grows in the wild seems to have promising active

compounds, which with adequate procedures implemented for their extraction could provide a high-added value raw material source for natural antioxidant constituents with a high potential for application in cosmetology and dermatology formulations. In recent years, different studies have been conducted to incorporate plants or herbal extracts for their therapeutic potential in the market as skincare products [14,15]. In the same way, phytochemical compounds have been evaluated in vivo and in vitro models to analyze their biological activity. However, it is necessary to consider the synergy of different extracts and evaluate the activity as a complex extract because it is possible that it could have a more effective impact.

To address this objective, two extractions were performed to obtain the most compounds and characterize them under different analytical techniques.

As a plant that grows in the wild, it was important to know if the phytochemical compounds are present at any time of the year, at least in this region of México. Procedures were applied in the same way under the same conditions, and according to Tables 1 and 2, the yield in the extraction seems to be very similar.

The dry extract of chloroform was partitioned with chloroform (CHCl3 Fc1) and methanol (MeOH Fc1), and CHCl3 Fc1 was analyzed by GC-MS. The results obtained and previously discussed in this work suggest the presence of BIS. According to Figure 1, two peaks were identified as 6-epi-shyobunol and (−)-α-bisabolol (BIS) at 2.5 and 97.5%, respectively. To confirm this result, BIS was purified and analyzed by 1H NMR. Figure 5 shows the spectra obtained and compared with a simulation of BIS in the software MestReNova. According to both analyses, the presence of this compound in *G. glutinosum* is confirmed for the first time. With the proposed methodology, BIS was extracted quickly and selectively with a good yield. So, it could be possible that the biological activity shown by this plant in skin problems can be attributed to BIS [30,31].

(−)-α-bisabolol (BIS), a monocyclic sesquiterpene alcohol, was isolated and identified for the first time from chamomile (*Matricaria chamomilla*) [27], and to date is mainly obtained from this natural source; although, it has also been isolated from the essential oil of other medicinal plants [31]. It is a compound that has been considered safe due to its low toxicity, and its effects have been widely studied in different models, indicating its potential beneficial actions [31]. BIS has a variety of biological activities, including antioxidant, gastroprotective, anti-infection and anticancer properties. In atopic dermatitis, it has been found to help attenuated pruritus and inflamed skin. Other results include improved facial texture, skin oiliness, hydrated skin, brightness and better appearance in patients who used it as a treatment [28–31].

Even though the phytochemistry of *G. glutinosum* is poorly described, and there is no report of suitable compounds that could be used as cosmetic ingredients besides BIS, some methoxylated flavones were identified. From the partitioned fraction of methanol (MeOH Fc1), a methoxylated flavones group was detected and analyzed. Two peaks at Rt of 11.9 and 13.5 min (Figure 3(3)) showed the same molecular ion at *m*/*z* 405.08, which consisted of the molecular formula C19H18O10 (5,7,2 ,4 -Tetrahydroxy-3,6,8,5 -tetramethoxyflavone and 5,7,4 ,5 -Tetrahydroxy-3,6,8,2 -tetramethoxyflavone). *m*/*z* 419.09 detected an Rt of 13.1 and 13.9 min (Figure 3(4)) and consisted of the molecular formula of C20H20O10 for two other methoxylated flavones (5,7,2 -Trihydroxy-3,6,8,4 ,5 -pentamethoxyflavone and 5,7,4 - Trihydroxy-3,6,8,2 ,5 -pentamethoxyflavone). Finally, another two peaks at Rt of 14.3 and 15.4 min (Figure 3(5)) showed the same molecular ion at *m*/*z* 433.11, assigned to the molecular formula C21H22O10 (5,7-Dihydroxy-3,6,8,3 ,4 ,5 -hexamethoxyflavone and 2-(5-Hydroxy-2,3-dimethoxyphenyl)-5-hydroxy-3,6,7,8-tetramethoxy-4H-1-benzopyran-4-one).

Most of these compounds have been previously reported in *G. glutinocum*. 5,7 dihydroxy-3,6,8,2 ,4 ,5-hexamethoxyflavone was isolated with (−)-17-hydroxy-neo-clerod-3-en-15-oic acid by Canales et al. from two samples of *G. glutinosum* obtained in Puebla and Hidalgo State (Mexico) [20,21]. Extracts obtained from these samples showed antimicrobial activity. In 2009, Serrano et al. [22] described the isolation of two methoxylated flavones, 5,7 dihydroxy-3,6,8-trimethoxyflavone and 5,7-dihydroxy-3,6,8,2 ,4 ,5 -hexamethoxyflavone,

which were responsible for the fungal activity against *Aspergillus niger*, *Candida albicans*, *Fusarium sporotrichum* and *Trichophyton mentagrophytes*. Some other flavonoids, such as quercitrin, quercetin, kaempferol, rutin and vitexin have been described from *G. glutinosum* [23]. All these compounds have demonstrated their biological activity as antifungal, antimicrobial and antioxidant compounds and these activities contribute to the medicinal properties, which are used in traditional medicine in the Tehuacan, Puebla (Mexico) region. More recently, a diterpene ent-labdene-type: ent-dihydrotumanoic acid (DTA) with antidiarrheal and antinociceptive effects was isolated.

The increase in knowledge of the damage that ultraviolet radiation can cause in carcinogenesis and aging has increased the use of skin care products, especially sunscreens. However, most of these products are made with synthetic molecules or chemical substances that cause dermal toxicity. Nowadays, different reports of the beneficial effects of plants and herbal products for the skin are available [33]. Most of the plants are rich in polyphenols, flavonoids and some other compounds with antioxidant activity that can protect the skin from the effects of ultraviolet radiation. These herbal extracts should be considered for use in herbal skincare products.

Antioxidant molecules, such as methoxylated flavones, can be used to reduce and neutralize free radicals and when combined with recognized natural compounds like BIS, could improve their biological activity, resulting in their use as potential extracts implemented in cosmetic, pharmaceutical and therapeutic formulations [15,33].

One of the important characteristics of this plant is its sticky property, which is why it receives the same name in many places: "planta pegajosa (sticky plant)". This characteristic could be related to the presence of oils, waxes or fatty acids. According to the UPLC-MS analysis, two families of hydroxy-C18 and -C20 fatty acids were identified. For the detected molecular ion at *m*/*z* 321.23 [M-H]<sup>−</sup>1, a molecular formula C20H34O3 was assigned, and at least six peaks correspond to this molecular weight indicating the presence of isomers of the identified 20-hydroxyicosa-(5,8,11)-trienoic acid (Figure 3(1)). In the same regard, for the molecular ion at *m*/*z* 319.21 [M-H]<sup>−</sup>1, four peaks were detected, indicating the presence of the same number of isomers that could be assigned to 20-hydroxyicosa-(5,8,11,14)-tetraenoic acid derivatives (Figure 3(2)).

The long-chain fatty acids present in the leaves of *G. glutinosum,* which are described for the first time in this work, could give the extract its waxy characteristic. Compounds such as waxes and oils extracted from plants have attracted attention for their properties to form films that can be used in cosmetic masks for skincare [34]. In this case, the presence of hydroxy long-chain fatty acids could help to keep the methoxylated flavones and the BIS in an aliphatic matrix for easier interaction with the skin. Anyway, functional groups such as hydroxyl and carbonyl groups in the long-chain fatty acids gave tunable properties to incorporate bioactive compounds for potentially sustainable alternatives over conventional products.

So, the presence of BIS, the antioxidant, the bacterial properties of the methoxylated flavones, and the aliphatic long-chain fatty acids are interesting mixtures for further investigation in the cosmetic industry of this extract obtained from *G. glutinosum*.

#### **5. Conclusions**

The use of cosmetics has become indispensable in our society. Although many plant products have been replaced by synthetic chemical compounds, the safety and efficacy of natural products have not been replaced, and in recent years, the preference for the natural has re-emerged. From an ethnobotanical study, a rapid and successful extraction of bio-compounds from *G. glutinosum* was conducted. From the chloroform extract, two partitions were obtained and analyzed. From the partition 1 (CHCl3 Fc1), the extraction and characterization of (−)-α-bisabolol as the main compound was conducted, with 97.5% of relative abundance. From partition 2 (MeOH Fc1), two groups of hydroxy-C18 and -C20 long-chain fatty acids are described for the first time in this plant. For the C20, the molecular ion at *m*/*z* 321.23 [M-H]−1, at least six peaks indicated the presence of isomers of the identified 20-hydroxyicosa-(5,8,11)-trienoic acid (49.7%). In the same regard, for the C18, the molecular ion at *m*/*z* 319.21 [M-H]−1, four peaks were assigned to 20-hydroxyicosa-(5,8,11,14)-tetraenoic acid derivatives (14.9%). On the other hand, under the basis of the UPLC-MS analysis, a set of methoxylated flavones are described. Two compounds with *m*/*z* 405.08, which consisted of the molecular formula C19H18O10 (5,7,2 ,4 -Tetrahydroxy-3,6,8,5 -tetramethoxyflavone and 5,7,4 ,5 -Tetrahydroxy-3,6,8,2 -tetramethoxyflavone) (8.1%). Two compounds with *m*/*z* 419.09 consisted of the molecular formula of C20H20O10 for two other methoxylated flavones (5,7,2 -Trihydroxy-3,6,8,4 ,5 -pentamethoxyflavone and 5,7,4 -Trihydroxy-3,6,8,2 ,5 -pentamethoxyflavone) (15.1%). Finally, there were another two compounds with *m*/*z* 433.11, assigned to the molecular formula C21H22O10 (5,7-Dihydroxy-3,6,8,3 ,4 ,5 -hexamethoxyflavone and 2-(5- Hydroxy-2,3-dimethoxyphenyl)-5-hydroxy-3,6,7,8-tetramethoxy-4H-1-benzopyran-4-one) (10.9%).

For those with a rapid, economical and efficient process of extraction, this extract could be eligible for further studies as ingredients for cosmetic purposes since they present a set of biocompounds with useful photoprotective, moisturizing, and skin-lightening properties.

**Author Contributions:** M.B.G.-P. and D.A.-B. conceived and designed the main ideas of this paper, carried out the GC-MS, UPLC-MS and DIESI-MS experiments, analyzed the experimental results, and wrote the paper. J.P.L.P. collected the plant samples and classified the plant. J.P.L.P., M.M.A.M. and I.A.-V. carried out the compounds extraction experiments and helped to discuss the results. I.A.-V. reviewed and edited the last version of the manuscript. The authors read and approved the final manuscript. Investigation, D.A.-B., J.P.L.P., M.M.A.M. and M.B.G.-P.; project administration, D.A.-B. and M.B.G.-P.; supervision, D.A.-B. and M.B.G.-P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Post-graduated Investigation Office of the National Polytechnic Institute (SIP-IPN, grants No. 20221534, 20232111, 20221454 and 20231544).

**Data Availability Statement:** All data is contained within the article.

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

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