**About the Editors**

#### **Antonella D'Anneo**

Antonella D'Anneo is an Associate Professor in Biochemistry at the Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo. She is also a Lecturer in Biochemistry for the Bachelor's degree in Biological Sciences, University of Palermo. She is a member of the Italian Society of Biochemistry and Molecular Biology (SIB). She is also the Associate Editor in Chief of *Cancer Management and Research* and Associate Editor for *Frontiers in Pharmacology*. She began her studies in the field of tumor biology and cell death mechanisms (apoptosis, necrosis, necroptosis, autophagy and anoikis), activated by natural and synthetic compounds, and biochemical pathways that can be activated in tumor cells. To complement her graduate training in biochemistry, she trained, as a visiting scholar, at Rangos Research Center (University of Pittsburgh, USA), taking part in projects concerning gene therapy approaches in order to determine the tolerization of diabetic patients against the cells of islet donors. More recently, her studies have focused on the identification of the nutraceutical properties (anti-tumor, anti-inflammatory, anti-obesity and antioxidant) of phytochemicals in order to disclose their biological activity and potential medicinal use. She is the author of 70 scientific publications in ISI-indexed journals.

#### **Marianna Lauricella**

Marianna Lauricella is an Associate Professor in Biochemistry at the Department of Biomedicine, Neuroscience and Advanced Diagnostics, School of Medicine, University of Palermo. She is a Lecturer in Biochemistry on the master's degree Course in Medicine, University of Palermo. She is a member of the Italian Society of Biochemistry and Molecular Biology (SIB). She is an Associate Editor of *BMC Cancer* and an Editorial Board Member of *The International Journal of Molecular Sciences*. Her research activity mainly concerns the study of the mechanisms governing the control of survival and death in cultured tumor cells. In particular, the aim of her research is to identify natural and synthetic compounds that can selectively induce cell death processes (apoptosis, autophagy, necroptosis). She also has expertise in the study of the role exerted by oxidative stress in inducing obesity and the anti-obesity effects of natural compounds in in vitro models of obesity. She is the author of over 75 scientific publications in ISI-indexed journals.

### *Editorial* **Advances in the Astonishing World of Phytochemicals: State-of-the-Art for Antioxidants**

**Marianna Lauricella 1,\* and Antonella D'Anneo 2,\***


In recent years, research on phytochemicals has underscored pleiotropic actions and medicinal and health-promoting properties which certainly deserve serious attention. Natural-derived molecules, such as phytohormones, glycosides, terpenoids, alkaloids, and phenolic compounds, offer a protective or preventative shield against many several pathological conditions such as aging, cardiovascular diseases, diabetes, obesity, cancer, asthma, and neurodegenerative disorders [1,2]. On the other hand, the multi-faceted potentials of phytocompounds isolated from different parts of plants or fruits stimulate the interest of the pharmaceutical, nutraceutical, and cosmetic industries. A main goal of these companies is to identify new and innovative phytomolecules to use as they are as natural reservoirs in plants or to appropriately modify them with the insertion of pharmacophore groups to design enhanced derivatives [3].

In the light of these considerations, we put together this Special Issue, titled "Advances in the Astonishing World of Phytochemicals: State-of-the-Art for Antioxidants", containing seventeen papers (fourteen research articles, one review, one comment, and a reply).

The scientific evidence reported in the SI analyzed both distribution and pleiotropic beneficial effects (antidiabetic, antitumor, antiflogistic, antibacterial, etc.) of some bioactive compounds with antioxidant properties. However, it also has to be considered that the relative abundance as well as the distribution of phytochemicals in plants or fruits is consistently affected by different parameters, such as environmental edaphic conditions, ripeness degree of fruits, and right season harvest [4].

In their contribution, Ali et al., for example, demonstrated that Australian fruits and spices such as mountain pepper berries (*Tasmannia lanceolata*), rosella (*Hibiscus sabdariffa*), lemon aspen (*Acronychia acidula*), and strawberry gum (*Eucalyptus olida*) represent a rich reservoir of bioactive phenolic metabolites (phenolic acids, flavonoids, isoflavonoids, tannins, stilbenes, lignans, and limonoids). Among these, the analysis provided evidence that both *Eucalyptus olida* and *Tasmannia lanceolata* possess the highest antioxidant and antidiabetic potential [5], a property that could be exploited in the development of specific biopharmaceuticals.

The effect of environmental conditions on the content and quality of phytochemicals was recently reported in studies performed on Amaranth, a leafy vegetable capable of growing under several salinity and drought-stress-induced conditions [6]. Salt stress has been demonstrated to enrich the amount of bioactive compounds with antioxidant properties. Indeed, the application of salt eustress conditions (25–100 mM NaCl) was able to boost the profile of microelements, macro-elements, phytochemicals, and phenolic acids in *Amaranthus gangeticus*, contributing to providing excellent quality in the end product for its antioxidant properties [7].

Studies performed on Romanian *Armoracia rusticana L*., a horseradish plant widely appreciated for its medicinal and aromatic properties, offered a complete profile of the

**Citation:** Lauricella, M.; D'Anneo, A. Advances in the Astonishing World of Phytochemicals: State-of-the-Art for Antioxidants. *Antioxidants* **2023**, *12*, 1581. https://doi.org/10.3390/ antiox12081581

Received: 19 July 2023 Revised: 28 July 2023 Accepted: 4 August 2023 Published: 8 August 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/).

low-molecular-weight metabolites of the plant grown in Romania. Nine categories of secondary metabolites (glucosilates, fatty acids, isothiocyanates, amino acids, phenolic acids, flavonoids, terpenoids, coumarins, and miscellaneous) were identified, and the development of phyto-engineered carrier systems capable of merging the biological properties of horseradish and kaolinite was proposed [8]. As a whole, the conclusion is that these systems could represent a possible controlled drug release system to apply to cancer-specific targeting.

In another study, Rani et al. explored the biological potential of dichloromethane and methanol root and shoot extracts of *Dryopteris juxtapostia*, a species belonging to the *Dryopteris* genus growing in the states of the north temperate zone. The study demonstrated that both extracts exerted radical-scavenging and anti-inflammatory and antitumoral effects in vitro as well as hepatoprotective actions in rats. *D. juxtapostia* root dichloromethane extracts exhibited the highest biological potential compared to other extracts, thus demonstrating the importance of using dichloromethane to obtain extracts enriched in phenolic components [9].

In addition, Vieira et al. demonstrated the anti-inflammatory effects of roots and flowers extracts of *E. purpurea*, a plant whose extracts are traditionally used to treat cold and flu. The study compared the effects of dichloromethanolic and ethanolic root and flowers extracts with alkylamide-rich extracts obtained by using the accelerated solvent extractor system, a green and innovative extraction technique. The authors concluded that all the extracts were capable of reducing the IL-6 levels as well as the intracellular levels of ROS/RNS in lipopolysaccharide-stimulated human-monocyte-derived macrophages. However, the alkylamide fractions possessed the strongest anti-inflammatory effects, thus evidencing these compounds as the main active extract components [10].

A fruit particularly rich in phytocompounds is tomato (*Lycopersicon esculentum* Mill.), a food largely consumed for its nutritive and nutraceutical properties [11]. Noteworthy, the different phytonutrient composition and antioxidant properties of the tomato are related to the different ripening times. On these bases, the study of Gambino et al. compared the different phytonutrients composition and properties of golden tomato (GT), a food product harvested at an incomplete ripening stage with respect to red tomato (RT), harvested at full maturation [12]. The authors demonstrated that GT contains a higher level of naringenin and chlorogenic acid, two polyphenols with antilipemic effects [13,14], than RT. Regarding biological activities, GT displays a better reducing power compared to RT [15]. Interestingly, GT oral supplementation in high-fed rats reduced body-weight gain and LDL cholesterol levels, as well as lowered oxidative stress markers both in the blood and liver, thus suggesting a potential of "GT" oral supplementation.

The biological properties of *Urtica dioica* (UD), *Matricaria chamomilla* (MC), and *Murraya koenigii* (MK), traditionally used in Ayurvedic medicine as nerve relaxants and cognition enhancers [16], were evaluated in the study of Shabir et al. [17]. Considering the effects of these plants on the nervous system, the authors investigated the ability of aqueous and ethanolic extracts of UD, MC, and MK to ameliorate the toxic effects of rotenone, a neurotoxic natural pesticide, in wild-type *Drosophila melanogaster*. The study evidenced the ability of plant extracts to exert neuroprotective effects on *Drosophila melanogaster* by alleviating rotenone-induced oxidative stress, enhancing locomotion, and restoring acetylcholine levels, thus suggesting a potential use of these extracts to treat neurological diseases. Of course, the right recovery of phytochemicals also depends either on the type of extraction techniques or solvents applied in the extraction procedure. This aspect was clearly addressed by Boyadzhieva et al., demonstrating a good recovery efficiency of phytochemicals from different parts (leaves, flowers, and stems) of *Gnaphalium viscosum* (Kunth, such as the antioxidants kaempferol, kaempferol-3-O-β-d-glucoside, and chlorogenic acid). Interestingly, for the first time, this study also demonstrated the presence in this species of leontopodic acids A and B, two highly potent antioxidants derived from glucaric acid [18].

In a study performed in yarrow (*Achillea millefolium L*.), a flowering plant commonly used in folk medicine to alleviate symptoms related to gastrointestinal discomfort [19], Villalva at al. used a supercritical antisolvent fractionation process [20] to obtain two different fractions containing polar phenolic compounds and monoterpenes and sesquiterpenes, respectively. Both the fractions explained the antibacterial effects observed against *Helicobacter pylori* strains. Furthermore, the extracted fractions exerted antioxidant and anti-inflammatory effects in *Helicobacter pylori*-infected human gastric AGS cells. From this study, we can conclude that yarrow extracts can be useful against *Helicobacter pylori* infection. The Villalva's data have been criticized by Franski and Beszterda-Buszczak [21]. Although they do not question the quality of the paper, these authors raised questions about the correctness of some compounds identified by Villalva et al. However, Villalva clarified all the doubts of Franski and Beszterda-Buszczak in a reply paper [22].

Nowadays, there is great interest in the bio-waste products of agriculture for the presence of bioactive healthy compounds [23,24]. Thinning young apples (TAPs) are usually discarded to guarantee the output and to increase the quality of harvested apples. However, it has been shown that TAPs contain more than 10-fold polyphenols with respect to harvested apples [25]. In their contribution, Ferrario et al. characterized the profile of polyphenols in TAP using a dual LC-HRMS metabolomic approach to identify a total of 68 polyphenols. According to this investigation, TAP fractions exert both antioxidant and anti-inflammatory effects by up-regulating the nuclear-factor-erythroid-2-related factor (Nrf2) signaling pathways and inhibiting NF-kB activation in cell models [26]. These results evidenced TAP as a source of bioactive molecules endowed with antioxidant properties.

The presence of bioactive compounds has also been Identified in marine environments. For example, seaweeds, such as red (*Rhodophyta*), green (*Chlorophyta*), and brown algae (*Phaeophyta*), which are not included in the diet of the Western world, are widely spread in Asian and Chinese nutrition for their high-quality profile in bioactive molecules as phenolic compounds, vitamins, pigments, and essential minerals. The use of a green pressured liquid extraction technique allowed Perez-Vazquez et al., under specific experimental conditions of temperature, type of used solvent, extraction time, and pressure, to recover a high yield of active biomolecules to exploit on both a pharmaceutical and food industrial scale [27].

Notably, a recent study of Liberti et al. demonstrated the antioxidant and antiinflammatory properties of sulfated exopolysaccharides (s-EPSs) and phycoerythrin (PE), two molecules naturally produced by the red marine microalga *Porphyridium cruentum*. In particular, s-EPSs were able to prevent GSH depletion and lipid peroxidation on a cell-based system but not in vitro, while PE showed high ROS scavenging capacity both in vitro and on a cell-based system. Interestingly, both the compounds were capable of inhibiting the pro-inflammatory enzyme COX-2 and promoting a fast scratch closure [28]. Altogether, the data obtained support the use of these compounds isolated by *P. cruentum* as anti-inflammatory components of medical patches.

The identification of phytomolecules with potential tailored applications represents a significant goal in the phytochemistry field. Particularly significant is the research discussed by Notaro et al. exploring the biochemical action of methyl gallate (MG), a gallotannin widely used in traditional Chinese phytotherapy to alleviate several cancer symptoms [29]. The findings reported by the authors shed light on the antitumor potential of MG. This phytocompound preferentially targeted HCT116 colon cancer cells, with respect to differentiated Caco-2 cells, an enterocyte-like cell model. In colon cancer cells, MG induced an oxidative injury sustained by ROS generation and endoplasmic reticulum stress as well as an upregulation in intracellular calcium content. In the first phase of treatment, oxidative events were accompanied by an autophagic process, that, for longer times of incubation, culminated in the apoptotic cell demise with DNA fragmentation and p53 and H2Ax activation. A particular role in the MG-induced mechanism was played by the oncosuppressor p53 protein. The conclusion of this research revealed the existence of an intertwined relationship between oxidative stress and p53 as a causative event in apoptotic cell death. Such a study paves the way to future investigations of MG alone or in combination treatment as a preventative or adjuvant phytocompound to apply in colon cancer treatment.

However, beyond these effects, bioactive compounds present in plants have also been demonstrated to play a protective role against oxidative injury, an aspect recently studied by Lv et al. in Caco-2 cells. The use of proanthocyanidins purified from kiwi leaves (*Actinidia chinensis*) counteracted both H2O2-induced oxidative damage as well as malondialdehyde increase [30]. Such an effect was accompanied with an upregulation of antioxidant systems (GSH-px, CAT, T-SOD) and the corresponding mRNA targets of Nrf2, the master regulator of the cellular stress response [31]. The conclusion of this interesting study is that the characterization of the antioxidant properties of kiwi leaves proanthocyanidins emphasizes their possible functional application either for a policy of circular economy or for sustainable industrial use.

The whole antioxidant activity of a sample cannot be ascribed only to a single bioactive component, but in many cases the overall potential is the result of the combinatorial effect of more components, acting in a synergistic, antagonistic, or additive manner. The comparative analysis of 10 phenolic acids (protocatechuic, gentisic, gallic, vanillic, syringic, p-coumaric, caffeic, ferulic, sinapic, and rosmarinic acid) used alone and in different combination mixtures provided evidence of the high antioxidant activity of gallic acid by a ferric reducing antioxidant power (FRAP) technique and a good oxygen radical absorbance capacity of rosmarinic acid by ORAC assays [32]. A relevant aspect of this study relied on the observation that hydroxybenzoic acid mixtures containing gentisic acid showed a clear synergistic action. These data strongly sustain the idea that the biological activity of a mixture, in some cases, cannot be ascribed to a single compound, but it has to be searched in the combination of compounds present and their ability to interact with each other.

We would like to share our gratitude to all authors who submitted their outstanding research to this Special Issue. Their manuscripts highlighted the role of natural-derived compounds with antioxidant potential action to apply as preventative or adjuvant molecules in the treatment of some chronic human diseases. Additionally, the identification of extraction techniques and solvents that can maximize the extraction of bioactive compounds is of great importance.

**Author Contributions:** Conceptualization, M.L. and A.D.; writing—original draft preparation, A.D. and M.L.; review and editing A.D and M.L. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** We thank all the authors who contributed to the research topic of this Special Issue and reviewers for the perceptive suggestions and comments. A particular acknowledgment is for all Editorial staff from *Antioxidants* journal for their huge support in the preparation of this Special Issue.

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

#### **References**


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### *Article* **Antioxidant, Alpha-Glucosidase Inhibition Activities, In Silico Molecular Docking and Pharmacokinetics Study of Phenolic Compounds from Native Australian Fruits and Spices**

**Akhtar Ali 1, Jeremy J. Cottrell <sup>1</sup> and Frank R. Dunshea 1,2,\***


**\*** Correspondence: fdunshea@unimelb.edu.au

**Abstract:** Native Australian fruits and spices are enriched with beneficial phytochemicals, especially phenolic compounds, which are not fully elucidated. Therefore, this study aimed to analyze native Australian mountain-pepper berries (*Tasmannia lanceolata*), rosella (*Hibiscus sabdariffa*), lemon aspen (*Acronychia acidula*), and strawberry gum (*Eucalyptus olida*) for phenolic and non-phenolic metabolites and their antioxidant and alpha-glucosidase inhibition activities. Liquid chromatography–mass spectrometry–electrospray ionization coupled with quadrupole time of flight (LC-ESI-QTOF-MS/MS) was applied to elucidate the composition, identities, and quantities of bioactive phenolic metabolites in Australian native commercial fruits and spices. This study identified 143 phenolic compounds, including 31 phenolic acids, 70 flavonoids, 10 isoflavonoids, 7 tannins, 3 stilbenes, 7 lignans, 10 other compounds, and 5 limonoids. Strawberry gum was found to have the highest total phenolic content (TPC—36.57 ± 1.34 milligram gallic acid equivalent per gram (mg GAE/g), whereas lemon aspen contained the least TPC (4.40 ± 0.38 mg GAE/g). Moreover, strawberry gum and mountain pepper berries were found to have the highest antioxidant and anti-diabetic potential. In silico molecular docking and pharmacokinetics screening were also conducted to predict the potential of the most abundant phenolic compounds in these selected plants. A positive correlation was observed between phenolic contents and biological activities. This study will encourage further research to identify the nutraceutical and phytopharmaceutical potential of these native Australian fruits.

**Keywords:** mountain pepper; rosella; strawberry gum; lemon aspen; flavonoids; anthocyanins; bioavailability; LC-MS/MS

#### **1. Introduction**

Diabetes mellitus is one of the leading causes of death around the globe [1] and is characterized by high blood glucose levels. Alpha-glucosidase (α-glucosidase) is the main enzyme with a significant role in hydroxylation, digestion, and absorption of sugars in the human body. Therefore, inhibiting α-glucosidase is an effective strategy for treating and minimizing type 2 diabetes. There is increasing interest in using natural sources to treat diabetes. Various nutraceuticals and bioactive compounds have been investigated to control/inhibit the complications of diabetes. Using phenolic metabolites is a therapeutic approach to suppressing the prevalence of pre- or post-diabetic conditions [1]. Therefore, detailed characterization and identification of phenolic metabolites is required to understand the potent role of polyphenols in food and human health.

Fruits, vegetables, herbs, spices, and medicinal plants contain large amounts of phytochemicals, including polyphenols. When they encounter living tissues, they exhibit a beneficial effect on human health [1]. Flavonoids are the largest subclass of polyphenols, with more than ten thousand compounds being reported [2]. According to nutritionists, foods high in polyphenols may reduce or remove the risk for certain malignancies, degenerative diseases, cardiovascular ailments, and chronic inflammation in humans [3].

**Citation:** Ali, A.; Cottrell, J.J.; Dunshea, F.R. Antioxidant, Alpha-Glucosidase Inhibition Activities, In Silico Molecular Docking and Pharmacokinetics Study of Phenolic Compounds from Native Australian Fruits and Spices. *Antioxidants* **2023**, *12*, 254. https:// doi.org/10.3390/antiox12020254

Academic Editors: Antonella D'Anneo and Marianna Lauricella

Received: 22 December 2022 Revised: 18 January 2023 Accepted: 19 January 2023 Published: 23 January 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/).

Excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) leads to oxidative stress in the body, the leading cause of the above-mentioned pathological conditions [4]. Tasmannia lanceolata, commonly known as the mountain pepper berry, is used in food flavoring and traditional medicine to treat venereal diseases, skin disorders, and stomachaches [5]. Lemon aspen is a pale/yellow, 2–2.5 cm in diameter fruit that is endemic to Queensland, Australia. It is traditionally used in curries and on meats as a seasoning, though syrups, sauces, and infused vinegar are also made from lemon aspen [6]. Strawberry gum and rosella are also used in traditional medicine.

Considering that bioactive phenolic compounds have strong antioxidant and antidiabetic potential, it was hypothesized that selected native Australian plants could have considerable bioactive potential. In this context, we comprehensively characterized native Australian mountain pepper berries, rosella, lemon aspen, and strawberry gum for their phenolic compounds' antioxidant and α-glucosidase inhibition potential. Previously, these native Australian plants had not been studied for radical scavenging and α-glucosidase inhibition activities. Therefore, total monomeric anthocyanin content (TMAC), total phenolic content (TPC), total condensed tannins (TCT), and total flavonoid content (TFC) were measured. Furthermore, antioxidant activities, through the ferrous ion chelating assay (FICA), ferric reducing antioxidant power (FRAP) test, 2,2- -diphenyl-1-picrylhydrazyl (DPPH) reducing power assay (RPA), 2,2- -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay, phosphomolybdate assay (PMA), and hydroxyl-radical scavenging assay ( •OH-RSA), were also measured in this study. The anti-diabetic potential of these selected native Australian plants was also measured through the α-glucosidase inhibition activity. Moreover, LC-ESI-qTOF-MS/MS was used to characterize and screen polyphenols from these native Australian plants. Furthermore, the binding affinities of most abundant phenolic metabolites in selected native Australian plants for the active sites of a-glucosidase (5NN8) were predicted using the in silico molecular docking. Nowadays, in silico molecular docking is widely used in drug discovery, which enables us to understand the behavior of drug molecules in the binding sites of the α-glucosidase protein and explains the basic biochemical processes [7–9]. Moreover, oral bioavailability, drug-likeness, absorption, distribution, metabolism, excretion, and toxicity of abundant phenolic compounds were computed to predict their suitability as therapeutic agents. This research explores the use of native Australian plants in the medicinal, pharmaceutical, food, and feed industries.

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

#### *2.1. Chemicals and Reagents*

Analytical, HPLC, and LCMS-grade chemicals were used as described [1,10].

#### *2.2. Preparation and Extraction of Phenolic Compounds*

Mountain pepper berries (whole dried), rosella (freeze-dried powder), and strawberry gum (finely ground) were purchased from the Australian superfoods Company (www.australiansuperco.com.au) accessed on 21 September 2021. Lemon aspen (freeze-dried powder) was purchased from Australian Creative Native Foods (www.creativenativefoods.com. au) accessed on 21 September 2021. The bioactive phenolic compounds from the selected native Australian fruits were extracted in triplicate by following the method of Ali et al. [1]. The extracts were stored at −20 ◦C, and all the analyses were conducted within a week.

#### *2.3. Measurement of Phenolic Contents and Biological Activities*

The TPC of selected native Australian plants was examined by following the method of Ali et al. [1]. A 25 μL sample extract or standard, 25 μL Folin-Ciocalteu reagent (25% in Milli-Q water) and 200 μL of H2O were mixed in a 96-well plate and incubated for 5 min at room temperature. Then, 25 μL of 10% sodium carbonate was mixed and again incubated for 60 min at room temperature in the dark. Gallic acid monohydrate (≥99%) in analytical grade ethanol (0–200 μg/mL) was used to generate standard curve at 765 nm. Then, the method of Sharifi-Rad et al. [11] was used to quantify the TFC of native Australian fruits and spices. The TCT and TMAC of selected plants were determined using the procedures of Ali et al. [1,10]. The DPPH and ABTS activities were measured using the methods of Chou et al. [12] and Zahid et al. [13]. The PMA, RPA and FRAP potential of these selected plants were quantified by adopting the methods of Ali et al. [10]. The FICA and the •OH-RSA potential of selected plants were quantified by adopting the methods of Bashmil et al. [14] and Ali et al. [10]. α-Glucosidase inhibition activity was determined by following our previously published method [1], and acarbose (Aca) was used as a reference drug (≥95%).

#### *2.4. LC-MS/MS Analysis*

LC-ESI-Q-TOF-MS/MS was used to analyze the untargeted phenolic metabolites from native Australian mountain pepper berries, rosella, strawberry gum, and lemon aspen by following the methods of Ali at al. [1,15]. The heatmap hierarchical clustering was conducted by using MetaboAnalyst 5.0 (www.metaboanalyst.ca) accessed on 7 November 2022.

#### *2.5. Molecular Docking and Pharmacokinetic Properties of Abundant Phenolic Compounds*

The pharmacokinetic properties of the most abundant phenolic compounds tentatively identified in the plants were predicted by following the methods of Ali et al. [16] and Daina et al. [17]. Oral bioavailability, absorption, distribution, metabolism, excretion, and toxicity of the abundant phenolic compounds were predicted. Moreover, in silico molecular docking was also conducted to predict the α-glucosidase potential of the selected phenolic compounds from native Australian fruits and spices, as described by Ali et al. [1]. Grid box dimensions were x = −12.95, y = −36.99, and y = 87.77 while docking ligands with a length lower than 20 Å.

#### *2.6. Statistical Analysis*

Minitab (version 18.0, Minitab, LLC, State College, PA, USA) and XLSTAT-2019.1.3 software were used for and analysis of variance (ANOVA), Pearson correlation, and a biplot analysis. The results of phenolic contents and their biological activities are represented as mean ± standard deviation.

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

#### *3.1. Measurement of Total Polyphenols (TPC, TFC, TMAC, TCT)*

Phytochemicals, especially plants' secondary metabolites, are vital for human health [18]. Phenolic acids and flavonoids are critical secondary bioactive metabolites with various health benefits. They are considered multi-functional metabolites, as metal chelators, hydrogen atom donators, free radical scavengers, and reducing agents [18].

In this study, we investigated Australian mountain pepper berries, rosella, lemon aspen, and strawberry gum for phenolic and non-phenolic compounds. TPC, TFC, TCT, and TMAC quantified in these native Australian plants are given in Table 1.

Total phenols represent phenolic acids, flavonoids, stilbenes, lignans, coumarins and derivatives, tyrosols, and other small molecules. In this context, strawberry gum was found to have the highest TPC (36.57 ± 1.34 mg GAE/g) of the selected Australian native plants. The TPC of strawberry gum was comparable to the previously quantified TPC of Australiangrown thyme (43.16 ± 1.54 mg GAE/g), basil (39.91 ± 1.39 mg GAE/g), and allspice (40.49 ± 1.92 mg GAE/g) [10,18]. Previously, the levels of phenolic compounds in Australian native lemon myrtle and Tasmanian pepper berry were found to be in the range of 16.9 to 31.4 mg GAE/g [19]. Moreover, the TPC of mountain pepper berries was comparable to the TPC reported by Cáceres-Vélez et al. [20] and Vélez et al. [21]. The concentrations of phenolic contents in mountain pepper berry, rosella, and lemon aspen are 2 to 3-fold higher than in Australian-grown cherries [22]. Previously, Lukmanto et al. [23] also measured the TPC of 8.63 mg GAE/g, which is comparatively higher than our results. The TPC of strawberry

gum is also comparable to that of villous amomum fruit (46.02 ± 1.12 mg GAE/g) and that of citron fruit (46.22 ± 1.01 mg GAE/g) reported by Liu et al. [24].


**Table 1.** Quantification of phenolic contents in Australian native fruits and spices.

Total phenolic content (TPC), total flavonoid content (TFC), total condensed tannins (TCT), total monomeric anthocyanin content (TMAC), cyanidin 3-glucoside equivalent (C3GE), gallic acid equivalent (GAE), quercetin equivalent (QE), catechin equivalent (CE). Values are presented as mean ± standard deviation (*n* = 3) per gram of dry weight. Values within the same column with different superscripts (a–d) are significantly different.

The highest TFC (15.69 ± 2.69 mg QE/g) was quantified in strawberry gum, and the lowest TFC (0.79 ± 0.04 mg QE/g) was quantified in lemon aspen. The highest TCT (8.05 ± 0.52 mg CE/g) was also measured in strawberry gum, and the lowest TCT (1.26 ± 1.13 mg CE/g) was measured in rosella. The TMAC was only measured in mountain pepper berry (0.17 ± 0.03 mg/g) and rosella (0.08 ± 0.02 mg/g). Previously, we measured higher amounts of total anthocyanins in the Davidson plum and quandong peach than berries [1]. Flavonoids are the most abundant class of phenolic compounds in fruits, herbs, and medicinal plants, and they have gained much interest due to their health properties. Previously, a limited number of studies have been conducted to investigate the total flavonoid contents in these plants. There are significant differences in total phenolics, and flavonoids found in each study conducted on these plants due to the aforementioned factors.

#### *3.2. Biological Activities of Native Australian Fruits and Spices*

Phenolic compounds include diverse antioxidant constituents present in plants that have various health effects. According to several studies, certain plants' antioxidant properties vary due to their diverse bioactive components and mostly depend on the extraction technique and method used to quantify them. Numerous studies have been carried out to estimate the antioxidant activities of plants from different geographical locations [25–30], but the information on native Australian plants is limited. Therefore, we conducted various antioxidant activity tests to understand the targeted antioxidant potential of native Australian fruits. Various antioxidant activity tests should help in understanding the potential of these native Australian herbs and medicinal plants.

In this study, seven in vitro antioxidant assays (DPPH, ABTS, FRAP, RPA, •OH-RSA, FICA, and PMA) were conducted, and α-glucosidase inhibition activity was tested, to measure the antioxidant and anti-diabetic potential of native Australian native mountain pepper berries, rosella, strawberry gum, and lemon aspen (Table S1, Figure 1).

DPPH and ABTS are the widely used in vitro antioxidant assays for total antioxidant potential measurement of plant extracts based on scavenging the free radicals in the biological system. The highest DPPH (49.70 ± 3.21 mg AAE/g) was measured in strawberry gum, and the lowest DPPH (24.94 ± 0.70 mg AAE/g) was quantified in sea lemon aspen. ABTS+ radical cation inhibition is based on the characteristic wavelength of 734 nm [31]. The ABTS values of strawberry gum (87.65 ± 3.17 mg AAE/g) and mountain pepper berries (85.60 ± 2.32 mg AAE/g) were higher than those of rosella (59.27 ± 1.50 mg AAE/g) and lemon aspen (46.18 ± 0.38 mg AAE/g). Some other studies also reported higher ABTS values for rosemary, oregano, and mint [18]. This indicates that strawberry gum has a higher antioxidant potential than mountain pepper berries, rosella, and lemon aspen. The Fe+3–TPTZ complex, which reduces the antioxidant compounds' ability to form an Fe+2–TPTZ complex

in the biological system, was evaluated through the FRAP assay [10]. The FRAP values of strawberry gum (26.57 ± 3.10 mg AAE/g) and rosella (14.30 ± 1.92 mg AAE/g) were higher than those of the other selected fruits and spices (*p* < 0.05). Previously, the highest FRAP values were found in rosemary (17.21 ± 0.54 mg AAE/g) and oregano (10.72 ± 1.44 mg AAE/g). Fenugreek was found to have the lowest value of FRAP (1.48 ± 1.21 mg AAE/g). Furthermore, Wojdyło et al. [32] also reported higher FRAP for rosemary than the other plants selected in our study.

**Figure 1.** Biological activities (2,2- -diphenyl-1-picrylhydrazyl (DPPH), 2,2- -azinobis-(3 ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric reducing antioxidant power (FRAP), reducing power assay (RPA), hydroxyl-radical scavenging assay (•OH-RSA), ferrous ion chelating assay (FICA), phosphomolybdate assay (PMA)) and α-glucosidase inhibition activity of native Australian rosella (R), mountain pepper berries (MPB), lemon aspen (LA), and strawberry gum (SBG); acarbose (Aca). The vales with letters (a–e) are significantly different from each other.

Excessive amounts of different reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxy radicals (•OH), and super-oxide radicals (O2 •), cause various pathologies. The •OH radicals cause lipid peroxidation and DNA damage due to high oxidative stress, and the daily consumption of antioxidant-rich fruits is crucial in order to protect the human body from these pathologies [18]. The highest •OH-RSA value (93.29 ± 2.20 mg AAE/g) was achieved by strawberry gum. This is vital because •OH scavenging inhibits lipid peroxidation by inhibiting the transition of oxidized metal ions [33,34]. The metal chelating ability of native Australian fruits and spices was estimated by using the ferrous ion chelating assay (FICA), and the highest FICA result (9.05 ± 0.27 mg EDTA/g) was achieved by strawberry gum. The principal antioxidant ingredients are flavonoids, according to a significant association between the antioxidant properties and flavonoids. These fruits and spices can contain different reducing agents which can bind with free radicals to terminate or stabilize the chain reactions in the

biological systems [35]. Thus, high reduction power for a fruit extract indicates high antioxidant capacity. Free radicals can be produced by metabolic processes within bodily tissues and brought from outside sources such as food, medications, and pollution. Natural antioxidants are increasingly being used as food additives to neutralize free radicals. This is due to their scavenging abilities and the fact that they are all-natural, non-synthetic items that are well-liked by consumers. Furthermore, α-glucosidase inhibition activity of strawberry gum (81.01 ± 4.6 %) and mountain pepper berries (65.78 ± 5.01 %) was quantified higher than acarbose (standard), rosella, and lemon aspen. Previously, Syabana et al. [36] quantified the IC50 of *Cosmos caudatus* (61.33 ± 1.21 μg/mL), *Etlingera elaitor* (53.13 ± 2.87 μg/mL), *Pluchea indica* (12.17 ± 0.18 μg/mL), and *Syzygium polyanthum* (11.76 ± 0.32 μg/mL). The inhibitory activity of strawberry gum (12.01 ± 1.2 μg/mL) was higher than the activity of *Cosmos caudatus* and *Etlingera elaitor* and comparable to the activity of *Pluchea indica* and *Syzygium polyanthum* (Table S1). The inhibitory activity levels of rosella (79.09 ± 7.52 μg/mL) and lemon aspen (83.07 ± 9.03 μg/mL) were lower than those of these plants (Table S1). Moreover, the inhibitory activity of strawberry gum was comparable to that of the methanolic extract of *Satureja cuneifolia* (10.66 μg/mL) reported by Taslimi et al. [37].

Fruits, herbs, spices, and medicinal plants are used as antioxidant sources in the human diet because they inhibit or deactivate the free radicals in the body [38]. Generally, phenolic molecules are regarded as the active antioxidant components in fruits, herbs, and medicinal plants, thereby having potent health benefits. They act as metal chelators, anti-radicals, hydrogen-ion donators, and reducing agents in the biological system [10]. It has been reported that there are many methods to measure a plant extract's total antioxidant potential due to the diverse nature of antioxidant compounds, mainly phenolic constituents [1,18]. The plant's bioactive compounds, mainly polyphenols, depend on the type of cultivar, area, and climatic conditions. There are several techniques to assess the antioxidant capacities of bioactive phenolic metabolites, each with its advantages and disadvantages [11,14,19]. Generally, no approach measures the exact antioxidant capacity of bioactive phenolic compounds because of the complexity of phenolic compounds and the variety of processes of reactions in the human body [39]. These results demonstrate that further research is required to identify and quantify the individual phenolic compounds in these selected native Australian plants. Thus, LC-MS/MS was used to elucidate plant extracts' structure, composition, and bioactive metabolites. The proper quantification and identification of individual phenolic compounds via the process of LC-MS/MS in these plants might help make the essential role of these bioactive metabolites in antioxidant activities understandable.

#### *3.3. Correlation Analysis*

Correlation analysis was executed between the phenolic contents (TPC and TFC) of the Australian native herbs and their antioxidant activities generated by the eight different assays (Table 2).


**Table 2.** Pearson correlation between phenolic contents and biological activities.

\* = α-glucosidase inhibition activity (%), values in bold are different from 0 with a significance level alpha = 0.1.

It is observed that a positive correlation (*p* ≤ 0.1) of TPC was observed with the TFC (*r* = 1.00), TCT (*r* = 0.99), FRAP (*r* = 0.92), •OH-RSA (*r* = 0.97), and RPA (*r* = 0.99); the TFC had a significant positive correlation with TCT (*r* = 0.99), DPPH (*r* = 0.98), FRAP (*r* = 0.92), •OH-RSA (*r* = 0.97), and RPA (*r* = 0.99). This appears to show a direct association between the phenolic compounds in the strawberry gum and the antioxidant processes of peroxyl inhibition, ferric chelation, and free radical scavenging. There was a strong correlation of flavonoids with hydroxyl inhibition, but there were lesser ones with the free radical scavenging, the phosphomolybdate assay outcome, and ferric ion chelation activity. This also indicates the diversity of phenolic and non-phenolic metabolites present in the extracts of native Australian plants. This may be connected to the fact that the flavonoid's ability to operate as an antioxidant often depends on where the hydroxyl group is located on the B-ring and whether it can provide a free radical, either a hydrogen or an electron [1]. Additionally, the experimental conditions, the mechanisms of the antioxidant reactions, and the synergistic or antagonistic effects of various compounds present in the reaction mixture can all impact the associations between antioxidant activity and phenolic compounds [15,18].

A biplot (Figure 2) exhibits that the higher TPC, TFC, and TCT in strawberry gum significantly contributed to all antioxidant activities except that shown by the phosphomolybdate assay. Furthermore, it is depicted that mountain pepper berries have higher concentrations of total monomeric anthocyanin than other plants. Interestingly, rosella and lemon aspen are negatively correlated with all biological activities, which indicates that these have low concentrations of phenolic compounds and flavonoids. Previously, it has been demonstrated that a greater number of OH groups in a flavonoid is favorable for biological activities. Furthermore, each ring's structural arrangement its number of hydroxyl groups, a catechol group in the B ring, and several double bonds in the C ring determine the antioxidant capacity of phenolic metabolites in extracts [40]. Numerous investigations of herbs and medicinal plants have shown a significant, positive association between phenolic content and antioxidant activity [41]. Previously, we reported a positive correlation between phenolic contents of herbs and spices and their antioxidant activities [10,18]. Additionally, two other studies showed that phenolic contents in native Australian fruits and other plants also had positive relationships with their biological activities [1,15].

#### *3.4. LC-MS Analysis*

Nutritionists and food scientists have concentrated on exploring the thorough characterization of fruits, spices, and medicinal plants in response to the growing interest in and understanding the antioxidant potential and associated health benefits of phenolic chemicals. The untargeted characterization and screening of phenolic compounds from Australian native fruits and medicinal plants (mountain pepper berries, strawberry gum, rosella, and lemon aspen) were achieved using LC-ESI-QTOF-MS/MS. In this context, a total of 143 phenolic and non-phenolic metabolites, including 31 phenolic acids, 70 flavonoids, 10 isoflavonoids, 7 tannins, 3 stilbenes, 7 lignans, 10 other compounds, and 5 limonoids, were tentatively characterized through the analysis of their MS/MS spectra (Table 3, Figures S1 and S2).

#### 3.4.1. Phenolic Acids

Phenolic acids are diverse plant metabolites from the secondary class produced via the phenylpropanoid pathway by shikimic acid [42]. They are broadly utilized in beauty, health, pharmacology, and medicinal industries due to their anti-aging, antioxidant, antimicrobial, anti-cancer, cardio-protective, antitumor, and anti-inflammatory properties [43].

**Figure 2.** Biplot analysis of phenolic contents and biological activities in native Australian lemon aspen (**LA**), rosella (R), strawberry gum (**SBG**), and mountain pepper berries (**MPB**).

Benzoic acid and Its Derivatives

Benzoic acid is the simplest aromatic carboxylic acid and has a range of derivatives. Eight hydroxybenzoic acids in total were tentatively identified in these native Australian plants. Compounds **1**, **3**, and **8** produced fragment ions at *m*/*z* 169, 153, and 137 after the loss of the glycosyl moiety [M−H−162]<sup>−</sup> from the precursor ions, respectively. Compounds **1**, **3**, and **8** were tentatively identified as gallic acid 4-*O*-glucoside, protocatechuic acid 4-*O*-glucoside, and 4-hydroxybenzoic acid 4-*O*-glucoside, respectively. Compounds **2**, **4**, and **5** generated product ions at *m/z* 125, 109, and 93 after the loss of CO2 (44 Da) from the parent ions, respectively [10]. Compounds **2**, **4**, and **5** were identified through pure standards as gallic acid, protocatechuic acid, and *p*-hydroxybenzoic acid. Compound **2** (gallic acid—C7H6O5) and compound **4** (protocatechuic acid—C7H6O4) were identified in mountain pepper berries and strawberry gum. Gallic acid 4-*O*-glucoside was only identified in mountain pepper berries, whereas benzoic acid (compound 4) was identified in all four fruits. The presence of the unique phenolic acid protocatechuic acid has been reported in many therapeutic plants [44]. It has said to possess several health benefits, including anti-inflammatory, antioxidant, anti-cancer, anti-ulcer, anti-diabetic, hepato-protective, and neuro-protective activities [44].


**Table**


**Table 3.** *Cont.* 


#### *Antioxidants* **2023**, *12*, 254

**Table 3.** *Cont.*

17


**Table 3.** *Cont.*


**Table 3.** *Cont.* Cinnamic Acids and Derivatives

The most prevalent phenolic acid class is hydroxycinnamic in fruits, herbs, and medicinal plants. Sixteen hydroxycinnamic acids were identified, and their fragmentation patterns were verified using MS/MS. The removal of CO2 and the hexosyl moiety from the parent ions is the primary way that phenolic acids exhibit the fragmentation pattern [10]. Rosmarinic acid, caffeic acid, sinapic acid, *p*-coumaric acid, 3-caffeoylquinic acid (chlorogenic acid), and cinnamic acid were confirmed through pure standards. The quinic acid derivatives **9** (*m*/*z* 349.0921), **20** (*m*/*z* 337.0934) **21** (*m*/*z* 367.1025), **22** (*m*/*z* 397.1141), and **29** (*m*/*z* 515.1197) are known as -feruloyquinic acid lactone, 3-*p*-coumaroylquinic acid, 3-feruloylquinic acid, 3-sinapoylquinic acid, and 1,5-dicaffeoylquinic acid, respectively. Compound 21 (3-feruloylquinic acid) was tentatively identified in rosella and lemon aspen at *m*/*z* 367.1029, which generated characteristic fragment ions of ferulic acid and quinic acid at *m*/*z* 193 and 191 in negative-ion mode. Compound **29** (1,5-dicaffeoylquinic acid) was detected in rosella and strawberry gum in negative mode. This was confirmed through MS/MS, where it produced fragment ions at *m*/*z* 353, 191, and 179 after the breakdown of precursor ions into 5-caffeoylquinic acid (*m*/*z* 353), quinic acid (*m*/*z* 191), and caffeic acid (*m*/*z* 179) units, respectively [10]. Previously, 1,5-dicaffeoylquinic acid was also identified in cumin [10]. Compound **10** at ESI− *m*/*z* 279.0503 was identified in lemon aspen and strawberry gum, which generated a product ion at *m*/*z* 163 (coumaric acid) after the loss of C4H4O4 (116 Da) from the precursor ion in the MS/MS scan. Therefore, compound **10** was putatively identified as *p*-coumaroyl malic acid. Rosmarinic acid produced a characteristic fragment at *m*/*z* 197 after the removal of a hexose moiety (162 Da), which further broke down into a caffeic acid unit (*m*/*z* 179) through the loss of H2O, and a caffeic acid (*m*/*z* 179) fragment at *m*/*z* 135 represents the loss of a CO2 [M−H−44]<sup>−</sup> unit [45,46]. Rosmarinic acid (compound **14**) is one of herbs' and medicinal plants' most plentiful phenolic acids. Compound **24** at ESI− *m*/*z* 163.0400 was identified in mountain pepper berries, rosella, and lemon aspen, which generated a product ion at *m*/*z* 119 after the loss of CO2 [M−H−44]<sup>−</sup> from the precursor ion (*m*/*z* 163.0400). Compound **24** was tentatively identified as *p*-coumaric acid. Compound **31** at ESI+ *m*/*z* 223.0605 generated product ions at *m*/*z* 205 and 147, and 119, after the loss of a unit of H2O (18 Da), the glycolic acid moiety (76 Da), and both from the precursor ion, respectively. In contrast, a product ion at *m*/*z* 119 is a specific fragment ion of *p*-coumaric acid. As a result, compound **31** was tentatively identified as *p*-coumaroyl glycolic acid in rosella and mountain pepper berries. Previously, Kadam et al. [47] also reported *p*-coumaroyl glycolic in *Lepidium sativum* seedcake.

#### 3.4.2. Flavonoids

Flavonoids are widely used in nutraceutical, pharmaceutical, and cosmetic industries due to their anti-carcinogenic, antimicrobial, anti-inflammatory, anti-mutagenic, and antioxidant properties. In this study, we tentatively identified seventy flavonoids (Table 2).

#### Anthocyanins

Anthocyanins are water-soluble, colored plant pigments. The main positions of their hydroxyls are 3, 5, and 7 in ring A and 3 and 5 in ring B [48]. The screening, identification, and characterization of anthocyanins in native Australian rosella and mountain pepper berries were conducted. This work identified nine anthocyanins using their MS/MS spectra (Table 2). The native Australian quandong peach and Davidson plum were used as reference plants to understand anthocyanins' structural and spectral characteristics further; these fruits are abundant in anthocyanins [1]. The removal of sugar units from anthocyanins (162 Da for hexoses, 150 Da for xyloses, 132 Da for pentoses, and 308 Da for the rutinoside moiety from the basic aglycone of corresponding anthocyanins) results in the formation of MS/MS product ions (303 Da for delphinidin, 331 Da for malvidin, 301 Da for peonidin, 317 Da for petunidin, and 287 Da for cyanidin) [1]. Compounds **33**, 35, and **36** at ESI<sup>+</sup> *m*/*z* 581.1526, 595.1660, and 449.0994 generated a characteristic fragment ion at *m*/*z* 287 (cyanidin). Thus, compounds **33**, **35**, and **36** were tentatively identified as

cyanidin 3-sambubioside, cyanidin 3-rutinoside, and cyanidin-3-O-glucoside, respectively. Compound **36** (cyanidin 3-*O*-glucoside) was identified in mountain pepper berries and rosella. Cyanidin-3-*O*-glucoside was quantified in grapes from 2.7 to 51.7 μg/mL by Oh et al. [49]. Compounds **32**, **38**, **39**, **40**, and **41** produced a distinctive fragment of delphinidin at *m*/*z* 303 in positive-ion mode (Table 2). Compounds **33**, **38**, and **39** were only identified in rosella. Due to their positively charged oxygen atom, anthocyanins have higher antioxidant activity than other flavonoids [50].

#### Flavanols

We identified monomeric flavanols in our samples, including epicatechin, epigallocatechin, and derivatives [48]. In this study, seven flavanols, including polymerized and derivative substances, were tentatively identified in mountain pepper berries, rosella, strawberry gum, and lemon aspen. Flavanols are also called catechins, having no double bond between C2 and C3, and there is no carbonyl group in ring C (C4) [51]. Compound **44** at ESI− *m*/*z* 305.0650 generated product ions at *m*/*z* 289, 169, and 125 from the ion precursor. Compound **44** was putatively identified as (−)-epigallocatechin (C15H14O7). They have been reported abundantly for their potent antioxidant and cardio-protective effects in tea and cocoa. Compound **46** at ESI− *m*/*z* 289.0711 was identified in strawberry gum, rosella, and mountain pepper berries, which produced product ions at *m*/*z* 245, 205, and 179 after CO2 loss [M−H−44]−, flavonoid A ring [M−H−84]<sup>−</sup> loss, and flavonoid B ring [M−H−110]<sup>−</sup> loss from the precursor ion, respectively. Compound **46** was tentatively identified as epicatechin (C15H14O6) [52]. These compounds are the building blocks of proanthocyanidins (condensed tannins). The most prevalent flavonoids: flavanols, and flavan-3-ols have a variety of chemical and biological properties.

#### Flavanones

Flavanones do not have double bond between C2 and C3, but they have a carbonyl ring at C4 in ring C [48]. Sixteen compounds were identified as flavanones. Compounds **49** (naringin 6- -malonate), **54** (6-geranylnaringenin), **56** (eriodictyol-7-*O*-glucoside), **59** (eriodictyol), **60** (naringenin), **62** (5,7-dihydroxyflavanone), **63** (Hesperidin), and 64 (3- ,4- ,5- -trimethoxyflavone) were only identified in strawberry gum; and compounds **52** (hesperetin 5-glucoside) and **58** (hesperetin 5- ,7-*O*-diglucuronide) were only identified in lemon aspen. Compound **61** (8-Prenylnaringenin) was only identified in mountain pepper berries. Compounds **51**, **52**, **55**, and **56** generated product ions at *m*/*z* 579, 301, 271, and 287 after the loss of a glycosyl moiety from their precursor ions, respectively. Therefore, compounds **51**, **52**, **55**, and **56** were tentatively identified as narirutin 4- -*O*-glucoside, hesperetin 5-glucoside, naringenin-7-O-glucoside, and eriodictyol-7-*O*-glucoside, respectively.

#### Flavones and Isoflavones

Flavones are characterized by a non-saturated C3 chain and have a double bond between C2 and C3 [48]. Sixteen compounds were characterized as flavones and flavanones in mountain pepper berries, rosella, strawberry gum, and lemon aspen. Compounds **67** (velutin), **72** (biochanin A 7-*O*-glucoside), and **79** (chrysin) were only identified in strawberry gum; and compounds **69** (azaleatin 3-arabinoside), **73** (Apigenin 6-C-glucoside), **75** (apigenin), **75** (chrysoeriol 7-*O*-glucoside), **77** (wogonin), and **78** (glycitein) were only identified in mountain pepper berries. Compounds **68** (diosmin), **71** (luteolin), **74** (apigenin), **76** (diosmetin), and **79** (chrysin) were identified via the MS/MS spectra of pure standards. Compounds **70** (syringetin-3-*O*-glucoside), **72** (biochanin A 7-*O*glucoside), **73** (apigenin 6-C-glucoside), and **75** (chrysoeriol 7-*O*-glucoside) generated product ions at *m*/*z* 299, 345, 271, and 299, respectively, after the loss of glycosyl moiety from their parent ions.

Flavonols, Dihydroflavonols, and Chalcones

Flavonols have a double bond between C2 and C3, and there is a carbonyl in ring C (C4) and a OH group at C3 [51]. These compounds have strong absorption at 340–380 nm. Eighteen compounds were identified as flavonols and dihydroflavonols. Compound **81** (limocitrin) was only identified in lemon aspen, and compounds **82** (myricetin 3-*O*-glucoside) and **97** (isorhamnetin 3-*O*-glucuronide) were only identified in rosella. Compounds **82** (*m*/*z* 479.0816), **92** (*m*/*z* 463.0842), and **94** (Kaempferol 3-*O*-glucoside) generated product ions at *m*/*z* **317** (myricetin), 301 (quercetin), and 285 (kaempferol) after the loss of a hexose moiety (162 Da) from the precursor ions, respectively. Compounds **82**, **92**, and **94** were putatively identified as myricetin 3-*O*-glucoside, quercetin-3-*O*-glucoside, and kaempferol 3-*O*-glucoside, respectively. Moreover, compounds **85**, **86**, **87**, **88**, **89**, **90**, **and 97** produced fragment ions at *m*/*z* 317 (myricetin), 285 (kaempferol), 301 (quercetin), 303 (dihydroquercetin), and 315 (isorhamnetin) after the loss of sugar moieties, including rhamnoside (146 Da), rutinoside (308 Da), arabinoside (132 Da), and glucuronide (176 Da), from their precursor ions. Compounds **83** (quercetin 3-(2-galloylglucoside) and **88** (quercitrin) were only identified in lemon aspen while compounds **80** (6-hydroxykaempferol 3,6-diglucoside 7-glucuronide), **86** (kaempferol 3-rutinoside), **87** (kaempferol 3-*O*-arabinoside), and **90** (quercetin 3-*O*-arabinoside) were only identified in mountain pepper berries. Compounds 84 (rutin), **92** (quercetin-3-*O*-glucoside), **93** (Isorhamnetin), **95** (myricetin), **96** (taxifolin), and **98** (quercetin) were identified through the MS/MS spectra of pure standards [53]. The resulting ions at *m*/*z* 300 and 271, which correspond to the loss of CH3 and CO2 from the precursor [1,18], were used to identify isorhamnetin (compound **93** at ESI− *m*/*z* 315.0504), which was identified in mountain pepper berries, lemon aspen, and strawberry gum. In addition to repairing iron-induced DNA oxidation, myricetin 3-*O*-rhamnoside (compound **85**) also inhibits the activity of digestive, lipid, fecal, and colonic bacterial enzymes and functions as an anti-allergenic, anti-obesity, and anti-cancer compound [54]. Flavonols are also frequently found in Australian native fruits and medicinal plants. According to a comparison of the flavonoid literature, the aglycone derivatives of kaempferol, myricetin, and quercetin are the most often found flavonols in these plants. These aglycone derivatives are renowned for having highly effective anti-diabetic properties. These aglycone compounds are eight times more potent than the diabetic medication acarbose, according to some research [55]. In many earlier investigations, quercetin and kaempferol were connected to rutinoside, galactosides, and glucosides; previously, these flavonoid-3-*O*-glycosides were not described in selected native Australian plants. Three phenolic compounds, **99**, **100**, and **101**, were only identified in strawberry gum.

#### 3.4.3. Isoflavonoids

Isoflavonoids differ from flavonoids, as the isoflavonoid skeleton was biogenetically engineered from the 2-phenylchroman skeleton. In isoflavonoids, ring A (phenyl ring) is fused with the C-ring (six-membered heterocyclic ring) and another phenyl B-ring at C3, whereas the B-ring is substituted at C2 position in flavonoids [15]. Ten phenolic compounds were identified as isoflavonoids. Compounds **103** (equol 7-*O*-glucuronide) and **107** (3- ,4- ,7 trihydroxyisoflavanone) were only identified in strawberry gum; and **109** (daidzein 7-*O*glucuronide) and **110** (3- -hydroxymelanettin) were only identified in rosella and lemon aspen. Compounds **103**, **106** and **109** generated product ions at *m*/*z* 241 (equol), 415 (daidzin), and 253 (daidzein) after the loss of [M−H−176] from their precursor ions, respectively. Compounds **103**, **106**, and **109** were tentatively identified as equol 7-*O*-glucuronide, daidzin 4- -*O*glucuronide, and daidzein 7-*O*-glucuronide, respectively. As per our knowledge, no previous research has been conducted in such a comprehensive way to identify these isoflavonoids in the selected Australian native plants.

#### 3.4.4. Tannins

Proanthocyanidins (condensed tannins) are condensed flavanols. Seven compounds were identified as tannins (proanthocyanidins, hydrolyzable and complex tannins) [56]. Compound **115** at *m*/*z* 865.2004 produced fragment ions at *m*/*z* 739, 713, and 695 in negative-ion mode. The daughter ion at *m*/*z* 739 formed after the loss of ring "A" because of the fission of the heterocyclic ring [M−H−126]<sup>−</sup> from the precursor ion, RDA (152 Da), and a water unit (18 Da) from the latter product ion (*m*/*z* 713). Compound **115** was putatively identified as the procyanidin trimer C1 in strawberry gum and rosella. Compound **116** at ESI− *m*/*z* 577.1353 was tentatively identified in mountain pepper berries, lemon aspen, and strawberry gum, which generated fragment ions at *m*/*z* 451, 425, and 289; C4, C5 and O-C2 showed cleavage of one pyran ring, which led to phloroglucinol molecule loss (A-ring) from the precursor ion [52], which resulted in product ions at *m*/*z* 451 [M−H−126]<sup>−</sup> and 425 [M−H−152]−. Compound **116** was putatively identified as the procyanidin B2. Previously, procyanidin B2 and procyanidin trimmer C1 were recognized in nutmeg and cinnamon [10]. They have been reported to have anti-cancer, antioxidant, cardio-protective, and anti-inflammatory activities [56,57]. Compounds **113** (2-*O*-galloylpunicalin) and **117** (punicafolin) were only identified in strawberry gum, and compound **114** (glucosyringic acid) was only identified in mountain pepper berries.

#### 3.4.5. Lignans and Stilbenes

Stilbenes are natural phytochemicals that contain a 1,2-diphenylethylene (a basic skeleton of stilbenoids), and lignans are a group of diphenol derivatives with dibenzylbutane skeleton structures [15]. Due to their diverse structural makeup and established advantages for human health, lignans and stilbenes are among the most studied secondary plant metabolites [15]. Ten metabolites that fit into these classes were putatively discovered in this investigation. A total of three stilbenes (piceatannol, polydatin, and piceatannol 3-*O*-glucoside) and seven lignans were tentatively identified in these selected Australian native fruits and medicinal plants. Compound **119** (piceatannol) resulted in a deprotonated precursor ion at *m*/*z* 243 that formed a fragment ion at *m*/*z* 225 following the removal of a water unit [M−H−H2O], and a second product ion at *m*/*z* 201 due to the neutral loss of C2H2O (42 Da) from the precursor ion. Previously, piceatannol was found in fenugreek and dill leaves [18] and has been reported to have strong antimutagenic, antioxidant, anti-inflammatory, and anti-cancer properties. Compound **127** at ESI+ *m*/*z* 299.1279 was putatively identified in mountain pepper berries and strawberry gum, which generated product ions at *m*/*z* 281, 187, and 165 after the loss of [M−H−H2O], [M−H−C6H8O2], and [M−H−C9H8O2], respectively, from the precursor ion. Compound **127** was characterized as enterolactone. Enterolactone has been acknowledged for its antioxidant [58] and anti-cancer activities [59]. Compounds **122** (sesamin), **124** (silibinin), and **128** (2-hydroxyenterodiol) were only identified in mountain pepper berries.

#### 3.4.6. Other Compounds

Ten compounds were identified as other compounds. Compounds **137** (carnosic acid) and **138** (mellein) generated product ions at *m*/*z* 287 and 135, respectively, after the loss of CO2 (44 Da). Compound **129** at ESI− *m*/*z* 125.0242 was identified in mountain pepper berries and generated product ions at *m*/*z* 107, 97, and 79 after the loss of H2O (18 Da) and CO (28 Da), and the removal of H2O after the loss of CO (18 Da). Compound **133** at ESI− *m*/*z* 161.0242 was identified in mountain pepper berries and rosella, which produced fragment ion s at *m*/*z* 133, 117, and 105 through the removal of [M−H−CO], [M−H−CO2], and [M−H−C2H2] from the precursor ion and the former product ion, respectively. Compound **133** was tentatively characterized as umbelliferone. Compound **134** (2-hydroxybenzaldehyde) was tentatively identified only in mountain pepper berries, which produced fragment ions at *m*/*z* 92 and 77 after the loss of CO (28 Da) and CO2 (44 Da), respectively, from the precursor ion. Compound **132** (1,2,4,6-tetragalloyl-*β* GREEK-D-glucopyranose) was only identified in strawberry gum; and compounds **129** (pyrogallol), **134** (2-hydroxybenzaldehyde), and **135** (*p*-coumaraldehyde) were only identified in mountain pepper berries. A total of five limonoids were putatively detected in these native Australian fruits and spices. Compounds **139** (limonin) and **143** (citrusin) were tentatively identified only in lemon aspen.

The screening and profiling of the phenolic compounds give an overall idea of antioxidation compounds in selected Australian native plants. Strawberry gum is an excellent source of phenolic compounds, especially flavonoids used in the food, feed, cosmetics, and pharmaceutical industries because several of them have already been shown to possess high antioxidation capabilities.

#### *3.5. Quantification/Semi-Quantification of Targeted Phenolic Compounds*

A total of 26 compounds were quantified in Australian native mountain pepper berries, strawberry gum, rosella, and lemon aspen, which are given in Table S2. Flavonoids are the most abundant class in these selected Australian native plants. Strawberry gum was found to have the highest concentration of flavonoids, and quercitrin had the highest concentration among them (1274.04 ± 43.78 μg/g). Myricetin 3-*O*-rhamnoside (394.71 ± 16.21 μg/g), 3- ,4- ,5- trimethoxyflavone (615.15 ± 21.63 μg/g), quercetin 3-*O*-arabinoside (371.54 ± 14.26 μg/g), quercetin 3-(2-galloylglucoside) (309.15 ± 20.38 μg/g), chrysin (35.52 ± 2.77 μg/g), and naringenin (24.72 ± 1.83 μg/g) were only quantified in strawberry gum. Chlorogenic acid (3-caffeoylquinic acid) is the most abundant phenolic acid in mountain pepper berries (134.05 ± 12.67 μg/g), and the lowest concentration of chlorogenic acid was quantified in strawberry gum. Previously, Konczak et al. [60] also quantified the higher concentration of chlorogenic acid in Tasmanian pepper berries. Protocatechuic acid was quantified in strawberry gum (63.56 ± 4.67 μg/g) and mountain pepper berries (44.57 ± 5.82 μg/g); *p*-hydroxybenzoic acid was quantified in rosella (11.74 ± 1.56 μg/g) and mountain pepper berries (21.91 ± 3.41 μg/g). The highest concentration of caffeic acid was found in mountain pepper berries (23.49 ± 1.92 μg/g), and the lowest concentration of caffeic acid was found in strawberry gum (15.51 ± 2.09 μg/g). Gallic acid (19.24 ± 3.12 μg/g) and *p*-coumaric acid (10.56 ± 1.35 μg/g) were found in mountain pepper berries. Gallic acid was also found in strawberry gum (23.54 ± 3.19 μg/g) and rosella (17.21 ± 2.17 μg/g). The highest concentration (39.52 ± 3.65 μg/g) of procyanidin B2 was found in strawberry gum, and the lowest concentration (11.32 ± 1.48 μg/g) was measured in lemon aspen. Rutin (56.61 ± 5.48 μg/g) was only found in mountain pepper berries. Previously, Konczak et al. [60] also found rutin in mountain pepper berries. The highest concentration of quercetin was found in mountain pepper berries (71.46 ± 4.52 μg/g), and the lowest concentration was measured in lemon aspen (18.31 ± 2.34 μg/g). The highest concentrations of isorhamnetin (26.83 ± 2.86 μg/g) and myricetin (23.67 ± 3.71 μg/g) were found in mountain pepper berries, and the lowest concentrations of isorhamnetin (12.52 ± 1.08 μg/g) and myricetin (13.16 ± 0.89 μg/g) were found in strawberry gum. A total of six anthocyanin compounds were also found in mountain pepper berries and rosella. Delphinidin 3-*O*-sambubioside (196.61 ± 17.91 μg/g) and cyanidin 3-rutinoside (142.98 ± 13.01 μg/g) were found in rosella; and delphinidin 3-*O*-sambubioside (59.67 ± 5.24 μg/g) and cyanidin 3-rutinoside (82.91 ± 7.25 μg/g) were found in mountain pepper berries. Cyanidin-3-sambubioside (72.21 ± 8.63 μg/g) and delphinidin 3-rutinoside (17.23 ± 1.61 μg/g) were only found in rosella.

Furthermore, hierarchical heatmap clustering (Figure 3) was conducted by using MetaboAnalyst 5.0 (www.metaboanalyst.ca) accessed on 7 November 2022.

It depicted in the heatmap that quercitrin, 3- ,4- ,5- -trimethoxyflavone, myricetin 3-*O*rhamnoside, quercetin 3-*O*-arabnoside, quercetin 3-(2-galloylglucoside), and epicatechin had higher concentrations than other quantified phenolic compounds in strawberry gum; and delphinidin 3-*O*-sambubioside, cyanidin 3-rutinoside, cyanidin 3-glucoside, cyanidin-3-sambubioside, cyanidin, epicatechin, and chlorogenic acid had higher concentrations in rosella. The highest concentrations of chlorogenic acid were found in mountain pepper berries and lemon aspen. Mountain pepper berries had higher concentrations of chlorogenic acid, epicatechin, cyanidin 3-rutinoside, quercetin, cyanidin, delphinidin 3-*O*-sambubiode, rutin, and protocatechuic acid.

#### *3.6. Molecular Docking*

In silico molecular docking was conducted to predict the roles of abundant phenolic compounds in α-glucosidase inhibition activity. The estimated binding geometry 2D and 3D structures of myricitrin and chlorogenic acid in α-glucosidase protein (5NN8) are given in Figure 4A,B; and the calculated binding energy, glide energy, and binding geometry 2D of selected phenolic compounds are given in Table S3 and Figure S3.

**Figure 4.** (**a**) The estimated binding geometry (2D (**left**) and 3D (**right**)) of myricitrin in 5NN8. The active side residues are named with three letters. (**b**) The estimated binding geometry (2D (**left**) and 3D (**right**)) of chlorogenic acid in 5NN8. The active side residues are named with three letters.

All compounds were properly docked in 5NN8. Myricitrin (Figure 4A) and quercitrin made two hydrogen bonds with ASP 282 (negatively charged) and one each with ASP 616 (negatively charged), ALA 284 (hydrophobic), and EDO 1024. They had double Pi–Pi stacking hydrophobically with PHE 525 and a single Pi–Pi bond with TRP 481. Chlorogenic acid (Figure 4B) made two hydrogen bonds with ASP 282, one hydrogen bond each with ASP 518 and ASP 404, and one with a water molecule; and it had π–π staking with the hydrophobic PHE 649. Quercetin 3-(2-galloylglucoside) made hydrogen bonds with ASP 282 (negatively charged), ASP 616 (negatively charged), ASP 518 (negatively charged), ASP 404 (negatively charged), ALA 284 (hydrophobic), ARG 600 (positively charged), PHE 525 (hydrophobic) and water molecules; and Pi–Pi bonds with TRP 481, PHE 525, and PHE 649. Moreover, delphinidin 3-rutinoside, delphinidin 3-sambubioside, and rutin made four hydrogen bonds (two with ASP 616, one with ASP 404, and one with ASP 518), six hydrogen bonds (two ASP 282 and one each with ASP 518, ASP 403, EDO 1024, and a water molecule), and six hydrogen bonds (ASP 282, ASP 404, ASP 518, ASP 616, ASN 524 and SER 523); they also had π–π stacking in one (TRP 481), two (TRP 481 and PHE 649), and four (two with PHE 649, one with TRP 481, and one with TRP 376) bonds, respectively. Acarbose made twelve hydrogen bonds (two each with ASP 518, ASP 404, ASP 282, and SER 523; three OH groups from water molecules, which further made hydrogen bonds with ASP 645 and ARG 281). Naringin made hydrogen bonds with ASP 282, PHE 525, LEU 678, EDO 1024, and ARG 281 and one Pi–Pi stacking interaction with TRP 481. Furthermore, diosmin made three hydrogen bonds with the negatively charged ASP 282 and one with the negatively charged ASP 616 (Figure S3). The binding energies of quercetin 3-(2-galloylglucoside), delphinidin 3-rutinoside, cyanidin 3-O-rutinoside, delphinidin 3-sambubioside, rutin, acarbose, cyanidin 3-rhamnoside 5-glucoside, delphinidin, procyanidin B2, myricitrin, 3-feruloylquinic acid, taxifolin, diosmin, quercitrin, chlorogenic acid, naringin, 3-*p*-coumaroylquinic acid, myricetin, quercetin, isorhamnetin, quinic acid, luteolin, (-)-epicatechin, hesperetin, and gallic acid in 5NN8 were calculated as − 11.09, − 11.08, −10.90, −10.38, −10.14, −9.65, −9.46, −8.48, −8.05, −7.59, −7.32, −7.13, −6.84, −6.72, −6.62, −6.40, −6.35, −6.28, −5.95, −5.68, −6.65, −5.52, −5.36, −5.28, and −5.15 kcal/mol, respectively (Table S3). From the given results, it is predicted that quercetin 3-(2-galloylglucoside) identified in strawberry gum has higher α-glucosidaseinhibiting activity than acarbose. Overall, flavonoids are predicted to have higher binding affinities than the other selected phenolic compounds. Interestingly, 3-feruloylquinic acid has a higher binding affinity than taxifolin, diosmin, quercitrin, naringin, myricetin, quercetin, isorhamnetin, and luteolin chlorogenic acid; and 3-*p*-coumaroylquinic acid has a higher binding affinity than myricetin, quercetin, isorhamnetin, luteolin, (-)-epicatechin, hesperetin, diosmetin, and naringenin (Table S3). In silico molecular docking is a prediction of possible interactions between target proteins (5NN8) and potential inhibitors. Therefore, it is critical to assess the inhibitory activities of individual purified phenolic compounds to establish the precise roles of individual bioactive compounds in the inhibition of α-glucosidase. Moreover, the insights into inhibitory mechanisms of bioactive polyphenolic compounds against α-glucosidase and other proteins involved in diabetic conditions can be revealed through advanced molecular dynamics techniques and free-energy calculations, and through inverse molecular docking [61].

#### *3.7. Pharmacokinetics Study of Selected Phenolic Compounds*

Using computational methods to test the potential drug metabolites helps reduce the number of experimental studies and improve the success rate in pharmacokinetics studies. Absorption, distribution, metabolism, excretion, and toxicological (ADMET) screening were also conducted to validate this study for drug discovery. The interaction of inhibitors with a target receptor cannot guarantee the suitability of phenolic metabolites as drugs for the target pathology. Therefore, ADMET screening of compounds is critical in drug discovery. Unfavorable characteristics of ADMET in the biological system are the main reasons for the failure of drug molecules during clinical experiments [7]. This study evaluated the most abundant phenolic compounds identified in selected plants for ADMET properties.

#### 3.7.1. Absorption and Distribution

The absorption of the phenolic compounds was predicted through the BIOLED-Egg method and using the pkCSM platform. The results of absorption are given in Figure 5 and Table S4 and Table S5.

**Figure 5.** Evaluation of abundant phenolic compounds through the BOILED-Egg method. The blue dots indicate molecules predicted to be expelled from the CNS by P-glycoprotein, and the red dots indicate molecules predicted not to be expelled from the CNS by P-glycoprotein. The egg-yolk area predicts the phenolic metabolites that will passively penetrate the blood–brain barrier. In contrast, the egg-white area predicts which phenolic compounds will be absorbed through the gastrointestinal tract.

Figure 5 predicts that cinnamic acid, coumarin, *p*-coumaric acid, *p*-hydroxybenzoic acid, chrysin, [6]-gingerol, and 3- ,4- ,5- -trimethoxyflavone pass through the blood–brain barrier; and gallic acid, protocatechuic acid, caffeic acid, pyrogallol, cyanidin, taxifolin, epicatechin, delphinidin, naringenin, genistein, phloretin, quercetin, diosmetin, isorhamnetin, limocitrin, and eriodictyol should be absorbed through the gastrointestinal tract. Moreover, the results predict that the cinnamic acid found in mountain pepper berries and strawberry gum will more readily cross the blood–brain barrier than other phenolic compounds (Table S4). 3- ,4- ,5- -Trimethoxyflavone (98.1%), coumarin (97.3%), cinnamic acid (94.8%), chrysin (93.8%), *p*-coumaric acid (93.5%), genistein (93.4%), [6]-gingerol (92.4%) naringenin (91.3%), xanthohumol (89.9%), cyanidin (87.3%), *p*-hydroxybenzoic acid (84%), pyrogallol (83.6%), and luteolin (81.1%) are predicted to have the highest human intestinal absorption. Coumarin is the only compound which is predicted to pass through the skin. It is worth noting that anthocyanin aglycones with sugar moieties are predicted to have no human intestinal absorption (Table S4). Therefore, we can predict that anthocyanins with sugar moieties may play a role in gut modulation after the breakdown through colonic

fermentation into their basic aglycones, or they will play a role as prebiotic polyphenols. Additionally, cinnamic (1.72), coumarin (1.65), 3- ,4- ,5- -trimethoxyflavone (1.39), *p*-coumaric acid (1.21), *p*-hydroxybenzoic acid (1.15), pyrogallol (1.12), naringenin (1.03), chrysin (0.95), [6]-gingerol (0.94), and taxifolin (0.92), are predicted to have the highest Caco-2 cell permeability. If the Caco2 permeability value is higher than 0.90, a compound is considered to have high Caco-2 permeability. Furthermore, the compounds which have Caco-2 permeability, gastrointestinal absorption, a good bioavailability score, and obey Lipinski's rule of five while not being able to pass through the BBB, not acting as P-gp substrates, and having poor skin permeability should be successful drugs [62].

Most of the flavonoids that are not absorbed in the gastrointestinal tract can be metabolized by gut microbiota into small phenolic metabolites, where they tend to be absorbed in the colon [48]. Flavonoids are bound to albumin and transported to the liver through the portal vein after absorption. However, the bioavailability of flavonoids is low due to the limited absorption, extensive metabolism, and rapid excretion [63].

#### 3.7.2. Drug-Likeness

The bioavailability radars of selected compounds were obtained by following the method of Daina et al. [17] to predict the drug-likeness to assess the oral bioavailability of compounds (Figure 6).

**Figure 6.** The pink area of the bioavailability radar represents the optimal range for each property. Radars of [6]-gingerol (**a**), 3- ,4- ,5- -trimethoxyflavone (**b**), naringin (**c**), quinic acid (**d**), gallic acid (**e**), and cyanidin (**f**) were obtained.

Figure 6 and Table S6 depict that no compound predicted oral bioavailability except quinic acid. To predict the oral bioavailability of selected compounds, six physiochemical properties (size, polarity, lipophilicity, flexibility, saturation, and solubility) were considered and analyzed through the bioavailability radar.

#### 3.7.3. Metabolism, Excretion, and Toxicity

Cytochrome P450 (CYP) plays a vital role in the metabolism of bioactive compounds (drugs) [63]. The predicted metabolism and excretion of the phenolic compounds are given in Table S7. Metabolism was predicted through the CYP model for substrate or inhibitor (CYP1A2, CYP2D6, CYP3A4, CYP2C9, and CYP2C19). Bioactive compounds that inhibit the CYP pathway may cause elevated concentrations of other bioactive compounds, resulting in higher toxicity of that compound and vice versa. Bioactive compounds with higher total clearance are predicted to have higher bioavailability and metabolism in the liver (Table S5). Virtual toxicological screening of the bioactive compounds is provided in Table S8. The predicted results indicate that all bioactive compounds do not inhibit the hERG 1 channel, and no compound predicted AIME toxicity, hepatotoxicity, skin sensitization, *Tetrahymena pyriformis* toxicity, or Minnow toxicity except 3- ,4- ,5- -trimethoxyflavone, which predicted toxicity in Minnow.

#### **4. Conclusions**

In this study, native Australian fruits and spices were comprehensively analyzed for polyphenols, and a total of 143 metabolites were identified. Twenty-six of these compounds were quantified. Strawberry gum had higher total phenolic content, antioxidant capacity, and α-glucosidase inhibition activity than rosella, lemon aspen, and mountain pepper berries. Furthermore, in silico molecular docking predicted that flavonoids have a significant role in the inhibition of α-glucosidase. Additionally, simulated pharmacokinetics predicted that all screening phenolic compounds from native Australian fruits and spices are safe and do not have any toxicity, and small phenolic metabolites such as phenolic acids have higher absorption in Caco-2 cells and the gastrointestinal tract than other phenolic compounds. This study demonstrates that strawberry gum has a significant medicinal and pharmaceutical potential that could be utilized in food, feed, cosmetic, and pharmaceutical industries with the further proved in vivo data.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox12020254/s1. Figure S1: Base peak chromatograms (BPC) of mountain pepper berries, rosella, lemon aspen, and strawberry gum in positive (black) and negative (blue) modes of ionization. Figure S2: MS/MS spectra of some selected compounds. Figure S3: Two-dimensional binding geometry of some selected compounds. Table S1: Antioxidant activities of native Australian fruits and spices. Table S2: Quantification/semi-quantification of phenolic metabolites in Australian native fruits and spices (μg/g). Table S3: The calculated binding energies of selected compounds. Table S4: Predicted absorption and distribution of selected compounds. Table S5: Pharmacokinetic properties of selected compounds. Table S6: Radar bioavailability properties of selected compounds. Table S7: Metabolism and excretion of selected compounds. Table S8: Predicted toxicity of abundant phenolic compounds.

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

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

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The supporting data are available in the Supplementary Materials.

**Acknowledgments:** We owe incredible thanks to William Nikolas, Asif Noor, and Swati Varshney from the Mass Spectrometry Proteomics Facility, Bio21 Molecular Institute, VIC, Australia, for providing their support in training and learning.

**Conflicts of Interest:** Authors declare no conflict of interest.

#### **References**

1. Ali, A.; Cottrell, J.J.; Dunshea, F.R. Identification and characterization of anthocyanins and non-anthocyanin phenolics from australian native fruits and their antioxidant, antidiabetic, and anti-alzheimer potential. *Food Res. Int.* **2022**, *162*, 111951. [CrossRef]


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### *Article* **Salt Eustress Induction in Red Amaranth (***Amaranthus gangeticus***) Augments Nutritional, Phenolic Acids and Antiradical Potential of Leaves**

**Umakanta Sarker 1,\* and Sezai Ercisli <sup>2</sup>**


**Abstract:** Earlier researchers have highlighted the utilization of salt eustress for boosting the nutritional and phenolic acid (PA) profiles and antiradical potential (ARP) of vegetables, which eventually boost food values for nourishing human diets. Amaranth is a rapidly grown, diversely acclimated C4 leafy vegetable with climate resilience and salinity resistance. The application of salinity eustress in amaranth has a great scope to augment the nutritional and PA profiles and ARP. Therefore, the *A. gangeticus* genotype was evaluated in response to salt eustress for nutrients, PA profile, and ARP. Antioxidant potential and high-yielding genotype (LS1) were grown under four salt eustresses (control, 25 mM, 50 mM, 100 mM NaCl) in a randomized completely block design (RCBD) in four replicates. Salt stress remarkably augmented microelements, proximate, macro-elements, phytochemicals, PA profiles, and ARP of *A. gangeticus* leaves in this order: control < low sodium chloride stress (LSCS) < moderate sodium chloride stress (MSCS) < severe sodium chloride stress (SSCS). A large quantity of 16 PAs, including seven cinnamic acids (CAs) and nine benzoic acids (BAs) were detected in *A. gangeticus* genotypes. All the microelements, proximate, macro-elements, phytochemicals, PA profiles, and ARP of *A. gangeticus* under MSCS, and SSCS levels were much higher in comparison with the control. It can be utilized as preferential food for our daily diets as these antiradical compounds have strong antioxidants. Salt-treated *A. gangeticus* contributed to excellent quality in the end product in terms of microelements, proximate, macro-elements, phytochemicals, PA profiles, and ARP. *A. gangeticus* can be cultivated as an encouraging substitute crop in salt-affected areas of the world.

**Keywords:** *A. gangeticus*; protein and dietary fiber; minerals; phytochemicals; HPLC-UV DPPH; ABTS+; PA profiles; NaCl

#### **1. Introduction**

Amaranth is a promising millennium vegetable with vast diversity [1–7]. It is an alternate source of nutrients because of its richness in vitamin C, minerals [8–15], vitamins [16–20], protein [21,22], dietary fiber [23–25], leaf pigments [26–42], phenolic compounds [43–58], and flavonoids [59–73] with strong antioxidants [74–86]. Amaranth has a noteworthy contribution as an antioxidant in food manufacturing owing to quenching reactive oxygen species (ROS) [87,88]. Wahid and Ghazanfar [89] reported that extreme salt enhanced the secondary plant metabolites, eventually accelerating plant protection apparatuses against ROS. Salinity enhances ROS production, which causes the oxidation of cellular components. ROS [90]. In plants, antioxidants (non-enzymatic), such as proteins, flavonoids, carbohydrates, carotenoids, and phenolic compounds, and enzymatic antioxidants are capable of ROS detoxification [90,91]. Hence, in human life, salt-tolerant plants could be considered a source of potent antioxidants. These compounds have extraordinary benefits to our food owing to quenching ROS and protecting against numerous diseases,

**Citation:** Sarker, U.; Ercisli, S. Salt Eustress Induction in Red Amaranth (*Amaranthus gangeticus*) Augments Nutritional, Phenolic Acids and Antiradical Potential of Leaves. *Antioxidants* **2022**, *11*, 2434. https:// doi.org/10.3390/antiox11122434

Academic Editor: Stanley Omaye

Received: 14 October 2022 Accepted: 29 November 2022 Published: 9 December 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/).

such as cancer, cardiovascular diseases, atherosclerosis, cataracts, retinopathy, emphysema, arthritis, and neuron-damaging diseases [88].

Taste, flavor, and color determine the suitability of foods. Recently, consumers are very much interested in coloring food products. These products have much interest in the nutritional, safety, and beautification aspects of customers as foods. The utilization of natural pigments is considerably increasing day by day. The selected *A. gangeticus* genotype had sufficient betalains with bright red-violet color. *Amaranthus* leafy vegetable is an exclusive origin of betalains with significant quenching capacity of free radicals [92]. In low-acid foods, betalains are preferable to be utilized as a food colorant. These have greater stability than anthocyanins for pH [93], have preferential utility in the promotion of health, act as anti-inflammatory compounds, and diminish the risk of cancers of the skin and lungs and cardiovascular diseases.

Amaranth is an extensively acclimated leafy vegetable due to diverse stresses, such as salinity [94–96] and drought [97], as well as having multiple uses. Salinity stress is a pioneer for the rapid augmentation of the quantity and quality of natural antioxidants through diverse factors, such as physiological, environmental, ecological, biological, biochemical, and evolutionary processes [98]. Very limited reports on the effect of salinity stress are available in terms of minerals, proximate, and bioactive compounds in different crops including leafy vegetables. Petropoulos et al. [99] reported the salinity-induced reduction of chlorophylls, fat, sugar, and carbohydrate and the augmentation of flavonoids, ascorbic acid (AsA), phenolics, proteins, and ARP in *Cichorium spinosum*. Different concentrations of sodium chloride enhanced the carotenoid content in buckwheat sprouts in comparison to the control [100]. Alam et al. [101] reported salt-induced amelioration of phenolics, ARP, and flavonoids in purslane. Ahmed et al. [102] recorded a salinity-induced increase in ARP and phenolics in barley. The influence of sodium chloride stress on the phytochemicals, nutrients, ARP, and PA profiles in *A. gangeticus* was studied for the first time. Based on our previous studies, the ARP genotype (accession LS1) along with high yield potential were selected. Therefore, the response of sodium chloride stress was assessed in *A. gangeticus* in terms of phytochemicals, nutrients, ARP, and PA profiles.

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

#### *2.1. Experimental Site, Conditions, and Plant Materials*

A high-yielding ARP genotype (accession LS1) was selected from among 120 genotypes from the Department of Genetics and Plant Breeding's collection. The seeds were sown in four replicates following a block design with complete randomization (RCBD) in plastic pots at the Bangabandhu Sheikh Mujibur Rahman Agricultural University (24◦23- N, 90◦08- E, 8.4 m.s.l., AEZ-28 [103,104]. Pots were filled with sandy loam soil. P2O5:K2O was applied @ 48:60 kg ha−<sup>1</sup> during the final land preparation. However, N was applied @ 46 kg ha−<sup>1</sup> in two equally split doses during the final land preparation and 10 days after the sowing of the seeds. Four salt treatments, 100 (severe sodium chloride stress, SSCS), 50 (moderate sodium chloride stress, MSCS), and 25 (low sodium chloride stress, LSCS) mM NaCl, and a control (normal water) were used in the study. Pots were regularly irrigated with normal water for 10 days after sowing (DAS). At 11 DAS, salt treatments were imposed and sustained until the edible stage (30 DAS). Pots were irrigated once a day using salt water (100, 50, and 25 mM NaCl) and normal water. *A. gangeticus* leaves were harvested at 30 DAS.

#### *2.2. Chemicals*

Acetone, HClO4, HNO3, Sb, dithiothreitol (DTT), CsCl, AsA, 2, 2-dipyridyl, Trolox, PAs, HPLC grade acetonitrile, acetic acid, gallic acid (GAA), NaOH, rutin, DPPH, H2SO4, Folin-Ciocalteu reagent, MeOH, ABTS+, AlCl3.6H2O, Na2CO3, CH3CO2K, and K2S2O8. All chemicals were bought from Kanto Chemical Co. Inc. (Tokyo, Japan) and Merck (Germany).

#### *2.3. Ash, Fiber, Moisture, Fat, Gross Energy, Carbohydrate, and Protein Estimation*

The ash, fiber, moisture, fat, gross energy, and protein were estimated by the AOAC method [105–107]. The mini-Kjeldahl method was followed to measure nitrogen (N). Protein was calculated by multiplying N with 6.25. Protein, ash, fat, and moisture (%) were deducted from 100 to estimate carbohydrates.

#### *2.4. Elements Estimation*

The leaves were dried in an oven at 70 ◦C temperature for 24 h. Mineral elements were determined from the ground leaf by digesting with HNO3 and perchloric acid [105,108]. Exactly 0.5 g of the leaf samples were digested with 400 mL HNO3 (65%), 40 mL HClO4 (70%), and 10 mL H2SO4 (96%). The absorbance was read at 213.9 (Zn), 285.2 (Mg), 766.5 (K), 279.5 (Mn), 248.3 (Fe), 258.056 (S), 422.7 (Ca), 880 (P), 589 (Na), 430 (B), 313.3 (Mo), and 324.8 (Cu) nm wavelengths using an atomic absorption spectrophotometer (AAS with flame) (Hitachi, Japan). Macro- and micro-elements were expressed in mg g−<sup>1</sup> and μg g−<sup>1</sup> FW.

#### *2.5. Beta-Carotene*

In a mortar and pestle, 500 mg leaves (fresh) were thoroughly mixed with 10 mL acetone (80%). The mixture was centrifuged at 10,000× *g* for 3–4 min for β-carotene determination [109]. After the separation of the filtrate in a flask, the final volume of 20 mL was maintained. The absorbance was taken at 510 and 480 nm by spectrophotometer (Tokyo, Japan). β-Carotene was expressed in fresh weight as mg 100 g<sup>−</sup>1.

#### *2.6. Ascorbic Acid (AsA) Estimation*

Fresh leaves were used to determine AsA and DHA. The sample was pre-incubated by dithiothreitol (DTT), which reduced DHA to AsA. With the reduction of AsA, Fe3+ converted to Fe2+. Fe2+ complexes were formed by reacting Fe2+ and 2, 2-dipyridyl [109]. The absorbance of the complexes was taken at 525 nm by a spectrophotometer (Hitachi, Japan) to measure AsA in mg 100 g<sup>−</sup>1.

#### *2.7. Samples Extraction and Determination of Total Polyphenols (TP), Total Flavonoids (TF), and Antiradical Potential (ARP)*

Leaves were dried in a shady place to avoid direct sunshine. The extraction was performed from both the ground dried and fresh leaves (30 d) separately with a mortar and pestle. Total polyphenols (TP) were measured from fresh leaves, while total flavonoids (TF) content and ARP were determined from dried leaves. A 90% MeOH solution 10 mL was added with 0.25 g samples in a capped bottle tightly. The mixture was placed for 1 h in a shaker (Tokyo, Japan) at 60 ◦C. The final filtrate was stored for TP, TF, and ARP estimation. TF and TP were estimated by the AlCl3 colorimetric method and the Folin-Ciocalteu reagent, respectively [105,110]. The absorbance at 760 and 415 nm with a spectrophotometer (Hitachi, Japan). TP and TF were measured as GAA and rutin equivalent μg GAE g−<sup>1</sup> of FW and μg RE g−<sup>1</sup> DW using standard GAA and rutin curves. The Trolox equivalent antioxidant activity (TEAC) of ARP was estimated by the DPPH reduction and the ABTS<sup>+</sup> assay [105,111]. ABTS<sup>+</sup> and DPPH reduction percentage equivalent to the control was measured for estimating the ARP using the equation:

$$\text{ARP} \left( \% \right) = \left( \text{Ac} - \text{As} / \text{Ac} \right) \times 100$$

where Ac denotes the control absorbance (150 μL MeOH for ARP (ABTS) and 10 μL MeOH for ARP (DPPH) instead of leaf extract) and As is the absorbance of the samples. The results were calculated as μg Trolox equivalent g−<sup>1</sup> DW.

#### *2.8. Samples Extraction and Determination of Phenolic Acids (PAs) by HPLC*

Fresh leaves (1 g) were extracted in MeOH (10 mL, 80%) containing CH3COOH (1%). The thoroughly homogenized mixture was kept in a 50 mL tightly capped test tube and placed in a shaker (Scientific Industries Inc., New York, NY, USA) for 15 h at 400 rpm. It was filtered in a 0.45 μm filter (MA, New York, USA) and centrifuged for 15 min at 10,000× *g*. The filtrate was used to estimate PAs. All extractions were repeated 3 times. The method of Sarker and Oba [112] was followed to determine PAs using HPLC. Shimadzu HPLC (Kyoto, Japan) was furnished with a binary pump, degasser, and detector. A column (150 × 4.6 mm, 5 μm; Shinwa Chemical Industries, Ltd., Kyoto, Japan) was used for the separation of PAs. Solvent B and solvent A (acetonitrile and 6% (*v*/*v*) acetic acid in water, respectively) were pumped for 70 min at 1 mL min<sup>−</sup>1. HPLC system was run using a gradient program with 0–15% acetonitrile for 45 min, 15–30% for 15 min, 30–50% for 5 min, and 50–100% for 5 min; 35 ◦C temperature in the column was maintained with a 10 μL volume of injection. For monitoring Pas continuously, the detector was set at 254 and 280 nm. The retention time and UV–vis spectra with their respective standards were compared for the identification of the compound. Pas was estimated as μg g−<sup>1</sup> FW.

Each PA was quantified using the corresponding standards of calibration curves. A total of 16 PAs were dissolved in MeOH (80%) 100 mg mL−<sup>1</sup> as stock solutions. Individual PAs were quantified using corresponding standard curves (10, 20, 40, 60, 80, and 100 μg mL<sup>−</sup>1) with external standards. Retention times, co-chromatography of samples spiked with commercially available standards, and UV spectral characteristics were utilized for identification and matching the PA.

#### *2.9. Statistical Analysis*

All the sample data of a trait were averaged for each treatment to obtain a replication mean [113–115]. The mean data of various traits were statistically and biometrically analyzed [116–118]. Data analysis and ANOVA were performed using Statistix software version 8.0, Tallahassee FL 32312, USA [119–121]. The means were compared at a 1% level of probability using Duncan's Multiple Range Test (DMRT). The results were reported as the mean ± SD of four separate replicates [122–124].

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

#### *3.1. The Response of Proximate Compositions to Sodium Chloride Stress*

Figure 1 represents the nutritional compositions of *A. gangeticus* under different salinity stresses. *A. gangeticus* leaves had a high moisture content like most leafy vegetables. Nevertheless, our study revealed that *A. gangeticus* leaves have copious ash, carbohydrates, dietary fiber, moisture, and protein. The constituents of these components were several times greater than *C. spinosum* [99]. The maximum moisture and fat were exhibited under the control treatment, whereas the minimum moisture and fat were observed under SSCS. Petropoulos et al. [99] reported a similar reduction in fat with the increase in salinity stress in *C. spinosum.* Moisture and fat were significantly reduced in the order: (control > LSCS > MSCS > SSCS) and (control > LSCS > MSCS = SSCS), respectively. Higher leaf dry matter obtained from leaves ensure lower moisture content. Hence, saltstressed *A. gangeticus* leaves confirmed greater dry matter in comparison to the control. The maximum dietary fiber, ash, carbohydrates, energy, and protein were recorded at SSCS, while the minimum dietary fiber, ash, carbohydrates, energy, and protein were noticed under the control. Similarly, Petropoulos et al. [99] reported higher ash and protein at the maximum and 8.0 and 6.0 dS m−1, than the control and minimum salinity in *C. spinosum*. Energy, protein, and dietary fiber contents were sharply augmented in the following order: control < LSCS < MSCS < SSCS, whereas ash and carbohydrates contents were statistically similar in the control and LSS levels and progressively augmented from MSCS to SSCS levels.

**Figure 1.** The response of ash, fiber, moisture, fat, gross energy, carbohydrate, and protein (g 100 g<sup>−</sup>1) to control, LSCS, MSCS, and SSCS in *A. gangeticus* accession; (n = 6), different letters in columns are varied significantly by Duncan Multiple Range Test (DMRT) (*p* < 0.01).

In LSCS, MSCS, and SSCS, dietary fiber, energy, carbohydrates, ash, and protein were increased by 17%, 2%, 2%, 9%, and 4%; 6%, 10%, 8%, 14%, and 19%; and 23%, 14%, 9%, 16%, and 29%, respectively, in comparison with the control condition (Figure 2).

**Figure 2.** Changes of ash, fiber, moisture, fat, gross energy, carbohydrate, and protein over control in *A. gangeticus* accession.

Dietary fiber has significantly acted in the remedy of constipation, increased digestibility, and palatability. Vegetarians and deprived rural communities in underdeveloped countries mostly trust *A. gangeticus* for protein. Since the low amounts consumed in a daily diet, the increments of energy content in the order of control < LSCS < MSCS < SSCS had no substantial influence on the energy balance in humans. The findings of *A. gangeticus* conformed with the outcomes of AT [91] and leaves of *Ipomoea batata* [125], respectively. They specified that it influences cell function, the fat covering the body's organs, and continues the temperature of the body. The fats of vegetables are prime sources of crucial fatty acids, such as Ω-6 and Ω-3. Fats perform a noteworthy contribution to the absorption, digestion, and transportation of vitamins A, E, K, and D.

#### *3.2. Sodium Chloride Impact on Minerals (Macroelements and Microelements) Composition*

*A. gangeticus* has abundant minerals (macroelements and microelements) (Figure 3). High levels of minerals were observed and corroborated with *A. tricolor* under normal cultivation practice in an open field [126]. *A. gangeticus* had higher Fe and Zn than *Manihot esculenta* leaves [127] and *Lathyrus japonicus*[128]. Jimenez-Aguiar and Grusak [129] also found abundant Zn, Cu, Mn, and Fe in different *A. spp.* They also found higher iron and copper compared with kale and higher Zn compared with leaf cabbage, *Spinacia oleracea*, and *Solanum nigrum*. The maximum Zn, Ca, Mo, Mg, Na, S, Cu, B, Mn, and Fe was noticed under the SSCS level, while the minimum levels Zn, Ca, Mo, Mg, Na, Cu, B, Mn, and Fe were reported under control conditions, and the lowest sulfur content was observed under the LSCS level. Zn, Ca, Mo, Mg, Na, Cu, B, and Mn were progressively augmented in the order control < LSCS < MSCS < SSCS. In contrast, potassium and phosphorus contents were drastically reduced in the order control > LSCS > MSCS > SSCS.

In LSCS, MSCS, and SSCS, Zn, Ca, Cu, Mo, Mg, Mn, B, and Na were augmented by −1%, 0.8%, 13%, −1%, 10%, 4%, 1%, and 6%; 21%, 16%, 29%, 24%, 46%, 67%, 24%, and 12%; and 30%, 34%, 67%, 52%, 72%, 100%, 81%, and 36%, respectively, in comparison with the control condition (Figure 4). In LSCS, MSCS, and SSCS, potassium and phosphorus content declined to 5%, 14%, 25%, and 3%, 36%, 42%, respectively, in comparison with the control condition (Figure 4).

**Figure 3.** Response of minerals concentration (**A**) macroelements and (**B**) microelements under control, LSCS, MSCS, and SSCS in *A. gangeticus* accession; (n = 6), different letters in columns are varied significantly by DMRT (*p* < 0.01).

**Figure 4.** Response of minerals (macroelements and microelements) over control in *A. gangeticus* accession.

Most of the minerals increased under different salt levels compared with control conditions, which were corroborated with minerals of *C. spinosum* under salinity stress [99]. Petropoulos et al. [99] reported sharp augmentation in calcium, magnesium, iron, manganese, zinc, and sodium content and a reduction in potassium content in *C. spinosum*. They stated that the application of fertilizer and treatments of salinity could be the reason for the amelioration of sodium content and suggested that the species utilized accumulated sodium to cope with the adverse effects of salinity. Iron content was statistically similar to the value of the control and LSCS levels, while iron content was progressively augmented under MSCS and SSCS levels by 12% and 62%, respectively. The lowest sulfur content was obtained from the LSCS levels, which differed significantly from the control condition. The sulfur content was gradually augmented under MSCS and SSCS levels by 20% and 51%, respectively (Figure 4).

#### *3.3. Impact of Salinity on Phytochemicals and ARP*

Polyphenols, beta-carotene, AsA, flavonoids, and ARP varied significantly under different sodium chloride stresses (Figure 5). Sodium chloride stress progressively augmented polyphenols, beta-carotene, AsA, flavonoids, and ARP in the following order: control < LSCS < MSCS < SSCS.

Beta-carotene, AsA, polyphenols, flavonoids, and ARP (DPPH and ABTS+) under LSCS, MSCS, and SSCS were predominately augmented by 12%, 4%, 5%, 7%, 6%, and 3%; 28%, 18%, 22%, 22%, 20%, and 19%; and 47%, 52%, 54%, 45%, 38%, and 41% than control, respectively (Figure 6).

The maximum polyphenols, beta-carotene, flavonoids, AsA, and ARP (DPPH and ABTS+) were recorded under SSCS. Conversely, the lowest polyphenols, beta-carotene, flavonoids, AsA, and ARP (DPPH and ABTS+) were confirmed under the control. Petropoulos et al. [99] reported the salinity-induced augmentation of flavonoids, ARP, AsA, and phenolics in *C spinosum*. Different concentrations of sodium chloride enhanced the carotenoid content in buckwheat sprouts in comparison with the control (Lim et al. [100]. Alam et al. [101] reported salt-induced amelioration of phenolics, ARP, and flavonoids in purslane. In barley, a similar salinity-induced increase of ARP and phenolics were stated.

**Figure 5.** Effect of salinity treatments (control, LSCS, MSCS, and SSCS) on phytochemicals composition in *A. gangeticus* accession. Flavonoids (μg RE g−<sup>1</sup> DW), AsA and beta-carotene (mg 100 g−<sup>1</sup> FW), ARP (DPPH and ABTS+) (μg TEAC g−<sup>1</sup> DW), and polyphenols (μg GAE g−<sup>1</sup> FW), (n = 6); different letters in columns are varied significantly by DMRT (*p* < 0.01).

**Figure 6.** Comparison of phytochemicals over control in *A. gangeticus* accession.

#### *3.4. Response of Salinity on PA Profiles*

The HPLC-identified PA values of *A. gangeticus* (accession LS11) under four sodium chloride stress were compared with PAs using the respective peaks of the compounds (Table 1). Sixteen PAs, including seven CAs and nine Bas, were confirmed in *A. gangeticus*. Three BAs [protocatechuic acid (PCA), β-resorcylic acid (β-RA), and gentisic acid (GA)] were identified as new compounds for the first time in *Amaranthus* leaves.


**Table 1.** Wavelengths of maximum absorption in the visible region (λmax), mass spectral data, retention time (Rt), and tentative identification of PAs in *A. gangeticus*.

BAs were the amplest among the two categories of acids, thereafter CAs in *A. gangeticus* (Figures 7 and 9). Salicylic acid (SA) was the most copious PAs across BAs thereafter GAA, GA, PCA, vanillic acid (VA), *p*-hydroxybenzoic acid (*p*-HBA), β-RA, and syringic acid (SYA) (Figure 7). BA contents in the *A. gangeticus* genotype under control conditions were superior to the BA content of *A. tricolor* [130]. Chlorogenic acid (CHA) was the most noticeable compound across CAs thereafter ferulic acid (FA), sinapic acid (SIA), *m*-coumaric acid (*m*-COA), *trans*-cinnamic acid (*Trans*-CA), and caffeic acid (CFA) (Figure 7). *A. gangeticus* had abundant CAs under control conditions. Seven CAs obtained were confirmed superior to CAs of *A. tricolor* [130]. Phenylalanine is the most extensively distributed PA in plant tissues, which are finally synthesized into CAs [131]. Identified Benzoic acids (BAs) have important biological activities. For instance, gallic acid and its ester derivatives ARE flavoring agents and preservatives in the food industry. There are diverse scientific reports on the biological and pharmacological activities of these phytochemicals, with emphasis on antioxidant, antimicrobial, anti-inflammatory, anticancer, cardioprotective, gastroprotective, and neuroprotective effects [132]. Vanillic acid exerts diverse bioactivity against cancer, diabetes, obesity, neurodegenerative, cardiovascular, and hepatic diseases by inhibiting the associated molecular pathways. Its derivatives also possess the therapeutic potential to treat autoimmune diseases, as well as fungal and bacterial infections [133]. Syringic acid shows a wide range of therapeutic applications in the prevention of diabetes, CVDs, cancer, and cerebral ischemia, as well as antioxidant, antimicrobial, anti-inflammatory, antiendotoxic, neurologic, and hepatoprotective activities [134]. High salicylate in diets has proven health benefits, such as lower risks of cancer, heart disease, and diabetes. Ellagic acid has been reported to have antimutagenic on bacteria and in mammalian systems as well. It has also shown strong antioxidant, anti-inflammatory, and anticarcinogenic activities, as well as a better preservative effect against oxidative stress when compared with vitamin E [135]. PCA is a major metabolite of anthocyanin. The pharmacological actions of PCA have been shown to include strong in vitro and in vivo antioxidant activity. In in vivo experiments using rats and mice, PCA has been shown to exert anti-inflammatory as well as antihyperglycemic and antiapoptotic activities [136]. β-resorcylic acid has antimicrobial activity [137]. Finally, gentisic acid possesses fibro growth factor inhibition, antimicrobial, antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective activities [138].

**Figure 7.** Impact of BAs concentrations (μg g−<sup>1</sup> FW) under control, LSCS, MSCS, and SSCS in *A. gangeticus* accession; (n = 6), different letters in columns are varied significantly by DMRT (*p* < 0.01).

Sodium chloride stress predominately augmented all the BA compositions. At SSCS, all the BAs displayed the maximum contents, while the minimum BA contents were obtained from the control treatment. From control to SSCS, VA, β-RA, *p*-HBA, and SYA ranged from 12.24 to 37.15, 8.26 to 16.48, 8.55 to 14.23, and 7.36 to 11.52 μg g−<sup>1</sup> FW, respectively (Figure 7). VA, β-RA, *p*-HBA, and SYA progressively augmented in the order: Control < LSCS < MSCS < SSCS (Figure 7). VA, β-RA, *p*-HBA, and SYA under LSCS, MSCS, and SSCS were predominately augmented by 20%, 28%, 204%; 14%, 79%, 100%; 15%, 46%, and 66%; and 8%, 30%, and 57% than control, respectively (Figure 8).

SA, GAA, and PCA contents had no statistical variations at the control and LSCS level; however, three acids were augmented remarkably from LSCS to SSCS with a range from 23.83 to 45.82, 15.76 to 22.46, and 11.95 to 23.42 μg g−<sup>1</sup> FW, respectively (Figure 7). In MSCS and SSCS, SA, GAA, and PCA contents were augmented by (37% and 92%), (12% and 43%), and (41% and 96%), respectively (Figure 8). GA and ellagic acid (EA) ranged from 12.68 to 22.58 and 5.08 to 6.55 μg g−<sup>1</sup> FW. GA and EA had no statistical variations between control and LSCS levels and between MSCS and SSCS levels; however, the contents of these acids were augmented remarkably from control condition or LSCS to MSCS or SSCS level (26% and 77%) (Figures 7 and 8).

All the CA contents were sharply augmented under sodium chloride levels. All the CAs showed the highest contents under the SSCS level, whereas the control treatment exhibited the lowest CA contents. From control to SSCS, CHA, *m*-COA, and *p*-coumaric acid (*p*-COA) ranged from 14.38 to 27.35, 7.87 to 21.36, and 4.16 to 8.75 μg g−<sup>1</sup> FW, respectively, (Figure 9). Identified Cinnamic acids (CAs) have important biological activities. For instance, Caffeic acid (CA) and its derivatives have antioxidant, anti-inflammatory, and anticarcinogenic activity [139]. Chlorogenic acid was effective in preventing weight gain, inhibiting the development of liver steatosis, and blocking insulin resistance induced by a high-fat diet [140]. *p*-coumaric acid decreases low-density lipoprotein (LDL) peroxidation, shows antioxidant and antimicrobial activities, and plays an important role in human health [141]. Ferulic acid has low toxicity and possesses many physiological functions (anti-inflammatory, antioxidant, antimicrobial activity, anticancer, and antidiabetic effects). It has been widely used in the pharmaceutical, food, and cosmetics industries [142]. Sinapic acid shows antioxidant, antimicrobial, anti-inflammatory, anticancer, and anti-anxiety activity [143]. Cinnamic acids have been identified as interesting compounds with antioxidant, anti-inflammatory, and cytotoxic properties [144].

**Figure 8.** Comparison of BAs composition over control in *A. gangeticus* accession.

**Figure 9.** Response of CAs composition (μg g−<sup>1</sup> FW) under control, LSCS, MSCS, and SSCS in *A. gangeticus* accession; (n = 6), different letters in columns are varied significantly by DMRT (*p* < 0.01).

CHA, *m*-COA acid, and *p*-COA were progressively augmented in the order of control < LSCS < MSCS < SSCS (Figure 9). In LSCS, MSCS, and SSCS, CHA, *m*-COA acid, and *p*-COA were predominately augmented by 13%, 42%, 90%; 25%, 74%, 171%; and 23%, 65%, 110% compared with the control condition, respectively (Figure 10). *Trans*-CA and SIA contents at control condition were statistically similar to the LSCS level; however, these two acids' contents were remarkably augmented from LSCS to SSCS with a range from 9.85 to 18.62 and 11.35 to 12.56 μg g−<sup>1</sup> FW, respectively (Figure 9). In MSCS and SSCS, *Trans*-CA and SIA contents were augmented by 41% and 89%; and 6% and 11%, respectively (Figure 10). FA and CFA ranged from 8.20 to 20.45 and 6.56 to 7.62 μg g−<sup>1</sup> FW, respectively. FA and CFA contents at the control condition were statistically similar to the LSCS level,

and at the MSCS level were statistically similar to the SSCS level. However, the contents of these acids were remarkably augmented from the control condition or LSCS to MSCS or SSCS level (47% and 16%) (Figures 9 and 10).

All the PA fractions were sharply and remarkably augmented under sodium chloride stress. All the PA fractions exhibited the highest contents under SSCS level, whereas the control treatment had the lowest PA fractions. From control to SSCS, total BAs, total CAs, and total PAs ranged from 105.71 to 200.21, 62.37 to 116.71, and 168.08 to 316.92, μg g−<sup>1</sup> FW, respectively (Figure 11).

**Figure 11.** Increase of PA fractions (μg g−<sup>1</sup> FW) (total BAs, total CAs, and total PAs) under control, LSCS, MSCS, and SSCS in *A. gangeticus* accession; (n = 6); different letters in columns are varied significantly by DMRT (*p* < 0.01).

**Figure 10.** Comparison of CAs over control in *A. gangeticus* accession.

Total BAs, total CAs, and total PAs were progressively augmented in the order control < LSCS < MSCS < SSCS (Figure 11). In LSCS, MSCS, and SSCS, total BAs, total CAs, and total PAs were predominately augmented by 7%, 52%, and 89%), (8%, 52%, and 87%), and (7%, 52%, and 89%), compared with control condition, respectively (Figure 12).

**Figure 12.** Comparison of phenolics acid fractions (total BA, total CAs, and total PAs) over control in *A. gangeticus* accession.

Petropoulos et al. [99] reported the salinity-induced augmentation of PAs in *C. spinosum*. Klados and Tzortzakis [145] showed a progressive increment of total PAs under increased sodium chloride stress in *C. spinosum*. Alam et al. [101] reported salt-induced amelioration of phenolics in purslane. Ahmed et al. [103] reported a salinity-induced increment of PA profiles in barley. In contrast, Neffati et al. [146] stated the reduction of PA profiles with an increment of sodium chloride concentrations in coriander.

The cost is very low to maintain salt stress by adding sodium chloride to the plants. Furthermore, we suggested cultivating in salt-prone areas where there are no salt susceptible crops grown successfully. So, those areas will be efficiently utilized for amaranth leafy vegetable cultivation to meet the demand for the leafy vegetable of that locality, as leafy vegetables are too susceptible to salinity stress as amaranth is a salinity-tolerant leafy vegetable with up to 200 mM salt concentration. It can produce enough biomass and perform optimal photosynthesis at 100 mM saline stress. Amaranth is highly tolerant to salinity. It can tolerate 200 mM NaCl [147]. As amaranth is salt tolerant, it increases all enzymatic and non-enzymatic antioxidants, and metabolites, to detoxify ROS and cope with salt stress.

#### **4. Conclusions**

Sodium chloride stress remarkably augmented the energy, ash, carbohydrates, protein, calcium, dietary fiber, magnesium, S, Fe, Mo, Mn, Na, Cu, B, Zn, and ARP of *A. gangeticus* leaves. All the nutrients, phytochemicals, PA profiles, and ARP of *A. gangeticus* leaves under MSCS and SSCS levels were superior to the control. It can be utilized as a valued product for human consumption and health benefits. Salt-treated *A. gangeticus* leaves had abundant nutrients, phytochemicals, PA profiles, and ARP. Phytochemicals, PA profiles, and ARP scavenge ROS that would be advantageous for human health benefits as these bioactive compounds have potent antioxidants. Furthermore, sodium chloride-stressed *A. gangeticus* contributed with excellent quality in the end users for nutrients, phytochemicals, PA

profiles, and ARP. It can be cultivated as a promising substitute crop in sodium chlorideaffected areas of the world.

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

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

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that are recorded in the current study are available in all of the tables and figures of the manuscript.

**Acknowledgments:** For its support of the research work, the author acknowledges the Department of Genetics and Plant Breeding, the Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh.

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

#### **References**


### *Article* **Romanian Wild-Growing** *Armoracia rusticana* **L.—Untargeted Low-Molecular Metabolomic Approach to a Potential Antitumoral Phyto-Carrier System Based on Kaolinite**

**Adina-Elena Segneanu 1,\*, Gabriela Vlase 1,2, Liviu Chirigiu 3, Daniel Dumitru Herea 4, Maria-Alexandra Pricop 5, Patricia-Aida Saracin <sup>3</sup> and S, tefania Eliza Tanasie <sup>3</sup>**


**Abstract:** Horseradish is a globally well-known and appreciated medicinal and aromatic plant. The health benefits of this plant have been appreciated in traditional European medicine since ancient times. Various studies have investigated the remarkable phytotherapeutic properties of horseradish and its aromatic profile. However, relatively few studies have been conducted on Romanian horseradish, and they mainly refer to the ethnomedicinal or dietary uses of the plant. This study reports the first complete low-molecular-weight metabolite profile of Romanian wildgrown horseradish. A total of ninety metabolites were identified in mass spectra (MS)-positive mode from nine secondary metabolite categories (glucosilates, fatty acids, isothiocyanates, amino acids, phenolic acids, flavonoids, terpenoids, coumarins, and miscellaneous). In addition, the biological activity of each class of phytoconstituents was discussed. Furthermore, the development of a simple target phyto-carrier system that collectively exploits the bioactive properties of horseradish and kaolinite is reported. An extensive characterization (FT-IR, XRD, DLS, SEM, EDS, and zeta potential) was performed to investigate the morpho-structural properties of this new phyto-carrier system. The antioxidant activity was evaluated using a combination of three in vitro, non-competitive methods (total phenolic assay, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay, and phosphomolybdate (total antioxidant capacity)). The antioxidant assessment indicated the stronger antioxidant properties of the new phyto-carrier system compared with its components (horseradish and kaolinite). The collective results are relevant to the theoretical development of novel antioxidant agent fields with potential applications on antitumoral therapeutic platforms.

**Keywords:** secondary metabolites; horseradish; mass spectra; kaolinite; phyto-carrier system; antioxidant activity

#### **1. Introduction**

*Armoracia rusticana G. Gaertn.*, *B. Mey*. & *Scherb* (*Armoracia rusticana* L.) from the *Brassicaceae* family has been part of traditional European medicine since ancient times. The first mention of the healing effects of this plant (analgesic, diuretic, and antiparasitic) occurs in *De Materia Medica* [1]. Dacian medicine recommends horseradish as an anti-inflammatory cure for colds, coughs, and migraines [1]. Currently, horseradish root is used globally and on a large scale in food, food preservation, and traditional medicine [1].

**Citation:** Segneanu, A.-E.; Vlase, G.; Chirigiu, L.; Herea, D.D.; Pricop, M.-A.; Saracin, P.-A.; Tanasie, S, .E. Romanian Wild-Growing *Armoracia rusticana* L.—Untargeted Low-Molecular Metabolomic Approach to a Potential Antitumoral Phyto-Carrier System Based on Kaolinite. *Antioxidants* **2023**, *12*, 1268. https://doi.org/10.3390/ antiox12061268

Academic Editors: Antonella D'Anneo and Marianna Lauricella

Received: 24 May 2023 Revised: 24 May 2023 Accepted: 6 June 2023 Published: 13 June 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/).

It is known that there is an interdependence between the content of phytoconstituents in horseradish and different abiotic factors (pH, humidity, temperature, light, etc.) [2,3]. Furthermore, various studies reported that the profiles of metabolites considered responsible for the aroma of horseradish differ, depending on the genotype and plant maturity [3,4].

Recent research has shown that horseradish has collective therapeutic properties: antimicrobial, antifungal, anti-inflammatory, antiviral, and antitumor activity [5–8]. This herb's notable pharmacological activity is due to the combined and synergistic action of its numerous secondary metabolites: glucosinolates, isothiocyanates, organo-sulfur compounds, flavonoids, terpenoids, phenolic acids, coumarins, amino acids, and fatty acids [6–10].

Recently, particular consideration has been given to advanced materials based on natural compounds, which feature extended-release, site-specific delivery and outperform the alternatives in terms of therapeutic activity (anti-tumor, antioxidant, antiviral, antimicrobial, neuroprotective, and anti-inflammatory) [11]. Various studies have investigated the isolation of glucosinolates, isothiocyanates, and organo-sulfur compounds, the main bioactive compounds of horseradish. It has been reported that their chemical stability and implicit bioavailability are influenced by time and temperature [12].

Among the foremost challenges related to new drug discovery from natural products are the composition and proportion differences of secondary metabolites resulting from the influence of biotic and abiotic factors [11,13]. Furthermore, the total synthesis of some phytoconstituents with sophisticated chemical structures and numerous chiral centers is demanding [14].

The use of natural products, especially those based on medicinal plants, has seen an upward trend across the whole world in recent years [15–17]. Although, for the majority of the population, herbal medicine is the primary strategy used in various ailments, recently, particularly in developed countries, natural products have begun to take an increasingly important place in modern civilization due to their high level of biocompatibility and weak side effects [15–17].

Several studies have reported the possible toxic effects of the different herbal products available on the market, which are mainly due to self-administration and exceeding the dosage [15–17]. The pandemic also contributed to this situation, and numerous deaths and severe complications were registered due to the effective lack of medicines [15–17]. Therefore, the most recent studies address the development of new plant-based materials with high performance, binding-site specificity, and controlled release [11,13]. Particular consideration is given to secondary metabolites with high levels of antioxidant, antimicrobial, antiviral, anti-inflammatory, neuroprotective, and antitumor activity [11].

On the other hand, clay therapeutic, food, and protective applications are an integral parts of human culture [18]. In Mesopotamia, Ancient Egypt, and Ancient Greece, clay was used for its anti-inflammatory, antiseptic, and wound-healing properties [18]. The great scholars of the ancient world, Hippocrates and Aristotle, were the first to create a classification of therapeutic clays according to their origin, chemical composition, and biological activity [18].

Recent studies have demonstrated that due to its outstanding physico-chemical properties, including small grain size (in the micrometric order), and large specific surface area (of approximately 100 m2/g), ensuring high adsorption, swelling, intercalation, and cationexchange capacity, mineral clays can be used as carrier materials or drug-delivery-system substrates or supports [19–21].

In addition, various studies have confirmed the biological properties of clay minerals and reported high chemical stability and the absence of toxicity in vivo. Currently, clay mineral applications are used as active agents or excipients in numerous pharmaceutical and dermato-cosmetic preparations [19–23].

Kaolinite, Al2Si2O5(OH)4, with a ratio of SiO2 to Al2O3 of approximately 1.18:1, consists of a two-dimensional layer of silica groups linked to a layer of aluminum groups. The

distance between the two layers is about 7.2 Å, and it has minor cation-exchange capacity. Furthermore, hydrogen bonds restrict the possibility of expansion or swelling between layers. The surface area is 10–30 m2/g. Due to its high chemical stability and inertness in vivo, kaolinite has numerous pharmaceutical applications (anti-inflammatory, antiviral, detoxification, hemostatic, antitumoral, protection against gastro-intestinal problems and skin damage, pelotherapy, detoxification, and others) [23–26].

Kaolinite increases the bioavailability of the drug through a controlled release and an oral administration route [23,25]. Many studies reported different drug-delivery systems based on clay minerals for use as in antioxidant, anti-inflammatory, antibiotic, antitumor, antimycotic, anticoagulant, antidiabetic, osteoporosis, and cardioprotective, applications, among others. The main benefits are the prolonged release, increased bioavailability, and minimized toxicity [24,25,27].

The most recent studies addressed the development of antitumoral and immunomodulation drug-delivery systems [24,25,27,28].

It is well-known that the excessive generation of reactive oxygen species (ROS) causes the onset of serious pathologies, including cancer [29–32]. Numerous studies investigated the use of antioxidants as a novel and potent approach to cancer prevention and treatment [29–32]. It is acknowledged that the excessive generation of reactive oxygen species (ROS) causes the onset of serious pathologies, including cancer [29–32]. Consequently, many studies have investigated the antioxidant function as a novel and robust approach to cancer prevention and treatment [29–32]. However, there are still many controversies regarding the effectiveness of antioxidants in cancer therapy [29–32]. Nevertheless, the most recent studies reported some possible factors that can significantly reduce their beneficial effects, such as low bioavailability and low transmembrane permeability, the absence of an adequate dosage, uneven distribution, and others [32].

Furthermore, the biological activity of a plant is the result of the synergistic action of the mixture or complex of secondary metabolites in different proportions [9,33,34].

The antioxidant activities of phytoconstituents are determined by several factors: diversity, climatic factors (temperature, humidity, pH, and soil chemical composition), and harvest maturity stage [35]. Additionally, antioxidant agents are grouped into several categories depending on their mechanism of action (direct or indirect), their source, and the physical-chemical properties of the biomolecule (size, solubility, and others) [36–38]. The efficiency of an antioxidant agent is influenced by several criteria: metabolism pathway, bioavailability, rate constant, concentration, the chemical structure of the biomolecule, and others [36,39].

The development of a successful phyto-carrier assembly relies upon the complementary and synergistic action of the carrier and the secondary metabolites. Furthermore the morpho-structural characteristics, chemical and thermal stability, and biological properties of the carrier have an essential role [13].

Consequently, the high-performance carrier system based on kaolinite development represents a novel multifunctional strategy that will overcome the limitations of the current therapeutic approach related to the drug resistance of cancer cells and ensure site-specific targeting and controlled release.

This study investigates, for the first time, the development and characterization of a phyto-engineered carrier system that accumulates the biological properties of horseradish and kaolinite. Furthermore, to the best of our knowledge, another novelty of this study is the identification of a complete low-molecular-weight metabolite profile of *Armoracia rusticana*, grown in the wild in Romania.

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

All used reagents were analytical grade. Methanol, chloroform, dichloromethane, and ethanol were acquired from Sigma-Aldrich (München, Germany) and used without further purification. The DPPH (2,2-diphenyl-1-picrylhydrazyl), β-carotene Type II, synthetic (≥95%), ascorbic acid, AgNO3, sodium citrate, sodium carbonate, Folin–Ciocalteu

phenol reagent (2 N), potassium persulfate, sodium phosphate, ammonium molybdate, and potassium chloride of 99% purity or higher were purchased from Sigma-Aldrich (München, Germany). Propyl gallate (purum) was purchased from Fluka (Buchs, Switzerland). The horseradish sample (leaves (28 cm in height) and roots (lengths of about 35 cm) were collected in November 2022 from the area of Timis County, Romania (geographic coordinates 45◦45- 59.99" N 21◦17- 60.00" E) and taxonomically authenticated at the University of Medicine and Pharmacy Craiova, Romania. Kaolinite was purchased from local market in Timisoara, Romania. The double distilled water (DDW) was used throughout the experiments.

#### *2.1. Phyto-Carrier-System Components' Preparation*

#### 2.1.1. Plant-Sample Preparation for Chemical Screening

The plant samples (roots and leaves) were cut and then quickly frozen in liquid nitrogen (180 ◦C). Subsequently, they were ground and sieved to obtain a particle size lower than 0.45 mm and then stored at −38 ◦C to prevent enzyme-mediated degradation of phytoconstituents, in a 100 mL conical flask containing 1.5 g dried plant sample and 15 mL of solvent (methanol/chloroform = 1:1). Subsequently, the mixture was subjected to sonication extraction for 30 min at 35 ◦C with a frequency of 60 kHz. The resulting solution was concentrated using a rotary evaporator, and the obtained residue was dissolved in 10 mL MeOH. The obtained extract was centrifuged (10,000 rot/min, 10 min), and the supernatant was filtered through a 0.2 μm syringe filter and stored at −25 ◦C until further analysis. All samples were prepared in triplicate.

#### 2.1.2. GC-MS Analysis

Gas chromatography was carried out on a GCMS-QP2020NX Shimadzu apparatus with a ZB-5MS capillary column (30 m × 0.25 mm id × 0.25 μm) (Agilent Technologies, Santa Clara, CA, USA), helium, flow of 1 mL/min.

#### 2.1.3. GC–MS Separation Conditions

The oven-temperature program started from 50 ◦C to 300 ◦C with a rate of 5 ◦C/minute, and it was finally kept at this temperature for 3 min. The temperature of the injector was 280 ◦C and the temperature at the interface was 230 ◦C. The compounds' mass was registered at 70 eV ionization energy starting after 3 min of solvent delay. The source of the mass spectrometer was heated at 235 ◦C and the MS quad was heated at 165 ◦C. The mass values of identified compounds were scanned from 50 amu to 570 amu. Compounds were identified based on their mass spectra, which were compared to the NIST0.2 mass-spectra-library database (USA National Institute of Science and Technology Software, (NIST, Gaithersburg, MD, USA). Furthermore, the calculated retention indices (RIs) for each compound were compared with the Adams indices in the literature (Table 1) [40].

#### 2.1.4. Mass Spectrometry

The MS experiments were performed using EIS-QTOF-MS (Bruker Daltonics, Bremen, Germany). The mass spectra were acquired in the positive ion mode in a mass range of 100–3000 *m*/*z*, scan speed was 2.0 scans/s, collision energy was 25–85 eV, and the temperature of source block was 80 ◦C. The identification of phytoconstituents was based on standard library NIST/NBS-3 (National Institute of Standards and Technology/National Bureau of Standards) (NIST, Gaithersburg, MD, USA). The obtained mass-spectra values and the identified secondary metabolites are presented in Table 2.

#### 2.1.5. Phyto- Carrier System Preparation

For each analysis, 2.5 g of sample was prepared from dried horseradish, and kaolinite powder was added (horseradish/kaolinite nanoparticles = 1:3) at room temperature (22 ◦C), ground, and homogenized for 10 min using a pestle and mortar.

#### *2.2. Characterization of the Phyto- Carrier System*

#### 2.2.1. Fourier-Transform Infrared (FTIR) Spectroscopy

Data collection was conducted after 20 recordings at a resolution of 4 cm−1, in the range of 4000–400 cm−1, on Shimadzu AIM-9000 with ATR devices (Shimadzu, Kyoto, Japan).

#### 2.2.2. XDR Spectroscopy

The X-ray powder diffraction (XRD) was performed using a Bruker AXS D8-Advance X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) equipped with a rotating sample stage, Anton Paar TTK low-temperature cell (−180 ◦C ÷ 450 ◦C), high vacuum, inert atmosphere, relative humidity control, and Anton Paar TTK high-temperature cell (up to 1600 ◦C). The XRD patterns were compared with those from the ICDD Powder Diffraction Database (ICDD file 04-015-9120). The average crystallite size and the phase content were determined using the whole-pattern profile-fitting method (WPPF).

#### 2.2.3. Scanning-Electron Microscopy (SEM)

The SEM micrographs were obtained with a SEM–EDS system (QUANTA INSPECT F50) equipped with a field-emission gun (FEG), 1.2 nm resolution, and energy-dispersive X-ray spectrometer (EDS) with a MnK resolution of 133 eV.

#### 2.2.4. Dynamic Light Scattering (DLS) Particle-Size-Distribution Analysis

The DLS analysis was carried on a Microtrac/Nanotrac 252 (Montgomeryville, PA, USA). Each sample was analyzed in triplicate at room temperature (22 ◦C) at a scattering angle of 172◦.

#### 2.2.5. Zeta-Potential Analysis

The zeta-potential analysis was conducted using an AMERIGO particle-size and zetapotential analyzer (Pessac, France), with six measurements/s. The main experimental conditions were as follows. Electrode distance: 5 mm; temperature: 25 ◦C; conductivity: 5.10 V; carrier frequency: 8210 Hz; reference intensity: 2660 kcps; applied field: 20.27 V/cm; and scattering intensity: 2850 kcps.

#### 2.2.6. Antioxidant Activity

The antioxidant activity of the newly phyto-carrier system was estimated using three different assays: a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay, a Folin– Ciocalteu assay, and phosphomolybdate assay (total antioxidant capacity).

The phyto-carrier system (0.25 g) and horseradish (0.3 g) samples were dissolved in methanol (10 mL and 12 mL, respectively). The mixtures were stirred at room temperature (22 ◦C) for 8 h, and then centrifuged at 10,000 rpm for 10 min. The supernatant was then collected for use in the antioxidant assays (2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay, Folin–Ciocalteu assay, and phosphomolybdate assay (total antioxidant capacity).

#### 2.2.7. Determination of Total Phenolic Content

The total phenolic contents in the newly phyto-carrier system and horseradish samples were determined spectrophotometrically according to the Folin–Ciocalteu procedure adapted from the literature [41].

A volume of 2 mL of Folin–Ciocalteu reagent (0.2 N) and 0.2 mL of each sample were vortexed and stored at room temperature (22 ◦C) for 8 min, in the dark. Sequentially, 2 mL sodium carbonate (7.5%) was added. Next, after two h of incubation at room temperature (vortexed in the dark) the absorbance was measured at 725 nm using a Tecan i-control, 1.10.4.0 infinite 200 Pro spectrophotometer with Corning 96 flat-bottomed clear polystyrol plates (Tecan, Männedorf, Switzerland). The phenol content was expressed in gallic acid equivalents (mg GAE/g sample) using a propyl gallate standard calibration curve between 1 mg/mL and 12.5 μg/mL in methanol [42].

Sample extract concentrations were calculated based on the linear equation obtained from the standard curve (y = 0.9873x − 0.0989).

#### 2.2.8. DPPH Radical-Scavenging Assay

The stock solution was prepared by dissolving 2 mg DPPH in 20 mL MeOH followed by dilutions for a calibration curve with a range of concentrations between 3.12 μg/mL and 0.1 mg/mL. Serial dilutions of ascorbic acid and β-carotene were used as positive standards and MeOH as a vehicle control sample. The ratio (*v*/*v*) of DPPH to samples was of 1:1. All samples were placed, in triplicate, in a 96-well plate and stored at 22 ◦C for 30 min in the dark. At 515 nm, the absorbance was determined on a Tecan i-control, 1.10.4.0 infinite 200 Pro spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland).

The obtained results were used to calculate the average and the inhibition percentage (Inh%) (Equation (1)).

$$\text{Inh}\%= \text{(A0}-\text{As)}/\text{A0}\times100\tag{1}$$

where:

A0 = vehicle control absorbance;

As—sample absorbance.

Further, the IC50 value was obtained from the inhibition percentage using the equation of a calibration curve generated for each sample and standard. The results were presented as Inh% versus concentration (μg/mL) [43].

#### 2.2.9. Phosphomolybdate Assay (Total Antioxidant Capacity)

The total-antioxidant-capacity assay of the new phyto-carrier system and horseradish samples was carried out by the phosphomolybdenum procedure using ascorbic acid as standard [44].

A volume of 5 mL reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) and 0.5 mL of each sample were placed into a water bath at 95 ◦C for 120 min. Next, the mixed solutions were cooled at room temperature (22 ◦C). The absorbance was measured at 765 nm using a UV-VIS Perkin-Elmer Lambda 35 (Perkin Elmer, Waltham, MA, USA).

A blank solution was used (5 mL reagent was added in 0.5 mL methanol, and then the mixture was incubated in the same experimental conditions (at 95 ◦C for 120 min, and then cooled at room temperature (22 ◦C)). Total antioxidant capacity was determined according to the following equation (Equation (2))

Total antioxidant capacity (%) = [(Abs. of control − Abs. of sample)/(Abs. of control] × 100 (2)

The results are presented as μg/mL of ascorbic acid equivalents (AAE).

#### 2.2.10. Statistical Analysis

All results were obtained with Microsoft Office Excel 2019. Data were used to calculate the average of three replicates for all samples, and all calibration curves and concentrations.

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

Plants contain an extensive range of categories of secondary metabolites, with complex chemical compositions [45,46].

In recent years, numerous studies addressed the phytochemical composition and pharmacological activities of metabolites from horseradish roots [4–8,47–54]. There are relatively few studies related to the phytoconstituents from horseradish leaves [1,7,8,55].

Nevertheless, a specific plant's biological activity is the synergistic action of whole phytoconstituent result. Furthermore, researchers have reported that various biotic or abiotic factors (stress, pathogens, and others) altered the metabolite balance and, implicitly, their variability and interrelation [56–58]. In addition, several other elements (the part of the plant used, the extraction process, and the solvent used) influence the type and proportion of bioactive compounds collected from plants [58–61]. Therefore, a plant extract's pharmacological activity differs from the experimental conditions, making it difficult to evaluate the relationship between chemical composition and therapeutic effect [58].

The chemical screening of the phytoconstituents from the horseradish sample was carried out via gas chromatography coupled with mass spectroscopy (GC-MS) and electrospray ionization–quadrupole time-of-flight mass spectrometry (ESI–QTOF–MS) analysis.

The gas-chromatography method coupled with mass spectroscopy (GC–MS) is the most convenient technique for secondary metabolites with relatively low molecular mass (volatile compounds, fatty acids, etc.), providing efficient separation and identification [62].

The GC–MS analysis (Figure 1) revealed the separation of several low-molecularweight metabolites from the horseradish sample.

The results are summarized in Table 1, which presents the tentative compound identification from the horseradish sample using GC–MS.




**Table 1.** *Cont.*

RI—retention indices calculated based upon a calibration curve of a C8–C20 alkane standard mixture.

The GC–MS analysis showed the presence of seventeen major components, accounting for 95.13% of the total peak area in the horseradish samples (Figure 1).

However, thermally unstable biomolecules require additional procedures (for instance, derivatization). Therefore, the mass-spectrometry method was selected for the metaboliteprofile screening [65].

#### *3.1. Mass-Spectrometric Analysis of Horseradish Sample*

The spectra revealed a complex combination of low-molecular-weight components, of which some were detected. The mass spectra of the identified metabolites were compared with those of the NIST/EPA/NIH Mass Spectral Library 3.0 database, in addition to a literature review [7,48,55,66]. The mass spectrum and the phytoconstituents identified by the ESI–QTOF–MS analysis are presented in Figure 2 and Table 2, respectively.

**Figure 2.** The mass spectrum of *Armoracia rusticana* L.

**Table 2.** The molecules identified through electrospray-ionization–quadrupole time-of-flight mass spectrometry (ESI–QTOF–MS) analysis.



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

44 166.21 165.19 C9H11NO2 phenylalanine amino acid [51]


**Table 2.** *Cont.*


**Table 2.** *Cont.*

The metabolite profile from the horseradish sample conducted through the GC–MS and mass spectroscopy corroborated the data reported in the literature [6–8,48–55,63–65].

#### *3.2. Screening and Classification of the Differential Metabolites*

The 90 secondary metabolites identified through mass spectroscopy were assigned to different chemical classes: glucosilates (18.9%), fatty acids (11.12%), isothiocyanates (8.9%), amino acids (8.9%), phenolic acids (6.67%), flavonoids (4.45%), terpenoids (3.34%), coumarins (2.23%), and miscellaneous. The assignment of the identified secondary metabolites into different chemical categories is presented in Table 3.

**Table 3.** Classification of bioactive secondary metabolites from the *Armoracia rusticana* L. sample in different chemical categories.



**Table 3.** *Cont.*


**Table 3.** *Cont.*

Figure 3 presents the classification chart of the phytoconstituents from the horseradish sample based on the data analysis reported in Table 3.

**Figure 3.** Phytoconstituent-classification bar chart for *Armoracia rusticana*.

According to Figure 3, *glucosinolates* are the largest category of phytochemicals, comprising about 19% of the total found in the horseradish sample. Recent studies demonstrated their antioxidant, anti-inflammatory, and antitumoral properties [7,66,67].

*Isothiocyanates* are a category of metabolites characteristic of cruciferous plants, with remarkable anti-cancer, anti-inflammatory, and neuroprotective effects [67,68].

Organo-sulfur phytoconstituents represented over 30% of all the metabolites identified in the horseradish sample. Various studies have shown that sulfur phytochemicals possess antioxidant, antiviral, antifungal, antibacterial, and antitumor properties [7,54,69,70].

*Fatty acids* represented more than 11% of the phytoconstituents identified in the horseradish sample. These secondary metabolites exhibit antioxidant, anti-inflammatory, cardio, and neuroprotective activities [71,72].

*Amino acids*: eight compounds were identified in the sample extract; the proportions of non-essential amino acids (proline, cysteine, asparagine, and tyrosine) and essential amino acids (threonine, lysine, phenylalanine, and arginine) were equal [73–75].

About one-third of the amino acids identified in the horseradish sample (arginine, phenylalanine, and proline) act as antitumor, neuroprotective, antiproliferative, and immunomodulating agents [71,74–77].

*Phenolic acids* are another class of phytochemicals with outstanding therapeutic properties (antioxidants, anti-inflammatory, antimicrobial, antidiabetic, antitumor, neuroprotective) [78,79].

The *Terpenoids* found in the horseradish samples were α-phellandrene, junipene, and menthol. Studies have reported that these have antitumor properties. Furthermore, menthol also acts as an antibacterial, antifungal, antipruritic, and analgesic agent [80–82].

*Flavonoids* are other category of secondary metabolites identified in the horseradish sample with notable pharmacological proprieties, including antioxidant, anti-inflammatory, antitumoral, and antimicrobial properties, as well as activities against neurodegenerative diseases (Alzheimer's) [73,79,83].

The two *coumarins* identified in the horseradish sample, scopoletin and esculetin, show exceptional therapeutic activity, with antioxidant, anti-inflammatory, antitumor, hepatoprotective, and antidiabetic properties, as well as activities against neurodegenerative diseases (Alzheimer's) [84,85].

Among the *miscellaneous* compounds identified in the horseradish sample, sphingosine exerts antitumoral, immunomodulatory, and neuroprotective activities [86–88]. Furthermore, glucogallin possesses antioxidant, anti-inflammatory, and antidiabetic properties [87].

The aromatic compounds of volatile metabolites (VOCs) identified in the horseradish sample are shown in Table 4 and Figure 4.




**Figure 4.** VOC-aroma profile of phytoconstituients identified in horseradish sample.

The predominant aromatic components of the investigated Romanian horseradish depend on different conditions (climatic conditions, maturity soil parameters, varieties, harvest time, and others) [4,7,47–49].

Their fragrances are unique, encompassing a purgent aroma with rocket and sulfuric, green, sweet-vanilla, and floral notes [4,7,47,48].

#### *3.3. Phyto-Carrier System*

The main challenges in the novel therapeutic approaches to cancer are the drug resistance of cancer cells, determined by the reduced retention interval, low permeability, the triggering of inactivation by the immune system, and the lack of specificity [89,90].

Hence, the development of an innovative phyto-carrier target system with cumulative and synergistic kaolinite and horseradish biological activity could make it possible to overcome the limitations related to vectorization, site-specific distribution, prolonged release, and membrane permeability.

#### *3.4. FT-IR Spectroscopy*

The use of FTIR is one of the most common analytical techniques, and it is considered fundamental in the analysis of complex carrier systems due to its features (sensitivity, flexibility, robustness, and specificity), allowing the investigation of interactions between biomolecules and mineral components [90].

The incorporation of the horseradish phytoconstituents into the pores of the kaolinite particles was successfully achieved and confirmed through FT-IR spectroscopy. Figure 5A presents the spectra of the horseradish, the kaolinite particles, and the new phyto-carrier system.

**Table 5.** The characteristic absorption bands attributed to secondary metabolites identified in *Armoracia rusticana*.


(**A**)

**Figure 5.** *Cont*.

**Figure 5.** (**A**) FTIR spectra of kaolinite, horseradish, and phyto-carrier system. (**B**) FTIR spectra of FT-IR absorption bands identified in the horseradish sample are presented in the following table (Table 5).

The FTIR peak of the kaolinite (Figure 5B) presented vibrational bands characteristic at 3686, 3652, 3619, and 3552 cm−<sup>1</sup> (attributed to the OH stretching vibrations), and 1117, 1066, 980, and 912 cm−<sup>1</sup> (associated with the Si-O stretching vibration) [100–103].

The data obtained and presented in Figure 5 confirm the successful development of the new phyto-carrier system.

The obtained IR spectra of the new phyto-carrier system incorporated peaks specific to the secondary metabolites from the horseradish at the following: 3330 cm−1, assigned to the OH group; 2760 cm−1, attributed to the O-H stretching in the amino acids; 2055 cm−<sup>1</sup> (N=C=S stretching of isothiocyanate); 1730 cm−<sup>1</sup> (C-H stretching by methylene groups); 1470 cm−<sup>1</sup> (C-H bending); 1367 cm−<sup>1</sup> (O-H bending); 1255 cm−<sup>1</sup> (C-O stretching); 1028 cm−<sup>1</sup> (C-N stretching); 959 and 925 cm−<sup>1</sup> (symmetric N-C-S stretch); and 680 cm−<sup>1</sup> (aromatic ring); and the characteristic absorption bands of the kaolinite [90–103].

In addition, the kaolinite absorption bands at 3686, 3652, 3619, and 3552 cm−<sup>1</sup> (attributed to O-H stretching vibrations) and the vibrational bands at 1117, 1066, 980, and 912 cm−<sup>1</sup> (associated with Si-O stretching vibration) were shifted to lower wavenumbers, indicating that this functional group was involved in the binding of the O-H, C-N, N-H, and C-O functional groups from the horseradish (Figure 5, Table 5) [90–103].

Moreover, several detectable changes occurred in the horseradish spectra, particularly the hydroxyl vibrations (O-H stretching and O-H bending), indicating that this functional group is involved in the binding of kaolinite [23–27,104].

#### *3.5. X-ray-Diffraction Spectroscopy*

The XRD technique was used to obtain information about the atomic structure of the phyto-carrier system and the raw materials.

Figure 6 displays the XRD patterns of the horseradish sample and the new phytocarrier system.

**Figure 6.** The overlapping XRD spectra of horseradish sample and new phyto-carrier system.

In the XRD spectrum of the new phyto-carrier system, the characteristic XRD peaks of the kaolinite and horseradish samples are easily observable. Hence, the absorption peaks at 2θ (degrees) values of 12◦, 25◦, 34◦, 36◦, and 51◦ can be assigned to a triclinic structure [105].

The XRD pattern of the horseradish sample (Figure 6) was in the range of 11.8–34.6◦, with large bands and weak peaks characteristic of amorphous phases, which can be attributed to the phytoconstituents from the horseradish (minerals, hydroxides, and fibers).

#### *3.6. Scanning-Electron Microscopy–Energy-Dispersive X-ray (SEM–EDX)*

Scanning-electron microscopy–energy-dispersive X-ray (SEM–EDX) is a versatile technique to investigate the morphologies, compositions, and microstructures of materials. In some complex materials, it allows the identification of the component phases through qualitative chemical analysis [106].

The morphological changes (the size, shape, and distribution of the particles) in the horseradish and kaolinite samples before and after the preparation of the new phyto-carrier system were investigated by using the SEM–EDX technique.

To acquire insights, the SEM micrographs were recorded at different magnifications. The obtained two-dimensional images are shown in Figure 7.

**Figure 7.** SEM images of kaolinite (**A**,**B**), horseradish (**C**,**D**), and phyto-carrier system (**E**,**F**).

The SEM micrograph of the kaolinite sample (Figure 7A,B) exhibited a heterogeneous size distribution of small anhedral and pseudo-hexagonal particles up to 5 μm in size [25].

It appears that the horseradish micrographs (Figure 7C,D) indicated the presence of a heterogeneous fibrous structure, with a thickness of about a few μm, with porous regions with irregular shapes. These porous regions allowed the arrest of the kaolinite particles.

The morphology of the phyto-carrier system (Figure 7E,F) indicated the presence of kaolinite particles both on the surface and in the porous areas of the horseradish sample. Changes in the sizes of the horseradish and kaolinite particles (reduction) were observed, which can be explained by the experimental conditions of the new phyto-carrier system preparation.

Accompanying the SEM spectra are EDX analyses on the elemental composition of the kaolinite and phyto-carrier investigated (Figure 8A,B).

**Figure 8.** (**A**) EDX composition of kaolinite sample; (**B**) EDX composition of the new phytocarrier system.

According to the data from the EDX (Figure 7A), the predominant element contents in the kaolinite sample were silica, aluminum, magnesium, calcium, potassium, iron, sodium, oxygen, and sulphur. Overall, the chemical analysis revealed the significant oxides SiO2, Al2O3, Fe2O3, MgO, CaO, Na2O, K2O5, and SO<sup>−</sup>3, which was in good agreement with the data reported in the literature [25].

The comparative analysis in Figure 8B highlights the presence of peaks corresponding to the kaolinite (Figure 8A) in the new phyto-carrier system. The EDX results confirmed the preparation of the new phyto-carrier system.

#### *3.7. Dynamic Light Scattering (DLS)*

Dynamic light scattering (DLS) is a fast and very efficient method for determining the sizes of particles and the particle-size distribution (PSD) in suspensions [107]. Particlesize measurement is established indirectly by using the intensity of the light-scattered fluctuations, yielding the rate of the Brownian motion [107].

The DLS method was used to obtain information about the average mean particle size of the phyto-carrier system and its raw components. The DLS results are displayed in Figure 9.

**Figure 9.** DLS patterns of kaolinite (**A**), horseradish sample (**B**), and phyto-carrier system (**C**).

The average diameter size of the kaolinite particles was 500.03 nm (Figure 9A), corroborating the SEM results. In the horseradish sample (Figure 9B), the average diameter of the particles was 100.2 nm.

The DLS pattern of the new phyto-carrier system (Figure 9C) exhibited two peaks that can be attributed to the kaolinite and horseradish particles, distributed in a narrow range. The mean diameter of the kaolinite in the phyto-carrier system was 277.5 nm. The second mean of the hydrodynamic diameter, associated with the horseradish particles, was about 186.4 nm. The fact that the average diameter of the horseradish particles in the phyto-carrier increased compared to that determined in the horseradish sample can

be attributed to the loading of the pores on the plant surfaces with the kaolinite particles, which was confirmed by the results of the SEM analysis.

Furthermore, the reduction in the mean size of the kaolinite particles from 500.03 nm (Figure 9A) to 277.5 nm (Figure 9C) was attributed to the experimental conditions for the preparation of the new phyto-carrier. In addition, Figure 9C shows well-dispersed particles of horseradish and kaolinite, which indicates the high stability of the new phyto-carrier system.

#### *3.8. Zeta Potential*

The zeta-potential method determines the charge of a particle in a suspension, providing an estimation of interactions between particles and the suspension stability.

The zeta-potential value of the kaolinite particles was −35.09 mV, indicating the high stability of the suspension, in good agreement with the data reported in the literature [108].

The zeta potential changed to −23.12 mV for the phyto-carrier system, indicating high biocompatibility.

#### *3.9. Screening of Antioxidant Activity*

For a specific herb, the total antioxidant capacity (TAC) is the outcome of the cumulative action of entire antioxidant classes from its composition [37]. The adequate investigation of the antioxidant activity of a plant requires an appropriate variety of tests to address the mechanism of action characteristic of each category of phytochemicals [37,38,109,110].

Various chemical (spectrometric, chromatographic, and electrochemical) and biochemical methods have been developed for the assessment of the antioxidant capacities of different biomolecules [37,38,109,110]. The most common are the in vitro tests, divided based on the reaction-mechanism type into hydrogen-atom transfer (HAT) and electron transfer (ET) methods [37,38,109,110].

The first category, HAT methods, includes the oxygen-radical-absorbance capacity (ORAC), the total radical-trapping-antioxidant parameter (TRAP), the total radicalscavenging-capacity assay (TOSCA), the chemiluminescent assay, β-carotene bleaching assays, and the inhibition of induced LDL oxidation [37,38,111–113].

The main ET methods (based on electron transfer) are the total phenolics assay (Folin–Ciocalteu reagent assay), the 2,2-Diphenyl-1-picrylhydrazyl radical-scavenging assay (DPPH•), the Trolox equivalence antioxidant-capacity assay (TEAC), the ferricion-reducing antioxidant-power assay (FRAP), the cupric reducing antioxidant capacity (CUPRAC) assay, the N,N-Dimethyl-p-phenylenediamine radical-scavenging assay (DMPD•+), and the 2,2-Azinobis 3-ethylbenzthiazoline-6-sulfonic acid radical-scavenging assay (ABTS•+) [37,38,109,110].

The choice of a particular method depends on criteria related to simplicity, sensitivity, associated costs, and reproducibility [37–39,111,112].

The biological activity of a plant varies depending on the complexity of the chemical composition and, implicitly, on the collective, complementary, and the synergistic actions of a variety of secondary metabolites. Moreover, the antioxidant activities of plants differ, depending on morphological parts, degree of maturity, and exogenous parameters (temperature, pH, humidity, and others) [37].

Hence, the antioxidant activity of the phyto-carrier system is a combined result of the complementary and synergistic actions of its components (horseradish and kaolinite). A total amount of ninety secondary metabolites from nine different chemical classes were identified in the horseradish sample. Consequently, to consider the antioxidant properties of the new phyto-carrier system more precisely, three different in vitro, noncompetitive methods were used (DPPH, Folin–Ciocalteu, and phosphomolybdate (total antioxidant capacity).

3.9.1. DPPH (1,1-diphenyl-2-picrylhydrazyl) Free-Radical-Scavenging Assay

The DPPH (2,2-diphenyl-1-picrylhydrazyl) is a fast, simple, low-cost, and accurate method based on a single electron transfer (ET)-type mechanism for the antioxidant assessment of plant extracts or other complex matrices. Furthermore, it is a highly frequently used assay to determine the free scavenging capacity of antioxidants based on the ability of compounds to act as free-radical scavengers or hydrogen donors [37–39,110–113].

Hence, the antioxidant activity of the new phyto-carrier system and its components were evaluated in relation to the antioxidant standards of β-carotene and ascorbic acid. It is noteworthy that different studies reported the presence of β-carotene and ascorbic acid in the chemical composition of horseradish [114,115]. The data obtained are presented in Table 6 and Figure 10.

**Table 6.** IC50 values for horseradish, the new phyto-carrier system, ascorbic acid, and beta-carotene.

**Figure 10.** Graphic representation of DPPH results expressed as IC50 (μg/mL).

The obtained IC50 values indicated that the antioxidant activity of the new phytocarrier system was higher than that of the horseradish sample, the kaolinite, and the ascorbic acid. For the new phyto-carrier system, the IC50 value was about half that of the horseradish sample. The increase in the antioxidant activity of the phyto-carrier system compared to the horseradish and kaolinite was in good agreement with the literature data [116,117]. The IC50 value for the beta-carotene standard can be explained by the experimental conditions (the low solubility of beta carotene in methanol) [118].

#### 3.9.2. Folin–Ciocalteu Assay

This assay is widely used as a fast, simple, precise, and inexpensive measure of total phenolics from natural products based on an oxidation/reduction-reaction mechanism (electron transfer) [38,39,41,119,120].

The total polyphenolic contents (TPCs) of the horseradish sample and phyto-carrier system were determined and the obtained results are presented in Table 7.

**Table 7.** Total polyphenolic contents in horseradish and the phytocarrier system.


According to the results, the total polyphenolic content identified in the new phytocarrier system was more than 39% higher than that of the horseradish sample. The higher

antioxidant capacity of the phyto-carrier system compared to the horseradish sample can be attributed to the synergistic action of the kaolinite and corresponds to the data reported in the literature [121].

#### 3.9.3. Phosphomolybdate Assay (Total Antioxidant Capacity)

Phosphomolybdate (total antioxidant capacity) is a frequently used and precise assay used to evaluate the total antioxidant potentials of plant extracts or other complex mixtures of biomolecules. It is based on the Mo(VI)-to-Mo(V) reduction of the presence of antioxidants [44].

The phosphomolybdate assay (total antioxidant capacity) was used to determine the total antioxidant potential of the prepared phyto-carrier system compared to those of the horseradish and ascorbic acid. The obtained experimental results are displayed in Table 8 and Figure 11.

**Table 8.** Total antioxidant potentials of phyto-carrier system and horseradish sample.

**Figure 11.** Graphic representation of phosphomolybdate (total antioxidant capacity) results expressed as μg/mL AAE.

The phyto-carrier system displayed a higher antioxidant activity than the horseradish sample. This result can be attributed to the synergistic and complementary action of the phytoconstituents in the horseradish and the antioxidant mechanism of the kaolinite [28]. In addition, the kaolinite potentiated the antioxidant activities of secondary metabolites in the horseradish sample [121].

#### **4. Conclusions**

In this study, a new phyto-carrier system with particular morpho-structural properties and high antioxidant activity was prepared. The low-molecular-mass-metabolite profiling and the VOC-aroma profile of the *Armoracia rusticana* grown in the wild in Romania were determined. The biological activities of each identified phytoconstituent category in the horseradish were discussed. The development of the horseradish–kaolinite carrier system was confirmed through FTIR, EDX, XRD, DLS, zeta-potential, and SEM studies. The size distributions of the kaolinite and horseradish particles were investigated through a DSL analysis. The kaolinite and the phyto-carrier system's stability levels in aqueous suspensions were determined using a zeta-potential analysis. A combination of assays (DPPH, Folin–Ciocalteu, and phosphomolybdate (total antioxidant capacity)) was used to evaluate the antioxidant properties of the proposed phyto-carrier system. The results demonstrated the significantly higher antioxidant activity of the phyto-carrier compared with its components (horseradish and kaolinite). However, further studies are required to investigate the biological activity, bioavailability, and biocompatibility of the new phytocarrier system. This study may motivate future research on therapies in the area of advanced antitumoral agents.

**Author Contributions:** Conception and study design: A.-E.S.; methodology: A.-E.S.; data acquisition: G.V., M.-A.P., D.D.H., P.-A.S. and S, .E.T.; analysis and data interpretation: L.C., G.V., M.-A.P. and D.D.H.; writing—original draft preparation: A.-E.S.; writing—review and editing: A.-E.S. and D.D.H.; investigation: G.V., M.-A.P., S, .E.T. and D.D.H. All authors have read and agreed to the published version of the manuscript.

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

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

**Informed Consent Statement:** Not applicable.

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

**Acknowledgments:** National Center for Micro and Nanomaterials (the Center is part of the Department of Science and Engineering of Oxide and Nanomaterials Materials of the Faculty of Applied Chemistry and Materials Science of the Politehnica University of Bucharest). This work was supported by a grant of the Ministry of Research, Innovation and Digitization CNCS–UEFISCDI, project number PN-III-P4-PCE-2021-1081 within PNCDI III (Contract no. 75/2022).

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

#### **References**


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### *Article Dryopteris juxtapostia* **Root and Shoot: Determination of Phytochemicals; Antioxidant, Anti-Inflammatory, and Hepatoprotective Effects; and Toxicity Assessment**

**Abida Rani 1, Muhammad Uzair 1,\*, Shehbaz Ali 2,3, Muhammad Qamar 4,\*, Naveed Ahmad 5, Malik Waseem Abbas <sup>6</sup> and Tuba Esatbeyoglu 7,\***


**Abstract:** An estimated 450 species of *Dryopteris* in the Dryoperidaceae family grow in Japan, North and South Korea, China, Pakistan, and Kashmir. This genus has been reported to have biological capabilities; however, research has been conducted on *Dryopteris juxtapostia*. Therefore, with the present study, we aimed to exploring the biological potential of *D. juxtapostia* root and shoot extracts. We extracted dichloromethane and methanol separately from the roots and shoots of *D. juxtapostia*. Antioxidant activity was determined using DPPH, FRAP, and H2O2 assays, and anti-inflammatory activities were evaluated using both in vitro (antiurease activity) and in vivo (carrageenan- and formaldehyde-induced paw edema) studies. Toxicity was evaluated by adopting a brine shrimp lethality assay followed by determination of cytotoxic activity using an MTT assay. Hepatoprotective effects of active crude extracts were examined in rats. Activity-bearing compounds were tentatively identified using LC-ESI-MS/MS analysis. Results suggested that *D. juxtapostia* root dichloromethane extract exhibited better antioxidant (DPPH, IC50 of 42.0 μg/mL; FRAP, 46.2 mmol/g; H2O2, 71% inhibition), anti-inflammatory (urease inhibition, 56.7% at 50 μg/mL; carrageenan-induced edema inhibition, 61.7% at 200 μg/mL; formaldehyde-induced edema inhibition, 67.3% at 200 μg/mL), brine shrimp % mortality (100% at 1000 μg/mL), and cytotoxic (HeLa cancer, IC50 of 17.1 μg/mL; prostate cancer (PC3), IC50 of 45.2 μg/mL) effects than *D. juxtapostia* root methanol extract. *D. juxtapostia* shoot dichloromethane and methanol extracts exhibited non-influential activity in all biological assays and were not selected for hepatoprotective study. *D. juxtapostia* root methanol extract showed improvement in hepatic cell structure and low cellular infiltration but, in contrast the dichloromethane extract, did not show any significant improvement in hepatocyte morphology, cellular infiltration, or necrosis of hepatocytes in comparison to the positive control, i.e., paracetamol. LC-ESI-MS/MS analysis showed the presence of albaspidin PP, 3-methylbutyryl-phloroglucinol, flavaspidic acid AB and BB, filixic acid ABA and ABB, tris-desaspidin BBB, tris-paraaspidin BBB, tetra-flavaspidic BBBB, tetra-albaspidin BBBB, and kaempferol-3-*O*-glucoside in the dichloromethane extract, whereas kaempferol, catechin, epicatechin, quinic acid, liquitrigenin, and quercetin 7-*O*-galactoside in were detected in the methanol extract, along with all the compounds detected in the dichloromethane extract. Hence, *D. juxtapostia* is safe, alongside other species of this genus, although detailed safety assessment of each isolated compound is obligatory during drug discovery.

**Citation:** Rani, A.; Uzair, M.; Ali, S.; Qamar, M.; Ahmad, N.; Abbas, M.W.; Esatbeyoglu, T. *Dryopteris juxtapostia* Root and Shoot: Determination of Phytochemicals; Antioxidant, Anti-Inflammatory, and Hepatoprotective Effects; and Toxicity Assessment. *Antioxidants* **2022**, *11*, 1670. https://doi.org/ 10.3390/antiox11091670

Academic Editors: Marianna Lauricella and Antonella D'Anneo

Received: 20 July 2022 Accepted: 24 August 2022 Published: 27 August 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/).

**Keywords:** cytotoxicity; hepatoprotective effects; HeLa cancer; inflammation; mass spectrometry; oxidation; prostate cancer; phytochemical

#### **1. Introduction**

Generation of free radicals in living systems is associated with intrinsic (stress) and extrinsic (alcohol, smoking, and radiation) factors, whereas antioxidant mechanisms help to neutralizing the negative impacts induced by oxidative stress [1]. Oxidative stress is a condition whereby imbalance occurs between reactive oxygen species (ROS) and the body's antioxidant system. Undue generation of ROS disrupts the normal functioning of important organs and can lead to the onset of many ailments, including inflammation, cancer, polygenic disorders, diabetes, and aging [2–4].

Pakistan, regardless of being an agricultural territory, is home to various ecological zones with many indigenous medicinal plant species [5,6]. However, limited research has been conducted to evaluate their pharmaceutical prominence due to the phytochemical potential of secondary metabolites [7]. Of 5700 reported medicinal plant species, Pakistan possesses 500-600 species, with only a few having been probed for biochemical assessment [8]. The *Dryopteris* genus of the Dryoperidaceae family of the North Temperate Zone comprises more than 450 species grown in Japan, North and South Korea, China, Pakistan, and Kashmir [9]. *Dryopteris ramose* and *Dryopteris cochleata* extracts, i.e., species from the aforementioned genus, have been reported to exhibit antioxidant activity [10,11]. *Dryopteris chrysocoma*, *Dryopteris blanfordii*, and *Dryopteris crassirhizoma* were reported to exhibit anti-inflammatory activities [12–14]. In other studies, *Dryopteris fragrans* and *Dryopteris crassirhizoma* extracts were found to possess anticancer potential [15,16]. However, *Dryopteris juxtapostia* extracts have not been explored for biological potential to date. Therefore, the aim of the present study is to explore phytochemical, radical-scavenging, anti-inflammatory, cytotoxic, and hepatoprotective potential of *Dryopteris juxtapostia* root and shoot extracts.

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

#### *2.1. Plant Material and Its Preparation*

*Dryopteris juxtapostia* (DJ) roots and shoot were collected from the Sawat area (Tehsil Matta upper swat. KPK., Village, Shukhdara, Biha, Charrma, Fazal Banda), Pakistan, and identified and authenticated at the Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan. The plant was assigned voucher no. tro-26609785. For the purpose of effective extraction, whole DJ root and shoot material was shade-dried for 15 days. Then, dried plant material was ground in a blender and weighed. Extraction was performed with dichloromethane and methanol in an orbital shaker in the dark for 48 h. The process was repeated three times, and the solvent was evaporated using a rotary evaporator (Heidolph, Schwabach, Germany) in to obtain semisolid plant material.

#### *2.2. Quantification of Total Phenolic and Flavonoid Contents*

Total phenolic contents were determined by Folin–Ciocalteu (FC) colorimetric assay using gallic acid as standard [17]. Absorbance was recorded at 765 nm using a spectrophotometer (UV-Vis 3000 ORI, Reinbeker, Germany), and values were recorded in triplicate using ethanol as a blank. Total flavonoid contents were determined using an AlCl3 assay [18]. Sample absorbance was read at 510 nm using a spectrophotometer (UV-Vis 3000 ORI). A quercetin standard curve was plotted; sample results are expressed as mg quercetin equivalents per gram (mg QE/g) of the dried weight.

#### *2.3. Antioxidant Activity*

DJ dichloromethane and methanol crude extracts of roots and shoots were evaluated for antioxidant activity using three assays i.e., DPPH, H2O2, and FRAP assays, as adopted by Qamar et al. (2021) [19]. Samples were prepared as 1 g/20 mL for all three

assays. In the DPPH and H2O2 assays, distilled water was used as a blank, with quercetin (125 μg/mL) as standard. The findings are reported as percent inhibition according to the following equation:

%Inhibition = [(absorbance of control − absorbance of sample/standard) ÷ absorbance of control] × 100

For the FRAP assay, ferrous sulphate was used for calibration. Results are expressed as Fe mmol/g.

#### *2.4. Anti-Inflammatory Activity*

#### 2.4.1. Urease Inhibition Assay (In Vitro)

Weatherburn's indophenol method was used to evaluate the urease activity by determining the ammonia production in the reaction mixture [20]. The reaction mixture comprising 25 μL jack bean urease enzyme, 55 μL buffer (100 mM urea), and 5 μL test compounds (0.5 mM) was incubated for 15 min in a 96-well plate at 30 ◦C. Urease activity was assessed by Weatherburn's method by measuring ammonia production using indophenol. In brief, 70 μL alkali (0.1% active chloride (NaOCl) and 0.5% NaOH *w/v*) and 45 μL phenol reagents (0.005% *w/v* sodium nitroprusside and 1% *w/v* phenol) were added to each well. After 50 min, the increase in absorbance was measured at 630 nm with a microplate reader (Molecular Device, Ramsey, NJ, USA). The reaction was performed in a triplicate run, with pH 6.8 and a final volume of 200 μL. Absorbance readings were processed with Max Pro software (Molecular Device, USA), and % inhibition was calculated using following equation:

$$\text{Percentage inhibition } (\%) = 100 - \left(\frac{\text{OD t}}{\text{OD c}}\right) \times 100$$

where OD t is optical density of the test well, and OD c is the optical density of the control. Thiourea was used as the standard urease inhibitor in this study.

2.4.2. Carrageenan- and Formaldehyde-Induced Paw Oedema (In Vivo)

A carrageenan-induced paw inflammation assay was employed to assess the painrelieving capabilities of DJ dichloromethane and methanol (root and shoot) extracts in rats according to Morris (2003) [21], with some modifications. The study was performed by adopting the parameters mentioned in the guidelines of the National Research Council [22] (NRC, 1996, Washington, DC, USA). The study was also approved by the departmental Committee pf Animal Care at BZU, Pakistan (approval number ACC-10-2019). Rats were divided into six groups (n = 5); animals in group 1 were provided with normal saline and designated the control group. Animals in group 2 were given standard indomethacin at a dose of 100 mg/kg body weight (b.w.) and designated the positive control. Rats in groups 3 and 4 were fed with the dichloromethane extract (200 mg/kg) of *D. juxtapostia* roots and shoots, respectively. Rats in groups 5 and 6 were fed with the methanol extract (200 mg/kg) of *D. juxtapostia* roots and shoots, respectively. One half hour after extract administration, the animals were injected with carrageenan into the plantar aponeurosis surface of the right hind paw. Any change in paw linear circumference was noted after 0, 1, 2, and 3 h using a plethysmometer (UGO-BASILE 7140, Comerio, Italy). An increase in paw circumference was taken as indicator of inflammation.

Likewise, a formaldehyde-induced hind-paw edema assay was used to examine the anti-inflammatory potential of DJ dichloromethane and methanol (root and shoot) extracts in mice, adopting the method of Brownlee with minor changes [23]. We divided animal into a total of six study groups; details are the same as those mentioned above for the carrageenan-induced paw inflammation assay. One half hour after extract administration, formaldehyde (100 μL, 4%) was injected into the plantar aponeurosis of each mouse's right paw, and changes in paw circumference were recorded after 0, 3, 6, 12, and 24 h.

#### *2.5. Brine Shrimp Lethality Assay*

The method described by Meyer et al. (1982) [24] was used to perform a brine shrimp lethality assay. Commercial salt was dissolved in distilled water to prepare artificial seawater in a rectangular plastic tray (22 × 32 cm) in the dark. Fifty milligrams of shrimp eggs (*Artemia salina*) obtained from Husein Ebrahim Jamal Research Institute of Chemistry (HEJ, Karachi, Pakistan) was scattered into the artificial seawater. Incubation lasted 48 h at 37 ◦C. Pasteur pipettes were used to collect hatched larvae. Dichloromethane and methanol extracts from roots and shoots of DJ were prepared at concentrations of 10, 100, and 1000 μg/mL. Samples with varying strengths were separately transferred to clean vials. Each incubation vial contained 1 mL artificial seawater (to a final volume of 5 mL) and 30 shrimp with pH 7.4 adjusted using 1N NaOH and incubated for 24 h at 26 ◦C. The shrimp survival rate was quantified in each vial, including the positive control (i.e., etoposide).

#### *2.6. Cytotoxic Activity*

To assess the cytotoxic potential of DJ dichloromethane and methanol extracts (root and shoot), we adopted the method described by Mosmann et al. (1983) [25]. Experimental samples of varying strengths (0.5–200 μg/mL) were prepared in 100 μL dimethylsulphoxide (1% *v/v*) in 96-well microtiter plates. After incubating the microtiter plates (37 ◦C, 48 h), 50 μL of the MTT solution (5 mg/mL) was added to each well. A microplate reader was used to check the reduction in MTT after a second incubation (37 ◦C for 4 h) by recording the absorbance at 570 nm. The untreated cells were used as a control against which to measure the effect of experimental extracts on the cell viability. The percent inhibition exhibited on the cell cultures by the test samples was computed using the following equation:

$$\text{Survival (\%)} = (\text{At} - \text{Ab}) / (\text{Ac} - \text{Ab}) \times 100$$

where At, Ab, and Ac indicate the sample, blank (complete media without cells), and control absorbance, respectively.

$$\text{Cell inhibition (\%)}=100-\text{cell survival (\%)}.$$

#### *2.7. Hepatoprotective Studies*

The active crude extracts of DJ roots and parts were subjected to hepatoprotective analysis following the OECD 423 (Organization for Economic Co-operation and Development) guidelines [26]. Preset parameters and guidelines of the National Research Council (1996, Washington, USA) were also considered. The institutional ethical committee of Bahauddin Zakariya University (BZU) Multan Pakistan approved the animal study under the title "Study of hepatoprotective potential of *Dryopteris juxtapostia*". The regimen presented in Table 1 was used to orally administer DJ plant extracts to the groups for ten days, but only the most biologically active extracts were considered for this analysis. All animals were treated according to the regimen presented in Table 2. After last dose, retro-orbital plexus blood was collected. Blood of animals in each treatment group was saved for lipid and protein analysis. Furthermore, serum samples were allowed to clot for 60–70 min at ambient room temperature to test for biochemical liver function markers, followed by centrifugation (2500 rpm at 30 ◦C) for 15–20 min.

#### 2.7.1. Assessment of Liver Functions

Serum glutamic-pyruvic transaminase (SGPT)/alanine aminotransferase (ALT), serum glutamic-oxaloacetic transaminase (SGOT)/aspartate aminotransferase (AST), alkaline phosphate, total bilirubin, total protein, and lipid profile were analyzed and quantified in each the serum from each group. The activity of serum transaminases (SGPT and SGOT) and blood lipid profile were examined using the Rietman and Frankel method [27]. Total protein was evaluated according to the Lowry procedure [13]. Total bilirubin (TB) and alkaline phosphate (ALP) were estimated using the methodologies described by Keiding et al. (1974) [28] and Tietz et al. (1983) [29], respectively.



**Table 2.** Carrageenan-induced edema in rat hind paw.


DJ, *Dryopteris juxtapostia*; DCM, dichloromethane; h, hours; MeOH, methanol; ns, non-significant. Values are presented as means ± S.D. of three measurements. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001.

#### 2.7.2. Histopathology of Liver

The liver of all experimental animals (rats) were excised and washed with normal saline. The cleansed liver tissues were separately preserved in 10% formalin solution (neutral) in air-tight, labelled jars. After eight days, the tissues were dehydrated using ethanol solution. The tissues were dried, embedded in paraffin, and sliced into 5 μm length segments. The liver sections were placed on a marked slide and dyed (hematoxylin–eosin (H & E) 400X). The prepared labelled slides were then observed under a photomicroscope (Olympus-CX23 Upright, Japan) for vacuolar degeneration, cellular infiltration, and necrosis of hepatocytes.

#### *2.8. LC-ESI-MS/MS Analysis of Active Crude Extracts*

Crude extracts exhibiting biological potential and outlined hepatoprotective effects were further subjected to mass spectrometry analysis using LC-ESI-MS/MS (Thermo Electron Corporation, Waltham, MA, USA) with the aim of tentative identification of activity-bearing compounds. Detection was carried out by adopting direct-injection-mode ESI (electron spray ionization) in both negative and positive modes. Range of mass, temperature of capillaries, and sample flow rate were maintained at *m/z* 50 to 1000, 280 ◦C, and 8 μL/min, respectively. Collision-induced energy generated during MS/MS analysis depended upon the nature/type of the parent molecular ion subjected to 10 to 45 eV. Furthermore, in order to ensure sufficient ionization and ion transfer, every compound was optimized for MS parameters. Similarly, for every analyte, the source parameters were unchanged but parent, whereas daughter signals were optimized either by analyte infusion or manually. Moreover, online mass data banks, software, and previously published literature were used for compound identification (www.chemspider.com, accessed on 12 December 2021).

#### *2.9. Statistical Analysis*

Study data are expressed as the mean (SEM) of three measurements. ANOVA was used to compare the differences between the control and treatment groups, and Dunnett's test was run using GraphPad Prism (Graph Pad Software V8, San Diego, CA, USA).

#### **3. Results**

*3.1. Phytochemical Constituents and Antioxidant Activity of Dryopteris juxtapostia (DJ) Crude Extracts*

The quantitative investigation recorded the maximum total phenolic contents in 100% DCM extract of *D. juxtapostia* roots, root methanol extract, shoot DCM extract, and shoot methanol extract as 222 ± 0.41 mg GAE/g, 163 ± 0.2 mg GAE/g, 109 ± 0.41 mg GAE/g, and 91.4 ± 0.2 mg GAE/g, respectively (Figure 1). In contrast, total flavonoid contents recorded in methanol extracts of *D. juxtapostia* roots and shoots, i.e., 83.7 ± 0.1 mg QE/g and 43.8 ± 0.3 mg QE/g, respectively, were higher compared to those of DCM root and shoot extracts, i.e., 51 ± 0.2 mg QE/g and 13.2 ± 0.5 mg QE/g, respectively.

**Figure 1.** Total phenolic and flavonoid contents of *Dryopteris juxtapostia* root and shoot crude extracts. Values are presented as means ± S.D. of three measurements.

The radical-scavenging ability of various *D. juxtapostia* crude extracts (shoot DCM extract, shoot methanol extract, root dichloromethane extract, and root methanol extract) was evaluated using various antioxidant assays, such as stable radical assay (DPPH), reducing assay (FRAP), and hydrogen peroxide (H2O2) inhibition assay. As shown in Figure 2, among all extracts, *D. juxtapostia* root DCM extract exhibited the lowest IC50 of 42.0 μg/mL against stable free radicals (DPPH), followed by root methanol extract, with an IC50 of 54.0 μg/mL. In contrast, in the present study, moderate activity was shown by *D. juxtapostia* shoot DCM and methanol extracts, with IC50 values of 59.0 μg/mL and 61.4 μg/mL, respectively. Quercetin was used as a standard antioxidant compound and exhibited remarkable activity, with an IC50 of 22.3 μg/mL.

*D. juxtapostia* root DCM extract demonstrated a higher reducing potential of 46.2 mmol/g, followed by shoot dichloromethane extract, root methanol extract, and shoot methanol extract, with a reducing potential of 31.1 mmol/g, 34.6 mmol/g, and 29.4 mmol/g, respectively. Quercetin was observed to have the highest reducing potential of 66.0 mmol/g.

In the hydrogen peroxide inhibition assay, *D. juxtapostia* root dichloromethane extract demonstrated 71.0% inhibition against delineated prominent activity relative to the stan-

dard quercetin (87.0% inhibition) and in contrast to root methanol extract (51.0% inhibition), shoot dichloromethane extract (32.1% inhibition), and methanol extract (34.2% inhibition).

**Figure 2.** Antioxidant activity of *Dryopteris juxtapostia* root and shoot crude extracts. D, 100% dichloromethane extract; M, 100% methanol extract; Q, quercetin (standard). Values are presented as means ± S.D. of three measurements.

#### *3.2. In Vitro Anti-Inflammatory Activity*

*D. juxtapostia* crude extracts were evaluated for possible antiurease activity at varying concentrations, i.e., 12.5, 25, and 50 μg/mL, using thiourea as a standard anti-inflammatory drug. The results presented in Figure 3 illustrate that *D. juxtapostia* root dichloromethane extract exhibited urease inhibition activity of 56.7% at 50 μg/mL, followed by root methanol extract, with moderate urease inhibition activity of 32.9% at 50 μg/mL. Similarly, the standard drug, i.e., thiourea, exhibited potent inhibition of 88.9% at 50 μg/mL. In contrast, *D. juxtapostia* shoot dichloromethane and methanol extracts evinced non-influential antiurease activity at all concentrations. The anti-inflammatory activity of *D. juxtapostia* root dichloromethane and methanol extracts was found to be consistent with total phenolic contents and antioxidant activity. Statistically, activity outlined by DJ root DCM extract was comparable to that of standard thiourea, with a non-significant difference (ns) observed between their activities, whereas the activity of DJ root MeOH (*p* < 0.01), DJ shoot DCM (*p* < 0.001), and DJ shoot MeOH *p* < 0.001) extracts was significantly lower when compared to standard thiourea (Figure 3).

#### *3.3. In Vivo Anti-Inflammatory Activity*

In the present study, the experimental crude extracts, including *D. juxtapostia* root dichloromethane and methanol extracts and *D. juxtapostia* shoot dichloromethane and methanol extracts were evaluated for possible in vivo pain-alleviating properties induced by carrageenan and formaldehyde at various concentrations, i.e., 50, 100, and 200 mg/kg (Table 2).

DJ root dichloromethane extract showed an antiedematous effect in a dose-dependent manner, with a maximum inhibition of 61.7% (*p* < 0.001) at 200 mg/kg after 3 h, in contrast to the control (normal saline). This is comparable to the anti-inflammatory effects of the standard anti-inflammatory drug indomethacin at a dose of 100 mg/kg, which altered inflammation by as much as 77.6% (*p* < 0.0001). In contrast, *D. juxtapostia* root methanol extract evinced moderate inhibition (43.9% at 200 mg/kg), whereas *D. juxtapostia* shoot extracts exhibited non-substantial activity when compared to the control. The in vivo anti-inflammatory properties of *D. juxtapostia* are in line with its in vitro anti-inflammatory, antioxidant, and phytochemical potential.

**Figure 3.** Urease inhibition activity (%) of various *D. juxtapostia* crude extracts. DCM, dichloromethane; DJ, *Dryopteris juxtapostia*; MeOH, methanol; ns, non-significant. Values are presented as means ± S.D. of three readings. \*\* *p* < 0.01. \*\*\* *p* < 0.001.

Akin to the previous model of inflammation, *D. juxtapostia* root dichloromethane extract, when administrated at a rate of 200 mg/kg, evinced inhibition of 67.3% (*p* < 0.001) after 24 h against formaldehyde-induced pain behavior in contrast to the control, i.e., normal saline. The activity was parallel to the anti-inflammatory effects of the standard anti-inflammatory drug indomethacin at a dose of 100 mg/kg, which altered inflammation by as much as 86.3% (*p* < 0.0001). Moreover, *D. juxtapostia* root methanol extract showed moderate inhibition of 45.1% at 200 mg/kg, whereas both extracts of the shoot portion, i.e., dichloromethane and methanol, exhibited non-substantial activity against formaldehydeintoxicated pain behavior (Table 3).

**Table 3.** Formaldehyde-induced edema in mouse hind paw.


DJ, *Dryopteris juxtapostia*; DCM, dichloromethane; MeOH, methanol; ns. non-significant. Values are presented as means ± S.D. of three measurements. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001.

#### *3.4. Brine Shrimp Lethality Assay*

Brine shrimp lethality assay is an imperative method for determining the preliminary cytotoxicity of experimental plant extracts and other substances based on their ability to kill laboratory-cultured larvae (nauplii). Such an assay is easy to use, inexpensive, and requires only a small amount of test material. In the present study, the cytotoxicity of various crude extracts of *D. juxtapostia* were evaluated at varying concentrations, i.e., 10, 100, and 1000 μg/mL, to compute % mortality. Dichloromethane extracts were observed to be more cytotoxic in comparison to methanol extracts in a dose-dependent manner (Table 4). In brief, *D. juxtapostia* root dichloromethane extract was found to be the most lethal of all investigated extracts, with 100%, 76%, and 10% mortality at 1000 μg/mL, 100 μg/mL, and 10 μg/mL, respectively, followed by standard etoposide (70% mortality at 10 μg/mL), *D. juxtapostia* shoot dichloromethane extract, *D. juxtapostia* root methanol extract, and *D. juxtapostia* shoot methanol extract. These findings revealed that *D. juxtapostia* root dichloromethane extract may contain some compounds that exert cytotoxic effects on certain cancer cells.


**Table 4.** Toxicity assessment of various *D. juxtapostia* crude extracts using a brine shrimp lethality assay.

DJ, *Dryopteris juxtapostia*; DCM, dichloromethane; MeOH, methanol. Values are presented as means ± S.D. of three measurements.

#### *3.5. Cytotoxic Activity of Various D. juxtapostia Crude Extracts Using MTT Assay*

*D. juxtapostia* root dichloromethane extract, root methanol extract, shoot dichloromethane extract, and shoot methanol extract were evaluated for possible anticancer potential using doxorobicin as a standard anticancer drug by MTT assay (Table 5). The MTT (3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity. Because for most cell populations, the total mitochondrial activity is related to the number of viable cells, this assay is broadly used to measure the in vitro cytotoxic effects of drugs on cell lines or primary patient cells.

*D. juxtapostia* root dichloromethane extract was not only found to anticipate a reduction in oxidative stress induced by DPPH, FRAP, and H2O2 but also yielded significant inhibition in cancer progression among both investigated cancer cell lines, i.e., HeLa human cervical and prostate cancer cell lines (PC3), with an IC50 of 17.1 μg/mL and 45.2 μg/mL, respectively. Moreover, *D. juxtapostia* root methanol extract demonstrated prominent inhibitory activity against the Hela cancer cell line, with an IC50 of 36.9 μg/mL, and moderate activity against human prostate cancer cell lines, with an IC50 of 98.3 μg/mL. The standard anticancer drug doxorubicin exhibited potent inhibition against both cancer cell lines, with IC50 values of 0.90 μg/mL (HeLa human cervical cancer cell line) and 1.90 μg/mL (PC3).


**Table 5.** Cytotoxic activity of various *D. juxtapostia* crude extracts at 30 μg/mL determined by MTT assay.

DJ, *Dryopteris juxtapostia*; DCM, dichloromethane; MeOH, Methanol. Values are presented as means ± S.D. of three measurements.

#### *3.6. Hepatoprotective Activity*

Histopathology results revealed normal hepatocytes with no inflammatory changes in the group given silymarin (group 3). However, the group treated with paracetamol (group 2) showed fatty changes, vacuolar degeneration, cellular infiltration, and necrosis of hepatocytes. Groups given *D. juxtapostia* root methanol extract at 300 mg/kg (group 6) and 500 mg/kg (group 7) showed improvement in cell structure, and low cellular infiltration was observed at a higher dose as compared to a lower dose. Groups 4 and 5 given *D. juxtapostia* root dichloromethane extract at doses of 300 mg/kg and 500 mg/kg, respectively, did not show any notable improvement in hepatocyte morphology, cellular infiltration, or necrosis of hepatocytes, as shown in Figure 4. As shown in Table 6, *D. juxtapostia* root methanol extract was found to be more effective in a liver function test, as well as total protein, and total lipid profile tests, in a dose-dependent manner as compared to *D. juxtapostia* root dichloromethane extract.


ALP, alkaline phosphate; SM, serum protein; SA, serum albumin; Gb, globulin; Tb, total bilirubin; SGPT/ALT, serum glutamic-pyruvic transaminase/alanine aminotransferase; SGOT/AST, serum glutamic-oxaloacetic transaminase/aspartate aminotransferase.

#### *3.7. Mass Spectrometry Analysis of Various Extracts*

*D. juxtapostia* root dichloromethane and methanol extracts showing notable biological activities were subjected to mass spectrometry analysis (ESI-MS/MS) to identify (tentative) compounds by comparing the mass spectra and their fragments with mass banks and previously published literature. In detail, albaspidin PP, 3-methylbutyryl-phloroglucinol, flavaspidic acid AB, flavaspidic acid BB, filixic acid ABA, filixic acid ABB, tris-desaspidin BBB, tris-paraaspidin BBB, tetra-flavaspidic BBBB, tetra-albaspidin BBBB, and kaempferol-3-*O*-glucoside were detected in DCM extract. All the aforementioned compounds were also detected in methanol extract, along with kaempferol, catechin, epicatechin, quinic acid, liquitrigenin, and quercetin 7-*O*-galactoside (Table 7). Compound (**A**) was previously identified in another species of the same genus called *Dryopteris crassirhizoma* [30]. Compounds (**B**–**I**) were also identified in the same genus in a species called *Dryopteris Adanson* [31]. Compounds (**J**–**M**) were identified according to recent literature reports [19,32,33].

**Figure 4.** Histopathological analysis of liver. DJDR, *Dryopteris juxtapostia* methanol root; DJMR, *Dryopteris juxtapostia* dichloromethane root.


**Table 7.** ESI-MS/MS analysis of various crude extracts.

#### **4. Discussion**

*D. juxtapostia* root dichloromethane and methanol extracts were found to have higher phenolic and flavonoid contents, respectively, as compared to shoot extracts (Figure 1). The identical potential of bioactive metabolites was reported in an previous study by Baloch et al. (2019) [34], wherein dichloromethane and methanol extracts of *Dryopteris ramose* belonging to the same genus as our experimental plant exhibited notable phenolic (184.2–199.2 mg GAE/g) and flavonoid (50.13–73.02 mg rutin equivalent (RE)/g) contents, supporting the findings of the present investigation. Another study revealed that *Dryopteris ramose* was high in total flavonoid contents using various solvents, including ethyl acetate extract (45.28 μg QE/mg), methanol extract (36.94 μg QE/mg), and water extract (25.69 μg QE/mg) [10]. Similarly, successive extracts of another species of the same genus, i.e., *Dryopteris cochleata* leaves, were reported to contain considerable amounts of total phenolic contents, with 17.7 μg GAE/g petroleum ether, 32.9 μg GAE/g chloroform, 43.4 μg GAE/g ethyl acetate, 90.4 μg GAE/g acetone, 30.86 μg GAE/g methanol, and 28.4 μg GAE/g water. Furthermore, successive extracts of *Dryopteris cochleata* leaves were also found to contain a considerable amount of total flavonoid contents, with 9.16 μg catechin-equivalent (CE)/g petroleum ether, 122.5 μg CE/g chloroform, 145.78 μg CE/g ethyl acetate, 146.9 μg CE/g acetone, 77.71 μg CE/g methanol, and 25.74 μg CE/g water [11].

In the present study, radical scavenging potential was found to align with total phenolic and flavonoid contents. *D. juxtapostia* root dichloromethane extract exhibited considerable antioxidant potential in all three assays as compared to other extracts. These findings are supported by the fact that experimental variables, such as the type of solvent, are important with respect to estimation of antioxidant activity [35–37], as in the present study, dichloromethane extract was found to contain a considerable amount of phytochemicals, in addition to considerable radical-scavenging activity. These findings are in line with those reported by Kathirvel and Sujhata (2016) [11], i.e., that acetone extract of *Dryopteris cochleata* leaves exhibited notable radical-scavenging potential as compared to other tested extracts, owing to its total phenolic and flavonoid contents. Numerous reports have highlighted the antioxidant potential of plants due to the presence of phenolic compounds [38,39]. Additionally, it has been determined that the antioxidant activity of phenolics and flavonoids is mainly a result of their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, and decomposing peroxides Osawa et al. [40]. The radical-scavenging potential of *Dryopteris ramose* crude extract (91.95%), methanol fraction (88.25%), water fraction (87.28%), and ethyl acetate fraction (69.97%) was recently reported by Alam et al. (2021) [10] using a DPPH assay. *Dryopteris affinis*, another species from the same *genus* as DJ was reported to comprise a reasonable amount of total phenolic contents of 112.5 mg GAE/g. *Dryopteris affinis* rhizome extract showed antioxidant potential in DPPH (IC50 of 4.60 μg/mL) and ABTS (22.35 μmol Trolox/g) assays, which is even superior to that of standard butylated hydroxytoluene (BHT), with an IC50 of 9.96 μg/mL [41]. Another study reported the

remarkable antioxidant potential of *Dryopteris ramose* dichloromethane (55.7% inhibition) and methanol extract (72.7% inhibition) in a DPPH assay parallel to standard quercetin (74.54% inhibition), further supporting the antioxidant potential of species belonging to this *genus.* A decade earlier, Kathirvel and Sujhata (2012) [42] reported that several extracts of *Dryopteris cochleata* leaves, i.e., acetone, ethyl acetate, methanol, chloroform, and water, exhibited reducing activities, with EC50 values of 243 μg, 327 μg, 378 μg, and 494 μg, respectively, in a dose-dependent manner. The current findings corroborate prior findings of antioxidant activity in components of the genus *Dryopteris*, although varying potency levels have been reported in the literature. Climate, geography, soil conditions, irrigation methods, harvesting timing, storage, transit facilities, drying procedures (shade drying, sun drying, oven drying, or freeze drying), the polarity of solvents, extraction methods, and extraction time could all play a significant role [19].

Urease is an enzyme that mediates the hydrolysis of urea, resulting in the production of ammonia and carbon dioxide, with its primary function being to protect bacteria in the acidic environment of the stomach [43]. Urease inhibitors have the potential to counteract urease's detrimental effects on living organisms. Urease inhibitors are effective against a variety of infections caused by urease secretion by *Helicobacter pylori*, including gastrointestinal disorders, such as gastritis, duodenal ulcers, peptic ulcers, and stomach cancer [44]. Antibiotic treatment can heal ulcers, prevent recurrence of peptic ulcers, and reduce the risk of stomach cancer in high-risk groups. However, resistance to one or more antibiotics, as well as other considerations, such as poor patient compliance, drug side effects, and the considerable expense of combination therapy, have resulted in concerns among consumers with respect to safety, cost-effectiveness, and availability [45]. Figure shows 3 that *D. juxtapostia* root dichloromethane extract exhibited notable urease inhibition activity, followed by root methanol extract, exhibiting moderate urease inhibition activity. The results of the present investigation cannot be compared with previous data, as no species from this genus has previously be explored for antiurease activity, although various studies have reported the anti-inflammatory potential of this genus in other assays. For example, *Dryopteris crassirhizoma* ethanol extract was reported to diminish the mediation of nitric oxide and prostaglandin production in lipopolysaccharide-stimulated RAW264.7 cells. It also downregulated the levels of mRNA expression of pro-inflammatory genes, such as inducible nitric oxide synthase, cyclooxygenase, and TNF-α [46]. Some compounds isolated from water extract of *Dryopteris fragrans* were reported to have nitric oxide production inhibition potential in lipopolysaccharide-induced RAW 264.7 macrophages, with IC50 values of 45.8, 65.8, and 49.8 μM, respectively [47]. Another study explored *Dryopteris filixmas* leaves and reported that aqueous extract inhibited the hemolysis of red blood cell membranes (56.45% inhibition at 6 mg/mL) parallel to the inhibition induced by standard drug acetylsalicylic acid (70% inhibition at 6 mg/mL) [48]. These reports support the anti-inflammatory results of the present investigation, indicating that species from the genus *Dryopteris* possess health-promoting potential.

Carrageenan-induced paw edema is considered a credible anti-inflammatory compound screening test. The development of carrageenan-induced paw edema is a biphasic reaction, with the first phase including the release of kinins, histamine, and 5-HT and the second phase involving the release of prostaglandins [49]. Similarly, formaldehydeinduced pain and edema are mediated by bradykinins and substance P during the early phase of formaldehyde injection, whereas a tissue-mediated response in the later phase is associated with the release of histamine, 5-hydroxytryptamine (5-HT), prostaglandins, and bradykinins [50]. In our study, *D. juxtapostia* root dichloromethane extract was found to inhibit inflammation in carrageenan-induced (*p* < 0.001) and formaldehyde-induced (*p* < 0.001) paw edema models in a significant manner, suggesting that anti-inflammatory activity may be accredited to the inhibition of inflammatory mediators during both phases of edema formation. Recently, *S. cumini* extract was recorded to contain a considerable amount of total phenolic, flavonoid, and antioxidant potential, in addition to showing remarkable anti-inflammatory activities against carrageenan-, formalin-, and PGE2-induced

intoxication [19]. Another group of researchers reported a direct relationship between phytochemical contents, antioxidant activity, and inhibition of inflammation [32], supporting the findings of current investigation. As previously stated, this is first report on the biological potential of *D. juxtapostia*, although many species of this genus have been found to offer inflammation aversion potential in the past. Ahmad et al. (2011) [12] reported that *Dryopteris chrysocoma* root extract exhibited significant (*p* < 0.001) inhibition of 51.1% when dispensed orally at a rate of 500 mg/kg after administration of formalin comparable to the standard aspirin. In the same study, significant inhibition (*p* < 0.001) of 57% was induced by *Dryopteris chrysocoma* root extract when administered at a rate of 500 mg/kg against carrageenan-induced paw edema, demonstrating the inflammation-averting potential of this genus. The inhibition was dose-dependent, and root extract was found to be more effective than leaf and stem extract. The findings are also in agreement with those reported by Khan et al. (2018) [13], i.e., that *Dryopteris blanfordii* extract showed significant analgesic activity (45% reduction in writhing, *p* < 0.01) when administered at a dose of 300 mg/kg, which is more than standard drug aspirin, exhibiting 40% inhibition at 150 mg/kg. In the same study, ethanolic extract of *Dryopteris blanfordii* exhibited significant (*p* < 0.01) inhibition against carrageenan-induced paw edema, with activity was parallel to the standard drug diclofenac sodium. Another species from the same genus, i.e., *Dryopteris crassirhizoma*, was reported to demonstrate significant antiallergic and anti-inflammatory activities by modulating the T-helper type 1 (Th1) and T-helper type 2 (Th2) response and reducing the allergic inflammatory reaction in phorbol myristate acetate (PMA)- and A23187-stimulated HMC-1 cells via NF-κB signaling in an ovalbumin (OVA)-induced allergic asthma model [51]. A decade earlier, radix methanolic extract of *Dryopteris crassirrhizoma* showed significant (*p* < 0.01) anti-inflammatory effects during the screening of almost 150 medicinal plants [52].

The findings of the brine shrimp assay revealed that *D. juxtapostia* root dichloromethane extract may contain some compounds that may have cytotoxic effects on certain cancer cells. As previously suggested by Mayer et al. (1982) [24], the brine shrimp lethality test can be used to predict compounds or extracts that may exhibit anticancer activity. These findings are in accordance with those reported by Baloch et al. (2019) [34], i.e., that dichloromethane extract of *Dryopteris ramosa* whole plant exhibited a very high potential for cytotoxicity, with an LD50 of 0.6903 μl/mL, which is 10 times more potent than etoposide, with an LD50 of 7.46 μl/mL. Another study reported *Dryopteris ramosa* to exhibit brine shrimp lethality potential, with an LD50 value of 47.64 μg/mL Alam et al. [10]. *Dryopteris affinis* rhizome and leaf methanol extracts were reported to exhibit moderate cytotoxicity in a brine shrimp assay, with LC50 values of 323.9 μg/mL and 85.5 μg/mL, respectively [41]. The methanol extract of *Dryopteris filixmas* leaves was also reported to exhibit potent cytotoxic activity, with an LC50 of 25.9 μg/mL, whereas the LC50 value of standard vincristine sulfate was 10.0 μg/mL [53].

*D. juxtapostia* root dichloromethane extract not only exhibited antioxidant, anti-inflammatory, and cytotoxic properties but also inhibited the proliferation HeLa human cervical and prostate cancer (PC3) cells (Table 6). Several species of the same genus have been reported to exhibit notable anticancer effects against different cancer cell lines [15,54,55], supporting the findings of the current investigation. The anticancer activity reported in the present study is consistent with total phenolics and flavonoids, anti-inflammatory effects, and cytotoxic potential. Anticancer activity of *Dryopteris cochleat* was previously believed to be associated with its phenolic and flavonoid contents [11,56]. *Dryopteris crassirhizoma* (50 and 100 g/mL) markedly inhibited the proliferation of PC-3 and PC3-MM2 cells without disturbing or inducing cytotoxicity toward normal spleen cells from BALB/C mice through the activation of caspase-3, -8, -9, bid, and PARP in PC3-MM2 cells [53].

#### **5. Conclusions**

The findings of the present investigation demonstrate the phytochemical, antioxidant, anti-inflammatory (in vitro and in vivo), and cytotoxic potential of *Dryopteris juxtapostia* (DJ) crude extracts, supporting the use of species belonging to this genus in traditional medicinal

systems. DJ root dichloromethane extract exhibited the highest biological potential in all aforementioned assays, followed by DJ root methanol extract. Both extracts were found to exert no toxicity in the livers of the tested animals when administered at dosis 300 and 500 mg/kg according to liver function test, total protein, and lipid profile. Mass spectrometry analysis showed that some phenolic compounds are responsible for the antioxidant, anti-inflammatory, and anticancer potential of DJ. Overall, the current study confirms the potential of DJ with respect to bioactivity and as a possible alternative therapeutic vector. This research also provides a database for future research to optimize extraction methods and solvents for the maximum extraction of polyphenols and/or flavonoids. Furthermore, preserving the bioactivity of polyphenols and optimizing their delivery also represents a future challenge. In conclusion, plant polyphenols and flavonoids may represent a significant complementary medicine for treatment of oxidation- and inflammation-induced physiological dysfunction. However, further clinical trials are required to establish the safety and efficacy of bioactive compounds.

**Author Contributions:** Conceptualization, M.U., A.R. and M.Q.; methodology, A.R.; software, M.Q.; validation, A.R., S.A. and M.Q.; investigation, A.R.; resources, M.U.; writing—original draft preparation, A.R., T.E., M.Q., N.A., M.W.A. and S.A.; writing—review and editing, T.E., N.A. and S.A.; visualization, T.E.; supervision, M.U.; project administration, A.R. and M.Q.; funding acquisition, T.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** The publication of this article was funded by the Open Access Fund of the Leibniz Universität Hannover, Germany.

**Institutional Review Board Statement:** All trials were conducted following preset guidelines of National Research Council (NRC, 1996, Washington, DC, USA). The study was approved by the departmental Animal Care Committee at BZU, Pakistan (approval number ACC-10-2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data is contained within the manuscript.

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

#### **References**


### *Article Echinacea purpurea* **Fractions Represent Promising Plant-Based Anti-Inflammatory Formulations**

**Sara F. Vieira 1,2, Samuel M. Gonçalves 2,3, Virgínia M. F. Gonçalves 4,5, Carmen P. Llaguno 6, Felipe Macías 6, Maria Elizabeth Tiritan 4,7,8, Cristina Cunha 2,3, Agostinho Carvalho 2,3, Rui L. Reis 1,2, Helena Ferreira 1,2,\* and Nuno M. Neves 1,2,\***


**Abstract:** *Echinacea purpurea* is traditionally used in the treatment of inflammatory diseases. Therefore, we investigated the anti-inflammatory capacity of *E. purpurea* dichloromethanolic (DE) and ethanolic extracts obtained from flowers and roots (R). To identify the class of compounds responsible for the strongest bioactivity, the extracts were fractionated into phenol/carboxylic acid (F1) and alkylamide fraction (F2). The chemical fingerprint of bioactive compounds in the fractions was evaluated by LC-HRMS. *E. purpurea* extracts and fractions significantly reduced proinflammatory cytokines (interleukin 6 and/or tumor necrosis factor) and reactive oxygen and nitrogen species (ROS/RNS) production by lipopolysaccharide-stimulated primary human monocyte-derived macrophages. Dichloromethanolic extract obtained from roots (DE-R) demonstrated the strongest antiinflammatory activity. Moreover, fractions exhibited greater anti-inflammatory activity than whole extract. Indeed, alkylamides must be the main compounds responsible for the anti-inflammatory activity of extracts; thus, the fractions presenting high content of these compounds presented greater bioactivity. It was demonstrated that alkylamides exert their anti-inflammatory activity through the downregulation of the phosphorylation of p38, ERK 1/2, STAT 3, and/or NF-κB signaling pathways, and/or downregulation of cyclooxygenase 2 expression. *E. purpurea* extracts and fractions, mainly DE-R-F2, are promising and powerful plant-based anti-inflammatory formulations that can be further used as a basis for the treatment of inflammatory diseases.

**Keywords:** *Echinacea purpurea* extracts; fractions; phenols/carboxylic acids; alkylamides; inflammation; human primary macrophages

**Citation:** Vieira, S.F.; Gonçalves, S.M.; Gonçalves, V.M.F.; Llaguno, C.P.; Macías, F.; Tiritan, M.E.; Cunha, C.; Carvalho, A.; Reis, R.L.; Ferreira, H.; et al. *Echinacea purpurea* Fractions Represent Promising Plant-Based Anti-Inflammatory Formulations. *Antioxidants* **2023**, *12*, 425. https://doi.org/10.3390/ antiox12020425

Academic Editors: Antonella D'Anneo and Marianna Lauricella

Received: 16 January 2023 Revised: 6 February 2023 Accepted: 7 February 2023 Published: 9 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/).

#### **1. Introduction**

Inflammation is crucial for the survival and maintenance of human health [1]. The inflammatory response is coordinated by the activation of several inflammatory signaling pathways in tissue-resident and recruited immune cells [2]. The main inflammatory signaling pathways associated with the initiation and progression of inflammation are nuclear factor-kappa B (NF-κB) [3], mitogen-activated protein kinase (MAPK) family (extracellular signal-regulated kinase (ERK), C-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 kinase) [4], cyclooxygenase (COX)-2 expression [5], and Janus kinase/signal transducers and activators of transcription (JAK/STAT) [6].

The dysregulation of the magnitude or duration of the inflammatory response can lead to chronic inflammation, which is characterized by the continuous infiltration of immune cells into the injured tissue [7]. Particularly, macrophages are key mediators of inflammation, orchestrating the immune response. Those cells are responsible for engulfing damaged cells and invading pathogens and present antigens to the adaptive immune system [8]. Once activated, macrophages release high levels of pro-inflammatory mediators, including reactive oxygen and nitrogen species (ROS/RNS) and cytokines (e.g., interleukin (IL)-6 and tumor necrosis factor (TNF)-α) [9–11]. These molecules allow for the communication between immune cells, regulating the intensity and duration of the inflammatory response. Hence, their suppression can be a valuable hallmark in the therapy of chronic inflammation where the immune system is overactivated.

The most severe and deleterious outcome of chronic inflammation is the continuous damage and destruction of tissues and organs, which leads to an increased risk of several pathologies (e.g., autoimmune disorders) [12,13]. The current treatment for chronic inflammation-associated diseases varies with their severity, but often it focuses on reducing the overactivity of the immune system. Available anti-inflammatory drugs include nonsteroidal anti-inflammatory drugs (NSAIDs, e.g., celecoxib), corticosteroids (e.g., dexamethasone), conventional disease-modifying anti-rheumatic drugs (cDMARDs, e.g., methotrexate), and biological (b) DMARDs (e.g., anti-IL-6 and anti-TNF-α) [14–16]. However, the prolonged administration of these drugs is frequently associated with several serious side effects. Those include disturbances in the gastrointestinal tract and an increased incidence of opportunistic infections and cancer [17,18]. Therefore, there is an urgent need to discover effective and safe anti-inflammatory drugs.

Plants have been an excellent resource of unique compounds with an important role in the development of many therapeutics [19]. Particularly, *Echinacea purpurea* formulations, recognized as safe by the World Health Organization, have been traditionally used as a potent immunomodulatory medicines [20]. Indeed, *E. purpura* extracts are employed to reduce oxidative stress and inflammation, as well as to prevent cold and flu. Moreover, the ability of *E. purpurea* to interact with immune cells is leading to new insights about its anticancer properties [21]. Other biological properties, such as antifungal, antiviral, and antibacterial activities, have also been reported [22]. Particularly, the antioxidant and anti-inflammatory activities of *E. purpurea* have been associated with its ability to reduce the production of ROS/RNS and pro-inflammatory mediators [23], decrease the infiltration of inflammatory cells [24], and block the receptors of the immune cell [25]. The anti-inflammatory properties have been attributed to alkylamides [26–31], polysaccharides [32–36], and caffeic acid derivatives [37,38]. More recently, sesquiterpenes have also been proposed as bioactive principles of *E. purpurea* [39]. However, other studies suggested that the antiinflammatory activity arises from the synergy between the different bioactive classes of compounds present in the *E. purpurea* extracts [40]. Additionally, the mechanism through which *E. purpurea* extracts exert anti-inflammatory activity is still unclear. Although few studies report the cellular mechanism of *E. purpurea* extracts, they are mainly developed in mouse-derived immune cells [32,39,40]. To the best of our knowledge, only two studies evaluated the mechanism of action of *E. purpurea* extracts using human-derived immune cells. Fast et al. prepared an aqueous extract that reduced the TNF-α production via the inhibition of Toll-like receptor (TLR) 1/2 in Pam3Csk4-stimulated human macrophages [33]. Chicca et al. reported that the standardized commercial tincture Echinaforce decreased the TNF-α production in part via cannabinoid type 2 (CB2) receptor signaling in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) [41]. Thus, the understanding of how a particular bioactive class of compounds present in *E. purpurea* extracts produces its effects in human-derived primary cells, which mimic the human cell environment, is urgently needed. Bioactivity-guided fractionation assays will help in the identification of substances responsible for the biological activity.

In a previous study, we demonstrated the potential of several *E. purpurea* extracts to reduce cytokine production and ROS/RNS levels in an LPS-stimulated macrophage cell line [42]. In this work, we aim to corroborate their anti-inflammatory effects with human primary monocyte-derived macrophages (hMDMs), investigate the bioactive principles, and explore the therapeutic targets. The three most promising extracts in the previous study dichloromethanolic extracts obtained from roots (DE-R), dichloromethanolic extracts obtained from flowers (DE-F), and ethanolic extracts obtained from flowers (EE-F)—were selected for this work [42]. The *E. purpurea* extracts prepared using a green and innovative extraction technique, the Accelerated Solvent Extractor (ASE), were fractionated by semipreparative high-performance liquid chromatography (HPLC) into a phenol/carboxylic acid rich fraction (F1) and an alkylamide rich fraction (F2) to identify the class of compounds responsible for the strongest bioactivity. Moreover, the chemical fingerprint of the bioactive compounds in the fractions was also evaluated by liquid chromatography–high-resolution mass spectrometry (LC-HRMS). The reduction in the production of pro-inflammatory cytokines (IL-6 and TNF-α), the decrease in intracellular ROS/RNS generation, and the downregulation of inflammatory signaling pathways (NF-κB, ERK1/2, p38, JNK/SAPK, STAT3, COX-2) were investigated in LPS-stimulated primary human monocyte-derived macrophages (hMDMs). LPS is an exogenous stimulus derived from the cell wall of Gramnegative bacteria that promotes the release of pro-inflammatory mediators (e.g., cytokines and ROS/RNS) [43]. After fractionation of *E. purpurea* extracts, it was observed that F2 enhanced the anti-inflammatory activity, suggesting that alkylamides are the bioactive compounds mainly responsible for this bioactivity. Interestingly, the further fractionation of alkylamides fraction demonstrated the existence of a possible synergistic effect between them. To the best of our knowledge, this is the first study demonstrating the anti-inflammatory effects of *E. purpurea*, mainly of dichloromethanolic extracts and their fractions, in LPS-stimulated hMDM, through the suppression of ERK1/2, p38, STAT3, and COX-2 inflammatory signaling pathways.

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

A scheme detailing the sequence of the methodology used in this work is illustrated in Figure 1.

#### *2.1. Reagents and Chemicals*

*E. purpurea* was purchased from Cantinho das Aromáticas (Vila Nova de Gaia, Portugal) in May 2017. The plants were transferred to soil and grown following a sustainable agriculture procedure (41◦37- 04.5-- N, 7◦16- 14.4-- W). After two years of cultivation, the flowers were collected in a full bloom phase (June and July 2019), and the roots, including rhizomes, were harvested in the autumn (October 2019). Flowers and roots were dried in the dark and stored at room temperature (RT) and protected from light and humidity until further use. HPLC-grade dichloromethane, acetonitrile (ACN) and HPLC-grade methanol were obtained from Fisher Scientific, Portugal. Dimethyl sulfoxide (DMSO) was purchased from VWR, Portugal. Roswell Park Memorial Institute (RPMI)-1640 media, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution 1 M, penicillin– streptomycin (10,000 U/mL), Dulbecco's phosphate-buffered saline (DPBS), formalin 10% (*v/v*), Quant-iT PicoGreen dsDNA Kit, Pierce Phosphatase Inhibitor Mini Tablets, PageRuler Plus Prestained Protein Ladder (10 to 250 kDa), Bolt Sample Reducing Agent, Bolt LDS Sample Buffer, Bis-Tris Bolt 8%, Bolt MES SDS Running Buffer, and iBlot 2 Transfer Stacks

(polyvinylidene fluoride, PVDF) were purchased from Thermo Fisher Scientific, Lisbon, Portugal. OctoMACS separator, human CD14 microbeads, MS columns, and human recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF) were obtained from Miltenyi Biotec, Bergisch Gladbach, Germany. AlamarBlue, Bio-Rad Protein Assay Dye Reagent Concentrate, and Tween 20 were purchased from Bio-Rad, Lisbon, Portugal. Human IL-6 and TNF-α DuoSet Enzyme-linked immunosorbent assay (ELISA) and DuoSet ELISA Ancillary Reagent Kit 2 were purchased from R&D Systems, Minneapolis, MN, USA. Ethanol, formic acid analytical grade, dexamethasone, Histopaque-1077, human serum, lipopolysaccharide (LPS, *Escherichia coli* O26:B6), radioimmunoprecipitation assay (RIPA) buffer, complete mini protease inhibitor cocktail tablets, bovine serum albumin (BSA), Tris-base, and high-purity standards of echinacoside, chicoric acid, caftaric acid, caffeic acid, chlorogenic acid, and cynarin were obtained from Sigma-Aldrich, Lisbon, Portugal. Echinacea isobutylamide standards kit, composed of undeca-2E/Z-ene-8,10 diynoic acid isobutylamide, dodeca-2E-ene-8,10-diynoic acid isobutylamide, and dodeca-2E,4E-dienoic acid isobutylamide, was acquired from ChromaDex, Los Angeles, CA, USA. High-purity standard dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide was obtained from Biosynth Carbosynth, Spain. Cellular ROS/Superoxide (O2 •−) detection assay kit and rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were acquired from Abcam, Boston, MA, USA. IRDye 800CW Goat anti-Rabbit IgG and IRDye 680RD Goat anti-Rabbit IgG secondary antibodies were obtained from LI-COR Biosciences GmbH, Bad Homburg, Germany. Rabbit NF-κB p65, rabbit p44/42 MAPK (ERK 1/2), rabbit p38 MAPK, rabbit SAPK/JNK, rabbit STAT3, rabbit COX-2, rabbit inducible nitric oxide synthase (iNOS), rabbit phospho-NF-κB p65, rabbit phospho-p38 MAPK, rabbit phospho-STAT3, rabbit phospho-SAPK/JNK, and rabbit phospho-p44/42 MAPK (ERK 1/2) were purchased from Cell Signaling Technology, Lisbon, Portugal. Sodium chloride was acquired from PanReac AppliChem, Lisbon, Portugal. Celecoxib was obtained from abcr GmbH, Karlsruhe, Germany. DAPI (4- ,6-diamidino-2-phenylindole) was purchased from Biotium, Fremont, CA, USA. Ultra-pure water was obtained from a Milli-Q® Direct Water Purification System (Milli-Q Direct 16, Millipore, Molsheim, France).

#### *2.2. Bioactive Compounds Extraction*

Dried flowers (F) or roots (R) were ground using an Analytical Sieve Shaker (AS200 Digit, Retsch, Haan, Germany) before extraction. Dichloromethanolic extracts (DE) and ethanolic extracts (EE) were prepared using an Accelerated Solvent Extractor 200 (ASE, Dionex Corp. Vigo, Spain), as previously described by Vieira et al. [42]. Briefly, the mixture of the plant material (2–5 g) with diatomaceous earth was placed and pressed into stainlesssteel extraction cells, presenting cellulose filters in the bottom. Two extraction cycles were carried out at constant pressure (1500 psi) for 30 min at the minimal operation temperature of the equipment (40 ◦C). The extract solutions were collected in vials, and then the organic solvent was evaporated using nitrogen. Once dried, all the extracts were stored at −80 ◦C until further use.

#### 2.2.1. Fractionation of Extracts

The chromatographic separation of the phenols/carboxylic acids and alkylamides was first optimized with standards by analytical HPLC. A stock solution of 1 mg/mL of all standards was prepared and stored in amber bottles at −80 ◦C. All standards were prepared in methanol, except the caffeic acid solution, which was prepared in ethanol. A standard mixture was prepared at a final concentration of 100 μg/mL for each. A LaChrom Merck Hitachi system equipped with a D-7000 Interface, an L-7100 Pump, an L-7200 autosampler, an L-7455 diode array detector (DAD), and an HPLC System Manager HSMD-7000 (Merck Hitachi, Tokyo, Japan), version 3.0, was used in the chromatographic analysis. The chromatographic separation was performed on a LiChrocart LiChrosphere 100 RP-18 (250 mm × 4 mm, 5 μm, Merck, Darmstadt, Germany). The gradient elution was optimized following the previous method reported by Pellati et al. [44], the mobile

phase being composed of water containing 0.1% formic acid and ACN (Supplementary Table S1). The flow rate was 1 mL/min, and the column was set at RT. The injection volume was 20 μL. The UV spectra were acquired in the range of 190 to 450 nm, and the peak integration was performed at 254 nm for alkylamides and 330 nm for caffeic acid and its derivatives.

**Figure 1.** Scheme of the experimental procedure used in this work. *Echinacea purpurea* root (R) and flower (F) extracts were obtained using an Accelerated Solvent Extractor (ASE). Three extracts were prepared: dichloromethanolic extracts obtained from roots (DE-R), dichloromethanolic extracts obtained from flowers (DE-F), and ethanolic extracts obtained from flowers (EE-F). Then, the extracts were fractionated into phenol/carboxylic acid fractions (F1) and alkylamide fractions (F2) by semipreparative high-performance liquid chromatography (HPLC). Both extracts and fractions were chemically characterized by liquid chromatography–high-resolution mass spectrometry (LC–HRMS). After, the whole extracts and fractions, at different concentrations, were added to lipopolysaccharide (LPS)-stimulated human monocyte-derived macrophages (hMDMs). These cells were isolated from human blood. Their cytocompatibility was evaluated through the metabolic activity and DNA amount determination. Their anti-inflammatory activity was validated by the decrease in interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels in the cell culture medium, as well as by the reduction in the intracellular generation of ROS/RNS/O2 •−. Moreover, inflammatory pathways, including ERK1/2, p38, NF-κB, COX-2, and STAT3, were analyzed to determine their mechanisms of action.

The optimized separation method was adapted for the fractionation of *E. purpurea* extracts by semi-preparative HPLC. In order to reduce the time consumed, the previous gradient method was optimized (Table 1). The flow rate was set at 2 mL/min. The injection volume of *E. purpurea* extracts varied between 200 and 400 μL. A Uptisphere WOD homemade semi-preparative column (250 mm × 10 mm, 5 μm, interchrom, Interchim, Montluçon, France) was used.


**Table 1.** Parameters of the optimized gradient method for semi-preparative HPLC.

The dry residues of DE and EE were dissolved in methanol (5 to 25 mg/mL) and centrifuged (10,000× *g*, 5 min; ScanSpeed Mini, Labogene, Lillerød, Denmark) to collect the supernatant. The DE and EE were fractionated into two main fractions: Fraction 1 (F1, 2–11 min) and Fraction 2 (F2, 11–20 min), defined as phenol and carboxylic acid fraction and alkylamide fractions, respectively. Fractions were obtained through the eluent collection. Briefly, the supernatants were injected in the LaChrom Merck Hitachi system equipped with a D-7000 Interface, an L-7100 Pump, an L-7200 autosampler, an L-7455 diode array detector (DAD) and an HPLC System Manager HSMD-7000, version 3.0. The chromatographic separation was performed on an Uptisphere WOD homemade semi-preparative column (250 mm × 10 mm, 5 μm, interchrom, Interchim, France). The mobile phase was composed of (A) water containing 0.1% formic acid and (B) ACN (Table 1). Only F2 (12.2–21.5 min) was recovered from DE-R. DE-F was fractionated into F2-i (12–14.6 min), F2-ii (14.6–16 min), and F2-iii (16–20 min). EE-F was fractionated into F1 (2–11 min) and F2 (11–20 min). The organic solvent was evaporated in a rotavapor (R210 Buchi, Switzerland), and then the fractions were freeze-dried (LyoQuest Plus Eco, Telstar, Terrassa, Spain) to remove the water. The crude fractions were stored at −80 ◦C until further use.

#### 2.2.2. Characterization of Fractions Composition by LC-HRMS Analysis

The LC-HRMS analysis of the fractions was performed according to the method described by Vieira et al. [42]. Briefly, the LC-HRMS analysis was performed on UltiMate 3000 Dionex ultra-high-performance liquid chromatography (UHPLC, Thermo Scientific, Lisbon, Portugal), coupled to an ultra-high-resolution quadrupole–quadrupole time-of-flight (UHR-QqTOF) mass spectrometer (Impact II, Bruker, Lisbon, Portugal). The chromatographic separation was performed on an Acclaim RSLC 120 C18 analytical column (100 mm × 2.1 mm i.d.; 2.2 μm, Dionex, Lisbon, Portugal). The mobile phase was composed of (A) water containing 0.1% formic acid and (B) ACN containing 0.1% formic acid. The gradient program was as follows: 0 min, 95% A; 10 min, 79% A; 14 min, 73% A; 18.3 min, 42% A; 20 min, 0% A; 24 min, 0% A; 26 min, 96% A. The LC-HRMS acquired data were processed using Bruker Compass DataAnalysis 5.1 software (Bruker, Lisbon, Portugal) to extract the mass spectral features from the sample raw data. Echinacoside, chicoric acid, caftaric acid, caffeic acid, chlorogenic acid, cynarin, undeca-2E/Z-ene-8,10-diynoic acid isobutylamide, dodeca-2E-ene-8,10-diynoic acid isobutylamide, dodeca-2E,4E-dienoic acid isobutylamide, and dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide were the standards used to confirm the identity of the compounds present in the fractions. The identification of these compounds in the *E. purpurea* fractions was confirmed by their retention time (*t*R, min), the mass-to-charge ratio (*m/z*) of the molecular ion, and MS/MS fragmentation patterns. Supplementary Table S2 presents the characteristics of standards obtained by LC-HRMS. The potential identity of the compound in which *tR* and MS data did not match with the

available standards were assigned by comparing the MS/MS spectra with the theoretical data MS/MS fragments and data in the literature [44–50].

#### *2.3. Preparation of E. purpurea Extracts and Fractions Solutions*

Stock solutions of DE-R, DE-F, and EE-F (30.0 mg/mL) and of F1 and F2 (60.0 mg/mL) were prepared in DMSO. Then, serial dilutions were made with complete RPMI (cRPMI, RPMI-1640 culture medium with 2 mM glutamine supplemented with 10% human serum, 1% penicillin/streptomycin, and 1% HEPES). The final concentrations of the samples in the well were 10, 50, and 100 μg/mL. The fractions were only tested in the highest concentration. The maximum concentration of DMSO in the well (0.33%) did not affect the cell viability.

#### *2.4. Human Monocytes*

#### 2.4.1. Ethics Statement

The in vitro experiments involved cells isolated from the peripheral blood of healthy volunteers at the Hospital of Braga, Portugal, approved by the Ethics Subcommittee for Life and Health Sciences (SECVS) of the University of Minho, Braga, Portugal (no. 014/015). Experiments were conducted according to the principles expressed in the Declaration of Helsinki, and participants provided written informed consent.

#### 2.4.2. Monocyte Isolation and Differentiation

Monocytes were isolated from the PBMCs of three different donors, as previously described by Gonçalves et al. [51]. Briefly, PMBCs were first subjected to a density gradient centrifugation using a Histopaque-1077 solution. The PBMC ring was carefully collected and washed twice with PBS. Then, the monocytes were isolated from PBMCs using positive magnetic bead separation with CD14 microbeads, according to the manufacturer's instructions. Isolated monocytes were resuspended in cRPMI. After, monocytes were seeded at a density of 1 × <sup>10</sup><sup>6</sup> cells/mL in adherent 24-well culture plates for 7 days in the presence of 20 ng/mL of recombinant human GM-CSF, at 37 ◦C, in a humidified atmosphere of 5% CO2. The culture medium was replaced every 3 days, and the acquisition of macrophage morphology was confirmed by visualization under an inverted microscope (Axiovert 40, Zeiss, Göttingen, Germany).

#### 2.4.3. Evaluation of Anti-Inflammatory Activity

The hMDMs were stimulated with 100 ng/mL of LPS in a fresh cRPMI medium. After 2 h, all *E. purpurea* extracts and fractions, at different concentrations (see Section 2.3), were added to the LPS-stimulated hMDMs and incubated for 22 h at 37 ◦C, in a humidified atmosphere of 5% CO2. Afterward, the culture medium was harvested (the triplicates were mixed and homogenized) and stored, aliquoted at −80 ◦C until cytokine quantification. The cells were washed with warm sterile DPBS, and the metabolic activity and DNA content were determined (see Section 2.4.4). Controls containing the same percentage of DMSO (see Section 2.3) in the maximal concentration of extracts/fractions were also tested. hMDM cultures stimulated or not with LPS were used as negative and positive controls for the production of pro-inflammatory mediators, respectively. Dexamethasone and celecoxib, prepared in ethanol (20 mM) and diluted with cRPMI (10 μM in the well), were used as positive controls of inhibition of the production of the pro-inflammatory mediators.

#### 2.4.4. Metabolic Activity and DNA Quantification

The metabolic activity of hMDM incubated with *E. purpurea* extracts and fractions was determined by the reduction of resazurin (blue) to resorufin (pink) by living macrophages using the alamarBlue assay [43]. These results are expressed in percentages related to the positive control.

The DNA concentration of macrophages was quantified using a fluorometric dsDNA quantification kit, according to the instructions of the manufacturer, as previously described by Vieira et al. [43]. DNA contents are expressed in relative concentrations of the positive control.

#### 2.4.5. Cytokine Measurement

The amounts of IL-6 and TNF-α were assayed using ELISA kits, according to the instructions of the manufacturer. The obtained values were normalized by the respective DNA concentration. The results are expressed in percentage relative to the positive control.

#### 2.4.6. Cellular ROS/RNS/O2 •− Detection Assay

Oxidative stress in the presence or absence of *E. purpurea* extracts and fractions was investigated using a cellular ROS/O2 •− detection assay kit, as previously described by Vieira et al. [42]. Briefly, LPS-stimulated hMDMs were treated with *E. purpurea* extracts and fractions, at 100 μg/mL, as mentioned before (see Section 2.4.3). After, the hMDMs were washed and labeled with the oxidative stress detection reagent (green, Ex/Em 490/525 nm) for the determination of total ROS/RNS, and O2 •− detection reagent (orange, Ex/Em 550/620 nm) for 1 h at 37 ◦C in the dark. These nonfluorescent detection reagents diffuse into cells, where they can be oxidized by ROS/RNS and O2 •−, converting to fluorescent probes. Then, the cells were fixed with 10% of formalin and the nucleus was labeled with DAPI in a ratio of 1:1000 in DPBS, for 10 min. The fluorescent samples were analyzed using a Fluorescence Inverted Microscope with Incubation (Axio Observer, Zeiss, Germany). The fluorescence intensity values, analyzed using ImageJ software (version 1.52a, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), were normalized against the number of nuclei. Changes in the fluorescence intensity relative to the positive control were related to an increase or decrease in the generation of intracellular ROS/RNS and/or O2 •−.

#### 2.4.7. Western Blot Analysis

LPS-stimulated hMDMs (5 × <sup>10</sup>5/well in 24-well plates) were treated with *E. purpurea* extracts and fractions, at 100 μg/mL, as previously described (see Section 2.4.3). After 24 h, the medium was removed, and the cells were washed with ice DPBS. Then, the cells were lysed in RIPA buffer containing a mixture of protease and phosphatase inhibitors at 4 ◦C for 30 min under shaking. Samples were collected and centrifuged (2000 rpm, 20 min). The supernatant was transferred to a new Eppendorf flask and the protein content was determined using the Bio-Rad Protein Assay, based on the method of Bradford. Bolt sample reducing agent and bolt LDS sample buffer were added to 30 μg of protein. Then, the samples were heated and denatured at 70 ◦C (20 min) and 95 ◦C (5 min). The centrifuged samples were loaded and separated on 8% precast polyacrylamide gels set on a Mini Gel Tank (Invitrogen, Thermo Fisher Scientific, Lisbon, Portugal). The proteins were transferred from the gel to a PVDF membrane using the iBlot 2 Gel Transfer Device (Invitrogen, Thermo Fisher Scientific, Lisbon, Portugal).

After blocking for 30 min at RT with 5% BSA in Tris-buffered saline Tween 20 (TBST), the membranes were incubated overnight at 4 ◦C with the following primary antibodies diluted in blocking solution: rabbit NF-κB p65 (1:1000), rabbit p44/42 MAPK (ERK1/2; 1:1000), rabbit p38 MAPK (1:1000), rabbit SAPK/JNK (1:1000), rabbit STAT3 (1:1000), rabbit COX-2 (1:500), rabbit iNOS (1:500), rabbit phospho-NF-κB p65 (1:1000), rabbit phospho-p38 MAPK (1:1000), rabbit phospho-STAT3 (1:2000), rabbit phospho-SAPK/JNK (1:1000), rabbit phospho-p44/42 MAPK (p-ERK1/2; 1:1000), and rabbit GAPDH (1:10,000). Afterwards, the membranes were washed three times for 5 min with TBST, and then IRDye 800CW Goat anti-Rabbit IgG or IRDye 680RD Goat anti-Rabbit IgG secondary antibodies, both diluted in TBST (1:15,000), were added and the samples were incubated for 1 h at RT in the dark. The Odyssey Fc Imaging System (LI-COR Inc., 2800, Lincoln, NE, USA) was used for image acquisition of the Western blots using near-infrared wavelengths of 700 or 800 nm. The intensity of the bands was quantified with Image Studio software (LI-COR, Inc. software version, Lincoln, NE, USA). The data were normalized to the housekeeping GAPDH.

#### *2.5. Statistical Analysis*

Results are expressed as mean ± standard deviation (SD) of 3 independent experiments with a minimum of 3 replicates for each condition. Statistical analyses were performed using GraphPad Prism 8.0.1 software (Boston, MA, USA). Two-way analysis of variance (ANOVA) and Dunnett's multiple comparisons or Sidak's multiple comparisons test was used for cell assays. Differences between experimental groups were considered significant with a confidence interval of 99% when *p* < 0.01.

#### **3. Results**

#### *3.1. Fractionation of the E. purpurea Extracts*

The optimized analytical method led to the successful separation of the ten studied standards (Figure 2A-i,ii). It was possible to clearly distinguish between phenols and alkylamides. The phenols, due to their high polarity, eluted first under reversed-phase conditions (from ≈ 5 to 28 min), while alkylamides, which are less polar, eluted later (from ≈ 30 to 37 min). To fractionate the extracts, a semi-preparative HPLC method was employed. The analytical method conditions were optimized to reduce the run time and increase the injection volume while maintaining the baseline separation of the two different fractions of interest. In the chromatogram obtained from the standard mixture (Figure 2B-i,ii), it is possible to observe a robust gap between phenol/acids and alkylamide fractions (from 10.6 min to 12.2 min), ensuring the successful separation between these two types of compounds.

The whole *E. purpurea* extracts were fractioned into F1 (phenol and carboxylic acid fraction) and F2 (alkylamide fraction). Different chromatogram profiles were observed for DE-R, DE-F, and EE-F (Figure 3). Accordingly, DE-R and DE-F showed higher absorbance values for alkylamides than phenols/carboxylic acids (Figure 3A-i,ii,B-i,ii). Moreover, the first extract seems to be more enriched with alkylamides. EE-F also presented phenols/carboxylic acids and alkylamides in their composition (Figure 3C-i,ii). Based on their chromatographic profiles, the *E. purpurea* extracts were fractionated. As phenols/carboxylic acids were not detected in DE-R, only the F2 fraction was harvested. DE-F presented defined alkylamide peaks, being possible their fractionation into three sections: F2 i showing three clear peaks, F2 ii presenting one perfect peak, and F2 iii displaying three main peaks. Finally, EE-F was fractionated into F1 and F2.

#### *3.2. Chemical Composition of the E. purpurea Fractions*

The identification of the bioactive compounds present in the *E. purpurea* fractions was performed by LC-HRMS (Table 2). Both product ions and relative intensities for standard fragments perfectly matched those obtained for the compounds in *E. purpurea* fractions. Supplementary Tables S3 and S4 include the retention times (*t*R), the precursor ions, and the product ions for phenols/carboxylic acids and alkylamides, respectively. Each extract and fraction exhibited different patterns of phenols/carboxylic acids and alkylamides. The identification of phenols/carboxylic acid compounds and alkylamides in whole DE-R, DE-F, and EE-F was comparable to our previous study [42]. Five phenols/carboxylic acids and twenty-three alkylamides were identified in all *E. purpurea* fractions. As expected, F1 only presented phenols/carboxylic acids, while alkylamides are just observed in F2. EE-F-F1 exhibited five phenols/carboxylic acids in its composition. DE-R-F2 presented the highest number of identified alkylamides (19 compounds), followed by EE-F-F2 (18 compounds), DE-F-F2 i (10 compounds), and DE-F-F2 ii and DE-F-F2 iii (4 compounds).

**Figure 2.** Analytical (**A**) and semi-preparative HPLC chromatograms (**B**) of standard mixture detected at 330 nm (phenols, **i**) and 254 nm (alkylamides, **ii**). Peak numbering on the chromatograms refers

to the following compounds: 1—caftaric acid (*tR*-A= 6.77 min); 2—chlorogenic acid (*tR*-A = 11.36 min); 3—caffeic acid (*tR*-A = 14.51 min); 4—cynarin (*tR*-A = 18.99 min); 5—echinacoside (*tR*-A = 22.72 min); 6—chicoric acid (*tR*-A = 24.03 min, *tR*-B = 9.47 min); 7—undeca-2E/Z-ene-8,10-diynoic acid isobutylamide (*tR*-A = 30.48 min, *tR*-B = 13.07 min); 8—dodeca-2E-ene-8,10-diynoic acid isobutylamide (*tR*-A = 31.17 min, *tR*-B = 13.89 min); 9—dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide (*tR*-A = 32.24 min, *tR*-B = 15.33 min); 10—dodeca-2E,4E-dienoic acid isobutylamide (*tR*-A = 34.59 min, *tR*-B = 19.44 min).

**Figure 3.** Semi-preparative HPLC chromatograms of DE-R (**A**), DE-F (**B**), and EE-F (**C**) at 19–24.6 μg/mL (100 μL), detected at 330 nm (phenols/carboxylic acids; **A-i,B-i,** and **C-i**) and 254 nm (alkylamides; **A-ii, B-ii,** and **C-ii**).


**Table 2.** Overview of the identified compounds (phenols/carboxylic acids and alkylamides) in *E. purpurea* extracts and fractions by LC-HRMS.


**Table 2.** *Cont.*

DE: dichloromethanolic extracts; EE: ethanolic extracts; R: roots; F: flowers; F1: phenol/carboxylic acid fraction; F2: alkylamide fraction; symbol "+" represents the presence of compound; symbol "-" represents the absence of compound. <sup>a</sup> Injected standards. E/Z stereochemistry is indicated here in accordance to existing literature [44–50], but it should be highlighted that without NMR spectra, it is not possible to conclusively distinguish between E and Z isomers.

DE-R-F2 presented the following alkylamides: dodeca-2E,4Z,10E-triene-8-ynoic acid isobutylamide, dodeca-2E,4Z,10Z-triene-8-ynoic acid isobutylamide, dodeca-2E,4E,10Ztriene-8-ynoic acid isobutylamide, dodeca-2Z,4E,10Z-triene-8-ynoic acid isobutylamide, dodeca-2E,4E,10E-triene-8-ynoic acid isobutylamide, undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide, undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide, dodeca-2E,4Zdiene-8,10-diynoic acid isobutylamide, dodeca-2E-ene-8,10-diynoic acid isobutylamide, trideca-2E,7Z-diene-10,12-diynoic acid isobutylamide, dodeca-2,4-diene-8,10-diynoic acid

2-methylbutylamide, dodeca-2Z,4Z,10Z-triene-8-ynoic acid isobutylamide, trideca-2E,7Zdiene-10,12-diynoic acid 2-methylbutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, dodeca-2E,4Z,10E-triene-8-ynoic acid 2-methylbutylamide or dodeca-2E-ene-8,10-diynoic acid 2-methylbutylamide, pentadeca-2E,9Z-diene-12,14-diynoic acid isobutylamide, dodeca-2E,4E,8Z-trienoic acid isobutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid 2-methylbutylamide, and dodeca-2E,4E-dienoic acid isobutylamide.

The fractionation of DE-F originated three different alkylamide fractions, namely (i) DE-F-F2 i composed of dodeca-2E,4Z,10E-triene-8-ynoic acid isobutylamide, dodeca-2E,4Z,10Z-triene-8-ynoic acid isobutylamide, dodeca-2E,4E,10E-triene-8-ynoic acid isobutylamide, undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide, undeca-2E/Z-ene-8,10-diynoic acid isobutylamide, dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide, dodeca-2E-ene-8,10 diynoic acid isobutylamide, trideca-2E,7Z-diene-10,12-diynoic acid isobutylamide, dodeca-2,4-diene-8,10-diynoic acid 2-methylbutylamide, and dodeca-2E,4Z,10E-triene-8-ynoic acid 2-methylbutylamide or dodeca-2E-ene-8,10-diynoic acid 2-methylbutylamide; (ii) DE-F-F2 ii constituted by dodeca-2,4,10-triene-8-ynoic acid isobutylamide isomer 1, trideca-2E,7Z-diene-10,12-diynoic acid 2-methylbutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, and pentadeca-2E,9Z-diene-12,14-diynoic acid isobutylamide; and (iii) DE-F-F2 iii that presented dodeca-2E,4E,8Z-trienoic acid isobutylamide isomer 1, dodeca-2E,4E,8Z-trienoic acid isobutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid 2 methylbutylamide, and dodeca-2E,4E-dienoic acid isobutylamide. It is important to highlight that none of the alkylamides was repeated in each sub-fraction of DE-F-F2, empathizing the efficiency of the separation method.

EF-F1 was composed of protocatechuic acid, chlorogenic acid, caffeic acid, chicoric acid, rutin, and rutin derivative. EF-F2 was constituted by dodeca-2E,4Z,10E-triene-8-ynoic acid isobutylamide, dodeca-2E,4Z,10Z-triene-8-ynoic acid isobutylamide, dodeca-2E,4E,10Ztriene-8-ynoic acid isobutylamide, dodeca-2E,4E,10E-triene-8-ynoic acid isobutylamide, undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide, undeca-2E/Z-ene-8,10-diynoic acid isobutylamide, pentadeca-2E,9Z-diene-12,14-diynoic acid 2-hydroxyisobutylamide, dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide, dodeca-2E-ene-8,10-diynoic acid isobutylamide, trideca-2E,7Z-diene-10,12-diynoic acid isobutylamide, dodeca-2,4-diene-8,10-diynoic acid 2-methylbutylamide, trideca-2E,7Z-diene-10,12-diynoic acid 2-methylbutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, dodeca-2E,4Z,10E-triene-8-ynoic acid 2 methylbutylamide or dodeca-2E-ene-8,10-diynoic acid 2-methylbutylamide, pentadeca-2E,9Zdiene-12,14-diynoic acid isobutylamide, dodeca-2E,4E,8Z-trienoic acid isobutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid 2-methylbutylamide, and dodeca-2E,4E-dienoic acid isobutylamide.

#### *3.3. Cytotoxicity of E. purpurea Extracts and Fractions*

The metabolic activity and the relative DNA concentration of LPS-stimulated hMDM in the absence or presence of *E. purpurea* extracts and fractions at different concentrations are presented in Figure 4. The cell metabolic activity and the DNA concentration were not affected by the presence of the DE, EE, and fractions at any tested concentration (Figure 4).

#### *3.4. Anti-Inflammatory Activity of E. purpurea Extracts and Fractions*

#### 3.4.1. Cytokine Production

The anti-inflammatory activity of *E. purpurea* extracts and fractions was evaluated by the decreased amounts of pro-inflammatory cytokines, namely IL-6 and TNF-α, in the cell culture supernatant of LPS-stimulated hMDM (Figure 5). Non-stimulated hMDM produced basal amounts of IL-6 (8.0 ± 10.5 pg/mL) and TNF-α (60.0 ± 36.0 pg/mL). As expected, LPS stimulation of hMDM led to a significant increase in the levels of these proinflammatory cytokines (IL-6: 19,139.0 ± 7850.8 pg/mL, TNF-α: 21,773.4 ± 9425.9 pg/mL). Dexamethasone (10 μM) effectively reduced the IL-6 and TNF-α production by 51.2 ± 6.5% and 38 ± 5.7%, respectively (Figure 5). As expected, celecoxib (10 μM) did not considerably decrease the IL-6 and TNF-α production (21.2 ± 17.2% and 4.6 ± 1.8%, respectively).

**Figure 4.** Metabolic activity (**A**) and relative DNA concentration (**B**) of LPS-stimulated human monocyte-derived macrophages (hMDMs) cultured in the presence of different concentrations of the *E. purpurea* extracts, fractions, and clinically used anti-inflammatory drugs (dexamethasone (DEX) and celecoxib (CEL), 10 μM) for 24 h. The dotted line represents the metabolic activity and DNA concentration of positive control (LPS-stimulated hMDM without treatment). There are no statistically significant differences in comparison to the positive control for each tested extract, fraction, DEX, and CEL. Statistically significant differences are 1 (*p* < 0.0133) and 2 (*p* < 0.0035) in comparison with a (DE-R vs. DE-R-F2) and b (DE-F vs. DE-F-F2) at the same concentration. CTL: control; DE: dichloromethanolic extracts; EE: ethanolic extracts; R: roots; F: flowers; F1: phenol/-carboxylic acid fraction; F2: alkylamide fraction.

When LPS-stimulated hMDMs were treated with the whole *E. purpurea* extracts, a significant decrease in the IL-6 amount in the culture supernatant was observed in a concentration-dependent manner (Figure 5A). Particularly, DE showed a higher ability to decrease IL-6 and TNF-α levels than EE. Moreover, the extracts obtained from roots more significantly reduced these two pro-inflammatory cytokines in comparison with the ones obtained from flowers. Indeed, 50 μg/mL of DE-R efficiently decreased the IL-6 production, being even more effective at 100 μg/mL (69.5 ± 10.0%). DE-F was only able to significantly decrease the IL-6 levels by 47.3 ± 6.2% at 100 μg/mL. A comparable significant IL-6 reduction was observed for EE-F over all tested concentrations, with the highest tested concentration displaying greater activity (35.2 ± 12.1%). DE-R was ≈1.5 and 2 times stronger than DE-F and EE-F, respectively. As extracts, all the fractions strongly decreased the IL-6 production, except the EE-F-F1 (25.1 ± 15.8%). Moreover, the bioactivity of DE-F and EE-F was significantly improved with their fractionation into F2. DE-R-F2 led to a more marked IL-6 reduction (84.3 ± 9.1%). DE-F-F2 i, DE-F-F2 ii, DE-F-F2 iii, and EE-F-F2 demonstrated similar bioactivity (62.7 ± 11.5%, 71.2 ± 12.3%, 68.5 ± 14.3%, and 71.6 ± 6.1%, respectively). Besides DE-R-F2 exhibiting a higher efficacy in IL-6 reduction, no significant differences were observed. Analyzing all the *E. purpurea* extracts and fractions, DE-R-F2 led to the strongest reduction in IL-6 production, followed by EE-F-F2 ≈ DE-F-F2

ii, DE-R ≈ DE-F-F2 iii, DE-F-F2 i, DE-F, EE-F, and EE-F-F1. Moreover, DE-R, DE-F, and all the F2 fractions demonstrated similar or higher bioactivity than dexamethasone.

**Figure 5.** IL-6 (**A**) and TNF-α (**B**) production by LPS-stimulated human monocyte-derived macrophages (hMDMs) cultured in the presence of different concentrations of the *E. purpurea* extracts, fractions, and clinically used anti-inflammatory drugs (dexamethasone (DEX) and celecoxib (CEL), 10 μM) for 24 h. Statistically significant differences are \* (*p* < 0.0363), \*\* (*p* < 0.0047), \*\*\* (*p* < 0.0003), and \*\*\*\* (*p* < 0.0001) in comparison to the positive control (LPS-stimulated hMDM without treatment) for all tested *E. purpurea* extracts, fractions, DEX, and CEL and 1 (*p* < 0.0428), 2 (*p* < 0.0018), 3 (*p* < 0.0003), and 4 (*p* < 0.0001) in comparison with b (DE-F vs. DE-R-F2) and c (EE-F vs. EE-F-F) at the same concentration. CTL: control; DE: dichloromethanolic extracts; EE: ethanolic extracts; R: roots; F: flowers; F1: phenol/carboxylic acid fraction; F2: alkylamide fraction.

Some extracts and fractions were also able to significantly decrease the TNF-α levels in LPS-stimulated hMDM cultures (Figure 5B). DE-R, at 50 μg/mL, significantly decreased the TNF-α production by 41.4 ± 4.9%. Conversely, DE-F and EE-F did not demonstrate the capacity to markedly decrease the TNF-α production (21.7 ± 10.5% and 11.6 ± 6.9%, respectively). DE-R was ≈1.7 and 3.3 times stronger than DE-F and EE-F, respectively. Regarding the fractions, only DE-R-F2 and DE-F-F2 i significantly reduced the TNF-α production by 53.1 ± 19.5% and 42.7 ± 1.4%, respectively. Indeed, the bioactivity of the DE-R and DE-F extracts was not significantly improved with their fractionation. DE-F-F2 ii, DE-F-F2 iii, and EE-F-F2 did not show an ability to significantly reduce the TNF-α production (31.6 ± 20.6%, 31.6 ± 18.2%, and 22.1 ± 27.0%, respectively). EE-F-F1 increased the TNF-α amount by 13.9 ± 5.7% in comparison to LPS-stimulated hMDM. Comparing the data obtained for all *E. purpurea* extracts and fractions, it is possible to conclude that DE-R-F2 exhibited the strongest reduction in TNF-α production, followed by DE-F-F2 i, DE-R, DE-F-F2 ii ≈ DE-F-F2 iii, EE-F-F2, DE-F, EE-F, and EE-F1. Moreover, DE-R, DE-R-F2, and DE-F-F2 demonstrated similar or higher bioactivity than dexamethasone.

#### 3.4.2. ROS/RNS/O2 •− Generation

The reduction in the intracellular levels of ROS/RNS and O2 •− in LPS-stimulated hMDM incubated with *E. purpurea* extracts and fractions at the maximal tested concentration are present in Figure 6 and Supplementary Figures S1 and S2. Non-stimulated hMDM produced basal levels of ROS and O2 •−, which were significantly increased by the stimulation with LPS (Figure 6 and Supplementary Figure S1). Dexamethasone (10 μM) effectively reduced the ROS/RNS generation, but no differences were observed with the positive control in the reduction in O2 •− (Figure 6). Conversely, celecoxib (10 μM) considerably decreased both intracellular ROS/RNS and O2 •− generation.

**Figure 6.** Fluorescence intensity of intracellular ROS/RNS (**A**) and O2 •− (**B**) production in LPSstimulated human monocyte-derived macrophages (hMDMs) in the absence or presence of *E. purpurea* extracts, fractions, and clinically used anti-inflammatory drugs (dexamethasone (DEX) and celecoxib (CEL), 10 μM) cultured for 24 h. Fluorescence intensity was measured using ImageJ software. The dotted line represents the basal levels of ROS/RNS and O2 •− in non-stimulated hMDM (negative control) and the dashed line corresponds to the amounts of ROS/RNS and O2 •− produced by LPSstimulated hMDM (positive control). Statistically significant differences are \*\*\* (*p* < 0.0002) and \*\*\*\* (*p* < 0.0001) in comparison to the positive control (LPS-stimulated hMDM without treatment) for each tested *E. purpurea* extract, fraction, DEX, and CEL, as well as 1 (*p* < 0.0453), 2 (*p* < 0.0023), and 4 (*p* < 0.0001) in comparison with b (DE-F vs. DE-R-F2) and c (EE-F vs. EE-F-F) at the same concentration. CTL: control; DE: dichloromethanolic extracts; EE: ethanolic extracts; R: roots; F: flowers; F1: phenol/carboxylic acid fraction; F2: alkylamide fraction.

The treatment of LPS-stimulated hMDM with the *E. purpurea* extracts drastically decreased the intracellular levels of ROS/RNS (Figure 6A and Supplementary Figure S2). EE-F demonstrated a strong capacity to decrease intracellular ROS/RNS production. EE-F was ≈4 and 5 times stronger than DE-R and DE-F, respectively. Regarding the fractions, all of them strongly decreased the intracellular ROS/RNS production. The fractionation of DE-F into DE-F-F2 i and DE-F-F2 iii significantly enhanced the reduction in intracellular ROS/RNS generation. However, the same trend was not observed for EE-F and its fractions. Indeed, the antioxidant activity was markedly decreased with the fractionation of EE-F into EE-F-F1 and EE-F-F2. DE-R-F2 and DE-F-F2 ii seem to present greater bioactivity than the whole extract, but no significant differences were observed. Analyzing all the *E. purpurea* extracts and fractions, EE-F demonstrated the most potent bioactivity, followed by DE-F-F2 i ≈ DE-F-F2 iii, DE-R-F2, DE-F-F2 ii, DE-R ≈ EE-F-F2, DE-F, and EE-F-F1. Moreover, all extracts and fractions were able to reestablish or decrease the levels to those observed in the non-stimulated hMDM.

As observed for ROS/RNS, the treatment of LPS-stimulated hMDM with the *E. purpurea* extracts significantly decreased the intracellular levels of O2 •- (Figure 6B and Supplementary Figure S2). The three extracts showed similar bioactivity in the reduction in O2 •− amounts. Additionally, all the fractions were also able to reduce the intracellular O2 •− a generation with comparable efficacy. Consequently, the fractionation of the extracts did not significantly improve their ability to reduce the intracellular O2 •− generation. Comparing all *E. purpurea* extracts and fractions, DE-F-F2 i exhibited the most powerful activity, followed by DE-R-F2, DE-R, EE-F, DE-F-F2 ii ≈ DE-F-F2 iii, DE-F, EE-F-F1, and EE-F-F2. Moreover, LPS-stimulated hMDM in the presence of all the extracts and fractions were able to reach similar or inferior levels of intracellular O2 •− to those observed in the non-stimulated macrophages.

#### 3.4.3. Therapeutic Targets

To understand the therapeutic targets responsible for the anti-inflammatory activity of the *E. purpurea* extracts and fractions, several pro-inflammatory signaling pathways were investigated by Western blot (Figure 7). Non-stimulated hMDM showed basal levels of ERK 1/2 (Figure 7A), p38 (Figure 7B), and NF-κB p65 phosphorylation (Figure 7C), but COX-2 (Figure 7D) and STAT3 (Figure 7E) expressions were not observed. The phosphorylation of all the studied inflammatory proteins was significantly enhanced in LPS-stimulated hMDM. Dexamethasone (10 μM) was able to significantly decrease the phosphorylation of all studied inflammatory proteins. Celecoxib (10 μM) also significantly reduced the phosphorylation of p38, STAT3, and the expression of COX-2, but no significant activity was observed for ERK 1/2 and NF-κB p65.

When LPS-stimulated hMDMs were treated with the whole *E. purpurea* extracts, a marked decrease in the activation of the ERK 1/2 signaling pathway was observed (Figure 7A). DE-R also efficiently decreased the phosphorylation of ERK 1/2, being its activity ≈8 and 9.5 times higher than DE-F and EE-F, respectively. As observed in extracts, the fractions reduced the phosphorylation of ERK 1/2, but DE-R-F2 and EE-F-F2 strongly suppressed the phosphorylation of this inflammatory protein. DE-F-F2 and EE-F-F1 demonstrated similar activity. Although F2 exhibited stronger bioactivity, no significant differences were observed compared to the whole extracts. Analyzing all the *E. purpurea* extracts and fractions, DE-R-F2 strongly suppressed the ERK 1/2 signaling pathway, followed by DE-R, EE-F-F2, DE-F-F2 iii, DE-F-F2 ii, DE-F-F2 i, DE-F, EE-F, and EE-F-F1. Moreover, LPS-stimulated hMDM in the presence of DE-R, DE-R-F2, DE-F-F2 ii, DE-F-F2 iii, and EE-F-F2 reached similar or lower levels of ERK 1/2 phosphorylation than non-stimulated macrophages.

Only DE-R was able to significantly reduce the activity of the p38 signaling pathway, being its bioactivity ≈2 and 2.7 times stronger than DE-F and EE-F, respectively (Figure 7B). DE-F and EE-F also led to decreased p38 phosphorylation, but no significant differences were observed. All the fractions significantly reduced the phosphorylation of p38, being DE-R-F2, DE-F-F2 iii, and EE-F-F1 the most promising. DE-F-F2 i, DE-F-F2 ii, and EE-F-F2 exhibited comparable bioactivity. Only the fractionation of EE-F into EE-F-F1 markedly reduced the p38 signaling pathway. Although other fractions presented increased bioactivity, no significant differences were observed in comparison with the whole extract. The comparison of all the *E. purpurea* extracts and fractions demonstrates a strong potential for DE-R-F2, DE-F-F2 iii, and EE-F-F1 in the reduction in p38 phosphorylation, followed by DE-F-F2 i ≈ DE-F-F2 ii ≈ EE-F-F2, DE-R, and DE-F ≈ EE-F.

Only EE-F was able to significantly reduce the activity of the NF-κB p65 signaling pathway, being its efficacy ≈1.1 times higher than DE-R and DE-F (Figure 7C). DE-F and EE-F showed a small ability to decrease the NF-κB p65 phosphorylation, with no significant differences. The fractionation of the whole *E. purpurea* extracts into fractions strongly improved the reduction in the NF-κB p65 signaling pathway. Indeed, all fractions markedly reduced the phosphorylation of NF-κB p65, being DE-R-F2, DE-F-F2 iii, and EE-F-F1 the most promising. DE-F-F2 i, DE-F-F2 ii, and EE-F-F2 demonstrated equivalent bioactivity. Comparing all the *E. purpurea* extracts and fractions, DE-R-F2, DE-F-F2 iii, and EE-F-F1 exhibited the most powerful bioactivity in the reduction in NF-κB p65 phosphorylation, followed by DE-F-F2 ii ≈ DE-F-F2 i ≈ EE-F-F1, EE-F, and DE-R ≈ DE-F. Moreover, all the fractions enabled LPS-stimulated hMDM to reach lower levels of ERK 1/2 phosphorylation than non-stimulated macrophages.

**Figure 7.** ERK 1/2 (**A1**,**A2**), p38 (**B1**,**B2**), NF-κB p65 (**C1**,**C2**), COX-2 (**D1**,**D2**), and STAT3 (**E1**,**E2**) signaling pathways downregulation of LPS-stimulated human monocyte-derived macrophages (hMDMs) cultured in the presence of *E. purpurea* extracts, factions, and clinically used anti-inflammatory drugs (dexamethasone (DEX) and celecoxib (CEL), 10 μM) for 24 h. Statistically significant differences are

\* (*p* < 0.0381), \*\* (*p* < 0.0071), \*\*\* (*p* < 0.0009), and \*\*\*\* (*p* < 0.0001) in comparison to the positive control (LPS-stimulated hMDM without treatment) for each tested *E. purpurea* extract and fraction, as well as DEX and CEL and 1 (*p* < 0.0358), 2 (*p* < 0.0023), 3 (*p* < 0.0002), and 4 (*p* < 0.0001) in comparison with a (DE-R vs. DE-R-F2), b (DE-F vs. DE-R-F2), and c (EE-F vs. EE-F-F) at the same concentration. CTL: control; DE: dichloromethanolic extracts; EE: ethanolic extracts; R: roots; F: flowers; F1: phenol/carboxylic acid fraction; F2: alkylamide fraction.

The treatment of LPS-stimulated hMDM with the whole *E. purpurea* extracts significantly suppressed the COX-2 expression (Figure 7D). Despite the similar bioactivity of the three extracts, DE-R showed to be ≈1.2 and 1.9 times stronger than DE-F and EE-F. The fractions decreased the COX-2 expression, but no significant differences were observed. Nevertheless, DE-R-F2, DE-F-F2, and EE-F-F2 presented comparable bioactivity, while EE-F-F1 showed a lower ability to reduce COX-2 expression. In this case, the fractionation of the whole *E. purpurea* extracts into fractions did not enhance the reduction in COX-2 expression by LPS-stimulated hMDM. Comparing all the *E. purpurea* extracts and fractions, DE-R and DE-F led to the most potent COX-2 suppression, followed by EE-F, DE-R-F2, DE-F-F2 ii, DE-F-F2 i, DE-F-F2 iii, EE-F-F2, DE-F-F2 iii, and EE-F-F1.

*E. purpurea* extracts also showed a strong capacity to reduce the activation of the STAT3 signaling pathway in LPS-stimulated hMDM (Figure 7E). DE-R efficiently decreased the phosphorylation of STAT3, being its activity ≈1.6 and 1.8 times higher than DE-F and EE-F, respectively. All the fractions also significantly reduced the phosphorylation of STAT3, but DE-R-F2, followed by EE-F-F2 and DE-F-F2 ii, strongly suppressed the phosphorylation of this inflammatory protein. DE-F-F2 i, DE-F-F2 iii, and EE-F-F1 presented less but similar bioactivity. Nevertheless, only DE-R-F2 and EE-F-F2 improved the bioactivity of the extracts, but no significant differences were observed. Analyzing all the *E. purpurea* extracts and fractions, DE-R-F2 was the most promising formulation in the reduction in STAT3 phosphorylation, followed by DE-R, EE-F-F2, DE-F, EE-F, DE-F-F2 ii, and DE-F-F2 iii ≈ EE-F-F1 ≈ DE-F-F2 i. Moreover, LPS-stimulated hMDM in the presence of DE-R and DE-R-F2 achieved similar or lower levels of STAT3 phosphorylation compared with non-stimulated macrophages.

The inflammatory proteins JNK/p-JNK and iNOS were also investigated, but no phosphorylation expression was detected in this study at 24 h of culture.

#### **4. Discussion**

The development of chronic pathologies, such as rheumatoid arthritis and osteoarthritis, is strongly correlated with persistent inflammation [12,13]. Additionally, most of the current treatments are associated with significant side effects, and thus, effective and safe therapies are urgently needed. As the initiation and progression of inflammation involve several inflammatory signaling pathways, new and safe entities that effectively modulate different molecular mechanisms are required.

This study used DE-R, DE-F, and EE-F since they exhibited the strongest anti-inflammatory properties in our previous work [42]. These three extracts presented different patterns of phenolic compounds, carboxylic acids, and alkylamides in their composition, comparable to our previous study [42]. Briefly, we identified a higher number of phenolic and carboxylic acids in EE-F (11 compounds) than in DE-R (4 compounds) or DE-F (3 compounds). Relative to the alkylamides, DE-R was the extract where more alkylamides were identified (24 compounds), followed by DE-F (20 compounds) and EE-F (19 compounds). As phenolic compounds, carboxylic acids, and alkylamides have different polarities, the extracts were fractionated into F1 (phenols/carboxylic acids) and F2 (alkylamides) fractions. Afterward, the anti-inflammatory activity of the cytocompatible extracts and fractions was investigated by their ability to decrease IL-6, TNF-α, and ROS/RNS levels in LPS-stimulated hMDM.

The three whole extracts drastically reduced the IL-6 levels in LPS-stimulated hMDM. This ability is important since IL-6 induces hematopoiesis, promotes the expansion and activation of T cells, stimulates B cell differentiation, and regulates neutrophil-activating chemokines, among other processes [6,11]. When the extracts were fractionated into F2, the

anti-inflammatory activity was considerably enhanced. On the other hand, the F1 obtained from EE-F did not significantly reduce the IL-6 amount. Therefore, it is possible to conclude that alkylamides are the main class of compounds responsible for the decrease in the IL-6 production by LPS-stimulated hMDM. Moreover, the bioactivity was increased when a high number of alkylamides was present, which involves possible synergistic effects. Specifically, DE-R-F2, composed of 19 alkylamides, demonstrated greater IL-6 reduction (84.3 ± 9.1%) than DE-F-F2 i (10 alkylamides, 62.7 ± 11.5%), DE-F-F2 ii (4 alkylamides, 71.2 ± 12.3%), or DE-F-F2 iii (4 alkylamides, 68.5 ± 14.3%).

DE-R was the only extract that significantly decreased TNF-α levels (41.4 ± 4.9%). Although the fractionation of extracts into fractions enhanced the bioactivity of DE-R-F2 (53.1 ± 19.5%) and DE-F-F2 i (42.7 ± 1.4%), no significant differences between whole extract and fractions were observed. Conversely, EE-F-F1 failed to decrease TNF-α production, and, consequently, its ability to recruit and enhance the differentiation and proliferation of the immune cells, as well as induce the transcription of several inflammatory genes [52]. These results strengthen the role of alkylamides as promising anti-inflammatory candidates. Furthermore, the bioactive pattern and the decrease in IL-6 and TNF-α levels obtained for LPS-stimulated hMDM are in agreement with our previous study with LPS-stimulated THP-1-derived macrophages [42]. These results support the correlation and similar behavior between the human cell line and primary cells. Nonetheless, it is important to stress that the bioactivity of the extracts was slightly lower in primary macrophages.

All the studied extracts and fractions strongly reduced the intracellular levels of ROS/RNS/O2 •− in LPS-stimulated hMDM, reaching similar or inferior levels to those of the non-stimulated macrophages. Particularly, the fractionated DE-F-F2 i and DE-F-F2 ii significantly reduced the intracellular ROS/RNS generation, suggesting that the alkylamides present in these fractions are directly involved in this bioactivity. Moreover, alkylamide fractions showed, in general, lower intracellular levels of ROS/RNS/O2 •−, suggesting that these compounds may be the main compounds responsible for this bioactivity. In this study, the fractionation of EE-F into EE-F-F1 dramatically diminished the capacity of intracellular ROS/RNS reduction, besides phenols/carboxylic acids present in *E. purpurea* extracts are considered strong antioxidants in in vitro assays [53,54]. These results are in agreement with our previous study, where *E. purpurea* extracts enriched in alkylamides presented the strongest intracellular ROS/RNS reduction [42]. It can be hypothesized that alkylamides may inhibit the direct production, mainly in the mitochondria, of these inflammatory mediators [55–57]. Alkylamides may also interfere with the transcription of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [58], as well as target the nitric oxide synthases (NOS) [59].

Different patterns were observed for the suppression of the inflammatory signaling pathways, demonstrating that extracts and fractions modulate different inflammatory mechanisms, including ERK 1/2, p38, NF-κB p65, COX-2, and STAT3, to diverse extents. The alkylamide fractions demonstrated stronger potential to drastically inhibit the inflammatory pathways, pointing out the main role of alkylamides in reducing inflammation. The synergistic effect between alkylamides was also observed since DE-R-F2 demonstrated strong bioactivity in general. We also demonstrated the downregulation of COX-2 expression in the presence of extracts, but not of fractions, in LPS-stimulated hMDM. Thus, a synergistic effect between the classes of compounds present in the whole extract should be required for the inhibitory effect of COX-2 expression.

The activation of the STAT3 signaling pathway in LPS-stimulated hMDM was strongly suppressed by extracts. DE-R-F2 was the only fraction that demonstrated higher bioactivity than the whole extract; however, no significant differences were observed. Nevertheless, once again, the alkylamides were the main compounds responsible for the reduction in this inflammatory pathway in LPS-stimulated hMDM. In this study, it was not possible to observe the inflammatory proteins p-JNK and iNOS, perhaps due to the occurrence of their expression at early time points [27,40].

The anti-inflammatory activity of *E. purpurea* preparations has been reported to be due to different alkylamides. Indeed, the major alkylamide found in *E. purpurea*, dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide, demonstrated minor anti-inflammatory effect compared to the alkylamide fraction [60]. Similarly to our results, Hou et al. reported that isolated chicoric acid did not show strong effects in the reduction in TNF-α levels in LPS-stimulated macrophages [27]. On the other hand, the isolated dodeca-2E,4E,8Z,10E/Ztetraenoic acid isobutylamide potentially decreased the expression of this protein in LPSstimulated primary human monocyte/macrophage-enriched PBMCs [61]. In fact, as previously reported, alkylamide fractions led to the robust inhibition of •NO production in LPS-stimulated RAW 264.7 macrophages [27,40,60]. Indeed, alkylamides, including dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide (present in DE-R-F2, DE-F-F2 i, and EE-F-F2), dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide (present in DE-R-F2, DE-F-F2 ii, and EE-F-F2), dodeca-2E,4E-dienoic acid isobutylamide (present in DE-R-F2, DE-F-F2 iii, and EE-F-F2), dodeca-2E,4Z,10Z-triene-8-ynoic acid isobutylamide (present in DE-R-F2, DE-F-F2 i, and EE-F-F2), dodeca-2E,4E,8Z-trienoic acid isobutylamide (present in DE-R-F2, DE-F-F2 iii, and EE-F-F2), and undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide (present in DE-R-F2), decreased the •NO production in RAW 264.7 macrophages [29]. To the best of our knowledge, only isolated phenols obtained from *E. purpurea* have been reported to have an anti-inflammatory effect. Chicoric acid was able to decrease the TNF-α, IL-1β, and IL-6 levels and the infiltration of inflammatory cells in streptozotocin (STZ)-induced diabetic C57BL/6J mice [24,37]. MTX-induced liver injury or chronic kidney disease in male Wistar rats pre-treated with chicoric acid reduced TNF-α, ROS, •NO, and malondialdehyde (MDA) levels [62,63].

Our results, together with the currently available evidence, suggest that alkylamides are powerful plant-based drugs, exhibiting strong pharmaceutical advantages to ameliorate the inflammatory process related to chronic diseases.

#### **5. Conclusions**

*E. purpurea* extracts efficiently decreased pro-inflammatory mediators (IL-6, TNF-α, and/or ROS/RNS) in LPS-stimulated hMDM, corroborating their anti-inflammatory effects. The fractionation of the whole extracts into alkylamide fractions drastically enhanced the bioactivity, evidencing these compounds as the main active principles. This study also showed that the combination of different phytochemical compounds exhibited high pharmacological properties. Particularly, an increased number of alkylamides demonstrated greater bioactivity. Moreover, alkylamides exert their anti-inflammatory activity through the reduction in ERK1/2, p38, NF-κB, and STAT3 inflammatory signaling pathways, and the downregulation of COX-2 expression. Therefore, *E. purpurea* extracts and fractions can revert and stop the hyperactivation of macrophages, reaching the desired homeostasis in chronic diseases and preventing damage of the surrounding cells and tissues. Consequently, these results point out the efficiency of *E. purpurea* extracts and fractions, particularly DE-R-F2, an alkylamide extract, as new, innovative, and powerful plant-based anti-inflammatory formulations in the modulation of the fate of macrophages in cases where the immune system is overactive. To the best of our knowledge, the anti-inflammatory activity of DE and DE fractions are studied here for the first time in LPS-stimulated hMDM, in which their therapeutic targets are reported. As the immune response involves both specific and non-specific mechanisms, further studies supporting the role of *E. purpurea* extracts and fractions in complex models of inflammation should be explored.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12020425/s1. Figure S1: Intracellular ROS/RNS and O2 •− production in LPS-stimulated human monocyte-derived macrophages in the absence or in the presence of clinically used anti-inflammatory drugs; Figure S2: Intracellular ROS/RNS and O2 •− production in LPS-stimulated human monocyte-derived macrophages in the presence of *E. purpurea* extracts or fractions for 22 h.; Table S1: Parameters of the optimized gradient method for analytical separation; Table S2: Properties of standards determined by LC-HRMS.; Table S3: Phenolic/carboxylic acidic compounds tentatively identified in *E. purpurea* fractions by LC-HRMS (negative mode).; Table S4: Alkylamides compounds tentatively identified in *E. purpurea* fractions by LC-HRMS (positive mode).

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

**Funding:** This work was financially supported by the FCT to the Ph.D. grants of S.F.V. (PD/BD/135246/2017 and COVID/BD/152012/2021), S.M.G. (SFRH/BD/136814/2018), and C.C. (CEECIND/04058/2018), and the projects PATH (PD/00169/2013), HEALTH-UNORTE (NORTE-01- 0145-FEDER-000039) and the NORTE 2020 Structured Project, co-funded by Norte2020 (NORTE-01- 0145-FEDER-000021).

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Subcommittee for Life and Health Sciences (SECVS) of the University of Minho, Braga, Portugal (no. 014/015). Approval on 14 December 2018.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available in the article and supplementary materials.

**Acknowledgments:** We acknowledge the professionals and donors at the Hospital de Braga (Braga, Portugal) for kindly providing buffy coats.

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

#### **References**


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### *Article* **"Golden" Tomato Consumption Ameliorates Metabolic Syndrome: A Focus on the Redox Balance in the High-Fat-Diet-Fed Rat**

**Giuditta Gambino 1,\*,†, Giuseppe Giglia 1,2,†, Mario Allegra 3,4, Valentina Di Liberto 1, Francesco Paolo Zummo 1, Francesca Rappa 1, Ignazio Restivo 4, Filippo Vetrano 5, Filippo Saiano 5, Eristanna Palazzolo 5, Giuseppe Avellone 4,6, Giuseppe Ferraro 1,3, Pierangelo Sardo 1,3,‡ and Danila Di Majo 1,3,‡**


**Abstract:** Tomato fruits defined as "golden" refer to a food product harvested at an incomplete ripening stage with respect to red tomatoes at full maturation. The aim of this study is to explore the putative influence of "golden tomato" (GT) on Metabolic Syndrome (MetS), especially focusing on the effects on redox homeostasis. Firstly, the differential chemical properties of the GT food matrix were characterized in terms of phytonutrient composition and antioxidant capacities with respect to red tomato (RT). Later, we assessed the biochemical, nutraceutical and eventually diseasemodifying potential of GT in vivo in the high-fat-diet rat model of MetS. Our data revealed that GT oral supplementation is able to counterbalance MetS-induced biometric and metabolic modifications. Noteworthy is that this nutritional supplementation proved to reduce plasma oxidant status and improve the endogenous antioxidant barriers, assessed by strong systemic biomarkers. Furthermore, consistently with the reduction of hepatic reactive oxygen and nitrogen species (RONS) levels, treatment with GT markedly reduced the HFD-induced increase in hepatic lipid peroxidation and hepatic steatosis. This research elucidates the importance of food supplementation with GT in the prevention and management of MetS.

**Keywords:** tomato-based products; metabolic syndrome; HFD; antioxidant capacity; phytonutrients

### **1. Introduction**

Tomato fruits *(Lycopersicon esculentum Mill.)* have increasingly grabbed attention as this food product, largely cultivated and consumed throughout the world, represents an invaluable source of bioactive compounds [1]. Noteworthy is that a wide plethora of nutritional substances are encountered in this food, i.e., antioxidants, such as flavonoids and naringenin, but also macronutrients, micronutrients, and organic and phenolic acids. Though the differential composition and quantity of discrete molecules depend on the cultivating conditions and could, hence, influence health-promoting activities, this still

**Citation:** Gambino, G.; Giglia, G.; Allegra, M.; Di Liberto, V.; Zummo, F.P.; Rappa, F.; Restivo, I.; Vetrano, F.; Saiano, F.; Palazzolo, E.; et al. "Golden" Tomato Consumption Ameliorates Metabolic Syndrome: A Focus on the Redox Balance in the High-Fat-Diet-Fed Rat. *Antioxidants* **2023**, *12*, 1121. https://doi.org/ 10.3390/antiox12051121

Academic Editor: Stanley Omaye

Received: 20 April 2023 Revised: 13 May 2023 Accepted: 16 May 2023 Published: 18 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/).

needs to be fully unveiled in terms of biochemical and nutritional characterization. In this context, "Golden Tomato" (GT) is a food product harvested at different degrees of ripeness and defined as "golden" due to the degree of coloring it possesses. A complete characterization in phytonutrients of this food product is deserved to fully explore its biological potential. As a matter of fact, previous research suggested a protective role for tomato-based products, modulating lipid profiles and positively influencing the development of cardiovascular diseases [2–5]. In particular, several clinical studies supported a role for different phytoconstituents of red tomato (RT) that can, for instance, improve the levels of antioxidant enzymes reducing lipid peroxidation rate in diabetic syndrome [6–9].

Metabolic syndrome (MetS) is a widely-diffused clustering of risk factors associated with obesity, cardiovascular disease, and alteration of oxidative status, conditions that could severely impact other comorbidities [10–14]. MetS is established if three or more of the following clinical conditions are present: hypertension, atherogenic dyslipidemia, increased visceral obesity, and hyperglycemia/insulin resistance [15,16]. From a mechanistic perspective, MetS development strongly relies on a vicious self-feeding cycle between chronic, low-grade inflammation and oxidative stress that predisposes individuals to cardiovascular diseases and type II diabetes. At the cellular level, oxidative stress consists of an increased production of reactive oxygen and nitrogen species (RONS) through the intervention of enzymatic, non-enzymatic, and/or mitochondrial pathways. In the background of the metabolic syndrome, NADPH oxidase emerges as a key ROS-producing enzyme [17]. Relevantly, RONS can interact with polyunsaturated fatty acids (PUFA), producing reactive lipid by-products (lipid peroxides and byproducts such as aldehydes) prone to interact with macromolecules such as proteins and DNA, leading to cellular dysfunction. At the same time, RONS overproduction modulates specific, intracellular kinase activities such as Jun N-terminal kinase and those related to NF-kB activation. These molecular events result in the increased phosphorylation of Ser/Thr residues of insulin receptor substrate (IRS-1) and lead to the impairment of both insulin signaling and glucose transport, frequently associated to MetS.

In animal models, MetS can be induced by employing a special diet regimen, i.e., a high-fat diet (HFD), that reproduces the complete clinical manifestations in terms of increased body weight, reduced food intake, glucose tolerance, and dyslipidemia [18–20]. Recently, our previous research revealed that specific systemic biomarkers of redox homeostasis are robustly predictive of the development of metabolic dysfunctions, strengthening the impact of oxidative-based alterations in MetS. A growing interest has arisen for the use of nutraceuticals contained in foods that can counteract MetS [21,22]. In this context, tomato stands out as a food of the Mediterranean diet representing an excellent source of nutrients and bioactive compounds, the concentrations of which are related to the prevention of chronic degenerative diseases such as cardiovascular disorders, cancer, and neurodegenerative diseases [11,23]. Numerous studies have already demonstrated the beneficial effect of tomato consumption on the lipid and glycemic profile [23,24]. Along these lines, we here evaluated the impact of the oral supplementation with GT, at an incomplete ripening stage, on the development of a multifactorial metabolic dysfunction, i.e., MetS, focusing on redox homeostasis. Since no studies so far have assessed the specific composition of GT as it is harvested at the ripening stage, we first investigated the different phytonutrients and antioxidant properties with respect to tomatoes harvested at full maturation. In detail, we characterized the food matrices of the tomato samples assessing the amount of phytonutrients present, but also the antioxidant properties via evaluation of radical scavenger activity for the purpose of discriminating between GT and RT. Secondly, we tested the impact of GT extracts in eventually counterbalancing MetS by exploiting a HFD rat model in order to evaluate the impact of this food product on oxidative-based impairments. Thus, we evaluated the influence of GT supplementation on altered body weight gain, glucose tolerance, lipid profile, and, even more importantly, on robust systemic and hepatic biomarkers of oxidative balance in MetS. This research could shed a novel light on the importance of food supplementation with GT in the prevention and management of MetS.

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

#### *2.1. Treatment of Tomato Samples*

The food matrix named "Golden Tomato" is the sample employed in the present research, which refers to a tomato harvested at the veraison stage and defined as "golden" on the basis of the degree of coloring it possesses. It is a tomato from the cultivar Brigade, grown in a sandy soil type with a northwest exposure and 650 m of altitude. Golden tomatoes were kindly provided by the manufacturer, Mr. Fabrizio Gioia (Company: "Azienda Agricola Fabrizio Gioia", Montemaggiore Belsito, PA, Italy).

The nutritional characterization of the red and golden tomato samples was carried out by determination of the following parameters: macronutrients (proteins, lipids, and carbohydrates) and micronutrients (mineral salts). The degree of ripeness was established by means of the colorimetric analysis of the fruit and based on the following analytical parameters: pH, Brix degree, acidity, and polyphenolic content.

Both tomato samples, after harvesting, were divided into aliquots (~1 kg), freeze-dried and vacuum-preserved at −20 ◦C while waiting to be analyzed and used for animal treatment.

#### 2.1.1. Chemical–Physical Analysis of Tomato Samples

The tomato samples were subdivided according to colorimetric grade and certain chemical–physical parameters. The colorimetric analyses were carried out using a colorimeter (CR-400, Minolta corporation, Ltd., Osaka, Japan) based on the CIELAB color space, also referred to as L\*a\*b\*, is a color space defined by the International Commission on Illumination (abbreviated CIE) in 1976. It expresses color as three values: L\* for perceptual lightness and a\* and b\* for the four unique colors of human vision: magenta, green, blue and yellow. These components were used to calculate hue angle (h◦) and chroma (C\*) as

$$\begin{aligned} \mathbf{h}^{\circ} &= 180^{\circ} + \arctan(\mathbf{b}^{\circ}|\mathbf{a}^{\circ}) \\ \mathbf{C}^{\circ} &= \sqrt{\left(\mathbf{a}^{\circ 2} + \mathbf{b}^{\circ 2}\right)} \\ \mathbf{h}^{\circ} = 180^{\circ} + \arctan\left(\mathbf{b}^{\circ}/\mathbf{a}^{\circ}\right) \text{ and } \mathbf{C}^{\circ} &= \left(\mathbf{a}^{\circ 2} + \mathbf{b}^{\circ 2}\right) 1/2 \end{aligned}$$

following established procedures [25,26].

The basic analytical parameters characterizing the red and golden tomatoes and their different degree of ripeness were moisture, ash, pH, Brix degree, and acidity [Metodi ufficiali di analisi per le conserve vegetali—Parte generale, Supplemento ordinario alla Gazzetta Ufficiale n. 168 del 20 luglio 1989]. In particular, the pH was determined with the help of a pHmeter instrument, as is stated in the Italian regulations and the Official Methods of Analysis, AOAC.

#### 2.1.2. Determination of Phytonutrients in Golden and Red Tomatoes by HPLC System

The analysis of certain phytonutrients that could differentiate the red from the golden tomato involved a phase of extraction with an organic solvent from the food matrix, a phase of separation, and, successively, identification and quantification in the HPLC system. A total of 200 mg of powdered dried GT and RT samples were weighed and extracted with the addition of 20 mL of Tetrahydrofuran (THF). The samples were then processed with Ultra Turrax for about 30" at 17,500 rpm, filtered with filter paper and, subsequently, with regenerated cellulose syringe filters (0.20 μm), and then loaded into autosampler vials and analyzed in HPLC.

For the HPLC analysis of tomato extracts, we used the UPLC-Q Exactive Orbitrap-HRMS system (Thermo Fisher Scientific™, Bremen, Germany) composed of a Dionex Ultimate 3000 liquid chromatograph coupled to a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer equipped with a heated electrospray ionization (HESI) ion source (detailed procedures are reported in Supplementary Materials).

#### 2.1.3. Determination of Total Polyphenolic Content and Antioxidant Properties

In general, the methods used to assess the antioxidant capacity of food can be divided according to their mechanism of action into two categories: methods based on hydrogen atom transfer (HAT) and those based on single-electron transfer (SET).

SET-based methods assess the ability of a compound defined as an antioxidant's potential to yield an electron by reducing the acceptor compound, which can be a metal. This property is quantified by the color change observed when the oxidant is reduced. These methods include the Ferric Reducing Antioxidant Power (FRAP) and the Folin– Ciocalteu methods. HAT-based methods measure the classical ability of an antioxidant to quench free radicals by hydrogen donation. The Crocin bleaching assay (CBA) is included among the HAT methods [27,28]. To evaluate the antioxidant properties of tomato samples, we used FRAP and Folin–Ciocalteu as SET methods and the Crocin bleaching assay as a HAT method. The analyses were performed on the methanolic extract obtained from fresh tomatoes (detailed procedures are reported in Supplementary Materials). The results were expressed as mean ± standard error of the mean (S.E.M.) of three replicates.

#### *2.2. Animals*

Male Wistar rats (4-week old) weighing 240–260 g were provided by Envigo S.r.l. They were housed two per cage and maintained on a 12 h on/off light cycle (8:00–20:00 h) at a constant temperature (22–24 ◦C) and humidity (50 ± 10%). During the acclimation period, animals were first fed with a standard chow diet providing 3.94 kcal/g and then divided into two homogenous groups with balanced weight. These groups were fed with standard laboratory food (NPD: normal pelletized diet, code PF1609, certificate EN 4RF25, Mucedola, Milan, Italy) or fed with HFD food with 60% of energy coming from fats (code PF4215-PELLET, Mucedola, Milan, Italy) in order to induce MetS, as assessed following criteria already established by previous literature [19,20]. Detailed description of the composition of the diet is included in Table 1, as in our previous study [20]. All rats had free access to food and water. Prior to starting the special diet, all animals were weighed. The experiment involved three stages—T0, T1, and T2—individuated according to procedures described in detail in Supplementary Materials. Animal care and handling throughout the experimental procedures were in accordance with the European Communities Council Directive (2010/63/EU). The experimental protocols were approved by the animal welfare committee of the University of Palermo, authorized by the Ministry of Health (Rome, Italy; Authorization Number 14/2022-PR), and conducted following the ARRIVE guidelines.


**Table 1.** Pellet composition is reported for both HFD and NPD. (SFA) Saturated Fat Acid, (MUFA) Monounsaturated Fatty Acid, (PUFA) Polyunsaturated Fatty Acid.

Experimental Groups

Each experimental group consisted of *n* = 6 animals, except for one group (NPD, *n* = 4). At T0, animals were initially subdivided in NPD or HFD on the basis of the diet administered for 8 weeks until induction of MetS. At T1, once MetS induction was verified, animals were divided into 4 groups according to the type of diet (NPD and HFD) and treatment administered (golden tomato or red tomato) until T2, which was reached 4 weeks after T1. In particular, the normal control was fed normal diet feed (NPD) until T2 and the second one (HFD group), representing the MetS control, was fed with the HFD diet throughout the trial (from T0 to T2). The control NPD and HFD groups were subjected i.p. to the same stress conditions as the treated group, since they received during the last month of the experiment, from T1 to T2, a volume of vehicle equal to the tomato solution administered to the treated groups. Furthermore, one group (HFD/GT) was treated 1 mL daily with golden tomato (GT) in the last month of the trial (T1-T2). Lastly, a group of rats was orally treated with red tomato (HFD/RT) at full maturation, at the same dose and under the same experimental conditions as HFD/GT group for 1 month, in order to verify eventual specific effects of red tomato on the redox homeostasis biomarkers of MetS.

#### *2.3. Preparation of the Orally Administered Tomato Solutions*

The amount of tomato administered was 200 mg/kg body weight, corresponding to a daily intake of 300 g for a man with an average weight of 70 kg. The dose was established on the basis of valid toxicity tests for red tomatoes in the literature [29,30] and considered to be over the minimum dose exerting an eventual biological effect when translated from animal studies to humans [31].

The dose was obtained by dissolving 50 mg of freeze-dried fresh tomato in solution with 50 mL of water. The volume of golden tomato solutions orally-administered daily using a syringe was 1 mL. The groups not receiving the tomato solutions took the same volume (1 mL) of plain water. No animals showed signs of toxicity or intolerance during the treatments.

#### *2.4. Biometric, Biochemical, and Oxidative Homeostasis Parameters Induced by MetS*

At the T2 time point, the effect of treatments on the experimental groups was evaluated on the MetS-induced alterations in terms of biometric, biochemical, and oxidative homeostasis parameters. At the end of the experimental procedures, all animals were sacrificed using 2% isoflurane anesthesia followed by cervical dislocation in accordance with authorized procedures. Plasma samples were collected for subsequent analyses to evaluate lipid homeostasis, oxidative stress parameters, and plasma antioxidant status. Hepatic samples were also collected for malondialdehyde (MDA), RONS, and GSH determination as well as for histological evaluations.

#### 2.4.1. Body Weight Gain

Body weight gain was evaluated at T2 for all animals after 4 weeks of nutritional treatments, calculating the Delta Body Weight (ΔBW) by subtracting the final rat weight from the initial weight recorded at T0.

#### 2.4.2. Glucose and Lipid Homeostasis assays

Glucose Tolerance Test (GTT), a diagnostic tool for diabetes and an indicator of metabolic efficiency and insulin resistance, was conducted at T2 following established procedures [20,32] to evaluate the effect of nutritional treatments on glucose metabolism in MetS. To assess the effect of GT supplementation on lipid homeostasis in MetS, after sacrifice, blood samples of each animal were collected by cardiac puncture. Detailed procedures are described in our previous paper [20]. In the plasma samples, triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) concentrations were quantified by commercial kits using the Free Carpe

Diem device (FREE® Carpe Diem; Diacron International, Grosseto, Italy). The data are expressed in mg/dL.

#### 2.4.3. Oxidative Stress Parameters and Plasma Antioxidant Status

Plasma redox balance was assessed using Diacron kits following detailed procedures already published [20]. To assess the prooxidant status, the dROM (Reactive Oxygen Metabolites, primarily hydroperoxides) and the LP-CHOLOX test were carried out, the former assessing the levels of hydroperoxyl free radicals and the latter the levels of circulating lipid peroxides and, in particular, oxidized cholesterol. In the plasma samples, hydroperoxides, lipoperoxides, and oxidized cholesterol were measured by commercial kits using the Free Carpe Diem device (FREE® Carpe Diem; Diacron International, Grosseto, Italy). Data from dROM tests are expressed in arbitrary units, namely, Carratelli units (UCARR). The normal range of the test results was 250–300 U.CARR (Carratelli Units), where 1 U.CARR corresponds to 0.08 mg/dL of H2O2 [33]. In the LP-CHOLOX test, LP-CHOLOX (lipoperoxides and oxidized cholesterol) levels are detected based on peroxides' ability to facilitate the oxidation of Fe2+ to Fe3+, which binds to an indicator mixture forming a colored complex detected by a spectrophotometer at 505 nm [34,35]. The results are expressed in mEq/L.

As for the plasma antioxidant status, the "BAP" test (Biological Antioxidant Potential) measures substances of an exogenous nature (ascorbate, tocopherols, carotenoids, and bioflavonoids) and substances of an endogenous nature (bilirubin, uric acid, and proteins) in plasma that have antioxidant potential and are capable of counteracting radical species. The analysis was performed using the Diacron kit by taking spectrophotometric readings at a wavelength of 505 nm as reported in a previous work and expressing the results as mmol/L [36]. Furthermore, the SHp test was used for evaluation of thiol groups to assess the reducing properties of tomato extracts that can counteract the oxidation of thiol groups and shift the balance in favor of reduced forms.

#### 2.4.4. MDA Assay

Evaluation of MDA levels in liver homogenates was performed according to Ohkawa et al. [37]. Briefly, the reaction mixture contained 0.2 mL of whole homogenate, 0.2 mL of 8.1% sodium dodecyl sulphate (SDS), 1.5 mL of acetic acid solution adjusted at pH 3.5 with NaOH, and 1.5 mL of 1% thiobarbituric acid (TBA) aqueous solution. The mixture was finally made up to 4.0 mL with distilled water and heated at 95 ◦C for 60 min. After cooling with tap water, 1.0 mL of distilled water and 5.0 mL of a n-butanol/pyridine solution (15/1, *v*/*v*) were added, and the mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the absorbance of the organic layer was measured at 532 nm. MDA levels were expressed as nmol MDA/g tissue, using 1,1,3,3,tetramethoxypropane as an external standard.

#### 2.4.5. RONS Assay

RONS levels were detected in liver homogenates using 2- ,7- dichlorodihydrofluorescein diacetate (H2DCF-DA) as previously reported [38]. Briefly, the whole homogenate was centrifuged at 3500 rpm for 10 min at 4 ◦C and 100 μL of the supernatant was mixed with 5 μL of H2DCF-DA (final concentration 10 μM). The mixture was incubated for 30 min at 37 ◦C protected from light and the fluorescence intensity was detected at 490 nm (excitation) and 540 nm (emission) by using a plate reader.

#### 2.4.6. GSH Measurements

Hepatic GSH/GSSG levels were measured in the whole homogenate by employing a glutathione colorimetric assay kit according to the manufacturer's instructions (Invitrogen, Milan, Italy).

#### *2.5. Histological Analyses*

Hepatic samples were immediately stored in paraformaldehyde for 48 h, then were moved to a 20% PBS/sucrose solution, and, after 1 week, to 10% PBS/sucrose solution. At last, the solution was removed and samples were stored at −80◦. Liver tissue sections (5 μm) were obtained from cryostat and stained with hematoxylin and eosin. Following staining, the slides were observed with an optical microscope (Microscope Axioscope 5/7 KMAT, Carl Zeiss, Oberkochen, Germany) connected to a digital camera (Microscopy Camera Axiocam 208 colour, Carl Zeiss). For the steatosis evaluation, a semiquantitative analysis was performed by two independent observers in a high-power field (HPF) (magnification 400×) and repeated for 10 HPFs.

#### *2.6. Statistical Analyses*

Statistical analysis was performed by using GraphPad Prism 9.02 (San Diego, CA, USA). Analyses of antioxidant composition between GT and RT samples were performed by an unpaired Student's *t*-test. Plasma glucose levels (GTT) were analyzed via a two-way repeated measures (RM) ANOVA, followed by Bonferroni post hoc test for significant differences for within- and between-subject comparisons, considering the effect of "time", "diet", and their interaction in the experimental groups. Values of ΔBW, GTT, TG, TC, AUC, MDA, and RONS levels and histological evaluations in liver were compared by a one-way ANOVA test followed by Bonferroni post hoc evaluations for differences between means and represented by scattered bar graphs, in which at least 4 animals per group were included for evaluation. Differences were considered significant when *p* < 0.05. The statistical power (g-power) was considered only if >0.75 and the effect size if >0.40. The results are presented as the mean ± standard error of the mean (S.E.M.), apart from GTT values at 0 and 120 min presented as box and whiskers plots.

#### **3. Results**

#### *3.1. Analytical, Nutritional, and Antioxidant Composition of Tomato Food Matrices*

The different ripening time determines the different phytonutrient composition and antioxidant properties of the tomato, making the red and golden tomato two different food matrices despite coming from the same cultivar and the same production conditions. In particular, the compounds identified in the two food matrices, together with the nutritional properties, micronutrient and organic acid composition are indicated in Supplementary Tables S1–S4. The chemical–physical characteristics used to distinguish the two groups of red (RT) and golden (GT) tomatoes are shown in Table 2.


**Table 2.** Analytical parameters of the two tomato samples expressed as percentages.

Based on the colorimetric analysis, four different colourings were considered with varying color gradations, evidenced by the C\* and h◦ parameters calculated with the specific equations given in the text (Section 2.1.1), from the red fruits (C\* = 25.11 and h◦ = 115.58) in a full ripening state to colorings of decreasing intensity up to the green fruits. Among these, only green (C\* = 32.08 and h◦ = 264.56), pre-veraison (C\* = 30.01 and h◦ = 264.72) and veraison tomatoes (C\* = 30.07 and h◦ = 264.68) were used to produce "golden" sample GT.

As described in Table 2, tomatoes at veraison stage (GT) have a slightly lower water content in absolute value than ripe tomatoes (RT), making all macronutrients slightly more concentrated. Furthermore, as might be expected, GT has a higher degree of acidity and lower pH than RT. From a nutritional viewpoint, GT and RT show minor differences in macronutrients and energy value provided (data reported in supplementary material) even if the reduced degree of ripeness makes the tomato poorer in vitamin C by 45% and in pro-vitamin A, expressed as beta-carotene, by 69%, as shown in Table 3. Regarding the polyphenolic content and antioxidant properties of tomato samples, the different antioxidant properties of GT and RT samples were evaluated via HAT and SET methods. GT exhibits a superior reducing power compared to RT, and this can be observed from results obtained by FRAP and Folin–Ciocalteu methods. In detail, the FRAP assay revealed a significant increase in GT versus RT samples (t = 6.18, df = 13, *p* < 0.0001, Figure 1A). Conversely, the CBA assay revealed a significant increase in RT versus GT samples (t = 3.48, df = 12, *p* = 0.0045, Figure 1B). Lastly, the analysis of total polyphenolic content revealed that GT samples contained significantly higher levels of polyphenols as in Figure 1C (t = 4.923, df = 4, *p* = 0.0079). The antioxidant properties outlined in GT and RT samples could be ascribed to the differential composition in phytonutrients as listed in Table 2. In particular, we observed that, in the GT, the levels of naringenin and chlorogenic acid are 57% higher for the former and 81% higher for the latter, respectively, which stand out compared to RT. Meanwhile, RT is characterized by an increased content in lycopene and beta-carotene.

**Table 3.** Antioxidant components in tomato samples. The concentrations of golden and red (GT and RT) tomatoes are expressed as mg/100 g (Dried Weight, DW).


**Figure 1.** Analyses of antioxidant properties of golden tomato and red tomato (GT and RT) samples. Evaluations using (**A**) Ferric Reducing Antioxidant Power (FRAP, mM/trolox), (**B**) Crocin Bleaching Assay (CBA, mM/trolox), and (**C**) total polyphenolic content (GAE mg/L) were reported and significant differences following unpaired *t*-test between GT and RT are indicated as (\*\*\*\*) *p* < 0.0001 and (\*\*) *p* < 0.01.

#### *3.2. Effects of GT Treatment on Body Weight in MetS*

At T2, after 4-week treatment with GT and RT, ΔBW was compared via one-way ANOVA followed by Bonferroni post hoc test. Significant differences in body weight were found in HFD/GT that reduced their weight gain versus HFD and versus NPD in MetS (F(2,13) = 3.17, *p* = 0.0014, g-power: 1.307; effect size: 0.769; Figure 2). In detail, following treatment with GT, rats reached a mean % weight increase relative to initial weight of 78.75 ± 11.91 with respect to HFD (90.35 ± 10.74%g).

**Figure 2.** Variation of body weight gain (ΔBW) versus the initial weight in HFD/GT, HFD, and NPD experimental groups. Statistical significance by one-way ANOVA followed by post hoc Bonferroni is indicated as (\*\*) for *p* < 0.001 versus HFD and (\*) for *p* < 0.005 versus NPD.

#### *3.3. Effects of GT Treatment on Glucose and Lipid Homeostasis in MetS*

The effects of nutritional treatments in MetS were tested on glucose homeostasis. To this purpose, GTT test was performed at the end of nutritional treatments and outlined significant differences between experimental groups. To begin with, we evaluated the AUC, as described in the methods section, by one-way ANOVA followed by a Bonferroni post hoc test that highlighted a significant reduction in HFD/GT group versus HFD and a significant increase versus NPD (F(2,10) = 37.43, *p* < 0.0001, g-power: 0.819; effect size: 2.33; Figure 3A), suggesting an improvement in glucose tolerance tests following GT supplementation, though not returning to baseline. Besides this, a two-way RM ANOVA followed by a Bonferroni post hoc test performed on GTT at 0 and 120 min revealed marked differences in plasma glucose levels for time (F(1,10) = 119.8, *p* < 0.0001), diet (F(2,10) = 12.01, *p* = 0.0022), and their interaction (F(2,10) = 9.44, *p* = 0.0050) in HFD/GT compared to HFD and NPD groups (Figure 3B). In accordance with previous data [39], no significant differences are observed between groups in fasting glucose levels while there are different trends over time after i.p. glucose administration. In particular, a significant reduction in plasma glucose was evidenced by post hoc Bonferroni tests due to GT treatment, since the HFD/GT group at 120 min reached lower values versus HFD. However, glucose tolerance was ameliorated but not compensated, since the HFD/GT group was still significantly higher than NPD, not returning to baseline.

As for the evaluation of lipid homeostasis at T2, we assessed the plasma concentrations of TG, TC, LDL, and HDL in the experimental groups. On the one hand, the one-way ANOVA performed on the plasma levels of TG did not point out significant differences between groups, as shown in Table 4. On the other hand, the plasma levels of LDL analyzed by one-way ANOVA were remarkably decreased following GT treatment in the HFD/GT group with respect to HFD, and returned to basal values that were not statistically different than the NPD group (F(2,13) = 12.59, *p* = 0.0009; g-power: 0.769; effect size: 1.30; Table 4). Total cholesterol was markedly increased in HFD/GT rats versus HFD and NPD group (F(2,11) = 16.39, *p* = 0.0005; g-power: 0.937; effect size: 1.688; Table 4). Importantly, HDL cholesterol was increased by GT treatment in HFD rats reaching significantly higher levels

with respect to HFD and NPD groups (F(2,11) = 20.84, *p* = 0.0002; g-power: 0.801; effect size: 1.358, Table 4).

**Figure 3.** GTT test in experimental groups at the end of experimental procedures. (**A**) Area under the curve (AUC). Plasma glucose levels (mg/dL) per unit of time (h) difference between HFD/GT, HFD and NPD groups. (**B**) Glucose levels in GTT. Plasma glucose level (mg/dL) differences between groups after 0 and 120 min of GTT. Statistical significance by one-way ANOVA followed by post hoc Bonferroni is indicated as (\*\*\*\*) *p* < 0.0001, (\*\*) *p* < 0.01, and (\*) *p* < 0.05.

**Table 4.** Biochemical parameters of lipid homeostasis triglycerides (TG), Total Cholesterol (TC), LDL Cholesterol, and HDL Cholesterol (mg/dL). Statistical significance for \* *p* < 0.05 versus HFD and for # *p* < 0.05 versus NPD groups.


*3.4. Effects of GT on Plasma Redox Homeostasis Biomarkers in MetS*

The antioxidant and prooxidant status was evaluated at time T2, after nutritional treatment, on plasma samples from all the experimental groups to explore the plasma redox balance in metabolic syndrome. Statistical analyses revealed that GT markedly modulated the antioxidant capacity of MetS animals.

In particular, a one-way ANOVA followed by post hoc test was conducted on mean values of SHp in HFD/GT that are higher than HFD, though still significantly reduced versus NPD group (F(2,13) = 27.09, *p* < 0.0001; g-power: 0.972; effect size: 1.869; Figure 4A). BAP values were reduced in both HFD/GT and HFD groups versus NPD (F(2,13) = 7.22, *p* = 0.0078; g-power: 0.905; effect size: 0.793; Figure 4B). Furthermore, statistical significance emerged from the analysis conducted on mean values of the prooxidant status, i.e., dROM and LP-CHOLOX levels. In detail, dROM levels were modified among the experimental groups, though post hoc analysis revealed a non-significant reduction induced by GT treatment versus HFD (F(2,13) = 6.15, *p* = 0.0132; g-power: 0.923; effect size: 0.809; Figure 4C).

**Figure 4.** Plasma redox homeostasis biomarkers between HFD/GT, NPD, and HFD groups at the end of experimental procedures. Antioxidant status evaluated by (**A**) SHp test for thiol group levels (mmol/L) and (**B**) Biological Antioxidant Potential (BAP test) levels (mmol/L). Prooxidant status evaluated by (**C**) dROM test for differences in ROM (primarily hydroperoxide) levels (UCARR) and (**D**) LP-CHOLOX test for differences in LP-CHOLOX (lipoperoxides and oxidized cholesterol) levels (mEq/L). Statistical significance of Bonferroni post hoc tests are indicated for (\*) *p* < 0.05, (\*\*) *p* < 0.01, (\*\*\*) *p* < 0.001 and (\*\*\*\*) *p* < 0.0001, as represented in the graphs.

Lastly, LP-CHOLOX levels were significantly reduced in the HFD/GT group versus HFD, returning to baseline since non-significant differences emerged with the NPD group (F(2,13) = 13.63, *p* = 0.0006; g-power: 0.776; effect size: 1.318; Figure 4D).

#### *3.5. Effects of GT on Hepatic Steatosis*

The histological evaluation performed on liver samples of the control NPD group showed an almost absent steatosis (average percentage of 3.2 ± 0.8) compared to the cases of the HFD group in which steatosis was found to be high (average percentage of 89.3 ± 1.7). In HFD liver tissue, macrovesicular steatosis with large lipid droplets was predominantly observed. In the liver samples of the GT group, the steatosis was microvesicular with an average percentage of 44.33 ± 14.5. Statistical evaluation by one-way ANOVA showed a significant decrease in the percentage of steatosis in HFD following GT supplementation versus HFD, though it was still significantly higher than NPD (F(2,12) = 6.59, *p* = 0.011; g-power: 0.99; effect size: 5.07; Figures 5 and 6).

**Figure 5.** Histological evaluation of hepatic steatosis. Differences in hepatic steatosis (%) between NPD, HFD, and HFD/GT groups. \*\*\*\* for *p* < 0.0001.

**Figure 6.** Histological features of liver tissue of experimental groups. Representative images of hematoxylin and eosin staining of liver tissue ((**A**,**B**): NPD; (**C**,**D**): HFD; (**E**,**F**): HFD/GT). (**A**,**C**,**E**): magnification 200×, scale bar 50 μm. (**B**,**D**,**F**): magnification 400×, scale bar 20 μm.

#### *3.6. Effects of GT on MetS-Induced Hepatic Oxidative Stress*

As shown in Figure 7A, the statistical analyses performed by one-way ANOVA on MDA levels in the liver showed significant differences between groups. In particular, GT significantly reduced the HFD-induced oxidative stress in the same tissue in comparison with the HFD group, though basal NPD values were not restored (F(2,9) = 124.2, *p* < 0.0001; g-power: 0.99; effect size: 5.19; Figure 7A).

**Figure 7.** Levels of hepatic oxidative stress in HFD/GT, NPD, and HFD groups at T2. (**A**) Malondialdehyde (MDA) levels, (**B**) reactive oxygen and nitrogen species (RONS) levels, and (**C**) GSH/GSSG levels. Statistical significance by one-way ANOVA followed by post hoc Bonferroni is indicated as \*\*\*\* for *p* < 0.0001, \*\*\* for *p* < 0.001 and \*\* for *p* < 0.01.

The evaluation of RONS in the liver highlighted that the treatment with GT managed to recover the oxidative status induced by HFD by reducing hepatic RONS levels. In particular, GT was able to reduce RONS levels even in comparison with the NPD group as shown by post hoc significance in Figure 7B (F(2,9) = 436.3, *p* < 0.0001; g-power: 1; effect size: 8.61). Finally and in accordance with this evidence, GT treatment also ameliorated the ratio of GSH/GSSG in the liver by significantly increasing its levels, although not to the control levels (Figure 7C, F(2,9) = 289.2, *p* < 0.0001; g-power: 1; effect size: 7.561).

#### *3.7. Effects of RT on MetS-Induced Systemic and Hepatic Oxidative Stress*

The HFD/RT group of rats was employed in this study to unveil its specific effect on redox homeostasis in our experimental model. Therefore, we focused on the assessment of systemic and hepatic oxidative stress in MetS following nutritional treatment with RT at full maturation.

To begin with systemic biomarkers, we discovered that RT treatment does not manage to rescue SHp values versus HFD as analyzed by a one-way ANOVA followed by a Bonferroni post hoc analysis (Figure 8A). In contrast, RT treatment significantly increases BAP values versus HFD, returning to the basal values of NPD group (F(2,13) = 8.01, *p* = 0.0054; g-power: 0.986; effect size: 0.968; Figure 8B). As for the evaluation of the prooxidant status, statistical significance emerged from the analysis conducted on mean values of dROM and LP-CHOLOX levels. In detail, dROM levels were markedly reduced by RT treatment versus HFD (F(2,13) = 21.02, *p* < 0.0001; g-power: 0.995; effect size: 1.72; Figure 8C) and LP-CHOLOX levels were significantly reduced in HFD/RT group versus HFD and also lower than the NPD group (F(2,13) = 32.79, *p* < 0.0001; g-power: 0.993; effect size: 2.15; Figure 8D).

**Figure 8.** Plasma redox homeostasis biomarkers between HFD/RT, NPD, and HFD groups at the end of experimental procedures. Antioxidant status evaluated by (**A**) SHp test for thiol group levels (mmol/L) and (**B**) Biological Antioxidant Potential (BAP test) levels (mmol/L). Prooxidant status evaluated by (**C**) dROM test for differences in ROM (primarily hydroperoxides) levels (UCARR) and (**D**) LP-CHOLOX test for differences in LP-CHOLOX (lipoperoxides and oxidized cholesterol) levels (mEq/L). Statistical significance of Bonferroni post hoc tests are indicated for (\*) *p* < 0.05, (\*\*) *p* < 0.01, (\*\*\*) for *p* < 0.001 and (\*\*\*\*) *p* < 0.0001, as represented in the graphs.

Regarding hepatic biomarkers of oxidative stress, our outcomes revealed that RT at full maturation is able to significantly reduce the HFD-induced MDA levels in hepatic tissue in comparison with the HFD group, though not restoring to basal NPD values (F(2,9) = 163.4, *p* < 0.0001; g-power: 0.999; effect size: 6.26; Figure 9A). The evaluation of RONS in the liver highlighted that the treatment with RT managed to recover the oxidative status induced by HFD by reducing hepatic RONS levels and restoring basal NPD values (F(2,9) = 399.5, *p* < 0.0001, g-power: 1; effect size: 8.30; Figure 9B). Lastly, GSH levels in RT-treated rats were higher than the HFD group, but still significantly different from control NPD rats (F(2,9) = 434.8 *p* < 0.001; g-power: 1; effect size: 9.04; Figure 9C).

**Figure 9.** Levels of hepatic oxidative stress in HFD/RT, NPD, and HFD groups at T2. (**A**) Malondialdehyde (MDA) levels, (**B**) reactive oxygen and nitrogen species (RONS) levels, and (**C**) GSH/GSSG levels. Statistical significance by one-way ANOVA followed by post hoc Bonferroni is indicated as (\*) for *p* < 0.05, (\*\*) for *p* < 0.01 and (\*\*\*\*) for *p* < 0.0001.

#### **4. Discussion**

GT is a food product harvested at an incomplete ripening stage and has a different nutritional and phytonutrient composition with respect to red tomato at full maturation. Not surprisingly, the two food matrices that we here analyzed for the first time differ to a great extent. To begin with, we revealed the higher acidity of GT samples compared to RT, which could account for a different bioavailability of phytonutrients [40] and for a different aggregation state of the polyphenolic compounds, which may explain the different antioxidant properties found in GT and RT food matrices. Indeed, we revealed the different phytonutrient composition that could be responsible for the higher antioxidant capacities of the GT in terms of reducing power compared to the RT. However, RT shows better radical scavenger activity than the GT evaluated by CBA. Among the phytonutrients, we demonstrated that GT has a higher naringenin and chlorogenic acid content than RT, which could be of striking importance since in vitro studies supported the beneficial effects of naringenin and of chlorogenic acid on MetS [23,41,42].

In the light of the intriguing antioxidant properties emerged by our evaluation of the GT food matrix, we orally administered GT extracts in an HFD rat model of MetS in order to further explore a putative protective role. Indeed, the HFD model was previously demonstrated to trigger an oxidative-dependent metabolic dysfunction that represents an undoubtedly valid model for the assessment of GT properties [20,43]. Our data revealed that one month of GT oral supplementation managed to reduce body weight gain in HFD rats. GT supplementation also counteracted the deleterious effect of the HFD on glucose tolerance. Nevertheless, the GT supplementation is able to ameliorate the glucose tolerance, but not to restore it to the basal values of the NPD group. Similarly to GT, some authors have shown that supplementation of naringenin or chlorogenic acid can improve but not normalize the manifestations of glucose tolerance [42,44,45]. Indeed, chlorogenic acid has anti-diabetic and anti-obesity properties by reducing glucose absorption in the small intestine through inhibition of the enzyme glucose-6-phosphate translocase, inhibiting the hepatic enzyme glucose-6-phosphatase, and increasing phosphorylation of AMP-activated protein kinase [46,47].

Noteworthy is that GT also reduced LDL cholesterol and increased HDL cholesterol versus both HFD and NPD rats. The same effect observed in our study was shown by administering 10 mg/Kg of chlorogenic acid to rats with hypercholesterolemia induced by the HFD diet [48]. This cholesterolemic-lowering effect could be due to the inhibitory action of naringenin and of chlorogenic acid on the enzyme HMG-CoA reductase already demonstrated [49–51]. On top of this, chlorogenic acid influences lipid metabolism by modulating the transcription of genes coding for lipogenic enzymes such as fatty acid synthase and acetyl-CoA carboxylase. In particular, it appears to induce down-regulation of LXR*α* and up-regulation of PPAR*α* [52].

As for the effect of GT supplementation on altered plasma biomarkers of antioxidant defenses in MetS, we here revealed that GT markedly modulated the systemic antioxidant capacity in vivo in the HFD model. The different composition in phytochemicals of the two food matrices allows to justify the behavior on plasma antioxidant status observed after administration of GT. In particular, the oral administration of GT to HFD rats was able to enhance SHp levels. GT is, in fact, composed of chlorogenic acid and naringenin, molecules with high reducing power, hence explaining the effect observed on thiol groups. Furthermore, the influence of GT supplementation on SH groups is supported by the result obtained in terms of reducing power, measured by FRAP in GT. We previously uncovered that SHp values negatively correlate with lipid profile biomarkers in MetS [20], thus showing that GT supplementation resets—in favor of thiolic groups—the MetS-induced shifted balance towards disulfide compounds. This could be due to a GT-mediated compensation that increases antioxidant defenses to counteract the excessive free radicals. It could appear counterintuitive that GT treatment did not manage to rescue BAP levels in HFD rats, though it further supports the specific effect induced by the reducing power of GT. In accordance with an improved antioxidant status, the oxidative biomarkers, i.e., dROMs and

LP-CHOLOX, were powerfully decreased by GT supplementation in HFD rats, showing a better protection from plasmatic lipid peroxidation products. In detail, GT extracts reduced both LP-CHOLOX levels and, not significantly, dROMs values, though basal levels of normally fed rats were not restored. The protective effect of GT supplementation on plasma lipoperoxidation and the plasma antioxidant barrier could be ascribed to naringenin, whose ability to reduce lipid peroxidation and normalize antioxidant defenses by increasing the activity of antioxidant enzymes in the liver has been demonstrated in the literature [41]. Reduced plasmatic levels of hydroperoxides and lipoperoxides by GT supplementation are thought to well-correlate with the improved glucose profile revealed by our study.

Relevantly, not only does GT supplementation reduce systemic oxidative stress but also significantly counteracts the HFD-induced hepatic production of RONS. Interestingly, these species have been shown to enhance MDA levels in the liver [53]. This reactive aldehyde has been demonstrated to irreversibly form adducts with macromolecules, modifying cell function and contributing to MetS development [53–55]. Consistently with the reduction of hepatic RONS levels, treatment with GT also markedly reduced the HFD-induced increase in hepatic lipid peroxidation and increased the GSH/GSSG ratio in the liver. These results are in line with previously reported evidence showing how RT supplementation has been demonstrated to exert powerful, anti-oxidative effects able to counteract the onset and development of several pathological conditions in humans, including MetS [56]. Notwithstanding the fact that BAP was not modified by GT treatment, our data clearly demonstrate an improvement of the hepatic redox state. This evidence might well be associated with the observed amelioration of the hepatic metabolic functions that are strongly dependent on the endocellular redox state [57]. Relevantly, the current experimental evidence demonstrates, for the first time in an in vivo model of MetS, the ability for GT to also reduce the HFDdependent hepatic oxidative stress and lipid peroxidation. In line with previously reported evidence [58], our data show that HFD-induced obesity is associated with the development of hepatic macrovesicular steatosis. Although tomato supplementation has already been reported to both ameliorate hepatic steatosis and reduce the risk of Nonalcoholic Fatty Liver Disease (NAFLD) development in rats [59], no data are available on the impact of GT treatment on hepatic dysfunction. Interestingly, our results show for the first time that GT consumption significantly improves hepatic steatosis. These effects on the HFD-induced liver structural damage are consistent and in agreement with the ability of GT to improve the plasma lipoprotein profile and to relieve hepatic oxidative stress.

In our study, we also tested the effects of red tomato at full maturation in vivo on the redox homeostasis biomarkers, considering that the effect of this food matrix has already been studied on dysmetabolism [30,60]. On this point, we uncovered that treating MetS rats with RT for one month is able to partially improve the systemic antioxidant endogenous barriers impaired by HFD, such as by increasing BAP levels and returning to basal control values, though not acting on the thiolic groups. Indeed, RT extracts appear to selectively target plasma antioxidant barriers in HFD rats, which could be due to the fact that RT is richer in carotenes, molecules capable of strongly influencing the BAP test. The effect exerted by RT on BAP test is in line with the antioxidant power measured by CBA in the RT food matrix. In contrast, the oral administration of GT to HFD rats was able to enhance SHp levels, but does not influence BAP levels in accordance with the outcomes of FRAP assay.

Even more evidently, RT reduced the prooxidant status in terms of lypoperoxides and hydroperoxides. Similarly, the hepatic oxidative stress biomarkers were positively modified by RT treatment in terms of MDA, RONS, and GSH levels. All these outcomes evaluated with crucial parameters of redox homeostasis confirmed the specific effects of RT in MetS, in accordance with previous literature [61], supporting the importance of tomato-based products in the prevention of oxidant-driven dysmetabolism.

A possible mechanism implicated in the GT-mediated protection exerted on HFDinduced liver damage could implicate adipokines (Figure 10). Indeed, alterations in adipokine levels are able to induce lipotoxicity and glucotoxicity in the liver with the development of steatosis through the involvement of TRP receptors [62]. Furthermore, according to data in the literature, naringenin—which we found to be specifically present in golden tomato—has been shown to act as a ligand for TRP receptors involved in many physiological processes that affect energy balance, inflammation, neuronal modulation, and oxidative stress [63–65]. It can be hypothesized that the reduction of free radicals observed in the liver and the regression of hepatic steatosis after treatment with the golden tomato could be associated with changes in the levels of the adipokines, leptin and adiponectin, as well as the involvement of TRP receptors, particularly TRPV1, as reported in the literature. This study opens up new possibilities for investigating the effect of golden tomato on the regression of NAFLD mediated by TRPV1 channels secondary to dietary activation of UCP2. Considering this, given the emerging clinical role of adipokines in cardiovascular disorders, the association of golden tomato with hypoadiponectinemia could suggest its role in the prevention of cardiovascular disorders.

**Figure 10.** Schematic representation of possible pathophysiological processes implicated in the protection by GT in MetS. The protection exerted by GT on body weight gain, systemic, and hepatic oxidative stress levels (i.e., SHp, lipoperoxides, MDA, RONS and GSH/GSSG) could hypothetically be due to an increase in adiponectin.

Furthermore, it has been shown that there is a correlation between alterations in the gut microbiota and the development of NAFLD [66,67]. In this view, the intake of golden tomato, extremely rich in fiber, could lead to the production of acetic acid, butyric acid, and propionic acid (SCFAs) important in intestinal homeostasis. SCFAs produced by fiber fermentation are able to influence the regulation of intestinal hormones such as gut trypsin peptide, glucagon-like peptide-1 (GLP-1), leptin, and peptide tyrosine–tyrosine (PYY) involved in energy balance and to maintain the integrity of the intestinal barrier by reducing the entry of substances that can generate metabolic inflammation responsible for metabolic disorders [68–70]. The results of the present study provide also an opportunity to further investigate the effect of golden tomatoes on the gut microbiota, focusing also on pre/probiotic compounds that could play a role [71], and the possible preventive action of this food in metabolic syndrome.

#### **5. Conclusions**

Our research for the first time discloses the potential of "Golden Tomato" oral supplementation, via a comprehensive characterization of its biochemical, antioxidant, and disease-modifying properties, exploiting an in vivo rat model of MetS. The current investigation hints at GT as a powerful novel nutritional modulator and functional food, similarly to more widely known bioactive molecules that can also influence brain processes [72]. Indeed, in the context of nutritional supplementation and healthy diet, antioxidant-functional foods could underpin a robust strategy for the prevention and management of a wide plethora of physiological conditions, theoretically inasmuch as adaptative responses could be implicated in the homeostatic balance of complex networks [73]. This research hence

substantiates the need for further investigations on tomato-based functional food, with a view to fully uncover GT nutritional application in food research.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12051121/s1, Table S1: Inclusion list of compounds sought and identified in the tomato samples analyzed; Table S2: Nutritional properties of tomato samples. The values are calculated on 100 g of edible part and are reported as the Mean ± SD of three repetitions; Table S3: Amount of micronutrients expressed in mg per 100 g of dry weight; Table S4: Quantity in mg of organic acids present in 100 g of dry product.

**Author Contributions:** Conceptualization: G.G. (Giuditta Gambino), G.G. (Giuseppe Giglia), G.F., P.S. and D.D.M.; methodology: G.G. (Giuditta Gambino), G.G. (Giuseppe Giglia), M.A., V.D.L., F.P.Z., F.R., I.R., F.V., F.S., E.P., G.A., P.S. and D.D.M.; validation, D.D.M. and G.G. (Giuseppe Giglia); formal analysis D.D.M., P.S., G.G. (Giuseppe Giglia), M.A., V.D.L., F.P.Z., F.R, I.R., F.V., F.S., E.P., G.A. and G.G. (Giuditta Gambino); investigation, D.D.M., P.S., G.G. (Giuseppe Giglia), M.A., F.P.Z., F.R., I.R., F.V., F.S., E.P., G.A., V.D.L. and G.G. (Giuditta Gambino); resources, D.D.M., G.F., P.S. and G.G. (Giuditta Gambino); data curation, D.D.M., P.S., G.G. (Giuseppe Giglia), M.A., F.P.Z., F.R. and G.G. (Giuditta Gambino); writing—original draft preparation, D.D.M., P.S., G.G. (Giuseppe Giglia), M.A., V.D.L., F.P.Z. and G.G. (Giuditta Gambino); writing—review and editing, all authors; supervision, D.D.M., P.S., G.G. (Giuseppe Giglia) and G.G. (Giuditta Gambino). All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partly supported by the project IN.PO.S.A-Grant Number: G66D20000170009, funded by PSR Sicilia (2014-2020)-16.1 and partly with FFR Funds provided by University of Palermo to Danila Di Majo and Giuditta Gambino.

**Institutional Review Board Statement:** The experimental protocols were approved by the animal welfare committee of the University of Palermo, authorized by the Ministry of Health (Rome, Italy; Authorization Number 14/2022-PR) and conducted following the ARRIVE guidelines.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on reasonable request from the corresponding author.

**Acknowledgments:** We thank Aten Center, in particular Rosa Pitonzo, and Gaetano Caldara and Riccardo Messina from the University of Palermo. We also thank Daniele Gallo, Alessandro Strano, Miriana Scordino, and Antonino Vitale, studying at the University of Palermo.

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

#### **References**


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## *Article* **Ethnopharmacological Effects of** *Urtica dioica***,** *Matricaria chamomilla***, and** *Murraya koenigii* **on Rotenone-Exposed** *D. melanogaster***: An Attenuation of Cellular, Biochemical, and Organismal Markers**

**Shabnam Shabir 1, Sumaira Yousuf 1, Sandeep Kumar Singh 2,\*, Emanuel Vamanu 3,\* and Mahendra P. Singh 1,\***


**Abstract:** Natural antioxidants derived from plants have been proven to have significant inhibitory effects on the free radicals of living organisms during actively metabolization. Excessive production of free radicals increases the risk of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and motor sclerosis. This study aimed to compare the ethnopharmacological effects of *Urtica dioica* (UD), *Matricaria chamomilla* (MC), and *Murraya koenigii* (MK) on the amelioration of rotenone-induced toxicity in wild-type *Drosophila melanogaster* (Oregon R+) at biochemical, cellular, and behavioral levels. Phytoextracts were prepared from all three plants, i.e., UD, MC, and MK (aqueous and ethanolic fractions), and their bioactive compounds were evaluated using in vitro biochemical parameters (DPPH, ABTS, TPC, and TFC), UV-Vis, followed by FT-IR and HPLC. Third instar larvae and freshly eclosed flies were treated with 500 μM rotenone alone or in combination with UD, MC, and MK for 24 to 120 h. Following exposure, cytotoxicity (dye exclusion test), biochemical (protein estimation and acetylcholinesterase inhibition assays), and behavioral assays (climbing and jumping assays) were performed. Among all three plant extracts, MK exhibited the highest antioxidant properties due to the highest TPC, TFC, DPPH, and ABTS, followed by UD, then MC. The overall trend was MK > UD > MC. In this context, ethnopharmacological properties mimic the same effect in *Drosophila*, exhibiting significantly (*p* < 0.05) reduced cytotoxicity (trypan blue), improved biochemical parameters (proteotoxicity and AChE activity), and better behavioral parameters in the organisms cotreated with phyto extracts compared with rotenone. Conclusively, UV-Vis, FTIR, and HPLC analyses differentiated the plant extracts. The findings of this research may be beneficial in the use of select herbs as viable sources of phyto-ingredients that could be of interest in nutraceutical development and various clinical applications.

**Keywords:** antioxidants; acetylcholinesterase; 1,1-diphenyl-2-picrylhydrazyl; HPLC; medicinal plants; oxidative stress

#### **1. Introduction**

The cellular redox status is determined by the balance between antioxidants and oxidants. The oxidative state of the cell is defined by an imbalance between these two, which can result in apoptosis or necrosis [1]. Reactive oxygen species (ROS) are primarily responsible for the high susceptibility of brain cells to oxidative stress [2]. Although oxygen is a relatively nonreactive substance, it can be metabolized in the body to create highly reactive free radicals, such as hydroxyl radicals (OH−), superoxide anions (O2 −), and many

**Citation:** Shabir, S.; Yousuf, S.; Singh, S.K.; Vamanu, E.; Singh, M.P. Ethnopharmacological Effects of *Urtica dioica*, *Matricaria chamomilla*, and *Murraya koenigii* on Rotenone-Exposed *D. melanogaster*: An Attenuation of Cellular, Biochemical, and Organismal Markers. *Antioxidants* **2022**, *11*, 1623. https://doi.org/10.3390/ antiox11081623

Academic Editors: Antonella D'Anneo and Marianna Lauricella

Received: 27 July 2022 Accepted: 17 August 2022 Published: 21 August 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/).

other reactive species [3]. These free radical species contribute to the pathophysiology of many neurodegenerative diseases, including amyloidosis, α-synucleinopathies, aging, and TDP-43 proteinopathies, which are caused by the inhibition of acetylcholinesterase [4]. Detoxification is aided by cellular defense mechanisms that involve endogenous antioxidants and antioxidant enzymes such as superoxide dismutase, glutathione reductase, lipid peroxidase, glutathione, and catalase [5]. The deterioration of these defense mechanisms damages significant cell biomolecules (lipids, DNA, and proteins) and ultimately leads to cell death [6].

Currently, conventional medicines or therapeutic drugs (levodopa or dopaminergic agonists) have only been used to treat motor symptoms by restoring neurotransmitters [7]. The extended use of these drugs, however, can have negative effects, such as fatigue and other motor difficulties [8]. Considering these limitations, there is a need for novel natural neuroprotective agents to halt or slow the progression of various neurodegenerative disorders [9]. According to cumulative evidence, nutraceuticals and other phytochemicals have been shown to have neuroprotective effects and alleviate the symptoms of neurodegenerative diseases by activating the PI3K/Akt/Nrf2 pathway by scavenging free radicals [10]. Herbal biomolecules have been used as phytomedicines in healthcare industries since the emergence of civilization; according to the World Health Organization (WHO), phytomedicine is used as a primary source of therapy by ~79% of the world's population [11]. *Urtica dioica* (UD), *Matricaria chamomilla* (MC), and *Murraya koenigii* (MK) have all been used as well-known cognition enhancers and nerve relaxants in the Ayurvedic medical system [12]. The phytochemicals of these three herbs, such as quercetin, polyphenols, alkaloids, reducing sugars, and vitamins, are known for their protective effects. UD has been extensively studied and has shown promising results in the treatment of prostate enlargement [13], colon carcinogenesis in rats [14], and protecting against hyperglycemia [15], hypertension, [16] and hypercholesterolemia [17]. Several studies have demonstrated the beneficial effects of MC against diabetes by regulating GLP-1, which is crucial in stimulating insulin gene transcription [18]. In addition, chamomile oil significantly decreased osteoarthritis [19], and may also help treat lung cancer [20]. Mondal et al. (2022) found that MK modulated various cellular programs and signaled cascades to intervene as an antioxidant in normal cells and as a pro-oxidant in lung carcinoma cells [21], protecting against liver damage caused by TPA [22]. Moreover, previous studies on the MK leaf fraction have observed its efficacy in treating hyperglycemia [23]. Although UD, MC, and MK have been used in several in vivo and in vitro experiments, there is still little evidence supporting of the therapeutic effect of these three herbs on cellular and neurological complications.

Considering the general protective and cognitive effects of UD, MC, and MK that have been reported in the literature. This study aimed to determine the bioactive compounds of these botanicals (UD, MC, and MK) through biochemical and analytical (UV-Vis, FTIR, and HPLC) methods. Furthermore, we attempted to investigate the cellular and neurological toxicities in a nontarget in vivo model of *D. melanogaster* through a widely used neurotoxic natural pesticide, rotenone (ROT). In addition, we also investigated its organismal effect on *Drosophila*, as it has been demonstrated to be a model framework of neurodegenerative diseases, particularly Parkinson's disease.

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

#### *2.1. Chemicals and Reagents*

Ellman's reagent (DTNB), 2,2- -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), gallic acid (C7H6O5), 1,-1-diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid (C6H8O6), sodium carbonate (Na2CO3), trichloroacetic acid (C2HCl3O2), sulfuric acid (H2SO4), ferric chloride (FeCl3), ethanol, gallic acid (C7H6O5), and aluminum chloride hexahydrate (AlCl3 6H2O) were obtained from Hi-Media (Mumbai, India). The solvent utilized for HPLC analysis was of HPLC grade, whereas all other organic solvents and chemicals were of analytical grade. Rutin, quercetin, rotenone, and acetylcholinesterase were obtained from

Sigma (Roedermark, Germany). Millipore grade water was used. Calorimetric analysis was performed using a Shimadzu UV-1601 spectrophotometer (Tokyo, Japan).

#### *2.2. Plant Materials Used in the Study*

The young leaves of *Urtica dioica* (UD) were collected from the local kitchen gardens and apple orchards of Sopore (Sopore, India) before the plants started developing seeds. *Matricaria chamomilla* (MC) flowers were procured from the Mediaroma Agro Producer Company Limited Kaskanj (Kasganj, India), and young leaves of *Murraya koenigii* (MK) were obtained from the garden maintained by Lovely Professional University (Phagwara, India). The identification and authentication of the medicinal plants were confirmed by a taxonomist from the Plant Sciences Division of the CSIR-Indian Institute of Integrative Medicine (IIIM) (Jammu, India).

#### 2.2.1. Drying, Processing, and Extraction of Samples

The collected plant leaves and flowers were washed under tap water and dried at room temperature for a week. After drying, all dry plant components were crushed to a fine powder with a mechanical grinder and then sieved through a 40-micron sieve to obtain fine particles. A 10% aqueous extract was prepared using five grams (±0.05) of the powdered sample mixed with 50 mL deionized water and steeping at 95–100 ◦C for 10–15 min. Then, the extracts were filtered by using Whatman No. 1 filter paper, and a 10% ethanolic extract was prepared using Soxhlet. Twenty grams (±0.05) of powdered plant samples were inserted into a Whatman 25 × 100 mm celluloid thimble by adding 200 mL of ethanol as a solvent at boiling temperature (70 ◦C). A dark green extract was obtained from UD and MK leaves, whereas the extract obtained from MC flowers was a pale yellow. The extract was then evaporated using a vacuum rotatory evaporator at 70 ◦C or less to remove the solvents. The crude extracts were weighed and stored at 4 ◦C in an airtight dark box for future assessment [24].

#### 2.2.2. Determination of Plant Yield

The percentage yields of UD, MC, and MK samples were calculated using the following formula [25]:

$$\mathbf{W\_2 - W\_1 \times 100} \tag{1}$$

where W2 signifies the weight of the extract including the container, W1 signifies the weight of the container itself, and W0 signifies the weight of the initial dried sample.

#### 2.2.3. Free Radical Scavenging Activity Using the DPPH Radical Assay

Antioxidant potential was measured using the 1,-1-diphenyl-2-picrylhydrazyl (DPPH) test [26]. A freshly prepared solution of DPPH (0.011 gm) was taken in 50 mL methanol for spectrophotometric measurements. The DPPH solution was further diluted with methanol, and the optical density (OD) was set between 0.8–1. Different concentrations of plant fractions were added to every 2 mL of DPPH mixture. Absorbance was measured at 517 nm using a Shimadzu UV-1601 spectrophotometer (Tokyo, Japan) after 30 min of incubation. Methanol was used as a blank, and DPPH was used as a control. Triplicate experiments were performed. Using the following equation, the radical scavenging activity was calculated as percent inhibition (1%) of the DPPH radical:

$$\text{DPPI inhibition (\%)} = \text{I} \{ \text{A}\_{\text{control}} - \text{A}\_{\text{sample}} \} / \text{A}\_{\text{control}} \} \times 100 \tag{2}$$

where Asample represents absorbance of the plant extract sample and Acontrol represents absorbance of the DPPH solution as a control.

#### 2.2.4. ABTS Radical Cation Decolorization Assay

The 2,2- -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization analyte was also used to assess the efficacy of plant fractions to scavenge free radicals, which is based on the reduction of ABTS radicals by antioxidants in plant extracts [27]. The radical cation formed when the ABTS stock solution (0.036 g in 10 mL methanol) was mixed with potassium persulfate (0.057 g in 10 mL methanol) at a 1:1 ratio. Then, the mixture was incubated in darkness for 16 h at ambient temperature. To attain an optical density (OD) of 0.8–1, the ABTS solution was further diluted with methanol. Every 2 mL of ABTS solution had extracts of various concentrations added to it. All samples were measured at 745 nm after 30 min of incubation. The percentage of scavenging activity was determined using the following equation:

$$\text{ABTS in inhibition (\%)} = \text{[(A}\_{\text{control}} - \text{A}\_{\text{sample}}) / \text{A}\_{\text{control}}] \times 100 \tag{3}$$

where Asample signifies absorbance of the plant extract sample and Acontrol signifies absorbance of the ABTS solution as a control.

#### 2.2.5. EC50 (Dose-Response Relationship)

EC50 is the half-maximal effective concentration of an antibody, drug, or toxicant that elicits a reaction halfway between the baseline and maximum, following a specific duration of exposure. Using CompuSyn software, data analysis for free radical scavenging activity and dose-response studies were performed to assess the potency of the selected herbs. A lower EC50 indicates greater radical scavenging activity [28]. SPSS software was used for statistical analysis.

#### 2.2.6. Determination of Total Phenolic Content (TPC)

The TPC of the plant fractions was measured by using Folin–Ciocalteu's colorimetric method [29]. Each plant fraction was coupled with 2.5 mL of the FC reagent (1:10 *v*/*v*) and vortexed. After 5 min, 2 ml of Na2CO3 (7.5%) were incorporated. Then, the solution was placed for approximately 90 min at room temperature before taking the OD at 760 nm by using a UV-Vis spectrophotometer. The results are given in mg GAE/g of dry weight. Triplicates of each sample were analyzed.

$$\mathbf{C} = \frac{\mathbf{c} \times \mathbf{V}}{\mathbf{m}} \tag{4}$$

where 'C' indicates the total phenolic component content in (mg g<sup>−</sup>1) plant extract in GAE, 'c' indicates the gallic acid concentration (mg mL<sup>−</sup>1), 'V' indicates the volume of extract in microliters (μL), and 'm' indicates the weight of crude plant extract in grams.

The correlation coefficient (R2) value was determined using the mean of three absorbance determinations for each concentration. The equation is shown below:

$$\mathbf{Y} = \mathbf{m}\mathbf{x} + \mathbf{c} \tag{5}$$

where 'Y' signifies extract absorbance, 'm' signifies the slope of the calibration curve, 'x' signifies extract concentration, and 'c' is the intercept. Concentrations of extracts were calculated using this regression equation. The phenolic content was estimated using the calculated value for each extract concentration.

#### 2.2.7. Determination of Total Flavonoid Content (TFC)

Total flavonoids were quantified using the aluminum chloride colorimetric technique [30]. The plant extracts were combined with methanol (1.5 mL), 100 μL of (10%) aluminum chloride followed by 0.1 mL of potassium acetate (1 M), and finally 2.8 mL of deionized water. The reaction mixture was placed for 40 min at ambient temperature, and the absorbance of the solution was obtained at 415 nm. Quercetin was used to create a calibration curve. The total flavonoid content was calculated in terms of quercetin equivalents (mg QE/g dry weight). Triplicate readings were taken for each plant sample.

$$\mathbf{C} = \frac{\mathbf{c} \times \mathbf{V}}{\mathbf{m}} \tag{6}$$

where 'C' is the total phenolic content in mg g−<sup>1</sup> plant extract in E, "c" reflects the quantity of quercetin determined by the calibration graph (mg/mL), 'V' shows the volume of plant extract in μL and 'm' is the weight of crude plant extract in grams. The absorbance of each concentration of the extract was measured using the procedure described above. Then, using the calculations above, the total flavonoid content was determined.

#### 2.2.8. Preliminary Qualitative Screening Analysis of Plant Extracts

Bioactive compounds such as polyphenols, reducing sugars, alkaloids, terpenoids, glycosides, flavonoids, saponins, and amino acids are mostly responsible for curative capabilities such as menstruation problems, muscular spasms, anemia, ulcers, hemorrhoids, inflammation, and wound healing [31]. The presence of phytochemicals is determined using conventional qualitative test methods, which include the following:


#### 2.2.9. UV-Visible Spectroscopic Analysis

The ultraviolet spectral data were obtained by a Shimadzu UV-1601 spectrophotometer (Tokyo, Japan). UV-Vis spectroscopy is concerned with the absorption of radiation in the ultraviolet and visible spectra and mostly used for quantitative analysis [35]. This radiation permits electrons in atoms or molecules to shift from lower to higher energy levels. The level of radiation absorbed is proportional to the number of molecules in the solution under specified conditions. Spectral data were used to demonstrate a link between absorption concentration and intensity. Quality control might thus be evaluated with a UV-Vis spectrometer without the need for expensive markers by establishing a library of spectrum data from actual raw samples [36]. Extracts of UD, MC, and MK were used for UV-Visible analysis. The samples were tested with a spectral range of 200–800 nm at 1 nm intervals at room temperature.

#### 2.2.10. FT-IR Spectroscopic Analysis

Fourier transform infrared spectrophotometry (FTIR) is one of the most potent instruments for detecting types of chemical bonds, molecular structures, and functional groups in substances. The absorbed wavelength of light is indicative of the chemical bond, as shown in the annotated spectrum. The chemical bonds of a molecule can be identified by interpreting the infrared absorption spectrum [37].

Ethanolic extracts of UD, MC, and MK were used for FTIR analysis. To make translucent sample discs, 10 mg of the crude plant extract sample was enclosed in 100 mg of KBr pellet. Then, the crude extract of each plant material was subjected to FT-IR spectroscopy (Perkin-Elmer Spectrum 2 with ATR and Pellet accessories). The samples were tested in the infrared band with a spectral range of 400–4000 cm−<sup>1</sup> and a resolution of ±4 cm−<sup>1</sup> at room temperature.

#### 2.2.11. HPLC Chromatographic Analysis

A Shimadzu Prominence I LC2030 Plus HPLC system (Kyoto, Japan) equipped with a Shimadzu LC 2030 UV-Vis detector was used to separate natural compounds from crude extracts of UD, MC, and MK. The standard external technique was used to perform HPLC analysis under isocratic conditions. Before running in the column, the mobile phase was degassed and filtered through a membrane using methanol and 0.5% acetic acid in water (90:10 *v*/*v*). Column C-18 (4.6 × 250 mm) with a 5 μm particle size was used and maintained at 25 ◦C temperature. Each injection volume was prepared at 20 μL and then injected into the HPLC. Samples were filtered using a 0.45 mm membrane filter (Millipore) before being put in vials, employing a 1.0 mL/min flow rate.

Spectral information was analyzed in the 200–400 nm region, and chromatograms were detected at a wavelength of 280 nm. Based on previous findings, the quantitative quantification of each bioactive component contained in the plant extracts was determined [38–40]. Peak identification was carried out by comparing the retention times of specific standards with those of the extract. The retention times of specific standards were compared with those of the extract to identify the peak.

#### *2.3. Fly Strain and Maintenance of Culture*

A wild-type *Drosophila melanogaster* strain (Oregon R+) that was kindly gifted by Dr. Anurag Sharma, Senior Assistant Professor, NITTE (Deemed to be University), Mangalore, India, and maintained on a standard *Drosophila* diet (containing maize powder, agar-agar, sugar, yeast, sodium benzoate, and propionic acid) was used for rearing. The flies were retained in a 12-h dark/light cycle at 24 ± 1 ◦C and 65–70% humidity levels. [41].

#### 2.3.1. Plant Concentration and Rotenone Exposure

To determine whether the treatment has any impact on the survival of the flies during the experimental period, preliminary studies were conducted with small numbers of flies, testing several concentrations (0.01, 0.025, 0.05, and 0.1%) of UD, MC, and MK. However, only one concentration, i.e., 0.1% per unit of the medium, was selected as the optimum concentration from the conclusive studies. Nevertheless, to evaluate the cellular and neurological protective properties of UD, MC, and MK, the concentration of rotenone (500 μM) used was selected based on our findings in *Drosophila* and those of other published studies [42–44].

#### 2.3.2. Treatment Schedule

For the experimental setup, flies/third instar larvae of *Drosophila* were divided into six groups. There were two groups of controls: Group I was fed the larval standard *Drosophila* food as a control, whereas Group II was fed food mixed with 0.1% DMSO as a vehicle control. Group III represented ROT (500 μM) treatment alone; Group IV comprised ROT with UD extract (0.1%); Group V consisted of ROT and MC extract (0.1%); Group VI was ROT cotreated with MK extract (0.1%). Larvae were permitted to feed either normally or with food that had been exposed to ROT or ROT+ fractions for 24 and 48 h. The flies were exposed for 120 h (5 days) and were assessed for jumping and climbing. We determined the modulatory effect of UD, MC, and MK fractions on rotenone-induced lethality, locomotor dysfunctions, inhibition of acetylcholinesterase, cellular toxicity, and proteotoxicity.

#### 2.3.3. Trypan Blue Dye Exclusion Assay

Dye exclusion was employed as described by Krebs and Feder (1997) with slight modifications [45]. This is a simple and quick method that distinguishes living and nonliving cells in tissue. It is used to detect cell death in the whole larvae and larval gut. At the end point of treatment, 5–10 larvae were thrice washed with 0.1 M phosphate buffer saline (pH 7.4), then whole or dissected midguts of larvae were incubated in trypan blue solution (0.2 mg/mL in 50.0 mM PBS, pH 7.4) for 15 min followed by three washes with 0.1 M phosphate buffer saline (pH 7.4). The larvae were analyzed by using a stereomicroscope; the images were acquired for trypan blue scoring, and were analyzed thoroughly.

#### 2.3.4. Homogenate Preparation

To obtain 10% homogenate/cytosol, the midgut of the third instar larvae was dissected from control (normal/untreated), DMSO, ROT, and ROT with phyto extract groups and homogenized in ice-cold 0.1 M phosphate buffer at a pH of 7.4, containing 0.15 M KCl. Following homogenization, the samples underwent centrifugation at 10,000× *g* at 4 ◦C for 15 min. After that, a nylon mesh sieve with a pore size of 10 mm was used to filter the supernatant, which was then used for various assays. [46].

#### 2.3.5. Protein Estimation

The method of Lowry et al. (1951) was used to measure the protein concentrations in the whole-body homogenates using bovine serum albumin as the standard [41].

#### 2.3.6. Acetylcholinesterase (AChE) Enzymatic Assay

AChE activity was measured as previously mentioned [47]. Briefly, the reaction was started by adding acetylthiocholine iodide (78 mM) to 0.1 M phosphate buffer with a pH of 8.0, which also contained 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB 10 mM) and sample (cytosolic) 0.01 mg protein. The change in absorbance was observed at 412 nm for three minutes. The amount of substrate hydrolyzed/min/mg protein was used to express enzyme activity.

#### 2.3.7. Measurement of Locomotor Deficits: Climbing Assay

The climbing assay was performed with some modifications, as previously described [48]. A vertical plastic tube measuring 18 cm in length and 2 cm in diameter was filled with twenty adult flies. During a 30 s time frame, flies followed tapping sounds until they reached the bottom of the vials; flies were scored if they crossed the 15 cm line. The average percentage of flies that cross the 15 cm line is represented by the climbing scores. The average number of flies above and below 15 cm (*n*top and *n*bot), expressed as a percentage of the total number of flies, determines the scores (*n*tot). The results are displayed in ±SD of the results from three independent observations. A performance index (PI) was calculated for each experiment and was given as follows: 1/2[(*n*tot + *n*top − *n*bot)/*n*tot].

#### 2.3.8. Jumping Assay

The jumping activity was performed to examine neuromuscular activity [49]. The speed of locomotor activity seems to have an impact on the threshold for the jumping reaction. One at a time, newly emerged flies were put into a labelled vial 1–10 cm, and the height the fly jumped from the bottom of the vial was recorded. The jumping activity was determined to be the mean number of jumps across five replicates. Five replicates of each group of 100 flies each were used.

#### *2.4. Statistical Analysis*

To analyze the UV-Vis, FT-IR, and HPLC profiles, professional assessment software was used (Digitized Quantitative Evaluation System of Herbal Medicine Chromatographic Fingerprints 4.0). EC50 analysis was performed using CompuSyn software (version 1.0). Chemometric data were expressed as the mean ± SEM, and using SPSS software (version 18), a two-way ANOVA and Tukey's test were used to compare n = 3 for significant differences. Significance was ascribed to a *p* value < 0.05.

#### **3. Results**

The results of the assays and analyses used to determine the feasibility of UD, MC, and MK extracts against rotenone-induced lethality, locomotor dysfunctions, inhibition of acetylcholinesterase, cellular toxicity, and proteotoxicity are presented in the subsections below.

#### *3.1. Analytical Assays*

#### 3.1.1. Percentage Yield of Bioactive Compounds

The results showed that MK leaf extraction yield was higher than that of UD and MC when distilled water was used as the extracting solvent (Table 1). The yield of ethanolic extraction was again higher in MK, followed by MC and UD. The findings also revealed that variation in the yield depends on the extraction solvent used. The variability in extract quantities from the plant materials used in this study could be due to the varying accessibility of extractable components caused by plant position.


**Table 1.** The percentage yield of plant extracts using aqueous and ethanolic extraction methods.

3.1.2. Antioxidant Potential of UD, MC, and MK

DPPH is widely used to evaluate the antioxidant and antiradical potential of plant fractions. The ability of antioxidants to scavenge DPPH radicals is assumed to be attributed to their hydrogen donating capabilities. The antioxidant potential of both aqueous and ethanolic extracts of UD, MC, and MK was measured using a free radical DPPH assay. As shown in Figures 1a and 2c, MK had the highest DPPH radical scavenging activity in both aqueous and ethanolic extracts, followed by UD and MC. Compared with aqueous extracts, ethanolic extracts of all three plants had the strongest scavenging efficacy.

ABTS is an unstable colored free radical that is used to investigate the antioxidant properties of both hydrophobic and hydrophilic antioxidants found in food extracts. The ABTS radical scavenging activity of the different extracts with different extraction solvents (aqueous and ethanol, Figures 1b and 2d) was found to be higher in MK, followed by UD and MC. These results suggest that MK has a higher efficacy in scavenging free radicals along with greater antiradical and antioxidant activity.

**Figure 1.** The effect of different concentrations of *U. dioica* (UD), *M. chamomilla* (MC), and *M. koenigii* (MK) in (**a**,**b**) aqueous and (**c**,**d**) ethanolic extractions on the (**a**,**c**) DPPH and (**b**,**d**) ABTS free radical scavenging assay. Data represent mean ± SD for n = 3. Statistically significance ascribed as \* *p* < 0.05 (intragroup) and # *p* < 0.05 (intergroup) compared with 0.5 mg/mL and 0.1 mg/mL of the respective groups.

### 3.1.3. EC50 Prediction Using Statistical Models

EC50 is a significant parameter for assessing antioxidant activity and can be used to compare the antioxidant capacities of different materials. The EC50 can be calculated, using various models, by interpolating data from a suitable curve or performing a nonlinear regression of the data using different components. The leaves of MK were found to have a higher antioxidant capacity due to its greater total phenolic and flavonoid content, with EC50 values of 0.33 and 0.10 mg/mL for aqueous and ethanolic extractions on DPPH scavenging and 0.51 and 0.07 mg/mL for aqueous and ethanolic extractions on ABTS radicals, respectively (Figure 2; Table 2).

**Table 2.** Estimated EC50 (mg/mL) of UD, MC, and MK were obtained by different models using DPPH and ABTS assays.


**Figure 2.** Dose-response profiles of the estimated EC50 (mg/mL) of (**a**,**c**) aqueous and (**b**,**d**) ethanolic extracts of *U. dioica* (UD), *M. chamomilla* (MC), and *M. koenigii* (MK) on the (**a**,**b**) DPPH and (**c**,**d**) ABTS assays.

#### 3.1.4. Phenolic and Flavonoid Potential of UD, MC, and MK

Individual aqueous and ethanolic extracts of UD, MC, and MK were quantified for total phenolic and flavonoid content. The total phenolic content (TPC) was calculated based on the gallic acid standard curve. In terms of aqueous extracts, MK (35.14 mg (GAE)/g) was found to have the highest TPC, followed by UD (26.08 mg (GAE)/g), then MC (24.01 mg (GAE)/g). The trend was the same in the case of ethanolic extracts: MK (48.93 mg (GAE)/g) was followed by UD (42.93 mg (GAE)/g) and was lowest in MC (40.5 mg (GAE)/g), as shown in Figure 3 and Table 3. The total flavonoid content (TFC) was calculated based on the quercetin standard curve. It was highest in MK leaves (9.64 mg (QE)/g), followed by MC flowers (5.46 mg (QE)/g), and lowest in UD leaves (5.45 mg (QE)/g) in the case of aqueous extracts, and the trend was the same for ethanolic extracts: MK (22.88 mg (GAE)/g), MC (12.64 mg (GAE)/g), and UD (12.48 mg (GAE)/g), as shown in Figure 3 and Table 3. The total amounts of phenolic and flavonoid content in the aqueous fractions of UD, MC, and MK were lower than in the ethanolic extracts. This may have been due to the aqueous solvent and extraction process employed. It has been shown that the solvent employed for extraction may be to blame for the quantity of phenolic content in the extract [24]. MK had the highest TPC and TFC levels compared with UD and MC in both aqueous and ethanolic extracts. The presence of hydroxyl groups in phenols allows them to scavenge free radicals, proving that they are the most essential phytochemicals.

**Figure 3.** Total (**a**) phenolic and (**b**) flavonoid contents of aqueous and ethanolic fractions of *U. dioica*, *M. chamomilla*, and *M. koenigii*. Data are shown as the mean ± SD for n = 3. Statistical significance is ascribed as \* *p* < 0.05 (intragroup) and # *p* < 0.05 (intergroup) of the respective groups.


*M. koenigii* Leaves 35.14 ± 3.0 48.93 ± 2.03 9.641 ± 2.5 22.88 ± 1.05


3.1.5. Preliminary Qualitative Screening Analysis of Plant Extracts

Phytochemicals are chemical molecules produced by plants due to their normal metabolic activities. These substances are referred to as secondary metabolites. There is still limited understanding of the benefits of plants because of a lack of raw data and experiments following proper scientific standards. Phytoconstituents such as amino acids, polyphenols, reducing sugars, alkaloids, terpenoids, glycosides, carbohydrates, and saponins are mostly responsible for plant curative capabilities, such as menstruation problems, muscular spasms, anemia, ulcers, hemorrhoids, inflammation, and wound healing. To confirm the existence of phytoconstituent substances, a phytochemical screening study was performed on the crude extracts of UD, MC, and MK, along with appropriate chemical tests. The presence of bioactive phytochemical substances such as phenols, flavonoids, alkaloids, tannins, carbohydrates, saponins, terpenoids, steroids, and glycosides are represented in Table 4**.**

**Table 4.** Preliminary qualitative screening of secondary metabolites of crude extracts of UD, MC, and MK.


Note: The presence of phytoconstituents is indicated by a '+' sign, whereas the lack of phytoconstituents is indicated by '−'.

#### 3.1.6. UV-Visible Analysis

Spectral data were observed in the range of 200–800 nm at intervals of 1 nm and showed maximum absorption at 277 and 321 nm in the case of UD, 266 and 323 nm for MC, and 286 and 316 nm for MK (Figure 4). With increasing concentration, absorption increased. This technique is a quantitative analysis of UD, MC, and MK and their absorption of radiation in the ultraviolet and visible spectra. This radiation enables electrons in atoms or molecules to shift from lower to higher energy levels. Under regulated conditions, the amount of radiation absorbed is proportional to the intensity of chemicals in the plant extracts. Spectral analysis showed peaks in crude extracts of UD, MC, and MK, indicating the presence of a variety of chemicals, particularly providing information about unsaturated bonds in conjugated or aromatic components.

**Figure 4.** UV-Visible spectra of (**a**) UD, (**b**) MC, and (**c**) MK fractions. Red arrows represent the peak of extract (conjugated or chemical bonds). The FT-IR absorption spectrum of (**d**) UD, (**e**) MC, and (**f**) MK with a scan range of 400–4000 cm<sup>−</sup>1.

#### 3.1.7. Fourier Transform Infrared Spectrophotometer (FTIR)

The functional groups of bioactive components present in plant extracts of UD, MC, and MK were identified using the FT-IR spectra depending on peak values in the IR radiation area. When the extract was run through FTIR, the functional groups of the constituents were segregated based on the ratio of their peaks. The existence of alkanes, amino acids, aldehydes, phenols, secondary alcohols, aromatic amines, ketones, and halogen compounds was verified by FTIR analysis (Figure 4).

In the ethanolic extracts of UD, MC, and MK, the band between 3500 and 3200 cm−<sup>1</sup> was assigned to an O–H stretching, indicating the presence of hydroxyl and phenolic groups. These groups are found in cellulose, hemicellulose, and lignin structures and could be related to the hydroxylated compounds (polyphenols) and moisture content of the extract. A minor intensity peak in the region of 2950–2960 cm−<sup>1</sup> showed an O–H (stretch), suggesting the existence of amide groups and alcohols, as well as C–H vibrations of the CH3 group. In the area of 1650–1600 cm−1, an acceptable N=O (stretch) vibration was recorded. The weak peaks in this area suggest the existence of C=C (stretch). Absorption in this region may indicate the existence of carbonyl groups. A doublet band was seen in the

fingerprint area between 1455 and 1370 cm<sup>−</sup>1, indicating the C-H (bending) vibration of the methyl group (–CH3) molecule. Furthermore, a broad peak was found between 1100 and 1000 cm−1, suggesting the presence of alcohol groups within the compound structure. This is most likely due to an alkoxy C–O (stretching) vibration. In addition, the UD, MC, and MK fractions showed aromatic C–H bonds between 720 and 620 cm−<sup>1</sup> in the infrared spectrum (Table 5).



Finally, the results revealed that the chemical structures of UD, MC, and MK are extremely polar (lignin (+)-neoolivil, 3,4-divanillyltetrahydrofuran, isolariciresinol, (−)-secoisolariciresinol, and pinoresinol), which stimulates the proliferation of human lymphocytes and has anti-inflammatory effects, as evidenced by the presence of wide peaks within the spectra.

#### 3.1.8. High-Performance Liquid Chromatography (HPLC)

The external standard technique was used to perform HPLC experiments under isocratic conditions. Ethanolic extracts of UD, MC, and MK were analyzed directly using the total extracts without any manipulation. The retention time of the chromatographic peaks of plant extracts was compared with reference standards (rutin and quercetin), and DAD spectra (200–400 nm) of existing literature were analyzed (Figure 5). Our findings revealed the presence of 24 compounds (Table 6) in the ethanolic extracts of UD, MC, and MK, including quercetin, coumaric acid, chlorogenic acid, gallic acid, apigenin, myricetin, ferulic acid, fumaric acid, rutin, isorhamnetin, kaempferol, etc.

In the ethanolic extract of UD, three classes of phenols were characterized: anthocyanin compounds (rosinidin 3-*O*-rutinoside; peonidin 3-*O*-rutinoside; and peonidin 3-O-(6- -O-coumaroyl glucoside), hydroxycinnamic acid derivatives (p-coumaric acid; chlorogenic acid; caffeoylquinic acid; and 2-O-caffeoylmalic acid), and flavonoids (rutin; isorhamnetin 3-O-rutinoside; quercetin; p-coumaroyl glucoside; kaempferol 3-O-rutinoside; kaempferol 3-O-and glucoside; and quercetin 3-O-glucoside).

The polyphenolic compounds found in the ethanolic fractions of MC flowers were identified as essential constituents, such as quercetin (quercetin-7-O-β-glucoside; quercetin-3-O-β-rutinoside; and quercetin-3-O-β-galactoside), apigenin (apigenin-7-O-7-glucoside; apigenin-7-O-apiosyl-glucoside; and apigenin-7-O-glucosyl-6- -acetate), luteolin (luteolin-7-O-β-glucoside; luteolin-4- -O-7-β-glucoside; and luteolin-7-O-β-rutinoside), isorhamnetin (isorhamnetin-7-O-β-glucoside), patuletin (patuletin-7-O-β-glucoside), eupatoletin, astragalin, chrysosplenol, and spinacetin. The MK ethanolic fraction was examined by HPLC-DAD, which permitted the identification of important components such as chlorogenic acid, quercitrin, citric acid, piperine, 7 p-coumaric acid, hesperidin, rutin, gallic acid, β-terpineol, ferulic acid, catechin, naringenin, D-α-pinene, di-α-phellandrene, dipentene, D-sabinene, caryophyllene, nicotinic acid, koenigine-quinone A, and koenigine-quinone B. All these secondary metabolites have been shown to have cerebrovascular protective, neuroprotective, and cardiovascular protective properties. In addition, it also acts as an anti-carcinogenic, anti-tumor, anti-inflammatory, antimicrobial, antiviral, and antibacterial agent and protects against oxidative stress-related diseases.

**Figure 5.** HPLC profiles acquired at 280 nm of (**a**) standard rutin and quercetin and ethanolic extracts obtained from (**b**) *U. dioica* leaves, (**c**) *M. chamomilla* flowers, and (**d**) *M. koenigii* leaves, showing different bioactive compounds. R stands for rutin, and Q stands for quercetin.

#### *3.2. Cellular Assays*

Cytotoxicity of Rotenone and Amelioration of Cytotoxicity through Bioactive Compounds UD, MC, and MK Determined by a Dye Exclusion Test (Trypan Blue) in Whole Larvae and Tissues of ROT-Exposed Organisms

To determine if exposure to ROT causes any tissue damage, we analyzed trypan blue staining in whole larvae and tissues of D. melanogaster (Figure 6). Of the larvae exposed to ROT, 95% showed blue staining in the whole larvae and their tissues (brain ganglia, salivary gland, midgut, and gastric caeca). ROT coexposed with MK exhibited significantly less blue staining than in the ROT + UD and ROT + MC groups in the whole larvae and the abovementioned tissues, respectively.


**Table 6.** Major

phytochemical

 compounds

 identified in ethanolic extracts *of U. dioica, M. chamomilla*,

 and *M. koenigii*.


**Table 6.** *Cont.*


**Table 6.** *Cont.*


**Table 6.** *Cont.*


**Figure 6.** Dye exclusion test through trypan blue staining in third instar larvae exposed to rotenone and cotreated with UD, MC, and MK, as shown in the upper panels. The lower panels show dissected third instar larvae stained with trypan blue. Seventy-two hour (±2 h) old larvae (early third instar) of D. melanogaster (Oregon R+) were exposed to ROT 500 μM alone or in combination with UD, MC, and MK for 48 h. Arrows of the upper panel show cytotoxicities in the whole larvae through trypan blue staining. Note: bg= brain ganglia, sg= salivary glands, pv= proventriculus, mg= midgut, mt= malpighian tubules, and hg = hind gut. The bar represents 100 μm. ROT= rotenone; UD = *Urtica dioica*, MC = *Matricaria chamomilla* and MK = *Murraya koenigii*.

#### *3.3. Biochemical Assays*

3.3.1. Decreased Protein Content in *D. melanogaster* Treated with Rotenone after 24 and 48 h

Third instar larvae of *D. melanogaster* exposed to 500 μM ROT exhibited a significant reduction (*p* < 0.05) in the total protein content of their tissues. After 24 h, the protein content in the larvae was reduced in the ROT group (9.34 ± 0.150 mg/mL) compared with the control group (12.88 ± 0.313 mg/mL). ROT coexposed with MK (11.63 ± 0.225 mg/mL) showed highest protein levels, followed by ROT + UD (10.05 ± 0.381 mg/mL), and ROT + MC (9.46 ± 0.174 mg/mL). After 48 h, in comparison with the control group (11.47 ± 0.328 mg/mL), the ROT treatment decreased the protein content in the larvae (7.37 ± 0.225 mg/mL). Increased protein levels were observed in ROT coexposed with MK (11.71 ± 0.263 mg/mL), followed by ROT + UD (9.07 ± 0.196 mg/mL), and ROT + MC (7.72 ± 0.213 mg/mL) (Figure 7).

3.3.2. Rotenone Inhibits AChE Activity in *D. melanogaster*, and This Effect Is Reversed by Phytoextraction

In this study, it was found that when the larvae were exposed to ROT for 24 h, they exhibited statistically significant (*p* < 0.001) inhibition of AChE activity compared with the control or DMSO, and ~60% reduced AChE levels were observed in this group. When ROT was coexposed with MK, AChE levels were improved, and only 9.7% inhibition was evident compared with the control. These elevated levels of AChE were significant when compared with the ROT-treated groups. The AChE levels in the ROT + UD and ROT + MC groups were also significantly improved (40.5% and 52.0% inhibition, respectively). Maximum inhibition of AChE levels was present in ROT-exposed organisms after 48 h (69.13% compared with control larvae), and the greatest improvement from ROTinduced toxicity was observed in the ROT + MK group (5.63% compared with control

groups). Significantly higher AChE levels were also observed in the ROT + UD and ROT + MC groups (38.15 and 47.45% inhibition, respectively) than in the ROT-treated group (Figure 8).

**Figure 7.** Total protein content in third instar larvae of *D. melanogaster* (Oregon R+) exposed to 500 μM rotenone for 24 and 48 h. Data represent the mean ± SD of three identical experiments made in three replicates. Significance is ascribed as \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. control or DMSO control. # = significance at \* *p* < 0.05 as compared with 500 μM rotenone. UD = *Urtica dioica*, MC = *Matricaria chamomilla* and MK = *Murraya koenigii*.

**Figure 8.** Acetylcholinesterase activity in the third instar larvae of *D. melanogaster* (Oregon R+) exposed to 500 μM ROT alone or in combination with UD, MC, and MK for 24 and 48 h. Data represent mean ± SD (n = 3); significance ascribed as \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. control or DMSO control. # is ascribed as significance at *p* < 0.05, ### *p* < 0.001 as compared with 500 μM rotenone. UD = *Urtica dioica,* MC = *Matricaria chamomilla* and MK = *Murraya koenigii*.

#### *3.4. Behavioral Assays*

3.4.1. Rotenone Affects Locomotor Behavior in *D. melanogaster*

After 30 s, the control and DMSO-treated flies demonstrated maximum climbing ability (only 11% and 12% reduction, respectively). The greatest reduction in climbing ability was observed in ROT-treated *Drosophila* (50.5%), and flies found it difficult to climb the plastic tube walls. The groups receiving ROT + phytoextracts exhibited varying levels of improvement in their climbing skills. All nutraceuticals improved the climbing ability of flies. Among the nutraceutical groups, ROT + MK (15%) exhibited the greatest improvement, followed by ROT + UD (26%), then ROT + MC (37%). To identify any significant differences, the mean ± SEM was compared using an unpaired Student's *t*-test. Significance was ascribed at *p* < 0.001 (Figure 9A).

**Figure 9.** (**A**) Jumping and (**B**) climbing activity of *D. melanogaster* (Oregon R+) flies exposed to ROT 500 μM alone or in combination with UD, MC, and MK for 120 h; significance is ascribed as \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. control or DMSO control. ## ascribed as significance at *p* < 0.01, ### *p* < 0.001 compared with 500 μM rotenone. UD = *Urtica dioica*, MC = *Matricaria chamomilla* and MK = *Murraya koenigii*.

3.4.2. Significant Changes in the Jumping Activity of ROT-Exposed Flies

We observed significantly decreased jumping behavior in the flies treated with ROT (62%) compared with the control and DMSO (22 and 25%, respectively). The groups receiving ROT + phytoextracts exhibited varying levels of improvement in their jumping skills. All nutraceuticals improved the jumping ability of flies. ROT + MK (31%) exhibited the greatest improvement, followed by ROT + UD (40%), and ROT + MC (49%). An unpaired Student's t-test was used to compare the mean ± SEM. Significance was ascribed at *p* < 0.001 (Figure 9A).

#### **4. Discussion**

The present study demonstrates the protective efficacy of UD, MC, and MK against rotenone-induced cellular, neurological, and organismal toxicity in a nontarget organism, *Drosophila melanogaster*. Unlike synthetic drugs, herbal medicines have a comprehensive structure of chemical elements. As a result, the methods of choice for identifying a 'botanical medicine' are primarily designed to obtain a unique fingerprint of certain plants that indicates the existence of quality-defining active chemical elements [50]. It is noteworthy that the Ayurvedic medical system has well-established the benefits of UD, MC, and MK for improving cognition, memory, and learning. To our knowledge, the current study is the first to report the comparative efficacy of UD, MC, and MK against various ROT-induced toxicities in *Drosophila*.

In this study, we examine how different aqueous and ethanolic fractions of UD, MC, and MK react to different radicals. Our results demonstrate that the ABTS activity in MK extracts did not differ significantly from the free radical scavenging potential measured by the DPPH assay because both assays use the same mechanism (single-electron transfer). Among the three extracts, MK displayed the greatest scavenging activity in both the DDPH and ABTS radical scavenging assay, as shown in Figure 1. Previous research has shown that MK has great antioxidant potential [51]. MK leaves had greater antioxidant capacities than UD leaves and MC flowers, owing to greater total phenolic and flavonoid content, with EC50 values for aqueous and ethanolic extracts in DPPH scavenging of 0.33 and 0.10 mg/mL, respectively, and EC50 values for aqueous and ethanolic extracts in ABTS scavenging of 0.51 and 0.07 mg/mL, respectively. Aqueous extracts of UD, MC, and MK had lower total phenolic and flavonoid concentrations their ethanolic counterparts. This could be due to the aqueous solvent and extraction method used. It was also discovered that the extraction solvent could be at fault for the amount of phenolic content in the extracted samples [52]. In both the aqueous and ethanolic fractions, the TPC and TFC were highest in MK, followed by UD, and MC had the lowest TPC values. This could be due to the extraction solvent used, as ethanolic extraction solvent was previously regarded to be the best for extracting total flavonoids [53]. Overall, MK exhibited the highest antioxidant properties due to the highest TPC, TFC, DPPH, and ABTS, followed by UD and MC. The aromatic leaves of MK have 11–21 pinnate leaflets that are each 2–4 cm (0.80–1.59 in) long and 1–2 cm (0.38–0.80 in) wide. It is known that MK leaves are a major source of phyto-carbazole alkaloids, comprising mahanimbine, koenine, girinimbine, murrayacine, koenidine, mahanine, and 8,8- -biskoenigine, with promising pharmacological activities [51]. Bioactive phytochemical substances of these three herbs, such as phenols, flavonoids, alkaloids, tannins, saponins, terpenoids, steroids, and glycosides, are primarily responsible for curative properties such as menstrual problems, muscular spasms, anemia, ulcers, hemorrhoids, inflammation, and wound healing [54].

UV-Vis spectral analysis was recorded in the range of 200–800 nm with intervals of 1 nm and showed maximum absorption at 277 and 321 nm for UD, 266 and 323 nm for MC, and 286 and 316 nm for MK. Beer's rule asserts that the amount of light absorbed at a specific frequency is proportional to the sample's composition absorption coefficient [55]. As a result, all spectral fluctuations caused by spectrophotometer and sample inaccuracies must be rectified before information processing. Peaks in crude extracts indicated the presence of a variety of ingredients or chemicals, particularly unsaturated bonds in conjugated or aromatic components [56].

A Fourier transform infrared spectrometer was used to determine the functional groups of the bioactive components in the plant fractions based on peak intensity in the infrared radiation (IR) region. The specific wavenumbers and intensities were determined in the range of 4000–400 cm−<sup>1</sup> [57]. Both stretching and bending vibration assignments were compared with data from the literature. Figure 5 represents the FTIR spectrum of UD, MC, and MK extracts in the form of KBr pellets and shows the presence of phenols, alcohols, ketones, nitro compounds, esters, carboxylic acids, ethers, aliphatic fluoro, alkenes, and aromatic rings. The broad absorption bands observed at 3358.28, 3327.40, and 3307.88 cm−<sup>1</sup> were attributed to the stretching of hydroxyl groups and H–bonding in alcohol or phenol groups [58]. The weak absorption peaks in alkanes were detected at 2923.25, 2921.90, and 2974.04 cm−1, which correspond to C–H stretching. N–H bends in primary amines were indicated by the high absorption peaks at 1698.70, 1622.77, 1601.79, and 1611.44 cm−1. C–C stretching in aromatic groups was assigned to the medium peaks at 1455.85 and 1404.99 cm<sup>−</sup>1. The rocking of the methyl group was assigned to the vibrational absorption bands at 1399.89 and 1396.51 cm−1. C-O stretching was represented by distinct bands at 1271.82 and 1275.04 cm−1. The C–N stretch in aliphatic amines was assigned to the thin peaks at 1055.84, 1074.42, 1043.61, and 1044.62 cm<sup>−</sup>1. The aromatic H out-of-plane bending had bands at 877.47, 720.06, 659.68, and 620.82 cm−<sup>1</sup> [59].

The HPLC technique is repeatable, sensitive, and reliable. The existence of 24 chemicals in the ethanolic fractions of UD, MC, and MK was identified using HPLC. In the UD ethanolic extract, three classes of phenols were characterized: anthocyanin compounds (rosinidin 3-O-rutinoside; peonidin 3-O-rutinoside; and peonidin 3-O-6- -O-coumaroyl glucoside), hydroxycinnamic acid derivatives (p-coumaric acid; chlorogenic acid; caffeoylquinic acid; and 2-O-caffeoylmalic acid), and flavonoids (rutin; isorhamnetin 3-O-rutinoside; quercetin; p-coumaroyl glucoside; kaempferol 3-O-rutinoside; and quercetin 3-O-glucoside), as described earlier [38]. The polyphenolic compounds found in the ethanolic fractions of MC flowers were identified by comparing with a previous study [60]. Our findings showed the existence of important constituents, such as quercetin (quercetin-7-O-β-glucoside; quercetin-3-O-β-rutinoside; and quercetin-3-O-β-galactoside), apigenin (apigenin-7-O-7-glucoside; apigenin-7-O-apiosyl-glucoside; and apigenin-7-O-glucosyl-6'-acetate), luteolin (luteolin-7- O-β-glucoside; luteolin-4'-O-7-β-glucoside; and luteolin-7-O-β-rutinoside), isorhamnetin (isorhamnetin-7-O-β-glucoside), patuletin (patuletin-7-O-β-glucoside), eupatoletin, astragalin, and spinacetin [61]. The MK ethanolic fraction was examined by HPLC-DAD, which permitted the identification of important components such as chlorogenic acid, quercitrin, citric acid, piperine, 7 p-coumaric acid, hesperidin, rutin, gallic acid, β-terpineol, ferulic acid, catechin, naringenin, D-α-pinene, di-α-phellandrene, dipentene, D-sabinene, caryophyllene, nicotinic acid, koenigine-quinone A, and koenigine-quinone B [62]. The various phytochemicals in the UD, MC, and MK extracts that are responsible for their antioxidant and protective potential have been well identified. UD has been extensively studied and has shown prominent results in the treatment of prostate enlargement [13], preventing colon carcinogenesis in rats [14], and providing a protective effect against hyperglycemia [15], hypertension [16], and hypercholesterolemia [17]. Numerous studies have shown that MC counteracts diabetes by controlling GLP-1, which is essential for promoting insulin gene transcription [18]. Chamomile oil has also been shown to significantly reduce osteoarthritis [19], and it may be useful in the treatment of lung cancer [20]. Mondal et al., 2022 found that MK modulates various cellular programs and signaling cascades to intervene as an antioxidant in normal cells, as a pro-oxidant in lung carcinoma cells [21], and protects against liver damage caused by TPA [22]. Additionally, earlier research on the MK leaf fraction reported its efficacy in the management of hyperglycemia [23]. Although UD, MC, and MK have been studied in some in vivo and in vitro experiments, there is little evidence for the effect of these three herbs on cellular and neurological complications.

Considering the general protective, organismal, or cognitive effects of UD, MC, and MK that have been reported in the literature, the present study treated third instar larvae and freshly eclosed flies with 500 μM ROT alone or in combination with UD, MC, and MK for 24 to 120 h. Following exposure, cytotoxicity assays (dye exclusion test), biochemical assays (protein estimation and acetylcholinesterase inhibition assays), and behavioral assays (climbing and jumping assays) were performed. ROT is a well-known generator of reactive oxygen species (ROS), which cause cellular damage and eventually lead to necrosis or programmed cell death. Lacking an effective antioxidant system, cells are unable to prevent the harm caused by ROS. *<sup>L</sup>*-DOPA appears to simply act as a dopamine precursor to restore endogenous dopamine deficits, as previous studies have shown that feeding the drug to flies did not reduce cell loss [63].

The cytotoxicity of rotenone and amelioration of cytotoxicity through the use of bioactive compounds (UD, MC, and MK) were determined through a dye exclusion test (trypan blue) in whole larvae and tissues of ROT-exposed organisms. Of the larvae exposed to ROT, 95% showed blue staining in the whole larvae and their tissues (brain ganglia, salivary gland, midgut, and gastric caeca). ROT combined with MK exhibited significantly less blue staining than the ROT + UD and ROT + MC groups in the whole larvae and the abovementioned tissues, respectively. This observation is supported by a previous study on ROT, which found that nutraceuticals significantly improved cell viability [64]. This protective property of UD, MC, and MK may be attributed to the presence of bioactive components responsible for quenching free radicals or due to the upregulation of antioxidative defense mechanisms.

To comprehend the unfavorable effects of ROT that increase cellular oxidant levels and cause proteins to undergo oxidative post-translational modifications, biochemical studies were carried out after giving treatment for 24 and 48 h. Third instar larvae of *D. melanogaster* exposed to 500 μM ROT exhibited a statistically significant decrease (*p* < 0.001) in the total protein content of their tissues. After 24 h, the protein content in the larvae was reduced in the ROT treated group compared with the control and DMSO control group. ROT coexposed with MK showed improved protein concentrations, followed by ROT + UD and ROT + MC. After 48 h, the trend remained the same in comparison with the control group, and the ROT treatment decreased the protein content of the larvae. Increased protein was observed in ROT coexposed with MK, followed by ROT + UD, and lowest in ROT + MC. This finding is consistent with previous research that found various pesticides led to reduced protein content in organisms [65].

Acetylcholinesterase (AChE) is a vital enzyme of the cholinergic system that modulates physiological processes, including memory and locomotor activities. It hydrolyzes acetylcholine to choline and acetate, thereby terminating cholinergic neurotransmission between synapses. In this study, it was found that when the larvae were exposed to ROT for 24 h, they exhibited a statistically significant (*p* < 0.001) inhibition of AChE activity compared with the control or DMSO and had ~60% reduced AChE levels. When ROT was coexposed with MK, AChE levels were improved, and only 9.7% inhibition was evident. AChE levels were improved to a lesser extent in the ROT + UD and ROT + MC groups compared with the control. These elevated levels of AChE were significant when compared with the ROT-treated groups. The maximum inhibition of AChE levels was present in ROT-exposed organisms after 48 h compared with control larvae, and the highest rescue from ROT-induced toxicity was observed in the ROT + MK group, followed by the ROT + UD and ROT + MC groups. We observed that inhibition of AChE in ROT-exposed organisms and nutraceuticals helps to rescue AChE levels, which is supported by previous observations [66,67].

An organism's behavior reflects its typical physiological activity. Climbing and jumping activities in this context reflect the physiological condition of the organism. Therefore, a high rate of locomotor deficits as evaluated by the climbing assay may indicate rotenoneinduced neurotoxicity. Due to their propensity to remain at the base of the plastic tube, flies with locomotor deficits do not appear to have normal leg coordination. This phenotypic expression has previously been attributed to the high energy needs of the muscles used for walking and flying, which are packed with mitochondria. Although speculative, uncoupled mitochondrial machinery may likely be to blame for the same underlying conditions of severe complex I inhibition. Surprisingly, MK > UD > MC were able to significantly rescue flies from worsening locomotor dysfunctions, showing that they may be able to protect by restoring the dopamine pool at the mitochondrial level. This finding supports previous research that showed a strong link between dopamine deficiency and locomotor dysfunction [68]. The adverse effect of the pesticide on the organism was shown by

the significant decrease in jumping behavior in exposed organisms, which was followed by an inhibition of AChE activity. Inhibition of AChE activity has previously been described as a sign of poor locomotor activity [69]. All nutraceuticals improved the jumping ability of flies. ROT + MK exhibited the highest rescue, followed by ROT + UD, and ROT + MC. In this context, the isolation of naturally occurring antioxidants have raised interest in plant biomass, which has proven to be rich in compounds. Plants possess the ability to biosynthesize a variety of non-enzymatic antioxidants that can reduce ROS-induced oxidative damage [70–73].

Taken together, the current study suggests that UV-Vis, FTIR, and HPLC analysis differentiates the extracts of UD, MC, and MK. The comparative account of these medicinal herbs revealed significant variation, which can be used to identify plants that have the most phytoconstituents to be used as phyto remedies for a variety of diseases. Based on our biochemical evidence, we conclude that short-term dietary feeding of UD, MC, and MK to *Drosophila melanogaster* has the propensity to attenuate ROT-induced oxidative stress because of its antioxidative properties and capacity to regulate antioxidant defenses. Additionally, their neuroprotective properties were demonstrated by their potential to significantly alleviate rotenone-induced oxidative stress, enhance locomotion, and restore AChE levels. Moreover, these findings reveal that these plants are medicinally important and should be studied further to locate bioactive compounds and determine their significance in pharmaceutical industries. In the future, we will work with advanced spectroscopic and nanotechnology-based investigations for the identification and structural elucidation of compounds present in UD, MC, and MK against ROT-induced toxicities.

#### **5. Conclusions**

Current research provides evidence that the antioxidant and antiradical activities of three ethnomedicinal plants collected from different geographical origins and varieties have statistically significant (*p* < 0.001) varied complex chemical mixtures. This investigation has provided preliminary information to determine the chemical composition of *U. dioica, M. chamomilla,* and *M. koenigii* using UV-Vis, FT-IR, and HPLC techniques. From the above investigations, it can be concluded that MK has higher anti-radical activity in aqueous as well as ethanolic extracts in DPPH, ABTS, TPC, and TFC assays among the screened plants. The presence of the O-H, C-H, N-H, C-O, C=O, C-C, C-N, N=C, S=O, and C=N groups were predicted by UV-Vis, FT-IR, and HPLC. Additionally, we conclude that shortterm nutritional feeding of UD, MC, and MK to *Drosophila* has the potential to reduce ROT-induced oxidative stress due to its antioxidative properties and capacity to regulate antioxidant defense mechanisms. They are promising plants for future research due to their antioxidant capabilities and might help slow or prevent the process of oxidative stress-related diseases. The findings of this study will be useful in the quality control of raw herbaceous material to verify their potential for phytopharmaceutical applications and health-promoting properties that could be used in drug discovery. However, further research is needed for a better understanding of their bioactivity, toxicity profile, and impact on the ecosystem and agricultural commodities.

**Author Contributions:** Conceptualization, M.P.S. and S.S.; methodology, M.P.S. and S.S.; software, S.S. and S.Y.; validation, M.P.S. and S.S.; formal analysis, M.P.S. and S.S.; resources, M.P.S.; writing original draft preparation, S.S., S.Y., M.P.S., E.V. and S.K.S.; supervision, M.P.S. All authors have read and agreed to the published version of the manuscript.

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

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

**Informed Consent Statement:** Not applicable.

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

**Acknowledgments:** We acknowledge Lovely Professional University for providing the infrastructure and reagents required for our research. The authors are also grateful to Anurag Sharma, NITTE (Deemed to be University), Mangalore, India, for kindly providing a gift of the *Drosophila* stock (Oregon R+).

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

#### **References**

