MOLECULAR DOCKING OF SPEARMINT PHYTOCOMPOUNDS AGAINST CYP21A2: IMPLICATIONS FOR PCOS THERAPY

Objective s : In this study, phytocompounds of spearmint that is known to have anti-androgenic activity are docked against a protein CYP21A2. This protein is also known as progesterone complex, one of the member cytochrome P450 enzymes; mutations in the genes encoding these proteins are causative factors of polycystic ovarian syndrome (PCOS). Methods: The study was based on computations using different phytochemicals of spearmint docking to a target protein CYP21A2 which causes hormonal imbalance leading to PCOS and hirsutism. Molecular docking was conducted using PyRx-virtual screening tool and Biovia discovery studio 2.0 to determine binding affinities of different phytochemicals to target protein. Results: The docking result revealed that bicyclogermacrene, cubebol, (-)-beta-bourbonene, alpha-bourbonene, and spathulenol showed highest binding affinities between –8.1 and –8.5 kcal/mol. Further, absorption, distribution, metabolism, excretion, and toxicity properties of these compounds are explored mainly to understand the possibility of developing potential drugs for PCOS. Conclusion: These bioactive compounds can be considered as potential agents that can be used with polyherbal plant extract to reduce the androgen levels in women suffering from PCOS.


INTRODUCTION
Polycystic ovarian syndrome (PCOS) can be considered the epidemic endocrine disorder of the 21 st century [1,2].It is affecting women of reproductive age, around the age group between 18 and 44.PCOS is also linked with irregularity in menstrual cycles, hyperandrogenism, alopecia, hirsutism, obesity, insulin (INS) resistance, anovulation, oligomenorrhea, and pre-diabetes [3,4].It is characterized by elevated androgen levels (hyperandrogenemia) leading to anovulation, microcysts in ovaries (polycystic ovaries) and can cause inhibition of follicular development.PCOS condition is diagnosed based on the National Institutes of Health consensus or Rotterdam criteria [5].PCOS is a multifactorial condition that is a combination of genetic and environmental factors, the latter factor predominantly involves poor dietary choices, increased stress levels among working women, and lack of physical activity [6].
Leaves of spearmint are known to have anti-androgenic properties and also reduce body weight in PCOS.Hyperinsulinemia and increased visceral adiposity result in the elevated production of androgen in ovaries.Reduction in body mass index of anovulatory obese women decreases testosterone concentration and INS resistance and reinstates ovulation.Mentha spicata also alleviates menstrual pain and hirsutism [14,15].
In this study, in silico virtual screening of phytocompounds from M. spicata is docked against CYP21A2 protein (progesterone complex), to analyze their binding affinities for potential drug candidates in reducing androgen levels in PCOS women.

Protein retrieval and preparation
The target structure should be determined experimentally by either nuclear magnetic resonance or X-ray crystallography, which can be downloaded from the protein data bank [PDB] database (https:// www.rcsb.org/structure/4y8w).The three-dimensional (3D) X-ray crystallographic structure of CYP21A2-progesterone complex (PDB ID-4Y8W) solved at 2.64 Å resolution was retrieved from RCSB PDB.The proteins underwent preparation through the following procedural steps.(a) Load the protein molecule downloaded in 3D simple document format (SDF) from PDB database, (b) hetero atoms, metal ions, water molecules, and cofactors are removed, (c) bound ligands removed, (d) B and C chains of the CYP21A2 protein are removed, and (e) save the novel protein file in PDB format.The protein was subjected to Ramachandran plot And Hydropathy plot analysis by employing the PDBsum (https://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/ pdbsum/GetPage.pl)and ExPASy respectively (https://web.expasy.org/protscale/).

Ligand selection and preparation
A total of 25 phytocompounds listed in Table 1 are isolated from M. spicata plant that has been reported to have anti-androgenic properties.These phytocompounds are obtained from Indian Medicinal Plants, Phytochemistry, and Therapeutic (IMPPAT) database (https://cb.imsc.res.in/imppat/formulations/Mentha%20spicata) and were selected for virtual screening and molecular docking.The 3D structures of these phytocompounds were obtained from the PubChem database in SDF.

Protein-ligand docking
Molecular docking protocol was accomplished through a flexible docking protocol.PyRx-Virtual Screening Tool is utilized for docking procedure with the following steps: (a) Load the protein molecule which is devoid of other ligands and hetero atom which was achieved through Biovia discovery studio, (b) convert the protein file from PDB to PDBQT format, (c) upload the 25 phytochemicals in the control panel of PyRx software, which were downloaded from the PubChem database, (d) minimize energy of ligands and convert all the ligands to AutoDock PDBQT format, (e) grid box is generated, ligands of interest are docked in this grid.Hence, it is important to maximize the grid box to cover most of the protein surface, and (f) docking results are obtained in excel sheet format consisting of values of binding affinities and root mean square deviation.

Screening of phytocompounds
Swiss absorption, distribution, metabolism, excretion, and toxicity (ADME) software of Swiss Institute of Bioinformatics was accessed on a web server that displays the submission page of Swiss ADME.This web tool was used to evaluate ADME behaviors of the phytoconstituents from M. spicata.The list of canonical Simplified Molecular Input Line Entry System (SMILES) of phytocompounds is made and the results are presented for every molecule in tables and Excel spreadsheets.An important segment of drug development is drug-likeness analysis which is used to identify the biological properties of drug candidates.

Protein preparation and analysis
The 3D structure of target protein (4Y8W) was retrieved from the PDB database.Later, by utilizing the software tool Biovia Discovery Studio, protein is subjected structural analysis and trimming protein with only one amino acid chain devoid of ligands and heteroatoms (Fig. 1).
The Ramachandran plot of 4Y8W depicted that most of the residues are congregated in favored regions with few irregularities for all the drug targets.The protein CYP21A2 (4Y8W) structures were solved by X-ray crystallography to resolution at 2.0Å and the protein had a normal G-factor score of 0.6 (Fig. 2).
The hydropathy plot indicates hydrophobic and hydrophilic tendencies of an amino acid sequence of A-chain of CYP21A2 protein (Fig. 3).

Selected phytocompounds of spearmint
Table 1 presents the name of 25 phytocompounds of M. spicata, which are identified using the IMPPAT database and their PubChem ID along with their canonical SMILES obtained from PubChem database.

Molecular Docking
The molecular docking of 25 phytocompounds Table 1 of M. spicata was docked against CYP21A2 protein (A chain of protein) also known as progesterone complex (PDB ID-4Y8W) using PyRx-virtual screening tool.After analyzing the active phytocompounds by binding free energy score and molecular interaction profile, out of 25 phytocompounds, only 5 (Bicyclogermacrene, Cubebol, (-)-beta-Bourbonene, alpha-Bourbonene, and Spathulenol) displayed the best binding affinity (ranging from -8.1 to -8.5 kcal/mol) and molecular interactions (Figs.[4][5][6][7][8].The specific target site for the receptor was set using the grid box with dimensions (61.5935 × 51.8689 × 64.7989) Å.The phytocompounds that have high binding affinity to target protein are called hit compounds due to their high-affinity scores.Thus, this filtered list of 5 phytocompounds was designated as druggable and was subsequently used for further studies (Table 2).Table 3 presents the physicochemical properties of phytocompounds bicyclogermacrene, cubebol, (-)-beta-bourbonene, alpha-bourbonene, and spathulenol, such as their molecular weight, number of atoms, fraction CSP3, number of rotatable bonds, molar refractivity, and

DISCUSSION
PCOS is associated with obesity, hyperglycemia, and INS resistance which correlate with elevated oxidative stress that comprises hyperandrogenemia environment in the ovary.In one study conducted in Turkey, men reported that consuming herbal tea of M. spicata or M. piperita caused diminished libido [15].This may be due to antiandrogenic properties of spearmint and peppermint.Studies show that spearmint decreases the oxidative stress and reduces cholesterol in type 2 diabetes.Leaf extract of spearmint contains phenolic compounds that remarkably enhance the antioxidant defence system and reduce levels of glucose and cholesterol in diabetic male rat [14].
PCOS is an oligogenic disorder, and it seems that the genes thymocyte selection-associated HMG BOX 33, DENN domain containing 1A, AMH, LHCGR, thyroid adenoma-associated gene, AMH receptor type 2, and INSR are important genes in the susceptibility of PCOS [16,17].The occurrence of modification in the genes homeobox 11 (gene codes for DNA-binding nuclear transcription factor) and homeobox A10 (protein coding gene) in women with PCOS affects the endometrial reception leading to infertility with implantation failure.Few studies also shown that polymorphism in FSHR gene is significantly associated with PCOS [16,18].
Many studies have validated that hyperandrogenism is one of the most distinguishable clinical features reported in patients with PCOS associated with heterozygous phenotypes with diverse genetic variants.These situations, involving deficiency of enzymes in the steroidogenesis pathway, are considered a prediction for PCOS [19].A subgroup of CYP genes encodes for enzymes involved in the steroidogenesis biosynthesis pathway.Biosynthesis of steroid hormones, inclusive of glucocorticoids, mineralocorticoids, androgens, progestins, and estrogen, are regulated by the enzymes, i.e., steroidogenic enzymes that comprise steroid hydroxysteroid dehydrogenases and certain CYPs enzymes [5,6,11,16].
In ovary steroidogenesis starts with alteration of cholesterol progresses sequentially to progestin, androgen, and estrogen, all of which are essential for subsequent synthesizing steroid hormones.These hormones are ultimately transported into blood circulation, where they Majority of research is focused on CYP11A1, CYP17A1, and CYP19A1 genes.The family of enzymes consisting of P450 enzymes is involved in the synthesis of cholesterol, steroids, lipids, and metabolism of drugs.CYP proteins are located in the network of endoplasmic reticulum, and these proteins catalyze the last step of steroid biosynthesis.Mutation in this gene may increase or decrease aromatase activity.In one study of letrozole, the drug was found to be an aromatase inhibitor, and it was approved for patients with hormone-responsive breast cancer.However, it has been studied for the induction of ovulation in women suffering from PCOS.The efficacy of drugs letrozole and anastrozole was studied for induction of ovulation, and there is no statistically significant difference in pregnancy rates.Mutation associated with CYP17A1 gene results in adrenal hyperplasia 17 alpha-hydroxylase deficiency also pseudohermaphroditism [6,11].Gene CYP11A1 codes for members of the CYPs family of enzymes.This protein is located in the inner membrane of mitochondria and it catalyzes the first step, conversion of cholesterol to pregnenolone in steroid hormone synthesis.The Tenascin X protein, retinitis pigmentosa, complement factor 4 (C4), and CYP21A2 genes are adjacent structurally, forming a genetic module of the RCCX complex.Mutation in CYP21A2 gene consequently results in 21-OH deficiency and also results in variable copy count of C4 gene, and the C4 gene encodes similar protein involved in classic complement activation [11,16].
The study of heritable changes in gene expression which is not encoded in the nucleotide sequence of DNA is known as epigenetics.Subjection to certain chemicals in environment (e.g., dietary substances, endocrine disruptors, and heavy metals) also behavioral exposures during early growth (e.g., child abuse) can lead to epigenetic modification.From many studies of cancers, there is conclusive evidence suggesting the link between human diseases and epigenetic dysregulation [20].Data Adiponectin is a protein hormone produced by adipocytes, and it has been reported that women with PCOS have decreased adiponectin.Insufficient levels of adiponectin are associated with INS resistance in murine models of obesity.One study concludes that increase in adiponectin is linked to weight loss rather than changes in macronutrient balance.Different macronutrient ratios through diet do not significantly alter adiponectin levels; instead hyperandrogenism is the main cause of adipose tissue dysfunction [21].Hence, it becomes an important factor to maintain androgen levels in the body of women with PCOS to regulate healthy hormonal balance in the body.Most of the symptoms of PCOS such as hirsutism, INS resistance, and irregular periods can be managed by lifestyle changes such as exercise and better healthier choices of plant-based food but it is not sufficient to battle the root cause of PCOS which remains unclear.PCOS women are more prone to mental health disorders such as high rates of depression and anxiety.Manifestation of physical and mental health disturbances reduces the quality of life [22].

Malakhed
It is important to understand and learn about antiandrogens to fight against hyperandrogenism in PCOS conditions.Many of the drugs are administered for this purpose that includes antidiabetic drugs to reduce INS resistance (e.g., metformin, sulfonylurea, biguanide, and clomiphene), aromatase inhibitors (e.g., letrozole), antiandrogens (e.g., spironolactone, flutamide, and finasteride), and oral contraceptives to restore ovulation in women who do not wish to become pregnant [1].These pharmaceutical drugs also come with side effects (bloating, liver inflammation, hypoglycemia, and allergic skin reactions) associated with them so now the focus has been shifting to herbal remedies such as herbal teas, essential oils, and polyherbal capsules.
Plant-derived antiandrogens are bioactive phenolic compounds that act as antagonist to androgens.Licorice (Glycyrrhiza glabra) is known to drop testosterone levels to normal which affects free testosterone in the body.Licorice root also known as sweet root has compound which is 50 times sweeter than sugar, it also contains phytoestrogens and other compounds that are thought to have endocrine effects.Licorice root also contains glycyrrhetinic acid and glycyrrhizin is known to have slight anti-androgen effect.Green tea (Camellia sinensis) comprises epigallocatechin which limits the conversion of 5-alpha reductase to testosterone to dihydrotestosterone. White peony (Paeonia lactiflora) has compound paeoniflorin which inhibits synthesis of testosterone and promoting the aromatase enzyme activity in the conversion of testosterone to estrogen [23].Other plants that are studied for their anti-androgenic effect are Reishi, Black cohosh, Chaste tree, and Saw palmetto.
Spearmint (M.spicata) has anti-androgenic properties which accounts for the reduction in free testosterone in blood.It is mainly used as an herbal remedy for women suffering from hirsutism.Spearmint does not affect the total dehydroepiandrosterone sulfate and total testosterone levels.In one clinical study, consumption of spearmint tea twice a day for a month resulted in decreased levels of androgens and gonadotropins in plasma [15].Bioactive compounds are also present in the food one consumes; these compounds are biological activities.
Focusing on innovative and cost-effective extraction procedures of bioactive compounds from food waste by incorporating microbiome

CONCLUSION
Overall, the favorable results from binding affinities and absorption, distribution, metabolism, excretion, and toxicity profile of 5 phytocompounds (bicyclogermacrene, cubebol, (-)-beta-bourbonene, alpha-bourbonene, and spathulenol) isolated from M. spicata plants could be explored as potential anti-androgenic agents to reduce symptoms of PCOS.However, in vitro and in vivo studies have to be performed to confirm findings of this study.Apart from spearmint, many other plants (such as licorice, chaste tree, and white peonies) are also known to have anti-androgenic effects.These plants and their extract have been studied and explored in detail to formulate polyherbal preparation as potential drugs for the treatment of PCOS.
There is a saying "you are what you eat" first quoted in 1826 by French author Jean Anthelme Brillant-Savarin, and certain nutrients affect health differently also gut microbiome varies depending on what one eats.It is important to make a lifestyle change along with these herbal remedies as a speedier elimination of PCOS.
Innovare Journal of Medical Science, Vol 12, Issue 1, 2024, 1-8 mining analyses suggest that abnormal DNA methylation patterns are connected with various disorders, including obesity, anemia, type 2 diabetes, cardiovascular disease, and numerous neurodevelopmental disorders indicating the importance of epigenetic regulation in the development of human diseases.Obesity and type 2 diabetes INS resistance is one of the main reasons for the development of PCOS.

Table 1 : Phytocompounds selected for docking studies S. No. Plant part Phytochemical name PubChem ID Canonical SMILES
SMILES: Simplified Molecular Input Line Entry SystemMalakhed

Innovare Journal of Medical Science, Vol 12, Issue 1, 2024, 1-8 topological
polar surface area.The molecular weight and number of atoms were less than 500 and 20, but molar refractivity and polar surface area were more than 50 and 20 o A 2 representing poor oral bioavailability 5 hit compounds.

Table 6
demonstrates pharmacokinetic values.According to results, cubebol and spathulenol showed high gastrointestinal absorption compared to other molecules.Except for bicyclogermacrene, all other compounds, cubebol, (-)-beta-bourbonene, alphabourbonene, and spathulenol, could cross the blood-brain barrier.All the 5 phytocompounds affected the liver CYPs enzymes such as CYP2C19, CYP2C9 (except for spathulenol), and CYP1A2 (except for bicyclogermacrene and cubebol).None of the phytocompounds affected CYP2D6 and CYP3A4 CYPs enzymes.However, skin penetration was better for all phytocompounds.Based on Table7, all 5 hit compounds obeyed the druglikeness filters, Ghose, Veber, and Egan.Cubebol and spathulenol obeyed Lipinski's rule of 5 and with one violation of Lipinski's filter of bicyclogermacrene, (-)-beta-bourbonene, and alpha-bourbonene.All 5 phytocompounds have one violation of Muegge filter.All compounds have a good bioavailability score of 0.55 which is more than 0.10 that is required for compound to be considered a potential drug candidate.Table8demonstrates that the filters for leadlikeness such as Pains filter and Brenk filter were obeyed by all 5 hit compounds bicyclogermacrene, cubebol, (-)-beta-bourbonene, alpha-bourbonene, and spathulenol (except for, cubebol violated Brenk filter).Synthetic accessibility values are moderate and can be considered for Investigational New Drug.