In silico analysis of Nigella sativa bioactive compounds as fertility potential in folliculogenesis disorders

Context : The capacity to get pregnant is known as fertility. Every woman has reproductive rights to be fertile. In reality, there are still many cases of infertility. Infertility is due to impaired folliculogenesis. Low GDF-9 and estrogen receptors can be used as biomarkers in folliculogenesis disorders. Nigella sativa is a plant with medicinal properties as an antidote and protects against toxicity in the lungs, heart, liver, digestive tract, and reproductive system. Aims : To identify the potential of the bioactive compounds N. sativa as activators of GDF-9 and estrogen receptor proteins on fertility in an in silico study approach. Methods : All ligands from N. sativa extract were from previous studies, and protein preparations were taken from PubChem database. The conversion of the sdf file to pdb on the ligand was carried out using the OpenBabel v2.3.1 software. The drug-like molecule properties of chemical compounds from N. sativa were identified through a drug-likeness test on the SwissADME server and using PyRx v9.9.0 software, LigPlot+ v.2.2 software, and PyMol v2.5 software to analyze and visualize the potential of N. sativa compounds against GDF-9 and estrogen receptor. Results : Compounds cis-13,16-docasadienoic acid and cholestan-3-ol, 2-methylene-, (3β ,5 α) - have the lowest bond affinity, hydrogen, and hydrophobic interactions, as well as the same position of amino acid interactions to control. Conclusions : Cis-13,16-docasadienoic acid and cholestan-3-ol, 2-methylene-, (3 β ,5 α) - can act as candidates for activating GDF-9 and estrogen receptors for fertility.


INTRODUCTION
Fertility is the ability to get pregnant (Zegers-Hochschild et al., 2017). Every couple must have the desire to get pregnant and have children. However, many couples still cannot get pregnant after marriage. Failure to achieve pregnancy after 12 months or more of regular unprotected sexual intercourse is called infertility (Datta et al., 2016). Infertile couples of childbearing age worldwide are estimated at around 50-80 million. Large-scale studies have shown that about half of all cases of infertility occur due to female factors, 20 to 30% male factors, and 20 to 30% due to common causes of both genders (Babakhanzadeh et al., 2020). Infertility is a reproductive health problem that affects human rights. Infertility causes deep human suffering, especially on the part of women. For women, the effects of infertility can destroy a marriage. Therefore, reproductive rights include the right to overcome infertility problems (Inhorn, 2009). One of the causes of infertility is folliculogenesis disorders (Patel et al., 2015).
The ovaries contain numerous primordial follicles. Some of these follicles enter the growth phase and progress through the primary and secondary follicle stages. Follicular granulosa cells proliferate to develop a multi-layered structure during follicular development. Then, a fluid-filled cavity forms within the follicle during the antral follicle stage. This fluid-filled cavity is called the antrum. In the antral follicle, the granulosa cells separate to form cumulus cells, which envelop the oocyte (Wigglesworth et al., 2015). Twoway communication between the oocyte and granulosa cells is necessary for ovarian function and fertility (Alam et al., 2018). Granulosa cells supply nutrients, metabolites, and molecular signals to oocytes, while oocytes promote granulosa cell proliferation, differentiation, and function. Oocytes regulate follicular development (Matzuk et al., 2002). Oocytes are required for early primordial follicle formation and regulate the specific characteristics and functions of cumulus and mural granulosa cells. During maturation, the oocyte secretes a factor that induces cumulus expansion, namely GDF-9 (Soyal et al., 2000).
GDF-9 is an important oocyte-derived factor that regulates ovarian function (Emori and Sugiura, 2014). GDF-9 stimulates granulosa cell differentiation, including inducing LH receptors and steroidogenesis (Durán-Pastén and Fiordelisio, 2013). According to Ito's research, mice with GDF-9 deficiency were found to be infertile because follicular development could not proceed beyond the primordial follicle stage (Ito et al., 2022). Besides GDF-9, there is a pro-tein essential in the process of folliculogenesis, namely Estrogen Receptor (ESR 1). It plays a crucial role in reproduction by functioning as a mediator of the hormone estrogen. Previous research findings suggested that abnormalities in ESR 1 could lead to infertility (Arnal et al., 2017;Rumi et al., 2017).
Nigella sativa L. (family Ranunculaceae) is an annual herb with many pharmacological properties (Tavakkoli et al., 2017). Muslims call this plant Habbatus Sauda, Alhabahat lsawda and Alkamoun Alaswad (Sahak et al., 2016). It also has medicinal properties as an antidote, protecting against toxicity in several organs, including the brain, kidneys, lungs, liver, heart, digestive tract, and reproductive system (Hannan et al., 2021). Based on previous research, administration of N. sativa extract can increase reproductive potential in male rats by increasing testosterone and LH levels in male rats (Sayed, 2019).
So far, research studies on the potential of N. sativa extract as a fertility drug in women, especially increasing estrogen receptors and GDF-9 have not been found in the literature review. This research will predict the potential of N. sativa extract as a fertility drug by increasing two fertility biomarkers, namely GDF-9 and ESR 1. The binding of certain compounds in N. sativa extract is expected to increase the activation of these two target proteins.

Protein preparation
GDF-9 and ESR 1 act as targets for N. sativa compounds. 3D structures with pdb files for both targets were obtained from Uniprot database (https://www.uniprot.org/). Water molecules and contaminant ligands on the target were removed via PyMol v2.5 software to optimize and prepare molecular docking (Rigsby and Parker, 2016).

Druglikeness identification
The drug-like molecule in chemical compounds from N. sativa was identified through a drug-likeness test on the SwissADME server (http://www.swissadme.ch/). Parameters such as Lipinski, Ghose, Veber, Eggan, and Bioavailability scores are used to determine drug-like molecules in candidate compounds. The drug-likeness test aims to identify the similarity of the properties of the query compound with the drug molecule (Daina et al., 2017).

Molecular docking
In this study, the ligand interaction from N. sativa with GDF-9 and ESR 1 was simulated through molecular docking simulations on PyRx v9.9.0 software. Docking simulation aims to identify the ability of ligand binding activity and interaction patterns on the target. Binding affinity is the negative energy that is formed when the ligand and target interact to form a stable molecular complex (Tripathi et al., 2021;Wijaya et al., 2021).

Chemical interaction
The LigPlot+ v.2.2 program was used to identify the chemical interactions of the ligand-protein complex in this investigation. Hydrogen and hydrophobic chemical bond interactions are shown in the software. This type of interaction in the ligand-protein complex is facilitated by weak binding to trigger specific biological responses, such as the level of activation and the stability of the molecular complex (Dibha et al., 2022).

Structural visualization
Ligand-target complexes are displayed in 3D structures using PyMol v2.5 software with color and structure selection methods. The colors shown are based on the ligand atomic composition and single protein structure. Structural types consisting of car-toons, sticks, and transparent surfaces with standard publications are used in this study (Rigsby and Parker, 2016).

Data analysis
The data analysis process was carried out in stages, including (1) drug-likeness, analysis of the similarity of all compounds using the Lipinski, Ghose, Veber, Eggan, Muegge methods, and the Bioavailability score was used to determine the drug in the candidate compound, (2) to determine the molecular docking of the compound with the lowest binding affinity, (3) after molecular docking, the compound with the lowest affinity was simulated to observe the molecular interactions between the compound and the two target proteins.

Drug-likeness
The identification of drug-likenes indicates that all compounds from N. sativa can be used for further analysis of their potential as fertility activators (Table  2).

Molecular docking
Molecular docking in this study aims to identify patterns of molecular interactions and assess the level of binding activity between compounds of N. sativa on target proteins, namely GDF-9 and ESR 1. The docking results show that the compounds from N. sativa consist of cis-13,16-docasadienoic acid and cholestan-3-ol, 2-methylene-, (3β,5α)-showed a more negative binding affinity than the other compounds, and the control (clomifene) ( Table 3). The ligandprotein complexes from the docking simulation results with the most negative binding affinity are displayed with stick structures, transparent surfaces, and cartoons with single staining (Fig. 3).

DISCUSSION
GDF-9 and ESR 1 are important proteins in the process of forming follicles in the ovaries. GDF-9 stimulates granulosa cell differentiation, including inducing LH receptors and stereoidogenesis (Durán-Pastén and Fiordelisio, 2013). While ESR 1 functions as a mediator of the hormone estrogen, which plays an important role in growth hormone in the reproductive system, triggers for ovulation, and also functions to maintain oocyte development. The ovarian follicles' hormone estrogen is secreted by the granulosa cells (Hamilton et al., 2017;Rumi et al., 2017).       Clomifene (control) 2800 -6.8 -6.4 Table 3. The results of chemical interaction analysis.

Ligan-protein Chemical interaction
cis-13,16-Docasadienoic acid_ESR 1 Hydrophobic: Leu525, Thr347, Ala350, Leu384, Gly521, Leu354, Glu380, Leu379, Trp383, Ile358, Lys529, Asp351 Hydrogen: Val376 Cholestan-3-ol, 2-methylene-, (3β,5α)-_ESR 1 Hydrophobic: Leu354,Glu380,Ile358,Lys362,Leu372,Gln375,Val376,Leu379,Trp383 cis-13,16-Docasadienoic acid_GDF-9 Hydrophobic: Glu442, Pro63, Tyr279, Glu444, Tyr275, Leu62, Phe66 Hydrogen: Asn112, Ser271, Asp269, Ala272 Cholestan-3-ol, 2-methylene-, (3β,5α)-_GDF-9 Hydrophobic: Lys67,Thr148,Lys441,Val150,Ser271,Gly60,Trp278,Ala274,Tyr275,Leu62,Ala64 In this study, we carried out molecular docking between compounds (ligands) from N. sativa and target proteins, namely GDF-9 and ESR 1 proteins, to see the potential of these ligands to affect the target protein. Identification of drug-likeness is used to determine the similarity of the properties of the query compound to the drug molecule with reference to physicochemical properties through parameters such as Lipinski, Ghose, Veber, and Eggan in SwissADME, compounds must meet at least one rule to be called a drug-like molecule. The ability of a medicine to circulate in the body is measured by its drug bioavailability, with an ideal score of >0.25 (Martin, 2005;Daina et al., 2017). According to the results of drug similarity identification, all of the N. sativa compound's ligands met the requirements for drug similarity parameters by satisfying at least one rule with a bioavailability score of 0.55 (Table 2) Molecular docking can evaluate the degree of ligand binding activity on the target and reveal patterns of molecular interactions. The ability of ligand activity refers to the level of the binding energy score. Binding affinity is the negative energy created when the ligand attaches to the target (Aini et al., 2022). The activity level of the ligand is determined by the value of the binding affinity that is formed when it interacts with the target. Negative values of binding affinity determine bond strength, more negative values indicate the strongest ligand bonds (Wijaya et al., 2021). Candidate compounds with more negative binding affinity values and binding to functional domains can act as activators on the target protein. In this study, cis-13,16-docasadienoic acid and cholestan-3-ol, 2methylene-, (3β,5α)-showed more negative binding affinity than the other compounds and clomifene (control). These results indicate that cis-13,16docasadienoic acid and cholestan-3-ol, 2-methylene-, (3β,5α)-in N. sativa extract have the potential to affect the physiological properties of the target protein because they have a higher binding ability than clomi-phene. Both of these compounds are predicted to trigger the activation of GDF-9 and ESR 1.
Docking visualization shows how the docking results interact between the ligand and the target protein. The interaction between ligands and amino acid residues can be visualized using this method. The interactions can occur in the form of non-covalent bonds, such as electrostatics, Van der Waals interactions, hydrogen bonds, and hydrophobic bonds. Hydrogen bonds develop in molecules due to the attraction between hydrogen atoms and other atoms with high electronegativity. Hydrogen bonds are the strongest bonds among other bonds. Meanwhile, hydrophobic bonds have an important role in the stability of the ligand to the receptor. Hydrophobic bonds tend to avoid watery environments and cluster on the inside of protein structures To reduce interactions with water, which could harm the protein structure (Lins and Brasseur, 1995;Dibha et al., 2022). Based on the research results, the molecular interactions showed that cis-13,16-docasadienoic acid and cholestan-3-ol, 2-methylene-, (3β,5α)-could form weak bond interactions consisting of hydrogen and hydrophobic. Cis-13,16-docasadienoic acid and cholestan-3-ol, 2methylene-, (3β,5α)-showed the same interaction position through Leu354, Glu380, Leu379, Trp383, Ile358, Val376 in the ESR 1 and GDF-9 domains through Tyr275, Leu62, Ser271. The interaction position on the same target of the two query compounds is predicted as a practical side that triggers target activation.
The limitation of this study is based on prediction or virtual screening. For this reason, it is necessary to study both in vitro and in vivo to obtain drug molecule candidates from N. sativa.

CONCLUSION
Chemical compounds from Nigella sativa consisting of cis-13,16-docasadienoic acid and cholestan-3-ol, 2-methylene-, (3β,5α)-can act as candidates for GDF-9 and ESR 1 activators because these two compounds have the binding affinity value is more negative than the control (clomifene) and forms weak bond interactions such as hydrogen and hydrophobic. Weak bonds can trigger ligand activity to affect the activation of both targets and then trigger a response in increasing fertility.