Potency of Vigna angularis against ERα through in silico studies

Vigna angularis, from the legume family, contains phytoestrogens. Phytoestrogens can activate estrogen receptors and are safer than estrogen. The study aims to analyze the potential of Vigna angularis as an activator of estrogen receptor-α (ERα) through in silico studies. The analysis of molecular docking used SAR (Structure Activity Relationship). A previous study has shown that Vigna angularis contains isoflavones such as Genestein (0.5%), daidzein (14.9%), glycitein (25.8%), formononetin (13%), and biochanin A (45.5%). The results of molecular docking to ERα show that genestein has a free energy binding value of -9.3 and the same amino acid structure, with a control level over estrogen of 66%. In conclusion, in silico studies have shown that genestein from Vigna angularis is the main component that activates the ERα.


INTRODUCTION
The use of estrogen as a hormone replacement therapy (HRT) is known to cause many side effects, such as an increased risk of endometrial and ovarian cancer (Beral et al., 2005;Beral et al., 2007). This condition leads to an increase in the consumption of phytoestrogen supplements as HRT agents, as these are safer (Patisaul et al., 2010).
Vigna angularis, from the legume family, is known to contain isoflavones such as genestein (Lee et al., 2005). Genestein is the isoflavone with the highest bioactivity, because genestein has a structure that is similar to estrogen and has more hydroxyl groups than other types of isoflavones. Genestein has 2 hydroxyl groups at the 5 th and 7 th bond positions on the A ring and one hydroxyl group at the 4 th position of the B ring (Ruffer and Kulling, 2006).
Estrogen is a steroid compound that works in the development and function of female reproductive organs. Estrogen consists of three compounds: estradiol, estrone and estriol. Estradiol is produced by the ovaries from testosterone in women during menarche until the pre-menopause stage. Estrone and estriol are produced by the supra-renal network from androstenedione. The potential of estradiol is stronger than that of estrone and estriol. Estradiol levels will decrease at menopause, associated with a loss of ovarian follicles, while estrone levels become more elevated (Fang et al., 2001).
To cause biological effects, estrogen binds to its receptors. There are two kinds of estrogen receptor: ERα and ERβ. Both include an intracellular receptor type and are antagonistic to each other. The two receptors are distributed differently in women (Lee et al., 2012).
Based on the above facts, this study aims to measure the activation potential of the active ingredient on ERα receptors through an in silico study.

METHODS
The experiment was carried out by the laboratory to test the potency of physicochemical compounds from Vigna angularis as activators of ERα through an in silico study. This research was conducted in the Biochemistry Laboratory of the Faculty of Medicine, University of Islam Malang and Bioinformatika of Brawijaya University of Malang.

Procedure
The active compound of Vigna angularis was determined based on the results of previous studies. Vigna angularis is known to contain flavonoids, phenolic compounds, tannins and alkaloids. Analysis of ethanol extracts from Vigna angularis using LCMS found the following active compounds: genestein, daidzein, biochanin A, formononetin, and glicitein (Damayanti et al., 2017). (https://pubchem.ncbi.nlm.nih.gov) with a CID that has been previously recorded. Target proteins were screened using HITPICK. The ERα structure was obtained from (https://pubchem.ncbi.nlm.nih.gov) with the ID 1a52. The activator reference estrogen (CID 5757) was taken from https://pubchem.ncbi.nlm.nih.gov. Next, the docking process was assessed using the PyRx program (autodock vina) (Dallakyan and Olson, 2015). Docking was specifically achieved with the compound as a ligand and with estrogen as the activator (CID 5757). Selected energy bindings with the smallest or most negative results showed the best complex conformation.

Potential analysis of
Docking results were stored and visualized using Ligandscout and PyMol (Delano, 2000). The docking results were visualized interactively to obtain interaction data between the two molecules (ERα and activator refence) using LigPlot (Wallace et al., 1995). The LigPlot results show the hydrophobic bonds and hydrogen bonds that occur in the complex. The activation potential is seen from the active site of the ERα that is bound by the active compound.

Target analyses and molecular docking of phytoestrogen to ERα
The results of potential protein measurements from Vigna angularis were computed as follows: To determine whether the compound can act as an analogue compound of estrogen, it was necessary to compare interactions with those of estrogen binding to ERα. Based on potential analyses, only genestein has 100% accuracy when binding ERα. Glycitein, formononetin and daidzein have greater accuracy against ERβ. Based on the molecular docking, estrogen as a control has a free energy binding value to ERα of -10.6. Genestein has the lowest free energy binding value against ERα of -9.3 compared to other isoflavones such as glicitein -8.4, biochanin A -7.0, formononetin -7.0, and daidzein -9.2.
The results of molecular docking interactions of some phytoestrogen compounds contained in Vigna angularis are as follows:    Phytoestrogens have antioxidant, anti-proliferative, anti-cancer, and anti-cholesterol activities. The isoflavones genestein and daidzein are the most well-known types of isoflavones that have an effect on health. In addition, there are other isoflavone groups found in bean crops such as formononetin and biochanin A (Boueä, 2003).
Phytoestrogens (isoflavones) have the ability to activate estrogen receptors 100-500 times lower than estrogens (17-β-estradiol) (Kuiper et al., 1998). Differences in the effects of phytoestrogens in some organs, especially reproductive organs, are influenced by differences in the distribution of receptors and differences in the ability of each type of phytoestrogen (Kuiper et al., 1998).

Potential analysis and molecular docking of phytoestrogen to estrogen receptor α
Computational approaches began to be widely used for the discovery of active ingredients. This process will shorten the time and cost necessary to test active ingredients prior to release on the market in the form of a drug (Srinivasa Rao and Srinivas, 2011). The active compound is predicted to have the ability to bind to the target protein and interact spontaneously Based on molecular docking results it was found that genestein has the smallest free energy among the phytoestrogens, and has the ability to bind estrogen receptor α through hydrogen bonds on the same amino acid with a control level of 66% and hydrophobic bonds on the same amino acid with a control level of 62.5%. Although other phytoestrogens are able to bind to the same amino acids as controls, their affinity for ERα is very low and their free energy binding is higher than controls; therefore, it is assumed that it is not able to form a stable bond so does not activate ERα.
Phytoestrogens such as genestein and daidzein prefer to interact with ERβ than ERα (Pettersson and Gustafsson, 2001 Based on these facts it can be computationally predicted that genestein has the best ability to bind to estrogen receptor α compared to other isoflavones. This was characterized by a targeted work on estrogen receptor α with 100% accuracy and a free energy of -9.8. Also, it has the same amino acids bound by hydrogen bonds type of 66% compared to estrogen control.