INTRODUCTION:

Infertility Queen or Polycystic ovary syndrome (PCOS) is a common and multifunctional disease associated with both endocrine and metabolic disorder. It affects approximately 4 – 18% of all reproductive- aged women in the world. PCOS is characterised by hyperandrogenism and ovarian abnormalities, resulting from a disruption in the hypothalamic-pituitary-ovarian axis [1]. Clinical manifestations includes oligoovulation, biochemical or clinical hyperandrogenism, hirsutism, male pattern baldness, acne, acanthonsis nigracans and polycystic ovaries. But PCOS has a long prodrome with detectable abnormalities that present as the metabolic syndrome like hyperinsulinemia, obesity, dyslipidemia related to decrease high density lipoprotein cholesterol and hypertriglyceridemia, hypertension, atherosclerosis, increased risk of development of type II diabetes and cardiovascular disease throughout the life of affected women.

PCOS has a heterogeneous etiology involving a variety of combination of reproductive, metabolic and genetic determinants. No single etiology has so far been found to have particular predictive power in explaining the occurrence of PCOS. PCOS is imbalance of female sex hormones. The ovaries are the part of female reproductive system along with uterus, fallopian tubes and vagina. The ovaries contain the lifetime supply of eggs. These eggs are immature and stored in tiny fluid filled follicles. Pituitary gland located at the base of brain produces hormones which direct the function of ovaries. Each month the pituitary gland secretes Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) in blood stream. After these hormones reach the ovaries several hundred immature eggs start maturing expanding the size of follicles which simultaneously secretes estrogen, the main female sex hormone. Once the level of estrogen reaches to certain level, the pituitary gland senses the surge of luteinizing hormone to the ovaries causing the most mature follicle to release the egg called ovulation. The free egg travels the fallopian tube where it awaits fertilization, eventually the remaining follicles and eggs dissolves. If the egg is not fertilized the lining of uterus sheds during the menstruation.

In PCOS the pituitary gland releases abnormally high amount of Luteinizing hormone in blood stream disrupting the normal menstruation cycle. As a result the follicle does not mature and ovulation does not occur which can lead to an ovulation. The immature follicle which does not dissolve remains as fluid filled sacs or cysts. These cysts lead to a hormonal imbalance because of an increased amount of testosterone. This can result in acne, an increase in facial and body hair and irregular periods. In addition there is high level of insulin hormone produced by pancreas. Insulin combines with luteinizing hormone and lead to excess production of male hormone testosterone in ovaries. Abnormally high amount of testosterone in the ovaries prevent ovulation which can lead to infertility. Along with it, PCOS raises the risk of Type II diabetes because of excess of insulin or insulin resistance, heart disease, high blood pressure, cholesterol abnormality and endometrial cancer [2].

Anethole is a type of aromatic compound that occurs widely in nature in essential oils. It contributes to the distinctive flavors of anise and fenne [3]. It is found to possess diuretic, analgesic, antipyretic and antioxidant activities. Essential oils are mainly concentrated in the mericarps and provide the unique aroma and taste [4].

The structure of anethole was drawn using the ACD Chem Sketch software. The 2D as well as the 3D structure of anethole can be obtained from PubChem.

Figure 1. 2D structure of anethole

MATERIALS AND METHODS:

1. Protein preparation:

The three dimensional crystal structure of insulin receptor substrate 1(IRS 1), follicle stimulating hormone (FSH) receptor and androgen receptor (PDB ID 1IRS, 1XUN & 1E3G) was downloaded from the RCSB Protein Data Bank.

2. Ligand preparation:

The chemical structure of the ligands was obtained from PubChem compound database. It was prepared by Chem Bio Draw and MOL SDF format of this ligand was converted to PDBQT file using PyRx tool to generate atomic coordinates.

3. Docking of anethole with different targets:

Molecular docking of anethole with insulin receptor substrate 1, follicle stimulating hormone receptor and androgen receptor were performed using Autodock4. The 3D structure of androgen receptor, insulin receptor substrate 1(IRS 1) and follicle stimulating hormone (FSH) receptor (PDB ID 1E3G, 1IRS & 1XUN) were acquired from RCSB (Research Collaboratory for Structural Bioinformatics).

ADMET, molecular and bioavailability scores:

The ADME (absorption, distribution, metabolism and excretion) properties were calculated by using smile notation in Swiss ADME web based tool [5]. The toxicity was calculated by using PROTOX web based tool [6]. The molecular properties and bioavailability scores were calculated by using Molinspiration web based tool.

Molecular docking:

Molecular docking is used to recognize and optimize drug candidates by examining and modelling molecular interactions between ligand and target macromolecules. Molecular docking are used to generate multiple ligand conformations and orientations and the most appropriate ones are selected [7].

Table 1: physicochemical properties of anethole

Formula	C₁₀H₁₂O
Molecular weight	148.20 g/mol
Num. heavy atoms	11
Num. arom. heavy atoms	6
Fraction Csp3	0.20
Num. rotatable bonds	2
Num. H-bond acceptors	1
Molar Refractivity	7.83
TPSA	9.23 Å²

Table 2 : Lipophilicity of anethole

Log P_o/w (iLOGP)	2.55
Log P_o/w (XLOGP3)	3.30
Log P_o/w (MLOGP)	2.67
Log P_o/w (SILICOS-IT)	2.79
Consensus Log P_o/w	2.79
Log P_o/w (WLOGP)	2.62

Table 3 : Water Solubility of anethole

Log S (ESOL)	-3.11
Solubility	1.15e-01 mg/ml ; 7.77e-04 mol/l
Class	Soluble
Log S (Ali)	-3.17
Solubility	1.00e-01 mg/ml ; 6.77e-04 mol/l
Class	Soluble
Log S (SILICOS-IT)	-2.98
Solubility	1.55e-01 mg/ml ; 1.04e-03 mol/l
Class	Soluble

RESULTS AND DISCUSSION:

Pharmaco Kinetics Properties:

There are many compounds with poor bioavailability shows less effective against disease. To solve this problem, predicting bioavailability properties will be great advantage for drug development. Hence using computer based methods like ADMET tools the molecular descriptors and drug likeliness properties was studied. Table 1- 6 gives the pharmacokinetic properties of the drug anethole.

Table 4: Pharmacokinetics of anethole

GI absorption	High
BBB permeant	Yes
P-gp substrate	No
CYP1A2 inhibitor	Yes
CYP2C19 inhibitor	No
CYP2C9 inhibitor	No
CYP2D6 inhibitor	No
CYP3A4 inhibitor	No
Log K_p (skin permeation)	-4.86 cm/s

Table 5: Druglikeness properties of anethole

Lipinski	Yes; 0 violation
Ghose	No; 1 violation: MW<160
Veber	Yes
Egan	Yes
Muegge	No; 2 violations: MW<200, Heteroatoms<2
Bioavailability Score	0.55

Table 6 : Bioavailability Scores of anethole

Molinspiration bioactivity score	v2018.03
GPCR ligand	-1.23
Ion channel modulator	-0.69
Kinase inhibitor	-1.31
Nuclear receptor ligand	-0.94
Protease inhibitor	-1.46
Enzyme inhibitor	-0.73

The various bioavailability scores are obtained from the SWISSADME web base tool

With the advent of genomics, proteomics, bioinformatics and technologies like crystallography, NMR, the structures of more and more protein targets are becoming available. So there is a need for computational tools that can identify and analyse active sites and suggest potential drug molecule that can bind to these sites. Docking is the computational determination of binding affinity between molecules (protein structure and ligand). Given a protein and a ligand we could find out the binding free energy of the complex formed by docking them.

Simple molecular and physicochemical descriptors like molecular weight (MW), molecular refractivity (MR), count of specific atom types and polar surface area (PSA) are described here. These properties are obtained from the SwissADME web based tool. From the table 1 it is clear that anethole is having 1 H-bond acceptors and there are 2 rotatable bonds.

The partition coefficient between n-octanol and water (log Po/w) is the classical descriptor for lipophilicity. SwissADME was used to predict the lipophilic character of the drug. SwissADME gives access to five freely available predictive models; i.e. XLOGP3, an atomistic method including corrective factors and knowledge-based library; WLOGP, their own implementation of a purely atomistic method based on the fragmental system of Wildman and Crippen; MLOGP, an archetype of topological method relying on a linear relationship with 13 molecular descriptors; SILICOS-IT, an hybrid method relying on 27 fragments and 7 topological descriptors; and finally iLOGP, our in-house physics-based method relying on free energies of solvation in n-octanol and water calculated by the Generalized-Born and solvent accessible surface area (GB/SA) model. iLOGP was benchmarked on two drug or drug-like external sets. The different lipophilicity of anethole is shown in the Table.2.

Having a soluble molecule greatly facilitates many drug development activities, primarily the ease of handling and formulation. Moreover, for discovery projects targeting oral administration, solubility is one major property influencing absorption. As well, a drug meant for parenteral usage has to be highly soluble in water to deliver a sufficient quantity of active ingredient in the small volume of such pharmaceutical dosage. Two topological methods to predict Water Solubility are included in SwissADME. From table.3 it is clear that anethole is soluble in all aspects.

The knowledge about compounds being substrate or non-substrate of the permeability glycoprotein (P-gp, suggested the most important member among ATP-binding cassette transporters or ABC-transporters) is key to appraise active efflux through biological membranes, for instance from the gastrointestinal wall to the lumen or from the brain. SwissADME enables the estimation for a chemical to be substrate of P-gp or inhibitor of the most important CYP isoenzymes. The pharmacokinetic properties of anethole was shown in the table.4. So from that it is clear that anethole is not a P-gp substrate.

Swiss ADME section gives access to five different rule-based filters, with diverse ranges of properties inside of which the molecule is defined as drug-like. The Lipinski (Pfizer) filter is the pioneer rule-of-five, Ghose (Amgen), Veber (GSK), Egan (Pharmacia) and Muegge (Bayer) methods are the five different rules. Any violation of any rule described here appears explicitly in the output panel. Anethole is having bioavailability score of 0.55 and it shows violations in certain aspects such as in Ghose and Muggee methods. It doesn’t shows any violations in the Lipinski rule of five, Veber and Egan methods (table.5).

Molecular docking:

Docking of small molecule compounds into the binding site of a receptor and estimating the binding affinity of the complex is an important part of the structure based drug design process. AutoDock Vina is a open-source program for drug discovery, molecular docking and virtual screening, offering multicore capability, high performance and enhanced accuracy and ease tof use. The parameters chosen for the docking can be judged by the docking tool’s ability to reproduce the binding mode of a ligand to protein, when the structure of the ligand–protein complex is known. Fig.2 shows the docked image of anethole with Insulin receptor substrate 1 receptor. Fig. 3 is the transparency inage of the docked molecule.

Figure 2 Docked image of anethole with IRS 1 receptor

Figure 3 Docked transparency image

Figure 4 Docked image of anethole with FSH receptor

Figure 5 Docked transparency image

The binding of anethole with FSH receptor was shown below. Anethole has better binding with androgen receptor compared to IRS1 receptor and FSH receptor.

Figure 6 Docking of anethole with androgen receptor

Figure 7 Docked transparency image

Fig. 6 shows the docked image of anethole with androgen receptor and fig. 7 shows the docked transparency image. Even though the anethole is seen outside the androgen receptor, the docking score of the compound shows better binding affinity with the androgen receptor. The docking score of anethole with the three receptors are shown in the table.7.

Table.7 Docking of anethole

Anethole docked with	Binding affinity(kcal/mol)
Androgen receptor	-6.0
Follicle Stimulating Hormone receptor	-5.6
Insulin receptor substrate 1 receptor	-4.2

From the above given table it is clear that anethole is having best docking capability with the androgen receptor with the FSH receptor and IRS1 receptor. The binding affinity for anethole with androgen receptor is shown to be -6.0 which is higher than -5.6 & -4.2 for FSH and IRS 1 receptors respectively.

CONCLUSION:

Computational tools may be helpful in finding the cause of this syndrome. PCOS is becoming more widespread in today’s scenario and their diversity is increasing at high pace thus an effective and efficient treatment is an urgent need of present times. The study shows that anethole is having best binding capacity with the androgen receptor. Because the binding affinity of anethole is greater for androgen receptor than that of FSH receptor and IRS 1 receptors. The binding affinity for anethole with androgen receptor is also greater. Thus we can conclude that anethole can be used for the treatment of PCOS.

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Received on 04.05.2019 Modified on 10.06.2019

Research J. Pharm. and Tech. 2019; 12(10):4958-4962.

DOI: 10.5958/0974-360X.2019.00860.6