INTRODUCTION:

Polycystic ovary syndrome (PCOS) is a multifaceted endocrine disorder among women in their reproductive period characterized by menstrual disorder, hyperandrogenism, hirsutism, and persistent absence of ovulation with multiple cysts in the ovary^1,2. It affects nearly 2.2-26% of women in their reproductive age in India³. The synergistic pathology of increased androgens and hyperinsulinemia in PCOS aggravates the risk for metabolic syndrome such as Impaired glucose tolerance (IGT), type 2 diabetes mellitus, obesity, dyslipidemia and nonalcoholic fatty liver disease which is mainly due to insulin resistance⁴.

It is well established that insulin manifests both hyperinsulinemia and ovarian hyperandrogenism leading to chronic anovulation⁵.

In excess insulin status, along with luteinizing hormone (LH) it has a stimulatory effect on steroidogenesis of granulosa cells by enhancing human chorionic gonadotropin (hCG)-leading to cyst formation in PCOS condition⁶. There are several studies which suggest that the insulin exerts its androgen-stimulating effect through the binding of the insulin receptors⁷. Insulin receptor substrate proteins (IRS-1 and IRS-2) act as receptors for the ligands such as insulin, other tyrosine kinase dependent growth factors and cytokines⁸. Recent studies indicate that polymorphisms in IRS receptors may increase the risk of type-2 diabetes mellitus in PCOS women since insulin mediated actions differ in insulin target tissues.

At present the management of PCOS includes lifestyle modifications in the form of dietary changes which includes reduced carbohydrate and fat intake, exercise for weight reduction and allopathic treatment consisting of anti-androgens, insulin sensitizing drugs and ovulation inducing drugs. But these treatments have side effects such as irregular menstruation, gastrointestinal disturbances, weight gain, and increased insulin resistance⁹. While developing alternative medication, plant-based drugs are used. Medicinal plants perform complex interventions through synergistic or antagonistic interactions with receptors¹⁰.

Asparagus racemosus (Liliaceae), commonly known as Shatavari, is present in India, Australia, Africa and Asia. It is consumed as a vegetable in many parts of the world. The shoots are edible and mostly used in soups, vegetable dishes and salads. In India, it has been used for the treatment of diarrhea, dysentery, nervous breakdown, rheumatism and microbial infection due to its various medicinal properties^11,12. Studies revealed that the phytochemical compound of A. racemosus constitutes oligosaccharides, flavonoids, steroidal saponins and sulphur – containing amino acids¹³. Studies have been reported for decreased intestinal propulsive movement in the methanolic extract, in castor oil induced diarrhea and fluid accumulation in rats. It has also been reported to decrease gastric emptying¹⁴. The meaning of Shatavari is “women who can have hundred husbands” implicating its potency to treat all women's health issues¹⁵. With this above scenario we made an attempt to study ameliorative activity of ethanolic root extract of A. racemosus, on PCOS associated hyperglycemia and hyper androgonemia through In vitro and in silico approach.

MATERIALS AND METHODS:

Preparation of crude extract:

The roots of the plant were shade dried, ground into coarse powder and stored in an airtight container. It was subjected to ethanol extraction in the ratio (1:3) by cold maceration method for three days. The extract was filtered, evaporated using a rotary evaporator and refrigerated.

In vitro antidiabetic activity:

The following antidiabetic activities were carried out. α-amylase enzyme inhibiting effect of the plant extract was studied by the method of Jayasri et al.¹⁶, inhibitory action of Glucose diffusion was calculated by the method of Gallagher et al.¹⁷, glucose uptake by yeast cells was performed by Cirillo¹⁸ method and measurement of glucose adsorption potential by plant extract was studied by the method of Ou et al.¹⁹.

In silico anti-hyperandrogenic activity:

It was carried out by molecular docking using patch dock analysis to find a fitting ligand of the plant compound as a competitive inhibitor for the receptor IRS 1 and IRS 2. The 3D structure of the receptors was taken from PDB (Protein data bank). The phytochemical compounds of the plant were known from the GC-MS studies of Hayes et al.,²⁰ Singh and Tiwari,²¹ Ahmad and Jain,²². Patch dock analysis was done for 14 compounds. The 2D structure of A. racemosus phytochemicals were retrieved from PubChem. Corina 3D converter was used to obtain its 3D structure. The score of ligand and receptor interaction was obtained from patch dock server. The best docking score was selected and inhibitory potential of the phytoconstituents of A. racemosus was analyzed by interactions with amino acid residues at the active site. It was visualized and confirmed by LIGPLOT.

Statistical analysis:

The values are reported as Mean ± SD (n = 3). the statistical analysis was done using ANOVA followed by Dunnet’s “t” test. Values of p<0.05 were considered.

RESULTS:

α Amylase inhibitory activity:

Figure 1 shows α amylase enzyme inhibitory activity of A. racemosus at different concentrations. From the present study, it was very prominent that the ethanolic extract of A. racemosus inhibited α amylase enzyme at a low concentration compared to the control Acarbose. The IC 50 value for the study is 367μg/ml.

Inhibitory activity of Glucose diffusion:

The inhibitory effect of glucose diffusion by the plant extract at different concentrations is shown in Figure 2. The ethanolic extract of A. racemosus inhibits the glucose diffusion at all given concentrations and an enhanced activity is observed at higher concentration of 1000 µg/ml. At this concentration the plant extract and the standard drug (Acarbose) showed almost similar inhibition of glucose diffusion.

Figure 1: Inhibitory activity at different concentrations of A. racemosus and Acarbose on α Amylase enzyme.

Each value represents the mean±SD (n = 3)

Figure 2: Inhibitory activities at different concentrations of A.racemosus and Acarbose on Glucose diffusion.

Each value represents the mean±SD (n = 3)

Measurement of Glucose uptake by yeast cells:

The percentage of glucose uptake by plant extract and the drug can be viewed from Figure 3. There was a concentration dependent increase of glucose uptake by the plant extract treated yeast cells. This shows that the phytochemicals present in the ethanolic extract of A. racemosus mimics the action of insulin thereby aids in the entry of glucose molecules inside the yeast cells.

Figure 3: Inhibitory activities of A.racemosus and Metronidazole on Glucose uptake by yeast cells.

Each value represents the mean±SD (n = 3)

Measurement of Glucose adsorption potential by plant extract:

Figure 4 shows the glucose adsorption potential of the plant extract. It was observed that the glucose adsorption capacity increases with increase in the glucose concentration. In the present study the plant extracts adsorbed glucose at both low and high concentration effectively. Irrespective of the glucose concentration, the drug was able to combine with glucose and thereby retard the absorption across the intestinal lumen.

In silico anti-diabetic activity:

Molecular docking performed for 14 compounds against the receptor IRS 1 and IRS 2 are tabulated with their corresponding docking scores obtained. (Table 1)

Figure 4: Glucose adsorption capacity of A. racemosus extract at different concentration of Glucose.

Each value represents the mean±SD (n = 3)

Table 1 - Docking score values of phytochemicals of Asparagus racemosus with IRS 1 and IRS 2

S.No	Compound Name	Binding energy expressed in (kJ mol^-1)
S.No	Compound Name	Insulin Substrate Receptor 1(IRS-1)	Insulin Substrate Receptor 2 (IRS-2)
1	Oligospirotinosides	-0.78	-1.02
2	Thiophene	-0.12	-7.07
3	Quercetin 3-O-Glucoronide	-0.32	-0.96
4	Racemosol	-0.01	-0.15
5	Schidigera Saponin	-0.57	-8.02
6	7-O-Beta-D-Glucopyranoside	-0.81	-1.30
7	9,10 Dihydrophenanthrane	--2.58	-6.10
8	Elatin	-0.68	-0.95
9	Hyperoside	-0.61	-0.57
10	Quercetin	-0.08	-0.44
11	Diosgenin	-0.28	-0.67
12	Rutin	-0.31	-0.65
13	Shatavarin 1	-0.46	-0.58
14	Shatavarin 4	-1.33	-0.91

The inhibitory activity of the receptors IRS 1 and IRS 2 with the phytochemicals was performed using Ligplot. From the 14 phytochemicals, Quercetin, Racemosol, Rutin, Hyperoside and Shatavarin I was selected based on their docking score. From Table 2 the interactions between amino acids, the H binding sites and hydrophobic contact sites between the ligands and receptor can be known, and visualized from Figure 5 and 6.

Table 2 - Interaction of IRS 1 and IRS 2 receptor with the phytochemicals of Asparagus racemosus

S. No	Compound	Insulin Receptor Substrate 1 (IRS1) receptor amino acid binding site		Insulin Receptor Substrate 2 (IRS2) receptor amino acid binding site
S. No	Compound	H bonding sites	Hydrophobic contact sites	H bonding sites	Hydrophobic contact sites
1	Quercetin	4	63	2	14
1	Quercetin	Lys 74 (A), Arg 75 (A), Phe 160 (A)	Asn 73 (A), Lys 74 (A), Arg 75 (A), Tyr 107 (A), Phe 160 (A), Lys 161 (A), Glu 162 (A)	Thr 286 (A), Glu 289 (A)	Asn 282 (A), Thr 286 (A), Glu 289 (A)
2	Racemosol	-	47	-	24
2	Racemosol	-	Lys 21 (A), Lys 23(A), Ser 24 (A), His 26 (A), Arg 28 (A), Tyr 46 (A), Arg 62 (A), Phe 93 (A)	-	Lys 203 (A), Pro 204 (A), Lys 205 (A), Phe 254 (A), Gln 273 (A)
3	Rutin	2	50	-	14
3	Rutin	Lys 74 (A), Asn 248 (A)	Lys 74 (A), Arg 75 (A), Ala 76 (A), Lys 81 (A), His 82 (A), Gln 103 (A), Val 244 (A), Gln 247 (A), Asn 248 (A), Glu 251 (A)	-	Lys 203 (A), Ser 251 (A), Phe 254 (A), Trp 271 (A)
4	Hyperoside	8	56	3	66
4	Hyperoside	Lys 15 (A), Ser 105 (A), Gln 112 (A), Arg 116 (A)	Arg 14 (A), Lys 15 (A), Ser 105 (A), Gln 108 (A), Gln 112 (A), Arg 116 (A)	Gln 199 (A), Asn 201 (A), Asp275 (A)	Gln 199 (A), Val 200 (A), Asn 201 (A), Gln 273 (A), Ala 274 (A), Asp 275 (A)
5.	Shatavarin I	1	72	-	51
5.	Shatavarin I	Arg 213 (A)	Gly 172 (A), Thr 176 (A), Arg 212 (A), Arg 213 (A), Phe 222 (A), Glu 224 (A), Pro 233 (A), Gly 234 (A), Glu 235 (A), Trp 237 (A)	-	Lys 205 (A), His 250 (A), Ser 251 (A), Glu 269 (A), Trp 271 (A), Gln 281 (A)

The ligands Quercetin, Racemosol, Rutin, Hyperoside and Shatavarin I had hydrophobic interactions between its amino acids, thus acting as an antagonist ligand for the receptor (IRS 1 and IRS 2). It also acts as a competitive inhibitor thereby exerting the anti-hyper androgenic effect.

(A) Quercetin

(B) RACEMOSOL

(C) Rutin

(D) Shatavarin I

(E) Hyperoside:

Figure 5: IRS 1 receptor binding with the phytochemicals of Asparagus racemosus

(A) Quercetin

(B) Racemosol

(C) Rutin

(D) Hyperoside

(E) Shatavarin I

Figure 6: IRS 2 receptor binding with the phytochemicals of Asparagus racemosus

DISCUSSION:

All over the world, the occurrence of diabetes mellitus is alarmingly increasing. PCOS also contributes significantly to this increased risk of early age type 2 diabetes mellitus (DM) especially in the women population due to peripheral insulin resistance^23. Insulin controls a wide variety of biological processes including several aspects of metabolism and growth. As a metabolic regulator, it controls carbohydrate, protein as well as lipid metabolism in terms of uptake of glucose and their utilization, protein synthesis, gene transcription and lipogenesis in the specific target tissues. It also acts as a mitogenic regulator in ovaries and controls the steroidogenesis and ovulation along with the insulin-like growth factor termed as insulin/insulin-like growth factor (IGF)-signaling system. Insulin performs these activities through their binding to cell surface receptors called Insulin-receptor substrates IRS-1 and IRS-2. These receptors belong to the class of tyrosine kinase family and they accept insulin, insulin-like growth factor I (IGF-I), epidermal growth factor, ﬁbroblast growth factor and several cytokines as their ligands²⁴. At molecular level the binding of the ligand to IRS receptors activates two different downstream signaling pathways namely phosphatidylinositol-3 kinase (PI3K) pathway which regulates the cellular metabolism and the mitogen-activated protein kinase (MAPK) pathway which regulates the cell growth²⁵.

Several studies have shown a close association between the defective insulin signaling and the pathophysiology of polycystic ovary syndrome (PCOS). These include hyperandrogenemia, (increased androgen production by ovaries) anovulation, hyperinsulinemia and insulin resistance²⁶. Moreover, insulin resistance is considered a restricted event, in the sense it will affect only the PI3K pathway, so that the metabolic response to insulin action is impaired. The enhanced activity of MAPK pathway, which is specific to insulin target tissues of the ovary, will not get affected²⁷. Moreover Welsh et al.,²⁸have also reported that changes in IRS occur as a result of hyperglycaemia, which becomes an etiology of diabetic complications. With this above implication it is clearly evident that PCOS women have metabolic complications including high blood glucose level due to insulin resistance as well as hyperandrogenemia because of the defective insulin signaling. In this context, we made an attempt to investigate the hypoglycemic activity of A. racemosus, especially acting on the enteric system to slow down the release of glucose into the circulation for the management of hyperglycemia due to PCOS. Also, we performed in silico docking analysis with the phyto molecules of A. racemosus to identify a suitable antagonistic ligand for the IRS1 and IRS 2 receptors to suspend the down streaming reactions leading to the complications of PCOS.

There are numerous natural sources which are being investigated for anti-diabetic activity and also for amylase inhibitory activity, a key enzyme in carbohydrate metabolism²⁹. In the present investigation, ethanolic extract of A. racemosus inhibited α amylase enzyme considerably. According to Vadivelan et al.,³⁰the total flavonoids and triterpenoids present in the plant extract have the potential to inhibit the digestion of carbohydrates by inhibiting α amylase enzyme and release of glucose. Moreover, studies performed by Hannan et al.,³¹ in aqueous and butanol extract of A. racemosus, indicated low glucose, as well as insulin release pattern could control the hyperglycemic condition. These inhibitory activities are helpful in ameliorating PCOS condition and its associated pathophysiology.

In increasing the blood glucose level, glucose diffusion from the enteric system plays a predominant role. There are plant extracts which slow down the glucose diffusion across the mucosal membrane of the intestine and act as hypoglycemic agents³². In the present investigation, ethanolic extracts of A. racemosus influences the glucose diffusion and contributed considerably for the hypoglycemic activity. Abdel-Wahab et al.,³³has studied the hypoglycemic effect of A. racemosus root extract in the diabetic rats and indicated the inhibition of glucose absorption as one of the tentative mechanisms for the glucose lowering effect. It may be expected that the decrease in the glucose diffusion may be due to the adsorption of the glucose by the phyto molecules which subsequently delay its release in the gastrointestinal tract thereby influencing the diffusion. Irrespective of the glucose concentration, the drug was able to adsorb the glucose and thereby retard the absorption across the intestinal lumen. Studies show the transport of sugars and glycosides across the yeast cell is mediated by stereo specific membrane carriers³⁴. Already Hannan et al.,³¹ have shown that ethanol, chloroform, and hexane root extracts of A. racemosus have insulinotropic activity in the sense that the phyto molecules present in the extract mimics the action of insulin thereby helping in the glucose uptake in the cells as evidenced by the yeast cells.

In various cellular substrates, insulin exerts the effects through insulin receptors, particularly IRS proteins intervene the responses of insulin through phosphorylation of tyrosine residue. As already mentioned, the downstream reactions due to this IRS signaling differ in different tissues. Corbould,³⁵ also in his studies mentioned the same findings, that the expression of IRS protein receptors differs between PCOs affected women and normal women in their adipocytes and muscles. With the clear understanding of hyperinsulinemia in PCOs women, it makes the ovarian theca cells produce androgen in excess leading to hyperandrogenism which is mediated through the binding and signaling of the MAPK pathway. We performed the in silico docking analysis to search an antagonist for IRS 1 and IRS 2 receptors, to inhibit the binding of insulin. This will reduce the ovarian hyperandrogenism directly and reduce the other interlinked symptoms like hypersecretion of LH, euglycemic hyperinsulinemia, in association with insulin resistance. We have identified five ligands Quercetin, Racemosol, Rutin, Hyperoside and Shatavarin I which have an antagonistic action against IRS receptor protein. These ligands can act as a competitive inhibitor for the insulin in binding to IRS receptors of theca cells in the ovary thereby inhibiting the mitogenic pathway and excess androgen secretion. Once hyperandrogenism was corrected, the interlinked pathology such as hyperinsulinemia, especially receptor binding action as well as altered LH: FSH ratio was also normalized to prevent the chronic tropic action elicited by these endocrine chemicals.

CONCLUSION:

From the above in vitro and in silico studies it is concluded that the ethanolic root extract has the ability to treat hyperglycemia and hyperandrogenism, an interrelated complicated pathophysiology occurring in PCOS condition. It may be expected that when hyperglycemia is directly managed through the regulation of enteric glucose, hyperinsulinemia can be controlled indirectly. Moreover, the selected phyto molecules present in the plant extract act as potential antagonistic ligands for the receptor IRS1 and IRS2 which regulates the synthesis of androgens in the ovary. Through their inhibition hyperandrogenism can be reduced. With the dual action of reducing hyperinsulinemia and hyperandrogenism the drug could be prescribed for the management of PCOS cure.

ACKNOWLEDGEMENT:

I thank Dr M.G.R Educational and Research Institute to help me do this project.

CONFLICT OF INTEREST:

All the authors enlisted in the manuscript have no conflict of interest.

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Received on 23.06.2021 Modified on 29.01.2022

Research J. Pharm. and Tech 2023; 16(5):2096-2102.

DOI: 10.52711/0974-360X.2023.00344