INTRODUCTION:

Diabetes mellitus is a multi-factorial disorder characterized by hyperglycemia, lipoprotein abnormalities and altered intermediary metabolism of major food substrates.¹ It is associated with the chronic complications such as cardiovascular diseases, neuropathy, nephropathy and diabetic retinopathy.² The management of the blood glucose level is a critical strategy in the control of diabetic complications.¹ Traditional plant treatments have been used for the therapy of diabetes mellitus.^3,4

The global diabetes prevalence in 2019 is estimated to be 9.3% (463 million people), rising to 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045.⁵Inhibition of carbohydrate digestive enzymes activity such as α-amylase and α-glucosidase leads to a reduction in starch hydrolysis which has beneficial effects on glycemic index control in diabetic patients.⁶Therapeutic drugs of alpha amylase and alpha glucosidase inhibitors such as acarbose, miglitol and voglibose have an array of side effects such as abdominal distension, bloating, flatulence, meteorism and possibly diarrhea.⁷ Gymnemic acid, an active phytochemical with antidiabetic properties in Gymnema sylvestre leaves^8,9was investigated for its inhibitory effect on α-amylase and α-glucosidase and compared with voglibose, a well- known synthetic drug.¹⁰

Materials and methods:

Sources of plant compound and enzymes:

Gymnemic acid can be extracted from Gymnema sylvestre leaves. Gymnemic acid IV was purchased from Sant herbals, Hungary. Porcine pancreatic amylase (A3176) was purchased from Sigma Aldrich. α-Glucosidase (75551) and p-Nitrophenyl α-glucopyranoside (12735) were purchased from SRL chemicals.

Assessment of alpha amylase inhibition:

The assay mixture containing 120μl of 0.06 M potassium phosphate buffer, 20μl of enzyme, 40μl of Gymnemic acid in concentration range from 20-100μg/ml, voglibose used as the positive control and 20μl of sodium chloride (act as activator) were incubated for 20 minutes at 37˚C. After incubation 40μl of substrate was added and distilled water was added to equal the volume of samples. The reaction mixture was incubated at 37˚C for 10 -15 minutes. The reaction was terminated with the addition of 40μl DNSA reagent and placed in a boiling water bath for 2-3 minutes, cooled and absorbance was measured at 540nm using ELISA reader. The control samples were prepared without any plant compounds. The % inhibition was calculated according to the following formula.^{11, 12}

Abs (540) control –Abs (540) Test

Inhibition (%) = -----------------------------------------------------------×100

Abs (540) Control

Assessment of alpha glucosidase inhibition:

The alpha glucosidase inhibitory activity was assessed according to the chromogenic method reported by Ranilla et al. with slight modifications. The mixture contained 20µl α-glucosidase (0.5 unit/ml), 120µl of 0.1 M phosphate buffer (pH 6.9) and 10µl of test sample at various concentrations. The mixed solution was incubated in 96-well plates at 37°C for 15 min. After pre-incubation, the enzymatic reaction was initiated by adding 20µl of 5mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9) and the reaction mixture was incubated for another 15 min at 37°C. The reaction was terminated by adding 80µl of 0.2 M sodium carbonate solution and then absorbance reading was recorded at 405 nm by microplate reader (BioTek XS2). The reaction system without plant phytochemical was used as control and the system without α-glucosidase was used as blank for correcting the background absorbance. The inhibitory rate of sample on α-glucosidase was calculated by the following formula. ^{13, 14}

Abs (405) control – Abs (405) Test

Inhibition (%) = ----------------------------------------------------------× 100

Abs (405) control

Calculation of IC₅₀:

The IC₅₀was calculated using linear regression equation in which the concentration of the sample is plotted in the y-axis and percent inhibition in the x-axis. From the equation y = a+bx, IC₅₀ values can be calculated using the following formula.

IC₅₀= 50-a

Docking study on α-amylase with Gymnemic acid and Voglibose:

The target alpha amylase (1dhk) structure was retrieved from RCSB/PDB structure database at (https://www.rcsb.org). The active sites of alpha amylase were predicted by using viewer lite tool. In the viewer lite tool, new hierarchy window was used to predict the active site of the alpha amylase. Unwanted amino acid chains, NAG residues and water molecules are deleted from the enzyme alpha amylase. Structure and smiles of voglibose drug were collected from drug bank at (https://www.drugbank.ca). Structure and smiles of the Gymnemic acid was retrieved from pub chem online tool and pub chem. ID is 14264063. Finally the chosen ligands (Gymnemic acid and voglibose) were docked with alpha amylase enzyme using patch dock server. Then the similarities of active amino acid binding sites of gymnemic acid and voglibose with α-amylase were analyzed using Ligplot software.

Docking study on α-glucosidase with Gymnemic acid and Voglibose:

The target alpha glucosidase (3wy1) structure was retrieved from RCSB/PDB structure database at (https://www.rcsb.org). The active sites of alpha glucosidase were predicted by using viewer lite tool. In the viewer lite tool new hierarchy window was used to predict the active site of the alpha glucosidase. Unwanted amino acid chains, and water molecules are deleted from the enzyme alpha glucosidase. Structure and smiles of voglibose drug were collected from drug bank at (https://www.drugbank.ca). The chosen ligands (Gymnemic acid and voglibose) were docked with alpha glucosidase enzyme using patch dock server. Then the similarities of active amino acid binding sites between gymnemic acid and voglibose with α-glucosidase were analyzed using Ligplot software.

Results:

Table I and Graph I represent the alpha amylase inhibitory activity of Gymnemic acid and voglibose. Concentration dependent inhibitory effect of alpha amylase was shown maximum in 100µg/ml in both Gymnemic acid and Voglibose with IC50 value of 68.12µg and 46.23µg respectively. Gymnemic acid shows less inhibitory effect when compared to that of voglibose drug.

Table I: Effect of Gymnemic acid and Voglibose on α-amylase activity

Concentration (µg/ml)	Gymnemic acid		Voglibose
Concentration (µg/ml)	% inhibition	(IC₅₀)	% inhibition	(IC₅₀)
20	15.60 ± 1.68	68.12	21.63 ± 0.78	46.23
40	22.47 ± 1.29		43.67 ± 1.76
60	49.13 ± 1.63		55.30 ± 1.25
80	56.63 ± 1.55		69.17 ± 1.89
100	63.40 ± 1.64		81.87 ± 1.33

Graph I: Percentage inhibition of porcine pancreatic amylase by Gymnemic acid and Voglibose

Table II and Graph II represent the α-glucosidase inhibitory activity of Gymnemic acid and Voglibose. Concentration dependent inhibitory effect of α-glucosidase was shown maximum in 100µg/ml in both Gymnemic acid and Voglibose with IC50 value of 65.77µg and 36.67µg. Gymnemic acid shows less inhibitory effect when compared to that of voglibose drug.

Table II: Effect of Gymnemic Acid and voglibose on α- Glucosidase activity

Concentration (µg/ml)	Gymnemic acid		Voglibose
Concentration (µg/ml)	% inhibition	(IC₅₀)	% inhibition	(IC₅₀)
20	20.67 ± 1.44	65.77	33.63 ± 1.64	36.67
40	28.80 ± 1.59		45.97 ± 1.55
60	40.10 ± 1.01		61.60 ±1.28
80	55.43 ± 1.50		81.43 ± 1.40
100	70.50 ± 1.32		91.20 ± 1.74

Graph II: Percentage of inhibition of yeast glucosidase by Gymnemic acid and Voglibose

In silico docking analysis of α-amylase with Gymnemic acid and Voglibose

Figure I: Docking image of α-Amylase binding with Gymnemic acid

Figure II: Docking image of α-Amylase binding with voglibose

Figure I and II represent the image of gymnemic acid and voglibose docked with α-amylase using patchdock server and viewerlite tool. The docking results shows 5746 score with α-amylase and gymnemic acid. 3616 is the docking score of α-amylase with voglibose. The binding of gymnemic acid and voglibose at the active sites of α-amylase were shown in table III using ligplot software. Table IV showed similar active binding sites of aminoacids between gymnemic acid and voglibose.

Table III: Amino acids interaction at the active sites of α-amylase between Gymnemic acid and Voglibose

Amino acid interaction at the active sites of α-amylase with Gymnemic acid	Amino acid interaction at the active sites of α-amylase with Voglibose

Table IV: Similar active site amino acid binding sites of alpha amylase with Gymnemic acid and Voglibose

Similar active amino acid sites of α-amylase with gymnemic acid

Similar active amino acid sites of α-amylase with voglibose

Lysine 261 (A)

Serine 270 (A)

Threonine 23(B)

Tryptophan 269(A)

Threonine 264 (A)

Leucine 237 (A)

Lysine 261 (A)

Serine 270 (A)

Threonine 23(B)

Tryptophan 269(A)

Threonine 264 (A)

Leucine237 (A)

Insilico docking analysis of α-glucosidase with

Gymnemic acid and Voglibose

Figure III: Docking image of α-Glucosidase binding with Gymnemic acid

Figure IV: Docking image of α-Glucosidase binding with voglibose

Figure III and IV show the docking image of gymnemic acid and voglibose with α-glucosidase using patchdock server and viewerlite tool. The docking results showed 5628 score with α-glucosidase and gymnemic acid. 3322 is the docking score of α-glucosidase with voglibose. The binding of gymnemic acid and voglibose at the active sites of α-glucosidase was shown in Table V using ligplot software. Table VI showed similar active binding sites of aminoacids between gymnemic acid and voglibose.

Table V: Amino acids interaction at the active sites of α-glucosidase between Gymnemic acid and Voglibose

Amino acid interactions at the active sites of α-glucosidase with Gymnemic acid

Amino acid interactions at the active sites of

α-glucosidase with Voglibose

Table VI: Similar active site amino acid binding sites of alpha glucosidase with gymnemic acid and voglibose

Similar active amino acid sites of α-glucosidase with gymnemic acid

Similar active amino acid sites of α-glucosidase with voglibose

Phenylalanine 397 (A)

Tyrosine 389 (A)

Arginine 400 (A)

Phenylalanine 147 (A)

Phenylalanine 166(A)

Valine 334 (A)

Alanine 229 (A)

Phenylalanine 397 (A)

Tyrosine 389 (A)

Arginine 400 (A)

Phenylalanine 147 (A)

Phenylalanine 166(A)

Valine 334 (A)

Alanine 229 (A)

Discussion:

Sankaradoss etal have established that Gymnemic acid showed maximum inhibitory activity on α-amylase with percentage of inhibition of 17.49% and 14.23% in the concentration of 5mg/ml and 10mg/ml respectively and concluded that the use of gymnemic acid instead of acarbose would greatly been effective in management of diabetes by lowering the rate of digestion and absorption of carbohydrates.¹ The combination of voglibose and gymnemic acid was experimentally investigated on the digestion and absorption of maltose in rat small intestine. Use of this voglibose drug induces gastrointestinal disturbance and hepatotoxicity. Gymnemic acid may diminishes the adverse effects not only by reducing the dosage of voglibose but also by suppressing the intestinal motility and inhibiting the growth of anaerobia which develops gastrointestinal problems. The authors have concluded the combination of voglibose and gymnemic acid would diminish adverse effect of voglibose in control of diabetes.¹⁵ According to the rapid screening of α-glucosidase inhibitors by bio-affinity ultrafiltration and high-performance liquid chromatography coupled with electrospray mass spectrometry (UF-HPLC-MS) method, the authors revealed 9 components were considered as potential α-glucosidase inhibitors ftom the extract of Gymnema sylvestre. Of these 9 components gymnemic acid is one of the components which possesess α-glucosidase inhibitor activity.¹⁶ Gymnemic acids possess anti-diabetic, anti-sweetener and anti-inflammatory activities.¹⁷ Oral administration of GA to experimental diabetic groups of rats showed significant reduction in the levels of fasting blood glucose and glycosylated hemoglobin and increased level of plasma insulin and hemoglobin.¹⁸ According to the earlier reports, gymnemic acid has also been perceived useful against obesity.¹⁹ Gymnemic acids slow up glucose absorption in the blood. Because the atomic arrangements of gymnemic acid was similar to that of glucose molecules which fills in the receptor location of taste buds of small intestine. Gymnemic acid molecules fill the receptors in the absorptive external layers of the small intestine which delays glucose absorption and leads to low blood sugar level.²⁰ Probably because of its structural resemblance to glucose molecules, this happens to inhibit α-amylase and α-glucosidase. Not only this triterpene glycoside from Gymnema sylvestre could inhibit the pancreatic α-amylase and α-glucosidase but also manifested to increase the GLUT2 protein levels and improved impaired insulin secretion of MIN6 cells which showed triterpene glycoside possess antidiabetic activity.²¹ Moreover, Gymnemic acid has the potential to maintain normoglycemia via the inhibition of Glycogen Synthase Kinase-3 activity for the development of active pharmacophore discovery and offering novel insights into the therapeutics for diabetes mellitus.²² The predicted activity of three gymnemic acid analogues viz., deacyl gymnemic acid, dihydroxy gymnemic triacetate and gymnemic acid IV was found comparable to the prior experimental data. The authors concluded that these structure activity relationship results might be of great chance in antidiabetic drug designing and discovery from natural products.²³ Gymnemic acids contain glucuronic acid that inhibits human sweet receptor T1R2+T1R3. The interaction between transmembrane domains of human T1R3 and the glucuronosyl group of gymnemic acids is predominantly required for the sweet suppressing effect. This provides further discrimination into drug design to alter sensitivity of sweet receptor.²⁴ Our present study also evidents the inhibitory activity of gymnemic acid on α-amylase and alpha glucosidase. Voglibose is used as the positive control. Insilico docking analysis between gymnemic acid and voglibose with α-amylase and α- glucosidase were carried out and active amino acid binding sites were analyzed by ligplot software. Similar amino acid binding sites were found in docking studies.

The present study establishes the inhibitory effect of gymnemic acid and voglibose. on α-amylase and α-glucosidase. Insilico docking study between gymnemic acid and voglibose with α-amylase and α-glucosidase show similar amino acid binding sites. Eventhough voglibose seems to be better α-amylase and α-glucosidase inhibitor, gymnemic acid has a multifactorial effect on glucose homeostasis. Hence it might be included as a combinatorial regime along with anti-diabetic drugs inorder to reduce the dosage of other synthetic drugs that have multiple side effects.

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Received on 11.08.2020 Modified on 22.09.2020

Research J. Pharm. and Tech. 2021; 14(9):4755-4759.

DOI: 10.52711/0974-360X.2021.00827