INTRODUCTION:

In current touch, the attention of the investigation has been inclined towards traditional medicinal plants and significant amount of evidence has been accumulated which scientifically justifies traditionally use of these plants.^[1,2] Many synthetic oral hypoglycaemic agents are available which may produce serious side effects and not suitable during pregnancy.^[3] This leads to the demand of natural products with ease of availability and fewer cost. Withania coagulans (Solanaceae) family has their use in Ayurvedic system of medicine for 3000 years in India.^[4,5]

Withania coagulans usually known as Indian cheese maker, paneerdodi or pannerdoda, vegetable rennet.^[6,7] Milk coagulation can be achieved by the berries of the shrub. They are used as blood purifier and used in nervous exhaustion, insomnia, impotence, dyspepsia, flatulent and intestinal infections.^[8] The teeth can be cleaned by chewing the twigs and for the relief of toothache the smoke of the plant can be inhaled. In the treatment of diabetes, the flowers of coagulans (stocks) Dunal are used. Anti- mutagenic potential of fruits extracts of Withania coagulans also have been reported.^[9]

Type 2 Diabetes mellitus (T2DM) consists of an array of dysfunction characterized by decreased insulin secretion and high blood glucose level.^[10]Despite rapid advancement in the diabetic therapeutics, it is expected to double by the year 2035 due to the predominance of DM.^[11,12]Hyperglycaemic state may be due to deceased production of insulin by the pancreas or the body does not respond properly to insulin that is produced.^[13] It is generally associated with several complications that arise secondary to diabetes such as neuropathy, nephropathy, retinopathy, cardiovascular complications etc.^[14]During DM glucose is abundantly available, which upon oxidation generates excessive free radicles and saturates body’s antioxidant defence (catalase, superoxide dismutase, glutathione etc).

These free radicals interact with the biomolecules and render them non-functional, thereby developing persistent oxidative stress in the body which aids in the development of secondary diabetic complications.^[15] Available anti-diabetic drugs are aimed on eradicating excessive glucose from the blood and have negligible effect on the mechanisms which leads to the development of various complications.^[16]Certain group of drugs for T2DM is represented by the inhibitors of alpha glucosidase which is responsible for the breakdown of oligo or disaccharides to monosaccharides.^[13,17]

In silico screening is a vital method employed for predicting preferred orientation of a molecule against the protein target α- glucosidase, PPAR-gamma, and DPP-4 were studied. This paper gives an overview on in-silico and in-vitro anti- diabetic activity of Withania coagulans targeting the majorly three proteins. Therefore, objective of the current study was to gain the mechanism through which it can aid in controlling diabetes through in vitro and in silico tools. In this study we evaluated the anti-diabetic potential of ethanolic extract of Withania coagulans fruits using the alpha glucosidase enzyme and the bioactive compounds responsible for the activity was predicted using in silico studies.

MATERIALS AND METHODS:

MATERIALS:

α- glucosidase used in the present study was procured from Sigma- Aldrich. Sodium phosphate buffer, p- nitrophenyl- α- D- glucosidase, sodium carbonate and Schrodinger- Glide- Maestero 2018 were used in Sri Ramachandra Institute of Higher Education and Research.

COLLECTION AND EXTRACTION OF PLANT MATERIAL:

The dried fruits of the above plant were procured from Natural remedies, Chennai, India. The species was authenticated by Prof. P. Jayaraman, Ph.D, Director, Institute of Herbal Botany PLANT ANATOMY RESEARCH CENTRE (PARC), Chennai. The plant material was ground into powder and the extract was prepared according to the methodology of Indian Pharmacopoeia (Anonymous, 1966) by cold maceration. 10g of dried powdered Withania coagulans (Dunal) was taken in a 250ml conical flask and 100ml of ethanol was added and kept at room temperature for 48hrs. It was then filtered and subjected to distillation. The final extract was obtained and used for the following assay.^[18]

INHIBITION ASSAY FOR ALPHA GLUCOSIDASE:

Alpha glucosidase inhibitory activity was performed using acarbose as a standard drug. 50μl of 0.2 M sodium phosphate buffer (pH 6.8) and 50μl of 0.1 U alpha glucosidase was taken. The various concentration of 50 μl of sample and standard (100, 200, 300, 400, 500μl) were taken and incubated at 37°C for 5 min. Then 50μl of p-nitrophenyl alpha- D-glucosidase was added, vortexed and incubated at 37°C for 30 min. The reaction was terminated by the addition of 800μl 0.1 M Na2CO3. α- glucosidase activity was determined spectrophotometrically at 405nm using a UV-vis spectrophotometer. It is quantified by measuring the quantity of p-nitrophenyl release from p- nitrophenylglucopyranoside.^[19] The % inhibition was calculated as follows

% Inhibition = (Control OD - Sample OD/Control OD) × 100

The IC50 value was defined as the concentration of the sample extract to inhibit 50% of α-glucosidase activity under assay condition. It was performed using Graphpad prism software.

IN SILICO DOCKING STUDY:

Protein structure for docking:

The 3D structure for alpha glucosidase [PDB ID: 5NN8], Peroxisome proliferator activated receptor [PDB ID: 3CS8] and Dipeptidyl peptidase [PDB ID: 2I03] were obtained from Protein Data Bank (http://www.rcsb. org/pdb). The Protein Preparation Wizard of Schrodinger-Maestro 2018 was used to prepare and refine the selected protein structure.^[20,21,22]

Ligand preparation:

The structure of the compounds and standards were retrieved from Pubchem databases ie; Withanolide A, Withacoagulin, Ajugin A and were prepared using Lig Prep wizard of Schrodinger-Maestro 2018.

Ligand docking:

The docking was carried out using the ligand docking wizard of Glide- Maestero 2018.

Statistical analysis:

Test were performed in triplicate. The mean values were calculated and expressed as Mean±SEM and differences between groups were considered to be statistically significant and the IC50 values were determined using Graphpad prism.

RESULTS:

In-vitro inhibition of alpha glucosidase:

Table 1 shows the ethanolic extract of the fruit of the W. coagulans (Dunal) was prepared in different concentration and was targeted against alpha- glucosidase inhibition. It was then compared against the standard acarbose.

In- silico inhibition of alpha glucosidase:

The ultimate goal of molecular docking is to find the optimal ligand/protein configurations as well as to consistently predict their binding free energy and the hydrogen bond interactions. Table 2 shows the results of molecular interaction with the specific protein alpha- glucosidase.

Table:1 Anti- diabetic potential of W.coagulans by alpha glucosidase inhibition assay

Sample

Concentration (μg/ml)

%Inhibition

IC50 (μg/ml)

Ethanolic extract of fruits of Withania coagulans

100

200

300

400

500

22.4±0.04

31.8±0.04

45.4±0.04

314.3

Acrabose (standard)

100

200

300

400

500

39.84±0.03

60.0±0.03

94.4±0.02

94.44±0.01

94.52±0.02

201.8

α- glucosidase inhibition of Withania coagulans and standard

Table 2 Results of docking interaction with different compounds

S: No	Protein	Ligand	Dockscore	H Bond
1.	α- glucosidase	Withanolide A Withacoagulin Ajugin	-4.48 -3.84 -1.68	4 2 2
2	Peroxisome proliferator activated receptor	Withanolide A Withacoagulin Ajugin	-5.97 -3.55 -5.09	- 1 2
3	dipeptidyl peptidase- 4(DPP-4).	Withanolide A Withacoagulin Ajugin	-4.16 -3.16 -1.46	1 2 1

DISCUSSION:

EXTRACT AND PERCENTAGE YIELD:

The extract of the powder of dried fruits of Withania coagulans was obtained according to the Indian Pharmacopoeia. The solvent used for the extract is ethanol. The percentage yield of the extract was found to be 0.19g.

Evaluation of Invitro Α- Glucosidase Activity:

Many serious side effects and complication is possible in the management of diabetes with insulin and synthetic oral hypoglycaemic drugs. Minimal or no side effects and low cost are the advantage of using Herbal remedies over synthetic ones. α- glycosidase and is responsible for the conversion of un-absorbable dietary polysaccharides into monosaccharides especially in the gut which are then absorbed and reaches the blood circulation thereby increasing the blood glucose level. Inhibition of these enzymes is associated with reduction in hyperglycaemia through reduction in dietary uptake of glucose.

This process is imitated in- vitro to screen the anti- diabetic potential of plant extract and this is the well- known and extensively used in-vitro anti-diabetic assays. In our study we screened for its anti- diabetic potential through inhibition of α-glucosidase and the results are depicted in table 1. The results showed that there is a concentration dependent activity of the enzymes. Hence a graded response is obtained.

IN SILICO ANTI- DIABETIC EVALUATION:

In-silico evaluation helps us to understand the ligand protein interaction and explore the binding sites. The withanolides, withacoagulin and ajugin have been choosen from fruits along. All the targets were validated before the docking studies. With target α- glucosidase withanolide A has highest docking score among the other compound 3H bonds with ASP91, ILC98, ARG331 and one with water molecule. Withacoagulin (-3.84) has 1H bonds with PRO94 and one with water. Ajugin had two hydrogen bond with TRP126 and CYS 127.

Target DPP-4: Withanolide A (-4.16) had 1H bond with TYR585. Withacoagulin had 2H bonds with GLU206 and ASN710. Ajugin with one hydrogen bond with AXD 1630.Target PPAR-γ: Withanolide A has no H bond and interaction. Withacoagulin had 3H bonds with LYS 261, ARG 280 and water molecule. Ajugin had hydrogen bond with water molecule.

CONCLUSION:

The ethanolic extract of withania coagulans showed greater IC50 value which was observed to be 314.3μg. From these results it shows that the plant extract of W. coagulans has appreciable potential to inhibit the enzyme and hence can be used as an additive drug in the management of diabetes. Hence it is concluded that on increasing the concentration of the extract, a significant IC50 value can be obtained. This will be helpful in finding more potent anti- diabetic drug from the natural resources. Withanolide A showed good interaction and docking score against α- glucosidase and not against DPP-4 and PPAR-γ. Hence withanolide A can be used as an anti- diabetic agent against α- glucosidase. In future withanolide A the constituent present in the fruits can be targeted against the alpha glucosidase enzyme.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGEMENT:

The authors would like to thank the management of Sri Ramachandra institute of higher education and research for providing the software and chemicals for successful completion of this research work.

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Received on 10.07.2019 Modified on 12.08.2019

Research J. Pharm. and Tech 2020; 13(2):631-635.

DOI: 10.5958/0974-360X.2020.00120.1