INTRODUCTION:

Diabetes mellitus is the metabolic disorder related to hyperglycaemia with perturbation of carbohydrates, lipids and protein metabolism be associated with defects in insulin secretion, insulin action or both.The increased level of blood sugar level eventually leads to micro and macro-vascular complication such as neuropathy, nephropathy, retinopathy, heart attack, strokes etc. Uncontrollable diabetes may prompts stupor, coma and if not treated demise because of ketoacidosis or from non ketotic hyperosmolar disorder. [1,7]The ubiquity of diabetes has expanded radically in adults and it set up a worldwide general wellbeing trouble .Diabetes is relied upon to influence in excess of 500 million adults worldwide by 2030.^[8]Right now there are several medications for the treatment of diabetes which have expensive and the available glucose –lowering therapy is mainly relays on altering the effects of the ‘ominous octet’ that lead to hyperglycaemia. Future bearings in diabetes care incorporate methodologies, for example, the 'bionic pancreas', stem cell therapy and focusing on the intestinal microbiome.^[9]All of these medications are as yet being refined, and it might be quite a few years before they are clinically helpful. Since plant based medicine has claimed less or no side effects, individuals turned their enthusiasms towards the herbal medicine.

Traditional and ethnic medicines plays a major role in the health services around the world. The genus Passiflora L belonging to the family Passifloracea and it is indigenous to South America , featuring over 500 species world-wide.^[10]Mostly they are tendril bearing vines with some being shrubs or tees. The Passiflora edulis Sims is the most commercialised species and different population of the species is distributed mainly according to the colour of fruit. Generally, they are known as passion fruit. The yellow coloured fruit is known as passiflora edulis Sims Var. flavicarpa (yellow passion fruit) and other population with purple colour fruit is named as Passiflora edulis Sims var. edulis. The plant yellow passion fruit have been used by common people and it has many folklore earlier claims for the treatment of anxiety, diuresis, hypertension, diabetes etc. The various parts of the plant has been used for the ailment of variety of disorders.^[11-13] Many literature studies have reported that the fruit of passiflora edulis Sims var. flavicarpa used as the remedial method for the lowering of blood glucose level, due to the presence of soluble pectin.^[14]. It also has been reported that leaves of yellow passion fruit contain bioactive compounds such as Orientin, Isoorientin, Isovitexin, Vicenin-2 and Vitexin. ^[15]

As human digestive tract can retain the monosaccharides type of glucose into the circulatory system, the enzyme alpha glucosidase situated in the surface film of the intestinal cells catalyses the cleavage of glucose from disaccharides to monosaccharides. Thus, it has been used as the one of the remedial target for the regulation of post prandial hyperglycaemia in type II diabetes. Several alpha glucosidase inhibitors such as acarbose, are utilised over the previous decades. All things considered, progressively productive alpha glucosidase inhibitors are promptly required because of low efficacy with high Ic50 values against enzyme with all known alpha glucosidase inhibitors. ^[16]

Now a days, Molecular docking is considered as one of the powerful technique to discover the potential ligand for known targets. The study mainly focused on binding modes which were essential for major structural interaction. The values of binding energy signifies the affinity of ligands towards the target.^[17-19]The present study is focused to evaluate the antidiabetic activity of ethanolic extract of yellow passion fruit leaves using alpha glucosidase enzyme and the interaction of bioactive compounds against alpha glucosidase enzyme using docking analysis.

MATERIALS AND METHODS:

M ATERIALS

The enzyme alpha glucosidase was procured from Sigma –Aldrich. The chemicals such as Sodium phosphate, P-nitrophenyl glucopyranoside, sodium carbonate and softwares such Schrodinger- Glide- Maestero 2018,Graph pad prism were used in Sri Ramachandra Institute of Higher Education and Research.

PLANT AUTHENTICATION

Fresh leaves of Passiflora edulis Sims Var. flavicarpa were obtained from Thalassery, Kannur district, Kerala in the month of February 2019 and pharmacognistically identified and authenticated by the Prof. P.Jayaraman Ph.D. ;Retd, Professor, Presidency college, Chennai

PREPARATION OF EXTRACT

The fresh leaves of Passiflora edulis Sims Var. Flavicarpa washed thoroughly with tap water and followed by distilled water to remove the dust particles and unwanted materials. Then the leaves are 50air dried under the shade about 10 days. The dried leaves are then subjected to grinding to make it as a coarse powder. The extraction were prepared using cold maceration method. The 10g of coarse powder was taken in a conical flask and it is soaked with 100 ml of ethanol and it is allowed to stand at room temperature for 72 hours with frequent agitation. It is done to increase the release of phytoconstituents in the solvent. It is filtered using whattsman filter paper. The filtrate was collected and incubated in room temperature for evaporation. Yield obtained was 140 mg.

ALPHA GLUCOSIDASE INHIBITION ASSAY

Alpha glucosidase inhibition assay was done using standard procedures with minor modifications.^[20] Alpha glucosidase inhibition assay was carried out by adding 50µl of 0.2M sodium phosphate buffer (PH6.8) and 50 µl of 0.1U alpha glucosidase in different test tubes. To this add 50 µl of sample and standard of different concentration (200, 400, 600, 800, and 1000 µl) were added and incubated at 37ºC for 5 minutes. Then, 50 µl of P-nitrophenyl glucopyranoside was added and vortexed for about 30 minutes at 37ºC. Finally, 800 µl of 0.1M sodium carbonate was added. The absorbance was measured at 405nm using UV-Visible Spectrophotometer. The alpha glucosidase inhibition assay was explicated by measuring the yellow colour Para-aminophenol released from P-nitrophenyl glucopyranoside. The acarbose was used as standard. The % inhibition was calculated as follows:

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

Concentration of extract bringing about 50% restraint of catalyst activity (IC50) were resolved graphically using Graph pad prism software.

INSILICO DOCKING STUDY:^[21]

Protein Preparation:

The three dimensional structure of alpha glucosidase (PDB ID-5NN8) was downloaded from the protein data bank in the pdb format and it retrieved it into the glide software. Then, the protein was prepared and refined by the protein preparation wizard of the Schrodinger- maestro 2018.

Ligand preparation:

The 3D structure of the ligands (Orientin, Isoorientin, Isovitexin, vicenin-2 and Vitexin) were downloaded from the PubChem database in sdf format. Then they were prepared using Lig prep wizard in Schrodinger –maestro 2018.

Ligand docking:

Finally, the ligand docking was performed using ligand docking wizard of glide –maestro 2018. Final scoring was performed based on the energy-minimising poses and it is exhibited as Glide score. The best docked pose with minimal glide score value was recorded for each ligands.

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 if p<0.5 and the IC50 were determined using Graph pad prism.

RESULTS:

In-vitro inhibition of alpha glucosidase:

Table 1 depicts the ethanolic extract of leaves of Passiflora edulis Sims Var. flavicarpa which was prepared in different concentration was targeted against the enzyme alpha glucosidase and then it was compared with the standard Acarbose.

Table 1: Anti –diabetic activity of Passiflora edulis Sims Var. flavicarpa and standard acarbose by alpha glucosidase inhibition assay.

Sample	Concentration	% Inhibition	IC50 (µg/ml)
Ethanolic extract of leaves of Passiflora edulis Sims Var. flavicarpa	200	0	507.6 µg/ml
	400	14.8±0.04
	600	44.5±0.04
	800	54.21±0.03
	1000	62.91±0.01
Acarbose (standard)	200	43.16±0.03	567 µg/ml
	400	59.38±0.01
	600	67.1±0.03
	800	84.23±0.03
	1000	96.23±0.01

Fig1: IC₅₀ of α-glucosidase by Passiflora edulis var.flavicarpa

Fig2:IC₅₀ of α-glucosidase by Acarbose

In-silico interaction of ligands against enzyme alpha glucosidase:

Molecular docking is carried out to find the binding energy and hydrogen bond interaction of ligands against target. Table.2, depicts the dock score and hydrogen bonding interaction of different bioactive compounds against the macromolecule alpha glucosidase.

Table 2: Results of docking interaction of protein with different bioactive compound

SI. NO.	TARGET	LIGANDS	DOCK SCORE	HYDROGEN BONDS
1	Alpha Glucosidase	Isoorientin	-8.67	5
2		Orientin	-7.39	4
3		Isovitexin	-7.01	5
4		Vicenin-2	-6.18	4
5		Vitexin	-5.25	3
6		Acarbose (standard)	-9.803	7

Fig3: Molecular docking of Isoorientin with alpha glucosidase

Fig4: Molecular docking of Orientin with α-glucosidase

Fig5: Molecular docking of Isovitexin with α-glucosidase

DISCUSSION:

In-vitro alpha glucosidase inhibitory activity assessment:

The plants have previously been used as a source of medicinal drug by folklore for the treatment of various disease. In spite of the fact that recovery may be slow, it has been used by many to cure various ailment, because of the minimal or less side effect of the herbal medicines.

Available literature depicts that, several plants have been used for the hypoglycaemic activity. The enzyme alpha glucosidase has the role to catalyse the cleavage of disaccharide form of glucose to monosaccharides. Thereby, the level of post prandial blood sugar increases, which results into the postprandial hyperglycaemia. Hence, inhibition of alpha glucosidase enzyme are needful in the control of post prandial blood glucose level.

The inhibitory activity of ethanolic extract of leaves of Passiflora edulis Sims Var. flavicarpa against the enzyme alpha glucosidase was investigated in the present study and the results are depicted in table.1. Hence, the result obtained from the data shows that ethanolic extract of leaves of yellow passion fruit is capable of inhibiting the enzyme alpha glucosidase in a concentration dependent manner.

In-silico anti-diabetic evaluation:

In-silico evaluation is the valid method which helps to explore the principle binding affinity of ligands with the protein having known three-dimensional structure. This technique is used to raise potential ligands for known targets. The target was validated before the docking studies. The bioactive compounds such as Isoorientin, Orientin, Isovitexin, vicenin-2 and Vitexin. Among this Isoorientin shows greater docking score (-8.67) and had 5 hydrogen bonds with ALA93, ASP91, GLY123 and CYS127. Orientin shows dock score of -7.39 and had four H bond with the amino acids ILE98, LYS96, ASP91, and GLN115.Isovitexin has dock score of -7.01 and five H bond with ALA93, ASP91, GLN124 and CYS127.Vicenin-2 shows moderate dock score of about -6.18 and had four H bond with ASP404, GLY651 and SER675. Vitexin shows least dock score (-5.25) compare to other bio-active compounds and had three hydrogen bonds with ASP282, ASP616 and ASP588.

CONCLUSION:

In the present study we carried out the in-vitro alpha glucosidase inhibition assay of ethanolic extract of leaves yellow passion fruit against diabetes and also the docking studies on bioactive compounds of leaves of yellow passion fruit against target alpha glucosidase.

The ethanolic extract of leaves of Passiflora edulis Sims Var. flavicarpa. Showed remarkable IC50 value, which was observed to be 507.6µg/ml. The result obtained from the data shows that, the ethanolic extract of leaves of passiflora edulis Sims Var. flavicarpa has marked potential to inhibit the enzyme in a concentration dependent manner. Hence, the extract of leaves of the Passiflora edulis Sims can be used as an additive alpha glucosidase inhibitor for the treatment of diabetes. Among the five bioactive compounds of the leaves of yellow passion fruit, the Isoorientin shows good interaction and greater docking score against alpha glucosidase. Hence, Isoorientin can be used as an anti- diabetic agent against alpha glucosidase enzyme. In future, Isoorientin the constituent present in the leaves of Passiflora edulis Sims Var. flavicarpa can be targeted alpha glucosidase enzyme.

CONFLICT OF INTEREST:

The authors declares no conflict of interest.

AKNOWLEDGMENT:

The authors would like to thank the management of Sri Ramachandra Institute Of Higher Education and research for providing the software and chemicals for the successful completion of this research work.

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Received on 29.07.2019 Modified on 16.10.2019

Research J. Pharm. and Tech 2020; 13(3): 1309-1313.

DOI: 10.5958/0974-360X.2020.00241.3