In vitro Studies on Basella rubra Different Extracts as Inhibitors of Key Enzymes Linked to Diabetes Mellitus

Enzyme, inhibiting carbohydrate metabolism and thereby decreasing glucose level is a class of drugs helpful in the management of type 2 Diabetes mellitus. Naturally existing α-amylase and α-glucosidase inhibitors from medicinally significant plants are shown to be effective in the management of postprandial hyperglycemia. In this investigation, leaf extract (BRLE), stem extract (BRSE), fruit extract (BRFRE) and flower extract (BRFLE) of Basella rubra were subjected to evaluate their antioxidant potential and their possible inhibitory effects on α-amylase and α-glucosidase. BRLE, BRSE, BRFRE, BRFLE (at concentration 100μg/ml) exhibited 65.78, 56.84, 63.1, 61.03% of α-amylase inhibitory activity respectively with IC50 values of 71.66, 89.69, 73.68, 80.37 μg/ml respectively. In the same way BRLE, BRSE, BRFRE, BRFLE (at concentration 100 μg/ml) exhibited 97.63, 92.79, 82.17, 92.71 % of α-glucosidase inhibition with an IC50 value of 26.97, 28.53, 41.30, 38.80 μg/ml respectively. Among the samples, the leaf extract of B. rubra registered higher content of total phenolics and flavonoids and also higher antioxidant activity in DPPH, nitric oxide and NBT radical scavenging assays. Though all the parts had shown potent inhibitory effects on α-amylase and α-glucosidase, the highest inhibitory potency was observed in the leaf extract of Basella rubra (p<0.001).


INTRODUCTION
Diabetic Mellitus (DM) is a disease which is caused due to the metabolic disorder of carbohydrate metabolism.The management of DM was mostly carried out by the treatment of oral hypoglycemic agents or antihyperglycemic agents and insulin.However due to their undesired effect the use of medicinal plants in the management of DM gained considerable interest.Thus the management of DM can be effected by inhibiting pancreatic enzymes like α-amylase and α-glucosidase which is responsible for the hydrolysis of carbohydrate and thereby causing postprandial hyperglycemia.Basella rubra (BR) belonging to the family Basellaceae is commonly called Malabar spinach and is native to the East Indies.It is a vigorous, climbing tropical vine that may be grown as leafy vegetable for its edible spinach-like stems and leaves or as an ornamental foliage vine.Leaves and stems are a good source of Vitamins A and C, calcium and iron.Basella rubra has been reported to have anti-microbial, 1 larvicidal, 2 hypoglycemic, 3 anti-ulcer, 4 analgesic, anti-pyretic, diuretic, 5 and anti-oxidant properties. 6Phytochemical investigation of this plant yielded different phytoconstituents like cardiac glycosides, saponins, tannins, flavonoids, terpenoids, carbohydrates, reducing sugars and basellasaponins A, B, C, and D. 7 The hypoglycemic effect of Basella rubra in streptpzotocin induced diabetic albino rats was reported. 8tural inhibitors of carbohydrate enzymes from plant sources put forth an attractive strategy for the control of postprandial hyperglycemia.This effort has been taken to investigate the α-amylase and α-glucosidase inhibitory activity of the leaf, stem, flower and fruit extracts of Basella rubra in the management of diabetes mellitus.

Collection of Plant Material
The plant material was collected from Hudco Colony, Peelamedu, Coimbatore and authentified by Mr. MUR-THY G.V.S, Scientist, Botanical Survey of India, Tamilnadu Agriculture University Campus, Coimbatore.A voucher specimen was prepared in the research laboratory and the voucher with no.PSGCP/DPC/03, is maintained for further reference.

Preparation of plant extracts (Leaf, Stem, Fruit and Flower)
The shade dried parts of the plant were powdered and subjected to defatting with petroleum ether.The marc is then subjected to cold maceration with 70% Hydroalcohol for 48 hr.Finally all the extracts were filtered and concentrated under reduced pressure to get various plant extracts namely Basella rubra Leaf Extract (BRLE), Basella rubra Stem Extract (BRSE), Basella rubra Flower Extract (BRFLE) and Basella rubra Fruit Extract (BRFRE).flavonoids, proteins, alkaloids, glycosides, saponins, tannins, volatile oils and mucilage. 9

Estimation of Total Phenols
Total phenolic content was determined in all the extracts by Folin-Ciocalteu method.This test is based on the oxidation of phenolic groups with phosphomolybdic and phosphotungstic acids.After oxidation, a green-blue complex is formed, which is measured at 750 nm.The total phenol content of a tested material can be related to the antioxidant activity shown by it. 10

Estimation of Total Flavonoids
Flavonoids present in the extracts were estimated by their characteristic absorption in the UV region and by their specific reaction with aluminium chloride and potassium acetate. 11

Antioxidant Assay
Antioxidant potential of different parts of Basella rubra was assessed by DPPH method, 12 Nitric oxide method 13 and Nitro blue tetrazolium method. 14

Alpha Amylase Inhibitory Assay
In vitro α-Amylase Inhibitory activity of alcoholic extract of various plant parts (leaf, stem, fruit, flower) of BR was carried out by using Kazeem et al 2013 method. 15In this assay various concentrations (20, 40, 60, 80, 100 μl) of different plant parts of alcoholic extract of BR was allowed to react with 250 μl of 0.02 m sodium phosphate buffer (pH 6.9) which contains α-amylase solution (0.5 mg/ml) (Hi media Rm 638 -α-Amylase from fungi).The content of the tubes was pre incubated at 25°C for 10 min after which 250 μl of 1% starch solution was added in 0.02 M sodium phosphate buffer (pH 6.9).The reaction mixture was incubated at 25°C for 10 min.The reaction was terminated by adding 500 μl of dinitro salicylic acid (DNS) reagent and further incubated in boiling water bath for 5 min and cooled to room temperature.The content of each test tube was diluted up to 5ml with distilled water and the absorbance was measured at 540 nm by Spectrophotometer.The reaction system without plant extracts was used as control and the system without α-amylase was used as blank for correcting the background absorbance.The percentage inhibition of α-amylase enzyme was calculated using the following formula.

Alpha Glucosidase Inhibitory Assay
In vitro α-Glucosidase Inhibitory activity of hydroalcoholic extract of various plant parts (leaf, stem, fruit, and flower) of BR was carried out by using Kazeem et al 2013 method.In this assay various concentrations (20, 40, 60, 80, 100 μl) of different plant parts of alcoholic extract of BR was allowed to react with 100 μl of α-Glucosidase (RM7067 α-Glucosidase from Saccharomyces species) (1 Unit/ml) in 20 mM phosphate buffer (pH 6.9).The content of the tubes was pre incubated at 25°C for 10 min.50 μL of p-nitropheynyl glucopyranoside was added to start the reaction.The reaction mixture was incubated at 25°C for 20 min.The reaction was terminated by adding 2 ml of 0.1 M sodium carbonate solution and finally made up to 5ml with distilled water.Then enzyme inhibition was (absorbance) measured at 405 nm by Spectrophotometer.The reaction system without plant extracts was used as control and the system without α-glucosidase was used as blank for correcting the background absorbance.The percentage inhibition of α-glucosidase enzyme was calculated using the following formula.

Statistical analysis
Statistical analysis was performed using GraphPad Prism statistical package (GraphPad Software, USA).The data were analyzed by One-way Analysis of Variance (ANOVA) method followed by Tukey's multiple comparison.The results were considered to be statistically significant when the P<0.05.All the results were expressed as mean ± SD for triplicate determinations.

Secondary metabolite
Estimation of the secondary metabolites showed significant content of total phenolic (25.9 GAE mg/g) and total flavonoid content (4.27 RE mg/g) in BRLE (Table 2).

Antioxidant potential
Four extracts namely BRLE, BRSE, BRFRE, BRFLE were subjected to in vitro antioxidant activity.Among the four extracts evaluated, hydro alcoholic extract of BRLE showed potent antioxidant property with the minimum IC 50 values in the scavenging of DPPH, Nitric acid and NBT assay.All the results were comparable with the standard.The IC 50 values of hydro alcoholic extract of BRLE were found to be 10.98, 62.5 and 48.24 against DPPH, Nitric oxide and NBT methods respectively (Table 3).

CONCLUSION
It is concluded by the study that the percentage inhibition of leaf extract of Basella rubra is more than that of the extracts of stem, flower and fruit.
The IC 50 value of leaf extract is less than that of stem, fruit and flower extracts of Basella rubra and hence it shows high alpha amylase and high alpha glucosidase inhibitory action.This activity may be due to the significant antioxidant property which may be due to high levels of phenolic and flavonoid content.However, further study is needed to isolate the active principle(s) in this plant which is responsible for this activity.

DISCUSSION
Diabetes mellitus is a common metabolic disorder that increases the postprandial glucose level which may lead to multiple organ damage and increase the risk of cardiovascular diseases which are the most common causes of death among people with diabetes.Management of blood glucose level is critical in the early treatment of diabetes mellitus and its complication.Sharp rise in the blood glucose level after food intake is aided by α-Amylase and α-Glucosidase enzymes, which break the carbohydrate to simple absorbable sugars. 16Synthetic enzyme inhibitors such as acarbose, voglibose, miglitol, are useful as oral hypoglycemic drugs for the control of post prandial hyperglycemia especially in type II diabetes patients. 17These inhibitors delay carbohydrate digestion and prolong the time taken for glucose absorption in intestine. 18Chronic usage of these inhibitors in conjunction with other antidiabetic drugs leads to GI side effects like abdominal discomfort, flatulence and diarrhea. 19everal plants have been identified as potential source of drugs in Indian system of medicine for the treatment of diabetes.Phenolic compounds, whose formation is associated with the normal metabolism of aerobic cells can protect the human body from free radicals.Such compounds are strong in antioxidants and can remove free radicals, chelate metal catalysts, activate antioxidant enzymes, reduce α-tocophenol radicals and inhibit oxidases. 20Derivatives of flavonoids have been found in many fruits and vegetables.Further, numerous studies have shown that majority of the antioxidant activities maybe from compounds such as flavonoids, isoflavones, flavones, anthocyanins, catechin and isocatechin rather than from vitamins C, E and β-carotene. 21Flavonoids have antioxidant activity and could therefore lower cellular oxidative stress. 22n the present study, BRLE showed a significant (p<0.001)inhibition of α-amylase and α-glucosidase activity at all concentrations tested and hence it can be used for the management of diabetes mellitus.
absorbance = Sample absorbance -Blank absorbance The concentration of extract resulting in 50% inhibition of enzyme activity (IC 50 ) was determined graphically using Microsoft excel.

Table 4 : Alpha Amylase Inhibitory Assay of various extracts of Basella rubra
Data are mean ± SD or % ± SD for triplicate measurements.