In Vitro Antidiabetic activity of Ethanol and Aqueous leaf extracts of Azima tetracantha Lam
B. Edwin Jose1, Dr. P. Panneerselvam2
1Department of Pharmaceutical Chemistry, SSM College of Pharmacy, Erode.
2Department of Pharmaceutical Chemistry, Bharath Institute of Higher Education and Research, Chennai
*Corresponding Author E-mail: meeraharsa23@gmail.com
Abstract:
The present investigation includes the preliminary screening of phytochemicals and evaluation of an in vitro antidiabetic activity of Azima tetracantha LAM extracts. Plant material was subjected to the extract preparation by soxhlet apparatus by using various solvents such as aqueous, ethanol, chloroform, petroleum ether. The various kinds of phytochemicals were detected and then in vitro antidiabetic activity of Azima tetracantha LAM ethanol and aqueous extracts were detected by using alpha amylase and alpha glucosidase enzyme in an in vitro model. The study reveals the presence of phytochemicals such as carbohydrate, tannin, flavonoids, phenols etc., Among the various extracts the aqueous and ethanolic extracts which contains the large number of phytoconstituents. The Azima tetracantha LAM extracts has been successfully inhibited both the enzymes in an in vitro model. The aqueous extracts of Azima tetracantha LAM inhibited the alpha amylase and alpha glucosidase enzymes as 48.59% and 64.99% respectively. The ethanolic extract of Azima tetracantha LAM inhibited the alpha amylase and alpha glucosidase enzymes as 65.34% and 58.48% respectively. From the results obtained in the current studies, the Azima tetracantha LAM extracts have a prominent antidiabetic property in an in vitro model and further studies can be carried out in an in vivo model and the isolation of active compound from Azima tetracantha LAM extract is needed.
KEY WORDS: Alpha amylase, Alpha glucosidase, Azima tetracantha LAM, Phytochemical.
INTRODUCTION:
Diabetes mellitus (DM) is an endocrine metabolic disorder of carbohydrate, fat, and protein that results from the defective insulin action or secretion and leads to hyperglycemia, abnormalities of lipoprotein, a defect in enzymes, damage to pancreatic beta cells induced through oxidative stress1. The micro and macro vascular complications, cerebrovascular diseases2,3 neuropathy, nephropathy and cardiovascular degeneration are the major complications associated with diabetes4,5. World Health Organization reported that over 150 million people all around the world were suffering from DM by the year 2000, and in the year 2010 the number had risen to 221 million and by the year 2025 around 300 million people will be suffering from diabetes. In urban areas, about 172 million people are diabetic, while in rural areas 119 million people are suffering from diabetes. By the year 2030, the expected difference is still to rise to 314 million people living in urban areas and 143 million in rural areas6.
DM is characterized by elevated blood glucose levels. Type I and II are generally two types of DM. Type I diabetes is insulin-dependent that resulted from less amount of insulin production due to a defect in insulin gene7. In type II diabetes there is an imbalance between absorption of blood sugar and secretion of insulin. Type II DM is also developed due to postprandial hyperglycemia. The postprandial rise in blood glucose level can be decreased by inhibiting the enzymes that hydrolyze dietary carbohydrates. α-Amylase is secreted by the pancreas and the salivary glands. Starch gets converted into alpha-limit dextrins, maltose and maltotriose by cleavage of α-D-1, 4 glucosidic linkages by α-amylase action8-10. The enzyme α-glucosidase present in the epithelial mucosa of the small intestine digests carbohydrates by hydrolyzing polysaccharide to monosaccharide which is absorbed through the intestinal tract11,12. Inhibition of α-amylase and α-glucosidase enzymes can slow uptake of dietary carbohydrates and suppress postprandial hyperglycemia. Gastrointestinal disorders such as bloating, visceral discomfort, diarrhoea, and flatulence are the common side effects caused by synthetic enzyme inhibitors, therefore, are less efficacious13.
Azima tetracantha (Salvadoraceae) is a well known medicinal herb, termed ‘Mulsangu’ in Tamil and 'Kundali' in Sanskrit. Root, root bark and leaves of Azima tetracantha (lam) are used with food as a remedy for rheumatism, diuretic and as stimulant14. Traditionally Indian medical practitioners use Azima tetracantha (lam) in inflammatory conditions, cough, asthma, small pox and diarrhoea15,16. The major phyto-constituents reported in Azima tetracantha (lam) are azimine, azecarpin, carpine, isorhamnitine-3-O-rutinoside, friedelin, lupeol, glutinol and β-sitosterol17,18. Azima tetracantha (lam) is reported to have antifungal19 antitumour20, antidiabetic21, antidiarrhoeal22 and hepatoprotective activities.
Azima tetracantha (lam) is a low, spinouts, highly branched bush, woody below but with pale green, herbaceous, almost quadrangular young branches. The leaves are in opposite to sub-opposite, decussate pairs. They are shortly petiolate, about 2x4cm long, entire, elliptic, acute, sharply mucronate, rigid, pale green with an acute base. Usually, there are two laterally placed spines in the axil of a leaf. The spines which morphologically represent the first pair of leaves of the auxiliary shoot are about three cm long, more or less, triangular in cross section, very sharp and with an indurate apex. The plant is dioeciously. The flowers are borne in the axils of leaves. Generally, there is cymes of three flowers in the axil of a leaf which is the upper branches, especially of the male plants become greatly reduced or even completely suppressed.
Uses:
The plant is used in indigenous medicines for rheumatism, microbial infections, diahorrea, inflammatory conditions, reduce lipid and as hepato-protective.
MATERIALS AND METHODS:
Collection of plants:
The aerial part (leaves) of Azima tetracantha (Lam) was collected from the Panayur area of Madurai, Tamilnadu as raw material, during the second week of February 2015 and a voucher specimen is stored in C.L. Baid Mehta College of Pharmacy (001/ATL/CLBP) and the plant material was authenticated by a renowned botanist. About 500 g of coarse powdered leaf in 2.5 L water is boiled, cooled and filtered. The filtrate is evaporated to dryness in desiccator and stored in refrigerator (Yield- 26.5% w/w). The aqueous extract of Azima tetracantha (lam) (AEAT) was subjected to preliminary phytochemical analysis23
Various extraction methods for isolation of constituents:
The whole plant will be subjected to shade drying and extraction with petroleum ether (60-80oC) chloroform, Ethyl acetate and 80% ethanol in soxhlet apparatus by simultaneous extraction each for 72 hours. Concentrate the solvents in vacuum. The crude solid obtained on evaporation are to be studied for preliminary qualitative phytochemical evaluation.
Phytochemical Screening:
The extract was subjected to phytochemical analysis to test the presence of carbohydrates, glycosides, alkaloids, flavonoids, tannins, sterols, and saponins in leaf extracts.
Pharmacological screening:
In vitro anti diabetic activity:
Alpha-amylase inhibition assay24,25:
A starch solution (0.1% w/v) was obtained by stirring 0.1g of potato starch in 100 ml of 16 mM of sodium acetate buffer. The enzyme solution was prepared by mixing 27.5 mg of alpha-amylase in 100 ml of distilled water. The colorimetric reagent is prepared by mixing sodium potassium tartarate solution and 3, 5-di nitro salicylic acid solution 96 mM. The starch solution is added to the both control and plants extract tubes and left to react with alpha-amylase solution under alkaline conditions at 25oC. The reaction was measured over 3 minutes. The generation of maltose was quantified by the reduction of 3, 5-dinitro salicylic acid to 3-amino-5-nitro salicylic acid. This reaction is detectable at 540 nm.
Preparations of test and standard solutions:
The Ethanol extract of Azima tetracantha (EEAT) and aqueous extract of Azima tetracantha (AEAT) and the standard Acarbose (20 mg each) were separately dissolved in 20ml of freshly prepared 5 % distilled DMSO. These solutions were serially diluted with freshly prepared distilled DMSO to obtain the lower dilutions.
Procedure:
The different concentrations (5-1000 micro gram/ ml) of EEAT and AECG, standard Acarbose were prepared 5% DMSO. 500 micro litre of test/standard was added to 500 micro ml of a-amylase (0.5 mg/ml) and was incubated for 10 mins at room temperature. Then added 500 micro ml of 1% l starch solution and incubated for another 10mins. After that 1ml of the 3, 5-dinitrosalicylicacid as a coloring reagent was added to the reaction mixture and heated in a boiling water bath for 5 min. After cooling, it was diluted with 10ml of distilled water. The absorbance was then measured at 540 nm against the reagent blank. The a-amylase inhibition was expressed as percentage of inhibition and the IC50 values determined by linear regression plots with varying concentration of fraction against percentage inhibition. The percentage inhibition was calculated using the formula given below.
% Inhibition =[ (100- Abs. of sample – Abs. of blank) X 100] Abs blank
Alpha-glucosidase Inhibition assay26:
The inhibitory activity was determined by incubating a solution of starch substrate (2% w/v maltose or sucrose) 1 ml with 0.2 M Tris buffer of pH 8.0 and various concentrations of plant extract for 5 min at 37°C. The reaction was initiated by adding 1 ml of alpha-glucosidase enzyme (1 U/ml) to it followed by incubation for 40 min at 35°C. Then the reaction was terminated by the addition of 2 ml of 6N HCl. Then the intensity of the colour was measured at 540 nm.
% inhibition =[ (100- Abs. of sample – Abs. of blank) X 100] Abs blank
Calculation of 50% Inhibitory Concentration (IC50):
The concentration of the plant extracts required to scavenge 50% of the radicals (IC50) was calculated by using the percentage scavenging activities at five different concentrations of the extract. Percentage inhibition (I %) was calculated by I %=(Ac-As)/Ac X 100, 8 where Ac is the absorbance of the control and As is the absorbance of the sample.
RESULTS:
IN VITRO ANTI DIABETIC ACTIVITY:
The study of in vitro anti diabetic activity has shown that ethanol and aqueous extracts. The results were expressed in terms of IC50, which is the concentration of the tested fraction to inhibit respective enzyme by 50 percentages. The ethanol extract of Azima tetracantha (EEAT) and aqueous extract of Azima tetracantha (AEAT), elicited a dose dependent inhibition of alpha amylase enzyme activity. The alpha amylase inhibitory effect of the EEAT and AEAT was studied at concentrations 10-100 micro gm/ml.
In vitro inhibition of α—amylase:
The Ethanol and aqueous extract of Azima tetracantha, elicited a dose dependent inhibition of a-amylase enzyme activity. The a-amylase inhibitory effects of EEAT was found to be ranging from 13.21 % to 65.34% when studied at concentration10-100 micro gm/ml. At the same concentration range the inhibitory effect of AEAT was found to be ranging from 10.76% to 48.59% whereas the effect of standard drug acarbose ranged from 20.25% to 74.12%.IC50 of EEAT was found to be 77.53 +or- 0.15 micro gm /ml, whereas AEAT showed at 123.5 +or-0.40 micro gm/ml. The IC50 of acarbose was found to be 55.00+or-0.25 micro gm/ml (Table 1)
Table: 1 In vitro - α- amylase inhibitory activity of extracts of Azima tetracantha
|
Test subtance |
Concentration (micro/ml) % inhibition |
IC50 |
|||||
|
10 |
20 |
40 |
60 |
80 |
100 |
||
|
Acarose (standard) |
20.25+or-0.02 |
26.75+or-0.05 |
38.17+or-0.05 |
45.42+or-0.03 |
58.24+or-0.02 |
74.12+or-0.05 |
55.00+or-0.25 |
|
EEAT |
13.21+or-0.04 |
19.90+or-0.05 |
30.09+or-0.05 |
38.21+or-0.02 |
46.38+or-0.05 |
65.34+or-0.03 |
77.53+or –0.15 |
|
AEAT |
10.76+or-0.03 |
15.13+or-0.04 |
24.71+or-0.04 |
28.90+or-0.05 |
39.42+or-0.05 |
48.59+or-0.02 |
123.5+or-0.40 |
In vitro inhibition of α-glucosidase:
The extract of EEAT and AEAT, elicited a dose dependent inhibition of a- glycosidase enzyme activity. The α-glucosidase inhibitory effect of the EEAT was found to be ranging from 10.14% to 58.48% when studied at concentrations 10-100 micro gram/ml. At the same concentration range the inhibitory effect of AEAT was found to be ranging from 17.65% to 64.99%. The IC50 of the EEAT was found to be 83.21+or- 0.28 micro gm/ ml, whereas AEAT showed at 95.88 +or – micro gram/ml. The IC50 of acarbose was found to be 66.64 + or – 0.51micro gm/ml. (Table 2)
Table: 2 In vitro α- gluocisidase inhibitory activity of extracts of Azima tetracantha
|
Test substance |
Concentrations (micro g/ml) inhibition |
IC50 |
|||||
|
10 |
20 |
40 |
60 |
80 |
100 |
||
|
Acarbose (standard) |
17.65+or–0.09 |
26.99+or – 0.08 |
35.18+or–0.05 |
42.84+or–0.06 |
53.73+or–0.06 |
64.99+or-0.09 |
66.64+or–0.25 |
|
EEAT |
10.14+or–0.03 |
19.48+or–0.03 |
32.24+or–0.05 |
36.11+or–0.04 |
48.62+or–0.07 |
58.48+or–0.05 |
83.21+or–0.15 |
|
AEAT |
9.23+or–0.05 |
17.10=or–0.07 |
26.21+or–0.07 |
35.84+or–0.06 |
44.32+or–0.06 |
53.67+or–0.09 |
95.88+or–0.40 |
DISCUSSION:
Globally the ratio of diabetic patients has increased considerably. New synthetic OHA agents, drugs derived from plants and moreover effective changes in diet plan are being designed by physicians all over the world for the treatment of DM. Several medicinal plants have the ability to reduce FBG level and in managing other complications related to diabetes27-29. Many phytoconstituents viz. carbohydrates, alkaloids, flavonoids, saponins, amino acids, steroids, peptides, terpenoids are plants derivatives. These phytoconstituents possess hypoglycemic, anti-hyperglycemic and glucose suppressive activities. The specific biochemical interaction is either by facilitating the release of insulin from pancreatic β-cells, hindering the absorption of glucose in the gut, invigorating glycogenesis in the liver and/ or increasing the utilization of glucose in the body 30-33.
Postprandial hyperglycemia is a major problem in diabetes mellitus and it can be controlled by inhibiting the enzymes α-amylase and alpha-glucosidase that hydrolyze dietary carbohydrates34-37.
The in vivo antidiabetic activity of ethanol and aqueous extracts of Azima tetracantha Lam was also evaluated. The extracts displayed a significant reduction in BGL of rats, thus revealing the antidiabetic nature of the phytocompounds. The results of the phyto compounds were compared to that of diabetic control and standard drug Acarbose and were statistically significant.
It is evident from the present work that the extracts of Azima tetracantha Lam has a potential in vitro antidiabetic activity, which is bioactivity of great relevance to diabetes mellitus complication therapy.
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Received on 11.10.2018 Modified on 30.10.2018 Accepted on 18.11.2018 ©A&V Publications All right reserved Research J. Science and Tech. 2019; 11(1):64-68. DOI: 10.5958/2349-2988.2019.00009.3 |
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