In vitro Alpha amylase and Alpha glucosidase enzyme inhibition of leaf extracts of Jatropha glandulifera Roxb.
Sathish M1*, Nandhini V1, Suresh R2
1Department of Pharmaceutical Chemistry, College of Pharmacy,
Madras Medical College, Chennai, Tamil Nadu, India.
2Greensmed Labs, Thoraipakkam, Chennai, Tamil Nadu, India.
*Corresponding Author E-mail: sathishpharmacy14@gmail.com
ABSTRACT:
Objective: To investigate the in vitro antidiabetic effect on the leaf extracts of Jatropha glandulifera Roxb. Methods: The Preliminary phytochemical screening was investigated on extracts using standard procedure. The in vitro α-amylase and α-glucosidase inhibition assay was carried out at different concentration such as 10, 20, 40, 80, 160, and 320µg/ml of extracts. The acarbose and voglibose were used as standards respectively. The absorbance was recorded by spectrophotometer. The percentage inhibition and IC50 value were calculated for both the assays. Results: The ethyl acetate extract of Jatropha glandulifera Roxb has shown good inhibition against α-amylase and α-glucosidase with an IC50 value of 21.6137 and 25.8257µg/ml respectively. The phytochemical investigation revealed that the ethyl acetate extract have various phytochemical constituents compared to other extracts like alkaloids, carbohydrates, saponins, Flavonoids and glycosides. These phytoconstitients may be responsible for observed anti diabetic activity. Conclusion: This study concludes that the ethyl acetate extract of Jatropha glandulifera Roxb exhibits better inhibitory activity against both α-amylase and α-glucosidase.
KEYWORDS: Antidiabetic, J.glandulifera Roxb., euphorbiaceae, in vitro, α-amylase, α-glucosidase.
INTRODUCTION:
Around 79% of diabetic adult found in the low and middle-income countries. IDF also estimated, the countries with the largest numbers of adults with diabetes are China, India and the United States of America in the year 20193. To control the diabetes, anti- hyperglycemic drugs are used by people but it causes various adverse effects on vital organs like heart, liver, kidney and brain etc4. Hence, there is an urgent need for alternative medicine to cure diabetes and their related complications. Many countries use herbal plants as medicine for treating diabetes5. Almost 70 % modern medicines in India are derived from natural products. The herbal products have been accepting a high demand for the diabetes management due to low cost, high availability and less side effects6.
The plant Jatropha glandulifera Roxb.(Tamil name: Kattamanaku) which belongs to Euphorbiaceae family widely found in the South Indian regions such as Tamil Nadu, Maharashtra and Kerala. The plant has greenish yellow flower and leaves are palmate, 3–5 lobed, margin serrate, serratures and stipules gland tipped7. The compounds such as Jatrophin, Jatropholone A, fraxetin, coumarino-lignan, 3,3- dimethylacrylylshikonin and acetylshikonin were isolated from this plant8,9. The leaf and roots extract of J. glandulifera showed in vitro antibacterial activity. In ayurveda and ethno-medicine it is reported for analgesic properties, inflammation, asthma and bronchitis10. Other related species of Jatropha reported on its bioactive compounds and their anticancer, antibacterial, antidiabetic activities11. Nevertheless, there are no previous studies reported on its antidiabetic activity, this current study was conducted to investigate the phytochemical screening and antidiabetic properties on leaf extracts of J.glandulifera Roxb using in vitro antidiabetic model such as α- amylase and α-glucosidase inhibition assay.
MATERIAL AND METHODS:
Collection of plant material:
The leaf of Jatropha glandulifera Roxb. was obtained from Sivanthipatti village, Tirunelveli district, Tamil Nadu in February 2020. It was identified and authenticated by V. Chelladurai, Former Research Officer-Botany, Central Council for Research in Ayurveda & Siddha, Government of India.
Figure 1 Plant of Jatropha glandulifera Roxb.12
Chemicals and Reagents:
All the chemicals and reagents were of AR grade and were procured from Sigma-Aldrich, Mumbai.
Preparation of plant extracts:
The fresh leaves of Jatropha glandulifera Roxb were dried under room temperature. Then the dried leaves were crushed and powdered by a mechanical grinder. The powdered leaves were passed through a 40-mesh sieve. Then it was extracted sequentially by hot continuous percolation method by Soxhlet apparatus, using different solvents such as petroleum ether (PE), chloroform (CH), ethyl acetate (EA) and ethanol (EOH). The extracts were collected and concentrated using a Rotary vacuum evaporator13.
Figure 2 Schematic representation of the study
Phytochemical screening of extracts:
All the extracts were investigated for the identification of phytoconstituents present in the leaves. The Preliminary qualitative test for the extracts was done by standard procedure14-17.
In vitro antidiabetic activity evaluation:
The petroleum ether, chloroform, ethyl acetate and ethanol extracts of J. glandulifera Roxb were evaluated for in vitro antidiabetic activity by α-amylase and α-glucosidase enzyme inhibition assay18.
α- amylase inhibition assay:
The amylase inhibitory effects of all extracts of J. glandulifera Roxb were evaluated by in vitro assay using acarbose as reference19. The α-amylase inhibitory activity of the test samples such as Pet ether (PE), chloroform (CH), ethyl acetate (EA) and ethanol (EOH) were carried out according to the standard method with minor modification20. 100µl of α-amylase solution (0.1 mg/ml) were mixed with different concentrations (10, 20, 40, 80, 160, and 320µg/ml) of test samples, standard (Acarbose), and control (without standard/test samples) and pre-incubated at 37°C for 15 min. Then, 100µl of starch solution was added to initiate reaction and incubation was done at 37°C for 60 min., then 10µl of 1 M HCl and 100µl of iodine reagent were added to the test tubes. The absorbance of the mixture was measured at 580nm. α-amylase inhibitory activity was measured using the formula:
% Inhibition = [(Abs Control - Abs Sample) / Abs Control] x 100
Where Abs Control = Absorbance of control; Abs Sample = Absorbance of test sample.
α- glucosidase inhibition assay:
The effect of the leaf extracts of J. glandulifera Roxb. on α-glucosidase inhibition activity was determined21. The substrate solution p-nitrophenyl glucopyranoside (pNPG) was prepared in 100mM phosphate buffer, and pH 6.8. 200µL of α-glucosidase was pre-incubated with different concentrations (10, 20, 40, 80,160 and 320) of the extracts for 10min. Then 400µL of 5.0mM (pNPG) as a substrate dissolved in 100mM phosphate buffer (pH 6.8) was then added to start the reaction. The reaction mixture was incubated at 37∘C for 20min and stopped by adding 1mL of Na2CO3 (0.1M). The yellow-colored reaction mixture, 4-nitrophenol, released from pNPG was measured at 405nm using UV-VIS spectrophotometer22. Voglibose was used as a positive control and the inhibitory activity of α-glucosidase was calculated using the following formula,
% Inhibition = [(Abs Control - Abs Sample) / Abs Control] x 100
where Abs Control = Absorbance of control; Abs Sample = Absorbance of test sample.
RESULTS:
Percentage yield of leaf extracts of Jatropha glandulifera Roxb.:
The appearance of concentrated and dried extracts of pet ether, chloroform, ethyl acetate and ethanol were observed and percentage yield was calculated for the extracts described in the table 1.
Table 1: Percentage yield and appearance of leaf extracts of J. glandulifera Roxb.
|
Extracts |
Appearance |
Percentage Yield (w/w) |
|
Pet ether |
Greenish brown |
8.5% |
|
Chloroform |
Dark green |
13..5% |
|
Ethyl acetate |
Yellowish green |
15.2% |
|
Ethanol |
Reddish brown |
20.5% |
Preliminary phytochemical screening:
As illustrated in the Table 2, Preliminary phytochemical screening revealed that the Pet ether extract consists of terpenoids, saponins and oils & fats. Chloroform extract showed the presence of alkaloids, carbohydrates, phytosterols, tannins, glycosides and oils & fats. Ethyl acetate extract showed the maximum number of constituents like alkaloids, carbohydrates, saponins, phytosterols, phenols, terpenoids, glycosides and flavonoids. The ethanol extract indicated the presence of alkaloids, tannins, phytosterols, flavonoids, glycosides and phenolic compounds.
Table 2: Preliminary phytochemical screening on leaf extracts of J. glandulifera Roxb.
|
Compounds |
PET Ether Extract |
Chloroform Extract |
Ethyl acetate extract |
Ethanol extract |
|
Alkaloids |
-VE |
+VE |
+VE |
+VE |
|
Carbohydrates |
-VE |
+VE |
+VE |
-VE |
|
Reducing sugars |
-VE |
-VE |
-VE |
-VE |
|
Saponins |
+VE |
-VE |
+VE |
-VE |
|
Phytosterols |
-VE |
+VE |
+VE |
+VE |
|
Phenols |
-VE |
-VE |
+VE |
+VE |
|
Tannins |
-VE |
+VE |
-VE |
+VE |
|
Flavonoids |
-VE |
-VE |
+VE |
+VE |
|
Proteins and Aminoacids |
-VE |
-VE |
-VE |
-VE |
|
Terpenoids |
+VE |
-VE |
+VE |
-VE |
|
Glycosides |
-VE |
+VE |
+VE |
+VE |
|
Fixed Oils and Fats |
+VE |
+VE |
-VE |
-VE |
Note: +VE- indicate the presence of phytoconstituent -VE- indicate the absence of phytoconstituent
Table 3: Effect of extracts of Jatropha glandulifera Roxb. on α- amylase inhibition assay
|
Conc.(µg/ml) |
Alpha Amylase Inhibition Assay (%) |
||||
|
Standard (Acarbose) |
PET Ether (PE) |
Chloroform (CH) |
Ethyl Acetate (EA) |
Ethanol (EOH) |
|
|
10 |
34.67439 |
28.15851 |
13.88653 |
22.19327 |
19.55896 |
|
20 |
67.78528 |
34.13670 |
47.37691 |
52.30676 |
34.3519 |
|
40 |
74.03322 |
51.14462 |
74.05007 |
75.36651 |
68.53974 |
|
80 |
81.27313 |
67.57815 |
79.53246 |
79.79938 |
77.37186 |
|
160 |
88.16091 |
77.78340 |
80.96336 |
86.45378 |
89.03698 |
|
320 |
91.78221 |
90.75897 |
91.29961 |
90.08118 |
90.76321 |
|
IC50 |
11.7293 |
36.6017 |
27.7014 |
21.6137 |
30.7750 |
α- amylase inhibition assay:
As described in the Table 3, all the extracts were subjected to α-amylase inhibition assay, it was observed that ethyl acetate extract (EA) has better α-amylase inhibition compared with other extracts with lowest IC50 value of 21.6137µg/ml. The IC50 value of standard Acarbose was found to be 11.7293µg/ml. The percentage inhibition of α-amylase of extracts and acarbose was represented in the Fig 3.
α- glucosidase inhibition assay:
As shown in the Table 4, all the extracts were evaluated for α-glucosidase inhibition assay, it was detected that ethyl acetate extract (EA) has better α-glucosidase inhibition compared with other extracts with minimum IC50 value of 25.8257µg/ml compared to other extracts. The IC50 value of standard Voglibose was found to be 10.6874µg/ml. The different concentration of dilutions of extracts and their percentage inhibition of α-glucosidase was represented in the Fig 4.
Figure 3 The percentage inhibition of α-amylase of extracts and std Acarbose (CH-chloroform extract, EOH- ethanol extract, EA- ethyl acetate extract and PE-pet ether extract)
Figure 4: The percentage inhibition of α-glucosidase of extracts and std Voglibose (CH-chloroform extract, EOH- ethanol extract, EA- ethyl acetate extract and PE-pet ether extract)
Table 4: Effect of extracts of Jatropha glandulifera Roxb. on α- glucosidase inhibition assay
|
Conc.(µg/ml) |
Alpha Glucosidase Inhibiton Assay (%) |
||||
|
Standard (Voglibose) |
PET Ether (PE) |
Chloroform (CH) |
Ethyl Acetate (EA) |
Ethanol (EOH) |
|
|
10 |
10.201042 |
14.594192 |
6.9992554 |
12.285927 |
14.445272 |
|
20 |
16.679077 |
36.783321 |
54.35592 |
58.600149 |
47.728965 |
|
40 |
69.918094 |
51.973194 |
63.067759 |
70.215934 |
58.302308 |
|
80 |
88.160834 |
61.131794 |
83.246463 |
74.013403 |
74.311243 |
|
160 |
92.256143 |
72.970961 |
85.927029 |
88.011914 |
78.927774 |
|
320 |
94.564408 |
79.076694 |
90.096798 |
92.107223 |
88.235294 |
|
IC50 |
10.6874 |
48.6008 |
29.8877 |
25.8257 |
33.5157 |
DISCUSSION:
The phytochemical investigation has revealed that the ethyl acetate extract on leaves of Jatropha glandulifera Roxb has maximum constituents than other extracts. These active constituents possess numerous pharmacological activities like antidiabetic, anticancer, antiatherosclerosis and other inflammatory diseases etc23.
The ethyl acetate extract of Jatropha glandulifera Roxb inhibited the activities of α-amylase and α-glucosidase with an IC50 value of 21.6137 and 25.8257 µg/ml respectively, suggesting the antidiabetic activity of the extract. Pancreatic α-amylase and intestinal α-glucosidase are two important carbohydrate digestive enzymes, which elevate the levels of postprandial blood glucose. The α-amylase begins the process of carbohydrate digestion by hydrolysis of 1,4 glycosidic linkages of polysaccharides (starch and glycogen) to disaccharides and α-glucosidase catalyse the disaccharides to monosaccharides which leads to post prandial Hyperglycemia. Hence the inhibitors of alpha amylase and alpha glucosidase are useful in the control of hyperglycemia as they delay carbohydrate digestion which consequently reduce postprandial plasma glucose level24. The Acarbose and Voglibose had strong inhibitory activities on α-amylase and α-glucosidase enzyme with IC50 value of 11.7293 and 10.6874 µg/ml respectively, but leads to undesired effects. Hence, the compounds from natural sources are essential to study the α-amylase and α-glucosidase enzyme inhibition activities for the control of postprandial Hyperglycemic state with minimal side effects25-27. Therefore, ethyl acetate extract shows good inhibition against both alpha amylase and alpha glucosidase compared to other extracts. Hence it possesses good antidiabetic activity.
CONCLUSION:
The leaves of Jatropha glandulifera Roxb. has the potent antidiabetic activity due to the presence of phytoconstituents. The ethyl acetate extract of J.glandulifera Roxb. exhibits promising role in antidiabetic activity by in vitro α-amylase and α-glucosidase inhibition assay. Further, the ethyl acetate extract will be subjected for isolation by column chromatography to detect the active constituents responsible for antidiabetic activity. Ethyl acetate extract will be subjected for in vivo anti diabetic activity.
ACKNOWLEDGEMENT:
The authors are grateful to the Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College, Chennai, for providing the facilities to carry out this research study.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 09.04.2021 Modified on 16.07.2021
Accepted on 03.09.2021 © RJPT All right reserved
Research J. Pharm. and Tech. 2022; 15(6):2493-2497.
DOI: 10.52711/0974-360X.2022.00416