Detection of Antioxidants in the Development of an Effective Polyherbal Combination in the Management of Diabetes

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1 South As. J. Biol. Sci. 1(2)2011:30-39 Research Article (ISSN ) Detection of Antioxidants in the Development of an Effective Polyherbal Combination in the Management of Diabetes *1 S.Vasugi, 2 A.S.Subhashini, 3 S.Balasubramanian and 4 D.Chamundeeswari 1 Department of Physiology, Sri Ramakrishna Dental College and Hospital, S.N.R. College Road, Avarampalayam, Coimbatore 2 Sri Ramachandra Medical College and RI, Sri Ramachandra University, Porur, Chennai 3 Department of Environmental Sciences, Bharathiar University, Coimbatore 4 Sri Ramachandra College of Pharmacy, Sri Ramachandra University, Porur, Chennai Abstract Diabetes Mellitus is known to involve oxidative stress. Many secondary plant metabolites have been shown to possess antioxidant activities, offering resistance against oxidative stress by scavenging the free radicals. The antidiabetic combination if it also possesses antioxidant property that would be more beneficial in the treatment of diabetes. The aim of the present study is to derive an efficient combination using the selected five plant extracts for diabetes mellitus with efficient antioxidant activity. The five plants selected were fruits of Momordica charantia (Cucurbitaceae), leaves of Aloe vera (Liliaceae), Annona squamosa (Annonaceae), Gymnema sylvestre (Asclepiadaceae) and the whole plant of Scoparia dulcis (Scrophulariaceae). Using the aqueous extracts of these five plants fifteen combinations were prepared. The antioxidant activities of the fifteen combinations were determined by 1, 1- diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay and Nitric oxide scavenging activity assay. Since no promising results were obtained, two modified combinations were arrived and were screened for antioxidant activity. The Inhibitory percentage and the IC 50 values for all the combinations were determined. Pearson regression analysis was done using SPSS statistical package for comparing the fifteen combinations and the modified combinations. The IC 50 and regression analysis of the modified polyherbal antidiabetic combinations (Momordica charantia, Aloe vera, Annona squamosa, Gymnema sylvestre and Scoparia dulcis in the ratio of 9: 8: 8: 9: 8 and 8: 7: 10: 10: 7) were found to have better antioxidant activity. Key words: Antidiabetic, Antioxidant, Regression coefficient analysis, DPPH radical scavenging assay, Nitric oxide scavenging activity * Corresponding author: 30 South As. J. Biol. Sci.1 (2): Vasugi et al.

2 Introduction Diabetes Mellitus (DM) is a metabolic disorder resulting from a defect in Insulin secretion, insulin action or both. Insulin Deficiency in turn leads to chronic hyperglycemia with disturbances in carbohydrate, fat and protein metabolism 1. Diabetes is one of the major causes of premature death worldwide. Every ten seconds a person dies from diabetes related causes mainly from cardiovascular complications 2. Free radicals induced by peroxidation have gained much importance because of their involvement in several pathological conditions such as cancer, diabetes, atherosclerosis, ischemia, liver disorder, neural disorder, etc 3. In diabetes mellitus, chronic hyperglycemia produces multiple biochemical sequelae, and diabetes-induced oxidative stress could play a role in the symptoms and progression of the disease 4. Several hypotheses have been put forth to explain the genesis of free radicals in diabetes. These include autoxidation processes of glucose, the non-enzymatic and progressive glycation of proteins with the consequently increased formation of glucose-derived advanced glycosylation end products (AGEs), and enhanced glucose flux through the polyol pathway 5. 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical was widely used as the model system to investigate the scavenging activities of several natural compounds. DPPH radical is scavenged by antioxidants through the donation of proton forming the reduced DPPH. This was one of the invitro antioxidant assay performed. Excess concentration of nitric oxide is associated with several diseases. Oxygen reacts with the excess nitric oxide to generate nitrite and peroxy nitrite anions, which act as free radicals 6. Antioxidants in the combinations show activity in competing with oxygen to react with nitric oxide and thus inhibit the generation of anions. This was the other invitro antioxidant assay performed. In this study we have selected five medicinal plants namely, fruits of Momordica charantia, leaves of Aloe vera, Annona squamosa, Gymnema sylvestre and the whole plant of Scoparia dulcis, all of them are known antidiabetic plants. Many of these plants are used in traditional ayurvedic medicine for the treatment of several diseases including diabetes. The objective of the present study was to investigate the antidiabetic efficacy of the combinations of these plants which was proved through invitro antidiabetic assays and further to determine their antioxidant activity. 31 South As. J. Biol. Sci.1 (2): Vasugi et al.

3 Materials and Methods Plant material Fruits of Momordica charantia (Cucurbitaceae), leaves of Aloe vera (Liliaceae), Annona squamosa (Annonaceae), Gymnema sylvestre (Asclepiadaceae) and the whole plant of Scoparia dulcis (Scrophulariaceae) were collected, identified and authenticated from the Botanical survey of India, Southern Regional center, Coimbatore (No.BSI /SC/5/23/09-10 /Tech 460). Extraction The medicinal parts of the plants except Momordica charantia were collected, shade dried and coarsely powdered. Momordica charantia was purchased from the local market at Coimbatore and authenticated. Aqueous extracts of the five plants were separately prepared by macerating the powder using distilled water with 1% chloroform for two days. The final extract obtained were filtered, concentrated and subjected to lyophilisation to obtain powdered extract which was stored separately for further use. Preparation of different combination Fifteen different combinations (C 1 to C 15 ) were prepared by varying the proportions of the five extracts as shown in Table 1. Each combination in five different concentrations (0.5, 1, 2, 4 and 8mg/ml) were assayed for antioxidant activity. Table1: Fifteen combinations with varying proportions of the five plants extract (C 1 C 15 ) Extract (mg) C 1 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 C 10 C 11 C 12 C 13 C 14 C 15 M.charantia A. vera A.squamosa G.sylvestre S. dulcis South As. J. Biol. Sci.1 (2): Vasugi et al.

4 In-vitro Antioxidant activity 1. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) assay Each extract of polyherbal combinations (0.1 ml) were added to 3ml of a 0.004% methanol solution of DPPH. Absorbance at 517nm was determined after 30 min. All the samples were assayed in triplicate and averaged Nitric oxide radical scavenging assay Various concentrations of each combination extract and sodium nitroprusside (10 mm) in phosphate buffer saline (0.025 M, ph 7.4) in a final volume of 3 ml were incubated at 25 o C for 150 min. Control experiments without the test compounds but with equivalent amount of buffer were prepared in the same manner as done for the test. Thereafter, 0.5 ml of incubation solution was removed and diluted with 0.5 ml Griess reagent (1% sulphanilamide, 2% O-Phosphoric acid and 0.1% naphthyethylene diamine dihydrochloride) and allowed to react for 30 min. The absorbance of the chromophore formed during diazotisation of nitrite with sulphanilamide and subsequent coupling with naphthylethylene diamine dihydrochloride was read at 546 nm. The percentage inhibition was calculated 8. Calculation of 50% Inhibitory Concentration (IC 50 ). Percentage inhibition (I%) was calculated using the formula, I% = (Ac As) 100 Ac where Ac is the absorbance of the control and As is the absorbance of the sample. After calculating the inhibitory percentage (I %) values, the concentration of extract required to cause 50% inhibition (IC 50 ) was determined using GraphPad Instat statistical software. Pearson regression coefficient analysis Regression analysis was done using SPSS statistical software to statistically compare and select two efficient combinations from the fifteen combinations. Results The percentage inhibitions showed by each combination in different concentrations with their IC50 values are shown in Table 3. The combinations possess significant antioxidant activity which was revealed through the DPPH radical scavenging activity and scavenging of Nitric oxide radical activity. Regression analysis values of the combinations at 2mg/ml concentration are shown in Table 4. A combination is considered to possess potent antioxidants if it possesses lower IC 50 and higher regression analysis values. Since no promising 33 South As. J. Biol. Sci.1 (2): Vasugi et al.

5 results were obtained, two modified polyherbal combinations MPHC 1 and MPHC 2 (shown in Table 2) were arrived and were screened for antioxidant activity. The Inhibitory percentage (I%), IC 50 and regression analysis were calculated and found to be better for these two combinations when compared with the fifteen combinations as shown in Table 3 and Table 4. Table 2: Modified polyherbal combinations MPHC 1 and MPHC 2 Extract MPHC 1 (mg) MPHC 2 (mg) M. charantia A. vera A. squamosa G. sylvestre S. dulcis Table 3: Percentage Inhibition of the Combinations with their Ic 50 Values Combinations C 1 C 2 C 3 C 4 Conc DPPH activity Scavenging of Nitric Oxide % Inhibition IC 50 % Inhibition IC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± South As. J. Biol. Sci.1 (2): Vasugi et al.

6 C 5 C 6 C 7 C 8 C 9 C 10 C 11 C 12 C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± South As. J. Biol. Sci.1 (2): Vasugi et al.

7 C 14 C 15 MPHC 1 MPHC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.68 All determinations were carried out in triplicates and the values are expressed as mean ± SEM. The IC 50 values of the combination MPHC 1 with DPPH radical scavenging activity and Nitric oxide radical scavenging activity were found to be 1.94 and 2.93 respectively. The IC 50 of the combination MPHC 2 with DPPH radical scavenging activity and Nitric oxide radical scavenging activity were found to be 1.28 and 2.74 respectively. The Regression analysis values were much higher for these two new combinations when compared to other polyherbal combinations in both the in-vitro assays. The regression analysis values of MPHC 1 at 2mg/ml concentration were found to be with DPPH radical scavenging activity and with Nitric oxide radical scavenging activity. The regression analysis values of MPHC2 at 2mg/ml concentration were with DPPH radical scavenging activity and with Nitric oxide radical scavenging activity. 36 South As. J. Biol. Sci.1 (2): Vasugi et al.

8 Table 4: Regression analysis AT 2mg/ml concentration Combinations Nitric Oxide Scavenging activity DPPH Radical Scavenging activity C C C C C C C C C C C C C C C MPHC MPHC Discussion Under physiological conditions, a widespread antioxidant defense system protects the body against the adverse effects of free radical production. The efficiency of this defense mechanism is altered in diabetes and, therefore, the ineffective scavenging of free radicals may play a crucial role in determining tissue damage 9. Plants often contain substantial amounts of antioxidants including alpha tocopherol (Vitamin E), carotenoids, ascorbic acid (Vitamin C), flavonoids and tannins which offer resistance against the oxidative stress by scavenging the free radicals, inhibiting the lipid peroxidation and by other mechanisms and thus prevent disease 10. And it has been suggested that antioxidant action may be an important property of plant medicines in diabetes. 37 South As. J. Biol. Sci.1 (2): Vasugi et al.

9 DPPH radical is scavenged by antioxidants through the donation of proton forming the reduced DPPH. The colour changes from purple to yellow after reduction, which can be quantified by its decrease of absorbance at the wavelength of 517nm. Radical scavenging activity increased with increasing percentage of free radical inhibition. From the present results, it may be postulated that the combinations reduced the radical to corresponding hydrazine when it reacts with hydrogen donors in antioxidant principles. Nitric oxide is a free radical generated by endothelial cell, macrophages, neurons etc, and involved in the regulation of various physiological process. In addition to reactive oxygen species, excess nitric oxide is also implicated in inflammation, cancer and other pathological condition 8. Oxygen reacts with the excess Nitric oxide to generate nitrite and peroxy nitrite anions, which act as free radicals. Plant extracts in all the combinations inhibited nitrite formation by competing with oxygen. Incubation of solution of sodium nitroprusside in PBS at 25 C for 150 min resulted in linear time dependent nitrite production, which is reduced by the extracts. Conclusion Based on the present studies exposed through 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) assay and Nitric oxide radical scavenging activity assay it can be concluded that modified polyherbal combinations MPHC 1 and MPHC 2 with Momordica charantia, Aloe vera, Annona squamosa, Gymnema sylvestre and Scoparia dulcis in the ratio of 9: 8: 8: 9: 8 and 8: 7: 10: 10: 7 respectively were found to be better antidiabetic combinations with potent antioxidants. Acknowledgement The authors are thankful to Dr.T.K.Ravi, Principal, Dr. M. Uma Maheshwari, Professor and Head of the Department of Pharmacology, Dr.M. Gandhimathi, Professor, Department of Pharmaceutical Analysis and Dr. R. Venkatasamy, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, Dr. K. Sumathi, Reader in biochemistry, Sri Ramakrishna Dental College and Hospital, Coimbatore, for their intense support to carry out this work. 38 South As. J. Biol. Sci.1 (2): Vasugi et al.

10 References 1. The Expert committee on the Diagnosis and classification of diabetes Mellitus (1997), Report of the expert committee on the Diagnosis and classification of Diabetes Mellitus. Diabetes care 20, Das, A.K., Rai, A world without diabetes and its complications: A preventive program. In: Jayaram BM (Ed.). Type 2 Diabetes and its complications: A preventive program. Microlabs Limited: Bangalore. pp Marx, J. L., Oxygen free radicals linked to many diseases. Science, Giugliano, D., Ceriello, Paolisso, G., Oxidative stress and diabetic vascular complications. Diabetes Care, 19, Oberley, L.W., Free radicals and diabetes. Free Radic. Biol. Med, 5, Sainani, G.S., Manika, J.S., Sainani, R.G., Oxidative stress: a key factor in pathogenesis of chronic diseases, Med Uptake, 1, Braca, A., Tommasi, N.D., Bari, L.D., Pizza, C., Politi, M., Morelli, I., Antioxidant principles from Bauhinia terapotensis. J. Nat. Prod. 64: Sreejayan, Rao, N.A., Nitric oxide scavenging by Curcuminoids. Pharm. Pharmacology 49: Wohaieb, S.A., Godin, D.V., Alterations in free radical tissue defense mechanism in streptozotocin-induced diabetes in rats. Effects of insulin treatment. Diabetes, 36, Youdim, K. A., Joseph, J. A., A possible emerging role of phytochemicals in improving age-related neurological dysfunction a multiplicity of effects. Free Rad Biol Med, 30, South As. J. Biol. Sci.1 (2): Vasugi et al.

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