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Experimental studies on antioxidant properties of calotropis gigantea
1.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 168 EXPERIMENTAL STUDIES ON ANTIOXIDANT PROPERTIES OF CALOTROPIS GIGANTEA SOLVENT EXTRACTS David Mandepudi1,2 , Bharath Kumar Ravuru3 , Bhavani Mandepudi2 1 Faculty of Biotechnology, Jawaharlal Nehru Technological University-Hyderabad, Hyderabad-500085, Andhra Pradesh, India 2 Department of Biotechnology, School of Engineering, Sir Padampat Singhania University, Udaipur-313601, Rajasthan, India 3 School of Biotechnology, Vignan University, Guntur – 522213, Andhra Pradesh, India ABSTRACT The active metabolites from plant sources had been explored and extracted to serve the societies as part of the medicine either internally or externally for the treatment of ailments. In the similar lines, the Calotropis gigantea, has been investigated for antioxidant properties using ethanol, methanol, chloroform and aqueous solvent extracts of leaves, buds and flowers with the help of popular antioxidant testing methods of DPPH and hydrogen peroxide free radical scavenging activities and an additional reducing power test. In the results, many of the solvent extracts were reaching to variable saturation levels beyond the respective extract concentrations of 5µg/ml. Further, comparable free radical scavenging activities for some of the solvent extracts like, ethanol and methanol leaves, buds & flowers extracts in addition to methanol, chloroform and aqueous extracts of leaves, buds and flowers were observed with respect to free radical scavenging activities of the standard antioxidants, ascorbic acid and quercetin in the tests. However the reducing power of these solvent extracts of Calotropis gigantea leaves, buds and flowers were very low in comparison to that of the standard antioxidants, ascorbic acid and BHT. So, the observed free radical scavenging activities of the solvent extracts may be due to the presence of the detected active metabolites like, alkaloids, tannins, polyphenols etc. Though these results favor the use of Calotropisgigantea as part of the traditional medicine due to their auxiliary antioxidant properties, similar studies may be carried out for the better utilization and management of the Calotropis species. INTERNATIONAL JOURNAL OF ADVANCED RESEARCH IN ENGINEERING AND TECHNOLOGY (IJARET) ISSN 0976 - 6480 (Print) ISSN 0976 - 6499 (Online) Volume 4, Issue 4, May – June 2013, pp. 168-180 © IAEME: www.iaeme.com/ijaret.asp Journal Impact Factor (2013): 5.8376 (Calculated by GISI) www.jifactor.com IJARET © I A E M E
2.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 169 Keywords: Antioxidant, Ayurveda, drugs of abuse, Prodrug, Polyphenols, Calotropis gigantean 1. INTRODUCTION The molecular agents that prevent the oxidation of other molecules either by stopping the transfer of electron or hydrogen are known as the antioxidants. But then, these antioxidant molecules get themselves oxidized often acting as reducing agents [1]. While the oxidation and reduction reactions are very important set of reactions in biological systems, the soldier like antioxidants play a vital role in the sustenance of the life [2-3] on the planet, Earth. On the other hand, the reactive species and free radicals were known to cause severe damage to the cellular components of the life systems especially of human and may lead to various ailments like early ageing, oxidative stress related with Alzheimer's disease,Parkinson's disease,diabetes, rheumatoid arthritis, and neuro degeneration in motor neuron diseases [2,3,4-13]etc. Since, these oxidation and reduction reactions were found to have the greater significance in the realm of biological systems with antioxidant mechanism, plants and animals established complex systems of antioxidants like glutathione, vitamins – C, D, A, K & E; enzymes like catalase, superoxide dismutase, peroxidases, β-carotene, lutein, lycopene, vitamin A, vitamin C, vitamin E, flavonoids, polyphenols, ascorbic acid [14-17] etc. to regulate and check the unnecessary burst of any reactive species [11] like hydrogen peroxide (H2O2), hypochlorous acid (HClO) and free radicals like hydroxyl radical (. OH) and superoxide anion (O2 - ). Owing to these facts, synthetic antioxidants like butylatedhydroxytoluene (BHT) and buytlatedhydroxyanisole (BHA)were also developed or derived in the form of health supplements or drug molecules to serve the purpose by various pharmaceutical and health care companies [7,18]. However the natural antioxidants are the best means for the human consumption and in the lines of acceptability for the environment without the creation of any xenobiotic compounds [19] by the synthetic processes. So, these facts elucidated the need to search for the novel and bioactive compounds [20] like antioxidants directing the scientists to investigate the plant based solvent extracts to isolate and identify the antioxidants that may be used as dietary supplements [21-23] or the drugs that can save the life. In such scenario, many plants like, Azadiractaindica, Meliaazedarach, Digitalis purpurea, Digitalis lanata, Daturastramonium, Atropa belladonna, Papaversomniferum, Catharanthusroseus, Colchicum autumnale, erythroxylon coca etc. were investigated for potential active metabolites likeazadiractin, cardenolides, parasympatholytic agents, analgesics, anticancer agents, antigout agent, local anesthetics etc. for the use of medicine and food supplements [20,24]. In the similar lines, the Calotropis sp. known for its poisonous nature for centuries and for medicine in the traditional medical practices [25-27] of several ethnic groups has been investigated for the possible active metabolites using the residual solvent extracts of Calotropis gigantea leaves, buds and flowers. 2. MATERIALS AND METHODS 2.1 Plant material & solvent extraction The Calotropis gigantia leaves, flowers and buds were collected [28] and processed to obtain the respective dry powders and these dry powders were added to the solvents of ethanol, methanol and chloroform and extracted the active metabolites by solvent extraction
3.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 170 procedure and named them as the solvent extracts of ethanol leaves (EL), ethanol flowers (EF), ethanol buds (EB); methanol leaves (ML), methanol flowers (MF), methanol buds (MB); Chloroform leaves (CL), chloroform flowers (CF), chloroform buds (CB). The aqueous extracts were also prepared by boiling the plant material (powder) individually and processed [28] to obtain the final extracts of Aqueous leaves (AL), Aqueous flowers (AF) and Aqueous buds (AB). These extracts were stored in the refrigerator until use in sterile bottles. 2.2 Antioxidant Activity of solvent extracts The extracted solvent extracts of leaves, buds and flowers of Calotropis gigantea were tested for the detection and estimation of antioxidant activity in terms of free radical scavenging activity using DPPH [29-32] and Hydrogen peroxide [33-34] scavenging assay methods. 2.3 The DPPH free radical scavenging activity The DPPH solution (3ml) of 0.02mg/ml in methanol was taken in three sets of clean & dry test tubes and the solvent extracts of 1 ml were added respectively with an increasing concentration in the range of (0-25µg/ml) in multiples of 5µg/ml. Similarly a standard antioxidant, ascorbic acid of 1 ml was also added separately to the 3 ml DPPH solution in three sets of test tubes and used as a reference for the assay. All those test tubes with the solutions were vortexes and incubated at room temperature for 30 minutes under dark conditions. The absorbance of these resulting solutions were recorded at 517nm using UV- Visible double beam spectrophotometer. So the assay was performed in triplicates and the results were expressed as mean values and the standard derivations were also calculated. The DPPH free radical scavenging activity of the standard, ascorbic acid and the solvent extracts were calculated using the equation (1). (% ) DPPH Free Radical Scavenging Activity (DFRSA) = ሺ ೞିሻ ೞ X100 (1) Where As is the absorbance of the DPPH in the absence of test solution (solvent extract/ascorbic acid) and Ae is the absorbance of the test solution. 2.4 Hydrogen peroxide free radical scavenging activity Estimation of antioxidant activity of enzyme, catalase [35] was adopted to detect and evaluate the hydrogen peroxide scavenging activity of solvent extracts of Calotropis gigantia leaves, buds and flowers. The solvent extracts were taken as test solutions of 0.4ml with varying concentrations in multiples of 5µg/ml in the concentration range of (5-25) µg/ml separately in three sets of clean and dry test tubes and then added 0.6ml of 40mM hydrogen peroxide solution (in 0.1M phosphate buffer of pH 7.4) to each test sample and incubated them for 15 min under dark conditions. Similarly, a standard antioxidant solution of quercetin was prepared and used for the assay following the above standard procedure with varying concentration in multiples of 5µg/ml in the concentration range of (5-25)µg/ml. After the incubation period, 1ml of dichromate solution in acetic acid (5% potassium dichromate aqueous solution mixed with glacial acetic acid in the ratio of 1:3) was added to each solvent extract test solution and standard quercetin solution separately and mixed them well. These test solutions were further incubated in boiling water bath for 10min, where the characteristic green color appeared in the test tubes. 2ml of deionized water (MilliQ water) was added to each test solution and the resultant solution was used for the measurement of absorbance at
4.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 171 620nm using UV-Visible double beam spectrophotometer. The control sample was prepared without any extract or standard antioxidant, containing only deionized water and the hydrogen peroxide as well as dichromate solutions in parallel with test sample& standard samples of quercetin. Hence the test was performed in triplicates and the measured absorbance at 620nm for each sample was taken as mean value and used this data for further analysis. The (%) hydrogen peroxide free radical scavenging activity of solvent extracts & the standard, quercetin were calculated as per the equation (2). (% ) Hydrogen peroxide Free Radical Scavenging Activity (HFRSA) = ሺ ೃିሻ ೃ X 100(2) Where AR is the absorbance of control sample, and Ae is the absorbance of extract or standard at 620nm. 2.5 Reducing power assay The solvent extracts of Calotropis gigantea leaves, buds and flowers were evaluated [36], where 1ml of the variable concentration of residual solvent extracts (0-1 mg/ml) were mixed with 2.5ml of 0.2M phosphate buffer (pH 6.6) and 2.5ml of 1% potassium ferricyanide (K3Fe CN6) solution respectively and then incubated the resulting solutions at 50o C for 20 minutes. Added 2.5ml of 10% trichloroacitic acid to each reaction mixture in the test tubes and then centrifuged at 3000g for 10 min where the supernatant of 1ml was withdrawn carefully and mixed with 2.5ml of distilled water and further added 0.5ml of 0.1% ferric chloride solution to get the final solution. The absorbance of the resulting solution for each test sample was measured using UV-Visible double beam spectrophotometer at 700nm. The ascorbic acid and BHT were taken as the standard antioxidants for positive controls following the above procedure and the experiments were performed in triplicates. The mean absorbance for each test sample was calculated and used the data for further analysis. 3. RESULTS The free radical scavenging activity of Calotropis gigantea leaves, buds and flowers were calculated using DPPH and hydrogen peroxide methods as the (%) reduction in the absorbance and used them to plot the free radical scavenging activity profiles of Calotropis gigantea leaves extracts, buds extracts and flowers extracts against their respective concentrations as represented in the Figures 1-6. These profiles in the figures also include the standard or reference antioxidant (Ascorbic acid for DPPH method and quercetin for H2O2 Method) profile, providing a means for the comparison of the solvent extract antioxidant profiles of Calotropis gigantea leaves, buds and flowers. The saturation free radical scavenging activities and the corresponding Effective Dosage for the 50% free radical scavenging activities (ED50) were noted for all the solvent extracts and represented in the Figures 7 & 8 with respect to DPPH and hydrogen peroxide methods. Similarly the reducing power of the residual solvent extracts of Calotropis gigantea leaves, buds and flowers were represented as the absorbance profiles against their respective variable concentrations as shown in the Figures 9-11 along with the absorbance profiles of standards, ascorbic acid and BHT. The reducing power of the standards were so high that the ascorbic acid test results were diluted to 5% and the BHT results were diluted to 12% for the convenience. However the data represented in the profiles and the figures were taken as the average of the triplicates.
5.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May Fig.1 DPPH free radical scavenging activity p Fig.2 DPPH free radical scavenging activity p Fig.3 DPPH free radical scavenging activity p International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 172 DPPH free radical scavenging activity profiles of Calotropis gigantea leaves extracts DPPH free radical scavenging activity profiles of Calotropis gigantea buds extracts DPPH free radical scavenging activity profiles of Calotropis gigantea flowers extracts International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN June (2013), © IAEME leaves extracts buds extracts flowers extracts
6.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May Fig.4 Hydrogen peroxide free radical scavenging activity of Fig.5Hydrogen peroxide free radical scavenging activity of Fig.6 Hydrogen peroxide free radical scavenging activity of International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 173 Hydrogen peroxide free radical scavenging activity of Calotropis gigantea extracts Hydrogen peroxide free radical scavenging activity of Calotropis gigantea Hydrogen peroxide free radical scavenging activity of Calotropis gigantea extracts International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN June (2013), © IAEME Calotropis gigantea leaves buds extracts Calotropis gigantea flowers
7.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May Fig.7 Variations in the saturation of DFRSA of radicals and the corresponding ED50 concentrations Fig.8 Variations in the saturation of HFRSA of peroxide radicals and the corresponding ED50 concentrations Fig. 9 Comparative reducing power of standard antioxidants, ascorbic acid and BHT International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 174 Variations in the saturation of DFRSA of Calotropis gigantea extracts on DPPH radicals and the corresponding ED50 concentrations Variations in the saturation of HFRSA of Calotropis gigantea extracts on hydrogen peroxide radicals and the corresponding ED50 concentrations Comparative reducing power of Calotropis gigantea leaves extracts along with the standard antioxidants, ascorbic acid and BHT International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN June (2013), © IAEME extracts on DPPH extracts on hydrogen leaves extracts along with the
8.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May Fig. 10 Comparative reducing power of standard antioxidants, ascorbic acid Fig. 11 Comparative reducing power of standard antioxidants, ascorbic acid and BHT 4. DISCUSSION The free radical scavenging activity of many plant based extracts and the standard antioxidants like ascorbic acid and quercetin were dependent on their concentration [37 however such free radical scavenging antioxidant properties of extracts on free radicals like that of DPPH were found to be saturating (for concentrations beyond 5µg/ml) as shown in the Figures 1 follows. 4.1 Antioxidant properties of Calotropis gigantea The leaves extracts of Calotropis gigantea values of free radical scavenging activity by 5µg/ml concentration whereas the standard antioxidant, ascorbic acid followed a gradually increasing free radical the saturation level at around 15µg/ml concentration. Conversely, the quercetin, the other standard antioxidant was found (Figure 4) to reach the saturation levels of free radical International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 175 Comparative reducing power of Calotropis gigantea buds extracts along with the standard antioxidants, ascorbic acid (AA) and BHT Comparative reducing power of Calotropis gigantea flowers extracts along with the standard antioxidants, ascorbic acid and BHT The free radical scavenging activity of many plant based extracts and the standard antioxidants like ascorbic acid and quercetin were dependent on their concentration [37 however such free radical scavenging antioxidant properties of Calotropis gigant extracts on free radicals like that of DPPH were found to be saturating (for concentrations beyond 5µg/ml) as shown in the Figures 1-6. The details of the results were discussed as Calotropis giganteaLeaves extracts Calotropis gigantea (Figures 1 & 4) reached the saturation values of free radical scavenging activity by 5µg/ml concentration whereas the standard antioxidant, ascorbic acid followed a gradually increasing free radical scavenging activity to the saturation level at around 15µg/ml concentration. Conversely, the quercetin, the other standard antioxidant was found (Figure 4) to reach the saturation levels of free radical International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN June (2013), © IAEME buds extracts along with the flowers extracts along with the The free radical scavenging activity of many plant based extracts and the standard antioxidants like ascorbic acid and quercetin were dependent on their concentration [37-41], Calotropis gigantea solvent extracts on free radicals like that of DPPH were found to be saturating (for concentrations 6. The details of the results were discussed as (Figures 1 & 4) reached the saturation values of free radical scavenging activity by 5µg/ml concentration whereas the standard scavenging activity to the saturation level at around 15µg/ml concentration. Conversely, the quercetin, the other standard antioxidant was found (Figure 4) to reach the saturation levels of free radical
9.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 176 scavenging activity on hydrogen peroxide by 5µg/ml concentration, but then its saturation free radical scavenging activity was only of about half the free radical scavenging activity saturation values of ascorbic acid (Figure 1). Similarly the Calotropis gigantea leaves extracts recorded the higher (twice) free radical scavenging saturation activities in case of ethanol, methanol & chloroform leaves extracts on DPPHradicals compared to that of hydrogen peroxide radicals; whereas the aqueous leaves extracts exhibited higher free radical scavenging activity on hydrogen peroxide compared to that of DPPH radicals (Figures 1, 4, 7 & 8). These results were also supported by the increasing reducing power of Calotropis gigantea leaves extracts as shown in Figure 9 where the leaves extracts reducing power profiles against the concentration were gradually increasing but then these profiles were very low in reducing power in comparison to that of the standard antioxidants, ascorbic acid and BHT. The effective dosage for 50% saturating free radical scavenging activity (ED50) as shown in the Figures 7 and 8 for various Calotropis gigantea solvent extracts, brought out ethanol leaves extract with lowest ED50 concentration of 2µg/ml on DPPHfreeradicals and aqueous leaves extract with highest ED50 concentration of 4µg/ml on hydrogen peroxide free radicals. 4.2 Antioxidant properties of Calotropis giganteaBuds extracts The free radical scavenging activity of Calotropis gigantea buds extracts were shown in the Figures 2, 7 & 4, 8 with respect to DPPH and hydrogen peroxide free radicals where the methanol buds extract recorded higher percentage of free radical scavenging activity on DPPH radicals (80%) and the least ED50 concentration was found as 2.2µg/ml with respect to hydrogen peroxide radicals. However the ethanol buds extract has shown decreasing activity on DPPH radicals beyond 5 µg/ml concentration whereas its converse profile was observed on hydrogen peroxide radicals, otherwise all other buds extracts exhibited the saturating free radical scavenging activity for the respective concentrations (>5µg/ml). These results were also supported by the moderate and decreasing reducing power of the Calotropis gigantea solvent extracts of leaves, buds and flowers as shown in the Figure 10. 4.3 Antioxidant properties of Calotropis giganteaFlowers extracts The Calotropis gigantea flowers extract free radical scavenging activity with respect to variation in their respective concentrations in the solvents were shown in the figures 3 & 6 where ethanol flowers extract exhibited higher and lower scavenging activities on DPPH (76%) and hydrogen peroxide (15%) radicals respectively. On the other hand aqueous flowers extracts had the least scavenging activity on DPPH (10%) free radicals and the higher scavenging activity on hydrogen peroxide (41%) free radicals. Conversely, the chloroform flowers extracts recorded moderately saturating free radical scavenging activity on both DPPH and hydrogen peroxide free radicals whereas these chloroform flower extracts reducing power profiles were found to exhibit lowest saturating reducing powers as shown in the Figure 11. In another interesting finding, the methanol flowers extracts recorded increasing free radical scavenging activity on hydrogen peroxide free radicals beyond the concentration of 5 µg/ml. However the reducing power of methanol flowers extract was found to be decreasing with increasing concentration whereas the reverse effects were found in case of aqueous flowers extracts. These observed free radical scavenging activities of the solvent extracts of Calotropis gigantea leaves, buds and flowers may be due to the bioactive compounds like tannins, polyphenols and alkaloids [42-43] etc. present as the residual components in these solvent extracts.
10.
International Journal of
Advanced Research in Engineering and Technology (IJARET), ISSN 0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME 177 5. CONCLUSIONS The study has provided an evidence for the antioxidant free radical scavenging activities of Calotropis gigantea leaves, buds and flowers extracts of ethanol, methanol, chloroform and aqueous solvent extracts. These results also demonstrated the comparable free radical scavenging activities for some of the solvent extracts like, ethanol leaves extract, methanol leaves extract, ethanol buds extract, methanol buds extract, ethanol flowers extract and methanol flowers extract on DPPH radicals with that of the standard antioxidant, ascorbic acid. Similarly, methanol, chloroform and aqueous extracts of leaves, buds and flowers also exhibited the comparable free radical scavenging activity on hydrogen peroxide radicals with that of the standard antioxidant, quercetin. However the reducing power of these solvent extracts of Calotropis gigantea leaves, buds and flowers were very low in comparison to that of the standard antioxidants like ascorbic acid and BHT. So these results support the use of Calotropis as part of the traditional medicine to a considerable extent as these extracts recorded the moderate antibiotic and wound healing properties [44-48] that may aid as part of the treatment to terminate the pathogens and their effects on animals and humans. ACKNOWLEDGEMENT Dr. M Lakshmi Narasu, Professor & Dr. ArchanaGiri, Assistant Professor, Faculty of Biotechnology, Jawaharlal Nehru Technological University, Hyderabad, Dr. ArchanaGajbhiye, Head, Department of Biotechnology, Sir Padampat Singhania University, Udaipur were acknowledged for their suggestions and critical comments on the research work. Mr. Ashok Ghosh, President, Ms. Rinu Ghosh, Vice President, Prof. P.C. Deka, Vice Chancellor and Prof. AchintyaChaudary, Dean, School of Engineering of Sir Padampat Singhania University, Udaipur were also acknowledged and thanked for their financial support and approvals for the research work. REFERENCES [1] J Chaudiere, and R. Ferrari-Iliou, Intracellular antioxidants: From chemical to biochemical mechanisms, Food and Chemical Toxicology, 37, 1999, 949–962. [2] T Finkel, and N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing, Nature,408, 2000, 239–247. [3] M Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, and J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, The International Journal of Biochemistry and Cell Biology,39, 2007, 44–84. [4] Y Christen, Oxidative stress and Alzheimer disease, The Americal journal of clinical nutrition, 71, 2000, 621S–629S. [5] L.A del Río, L.M. Sandalio, J.M. Palma, P. Bueno, and F.J. Corpas, Metabolism of oxygen radicals in peroxisomes and cellular implications, Free Radical Biology & Medicine,13, 1992, 557–580. [6] D.C Hooper, G.S. Scott, A. Zborek, T. Mikheeva, R.B. Kean, H. Koprowski, and S.V. Spitsin, Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis,The FASEB Journal,14, 2000, 691–698.
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