In vitro Antioxidant activity of stem and leaves extracts of Hedera nepalensis K. Koch

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1 International Journal of Biosciences IJB ISSN: (Print), (Online) Vol. 7, No. 2, p , 2015 RESEARCH PAPER OPEN ACCESS In vitro Antioxidant activity of stem and leaves extracts of Hedera nepalensis K. Koch Muhammad Romman 1, Samin Jan 1, Muhammad Hamayun 2, Izhar Ahmad 1, Sher Wali 4* 1 Department of Botany, Islamia College Peshawar, Khyber Pakhtunkhwa, Pakistan 2 Department of Botany, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan 3 Department of Botany, Shaheed Benazir Bhutto University Sheringal, Dir Upper, Khyber Pakhtunkhwa, Pakistan Key words: Antioxidant, Hedera nepalensis, 1,1-diphenyl-2-picrylhydazyl, hydrogen peroxide. Article published on August 09, 2015 Abstract Two in vitro methods viz 1,1-diphenyl-2-picrylhydazyl free radical scavenging assay and hydrogen peroxide scavenging assay were used for the antioxidant activity of the Hedera nepalensis leaves and stem methanol, ethanol, ethyl acetate, dichloromethane, petroleum ether extracts. The 1, 1-diphenyl-2-picrylhydazyl scavenging activity was studied at the absorbance of 517nm. The absorbance was due the number of the electrons taken up. At increase in concentration absorbance was reduced gradually. Concentration of 0.1 mg/ml exhibited 42.72, 26.36, 46.36, and scavenging activity for leaves extracts respectively. Similarly concentration of 0.1 mg/ml exhibited 34.02, 45.13, 31, 53, and 61 scavenging activity for stem extracts respectively. However, DPPH scavenging activity of ascorbic acid at this concentration exhibited marked scavenging activity of and 76 respectively.the hydrogen peroxide scavenging activity was studied at the absorbance of 285nm with average time of 10 minutes incubationwas studiedfor leaves methanol, ethanol, ethyl acetate, dichloromethane, petroleum ether extracts. Concentration of 0.1 mg/ml exhibited 50, 54.54, 47.27, 60, and hydrogen peroxide scavenging activityfor leaves extracts respectively. Similarly concentration of 0.1 mg/ml exhibited 50, 77, 74, and 72 hydrogen peroxide scavenging activity for stem extractsrespectively. However, hydrogen peroxide scavenging activity of ascorbic acid at this concentration exhibited marked scavenging activity of and 85. The results conclude that the extracts of the H. neplalensis has the potential of antioxidant and be a natural source to treat pathological diseases. * Corresponding Author: Sher Wali sherwali@sbbu.edu.pk 19 Romman et al.

2 Introduction Reactive oxygen species Cascade of reactive oxygen species (ROS) in the mitochondria of human cell include such as superoxide (O2 ), hydroxyl (OH - ) and peroxyl (.OOH, ROO.) are the sources of oxidative stress (Knight, 1998; Burns et al. 2001). ROS in increased amount harm surrounding tissues either by powerful direct oxidizing action or indirectly with hydrogen peroxide (H2O2) and (OH) radicals formed from O2 - which initiates lipid peroxidation resulting in membrane destruction (Lewis, 1989).ROS from both endogenous and exogenous sources may be involved inthe etiologies of such diverse human diseases as arteriosclerosis, ischemic injury, cancer and neurodegenerative diseases, as well as in processes like inflammation and ageing (Halliwell and Gutteridge, 1998; Good et al., 1996; Gassen and Youdim, 1997). Human antioxidant enzymology Human cells can prevent itself by some of the mechanisms like superoxide dismutase, catalase, glutathione reductase, tocopherol and ascorbic acid (Niki, 1994). Antioxidant supplement of natural origin is plant possessing agents that can scavenge these reactive oxygen species and may be helpful in preventing various diseases (Sakat, 2010). Activities of H. nepalensis The extract of H. nepalensis is antitussive, antispasmodic and anti-inflammatory activity (Braudet, 1967). The triterpenoid saponins of H. helix indicated antifungal activity (Favel et al,.1994). Aims of the study H. nepalensis has been subjected for the evaluation of its antioxidant activity. The aim of the present study was to investigate the in vitro antioxidant activity of methanol, ethanol, ethyl acetate, DCM, petroleum ether extract of H. nepalensis. Material and methods Extract preparation The plant material collected in June from District Malakand was dried and thenmixed with methanolfor 5 days. The extract was filtered and the solution was then exposed to rotary evaporator and methanol was removed. Residue was again mixed with methanol for 5 days andthe extract was separated from plant residue and exposed to rotary evaporator. The remaining residue was placed at room temperature for further dryness. Fractionation Two hundred grams of the crude extract was dissolved in distilled water. This aqueous extract was then subjected to fractionation with different solvents. Various solvents likemethanol, Ethanol, Ethyl Acetate, Dichloromethane (DCM) and Petroleum ether wereused. The aqueous extract and organic solvents were poured into a separating funnel and were shake vigorously. The two layered were formed. In which one was that of aqueous layer and the other was that of organic layer. Both were collected in separate flasks. The process was repeatedfour times and the different solventsextracts were concentrated using Rotary Evaporator under reduced pressure at temperature C. Antioxidant assay Two in vitro methods viz DPPH free radical scavenging assay and hydrogen peroxide scavenging assaywere used for the antioxidant activity of the plant extracts. Dpph free radical scavenging activity The antioxidant activity was determined according to the protocol of Ahmad et al., The free radical scavenging activity of ethanol extracts of the plants were measured in terms of hydrogen donating or radical scavenging ability using the stable radical 1, 1- diphenyl-2-picrylhydazyl (DPPH) mg of DPPH was dissolved in 100 ml of ethanol to give 100µM solution. With 0.5 ml of extract solutionin ethanol (0.1g/100ml) was added to 1 ml of DPPH solution separately. These solution mixtures were kept in dark for 30 min (incubation period) at room temperature. After 30 minutes, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture 20 Romman et al.

3 indicated higher free radical scavenging activity. All tests were carried out in triplicate. Finally the radical scavenging activity was calculated as percentage of DPPH discoloration using the equation; % scavenging DPPH free radical = 100 (1-AE/AD). Where AE is absorbance of the solution, when extract has been added at a particular level and AD is the absorbance of the DPPH solution with nothing added (blank, without extract). All the tests and analysis were performed in triplicates and averaged. Hydrogen peroxidefree radical scavenging activity The methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts of H. nepalensis to scavenge hydrogen peroxide were subjected to spectrophotometer for determination at 285nm using the protocol of Beers and Sizer, Hydrogen peroxide solution of 2mM was prepared in phosphate buffered saline (PBS) at ph 7.4. Various concentrations of the test extract such as mg/ml, 0.01mg/ml, 0.02 mg/ml, 0.05 mg/ml, and 0.1mg/ml were added to hydrogen peroxide solution. The absorbance was measured through spectrophotometer and was taken after 10 minutes against blank solution containing test extract in phosphate buffered saline without hydrogen peroxide. All the tests and analysis were performed in triplicates and averaged. Results and discussion Dpph radical scavenging activity The DPPH scavenging activity was studied at the absorbance of 517nm. The absorbance was due the number of the electrons taken up. At increase in concentration absorbance was reduced gradually. The free radical scavenging activity of leaves methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts is given in the table 1. Table 1. DPPH radical scavenging activity of Ethyl acetate, petroleum ether, DCM, Ethanol and Methanol Leaves extracts of H. nepalensis and standard Ascorbic acid. Compound Concentration (mg/ml) Absorbance at 517nm Inhibition %age Control ± Et. ac. E ± Pet. Ether. E ± DCM.E ± Ethanol. E ± Methanol. E ± Ascorbic acid ± Each value represents the mean ± standard deviation of triplicate analysis. Table 2. DPPH radical scavenging activity of Ethyl acetate, petroleum ether, DCM, Ethanol and Methanol Stem extracts of H. nepalensis and standard Ascorbic acid. Compound Concentration (mg/ml) Absorbance at 517nm Inhibition %age Control ± Et. ac. E ± Pet. Ether. E ± DCM.E ± Ethanol. E ± Methanol. E ± Ascorbic acid ± Each value represents the mean + standard deviation of triplicate analysis. Concentration of 0.1 mg/ml exhibited 42.72, 26.36, 46.36, and scavenging activity respectively. While free radical scavenging activity of stem methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts is given in the table 2. Concentration of 0.1 mg/ml exhibited 34.02, 45.13, 31, 53, and 61 scavenging activity respectively. However, DPPH scavenging activity of ascorbic acid at this concentration exhibited marked scavenging activity and 76 respectively. 21 Romman et al.

4 Table 3. Hydrogen peroxide radical scavenging activity of Ethyl acetate, petroleum ether, DCM, Ethanol and Methanol Leaves extracts of H. nepalensis and standard Ascorbic acid. Compound Concentration (mg/ml) Absorbance at 285nm Inhibition %age Control ± Et. ac. E ± Et. ac. E ± Et. ac. E ± Et. ac. E ± Et. ac. E ± Pet. Ether. E ± Pet. Ether. E ± Pet. Ether. E ± Pet. Ether. E ± Pet. Ether. E ± DCM.E ± DCM.E ± DCM.E ± DCM.E ± DCM.E ± Ethanol. E ± Ethanol. E ± Ethanol. E ± Ethanol. E ± Ethanol. E ± Methanol. E ± Methanol. E ± Methanol. E ± Methanol. E ± Methanol. E ± Ascorbic acid ± Each value represents the mean + standard deviation of triplicate analysis. Table 4. Hydrogen peroxide radical scavenging activity of Ethyl acetate, petroleum ether, DCM, Ethanol and Methanol Stem extracts of H. nepalensis and standard Ascorbic acid. Compound Concentration (mg/ml) Absorbance at 285nm Inhibition %age Control ± Et. ac. E ± Et. ac. E ± Et. ac. E ± Et. ac. E ± Et. ac. E ± Pet. Ether. E ± Pet. Ether. E ± Pet. Ether. E ± Pet. Ether. E ± Pet. Ether. E ± DCM.E ± DCM.E ± DCM.E ± DCM.E ± DCM.E ± Ethanol. E ± Ethanol. E ± Ethanol. E ± Ethanol. E ± Ethanol. E ± Methanol.E ± Methanol.E ± Methanol. E ± Methanol. E ± Methanol. E ± Ascorbic acid ± Each value represents the mean + standard deviation of triplicate analysis. 22 Romman et al.

5 Fig. 1. DPPH Radical Scavenging Activity of H. nepalensis Leaves extracts. Fig. 4. Hydrogen peroxide Radical Scavenging Activity of H. nepalensis Stem extracts. The free radical scavenging activity of leaves methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts is given in the table 4. Concentration of 0.1 mg/ml exhibited 50, 77, 74, and 72 scavenging activity respectively. However, DPPH scavenging activity of ascorbic acid at this concentration exhibited marked scavenging activity and 85. Fig. 2. DPPH Radical Scavenging Activity of H. nepalensis Stem extracts. Hydrogen peroxide scavenging activity The hydrogen peroxide scavenging activity was studied at the absorbance of 285nm with average time of 10minutes incubation. The hydrogen peroxide radical scavenging activity of leaves methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts is given in the table 3. Concentration of 0.1 mg/ml exhibited 50, 54.54, 47.27, 60, and scavenging activity respectively. References Knight JA. Free radicals: their history and current status in aging and disease Annals of Clinical & Laboratory Science 28, Burns J, Gardner PT, Matthews D, Duthie GG, Lean ME, Crozier A Extraction of phenolics and changes in antioxidant activity of red wines during vinification. Journal of Agricultural and Food Chemistry 49, Lewis DA Anti-flamatory drugs from plant and marine sources. Birkhause Verlag, Basel. Halliwell B, Gutteridge JMC Free radical in biology and medicine, 3rd Edition Chapter 3. Oxford University Press, London. Fig. 3. Hydrogen peroxide Radical Scavenging Activity of H. nepalensis Leaves extracts. Pappolla MA, Chyan YJ, Omar RA, Hsiao K, Perry G, Smith MA, Bozner P Evidence of neuronal oxidative damage in Alzheimer's disease. The American Journal of Pathology 149(1), Romman et al.

6 Good PF, Werner P, Hsu A, Olanow CW, Perl DP Evaluation of neuronal oxidative damage in AlzheimerÕs disease. The American Journal of Pathology 149, Gassen M, Youdim MB The potential role of iron chelators in the treatment of Parkinson s disease and related neurological disorders. Pharmacology & Toxicology 80, Niki E, Shimaski H, Mino M Antioxidantism-free radical and biological defense. Gakkai Syuppn Center, Tokyo Sakat S, Archana RJ, Manoj G In vitro Antioxidant and Antiinflammatory Activity of methanolextract of Oxalis corniculata linn. International Journal of Pharmacy and Pharmaceutical Sciences 2(1), Braudet P Antitussive, Antispasmodic and Anti-inflamatory triterpenic extract from Hedera helix. Fr. M (Cl. A61K, (07g) 28 Oct Appl. 24 May, 1967, 6 p Favel A, Steinmetz MD, Regli P, Vidalollivier E, Elias R, Balansard G In Vitro Antifungal Activity of Triterpenoid Saponins. Planta Medica 60, Ahmad N, Fazal H, Abbasi BH, Farooq S Efficient free radical scavenging activity of Ginkgo biloba, Stevia rebaudiana and Parthenium hysterophorous leaves through DPPH (2, 2-diphenyl- 1-picrylhydrazyl). International Journal of Phytomedicine 2, Beers RF, Sizer IW Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry 195, Romman et al.

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