International Journal of PharmTech Research CODEN (USA): IJPRIF ISSN : 0974-4304 Vol.2, No.4, pp 2219-2223, Oct-Dec 2010 Evaluation of antioxidant and invitro cytotoxicity of crude fractions from the roots of Xanthiuim Strumarium S. Ishwarya 1, Mukesh Kumar Singh 2 * 1 Department of Pharmaceutical Biotechnology, S R M College of Pharmacy, SRM University, Kattankulathur 603203. Chennai (T.N.), India. 2 Rungta College of Pharmaceutical Science and Research Kohka road, Kurud, Bhilai-491024 (C.G.) India *Corres. author: mukeshbiotech09@gmail.com Phone No: O9691699320 Abstract: The present study was aim to explore the possible biological activity of the medicinal herb, xanthium strumarium. The chloroform and methanolic extracts of the plant were screened for the antimicrobial, antioxidant and cytotoxicity activity. The extracts were evaluated for their free radical scavenging activity by DPPH method and IC 50 value showed extracts having good free radical scanvenging activity. The invitro antiproliferative activity of crude extract from the roots xanthium strumarium. It showed potent cytotoxic activity against the HEP-2 cell lines. The plant extract showed a better antimicrobial activity against the gram positive and gram negative microorganism. Key words: Xanthiuim strumarium, DPPH method, Anti microbial activity, Cytotoxic activity, Thymidine assay, HEP-2 cell lines. Introduction: Xanthium Strumarium belongs to the family Asteraceae which is an important indigenous medicinal plant for treatment of Diaphoretic, Sedative, Sudorific, Sialogogue and it is considered useful in that treatment of long standing malaria. Roots are useful in strumous diseases and cancer. Fruits were reported to posses cooling and demulcent properties when given in small pox. 1. Antioxidant compound in food play an important role as a health protecting factor. Scientific evidence suggests that antioxidants reduce the risk for chronic diseases including cancer and heart disease. Antioxidants help to neutralize free radical, which are unstable molecules that are linked to the development of a number of degenerative diseases and condition including cancer, cardiovascular disease, cognitive impairment immune dysfunction, cataract and macular degeneration. 2,3 Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide and hydroxyl radical are closely involved in human diseases such as Alzheimers disease, aging, cancer, inflammation, rheumatoid, arthritis, and atherosclerosis. 4,5 Literature showed that there were no reports are available exclusively on antioxidant properties and cytotoxicity activity of the roots of this medicinal herb. Several analytical method have been proposed for determining the total antioxidant activity of biological extracts in order to evaluate the total antioxidant capacity of biological samples. The objective of this work was to investigate the antioxidant and invitro cytotoxicity activity of crude fractions from the roots of Xanthium Strumarium.
Mukesh Kumar Singh et al /Int.J. PharmTech Res.2010,2(4) 2220 Material and method: Collection of plant material The plant Xanthium Strumarium was collected in and around Tambram, Chennai and was identified and authenticated by prof. Jayaraman a renowned Indian botanist. The root was separated, washed with water, Air-dried and made into a coarse powder this powdered root was used for further studies. Prepartion of Xanthium Strumarium root extracts: Plant material was extracted with increasing polarity by two different methods. 6 Cold Extraction Three liters of hexane was added to the 300g of plant material in a 5L round bottom flask. The mixture was shaken well and left to stand overnight at room temperature. The solvent was distilled of and the procedure was repeated for three times. The solvent was then changed from hexane to chloroform. 3 L of chloroform was added to the plant material and the procedure was repeated as above. The crude extract was collected and kept separately. Hot Extraction The solvent was finally changed from chloroform to the highest polar solvent methanol. 3 liters methanol was added to the plant material and the mixture was boiled on a heating mantle for 48hrs using Soxhlets apparatus. The solvent was distilled off and crude extract thus obtained was kept separately. Antimicrobial Assay: Antimicrobial studies in terms of minimum inhibitory concentration (MIC) of obtained product against various strains of bacteria were carried out by two-fold serial dilution techniques. For this Seed broth was adjusted, between 10 5 and 10 6 cfu / ml from standardized stock culture. A series of six assay tubes for each organism was taken, to the first tube 0.2ml of stock solution (1mg/ml) and 1.8ml of seeded broth was added, then from the first tube 1ml was added to 1ml of seeded broth, like wise two-fold serial dilution was carried out up to 6 th tube and incubated at 37 0 c for 24 hrs for bacteria and 27 0 c for 24 48 hrs for fungi. After incubation it was observed for the lowest concentration of the compound which completely inhibits the growth of microorganism and it was noted as the minimum inhibitory concentration. 7 Determination of antioxidant activity: The antioxidant activity of Xanthiuim Strumarium was measured in terms of hydrogen donating or radical scavenging ability using a stable DPPH method. 8 The assay was carried out in a 96-well microtitre plate. To 200 µl of DPPH solution, 10µl of each of the test sample or the standard solution was added separately in the wells of microtitre plate. The final concentration of the test and the standard solution used are 1000µg/ml, 500 µg/ml, 250 µg/ml, 62.5 µg/ml, 31.5 µg/ml and 15.62 µg/ml. the plates were incubated at 37 0 c for 30 minutes and the absorbance of each solution was measured at 490 nm. Using ELISA reader against the corresponding test and standard blanks and the remaining DPPH was calculated. The IC 50 is the concentration of sample required to scavenge 50% of DPPH free radicals. Cytototoxic studies: HEP-2 cells lines were purchased from National chemical laborites (NCL), Pune. Cells were culture in DMEM medium and incubated at 37 0 C with 5% Co 2 and cell were periodically checked for any morphological changes and contamination. The cytotoxicity assays were performed according to the [ 3 H] Thymidine labeling of cells. 9 Cells were seeded in 12-well plates and T-25 flasks at a concentration of 0.5 million cells/ml and treated for specific time periods with plant extracts. The were then harvested and processed for [ 3 H] Thymidine. After seeding the cells in the plates, and allowing them to attach overnight, 1µ of [ 3 H] Thymidine was added to each of the wells. Cells were incubated for at 48 hrs. Cultures were harvested by trypsinisation after aspiration of the media. The trypsin was diluted out with fresh media and the cells were centrifuged to remove the supernatant. 0.5 ml of ice-cold 10% TCA was added to the cells and the tubes were placed at 4 0 c for 1 hrs. The cells were then centrifuged and washed twice with 5% TCA solution. The cell pellet was finally solubilized with 300µl of 0.1 N NaOH with 0.025 SDS. The cell pellets were incubated for 2-3 hrs in a water bath at 60 0 c. 50µl of this solution was spotted on a glass fibre, air dried and the radioactivity was measured in a liquid scintillation counter, after adding 5 ml of scintillation cocktail. The results were expressed as counts per minute/milligram of protein (cpm/mg protein). Dose response study: The different solvent fractions of both the plants were added to the cells. In a six well plate 20 µl of sample was added in each well. As control the same volume of the different solvents was added. The different dilution of the ethyl acetate fraction of both extracts like 100µg, 75 µg, 50 µg, 25 µg, and 12.5 µg, was carried out. The wells were terminated at different time points time points from day 1 and day 6 after treatment with the plant extract in the respective experiments. Time course analysis: To understand the kinetics of the cytotoxic effect a time course analysis of the effect of the plant on HEP- 2 cells was performed. The amount of [ 3 H] Thymidine
Mukesh Kumar Singh et al /Int.J. PharmTech Res.2010,2(4) 2221 incorporated in to HEP-2 cells exposed to both compounds were analyzed. The time points chosen were 24hrs and 48 hrs. The treated HEP-2 cells were processed and counted as mentioned. Table -1 Plant extracts Salmonella typhi Streptococcus Staphylococcus Proteus mirabilis pneumonia aureus C 2 0.250 1 2 C-1 2 2 0.250 1 C-2 2 1 2 1 C-3 1 1 1 1 C-4 0.250 1 2 0.250 C-5 0.125 0.250 0.5 0.5 C-6 0.5 0.5 1 2 M 1 2 2 1 M-1 0.5 0.125 0.250 1 M-2 0.5 0.5 0.250 0.125 M-3 0.125 0.125 0.125 0.125 M-4 0.125 0.125 0.125 0.125 M-5 0.250 0.125 0.250 0.125 Where C = chloroform crude extract, C-1 to C-5 fraction of chloroform extract. M- Methanolic crude extract, M-1to M-5 fraction of methanolic extract. Table-2 Crude extract and their fraction IC 50 Values ± Standard error value (µg/ml) C 10.28±0.69 C-1 51.05±3.61 C-2 63.83±0.23 C-3 158.87±4.30 C-4 180.40±2.69 C-5 123.00±5.31 C-6 119.31±5.03 M 40.40±0.19 M-1 90.70±2.15 M-2 184±2.69 M-3 89.91±5.16 M-4 155.87±4.30 M-5 63.83±0.23 Ascorbic acid 54.17±9.27 Rutin 43.60±1.79
Mukesh Kumar Singh et al /Int.J. PharmTech Res.2010,2(4) 2222 Results and Discussion: Determination of antimicrobial assay: The determination of the MIC by means of the liquid dilution (Table 1) showed that the plant extracts tested exhibited an antimicrobial effect against some of the four tested microorganisms. The C and C-1 extracts showed good MIC against Staphylococcus aureus. C-4 extract showed good antimicrobial activity on Salmonella typhi and Proteus mirabilis. Whereas M-3 AND M-4 showed good MIC of 0.125µg/ml against all the tested organisms. Determination of antioxidant Activity: The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability (Baumann et al; 1979). The chloroform crude extracts(c) showed highest antioxidant activity. The other methanolic crude extract showed good antioxidant activity. Whereas crude fractions of chloroform and methanolic showed poor antioxidant activity (Table-2). The IC 50 value of chloroform crude extract was found to be 10.28µg /ml and methanol was found to 40.40µg /ml and that of Ascorbic acid and Rutin was 54.17µg/ml and 43.60µg /ml respectively and is inversely related to antioxidants capacity. Cell proliferation assay: The root extracts showed potent cytotoxic activity against the HEp-2 cell lines tested. The crude methanolic and chloroform extract was evaluated for their cytotoxic activity on HEp-2 cell line. The extract was tested at the range 12.5-100 µg /ml. After 24 hrs and 48 hrs of incubation the percentage of cell proliferation assay was determined and showed 59.53% and 65.51% for chloroform extract and 62.98% and 70.15% for methanolic extract at the concentration of 100 µg /ml (fig.1& 2). The time course analysis of the effect of plant extracts on the cancerous HEp-2 cells was studied along with the dose response studies, which showed dose dependent destruction of the monolayer and morphological changes of the cell line (fig.3). The result showed both methanolic and chloroform extracts have more potent cytotoxicity against the tested cell lines. Cell proliferation assay: Fig. 1 Cell proliferation assay for C after 24 and 48 hrs Fig.2 Cell proliferation assay for M after 24 and 48 hrs.
Mukesh Kumar Singh et al /Int.J. PharmTech Res.2010,2(4) 2223 Fig.3 Dose response study and time course analysis Antimicrobial studies of chloroform and methanolic crude extracts along with their fractions gave good results against Staphylococcus aureus, Salmonella typhi, proteus mirabilis tested with MIC values nearly 0.125µg/ml. The IC 50 shows that the plant extracts have good free radical scavenging activity indicating good antioxidants activity. The chloroform crude showed highest antioxidants activity, whereas methanolic crude extract showed moderate activity. The root extracts showed potent cytotoxic activity against the HEP-2 cell lines tested. The time course analysis of the effect of plant extracts on the cancerous cell lines was studied along with the does response studies, which showed does dependent destruction of the monolayer and morphological changes of cell line. These studies on the antimicrobial, antioxidants and cytotoxic studies gave optimistic results, which insist the importance of further studies on Xanthium strumarium root. Extensive research should be done for exploiting better results on this plant as the studies done under this by me preliminary. Further processing of this plant may gift the world a potent drug for various ailments. References: 1. Talakal TS., Dwivedi SK., and Sharma SR., Journal Ethanopharmacology., 1995, 49, 141-145. 2. Leborgne L., Maziere JC., Maziere C., and Andrejak M., Oxidative stress, atherogenosis and cardiovascular risk factors, Archivies des Maadies du Coeur et des vaisseaux., 2002, 95(9), 805-814. 3. Folts JD., Potential health benefits from the flavanoids in grape products in vascular disease, Advances in Experimental Medicine and Biology., 2002, 505, 95-111. 4. Freeman BA., Biological sites and mechanism of free raical production, In D.Armstrong, R.Sohal, R.G. Culter, & T.Slater (Eds.), Free radicals in molecular biology,aging,and disease.,1984,43-52. 5. Squadrito G. L., & Pryor W.A., Oxidative chemistry of nitric oxide, the roles of superoxide, peroxynitrite, and carbon dioxide, Free radical biology and medicine., 1998, 25, 392-403 6. Chng Jun YI., Kuruppiah Pillai Manoharan and Yang Daiwen, Isolation characterization and screening of anticancer metabolites from natural product, National university of Singapore. 7. Soroush Sardari., Gholamreza Amin., Ronald G., Micetich and Mohsen Daneshtalab., Pharmaceutical biology., 1998, 36, 180-188. 8. Mgammal-Eldeen A., Kawasthy SA., Ibrahim LF., Shabana MM., and Negoumy EI SI.,Journal of natural remedies., 2004, 4/1, 81-96. 9. Kenneth C., Norbury and Isaiah J., Fidler., Journal of Immunological methods.,197, 7 (1), 109-121. 10. Baumann J., Wrung., Bruchlausen FV., Prostaglandin synthetase inhibiting oxygen radical scavenging properties of some flavanoids and related phenolic compounds, Naunyn- Schmiedebergs Archieves of Pharmacology., 1979, 308,R27. *****