LEAVES OF CYCAS REVOLUTA: POTENT ANTIMICROBIAL AND ANTIOXIDANT AGENT

World Journal of Science and Technology 2011, 1(10): 11-20 ISSN: 2231 2587 www.worldjournalofscience.com LEAVES OF CYCAS REVOLUTA: POTENT ANTIMICROBIAL AND ANTIOXIDANT AGENT Manoj K. Mourya 1*, Archana Prakash 2, Ajay Swami 5, Gautam K. Singh 1 and Abhishek Mathur 3, 4 * 1 Dept. of Biochemistry, Sai Institute of Paramedical & Allied Sciences, Dehradun (U.K), India. 2Dept. of Biochemistry, HIHT University, Jolly Grant, Dehradun (U.K), India. 3 Dept. of Biotechnology, Dev Bhoomi Group of Institutions (DBGI), Dehradun (U.K), India. 4 Institute of Transgene Life Sciences (ITLS), Lucknow (U.P), India. 5 Dept. of Botany, Chinmaya Degree College, Hardwar (U.K), India. Corresponding author e-mail: mathurabhishek37@yahoo.com Abstract Plants have served as a source of new pharmaceutical products and inexpensive starting materials for the synthesis of many known drugs. Besides angiosperms, gymnosperms are forming the important medicinal flora of Indian forests. These are the mega-plants forming the cultural and economic heritage of the country. In the present investigation the hydro-alcoholic and chloroform extracts of the leaves of Cycas revoluta plant were prepared in order to assess the antimicrobial and antioxidant potential of the plant. The hydro-alcoholic extracts of leaves of the plant showed promising antimicrobial and antioxidant activities. Hydro-alcoholic extracts of leaves of the plant showed potent antimicrobial activity against E.coli, Klebsiella pneumoniae and Saccharomyces cerevisae while chloroform extracts showed almost similar antimicrobial activity against E.coli and Saccharomyces cerevisae. It was observed that Lactococcus sp., Streptococcus pyogenes, Aspergillus niger and Candida albicans are the most resistant organisms against both the hydro alcoholic and chloroform extracts of leaves of the plant. The extracts showed almost similar pattern of antioxidant activity. Hydro-alcoholic extracts showed significant antioxidant activity in comparison to chloroform extracts. The results of the study thus further emphasized on the isolation and characterization of novel molecules present in leaves responsible for pharmacological activities. Keywords: Cycas revoluta, leaves, antimicrobial activity, antioxidant activity. Introduction The Gymnosperm, Cycas revoluta is from the cycad family, an ancient group of plants dating back to the age of the dinosaurs. This plant will produce a symmetrical rosette of glossy, stiff fronds with re-curved edges. As it grows, it slowly develops a thick, wooden trunk, which can produce basal shoots, later forming multiple crowns. According to Systemic position, Cycas plant occurs in Division, Cycadophyta; Class, Cycadopsida; Order, Cycadales and Family, Cycadaceae. Taxonomic classification of Cycas revoluta and other related species were done (Hill et al., 2004; Hill and Stanberg, 2009). Underutilized plants are those species with underexploited potential for contributing to food security, health (nutritional/medicinal), income generation and environmental services (Anonymous, 2006). Underutilized plant species have a potential, not fully exploited, to contribute to food security and poverty alleviation. Promotion of their use should be done in a sustainable fashion (Hoeschle-Zeledon and Bordoni, 2003). Freiberger et al. (1998) investigated the nutritional role of wild underutilized plants in the

Nigerian diet. Pugalenthi et al. (2005) studied the nutritional potential of an underutilized legume Mucuna pruriens var. utilis and current state of its utilization as food/feed for both human beings and livestock throughout the world. Bissa et al., 2008 determined the antibacterial potential of gymnosperms. Rao and coworkers, 2007 determined the antiviral activity of coralloid root of Cycas revoluta extract against some viruses of tomato plant. Hill and coworkers, 2004 categorized the different species of Cycas revoluta. Chiang et al., 2009 determined the paraphyly of organelle DNAs in Cycas species due to ancient ancestral polymorphisms. Economic value of underutilized plant species in Ayubia National Park were determined (Ahmad et al., 2007). Medicinal plants and phytomedicines linking plant Biochemistry and Physiology to Human health (Briskin, 2000). Dar and Dar (2006) reviewed gymnosperms as Wealth of Kashmir Himalaya. Several active constituents are present in bark, leaves, fruits and roots of gymnosperms (Dhar, GL, 1966; 1975; Dhar, U, 1978; Dar and Dar, 2006). Farnsworth and Fabricant (2001) investigated the importance of plants in Traditional Medicine for Drug discovery. Hill and Stanberg (2009) described the systemic position of Cycas species. In the present investigation in vitro antimicrobial and antioxidant activities of Cycas leaves were determined in order to evaluate the pharmacological importance of the gymnosperms. Materials and Methods Plant Materials The leaves of the plant, Cycas revoluta (Sago-Palm) were collected from the local gardens of Forest Research Institute (FRI), Dehradun. Chemicals Analytical reagent (AR) grade ethanol and chloroform were purchased from Ranchem Pvt. Ltd., India. Nutrient agar/broth and Sabouraud s Dextrose agar/broth were purchased from Hi Media Pvt. Ltd., Mumbai, India. Positive controls, Chloramphenicol and Fucanazole were obtained as the gift samples from Ranbaxy Pvt. Ltd., India. The chemicals and reagents used for assessing antioxidant activity and phytochemical screening were purchased from CDH and Ranchem Pvt. Ltd., India. Preparation of Plant extracts The leaves of the plant were separated, washed with distilled water, dried under shade and pulverized. The method of Alade and Irobi (1993) was adopted for preparation of plant extracts with little modifications. Briefly 20 g portions of the powdered plant material were soaked separately in different solvents viz. hydro-alcohol (50 % v/v) and chloroform on the basis of increasing polarity for 72 h. Each mixture was stirred every 24 h using a sterile glass rod. At the end of extraction, each solvent was passed through Whatmann filter paper No. 1 (Whatmann, England) The filtrates obtained were later concentrated in vacuo using water bath at 30 0 C. Determination of antimicrobial activity Culture Media For antibacterial test, Nutrient agar/broth and Sabouraud s dextrose agar/broth of Hi Media Pvt. Bombay, India were used for antifungal test. Inoculum The bacteria were inoculated into Nutrient broth and incubated at 37 0 C for 18 h and suspension was checked to provide approximately, 3X10 5 cfu/ml. The same procedure was done for fungal strains and there strains were inoculated into Sabouraud s dextrose broth but the fungal broth cultures were incubated at 48-72 h. Microorganisms used The pure cultures of test organisms E.coli, Klebsiella pneumoniae, Streptococcus pyogenes, Lactococcus sp., Aspergillus niger, Saccharomyces cerevisae and Candida albicans were procured from Microbiology Laboratory, Roorkee Research & Analytical Lab., Roorkee (Uttarakhand), India. Determination of diameter of zone of inhibition by well diffusion method The agar well diffusion method (Perez et al., 1993) was modified. Nutrient agar medium (NAM) was used for bacterial cultures. The culture medium was inoculated with the bacteria separately suspended in nutrient broth. Sabouraud s dextrose agar/broth was used for fungal cultures. The culture medium was inoculated with the fungus separately suspended in Sabouraud s dextrose broth. A total of 8 mm diameter wells were punched into the agar and filled with leaves extracts and solvent blanks. Solvents, chloroform and hydro-alcohol were used as negative controls. Standard antibiotic

(Chloramphenicol, 1 mg/ml) was simultaneously used as the positive control. The plates were then incubated at 37 0 C for 18 h. The antibacterial activity was evaluated by measuring the diameter of zone of inhibition observed. For assaying, antifungal activity of plant extracts, Sabouraud s dextrose agar/broth medium plates were used. The same procedure as that for determination of antibacterial property was adopted and then after, the diameter of zone of inhibition was observed after 48-72 h. Fucanazole (1 mg/ml) was used as standard for determination of antifungal activity. The procedure for assaying antibacterial and antifungal activity was performed in triplicates to confirm the readings of diameter of zone of inhibition observed for each of the test organism. Determination of Minimum Inhibitory Concentration (MIC) MIC values of potent plant extracts was determined by the method adopted by Vollekova et al., 2001 and Usman et al., 2007, with some modifications. Plant extract was prepared in highest concentration (200 µg/ml) in sterile distilled water and is serially diluted with N-saline (0.85 % NaCl) and similar quantity of bacterial suspension was added to different test tubes and incubated for 48 h. The inhibition of turbidity appeared in the minimum dose at which total growth of bacteria gets killed is known as minimum lethal concentration (MLC) while little turbidity appeared in the minimum amount of dose of plant extract which inhibits the growth of bacteria is known as Minimum Inhibitory Concentration (MIC). Determination of in vitro antioxidant activity of the extracts Superoxide Anion Radical Scavenging Activity Superoxide Anion Radical Scavenging Activity was measured with some modifications (Duan et al., 2007). The various fractions of plant extracts were mixed with 3 ml of reaction buffer solution (ph, 7.4) containing 1.3 µm riboflavin, 0.02 M methionine and 5.1 µm NBT. The reaction solution was illuminated by exposure to 30 W fluorescent lamps for 20 minutes and the absorbance was measured at 360 nm using a spectrophotometer. Ascorbic acid was used as positive control and the reaction mixture without any sample was used as negative control. The superoxide anion radical scavenging activity (%) was calculated as: A o A S X 100 A o Where, A O = absorbance of positive control; A S = absorbance of sample Total Antioxidant Activity Determination of the Extracts Total antioxidant capacities of the plant extracts and Ascorbic acid were determined by the method of Pan et al. (2008). An aliquot (0.1 ml) of each of these fractions were combined with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate) separately. The tubes were capped and incubated at 95 C for 90 minutes. After the samples were cooled to 25 C, the absorbance was measured at 695 nm against reagent blank containing no test samples/ extracts. The blank contained 1 ml of reagent solution without the sample. The total antioxidant activity was expressed as the absorbance value at 695 nm. The higher absorbance value indicates the greater antioxidant activity. Determination of Total Phenolic Content (TPC) of the Extracts The Total Phenolic Content of each fraction obtained from different extracts was determined (Singleton and Rossi, 1965) and the phenolic content was expressed as µg/g Gallic acid equivalents. In brief a 100 µl aliquot of the sample was added to 2 ml of 0.2 % (w/v) Na 2CO 3 solution. After two minutes of incubation, 100 µl of 500 ml/l Follin-Ciocalteu reagent added and the mixture were then allowed to stand for 30 minutes at 25 0 C. The absorbance was measured at 750 nm using a UV-VIS Systronics spectrophotometer. The blank consist of all reagents and solvents but no sample. The Total Phenolic Content (TPC) was determined using the standard Gallic acid calibration curve and was expressed as mg/g Gallic acid equivalents. Phytochemical screening of the extract The portion of the dry extracts was subjected to the Phytochemical screening using the method adopted by Trease and Evans (1983) and Harbourne (1983). Phytochemical screening was performed to test for alkaloids, saponin, tannins, flavanoids, steroids, sugars, cardiac glycosides and anthraquinones (Sofowora, 1993). Test for alkaloids The 0.5 g of the plant extract was dissolved in 5 ml of 1% HCl and was kept in water bath for

about 2 minutes. 1ml of the filtrate was treated with Dragendroff s reagent Turbidity or precipitation was taken as indicator for the presence of alkaloids. Test for Tannins About 0.5 g of the sample was dissolved in 10 ml of boiling water and was filtered. Few ml of 6% fecl 3 was added to the filtrate. Deep green colour appeared confirmed the presence of Tannins (Trease and Evans, 1983). Test for Flavanoids About 0.2 gm of the extract was dissolved in methanol and heated for sometime. A chip of mg metal was introduced followed by the addition of few drops of conc. HCl. Appearance of red or orange color was Indicator of the flavanoids. Test for Saponin About 0.5 g of the plant extract was stirred with water in the test tube. Frothing persists on warming was taken as evidence for the presence of saponin. Test for Steroids Salkowski s method was adopted for the detection of steroids. About 0.5 g of extract was dissolved in 3 ml of chloroform and filtered. To the filtrate, conc. H 2 SO 4 was added to form a lower layer. Reddish brown color was taken as positive for the presence of steroids ring (Agarwal et al., 2011). Test for Cardiac glycosides About 0.5 g of the extract was dissolved in 2ml of glacial acetic acid containing 1 drop of 1% Fecl 3. This was under laid with conc. H 2 SO 4. A brown ring obtained at the interphase indicates the presence of deoxy sugar. A violet ring appeared below the ring while in the acetic acid layer a greenish ring appeared just above ring and gradually spread throughout this layer. Test for reducing Sugars 1ml each of Fehling s solutions, I and II was added to 2 ml of the aqueous solution of the extract. The mixture was heated in a boiling water bath for about 2-5 minutes. The production of a brick red precipitate indicated the presence of reducing sugars. Test for Anthraquinones 5ml of the extract solution was hydrolyzed with dil/conc. H 2SO 4. 1 ml of dilute ammonia was added to it. Rose pink colour confirmed the presence of anthraquinones. Results Antimicrobial activity In the present investigation, hydro alcoholic and chloroform extracts of leaves of Cycas revoluta were screened for antimicrobial activity against some pathogenic micro organisms by well diffusion method. The hydro alcoholic extracts of the leaves of the plant showed potent activity against Klebsiella pneumoniae (zone of inhibition: 15.0 mm) followed by Saccharomyces cerevisae (zone of inhibition: 14.0 mm) and E.coli (12.0 mm). Hydro alcoholic extracts of leaves of the plant showed potency against Klebsiella pneumoniae (MIC: 0.8 mg/ml; MLC: 0.9 mg/ml), E.coli (MIC: 0.8 mg/ml; MLC: 0.9 mg/ml) and Saccharomyces cerevisae (MIC: 0.7 mg/ml; MLC: 0.8 mg/ml). Hydro alcoholic extracts showed no activity against Lactococcus sp., Streptococcus pyogenes, Aspergillus niger and Candida albicans. Hexane extracts showed almost similar potency against Saccharomyces cerevisae (zone of inhibition: 12.0 mm) and E.coli (zone of inhibition: 11.0 mm). Hexane extracts showed no activity against Lactococcus sp., Klebsiella pneumoniae, Streptococcus pyogenes, Aspergillus niger and Candida albicans. The results are shown in Table 1 and 2; Figure 1 (a) and (b). Antioxidant activity In vitro antioxidant activities of hydro alcoholic and chloroform extracts were determined by Superoxide anion radical scavenging activity, Total antioxidant activity and Total Phenolic Content. Hydro alcoholic extracts showed potent antioxidant activity in comparison to chloroform extracts. Hydro alcoholic extracts showed prominent superoxide anion radical scavenging activity (77.0 %) followed by chloroform extracts (75.0 %). The results are shown in Table 3 and Figure 3. The findings also support the results of Total antioxidant activity. Hydro alcoholic extracts (A695-0.161) showed prominent antioxidant activity in comparison to chloroform extracts (A695-0.07). The results are shown in Table 4 and Figure 4. TPC content also validates the above findings. TPC content in hydro alcoholic extracts (92.30 mg/g gallic acid) was found to be prominent in comparison to that of chloroform extracts (83.56 mg/g gallic acid). The results of TPC are shown in Table 5 and Figure 5.

Table 1: Antimicrobial activity of the solvent extracts of leaves of Cycas revolute Plant Cycas revoluta Part of the plant used Leaves Solvent Extract Hydro alcoholic Diameter of zone of inhibition (mm) ECO LAC KLB SP AN CA SC 12.0 NA 15.0 NA NA NA 14.0 Chloroform 11.0 NA NA NA NA NA 12.0 Chloramphenicol ---------- --------- 36.0 37.0 25.0 53.0 NT NT NT (1 mg/ml) Fucanazole (1 mg/ml) --------- --------- NT NT NT NT 25.0 26.0 28.0 ECO: E.coli; LAC: Lactococcus; KLB: Klebsiella pneumoniae; SP: Streptococcus pyogenes; AN: Aspergillus niger; CA: Candida albicans; SC: Saccharomyces cerevisae NT: Not Tested; NA: No Activity Antimicrobial activity of leaves extracts of Cycas revoluta Diameter of zone of inhibition (mm) 60 50 40 30 20 10 0 Hydro --------- ECO LAC KLB SP AN CA SC Pathogens affected ECO: E.coli; LAC: Lactococcus; KLB: Klebsiella pneumoniae; SP: Streptococcus pyogenes; AN: Aspergillus niger; CA: Candida albicans; SC: Saccharomyces cerevisae Fig 1 (a). Antimicrobial activity of the solvent extracts of leaves of Cycas revoluta Fig 1 (b). Antimicrobial activity of the solvent extracts of leaves of Cycas revoluta

Table 2. MIC & MLC values of the hydro alcoholic extracts of leaves of Cycas revoluta ECO: E.coli; KLB: Klebsiella pneumoniae; SC: Saccharomyces cerevisae Plant/Extract MIC/MLC values Pathogens affected Cycas revoluta leaves hydro Minimum Inhibitory ECO KLB SC alcoholic extract Concentration (MIC) 0.8 0.8 0.7 (mg/ml) Minimum Lethal Concentration (MLC) (mg/ml) 0.9 0.9 0.8 Table 3. Superoxide Anion Radical Scavenging Activity of extracts of leaves of Cycas revolute Plant/ Sample Cycas revoluta leaves Solvent Extract Percent Superoxide Anion Radical Scavenging Activity Hydro alcoholic 77.0 Chloroform 75.0 Percent scavenging activity of solvent extracts of Cycas revoluta leaves %scavenging activity 77 76.5 76 75.5 75 74.5 74 Percent Superoxide Anion Radical Scavenging Activity Solvent extracts Hydro alcoholic Chloroform Figure 3. Superoxide Anion Radical Scavenging Activity of extracts of leaves of Cycas revolute Table 4. Total Antioxidant Activity of extracts of leaves of Cycas revolute Plant/ Sample Solvent Extract Total Antioxidant Activity (A695) Cycas revoluta leaves Hydro alcoholic 0.161 Chloroform 0.07

Total Antioxidant Activity of solvent extracts of Cycas revoluta leaves Total Antioxidant Activity (A695 nm) 0.2 0.15 0.1 0.05 0 Solvent extracts Solvent Extract Hydro alcoholic Chloroform Fig 4. Total Antioxidant Activity of extracts of leaves of Cycas revolute Table 5. TPC (mg/g) gallic acid in extracts of leaves of Cycas revolute Plant/ Sample Solvent Extract Total Phenolic Content (mg/g) gallic acid equivalents Cycas revoluta leaves Hydro alcoholic 92.30 Chloroform 83.56 TPC (mg/g) Gallic acid equivalents of leaves of Cycas revoluta TPC (mg/g) gallic acid eq. 100 80 60 40 20 0 Solvent extracts Solvent Extract Hydro alcoholic Chloroform Fig 5. TPC (mg/g) gallic acid in extracts of leaves of Cycas revoluta Phytochemical screening Various conventional methods were followed to determine qualitatively the presence of alkaloids, flavanoids, steroids, saponins, glycosides, tannins, anthraquinones and sugars in the hydro-alcoholic and chloroform extracts of leaves of Cycas revoluta. The results are recorded in Table 6 and Figure 6 (a) and (b).

Table 6. Phytochemical constituents of leaves extracts of Cycas revoluta Cycas revoluta leaves extracts Hydro alcoholic Alkaloid Flavanoids Steroids Saponins Glycosides Tannins Anthraquinone Sugars + - + - - + - - Chloroform - - - + - + - + +, present; -, absent Fig 6 (a). Phytochemical constituents of hydro alcoholic extracts of leaves of Cycas revolute Fig 6 (b). Phytochemical constituents of chloroform extracts of leaves of Cycas revoluta

Discussion and Conclusion The potential for developing antimicrobial drugs from higher plants appears rewarding as it will leads to the development of a phytomedicines to act against microbes. Plant based antimicrobial have enormous therapeutic potential as they can serve the purpose with lesser side effects that are often associated with synthetic antimicrobials. Oxidation is one of the most important processes, which produce free radicals in food, chemicals and even in living systems. Free radicals have an important role in the processes of food spoilage, chemical material degradation and also contribute to more than one hundred disorders in humans. The present study validates the use of extracts of leaves of Cycas revoluta as potent antimicrobial and antioxidative agent. The results showed that that hydro alcoholic extracts antimicrobial and antioxidant agent. Hydro alcoholic extracts of leaves of the plant showed potency against Klebsiella pneumoniae (MIC: 0.8 mg/ml; MLC: 0.9 mg/ml), E.coli (MIC: 0.8 mg/ml; MLC: 0.9 mg/ml) and Saccharomyces cerevisae (MIC: 0.7 mg/ml; MLC: 0.8 mg/ml). Hexane extracts showed almost similar potency against Saccharomyces cerevisae (zone of inhibition: 12.0 mm) and E.coli (zone of inhibition: 11.0 mm). The aim of the study to reveal potent active constituents in the hydro-alcoholic and chloroform extracts thus is justified. In the present study the results supported that the leaves of Cycas revoluta are potent antimicrobial (Bissa et al., 2008; Yagi et al., 2002) and antioxidant agent (Rahmatullah et al., 2010). From the present investigation it was observed that Lactococcus sp., Streptococcus pyogenes, Aspergillus niger and Candida albicans are the most resistant organisms against both the hydro alcoholic and chloroform extracts of leaves of the plant. Most sensitive organisms found against the extracts of the plant were E.coli, Klebsiella pneumoniae and Saccharomyces cerevisae. The use of these extracts in the treatment of pathogenic diseases associated with the infection of these pathogens is validated, scientifically supported by the results obtained in this work. The studies may lead to the formulation of antibiotic which may be used against the treatment of diseases associated with these microbial infections. References 1. Agarwal, A., Singhvi, I.J., Bele, D., Sharma, K., Gupta, S.K., Karwani, G., Kumawat, M., (2011). Evaluation of steroids in face creams of different marketed brands. International J. Pharmacy & Technology, 3(2), 2480-2486. 2. Ahmad, S.S., Javaid, S., (2007). Exploring the economic value of underutilized plant species in Ayubia National Park. Pak. J. Botany, 39(5), 1435-1442. 3. Alade, P.I., Irobi, O.N., (1993). Antimicrobial activities of crude leaf extracts of Acalypha wilkensiana. Journal of Ethnopharmacology, 39, 171-174. 4. Anonymous., (2006). ICUC. Annual report 2005-2006, Colombo, Sri Lanka, pp.16. 5. Bissa, S., Bohra, A., Bohra, A., (2008). Antibacterial potential of three naked seeded (gymnosperm) plants. Natural Product Radiance, 7(5), 420-425. 6. Briskin, D.P., (2000). Medicinal plants and phytomedicines. Linking Plant Biochemistry and Physiology to Human health. Update on Phytomedicines, 124, 507-514. 7. Chiang, Y.C., Hung, K.H., Moore, S.J., Ge, X.J., Huang, S., Hsu, T.W., Schaal, B.A., Chiang, T.Y., (2009). Paraphyly of organelle DNAs in Cycas Sect. Asiorientales due to ancient ancestral polymorphisms. BMC Evolutionary Biology, 9(161), 1-19. 8. Dar, A.R., Dar, G.H., (2006). The Wealth of Kashmir Himalayas- Gymnosperms. Asian Journal of Plant Sciences, 5(2), 251-259. 9. Dhar, G.L., (1966). The distribution of coniferales in India Part I. Pinaceae. Kashmir Sci., 3, 33-42. 10. Dhar, G.L., (1975). Sesuiadendron giganteum-a report from Kashmir. The Indian Forester, 101; 562-564.

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