In vitro Evaluation of the Efficacy of Leaf and its Callus Extracts of Cardiospermum halicacabum L. On Important Human Pathogenic Bacteria

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1 Advances in Biological Research 2 (-2): 34-38, 2008 ISSN IDOSI Publications, 2008 In vitro Evaluation of the Efficacy of Leaf and its Callus Extracts of Cardiospermum halicacabum L. On Important Human Pathogenic Bacteria 2 H.V. Girish, M.S. Sudarshana and E. Rati Rao Plant Biotechnology Laboratory, Department of Studies and Research in Botany, University of Mysore, Manasagangotri, Mysore , Karnataka, India 2 Microbiology Laboratory, Central Food Technological Research Institute, Mysore , Karnataka, India Abstract: The investigation is aimed to carry out the antimicrobial activities of different solvent extracts of leaf and leaf derived callus extracts of Cardiospermum halicacabum L. (Sapindaceae). The leaf segments of C. halicacabum were cultured on MS medium supplemented with auxins and cytokinins alone. Maximum callus was recorded on medium containing BAP. The antibacterial properties of leaf and its callus extracts were screened against ten human pathogenic bacteria by cup diffusion method. Powdered leaf and leaf derived callus material was subjected to extraction of aqueous and with different organic solvents viz., petroleum ether, chloroform, methanol and ethanol using soxhlet apparatus. All the solvent extracts were evaporated to dryness using rotary flash evaporator. Dry residues was dissolved in methanol, and tested for antibacterial activity. Among all the solvents tested, significant inhibitory activity was observed in ethanol extract of both leaf and leaf derived callus followed by methanol. It also observed that the activity was more pronounced on Gram-positive bacteria than Gram-negative bacteria. Key words: Antibacterial activity Cardiospermum halicacabum Callus INTRODUCTION of extracts of medicinal plant between chemical composition, biological and therapeutic activities. Traditional herbalists in India use a variety of herbal Medicinal herbs are moving from fringe to preparations to treat different kinds of ailments including mainstream use with greater number of people seeking many microbial infections. The uneven distribution of remedies and health benefits free from side effects. health personnel in rural and urban areas, the rural Recently considerable attention has been paid to utilize dwellers are virtually left with no alternative other than to eco-friendly and bio-friendly plant based products for the patronize the herbal practitioners. This has been the case prevention and cure of different human diseases including even before the introduction of antibiotics and modern microbial infection [2]. Biological studies are very much drugs. In most cases, the herbal practitioners are ignorant essential to substantiate the therapeutic properties of of the pharmacological and toxicological values of their medicinal plants and drugs mentioned in Ayurveda on medications []. It is a necessity from the scientific point scientific lines. The plant kingdom represents an of view, to establish a rational relationship between extraordinary reservoir of novel molecules. The potential chemical composition, biological and therapeutic of higher plants, as source for new drug is thus still activities. The search for biologically active compounds largely unexplored [3]. from natural sources has always been of great interest to The use of plants, plant extract provides the scientist looking for new sources from drugs useful in foundation to modern therapeutic sciences and thus infectious diseases. In recent years a number of studies enabled the man to establish the empirical system of have been reported, dealing with antimicrobial screening medicine. In view of the commercial importance given to Correspondence Author: M.S. Sudarshana, Plant Biotechnology Laboratory, Department of Studies and Research in Botany, University of Mysore, Manasagangotri, Mysore , Karnataka, India 34

2 Advan. Biol. Res., 2 (-2): 34-38, 2008 the secondary metabolites in recent times, efficient sterile distilled water in a warring blender (Waring production of bioactive compounds by tissue culture international, New Hartford, CT, USA) for 0-5 min. technology has gained popularity [4]. The macerate was first filtered through double layer Since the secondary metabolites often have a muslin cloth then centrifuged at 3500 rpm for 30 min. complex stereo structure with many chiral centers, which The supernatant was filtered through Whatman No. may be essential for biological activity, many of these filter paper and sterilized at 20 C for 30 minutes. The cannot be synthesized economically on a commercial extracts were preserved aseptically at 5 C for further basis. Moreover the continuous and non-organized use [3]. exploitation has resulted in many plants becoming rare and some even became extinct. To overcome this Soxhlet Extraction: Thoroughly washed leaves and limitation, biotechnologists suggested the use of cell proliferated callus of C. halicacabum were dried in shade and tissue culture technology rather than to use the and then powdered with the help of Waring blender. Ten whole plant for the extraction of certain secondary grams of shade-dried leaf and callus powder was filled in metabolites [5]. a thimble and extracted with 00 ml of petroleum ether, Cardiospermum halicacabum Linn. (Sapindaceae) is chloroform, methanol and ethanol successively up to an herbaceous climber [6], commonly used in the 48 h. Each of the solvent extracts was concentrated treatment of rheumatism, lumbago, earache, fever [7, 8]. separately under reduced pressure [4]. After complete Reports are available on analgesic, anti-inflammatory and solvent evaporation, one gram of each concentrated vasodepressant activities [9, 0, ]. Literature survey on solvent extract was dissolved in 9 ml of methanol and this plant revealed efforts have not been made towards used in antibacterial assays. the study of antibacterial activity of C. halicacabum leaf and its callus on human pathogenic bacteria. Test Microorganisms: Authentic pure cultures of human pathogenic bacteria like Bacillus cereus, Bacillus subtilis, MATERIALS AND METHODS Escherichia coli, Klebsiella sp. Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Shigella sp. Explants Preparation: Healthy, disease free leaves of Staphylococcus aureus, and Yersinia enterocolitica, two-month old C. halicacabum were collected from were obtained from Central Food Technological Research Mysore, Mysore district, Karnataka (India). The leaves Institute, Mysore, Karnataka (India). were cut into 0.5- cm segments. These explants were washed both in liquid detergent Tween 20 for 3-4 minutes Antibacterial Activity Assay: Antibacterial activity of and in running tap water for 0 minutes. The washed aqueous and solvent extracts of both leaf and callus explants were surface sterilized with 0.% mercuric extracts was determined by the cup diffusion method on chloride solution for 3-4 min. followed by 4 to 5 rinses in nutrient agar medium [5]. cooled sterile distilled water and were inoculated on the culture medium. Aqueous Extract: The organism to be tested was inoculated into sterile nutrient agar. After incubation Media Preparation, Callus Initiation and its period of 24 h at 37 C, a loop of inoculum was transferred Proliferation: Murashige and Skoog [2] medium into 5 ml of nutrient broth and incubated for 2 h at 37 C supplemented with auxins viz., 2.4-D, NAA, IAA and served as fresh suspension inoculum. Wells (5 mm cytokinins viz., BAP and Kn alone at different diameter) were made in sterile nutrient agar plate using concentrations was used for callus initiation. The cultures cork borer and inoculum containing 06 CFU ml of test were incubated under cool fluorescent light with 3000 lux bacteria were spread on the sterile nutrient agar medium for 6 h at a temperature of 25±2 C and 70±0% relative in Petridishes was uniformly smeared with test culture. humidity. Each experiment had 0 replicates and repeated Then 25 µl of aqueous extract of both leaf and callus was at least thrice. The proliferated callus cultures were sub introduced into each of the wells. The treatment also cultured and maintained for days on the same includes 25 µl of sterilized distilled water as control. All medium supplemented with the same growth regulator. the plates were incubated for 24 h at 37 C and zone of inhibition if any around the well was measured in Preparation of Extracts millimeter (mm). For each treatment three replicates were Aqueous Extract: Ten grams of both fresh leaf and leaf maintained. The data was subjected to statistical analysis derived callus were macerated separately with 20 ml of of variance. 35

3 Advan. Biol. Res., 2 (-2): 34-38, 2008 Solvent Extract: One gram of both leaf and its callus concentrated solvent extract of petroleum ether, chloroform, methanol and ethanol were dissolved in 9 ml of methanol. The sterile nutrient agar medium in Petridishes was uniformly smeared with test culture. Wells (5 mm) were made in each Petridish to which 25 µl of solvent extracts dissolved in methanol was introduced. For each treatment three replicates were maintained. Methanol was served as control. Antibiotics: Streptomycin (0 mcg disc ) generally used in the management of human pathogenic bacteria was selected for antibacterial activity assay for comparative evaluation. RESULTS Antibacterial Activity Assay: The antibacterial activity of aqueous and solvents extracts of C. halicacabum leaf and leaf derived callus against human pathogenic bacteria both Gram-positive and Gram-negative bacteria at 25 µl concentration are presented in Table. The results revealed that among ten pathogenic bacteria only Bacillus subtilis and Bacillus cereus belongs to Gram-positive bacteria showed susceptible for leaf aqueous extract when compared to Staphylococcus aureus. In Gram-positive bacteria Bacillus subtilis showed maximum inhibition. Where as Gram-negative bacteria showed least susceptible for leaf aqueous extract. As in the case of leaf derived callus extract did not show any activity. The different solvents viz., petroleum ether, chloroform, methanol and ethanol of leaf and leaf derived callus extracts were tested for antibacterial activity. Among the four solvents tested, all the test pathogens were highly sensitive to ethanol extract followed by methanol and chloroform extract. Petroleum ether extract did not show antibacterial activity against test pathogens. Among the test bacteria Bacillus sp. and Staphylococcus aureus were highly susceptible for leaf and leaf derived callus extracts. The data of inhibitory effect of different solvent extracts revealed that high inhibitory activity was observed in ethanol extract of both leaf and leaf derived callus extracts. DISCUSSION Review of literature reveals lack of information on the antibacterial potential of C. halicacabum leaf and leaf derived callus extracts. Aqueous and different solvent extracts of leaf and leaf derived callus extracts of C. halicacabum evaluated for antibacterial potential, revealed significant activity of ethanol extracts against Gram-positive bacteria followed by methanol extracts. None of the earlier reports have demonstrated the antibacterial potential of this plant leaf callus extracts [8, 0, ]. In the present investigation the antibacterial activity of this leaf callus extracts has been demonstrated for the first time. Table : Antibacterial activity of aqueous and different solvent extracts of leaf and leaf derived callus of Cardiospermum halicacabum against some human pathogenic bacteria at 25 µl Leaf extracts Callus extracts Sl. Pathogens Aqueous Petroleum Aqueous Petroleum Streptomycin No. Methanol Control Extract Ether Chloroform Methanol Ethanol Extract Ether Chloroform Methanol Ethanol (0mcg disc ) Bacillus cereus ±0.00 ±0.27 ±0.00 ±0.00 ±0.00 ±0.4 ±0.00 ±0.00 ±0.00 ±0.00 ±0.00 ± Bacillus subtilis ±0.00 ±0.27 ±0.00 ±0.24 ±0.36 ±0.36 ±0.00 ±0.00 ±0.00 ±0.27 ±0.27 ± Escherichia coli ±0.00 ±0.00 ±0.00 ±0.27 ±0.27 ±0.4 ±0.00 ±0.00 ±0.00 ±0.00 ±0.27 ± Klebsiella sp ±0.00 ±0.00 ±0.00 ±0.24 ±0.24 ±0.00 ±0.00 ±0.00 ±0.00 ±0.00 ±0.27 ±0.4 5 Proteus mirabilis ±0.00 ±0.00 ±0.00 ±0.00 ±0.00 ±0.27 ±0.00 ±0.00 ±0.00 ±0.4 ±0.00 ± Pseudomonas aeruginosa ±0.00 ±0.00 ±0.00 ±0.00 ±0.4 ±0.27 ±0.00 ±0.00 ±0.00 ±0.2 ±0.00 ± Salmonella typhi ±0.00 ±0.00 ±0.00 ±0.47 ±0.27 ±0.00 ±0.00 ±0.00 ±0.00 ±0.24 ±0.36 ± Shigella sp ±0.00 ±0.00 ±0.00 ±0.24 ±0.24 ±0.24 ±0.00 ±0.00 ±0.00 ±0.00 ±0.00 ± Staphylococcus aureus ±0.00 ±0.00 ±0.00 ±0.00 ±0.36 ±0.36 ±0.00 ±0.00 ±0.00 ±0.4 ±0.47 ± Yersinia enterocolitica ±0.00 ±0.00 ±0.00 ±0.00 ±0.00 ±0.27 ±0.00 ±0.00 ±0.00 ±0.27 ±0.4 ±0.00 Zone of inhibition (Mean three replicate±standard error). When subjected to analysis of variance (ANOVA) p<

4 Advan. Biol. Res., 2 (-2): 34-38, 2008 The ethanol extracts of C. halicacabum leaf and their secondary metabolites, very few reports are leaf-derived callus were most active, and promising result on in vitro derived callus. In the current study therefore, were obtained, which all to a varying extent inhibited the the main emphasis was on the potentialities of the use of growth of the bacteria. Other tested extracts also inhibited in vitro derived callus of C. halicacabum for antibacterial the growth of number of test organisms but to a lesser activity have been demonstrated for the first time. extent and mainly active against Gram-positive bacteria. Since several Staphylococcus strains are reported to ACKNOWLEDGMENT express drug resistance [6] and natural plants were shown to be good anti-staphylococcus activity [7]. In The authors thank the Department of Studies and the study, among extraction assayed, the ethanol extract Research in Botany, University of Mysore and Central of leaf and its callus showed significant inhibition against Food Technological Research Institute for providing Staphylococcus aureus. The result of the present study facilities supported that, plant extract possess compounds with antimicrobial properties that can be used as antimicrobial REFERENCES agents in new drugs for the therapy of infectious diseases caused by microorganisms.. Olukoya, D.K., N. Idika and T. Odugbemi, 993. Successful prediction of botanical compounds from Antibacterial activity of some medicinal plants from plant materials is largely dependent on the type of solvent Nigeria. J. Ethnopharmacol., 39: used in the extraction procedure. Traditional healers use 2. Dubey, N.K., R. Kumar and P. Tripathi, Global primarily water as a solvent, but in our studies we found promotion of herbal medicine: Indian opportunity. that plant extracts in organic solvents (ethanol and Current Sci., 80: methanol) provide more consistent antibacterial activity 3. Hostettaman, K. and J. Walfender, 997. The Search compared to extracted in water. Our finding also for Biologically Active Secondary Metabolites. supported by several workers [8,9,20,2] has been Pesticide Sci., 5: generally reported that water extracts of plants do not 4. Vaniserce, M., C. Lee, S.M. Nalawade, C.Y. Lin and have much activity against bacteria. Eloff [22] reported H. Tasy, Studies on the production of some that methanol and ethanol were the most effective solvent important secondary metabolite from medicinal plants for plant extraction than n-hexane and water. Similarly, in by Plant tissue culture. Bot. Bull. Acad. Sin., 45: -22. our study ethanol and methanol extracts exhibited higher 5. Rajendra, K. and D Souza, Secondary activity than other solvents in both leaf and leaf derived metabolite of Ayurvedic plants in vitro, Plant. callus extracts. Our results indicated that ethanol and Biotechnology Recent Advances. Trivedi, P.C. (Ed.) methanol is ideal solvents to extracts antimicrobial Panima Publishing Corporation. New Delhi, compounds found in leaf and leaf derived callus. pp: In general, the cell walls of Gram-negative organisms, 6. Joshi, S.K., B.D. Sharma, C.R. Bhatia, R.V. Singh and which are more complex than Gram-positive ones, act as R.S. Thakur, 992. The Wealth of India Raw Materials, a diffusional barrier and making them less susceptible to Vol. III. Council of Scientific and Industrial Research the antibacterial agents than the Gram-positive bacteria publication, New Delhi, pp: [23, 24]. In spite of this permeability difference, however, 7. Nadkarni, K.M., 976. Indian Materia Medica. Popular ethanol extracts of leaf and leaf derived callus extracts Book Depot, Bombay, pp: 27. have still exerted some degree of inhibition against 8. Asha, V.V. and P. Pushpangadan, 999. Antipyretic Gram-negative organisms as well. activity of Cardiospermum halicacabum. Indian J. Plant extracts have great potential as antimicrobial Exp. Biol., 37: compounds against test bacteria. It also indicated the 9. Gopalakrishnan, C., R. Dhananjayan and L. possible utilization of plant biotechnology towards the Kameswaran, 976. Studies on the pharmacological development of an effective in cultured derived callus, actions of Cardiospermum halicacabum. Indian J. which were potentially bioactive compounds. However, in Physiol. Pharmacol., 20: the present study the demonstration of bioactivity in 0. Sadique, J., T. Chandra, V. Thenmozhi and V. Elango, in vitro derived callus in comparison to that of native 987. Biochemical modes of action of Cassia plant has been presented. Although, numerous reports occidentalis and Cardiospermum halicacabum in the have appeared on the antibacterial activity of plants and inflammation. J. Ethnopharmacol., 9:

5 Advan. Biol. Res., 2 (-2): 34-38, Paakkari, H., 994. Epidemiological and financial 9. Sanshes, N.R., D.A.G. Cortez, M.S. Schiavini, aspects of the use of non-steroidal anti-inflammatory C.V. Nakamura and B.P. Filho, An Evaluation of analgesics. Pharmacol. Toxicol., 75: Antibacterial Activities of Psidium guajava (L). 2. Murashige, T. and F. Skoog, 962. A revised medium Brazilian Arch. Biol. Technol., 48: for rapid growth and bioassays with tobacco tissue 20. Aboaba, O.O., S.I. Smith and F.O. Olude, culture. Physiol. Plant., 5: Antibacterial effects of edible plant extracts on 3. Gupta, V.P. and D.R.K. Govindaiah, 996. Plant Escherichia coli 057:H7. Pak. J. Nut., 5: extracts: A non-chemical approach to control 2. Durmaz, H., E. Sagun, Z. Tarakci and F. Ozgokce, Fusarium diseases of mulberry. Current Sci., Antibacterial activities of Allium vineale, 7: Chaerophyllum macropodum and Prangos 4. Harborne, J.B., 998. Phytochemical methods: A guide ferulacea. African J. Biotechnol., 5: to modern techniques of plant analysis. 3rd Edn. 22. Eloff, J.N., 998. Which extractant should be used for Chapman and Hall Pub. London.UK. the screening and isolation of antimicrobial 5. Anon, 996. Pharmacopoeia of Indian (The Indian components from plants?. J. Ethnopharmacol., 60: -8. Pharmacopoeia) 3rd edition. Governament of India, 23. Nostro, A., M.P. Germano, V. D Angelo, A. Marino New Delhi. Ministry of Health and family welfare. and M.A. Cannatelli, Extraction methods and 6. Boer, H.J., A. Kool, A. Broberg, W.R. Mziary, bioautography for evaluation of medicinal plant I. Hedberg and J.J. Levenfors, Anti-fungal and antimicrobial activity. Lett. Applied Microbiol., anti-bacterial activity of herbal remedies from 30: Tanzania. J. Ethnopharmacol., 96: Tadeg, H., E. Mohammed, K. Asres and T. Gebre- 7. Lechner, D., M. Stavri, M. Oluwatuyi, R. Pereda- Mariam, Antibacterial activities of some Miranda and S. Gibbons, The anti- selected traditional Ethiopian medicinal plants used in staphylococcal activity of Angelica dahurica the treatment of skin disorders. J. Ethnopharmacol., (Bai Zui). Phytochemistry, 27: : Martin, G.J., 995. Ethonobotany: A Methods Manual. London: Chapman and Hall. 38

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