ANTI BACTERIAL POTENTIAL OF LESSER KNOWN HIMALAYAN

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1 NSave Nature to Survive 11(4): , ANTI BACTERIAL POTENTIAL OF LESSER KNOWN HIMALAYAN AN MEDICINAL PLANTS VIZ, BRYOPHYTES KAVITA NEGI* AND PREETI CHATURVEDI Department of Biological Sciences, C.B.S.H., G.B. Pant University of Agriculture andtechnology, Pantnagar , Uttarakhand, INDIA KEYWORDS Plagiochasma appendiculatum Anaectangium thomsonii Plagiothecium denticulatum MIC Received on : Accepted on : ABSTRACT The antibacterial activity of organic and aqueous extracts of three bryophytes viz., Anoectangium thomsonii Mitt., Plagiothecium denticulatum(hedw.) Schimp. (mosses) and Plagiochasma appendiculatum Lehm. & Lindenb (liverwort) of Kumaon Himalayas were studied using agar well diffusion and microbroth dilution methods against both gram negative (Escherichia coli and Pseudomonas aeruginosa, Salmonella enterica) and gram positive (Bacillus subtilis, B. cereus) bacteria. The plant extracts were prepared by cold as well as hot extraction methods. All the extracts showed varied degree of potent antibacterial activities in dose-dependent manner. Maximum zone of inhibition was shown by ethanolic extracts of A. thomsonii, P. denticulatum and P. appendiculatum against E. coli, P. aeruginosa and S. enterica respectively. The bioactivity of all the plant extracts in most of the solvents, was comparable to the broad range antibiotic, chloramphenicol. The results clearly proved the biocontrol potential of tested extracts to inhibit clinical strains of pathogenic bacteria. *Corresponding author INTRODUCTION The use of antibiotics is one of the most common means of treatment of human diseases. However, prolonged use of drugs slowly develops resistance in microbial pathogens. In addition, it also causes hypersensitivity, immune system disorder, allergic reaction and toxicity. Therefore, in recent times, major search for antimicrobial agents has been shifted to plants(asakawa,2008). Conservative estimates suggest that about 10% of all flowering plants on earth have at one time, been used by local communities throughout the world but only 1% have gained recognition by modern scientists(kafaru,1994). Lower plants, particularly bryophytes are rarely been investigated scientifically for therapeutic potential though the Chinese, Europeans, and North Americans have used bryophytes as traditional medicine since time immemorial. The bryophytes, with > species worldwide, are the most diverse group of land plants except for the flowering plants(mishler,2001). Bryophytes are used in China for treatment of ulcer, bronchitis, skin diseases and burns(asakawa,1998). Plagiochasma appendiculatum is ethnomedicinally known to be used by the Gaddi tribe of Himachal Pradesh for treatment of skin infections(kumar et al., 2000). The bioactivity of bryophytes is due to several compounds like terpenoids, phenolics, flavonoids, glycosides and fatty acids. Most of the sesqui- and di- terpenoids (bibenzyls, bis-bibenzyls, benzoates, cinnamates and phenols etc.) found in liverworts are enantiomers of those found in higher plants. At present, over 400 new compounds have been isolated from bryophytes which are responsible for different unique activities(asakawa,2008). Despite their good diversity, ethnomedicinal use and nature of unique compounds, their medicinal importance is negligibly explored. There is not much information available in literature on therapeutic uses of bryophytes. The present communication deals with the preliminary investigation of the in vitro antimicrobial potential of crude extracts of three bryophytes viz., A. thomsonii, P. denticulatum and P. appendiculatum against important bacterial pathogens with an idea to find an alternative therapeutic source. MATERIALS AND METHODS Fresh plants of Plagiothecium denticulatum (Hedw.)Schimp. was collected from Ranikhet(N 79º " and E 29º ") at an altitude of 1780m, Anaectangium thomsonii Mitt. from Mukteshwar (N 29º and E 79º ) at an altitude of 2164m and Plagiochasma appediculatum Lehm. and Lindenb. were collected from Cheena peak in Nainital (N 29º "and E 79º ") at an altitude of 2234m, in June The plants were identified by Dr. S. D. Tewari, Kumaon University and authenticated by consulting NBRI Herbarium Lucknow. Voucher specimens were submitted to the Department of Biological Sciences, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, India. 2231

2 KAVITA NEGI AND PREETI CHATURVEDI Preparation of plant extract The plant thalli with rhizoids were thoroughly washed under running tap water, shade dried, pulverized and extracted by cold extraction(ce) (10 g/100 ml) in 80 % of the solvents viz ethanol, methanol, acetone and water. The residue which was left over after cold extraction was again used for hot extraction (SE) by Soxhlet apparatus. The percentage yields of crude extracts of bryophytes (in different solvents) are given in Table 1. The extracts were filtered and concentrated using rotary evaporator(biogen). Different concentrations of the crude extracts (100, 400, 700 and 1000 μg ml -1 ) were prepared and used for further study. All the test bacteria viz., Escherichia coli(mtcc 443), Pseudomonas aeruginosa(mtcc 424) (Gram ve), Bacillus subtilis(mtcc 441) and B. cereus(mtcc 430) (Gram+ve) were obtained from Institute of Microbial Technology, Chandigarh. Salmonella enterica(gram ve) was kindly provided by Microbiology Lab, College of Veterinary Sciences, G.B. Pant University of Ag.and Tech., Pantnagar. The bacterial cultures were revived by incubating for 24 hrs in nutrient broth(nb)at 37 ± 2ºC and then 1ml. of culture was transferred in nutrient agar(na) and incubated again for 24 hrs. Wells were made by using sterile cork borer(6mm). Antibacterial assay was done by agar well diffusion method (Kumar and Chaudhary,2010). 40 µl extract of different conc. in different solvents(organic as well as aq.) was pipetted out into the wells and plates were incubated at 37ºC for 24 hrs. Microbial growth was determined by measuring the diameter of zone of inhibition (ZI) in mm. Antibiotics viz., streptomycin, tetracycline and choloramphenicol were used as positive control and respective solvents were used as negative control. Broth dilution test was used to determine the minimum inhibitory concentration (MIC) of different extracts. Overnight culture of test bacteria grown in nutrient broth was diluted 100 folds in NB(100 μl of inoculum suspension ( CFU/ ml) in 10 ml NB)(Gahtori et al.,2011). Test solutions were prepared by two fold serial dilution of stock solutions (1000ì g ml -1 ) and were added in test tubes already containing broth media. All tubes with bacterial cultures were incubated at 37ºC for 24 hrs and visible turbidity and optical density of cultures were determined at 620 nm using UV-Vis spectro photometer(genesys). The lowest concentration that inhibited visible growth of tested organisms was recorded as MIC and test culture without any visible microbial growth was taken as minimum bactericidal concentration (MBC). All the experiments were performed in triplicates. Values were represented as mean ±SE. ANOVA (three way analysis of variance) revealed significant differences between different concentrations of different solvents using SPSS 15.0 at P < 0.05(Snedecor and Cochran, 1989). RESULTS AND DISCUSSION The yield of plant extract is a very essential aspect for its pharmacological use. In most of the cases, the hot extraction method(se) was found more productive than cold extraction(ce), except ethanolic extraction of P. denticulatum(ce = 3.0 % and SE=2.5 %)and A. thomsonii(ce = 1.8% and SE =1.0%)(Table 1). Among the tested solvents, alcoholic extracts had better efficacy in terms of higher percent yield(table 1). A stronger and broader spectrum of antimicrobial activity was also observed in ethanolic and aq. extracts of all the bryophytes (Table 2, 3). However, Bissa(2015) observed maximum antibacterial activity in petroleum ether extract of Ephedra foliata. In the present study, P. appendiculatum proved most efficient followed by A. thomsanii and P. denticulatum. Among all the test organisms, S. enterica was found to be most sensitive(zone of inhibition=27.66mm), P. aeruginosa(24.33mm) came next, followed by E. coli (23.66mm), B. cereus (23mm) and B. subtilis(22.66mm) to ethanolic extract of P. appendiculatum(table2). The ZI produced by most of the organic and aq. extracts was greater than ZI produced by choloramphenicol. ZI obtained in different extracts was more or less similar to ZI produced by tetracycline. However, ZI of alcoholic extracts was higher than ZI of streptomycin against P. aeruginosa and S. enterica. Singh et al. (2011) also reported that the ethanolic extract of P. appendiculatum was more inhibitory to gram negative than to gram positive bacteria. In the present study, P. appendiculatum showed most potent activity against S. enterica followed by E. coli and P. aeruginosa(all Gram negative bacteria). Significant antimicrobial activity of ethanolic and aq. extracts of P. appendiculatum has also been found against number of bacterial and fungal microorganisms. The most potent activity was found in ethanolic solvent with increased inhibition at higher levels of extract concentration(singh et al.,2006). Mewari et al.(2007) also reported potent antimicrobial activity of alcoholic extracts of mosses. Toppo et al.(2012) also observed greater activity of methanolic extract. The present study also revealed similar trends. Increased inhibition in ethanolic extract might be due to efficient extraction of bioactive compounds viz., flavonoids, steroids, terpenoids and other polyphenolic compounds in ethanol. Varying degree of dose - dependent antibacterial activity was observed in all the extracts of P. denticulatum against different microorganisms except acetonic and aq. extracts against P. Table 1: Percentage yield of plant extracts in different solvents. Solvents Percent yield(%) Name of Species Acetone Ethanol Methanol aqueous CE SE CE SE CE SE CE SE P.appendiculum P. denticulatum A. thomsonii CE = Cold extraction, SE = Soxhlet extraction 2232

3 ANTI BACTERIAL POTENTIAL OF LESSER KNOWN HIMALAYAN MEDICINAL PLANTS Table 2 : Antibacterial activity of bryophytes in different solvents (expressed as zone of inhibition) against Gram (-ve) and Gram (+ve) bacteria. Zone of inhibition (mm) P. appediculatum P. denticulatum A. thomsonii, S T C EC Concentration Acetone Ethanol Methanol aqueousr Acetone Ethanol Methanol aqueous Acetone Ethanol Methanol aqueous 100 μg/ ml 14.66± ± ± ± ± ± ± ± ± ± ± ± μg/ ml 16± ± ± ± ± ± ± ± ± ± ± ± μg/ ml 17± ± ± ± ± ± ± ± ± ± ± ± μg/ ml 18.33± ± ± ± ± ± ± ± ± ± ± ± BC 100 μg/ ml 11.33± ± ± ± ± ± ± ± ± ± ± ± μg/ ml 11.66± ±0.0 16± ± ± ± ± ± ± ± ± ± μg/ ml 13.66± ± ± ± ± ± ± ± ± ± ± ± μg/ ml 14.66± ± ± ± ± ± ± ± ± ± ± ± BS 100 μg/ ml 15.33± ± ± ± ± ± ± ± ± ± ± ± μg/ ml 16.33± ± ± ± ± ± ± ± ± ± ± ± μg/ ml 17.66± ± ± ± ± ± ± ± ± ± ± ± μg/ ml 18.66± ± ± ± ± ± ±0.0 22± ± ± ± ± PA 100 μg/ ml 18± ± ± ± ± ± ± ± ± μg/ ml 18.66± ± ± ± ± ± ± ± ± ± μg/ ml 19.66± ± ± ± ± ± ± ± ± ± μg/ ml 21± ± ± ± ± ± ± ± ± ± SE 100μg/ ml 20± ± ± ± ± ± ± ± ± ± μg/ ml 21.33± ± ± ± ± ± ± ± ± ± μg/ ml 22.33± ± ± ± ± ± ± ± ± ± μg/ ml 24± ± ± ± ± ± ± ± ± ± SEm. CD at 5% SEm. CD at 5% SEm. CD at 5% A B C AB * * * BC AC * * * ABC Cv , No activity. *, Significant, Data are given as zone of inhibition ± SEM. of triplicates experiments. EC: Escherichia coli, BC: B. cereus., BS: Bacillus subtilis., PA: Pseudomonas aeruginosa., SE: Salmonellaenterica., S: Streptomycin (40 ì g/well); T: Tetracycline(40 ì g/well); C: Choloramphenicol (40 ì g/well) 2233

4 KAVITA NEGI AND PREETI CHATURVEDI Table 3 : Minimum inhibition concentration (MIC;μg ml 1 ) and minimum bactericidal concentration (MBC; μg ml 1 ) of different organic extracts against Gram (-ve) and Gram (+ve) bacteria. S. No. Plant extract E. coli B. cereus B. subtilis P. aeruginosa S.enterica MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC 1 PA Acetone Ethanol Methanol aqueous PD Acetone Ethanol Methanol aqueous Acetone AT Ethanol Methanol aqueous , No activity. PA = P. appediculatum, PD = P. denticulatum, AT = A. thomsonii. aeruginosa and S. enterica respectively(table 2). The ethanolic extract of P. denticulatum was found to be most active against P. aeruginosa(zi=26.33mm) followed by E.coli(ZI=23.66mm) and B. cereus (22.66 mm). All the organic and aq. extracts were found to be more effective than chloramphenicol as indicated by the higher value of ZI. Compared to tetracycline, all the plant extracts exhibited lower ZI against all the pathogens. The ZI of different extracts of P. denticulatum was found to be less than ZI of streptomycin except in ethanolic and methanolic extracts against E. coli and, ethanolic extract against P. aeruginosa. The moss A. thomsonii is widely distributed calcicole moss of the Kumaon Himalaya region. All the extracts of A. thomsanii were found to be biologically active against almost all microorganisms except for acetonic and aq. extracts against P. aeruginosa and S. enterica respectively(table2). Highest inhibition was shown against E. coli (26.67mm) followed by P. aeruginosa(23.33mm), S. enterica and B. subtilis(20.67mm). ZI was found to be significantly higher than the ZI exhibited by choloramphenicol. ZI was less than the ZI of streptomycin except in ethanolic extract against E. coli(zi=26.67mm). Here also, all the plant extracts exhibited smaller ZI compared to tetracycline. The inhibitory activity of extracts clearly indicated greater antibacterial activity than that of choloramphenicol against different microorganisms. Thus, both the mosses showed higher inhibition against Gram negative than against Gram positive bacteria. This is consistent with the study of antibacterial activity of some mosses against P. aeruginosa(savaroglu et al.,2011). Leptodictyum riparium extract was also found to be more active against gram negative than against gram positive bacteria(castaldo Cobianchi et al.,1988). Similarly, acetone extract of Rhynchostegium riparioides(hedw.) was also more active on Gram negative bacteria(basile et al., 1998). The MIC/MBC values of all the bryophyte extracts against different microorganisms ranged from 1.95 to 250 μg/ml -1 (Table3). The results of MIC experiments were in line with the results of antibacterial activity(zi). The present study clearly indicated that both liverworts and mosses showed more inhibition against gram negative microorganisms than against gram positive microorganisms. The ethanolic extracts of all the bryophytes showed broad spectrum antibacterial activity against all the tested microorganisms. The most sensitive bacteria was S. enterica (ZI =27.66±0.88) to P. appendiculatum followed by P. aeruginosa(26.33±0.33) to P. denticulatum and E. coli(26.67±0.88) to A. thomsonii. In contrast, the conventional antibiotics are more active against gram positive bacteria than against gram negative bacteria. (Basile et al., 1999). This is because the outer membrane of gram-negative bacteria contains extra layer of lipopolysachcharide membrane (LPS), which is negatively charged and gets stabilized by attachment of cations (Fe +2, Mg +2 etc.) and therefore antibiotics show resistance against gram negative bacteria. The low molecular weight antimicrobial compounds (phytoalexin or flavonoids) are synthesized at the site of infection in response to microbial activity(samanta et al., 2011; Bajaj,1996). In bryophytes, the flavonoids may interact with LPS and get transported by the porins because of their small size and act like chelating complex by addition of cations, to cause destruction of LPS. Due to this, small amount of extract having flavonoids can kill the bacteria(bodade et al., 2008, Olukoya et al., 2003). All the tested bryophytes in the present study showed significantly better antibacterial potential than the antibiotic(chloramphenicol) and were well comparable with the broad spectrum antibiotics viz., streptomycin and tetracycline. ACKNOWLEDEGEMENT We are indebted to UCOST for providing the necessary funds. We are thankful to Dr. S.D. Tewari for his kind help in identification of plants. The kind help rendered by Dr. V. Nath and Dr. A.K. Asthana, NBRI, Lucknow is also duly acknowledged. 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