Invitro Antimicrobial Activity of Four Indigenous medicinal plants belonging to Bapatla, A.

P
M K R Konduri, K B Uppuluri*, R Chintha, S Shaik Mulla and R Peruri Department of Biotechnology, Bapatla Engineering Collge, Bapatla, Guntur, A.P, India-522101

*Corresponding author: Kiran Babu Uppuluri, E mail: kinnubio@gmail.com, Phone: +918643211335 (off), +91-9703506797(mobile), Fax: + 91-8643224246

Abstract
The antimicrobial activity of leaf extracts of four indigenous medicinal palnts (Cassia auriculata, Psidium guajava, carica papaya, and Adathoda vasica, of families Caesalpinaceae, Solanaceae, caricaceae and Acanthaceae) were evaluated on four bacterial strains

(staphylococcus aureus (MTCC 96), Escherichia coli (MTCC 738), Enterobacter aerogenes (MTCC 2990) and Serratia marscens (MTCC 2645)). The Methanol, Ethyl acetate and Aqueous extracts were obtained by soxhlet apparatus and antimicrobial activity was found using disc diffusion method. Results of phytochemical analysis indicated that out of 12 extracts (from four plants and three solvents) ethyl acetate extract of Adathoda vasica exhibited maximun antimicrobial activity against all the four tested microorganisms. Ethyl acetate extract of Adathoda vasica showed maximum inhibition (31mm) zone towards the microorganism stahphylococcus aureus and 23mm, 18mm, 15mm zones of inhibition against Enterobacter aerogenes, Serratia marscens and Escherichia coli respectively. Methanol extracts of Psidium guajava and Carica papaya also found to be effective against E. coli with zones of inhibition 27mm and 25 mm respectively. The antibacterial activity of active extracts was compared with the antibiotic, Streptomycin. Comparative studies with antibiotic streptomycin showed that ethyl acetate extract of Adathoda vasica against S. aureus, methanolic extracts of psidium guajava

and carica papaya against E.coli were found to be more effective compared to streptomycin. This study demonstrates potentials of all above plants as source of antimicrobial activities that could be harness for use in the health care delivery process. Key words: Adhatoda vasica , Psidium guajav ,Carica papaya, Antimicrobial activity, Staphylococcus aureus

Introduction Nature has been a source of medicinal agents for thousands of years and an impressive No. of modern drugs has been isolated from natural source, many based on their use in Traditional medicine. Various medicinal plants have been used for years in daily life to treat diseases all over the world1. The use of herbs and medicinal plants as the first medicine is a universal phenomenon. Every culture on earth, through written or oral tradition, has relied on the vast variety of natural chemistry found in healing plants for their therapeutic properties. One of the earliest records of herbal medicine is the use of chanlonoogra oil from species of Hydrocarpus guartin, which was known to be effective for the treatment of leprosy in china between 2730 and 300 BC
2,3

defined

medicinal plant as any plant in which one or more of its organs contain substances that can be used for therapeutic purpose or as precursors for pharmaceutical synthesis. Herbal medicine is still the main stay of about 75% to 80% of the whole population mainly in developing countries, for primary health care because of better Cultural acceptability, better Compatibility with human body and feral said effects4 .However there has been a revival of interest in herbal medicines and their use in the development nations. India is a varietal emporium of medicinal plants and is one of the richest resources of medicinal plants. It exhibits a wide range in topography and climate, which has a varying (or) its visitation and floristic composition. More ever the agro climate conditions are conducive for interdicting and demonstrating new exotic plant verities[5]. Infection diseases are the leading causes of death worldwide. The use of antibiotics has revolutionized the treatment of various bacterial infections; however their indiscriminate use has

led to an alarming increase in antimicrobial resistance among microorganisms

[15]

. And also

recent appearance of strains with reduced susceptibility to antibiotics raises the specter of untreatable bacterial infections and adds urgency to the search for new infection fighting strategies[16]. One way to prevent antibiotic resistance of pathogenic species is by using new compounds that are not based on existing synthetic antimicrobial agents [17]. Many infectious diseases have been known to be treated with herbal remedies throughout the history of mankind. Natural products either as pure compounds or as standardized plant extracts provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity. Thus, it is anticipated that phytochemicals with adequate antibacterial efficacy will be used for the treatment of bacterial infections[18]. Since time immemorial, man has used various parts of plants in the treatment and prevention of various ailments[19].In the recent years, secondary plant metabololites (phytochemicals), previously with unknown

pharmacological activities, have been extensively investigated as a source of medicinal agents

[20]

We chose four different medicinal plants used in folk medicine to determine their antimicrobial activity: Cassia auriculata, psidium guajava, carica papaya, and Adathoda vasica. In general these plants are used in folk medicine in the treatment of skin disease, infected wounds and nervous disorders. Evidently there are not sufficient studies that confirm the antimicrobial properties of these plants. This study looks into the Invitro antimicrobial activity of these plants against four pathogenic microorganisms that cause the most common cases of water and food born infections. Materials and methods

Plant materials Cassia auriculata, Psidium guajava, carica papaya, and Adathoda vasica were obtained from Bapatla; a rural area in Guntur district, Andhra Pradesh, India. These are the plants commonly used by the local people of Bapatla for the treatment of wound infections Taxonomic identification of plants was done at Department of Botany, Bapatla College of Arts and Sciences, Bapatla, Guntur district, Andhra Pradesh, India based on physical (Macro and micro) characters. Fresh plant material was washed under running tap water, air dried and then homogenized to fine powder and stored in airtight bottles. Preparation of extracts The powdered plant materials (50g of each) were separately extracted with 200 ml of methanol and 200 ml ethyl acetate using a soxhlet apparatus for 8 h. The obtained solvent extracts were filtered through a rotary evaporator and dried using freeze dryer. For aqueous extracts, the powdered plant materials (50g of each) were separately extracted with 100 ml water by using a shaking water bath at 70oC for 2 h. The obtained aqueous extracts were filtered through a rotary evaporator and dried using freeze dryer. All the extractions were repeated thrice. The dried extracts were stored at -200 C until used. Microorganisms Staphylococcus aureus (MTCC 96), Escherichia coli (MTCC 738), Enterobacter aerogenes ( MTCC 2990) and Serratia marscens (MTCC 2645) were obtained from MTCC, Chandigarh, India. All the cultures were grown overnight in nutrient medium and stored on nutrient agar slants at 4o C until further use.

Phytochemical Analysis A small portion of the dried extracts were used for phytochemical screening test[6,7].The method of Harboune was implemented to identify the general phytochemical groups of compounds in the extracts as follows: Alkaloids (200 mg plant material in 10 ml methanol, filtered) 2ml of filtrate + 1 % HCl + steam, 1 ml filtrate + 6 drops of Mayers reagent/wagners reagent/Dragendorffs reagent, creamish precipite/brownish-red prcipite/orange precipitate indicated the presence of respective alkaloids. Tannins (200 mg plant material in 10 ml distilled water, filtered); a 2ml filtrate + 2 ml ferric chloride, blue black precipitate indicates the presence of Tannins. Terpenoids (Liebermann- burchard reaction: 200 mg plant material in 10 ml ethanol, filtered); a 2ml of filtrate + 2 ml acetic anhydride + Conc.H2SO4, blue-green ring indicates the presence of Terpenoids. Flavanoids (200mg plant material in 10 ml ethanol, filtered); a 2ml filtrate + conc. HCl, magnesium ribbon pink tomato red color indicated the presence of Flavanoids. Saponins (Frothing test; 0.5 ml filtrate + 5 ml distilled water); frothing persistanec indicates the presence of Saponins. Antibacterial assay: Antibacterial assay was performed by agar disc diffusion method
[8]

.The

nutrient agar was inoculated with 100 l of the inoculums (1x105 cfu/ml) and poured into Petri plate. Sterilized filter paper discs (Whatman No.1) with the diameter of 7mm were impregnated with 100 l of each extract of different concentrations viz. 2, 5, 10, 15 mg/ml. The impregnated paper discs were subsequently placed on inoculated agar plates, incubated overnight at 370 C. Microbial growth was determined by measuring the diameter of zone of inhibition. For each bacterial strain, controls were maintained where pure solvents were used instead of the extract. The effects were compared with that of standard antibiotic streptomycin at a concentration of 1

mg/ml

[9]

. The experiment was done three times and the mean values are presented. The

experiment was done three times and the mean values are presented. Minimal inhibitory concentration: A quantity of 0.05 g of each extract was dissolved in 10 ml nutrient broth to yield an initial concentration of 5mg/ml. subsequently serial dilutions were made from the stock of 10 ml containing 5mg/ml. nutrient broth was used to obtain the following concentrations 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg/ ml. one ml of a standardized inoculums of each test organisms was introduced into each extract-nutrient broth mixture and then incubated at 37o C for 24 h. The lowest concentration of the extract that inhibited the test organisms was recorded as the MIC. Results and discussion The results of the phytochemical screening of each plant extract are shown in Table 1. Tannins, alkaloids, flavanoids, phenolics, trepenoids are found to be present in ethyl acetate extract of all plants used in the study. Saponins are found to be present only in ethyl acetate extract of Cassia auriculata. Methanolic extracts of all four medicinal plants showed the presence of alkaloids, terpenoids, flavanoids, Saponins. Tannins are found to be present in methanolic extract of psidium guajava. Presence of tannins in psidium gauajava was also reported in previous studies
[9]

. Previous reports also revealed the presence of tannins, flavanoids, terpenoids and alkaloids in

all the medicinal plants used in this study [10]. The results of antimicrobial screening of the crude extracts of all plants are shown in Table 2. From Table 2 it was shown that ethyl acetate and methanolic extracts of all plants showed anti microbial activity against all microorganisms used in the study. Whereas aqueous extract of psidium guajava showed antimicrobial activity against E. coli, remaining microorganisms were

found to be resistant to all aqueous extracts. Ethyl acetate extract of Adathoda vasica showed maximum antimicrobial activity (31mm, zone of inhibition) against Staphylococcus aureus. Methanolic extracts of Psidium guajava and Carica papaya showed maximum antimicrobial activity , 28 mm, 25mm zones of inhibition respectively against E.coli. All the three extracts showed maximum activity at 15 mg/ml. Carica papaya is the traditional medicinal plant in tropics
[11]

. Former studies with Carica papaya revealed the anti parasitical activity against

Ichthyophthirius multifilis[12]. Herbs that have tannins as their main component are astringent in nature and are used for treating intestinal disorders such as diarrhea and dysentery[13].one of the largest groups of chemical produced by plants are the alkaloids and their amazing effect on humans has led to development of powerful pain killer medications
[14]

. Comparative studies

with antibiotic streptomycin have shown that ethyl acetate extract of Adathoda vasica , methanol extracts of Psidium guajava and Carica papaya are found to be effective against S aureus, and E. coli than streptomycin, which showed only 25 mm and 23mm zones of inhibition against S. aureus and E.coli at 1mg/ ml concentration. The MIC of ethyl acetate extract of Adathoda vasica against the tested organisms varied between 0.2 mg/ml and 0.5 mg/ml. The MIC of ethyl acetate extract of Cassia auriculata against tested organisms varied between 0.6 mg/ml and 0.9 mg/ml. The MIC of ethyl acetate extract of Carica papaya varied between 0.3 mg/ml and 0.5 mg/ml, while that of Psidium guajava varied between 0.4 mg/ml and 0.7 mg/ml. The MIC of methanol extract of Adathoda vasica against the tested organisms varied between 0.4mg/ ml and 0.7 mg/ ml. the MIC of methanol extract of Cassia auriculata against tested organisms varied between 0.4 mg/ml and 1.0 mg/ml. The MIC of methanol extract of Carica papaya varied between 0.3 mg/ml and 0.6 mg/ml, while that of Psidium guajava varied between

0.2 mg/ml and 0.6 mg/ml. The standard streptomycin had MIC values varying between 0.1 mg/ ml and 0.5 mg/ml. These results indicate that antimicrobial activity of A. vasica, Carica papaya and P. guajava extracts almost comparable to standard antibiotic streptomycin as shown in Table 3. The results obtained indicated the existence of antimicrobial compounds in the crude ethyl acetate and methanol extracts of these plants and some showed a good correlation between the reported uses of these plants in traditional medicine against infectious diseases. For example the inhibition of S. aureus and E.coli by the extracts of A. vasica, carica papaya and P. guajava has justified their use in for the treatment of wound infection in the traditional medicine. Conclusion All the extracts showed varying degrees of antimicrobial activity on the microorganisms tested. Some of these plants were found to be effective than traditional antibiotics like streptomycin to combat pathogenic microorganisms studied. The chance to find antimicrobial activity was more apparent in ethyl acetate and methanol than water extracts of the same plants. Two species (A. vasica and P. gaujava) presented the lowest MIC similar to antibiotic standard. These plants could be a source of new antibiotic compounds. Further work is needed to isolate the secondary metabolites from the extracts studied in order to test specific antimicrobial activity. Acknowledgements The authors are grateful to Bapatla Engineering College, Bapatla, A.P for giving financial assistance to carry out the research work. References

1.Srivastava, J., lambert, J., Vietmeyer, N. Medicinal plants: An expanding role in development. World Bank Technical Paper. No.320. 2.Le Strange, J. N., The useful plants of West Tropical Africa, The crown agents, London, 325 (1977) 3.Sofowara, A., Traditional Medicine: Practice in West Africa, Ibadan university press, Ibadan, Nigeria, 2nd edition, 3-26 (1984). 4.Parekh, J., Nair, R., chanda, S. Preliminary screening of some folklore medicinal plantsfrom western India for potential antimicrobial activity. Indian J. Pharmacol, 37: 408-409 (2005). .5.Martins, A. P., Salguelro, L., Gonclaves, M. J etal. Essential oil composition and antimicrobial activity of three Zingiberaceae from S.Tomee principle. Planta Med, 67: 580-584 (2001) 6.Trease, G. E and Evans, W. C., Textbook of pharmacognosy, Blliere, Tindall, London, 12th edn, 57-59, 343-383 (1983). 7.Harbourne, J. B., Phytochemical Methods A guide to modern technique of plant analysis, Chapmann and Hall, London, 271 (1973) 8.Bauer, R. W., Kirby, M. d. K., Sherris, J. C., Turck, M. Antibiotic susceptibility testing by standard disc diffusion method. Americal journal of Clinical Pathology, 45: 493-496 (1966). 9.Akinpelu, D. A. and Onakoya, T. M. Antimicrobial activities of medicinal plants used in folklore remedies in south western. African Journal of Biotechnology, 5 (11): 1078-1081 (2006).

10.Duraipandiyan, V., Ayyanar, M., Ignacimuthu, S. Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil nadu, India. BMC Complementary and Alternative Medicine, 6: 35, (2006). 11.Ghosal, S., Singh, S. Bhattacharya, S. K. Alkaloids of Mucuna pruriens. Chemistry and pharmacology. Planta. Med, 19: 279-284 (1971). 12.Ekanem, a. P., Obiekezie., Kloas, W., Knopf, k. Effects of crude extracts of Mucuna pruriens (Fabaceae) and Carica papaya (Caricaceae) against the protozoan fish parasite Ichthyophthirius multifiliis. 92: 361-366 (2004). 13.Dharmananda, S. gallnuts and the Uses of Tannins in Chinese Medicine A paper delivered at Institute for Traditional Medicine, Portland, Oregon (2003) 14.Raffaul, R. F., A guide to Their Discovery and Distribution, Hawkworth Press, Inc, New York, 35 (1996). 15.Westh, H., Zinn, C. S., Rosdahl, V. T etal. An international muticenter study of antimicrobial consumption and resistance in staphylococcus aureus isolate from 15 hospitals in 14 countries. Microb. Drug. resist. 10: 169-176 (2004) 16.Rojas, R., Bustamante, B., Bauer, J etal. Antimicrobial activity of selected Peruvian medicinal plants. J. ethnopharmcol, 88: 199-204 (2003). 17.Shah PM : The need for new therapeutic agents; what is in the pipe line? Clinical Microbiology and Infection. 2005, 11:36-42 18..Balandrin, M. F., kjocke, A. J., Wurtele, E etal. Natural plant chemicals: sources of industrial and mechanical materials. Science, 228: 1154-1160 (1985)

19.Tanaka, H., Sato, M., Fujiwara, S. Antibacterial activity of isoflavonoids isolated from Erythrina variegate against methicillin resistant Staphylococcus aureus. Lett. Appl. Microbiol, 35: 494-498 (2002). 20.Krishnaraju, A. V., Rao, T. V. N., Sundararaju, D etal. Assessment of bioactivity of Indian medicinal plants using brine shrimp (Artemia salina) lethality assay. Int. J. Appl. Sci. Eng, 2: 125-134 (2005).

Table 1: Phytochemical screening of leaf extracts of medicinal plants used in the study.

Plant Species Adathoda vasica

Solvents used Tannins Ethyl acetate + Methanol + Aqueous _ Ethyl acetate Methanol Aqueous Ethyl acetate Methanol Aqueous Ethyl acetate Methanol Aqueous + + _ + +++ _ + ++ _

Saponins _ _ _ _ _ _ _ _ _ _ _ _

Flavanoids Terpenoids +++ + ++ + _ + ++ _ + + + + + _ _ + + + _ + _ _ _ +

Alkaloids Glycosides ++ _ _ _ + + _ _ + + _ _ ++ ++ + + _ + + _ + _ _ _

Cassia auriculata

Psidium guajava Carica papaya

Table 2: Comparative Studies of Antimicrobial activity of leaf extracts and streptomycin. Plant name Solvents Aqueous extract Ethyl acetate Concentartion (mg/disc) Crude 2 5 10 15 2 5 10 15 Crude 2 5 10 15 2 5 10 15 Crude 2 5 10 15 2 5 10 15 Crude 2 5 10 15 2 5 10 15 20g/disc Zone of Inhibition (mm) E.coli S.aureus E.aerogenes 0 0 0 3 5 5 4 6 7 12 26 18 15 31 23 2 2 2 3 2 2 5 4 2 10 7 5 0 2 2 5 6 2 2 5 5 0 3 7 7 10 3 12 23 27 0 3 3 5 8 5 13 18 25 20 0 0 2 2 3 0 0 0 0 0 3 3 3 5 0 3 7 10 0 5 8 11 14 2 2 6 10 18 0 0 2 2 2 0 0 0 0 0 2 5 5 8 2 5 7 13 0 0 2 2 5 2 2 7 8 23 S.marscens 0 3 3 16 18 2 3 8 11 0 0 2 2 2 0 0 0 0 0 0 5 5 7 2 2 5 8 0 0 2 2 5 1 3 6 7 21

Adathoda vasica

Methanol

Aqueous extract Ethyl acetate

Cassia auriculata Methanol

Aqueous extract Ethyl acetate

Psidium guajava

Methanol

Aqueous extract Ethyl acetate

Carica papaya

Methanol

Streptomycin

Table 3: The MIC of Methanolic leaf extract of medicinal plants used in the study. Microorganism S.aureus E.coli S.marsecens E.aerogenes MIC(mg/ml) Streptomycin 0.25 0.1 0.5 0.5

A. vasica 0.40 0.55 0.45 0.7

P.guajava 0.35 0.20 0.50 0.60

C.auriculata 0.75 0.40 1.0 1.0

C.papaya 0.45 0.30 0.45 0.6

Table 4: The MIC of Ethyl acetate leaf extract of medicinal plants used in the study. Microorganism S.aureus E.coli S.marsecens E.aerogenes MIC(mg/ml) Streptomycin 0.25 0.1 0.5 0.5

A. vasica 0.2 0.35 0.50 0.50

P.guajava 0.55 0.40 0.70 0.65

C.auriculata 0.85 0.60 0.85 0.90

C.papaya 0.45 0.30 0.50 0.50