Subhadip Hajra et al.

/ Journal of Pharmacy Research 2011,4(7),2432-2435

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

Assessment of antimicrobial activity of Cassia fistula and Flacoartia indica leaves

Subhadip Hajra*, Archana Mehta and Pinkee Pandey Lab of Microbiology, Department of Botany, School of Biological and Chemical Sciences,Dr. H. S. Gour Central University, Sagar (M.P). 470003, India

Received on: 12-04-2011; Revised on: 18-05-2011; Accepted on:21-06-2011 ABSTRACT

In the present study, we evaluated the antibacterial activity of alcoholic, acetone and aqueous extracts of Cassia fistula and Flacoartia indica leaves against some pathogenic bacterial strain namely Escherichia coli (MTCC 40), Pseudomonas aeruginosa (MTCC 424), Klebsiella pneumoniae (MTCC432) Staphylococcus aureus (MTCC 96), Bacillus subtilis (MTCC 619), Salmonella typhimurium (MTCC 98) and Aeromonas culicicola (MTCC 3249) as well as some fungal strain such as Aspergillus niger , Aspergillus flavus, Penicilium crysogenum, Rhizopus stolonifer and Fusarium oxosporium. Disc diffusion and Minimum Inhibitory Concentration (MIC) method were carried out to assess the antimicrobial activity. Streptomycin and Nystadin were used as the reference standard. Acetone extract of C. fistula leaves showed most significant antibacterial activity against all the tested organisms; most susceptible bacteria are Pseudomonas aeruginosa and Escherichia coli (zone of growth inhibition was 14.30.45mm and 13.50.08mm) while most resistant bacteria is Staphylococcus aureus (zone of growth inhibition was 10.00.22 mm) at the concentration of 200g/ml. The ethanolic extract of F. indica leaves showed most susceptible activity against Staphylococcus aureus and Bacillus subtilis (zone of inhibition was 19.00.04mm and18.40.24mm) respectively at a concentration of 200g/ml while Pseudomonas aeruginosa was the most resistant bacterial strain (zone of inhibition was 15.20.03mm). All the extract showed moderate antifungal activity except Fusarium oxosporium and R. stolonifer. Phytochemical analysis showed mainly the presence of alkaloids, tannins and flavonoids which may the active compounds. The result justifies the use of the plant in folk medicine. Key words: Cassia fistula, Flacoartia indica, Disc diffusion, Streptomycin, Nystadin and phytochemical.

INTRODUCTION
India is a richest source of medicinal plants in the world in regard to genetic resources of medicinal plants. The agro-climatic conditions are favorable for introducing new exotic plant varieties [1]. In India, Herbal medicines have been the basis of treatment and cure for various diseases in traditional practiced such as Ayurveda, Unani and Siddha [2]. Many infectious diseases are known to be treated with herbal remedies throughout the history of mankind. Recently, multiple drug resistance pathogenic microorganisms have been developed due to indiscriminate use of commercial antimicrobial drugs [3]. Increasing development of drug resistance in human pathogens as well as the appearance of side effect of synthetic drugs need to developed new antimicrobial drugs from natural sources. This situation has forced to search new antimicrobial substances in various sources like medicinal plants. Cassia fistula Linn. Commonly known as Banderlathi native to India, the Amazon and Sri Lanka belongs to plant family Leguminosae . It is a medium sized tree of about 10-15 meter tall. It is planted as an ornamental tree in homesteads and along the roadside. Leaves fall during cold weather and the early part of hot season. The leaflets are 4 - 8 pairs, opposite, dark-green and shining above. In the Indian literature, this plant has been described to be useful against skin diseases, liver troubles, haematemesis, pruritus, leucoderm and diabetes [4, 5]. C. fistula extract is used as an anti-periodic agent and in the treatment of rheumatism [6] and the leaf extract is also indicated for its antitussive and wound healing properties [7]. Plants also showed Antitumor [8], hepatoprotective [9] and antioxidant activity [10]. Flacourtia indica (Burm. f.) Merr. Commonly known as Baichi or Katai, belongs to the plant family Flacourtiaceae, is an indigenous medicinal plant widely distributed in Bangladesh and India [11]. This plant has been reported as an effective remedy for the treatment of a variety of diseases such as paracetamol induced hepatotoxiciy [12]. Fruits are used as appetizing and digestive, diuretic, in jaundice and enlarged spleen. Barks are used for the treatment of intermittent fever. Roots are used in nephritic colic and gum is used in cholera [13]. The present study was undertaken to evaluate the antibacterial potential of alcoholic, acetone and aqueous extract of Cassia fistula and Flacourtia indica leaves against some pathogenic microbial strain. 1.MATERIALS AND METHODS 1.1.Preparation of plant material Fresh leaves of Cassia fistula and Flacourtia indica were collected from the Grapery forest, Sagar (M.P), India. The leaves were shade dried and ground into fine powder using mechanical grinder. The powdered leaves (33gm) were soaked in 500 ml of 75% of ethanol, methanol respectively where as acetone and distilled water were used as pure and kept for 72h in a shaker. Then the extract were filtrated on filter paper and concentrated to dryness and stored at 4 0C for further studies. The extract was dissolved in Dimethyl sulfoxide (DMSO) under aseptic condition to prepare the desired dilutions. 1.2.Test microorganisms Cultures of Escherichia coli (MTCC 40) , Pseudomonas aeruginosa (MTCC 424), Klebsiella pneumoniae (MTCC432) Staphylococcus aureus (MTCC 96), Bacillus subtilis (MTCC 619), Salmonella typhimurium (MTCC 98) and Aeromonas culicicola (MTCC 3249) as well as fungi such as Aspergillus niger , Aspergillus flavus, Penicilium crysogenum, Rhizopus stolonifer were collected from the Institute of Microbial Technology, Chandigarh, India. 1.3.Evaluation of antimicrobial activity The antimicrobial assay of different solvent extract was performed by agar disc diffusion method [14, 15]. The microorganisms was activated by inoculating a loopful of the strain in the nutrient broth (30 ml) and Potato Dextrose Broth, incubated for 6 h to maintain McFarland standard turbidity (10 6cells/ ml). Then 0.1 ml of inoculums was inoculated into the molten Muller Hinton agar (Hi-media) and Potato Dextrose Agar media, spread uniformly into the Petri plate. Then the test compound (40l) was introduced on the disc (6mm) (Hi-Media) and allowed to dry. Then the disc was impregnated on the seeded agar plate. Dimethyl sulfoxide (DMSO) was used as a negative control while streptomycin and Nystadin was used as a positive control. The plates were done in triplicates and were incubated at 37C for 24 h for bacterial strain while for fungal strain incubated at 28C for 48 h. The antimicrobial activity was taken on the basis of diameter of zone of inhibition. The mean of three readings is presented. 1.4.Determination of minimum inhibitory concentration (MIC) The agar dilution method was used to determine the minimum inhibitory concentration (MIC) of all the solvent extract. Different solvent extracts were dissolved in Dimethyl sulfoxide (DMSO) and prepared the desired dilution. 1 ml each of the extracts was added to sterile molten nutrient agar and PDB media, mixed thoroughly and poured into pre-labeled sterile Petri dishes, a loop full of the standardized microbial culture was used to inoculate the plates which were incubated at 37C for 24 h and 28C for . Growth of organisms on each concentration was checked to determine the minimum concentration that inhibits growth of test organism.

*Corresponding author.
Subhadip Hajra Lab of Microbiology Department of Botany School of Biological and Chemical Sciences Dr. H.S. Gour Central University Sagar (M.P). India Tel.: 09752717291 E-mail:dip.microworld@gmail.com

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1.5.Determination of minimal bactericidal concentration (MBC) Samples were taken from the nutrient agar plates that showed no visible growth after 24 h incubation and sub cultured into freshly prepared sterile nutrient agar and PDA medium. The least concentration that did not produce growth after 24 h was regarded as the MBC. 2.RESULT AND DISCUSSION Results of the antimicrobial screening of the different concentrations of the extracts have been tabulated in Fig. 1, 2, 3, 4, 5, 6, 7, 8 and table 3, 4. The results showed that increase in concentration of extract increased the zone of growth inhibition. The four different solvent extracts of C. fistula leaves, highest bacterial zone of growth inhibition was exhibited by the acetone extract as compared to alcoholic and aqueous extract. Aqueous extract did not inhibit the growth of E. coli, K. pneumoniae and Bacillus subtilis at any of the tested concentrations. Ethanolic extract was most susceptible against Staphylococcus aureus and Bacillus subtilis (zone of growth inhibition was 14.00.34mm and 13.10.21mm) while most resistant bacterial strain is E. coli (zone of growth inhibition was 08.30.43mm) at the concentration of 200g/ml. Methanolic extract was most susceptible against Staphylococcus aureus and Pseudomonas aeruginosa (zone of growth inhibition was 13.70.35mm and 11.40.29mm) while most resistant bacterial strain is B.
Table 1: Antibacterial potential of Streptomycin and DMSO
Test Organisms E. coli S. typhimurium K. pneumonia S. aureous B. subtilis P. aeruginosa A. culicicola Streptomycin (+ control) 26.5 0.34 24.24 0.32 27.67 0.09 28.45 0.18 25.00 0.25 22.78 0.21 28.09 0.46 DMSO (-control) 00 00 00 00 00 00 00

Zone of inhibition (mm)

16 14 12 10 8 6 4 2 0
E.c oli

Antibacterial activity of acetone extract of C. fistula l e a v e s

25 g/ml 50 g/ml 100 g/ml 200 g/ml

S. typ hi K. pn eu mo nia B. su bti lis P. aer ug ino sa S.a ure us A. cu lic ico la

Concentration g/ml

Fig.3.Antibacterial activity of acetone extract of C. fistula leaves

Zone of inhibition (mm)
12 10 8 6 4 2 0
E.c oli S.a ure us S. t yp hi K.p ne um on ia P. ae rug ino sa A. cu licic ola B. su bt ilis
Antibacterial activity of water extract of C. fistula leaves

25 g/ml 50 g/ml 100 g/ml 200 g/ml

Zone of inhibition (mm)

Table 2. MIC values of alcoholic, acetone and aqueous extracts of C. fistula and F.indica leaves against bacteria tested
Bacteria *Minimum Inhibitory Concentration (g/ml) Ethanolic Methanolic Acetone Aqueous C. fistula F. indica C.fistula F.indica C. fistula F. indica C. fistula F.indica 5.75- 6 6.25 6.0 5.75 6.25 ND ND 2.50-3 3.25 3.0 2.5 2.25 3.75 4.0 6.50 6.25 6.25 6.25 ND ND ND 5.25 ND 5.5 5.0 2.0 4.25 4.25 4.75 ND 5.5 6.50 5.5 4.25 4.75 6.5 ND ND 5.5 6.25 ND 6.25 ND ND ND ND ND ND ND ND ND ND ND ND ND ND

Concentration g/ml Fig.4. Antibacterial activity of aqueous extract of C. fistula leaves

Antibacterial activity of ethanolic extract of F. indica leaves

E. coli S. typhimurium K. pneumonia S. aureous B. subtilis P.aeruginosa A. culicicola

20 18 16 14 12 10 8 6 4 2 0
E.c oli S.t yp hi K. pn eu m on ia S.a ure us B. su bti lis P.a eru gin osa A. cu lici co la

25 g/ml 50 g/ml 100 g/ml 200 g/ml

* = Mean of 3 determinations; ND = Not Determined since there was no inhibitory activity

A n t i b a c t e r i a l a c t i v i t y o f a c e t o n e e x t r a c t of F. indica l e a v e s

16
Zone of inhibition (mm)

14 12 10 8 6 4 2 0
E.c oli S. t yp hi K. pn eu mo nia S.a ure us P. ae rug ino sa B. su bt ilis A. cu lici co la

25 g/ml 50 g/ml 100 g/ml 200 g/ml

Concentration g/ml

Fig.5. Antibacterial activity of ethanolic extract of F. indica leaves

Zone of inhibition (mm)
25 20 15 10 5 0
E.c oli S. typ hi K. pn eu m on ia S.a ure us B. su bti lis P.a eru gin osa A. cu lici co la
Antibacterial activity of Methanolic extract of F. indica leaves

25 g/ml 50 g/ml 100 g/ml 200 g/ml

Concentration g/ml

Fig.1.Antibacterial activity of acetone extract of F. indica leaves

Antibacterial activity of methanolic extract of C. fistula l e a v e s

Concentration g/ml

Zone of inhibition (mm)

Fig.6.Antibacterial activity of Methanolic extract of F. indica leaves

16
Zone of inhibition (mm)
Antibacterial activity of acetone extract of F. indica l e a v e s

16 14 12 10 8 6 4 2 0
E.c oli S. t yph i K.p ne um on ia S.a ure us B. su bt ilis P.a eru gin osa A. cu lici co la

25 g/ml 50 g/ml 100 g/ml 200 g/ml

14 12 10 8 6 4 2 0
S. typ hi K. pn eu m on ia E.c oli S.a ur eu s P. ae ru gin os a A. cu lici co la B. sub tilis

25 g/ml 50 g/ml 100 g/ml 200 g/ml

Concentration g/ml

Concentration g/ml

Fig.2.Antibacterial activity of methanolic extract of C. fistula leaves

Fig.7.Antibacterial activity of acetone extract of F. indica leaves

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Zone of inhibition (mm)
Antibacterial activity of water extract of F. indica l e a v e s

18 16 14 12 10 8 6 4 2 0
E.c oli S.t yp hi K. pn eu mo nia S.a ure us B. su bti lis P. ae rug ino sa A. cu lici co la

25 g/ml 50 g/ml 100 g/ml 200 g/ml

typhimurium (15.20.03mm and14.20.04mm) while Pseudomonas aeruginosa was the most resistant bacterial strain. Aqueous extract was showed moderate activity against Escherichia coli and Salmonella typhimurium while extract showed no growth of inhibition against Klebsiella pneumoniae, Pseudomonas aeruginosa and Aeromonas culicicola at any of the tested concentration. Methanolic extract of F. indica leaves showed better antifungal activity as compared to other solvent extracts. The methanolic extract showed most susceptible activity against R. stolonifer (Zone of growth inhibition was 13.2 0.27mm) at the concentration of 200g/ml while aqueous extract did not showed any activity against P. chrysogenum at any of the concentrations. Based on our results, it is concluded that both the plant extract showed antimicrobial activity at the concentration dependent manner. Organic solvent extracts of both the plants showed better activity as compared to aqueous extracts and have great potential as antimicrobial compounds against microorganisms and can be used in the treatment of infectious diseases caused

Concentration g/ml

Fig.8.Antibacterial activity of aqueous extract of F. indica leaves

Fungal strain

Table 3. Antifungal activity of alcoholic extracts of C. fistula and F.indica leaves against different fungal strain
*Zone of inhibition (g/ml) Ethanolic Methanolic C. fistula Concentration (g/ml) F. indica Concentration(g/ml) C. fistula Concentration (g/ml) F. indica Concentration(g/ml) 25 50 100 200 25 50 100 200 25 50 100 200 25 50 100 200 ND 3.2 6.2 2.8 4.1 4.1 5.1 8.2 4.9 6.2 7.2 8.9 11.5 7.1 8.6 9.4 10.2 13.2 9.6 10.1 ND 2.2 4.4 ND 6.2 04.4 04.8 6.2 ND 8.5 06.3 07.4 8.5 5.4 10.2 9.2 11.5 12.2 7.2 13.2 ND ND 4.5 ND ND ND 3.2 7.2 ND 5.2 5.1 5.1 9.1 ND 7.1 7.2 7.2 11.5 5.1 8.9 ND 4.2 6.2 5.2 5.4 ND 6.8 8.2 7.1 5.2 4.2 7.9 11.4 9.1 8.2 7.2 10.2 13.2 10.3 9.1

A.Niger A.Flavus R. stolonifer P. chrysogenum F. oxosporium

* = Mean of 3 determinations; ND = Not Determined since there was no inhibitory activity

Table 4. Antifungal activity of acetone and aqueous extracts of C. fistula and F.indica leaves against fungal strain
Fungal strain Zone of inhibition (mm) Acetone Aqueous C. fistula Concentration (g/ml) F. indica Concentration(g/ml) C. fistula Concentration (g/ml) F. indica Concentration(g/ml) 25 50 100 200 25 50 100 200 25 50 100 200 25 50 100 200 ND ND 4.1 5.1 ND ND ND 5.2 7.2 6.3 ND ND 6.6 9.9 8.6 ND 6.1 8.9 12.1 10.2 ND 3.7 4.2 ND 4.1 ND 5.8 5.2 ND 5.2 ND 8.2 7.4 6.1 7.4 ND 10.1 9.2 8.2 10.2 ND ND ND ND 4.4 ND ND ND ND 6.2 5.2 4.1 6.2 ND 8.4 8.2 7.2 9.1 ND 10.2 ND ND ND ND 3.2 ND ND ND ND 5.2 3.1 4.2 6.3 ND 7.7 5.2 6.2 8.9 ND 8.2

A.A. niger A.A. flavus R. stolonifer P. chrysogenum F. oxosporium

* = Mean of 3 determinations; ND = Not Determined since there was no inhibitory activity Table 5. MIC values of alcoholic, acetone and aqueous extracts of Cassia fistula and F. indica leaves against tested fungal strain
Fungas Ethanolic C.fistula F.indica A.niger A.flavus R. stolonifer P. chrysogenum F. oxosporium ND 25-50 25 25-50 20 75-100 25 10-25 75-100 15-20 *Minimum Inhibitory Concentration (mg/ml) Methanolic Acetone C.fistula F.indica C.fistula F.indica ND 50-75 25 175-200 35-50 ND 25 25 25 20 ND ND ND 20 50 ND 20-25 ND 75-100 20 Aqueous C.fistula F.indica ND 75-100 ND ND 25 ND 100-150 ND 75-100 25

*=Mean of 3 determinations; ND = Not Determined since there was no inhibitory activity subtilis (zone of growth inhibition was 10.20.23mm) at the concentration of 200g/ml. Acetone extract showed most significant antibacterial activity against all the tested organisms; most susceptible bacteria are Pseudomonas aeruginosa and Escherichia coli ( zone of growth inhibition was 14.30.45mm and 13.50.08mm ) while most resistant bacteria is Staphylococcus aureus (zone of growth inhibition was 10.00.22 mm) at the concentration of 200g/ ml. In case of antifungal activity ethanolic extract showed most susceptible activity against R. stolonifer (zone of growth inhibition was 14.219 mm) while aqueous extract did not showed any activity against P. chrysogenum at any of the tested concentrations. Phytochemical studies indicates that presence of tannins, saponins and alkaloids and flavonoids in the extract inhibit the growth of the test bacterial strain [16]. Tannins inhibit the cell protein synthesis by the formation of irreversible complexes with proline-rich proteins [17]. This activity was exhibited against test organisms with the plant extracts. On the other hand ethanolic extract of Flacoartia indica leaves exhibited a significant antibacterial activity against all the tested organisms as compared to methanolic, acetone and aqueous extract. All the extract showed concentration dependent activity against all the tested organisms. The ethanolic extract of this plant showed most susceptible activity against Staphylococcus aureus and Bacillus subtilis (zone of inhibition was 19.00.04mm and 18.40.24 mm) respectively at a concentration of 200g/ml while Pseudomonas aeruginosa was the most resistant bacterial strain (zone of inhibition was 15.20.03mm). Methanolic extract showed significant antibacterial activity against all the tested organisms except Salmonella typhimurium . Acetone extract was most susceptible activity against Bacillus subtilis and Salmonella by resistant microorganisms. The MIC of the all the extracts against all the tested organisms were tabulated in (Table: 2 and 5). In conclusion, the present study proved that antimicrobial potential of C. fistula and F. indica leaves. Further studies are necessary to isolate and reveal the active compounds of the extract. ACKNOWLEDGEMENTS The authors are grateful to Head, Department of Botany, Dr. H. S. Gour Central University, Sagar (M.P) for providing the laboratory facilities and University Grant Commission (UGC), New Delhi, India, for providing financial assistance. REFERENCES
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Source of support: UGC, New Delhi, India; Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 7. July 2011

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