ANTIMICROBIAL ACTIVITY OF ETHANOLIC AND AQUEOUS EXTRACT OF CAESALPINIA PULCHERRIMA FLOWERS

ANTIMICROBIAL ACTIVITY OF ETHANOLIC AND AQUEOUS EXTRACT OF CAESALPINIA PULCHERRIMA FLOWERS

Pushpendra S. Dhaked*, Sunil N. Kshirsagar and D. M.Sakarar

Department of Pharmacology, Sudhakarrao Naik Institute of Pharmacy, Pusad- 445204, Maharashtra, India

ABSTRACT

Caesalpinia pulcherrima (Linn.) Popularly known as Peacock flower in India belongs to family Caesalpiniace. The objective of the present work was to evaluate the in-vitro antimicrobial potency of the ethanolic and aqueous extract of Caesalpinia pulcherrima (Linn.) flower (CPF) using agar plate disk diffusion method against Escherichia coli, bacillus subtilis and streptococcus aureus. Cephalosporin (5µg/ml) use as a Standard drug. Results were subjected to minimum inhibitory concentration assay by two fold dilution method. Ethanolic extract of Caesalpinia pulcherrima was more effective against Escherichia coli, bacillus subtilis than aqueous extract and cephalosporin. While in case of streptococcus aureus aqueous extract is more effective than ethanolic extract and cephalosporin. Thus, the present study demonstrate that the traditional claim of Caesalpinia pulcherrima (Linn.) flower as an antimicrobial has been confirmed as the aqueous and ethanolic extracts displayed activity against the different microorganism  used in study

S. aureus

INTRODUCTION: Natural products perform various functions, and many of them have interesting and useful biological activities ¹. Despite of tremendous progress in human medicines, infectious diseases caused by bacteria, fungi, viruses and parasites are still a major threat to public health. There impact is particularly large in developing countries due to relative unavailability of medicines and the emergence of widespread drug resistance ².

During the last two decades, the development of drug resistance as well as the appearance of undesirable side effects of certain antibiotics ³ has lead to the search of new  antimicrobial agents mainly among plant extracts with the goal to discover new chemical structures, which overcome the above disadvantages ⁴ Current research on natural molecule and products primarily focuses on plants since they can be sourced more easily and be selected based on their ethno-medicinal uses ⁵.

With the rising prevalence of microorganism showing resistance to antibiotics, there is an urgency to develop new antimicrobial compounds. Since antiquity, plants have been used to treat common infectious diseases.

The healing potential of many plants have been utilized by Indian traditional medicines like Siddha,  Ayurvedha and Unani. Being nontoxic and easily affordable, there has been a resurgence in the consumption and demand for medicinal plants ⁶.

Caesalpinia pulcherrima (Linn.) Swartz (Leguminosae) commonly known as red bird of paradise (Peacock flower) is a medicinal herb used in the treatment of various diseases ⁷. The different parts of this herb have been used in common remedies for treatment of a number of disorders including pyrexia, menoxenia, wheezing, bronchitis and malarial infection ⁸ The plant is rich in many pharmaceutical active ingredients like flavonoids, aritonoids, glycosides and sterols ⁹.

MATERIAL AND METHOD:

Plant: The fresh flowers of Caesalpinia pulcherrima (Linn.) were collected in the month of September 2010 from its natural habitat at mudkhed village in Nanded region, Maharashtra, India. The plant was authenticated by Dr. A. Chaturvedi of Botany Department; RTM Nagpur University, Nagpur India. A voucher specimen (No: 9439) was deposited at Herbarium, Department of Botany, RTM Nagpur University Nagpur.

Material: Ethanolic and Water extracts of Caesalpinia pulcherrima (Linn.) flower, Cephalosporin, Escherichia coli, Bacillus subtilis, staphylococcus aureus.

Preparation of Extracts of Caesalpinia pulcherrima (Linn.) flower: The Caesalpinia pulcherrima (Linn) Flowers were dried under shade and undergone crushing in electric blender to form powdered and subjected to successively extraction by Pet. ether and ethanol using Soxhlet’s extractor. The percent yield of ethanolic extract was 24.8% w/w and petroleum ether (60 Grade) extract yield 6.1% w/w. Both the extracts were concentrated by evaporation at room temperature and were used for pharmacological studies. Powdered material of Caesalpinia pulcherrima (Linn.) flower was kept for maceration with 1000 ml of distilled water for 7 days. The Aqueous extract was double filtered by using muslin cloth and Whatman no. 1 filter paper and concentrated by evaporation on water bath. The extract was dried and used as a powder. The percentage yield of extract was found to be 13.56 percent.

Preparation of extract/drug stock solution: The stock solution of Caesalpinia pulcherrima flower extract was prepared on each occasion by careful weighing and dissolving in suitable volume of Dimethylsulphoxide (DMSO) to get a concentration of 100 mg/ml. A tablet of cephalosporin was dissolved in appropriate volume of water to get 5 mg/ml of stock solution.

Culture media: The media employed for the study was: Nutrient agar.

Test microorganisms: Clinical isolates of Escherichia coli, Bacillus subtilis, staphylococcus aureus were obtained from the Department of Pharmaceutical Microbiology, University of Nigeria, Nsukka.

Sterilization of materials: The petri dishes and pipettes packed into metal canisters were appropriately sterilized in the hot air oven at 170°C for 1 h at each occasion. Solution of the extract and culture media were autoclaved at 121°C for 15 min.

Preparation of culture media: All culture media were formulated according to manufacturers’ specification. Basically for nutrient agar, this involves appropriate weighing of nutrient agar, distributing into bijou bottles (in 50 ml) and then sterilization using autoclave at 121°C, 151 b/sq. inch for 15 min; then allowed to cool to 45°C before pouring into the agar plate. The pH of the agar medium was maintained at 7.4.

Maintenance and standardization of test organisms: The organism (E. coli, B. subtilis, S. aureus) was maintained by weekly sub culturing on nutrient agar slant. Before each experiment, the organism was activated by successive sub culturing and incubation. Standardization of the test microorganism was according to previously reported method ¹⁰.

In vitro determination of antibacterial activity: Stock cultures were maintained at 4°C on slants of nutrient agar. Active cultures for experiments were prepared by transferring a loopful of colonies from the stock culture to peptone water and incubated for 4h at 37°C. Antibacterial activity was determined by agar disc diffusion method ¹¹. The medium was sterilized by autoclaving at 120°C (15 lb/in2). About 30 ml of the medium (nutrient Agar Medium) with the respective strains of bacteria was transferred aseptically into each sterilized Petri plate.

The Plates were left at room temperature for solidification. Each plate, a single well of 6 mm diameter was made using a sterile borer. The extracts were freshly reconstituted with suitable solvents (Dimethyl Sulphoxide) and tested at various concentrations. The samples were placed in 6-mm diameter well. Antibacterial assay plates were incubated at 37±2°C for 24 h, Standard disc (6 mm diameter) with cephalosporin (5μg/ml) was used as a positive control for antibacterial activity Plates were kept in laminar flow for 30 minutes for pre diffusion of extract to occur and then incubated at 37^oC for 24 hours. Resulting zone of inhibition was measured using a Hi media zone scale ¹².

Determination of Minimum Inhibitory Concentration (MIC): The minimum inhibitory concentration values were determined by broth dilution assay. Varying concentrations of the extracts (100, 50, 25, 12.5, 6.25, 3.125, 1.56 mg/ml) were prepared. 0.1mL of each concentration was added to each 9mL of nutrient broth containing 0.1mL of standardized test organism of bacterial cells.

The tubes were incubated at 37°C for 24h. Positive controls were equally set up by using solvents and test organisms without extracts. The tube with least concentration of extract without growth after incubation was taken and recorded as the minimum inhibitory concentration ¹³.

RESULTS AND DISCUSSION: In the present study, antimicrobial activity of was evaluated against E. coli,  B. subtilis and S. aureus was evaluated by agar well diffusion method. Ciprofloxacin was used as standard and distilled water was used as negative control. As revealed from Table 1, the Caesalpinia pulcherrima aqueous extract and ethanolic extract were found to be effective against  Escherichia coli, bacillus subtilis and streptococcus aureus with zone of inhibition. Ethanolic extract of Caesalpinia pulcherrima was more effective against Escherichia coli, bacillus subtilis than aqueous extract and cephalosporin. While in case of streptococcus aureus aqueous extract is more effective than ethanolic extract and cephalosporin. Minimum inhibitory concentration of the active extracts is shown in Table 1.

TABLE 1: ANTIMICROBIAL ACTIVITY OF CAESALPINIA PULCHERRIMA (LINN) FLOWER.

Results were subjected to minimum inhibitory concentration assay by two fold dilution method. Lower Minimum inhibitory concentration of the extract shown more zone of inhibition against the bacterial species. Lower MIC is shown by the ethanolic extract of caesalpinia pulcherrima.  Results of these kinds herald an interesting promise of designing a potentially active antibacterial synergized agent of plant origin. There might be several reasons for the lower antibacterial activity shown by the plant extracts as suggested by Parekh and Chanda ¹⁴, either the plant part used or the type of extraction might have resulted in the lower activity in this study, or the time of collection of herbal material and climate, which might, inturn, affect the amount of active constituents in the plant material.

The difference in potency shown by the plant extracts may be due to different sensitivity of food associated strains, difference in concentrations, methods of extraction used in the study and the little diffusion properties of these extracts in the agar as suggested by El- Astal et al ¹⁵. Although the plant extracts in the present study have shown higher antimicrobial activity, they still might be considered as potential drug agents for curing infectious diseases.

ACKNOWLEDGEMENTS: The authors are grateful thanks to Miss Dr. D. V. Dhanorkar, Head of department of microbiology in Institute of sudhakarrao institute of pharmacy pusad, Amravati University, Amravati for providing suggestion to carry out research work.

REFERENCES:

1. Galal M., A.K. Bashir, A.M. Salih, and S.E.I., Adam, 1991. Activity of water extracts of Albiziaanthelmintica and lebbek backs against exprimental Hymenolepis diminuta infection in rats. J. Ethnopharmacol., 31: 333-337.
2. Zampini IC, Cuello S, Alberto MR, Ordonez RM, Almeida RD, Solorzano E, Isla MI, 2009. Antimicrobial activity of selected plant species from the Argentine puna against sensitive and multiresistant bacteria. Journal of Ethnopharmacology, 124: 499-505.
3. Okemo PO, Bais HP, Vivanco JM, 2003. In vitro activities of Maesa lanceolata extracts against fungal plant pathogens. Fitoterapia, 74: 312-316.
4. Bouamama H, Noel T, Villard J, Benharref A, Jana M, 2006. Antimicrobial activities of the leaf extract of two Moroccan Cistus L species. Journal of Ethnopharmacology, 104: 104-107.
5. Arora DS, Kaur GJ, 2007. Antibacterial activity of some Indian medicinal plants. Journal of Natural Medicine, 61:313-317.
6. Jayashree, A. and  Maneemegalai, S. 2008 . Studies on the antibacterial activity of the extracts from Tridex procumbans L and Ixoracoccinea L, Biomedicine 28: 190- 194
7. Srinivas KVNS, Rao YK, Mahender I, Flavonoids from Caesalpinia pulcherrima, Phytochemistry, 2003, 63, 789-793.
8. Chiang LC, Chiang W, Liu WC, Lin CC, In vitro antiviral activities of Caesalpinia pulcherrima and its related flavonoids, J. antimicrob Chemother, 2003, 52, 194-198.
9. Krag K, Plants used as contraceptives by the North American Indians: an ethnobotanical study, Botanical Museum, Cambridge, MA: Harvard University (1976)
10. Chinwuba GN, Chiori Go, Ghobashy AA, OkorE VC Determination of Synergy of antibiotic combination by overlay inoculum susceptibility Research. (1991) 41:14 –150
11. Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Turck, M. 1966. Antibiotic susceptibility testing by a standardized single disk method, American J of Clinical Pathology 45: 493-496.
12. Khan NH, nur-E Kamal MSA, Rahman M. Antibacterial activity of Euphorbia thymifolia Indian J Med Res., 87, 1988, 395-397
13. Atata, R.F., Sani, A. and Ajewole, S.M. 2003. Effect of stem bark extracts of Enantia chloranta on some clinical isolates,Biokemistri 15(2): 84-92.
14. Parekh J, Chanda SV. In vitro antimicrobial activity and phytochemical analysis of some Indian medicinal plants. J. Biol.,31, 2007, 53-58.
15. El-Astal ZY, Ashour AA and Kerrit A. Antimicrobial activity of some medicinal plant extracts. West Afr. J. Pharmacol. Drug Res., 19, 2003, 16-21.
    

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