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Journal of Pharmaceutical and Allied Sciences

JOPHAS
ANTIBACTERIAL ACTIVITY OF CRUDE EXTRACT OF OCIMUM GRATISSIMUM (AFRICAN BASIL) ON CLINICAL STRAINS OF ESCHERICHIA COLI, SALMONELLA TYPHI AND KLEBSIELLA PNEUMONIAE ADEWUMI, A.A.J., *AINA, V.O., SARATU ABDULSALAM AND NKECHI, L. U. DEPARTMENT OF APPLIED SCIENCE, COLLEGE OF SCIENCE AND TECHNOLOGY, KADUNA POLYTECHNIC, KADUNA NIGERIA. *Corresponding Author E-mail: vocwummi2006@yahoo.com
ABSTRACT Ocimum gratissimum is a local plant used traditionally in Nigeria to treat ailments such as diarrheoa, pneumonia, fever etc. The antibacterial properties of ethanolic extract of Ocimum gratissimum against Escherichia coli, Salmonella typhi and Klebsiella pneumoniae were investigated using agar well/cup plate diffusion method. The percentage yield of the extract was calculated to be eighty one percent (81%) after the extraction. The result of the phytochemical screening revealed the presence of alkaloids, cardiac glycosides, saponins, tannins and steroids while flavonoids were absent. The antibacterial activity of ethanolic extract of Ocimum gratissimum revealed the greatest zone of inhibition at a concentration of 1000 g/ml against Escherichia coli, followed by Salmonella typhi. The least zone of inhibition was observed against Klebsiella pneumoniae. At 100 g/ml, Ocimum gratissimum exhibited the greatest zone of inhibition against Escherichia coli, followed by Salmonella typhi while Klebsiella pneumoniae had the least zone of inhibition. The MIC was determined using tube dilution technique and was found to be 100 g/ml while the MBC was 1000 g/ml of the extract. Keywords: Minimum inhibition concentration, Ocimum gratissimum, Escherichia coli, zone of inhibition, antibacterial

INTRODUCTION Medicinal plants have been used for centuries before the advent of orthodox medicine. Leaves, flowers, stems, roots, seeds, fruits and barks can all be constituents of herbal medicines. The medicinal values of these plants and plant parts lie in their component phytochemicals, which produce definite physiological actions on the human body. The most important of these phytochemicals are alkaloids, tannins, flavonoids, and phenolic components (1). Ocimum gratissimum (African basil) is a herbaceous perennial herb, woody at the base and belongs to the family Lamiaceae.

Ocimum gratissimum is called Efinrin by the Yorubas of the South Western part of Nigeria, Nchanwu by the Igbos and Daidoya by the Hausas. It has been reported to contain terpenoids, eugenol, thymol, saponins and alkaloids (2). Ocimum gratissimum is used in most local dishes/foods to achieve a variety of purposes in Nigeria. Nakaruma et al. (3) had studied the interaction of the plant with conventional antibiotics.
Journal of Pharmaceutical and Allied Sciences Vol. 8 No. 3 (2011) ISSN: 1596-8499 Website: http://ajol.info/index.php/jophas

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The whole plant and the essential oil have many applications in traditional medicine, especially in Africa and India. In folk medicine, it is used in the treatment of upper respiratory tract infections, diarrhea, headache, ophthalmia, skin disease, pneumoniae, cough, fever, conjunctivitis, gonorrhea, mental illness, high fever and influenza (4). Ocimum gratissimum had been reported earlier by Abdulrahman (5) to have in vitro antifungal activity against some dermatophytes. Recent studies on Ocimum gratissimum carried out by Elijoba (6) proved that the plant extract can be a source of medication for people living with Human Immuno Deficient Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS). The aim of the present study is to determine the active component(s) of Ocimum gratissimum leaf through phytochemical screening and to evaluate its antibacterial activities against Escherichia coli, Klebsiella pneumoniae and Salmonella typhi. MATERIALS AND METHODS Sample Collection and Handling Fresh Ocimum gratissimum leaves were collected from Kaduna Polytechjnic Staff Quarters, Kachia Express Road, Kaduna in April, 2010. This study was carried out between April-October, 2010 in the Microbiology Laboratories of the Department of Applied Sciences, Kaduna Polytechnic, Kaduna-Nigeria. Authentication and confirmation of the taxonomic identity of the plant material was carried out using voucher specimen in the herbarium of Biology/Microbiology Unit of Kaduna Polytechnic. The leaves were air-dried in an aerated room for 2 weeks. They were further pulverized using a mortar and pestle. The powder was stored in an air-tight bottle until required (7). 1349

Soxhlet Extraction Powdered leaves of Ocimum gratissimum (5 g) were weighed using a weighing balance. The weighed sample was then wrapped and tied in a clean white cloth which was then placed into the thimble of the soxhlet apparatus. The boiling flask of the soxhlet apparatus was filled with 250 cm3 of ethanol solvent and allowed to reflux for 3 hours. Thereafter, the thimble was carefully removed when the boiling flask was almost free of ethanol which implies solvent recovery. This was then transferred into a conical flask placed on a water bath to concentrate and yield the crude extract. The crude extract yielded was allowed to cool, and the weight noted. Bacterial Isolates The bacterial isolates namely Escherichia coli, Salmonella typhi and Klebsiella pneumoniae isolated from gastroenteritic patients were collected from Microbiology Department Laboratory of 44 Nigerian Army Reference Hospital, Kaduna, Kaduna State. The pure isolates obtained were sub-cultured, authenticated and identified by Gram staining technique prior to use. Preparation of Culture Media Nutrient agar powder (7 g) was weighed and dissolved in 250 cm3of distilled water. The homogenous mixture was sterilized by autoclaving at 1210C for 15 minutes. The slant cultures of the organisms were prepared aseptically using the sterile nutrient agar and incubated at 370C for 24 hours to obtain the pure culture of the bacterial isolates used for the bioassay. Preparation of Nutrient Broth Nutrient broth (3.2 g) was weighed, dissolved in 250cm3 of distilled water in a conical flask and mixed homogenously. A 10 cm3 volume of the homogenous mixture was transferred into 4 different

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test tubes and was inoculated at 1210C for 15 minutes (8). Preparation of Overnight Broth Culture A loopfull of the test organisms from the stored slant culture was separately and aseptically introduced into the sterile nutrient broth in the test tubes. These were incubated at 370C for 24 hours. The overnight broth cultures were used to prepare the inocula.
Preparation Solution of McFarland Standard

sterile saline was allowed to cool and 0.01 ml of each overnight broth culture of the bacterial isolates was suspended into separate test tubes containing the sterile normal saline. These served as the standard inocula which were used for the antibacterial activity testing and for determination of minimum inhibitory concentration (MIC). Antibacterial Activity Testing Several concentrations of the crude ethanolic extract of the sample were prepared as follows: To prepare 1000 l/ml, 0.2 g of the extract was accurately weighed and dissolved into 2 ml of sterile distilled water. This served as the stock solution for testing at 1000 l/ml. For concentration at 100 l/ml, 0.1 ml from the stock solution of 1000 l/ml was placed in another separate test tube and diluted with 0.9 ml of sterile distilled water, serving as the stock solution for testing at 100 l/ml. The antibacterial test of the leaf extract was carried out on the test bacteria using the agar-well diffusion inhibition test. Three wells (holes) of about 6.0 mm diameter were aseptically punched on the agar-plate using a sterile cork borer allowing at least 30 mm between adjacent wells and between peripheral wells and the edge of the Petri dish. A 0.1 ml volume of the leaf extract was then introduced into the wells in the plates. A control well was made in the centre with 0.01 ml of the extracting solvent. The plates were then incubated at 370C for 24 hours for the test bacteria. Duplicate excercises were conducted in each case. Determination of Minimum Inhibitory Concentration (MIC) The concentrations, 1000 l/ml and 100 l/ml were prepared for each extract using the stock solution. To 9 ml of each labeled sterile broth, 1 ml of the concentrated stock solution of the extract was added to make up to 10 ml. A loopfull of the inocula of the test organisms was aseptically inoculated into the broth

McFarland equivalent turbidity standard (0.5) was prepared by adding 0.6 ml of 1% barium chloride solution (BaCl2. 2H2O) to 99.4 ml of 1% sulphuric acid solution (H2SO4) and mixed thoroughly. A small volume of the turbid solution was transferred to a capped tube of the same type that was used to prepare the test and control inocula. This was then stored in the dark at room temperature (250C). Exactly 0.5 McFarland gives an equivalent approximate density of bacterial 1 x 10-8 cfu (Colony forming units). Inoculum Preparation by Direct Colony Suspension Method A small volume of sterile water was poured inside a test tube to which general colonies of the test organisms taken directly from the plate were emulsified and the suspension was adjusted to match the 0.5 McFarland standard solutions which had a similar appearance of an overnight broth culture by adding distilled water (9). Preparation of the Inocula The previously prepared overnight broth culture of each bacterial isolate was used to prepare the inocula by diluting with sterile saline solution. The sterile normal saline was prepared by weighing 0.85 g of sodium chloride (NaCl) and dissolving in 100 cm3 of sterile distilled water. Ten (10) ml each, of the solution was transferred into clean different test tubes and was autoclaved at 1210C for 15 minutes. The

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RESULTS Table 1: Percentage yield of the Ethanolic Plant Extract Plant Material Initial Weight Weight After % yield of sample(s) Extraction(g) of extract Ocimum gratissimum (African basil) Table 2: 50 40.5 81

Phytochemical Analysis of Ethanolic Crude Extract of Ocimum gratissimum (African basil) leaf Plant Bioactive Result Component Alkaloids Cardiac glycosides Flavonoids Saponins Steroids Tannins + + + + +

KEY/LEGEND + = Presence of Bioactive Component = Absence of Bioactive Component Estimation of the Bacterial Suspension using the McFarland Nephelometer Standard Test Microorganism Result Escherichia coli 30 x 108 cfu Salmonella typhi 21 x 108 cfu Klebsiella pneumoniae 18 x 108 cfu Table 4: Antibiogram of the Extract of Ocimum gratissimum against the test Microorganism of the concentration 1000 l/ml 20 16 10 Table 3:

Mean Zone of Inhibition of Plant Extract in respect used (mm) Test Microorganism 100 l/ml Escherichia coli 15 Salmonella typhi 11 Klebsiella pneumoniae 7

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Ocimum gratissium at 100 l/ml had the least antibacterial activity against Esherichia coli, Salmonella typhi and Klebsiella pneumoniae. The highest antibacterial activity was observed against Esherichia coli and Salmonella typhi at concentration of 1000 l/ml.

Table 5:

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Extract of Ocimum gratissimum Concentration of the Extract 100 l/ml 1000 l/ml No turbidity Little turbidity Turbid
No growth observed on solid media No growth observed on solid media Growth was observed on solid media

Test Microorganism Esherichia coli Salmonella typhi Klebsiella pneumoniae KEY: Turbid No turbidity Growth on solid media Lack of growth on Solid media

= = = =

Presence of microorganisms Inhibitory effects on the growth of microorganisms Ineffective action of the extract media MBC

Absence of growth which was indicated by lack of turbidity in the tube was observed in tubes at the concentration of 100 l/ml of Ocimum gratissimum against Esherichia coli and Salmonella typhi at concentration of 1000 l/ml against Ocimum gratissimum. The lowest concentration of MIC tubes that indicated no visible growth on solid media was regarded as minimum bactericidal concentration, while the lowest concentration that inhibits growth was regarded as minimum inhibitory concentration.

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mixture. This procedure was carried out for both concentrations of extract. The test tubes were plunged with sterile cotton wool and incubated at 370C for 24 hours. Determination of Minimum Bactericidal Concentration (MBC) MBC was determined by sub-culturing the test dilution on fresh solid medium and further incubated at 370C for 24 hours. The lowest concentration of MIC tubes that indicated no visible growth on solid media was regarded as minimum bactericidal concentration, while the lowest concentration that inhibits growth was regarded as minimum inhibitory concentration (10). Phytochemical Screening of the Extracts This was carried out using the method of Trease and Evans (11) for saponins, tannins, flavonoids, steroids, cardiac glycosides and alkaloids.

inhibition of the Ocimum gratissimum extracts against test isolates was observed to have a direct relationship with the concentration of the extract. The least concentration had not visible effect against the isolates while the highest concentration had the greatest zone of inhibition (Table 4). This is in agreement with the reports of Ekwenye and Elegalam (12). CONCLUSION Ocimum gratissimum leaf extract has phytochemical compounds with antibacterial activity. The plant extract contains glycosides, tannins, steroids, with alkaloid only present in the leaves and stem. The leaf extract had broad spectrum of inhibitory activity at both 1000 l/ml and 100 l/ml concentrations against Escherichia coli and Salmonella typhi which are both Gram negative organisms. ACKNOWLEDGEMENT The co-authors wish to thank the Head of Department of Applied Sciences, Kaduna Polytechnic, Kaduna-Nigeria for allowing access to the Laboratories and reagents. REFERENCES
1. Afolabi, C., Emmanuel, E.M., Ibukun, A., & Farombi, O. (2007): Phytochemical Constituent and antioxidant activity of extract from the leaves of Ocimum gratissimum. Scientific Research and Essay. 2(5)_: 163-166. Gill, L.S (2000): Ethnomedical uses of plants in Nigeria. Ibadan University press p. 276. Nakaruma, C.V., Nakaruma, T.U., Bando, E., Melo, A.F.N & Cartez, D.A.G (2002): Antibacterial Activity of Ocimum gratissimum L. essential ail. Plantamedica. 94:675-678. Begun, J. (2002): Studies on essential ails for their Antifungal properties. Preliminary screening of 35 essential ails. Journal of Science, Ind. Res. 28: 25-34. Abdulrahman, F. (1999): Studies in Natural Products. The Moraceae in

DISCUSSION The result of phytochemical screening, presented in Table 2 revealed the presence of glycosides, saponins, steroids, alkaloids and tannins as the secondary metabolites. Gill (2), reported that phytochemical screening of a plant is the basic feature that provides means of identifying authentic plant material that could be used in treating common ailments. The result of antibacterial study of the extract showed that the crude extract exhibited significant activity against the test microorganisms Escherichia coli, Salmonella typhi and Klebsiella pneumoniae. The activity exhibited by the extract may be as a result of the bioactive compounds present in the plant. The antibacterial activity was high against Escherichia coli, and Salmonella typhi and will be very useful in identifying the plant extract that can be used to treat infection due to these organisms. The zone of

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African Tradition Medicine and Management of Psychiatry in Bornu State. In: Unpublished M.Sc Thesis, Department of Chemistry; University of Maiduguri. Elijoba, A.A (2000): Studies on the antidiarrhoea activity of Ocimum gratissium and guava. University of Ile-Ife Press pp. 112-113. Sofowora, L.A. (2003): Medicinal Plants and Traditional Medicine in Africa. Spectrum Books Ltd, Ibadan. 55-71. Chitwood, .J. (2003): Phytochemical based strategy for nematode control. Annual Review of Phytopathology 40:221-249. Boyd, M.Y., Hallock, J., Cardellina, K. Manfredi., J. Crogg, D., Thomas, D., & Jato, J. (2001): Anti-HIV Michelamines from Ancistrocladus

korupensis Medicinal Chemistry. 37:17401745. 10. El-Said, F. Sofowora, E.A., Malcolm, S.A. and Hofer, A. (2000): An investigation into the efficiency of Ocimum gratissium as used in Nigerian native medicine. Planta Medica. 17: 195-200. 11. Trease, G.E. & Evans, W.C (1978): Pharmacogsy. 11th Edition, Baillirer Tindall Ltd, London p. 784. 12. Ekwenye, U.N. & Elegalam, N.N (2005): Antibacterial activity of ginger (Zingiber officinals Rosoe) and garlic (Allium sativum L.) extracts on Escherichia coli and Salmonella typhi. Journal of Molecular Medicine and Advanced Science 1(4): 411-416. Gill, L.S. (2000): Ethnomedical uses of plants in Nigeria. Ibadan University press p. 276.

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