SEMINAR ON Antibacterial activity of crude seed extracts of Buchholzia coriacea E. on some pathogenic bacteria.

Antibacterial activity of crude seed extracts of Buchholzia coriacea E. on some pathogenic bacteria. ABSTRACT The antibacterial efficacy of hot water and methanol extracts of dried seeds of Buchholzia coriacea against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Bacillus cereus, and Vibrio cholerae were determined using the Agar-gel diffusion method. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and phytochemistry of the extracts were also evaluated. Results obtained showed that the methanol extracts of the dried seed was potent, inhibiting the isolates with diameter zone of inhibition ranging from 7.0 -35.0 mm. The extracts inhibited the growth of the bacterial isolates in a concentration dependant manner with MICs ranging between 9.3 50 mg/ml, and MBCs of 4.1 -17.4 mg/ml. Phytochemical analysis of dried seed extracts revealed the presence of alkaloids, anthraquinones, carbohydrates, cardiac glycosides, flavonoids, glycosides, resins, saponin, steroidal rings, steroidal terpenes and tannins. The findings from this study could be of interest and suggests the need for further investigations in terms of toxicological studies and purification of active components with the view to using the plant in novel drug development. Key words: Antibacterial activity, phytochemical analysis, Buchholzia coriacea, bacterial isolates. INTRODUCTION Traditional medicine is widespread throughout the world and it can be described as the total combination of knowledge and practices, whether explicable or not, used in diagnosing, preventing or eliminating a physical, mental or social disease and which may rely exclusively on past experience and observation handed down from generation, verbally or written (Sofowora, 1984). Medicinal plants has been defined by WHO consultative group as any plant which in one or more of its organs contains substances that can be used for therapeutic purposes or which are precursors for the synthesis of useful drugs (Andrews, 1982). For many years medicine depended exclusively on leaves, flowers and barks of plants; only recently have synthetic drugs came into use and in many instances, there are carbon copies of chemicals identified in plants (Conway, 1973). In orthodox medicine, a plant may be subjected to several chemical processes before its active ingredient is extracted, refined and made ready for consumption while in traditional medicine a plant is simply eaten raw, cooked or infused in water or native wine or even prepared as food (Conway, 1973). Buchholzia coriacea E. (Capparidaceae) is a forest tree with large, glossy, leathery leaves and conspicuous cream white flowers in racemes at the end of the branches. The plant is easily recognized by the compound pinnate leaves and the long narrow angular fruits containing large, usually aligned seeds. In Nigeria the plant has various common names including; ‘Ovu’ (Bini), and ‘Aponmu’ (Akure). B. coriacea is found widely distributed in other African countries such as Ivory Coast and Gabon (Keay et al., 1964; Koudogbo et al., 1972). The plant’s fruit is about 5 inches long and 2 -3 inches in diameter and resembles avocado pear, yellowish when ripe with a yellow flesh containing a few large, blackish seeds about 1 inch long. They are edible and taste peppery. It has been used for years to meet a variety of illnesses; since it has been used continually over many generations it is likely that the kola (seed) actually has an effect against illnesses. The leaves and stem bark of Buchholzia in various formulations, decoctions and concoction exhibit antihelmintic, antimicrobial and cytotoxicity effects on microorganisms (Ajaiyeoba et al., 2001; Ajaiyeoba et al., 2003; Nweze and Asuzu, 2006; Ezekiel and Onyeoziri, 2009). In Ghana fresh bark of the plants were used for earache (Irvine, 1961). Despite the various reports, information on the antibacterial properties of seeds of the plants on gastrointestinal pathogens is scare. The study was therefore undertaken in order to evaluate the antibacterial activities and phytochemical profile of the crude extracts of seeds of B. coriacea on some gastrointestinal bacterial pathogens. BUCHHOLZIA CORIACEA The pictures of Buchholzia coriacea tree, leaves and seeds. It has a dense crown, large glossy leathery leaves arranged spirally and clustered at the ends of the branches, and conspicuous cream-white flowers in racemes at the end of the branches 10. In Gabon the plant Buchholzia coriacea is sometimes cultivated as a medicinal and fetish plant 11. Description Taxonomy Profile Family: Capparaceae Juss Order: Brassicales Bromhead Genus: Buchholzia engl Class: Eqissetopsida c. Agardh Specie: Coriaceae Common and Local Names Common names of Buchholzia coriacea include; wonderful cola, musk tree, Cola pime and Elephant cola. It is called; ‘Ndo’ in Mende (Sierra Leone), ‘Doe-fiah’ in Kru-basa (Liberia), ‘Eson-bese’ in Akan-asante (Ghana), ‘Banda’ in Munga (West Cameroons), ‘Esson bossi’ in Central Africa, ‘Kola Pimente’ in French, ‘6wi’ in Edo State, ‘Okpokolo’ in Igbo, ‘Uwuro’ and ‘Aponmu’ in Yoruba (Nigeria) Ethnomedicinal Uses Buchholzia coriacea has multiple medicinal values. These seed gave it its common name (wonderful kola) because of its usage in traditional medicine. The plant parts commonly eaten are the seeds which are either cooked or eaten raw 11. In Africa, it is useful in treatment of hypertension and also prevents premature aging. It is a brain food which promotes memory. In Africa, wonderful kola has the ability to stop migraine headache when applied on the forehead. The stem bark extract is applied as an enema to treat back pain. Non specified bark preparations are also applied externally against pleurisy, rheumatism, conjunctivitis, smallpox, scabies and other skin complaints. Leaf decoctions are used to treat sterility in women. Leaf infusions are applied to the eyes against filarial nematodes, and powdered or pulped leaves are applied to treat fever, ulcers, boils and haemorrhoids. Ground fruits are applied as anodyne. Fruit kernels are chewed to treat angina and nose bleeding, and fruit extracts are taken as anthelmintic. Fruit scrapings are administered to treat asthma and cough. Seed preparations are taken to treat; fever, diabetes, hypertension, cough, psychiatric disorders and impotence. Seed pulp is applied to snakebites. Seed oil is taken against menstruation problems and gastro-intestinal complaints. The bark is used as an ingredient in the preparation of arrow. The seeds which have a peppery taste are used as a substitute of capsicum pepper. In Côte d’Ivoire the seed is chewed as a substitute for kola nuts. The wood is sometimes used in house construction 13, 15 . ETHNO-PHARMACOLOGICAL PROPERTIES Anti-diabetic properties The methanol seed extract of B. coriacea had been shown to elicit hypoglycaemic effects, which exhibited synergistic actions with metformin, a standard oral hypoglycaemic agent. Oral administration of B. coriacea at 100, 200, 400 mg/kg doses per oral (po) exhibited percentage blood glucose reduction (PBGR) of 37.73, 12.30 and 11.30% respectively after 4 hours treatment. The combination of extract (100 mg/kg) and metformin (100 mg/kg) gave a PBGR at 4th and 7th day treatment of 73.4 and 72.2% respectively 16. In streptozotocin-induced diabetic rat’s model, oral treatment with 150, 300 and 600 mg/kg of methanol fruit extract of Buchholzia coriacea caused significant dose dependent decrease in fasting blood glucose values. The serum concentration of catalase and reduced glutathione were significantly higher in rats treated with Glibenclamide (2 mg/kg) and various doses (150, 300 and 600 mg/kg) of the extract in contrast to the values in negative control rats. The extract also decreased serum triglyceride and total serum cholesterol levels. The fruit extract dose dependently reduced lipid peroxidation in diabetic rats 17. Study carried out by Adisa, et al., (2011) to evaluates the possible hypoglycemic activity and ameliorative effects of oral administration of ethanol extracts (EEBC) and butanol fraction (BFBC) of Buchholzia coriacea seeds in streptozotocin (STZ)-induced diabetic mice and rats showed significantly decreased (P<0.05) fasting blood glucose (FBG) in hyperglycemic mice and normoglycemic rats within 4 and 12 h, respectively after extract administration. Administration of the extract and glibenclamide caused a significant (P<0.05) reduction in FBG and serum alanine aminotransferase and aspartate aminotransferase levels as well as serum creatinine, urea, total cholesterol, triglyceride and thiobarbituric acid reactive species (TBARS) products in STZ-induced diabetic rats in diabetic rats. Treatment with extract and glibenclamide significantly increased serum superoxide dismutase activity. Adisa et al (2011) proposed that B. coriacea seeds contain a potent hypoglycemic and antioxidant agent suggested to be a flavone glycoside concentrated in BFBC which may find clinical usefulness in ameliorating diabetes-induced secondary complications 18. MATERIALS AND METHODS Plants collection The seeds of B. coriacea were collected from Awka, Nigeria and were authenticated by a Taxonomist at the International Institute of Tropical Agriculture (IITA) Ibadan, Nigeria. The seeds were air-dried for 5 days to constant weight, cut into pieces and grinded into powder using a sterile electric blender. The powder was then used for extraction of bioactive components. Extraction of plant material Aqueous (water) and organic (methanol) solvents were used for extraction of the active components of the plant part. For aqueous extraction, hot water extraction method as described by Asuzu (1986) was used. 20 g of each of the grounded seeds were extracted by successive soaking for 2 days using 40 ml of hot distilled water in a 250 ml sterile conical flask. The extracts were filtered using Whatman filter paper and the filtrates concentrated in vacuum at 60°C. The concentrated filtrate, now the extracts were then stored in universal bottles in the refrigerator at 4°C prior to use. For organic extraction, 25 g of the powdered plant part was extracted in 250 ml of 95% methanol for 6 h using the soxhlet apparatus as described by Harbone (1993). The volatile oil obtained was concentrated by evaporation using water bath at 100°C for 1 h. Preparation of crude extract Each of the extracts were reconstituted by dilution (methanol crude extract in 50% Dimethylsulphoxide (DMSO) and aqueous extracts in sterile distilled water) to various concentrations of 250, 200, 150, 100 and 50 mg/ml) as described by Akujobi et al. (2004) and used for antibacterial susceptibility testing. Photochemical screening This was carried out according to the methods described by Trease and Evans (1989). Test bacteria Clinical isolates of Bacillus cereus, Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Vibrio cholerae used for this work were collected from the Bacteriology Laboratory Center, University of Nigeria, Teaching Hospital, Enugu. The bacterial isolates were further purified by subculturing each isolate onto fresh plates of Nutrient Agar (NA). The pure isolates were identified using standard biochemical methods (Holt et al., 1994) and then maintained as described by Cruickshank et al. (1980). Determination of antibacterial susceptibility of extracts This was carried out using the agar-gel diffusion method as described by Osadebe and Ukwueze (2004). In this method, broth culture of the test isolates (0.1 ml) containing 1 x 105 cells/ml of organism was aseptically inoculated by spreading evenly onto the surface of NA plates using a bent sterile glass rod. Six wells (5.0 mm diameter) were then made in the plates using a sterile cork borer. The fifth and sixth wells served as negative and positive control. The sterile distilled water served as the negative control, ciprofloxacin used as the positive control. The bottom of the wells 1 -4 was sealed with one drop of the sterile nutrient agar to prevent diffusion of the extract under the agar. Fixed volumes (0.1 ml) of the extracts were transferred into the wells 1 -4 using a sterile Pasteur pipette. The control wells were filled with 0.1 ml of distilled water and ciprofloxacin. The plates were allowed on the bench for 40 min for pre-diffusion of the extract (Esimone et al., 1998) and then incubated at 37°C for 24 h. Antibacterial activity of the extracts were determined by measurement of the resulting zone diameters of inhibition (mm) against each test bacteria using a ruler. The experiment was carried out in triplicates and the mean values of the results were taken as antibacterial activity (Abayomi, 1982; Junaid et al., 2006). Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The MIC and MBC of the potent extracts was determined according to the macro broth dilution technique (Boron and Fingold, 1990). Standardized suspensions of the test organism was inoculated into a series of sterile tubes of nutrient broth containing dilutions (250, 200, 150, 100 and 50 mg/ml) of leaf extracts and incubated at 37°C for 24 h. The MICs were read as the least concentration that inhibited any visible growth (absence of turbidity) of the test organisms. For MBC determination, a loopful of broth from each of the tubes that did not show any visible growth (no turbidity) during MIC determination was subcultured onto extract fresh free NA plates, and further incubated for 24 h at 37°C. The least concentration, at which no visible growth was observed, was noted as the MBC. RESULTS AND DISCUSSION Results of preliminary phytochemical screening of the seed extracts of B. coriacea are shown in Table 1. Results showed the presence of alkaloids, anthraquinones, carbohydrates, cardiac glycosides, flavonoids, glycosides, resins, saponin, steroidal rings, steroidal terpenes and tannin. The presence of phytochemicals in the seed extracts (Table 1) showed that the extracts possess antibacterial properties. These results are in agreement with similar study by Ajaiyeoba et al. (2003). Table 2 shows the results of antibacterial effects of seed extracts of the plant against the test bacteria. Results showed that the activity of the extracts against the test bacteria decreased with decrease in the concentration with the methanol extracts demonstrating higher activity (35 mm, 250 mg/ml,) than the hot water extracts (3 mm, 50 mg/ml). This could be because the Mbata et al. 003 Conclusion Results of the study showed that seed extracts of B. coriacea possessed phytochemical substances that can be used as components of new antimicrobial agents. Therefore there is need for further investigations in terms 004 J. Dev. Biol. Tissue EnREFERENCES Abayomi S (1982). The State of Medicinal Plants Research in Nigeria. University of Ife Press p. 200 Ajaiyeoba EO, Onocha PA, Olarenwaju OT (2001). In-vitro antihelmintic properties of Buchholzia coriacea and Gynandropsis gynandra extracts. Pharm. Biol. 39 (3): 217-22. Ajaiyeoba EO, Onocha PA, Nwozo SO, Sama W (2003). 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