Effect of Aqueous Extract of Acacia nilotica on Microbial and Castor Oil Induced Diarrhoea

The seeds of Acacia nilotica were evaluated for in vitro bioactivity against Escherichia coli and Salmonella typhi using agar dilution method. The aqueous seeds extract exhibited bactericidal effects against E. coli and S. typhi at 1mg/ml. The minimum inhibitory concentration (MIC) of aqueous extract of A. nilotica was 0.5mg/ml for both organisms. Five (5) Albino rats of both sexes weighing between (85-125g) were used for each experiment and diarrhoea was induced using Castor oil. The extract showed remarkable anti-diarrhoea activity which was dose dependent at 100, 200 and 400mg evidenced by decrease in volume of intestinal content significantly different at P<0.05 among the treatment. Anti-diarrhoea activity of the extract was comparable to 2mg/kgbw loperamide (standard drug) at 400mg/kgbw. The results suggest that aqueous extract of A. nilotica could be useful in the treatment of diarrhoea.


Introduction
Diarrhoea refers to a familiar phenomenon of unusually passage of loose or liquid stools about three times or more per day (WHO, 2013). The severity of diarrhoea is determined by the size and number of stools passed within a period of time, severe diarrhoea could pose risk of dehydration which may be a feature of chronic disease as body lose large amount of fluid quickly (Webmd, 2012).
Drugs such as Loperamide hydrochloride, Chloramphenicol, Metronidazole and Kaopectate useful in the treatment of diarrhoea are associated with depression of bone marrow in iron utilization, anaemia, leucopenia, unpleasant mouth taste, nausea, vomiting, constipation, dizziness and loss of appetite amongst other (Mohammed et al., 2009;Sanders et al., 2007). Since diarrhoea chemotherapy is beset with multitude of problems such as drug toxicity, adulteration and increase in drug resistance, there is an urgent need to search for new drugs that are devoid of these problems. Plants are sources of these new drugs, therefore it is necessary to carry out preliminary investigation on a medicinal plant such as A. nilotica with antidiarrhoea activity on microbial and castor oil induced diarrhoea. A. nilotica has been used as medicinal plants in parts of Northern Nigeria, West Africa and other parts of the world generally (Okoro et al., 2014). Bark of A. nilotica had been reported useful in the treatment of haemorrhage, tuberculosis and diarrhoea (New 1984). The root had been reported useful as an aphrodisiac and the flower used for treating syphilis lesions (New 1984). The ethyl acetyl fraction of A. nilotica pods was reported to contain anti-diarrhoea property (Sanni et al. 2010). In South Africa, bark of A. nilotica is useful to treat cough locally, the leave is believed to have cured potency while the bark, leaves, gum and pods are useful together as astringent because of the tannin they posses (Krause, 2005). Thus, A. nilotica seeds were selected in this research for study of its anti-diarrhoea activity in in vivo and in vitro models.

Materials and Methods
Plant Collection and Extract Preparation: The seeds of Acacia nilotica were collected from National Institute for Pharmaceutical Research and Development (NIPRD) Abuja. The plant was authenticated by ethno botanists in Herbarium Department of NIPRD. NIPRD (2009) Protocol for extraction of plant materials was used for extraction of seeds of Acacia nilotica. The seeds were washed, rinsed and air dried at a room temperature (28±2°C) in a shade for a period of two weeks. The seeds were blended into powder using blender and the powdery form was used for herbal preparation. One hundred gram (100g) of the powdered seeds of the plant sample was extracted in 800ml of sterile distilled water by refluxing for six hours. The resulting mixture was then filtered with muslin cloth and the filtrate was then evaporated to dryness using a rotary evaporated at 50°C. The dried extract was kept in a sterile film plastic and stored in a refrigerator until required for antimicrobial and anti-diarrhoea assay.
Laboratory Animals: Albino rats (85-125) of either sex were obtained from National Institute for Pharmaceutical Research and Development (NITRD) Vom, Plateau State. The animals were maintained in a well ventilated room in appropriately design metallic cages bedded with dry clean wood shavings. The animals were fed with standard NIPRD formulated feed and had access to clean water from Abuja municipal waste water supply. The experimental rooms were clean and disinfected regularly. Soiled wood shavings were replaced often. The feed, water containers and animal cages were washed regularly.
Experimental designs: Tijani et al. (2009) method for experimental designs of animals was used. The experimental animals were twenty five albino rats of either sex, the animals were randomized into five groups, A, B, C, D and E with each group consisting of five animals. Individual animals were weighed and randomized into cages and code was used to differentiate each. The randomization is to ensure an average distribution of weight. The animals were fasted for 24 hours and were allowed free access to water. One gram (1g) of the extract was weighed and dissolved into 10ml of distilled water to obtain 100mg of the aqueous extract. Group A was set as a negative control and receive no treatment, group E was set as a positive control and was given 2mg loperamide (a standard drug for treatment of diarrhoea) While group B, C and D were given 100mg, 200mg and 400mg of the aqueous extract two hours before castor oil was given to them.
Induction of Diarrhoea: Diarrhoea was inducted into the animals using castor oil after the animals have fasted for 24hours but allowed free access to water using Abdullahi et al. (2003) method. 1ml of castor oil was given to each animal in each group (A-E).
Standardization of Organisms: A loopful of Escherichia coli and Salmonella typhi, pure culture obtained from NIPRD was inoculated in 5ml of sterile broth and then incubated for 24hours. After incubation, 0.2ml of the inoculums was inoculated into 20ml of fresh nutrient broth. The incubation was done for 3-5 hours to obtain approximately 10 6 cfu/ml of the organism. A loopful of the standardized culture was used for antimicrobial assay (Babayi et al., 2004).
Screening of the extract for antimicrobial activity (MIC): Mueller Hilton Agar was prepared according to manufacturer's direction. One mill (1ml) of the standardized test organisms was inoculated into the molten agar poured into sterile Petri-dishes which was allowed to get at room temperature. Eight Millimetres (8mm) diameter sterile cork borer was used to make wells which base was covered with molten agar. Different concentration of the prepared extract was introduced into the wells and was allowed to pre diffuse for 30minutes. The plates were then incubated at 37°C for 24 hours, after which the inhibition zone was measured and recorded in millimetre (Mm).

Screening of the extract for Minimum Inhibitor Concentration (MIC): Minimum
Inhibitory Concentration (MIC) of the seeds aqueous extract of Acacia nilotica was determined using agar dilution method of NIPRD (2006). 200mg, 100mg, 50mg, 25mg, 12.5mg and 6.25mg of the extract was reconstituted in 5ml of sterile distilled water and vortexed for homogeneity. One mill (1ml) each of the reconstituted extract was added to Petri-dishes containing 19ml of sterile molten agar, these plates were prepared in triplicates, allowed to cool and were labelled with appropriate test organisms. The plates were streaked with a loopful of standardized test organisms (Salmonella typhi and Escherichia coli). Control plates included organisms' viability control (OVC), Extract sterility control (ESC) and medium sterility control (MSC). The plates were then incubated at 37°C for 24hours.

Screening of the extract for Minimum Bacteriocidal Concentration (MBC):
Minimum bactericidal Concentration was tested using agar dilution method of NIPRD, (2006). Fresh 19ml of nutrient agar containing no extracts were prepared and allowed to solidify. These plates without incorporated extract were subsequently inoculated with MIC plates of aqueous extract of A. nilotica showing no visible growth using sterile swab stick. Control plates were included and the plates were incubated at 37°C for 24hours.
Statistical Analysis: All experiments were carried out in triplicate. Data obtained were analyzed by one way analysis of variance and means were compared by Duncan Multiple Range Test (SPSS 20.0 version). Differences were considered significant at P ≤ 0.05.

Results
Antimicrobial activity of aqueous extract of Acacia nilotica: The in-vitro bioactivity of Acacia nilotica aqueous extract against Escherichia coli and Salmonella typhi are shown in Table 1. The crude seeds extract exhibited bactericidal effects against E. coli and S. typhi at 100mg/ml. The MIC of A. nilotica was 50mg/ml for both organisms Effects of aqueous extract of Acacia nilotica on castor oil induced diarrhoea: The aqueous extract of A. nilotica seeds was found to be effective against castor oil induced diarrhoea on experimental rats at various doses 100, 200 and 400mg/kg Table 2. The extract produced a profound decrease in intestinal transit at 100 and 200mg/kg when compared with control and anti-diarrhoeal activity was more evident at 400mg/kg when compared with control. The activity at 400mg/kg of the extract was compared with 2mg/kgbw loperamide (Positive control).

Discussion
The extraction of bioactive components from medicinal plants facilitates pharmacological study leading to synthesis of more active drugs with reduced toxicity (Gandhamathi et al., 2009 (New, 1984). The sensitivity of E. coli and S. typhi to stem bark and root methanolic extract of A. nilotica had been reported (Okoro et al., 2010). The leaves and the bark methanolic extract of A. nilotica were reported to contain potent antimicrobial property against E. coli (Kshipra et al., 2012). Diarrhoea can occur when a poorly absorbed, osmotically active substance is ingested (WHO, 06). Castor oil (1ml) treatment in rats was accompanied with watery and frequent defecation because Castor oil prevents reabsorption of water, thus making up the intestinal content and causing diarrhoea (Mekeon 1999). Oral administration of seeds aqueous extract of A. nilotica exhibited a significant dose dependent anti-diarrhoea activity as the result at 400mg/kgbw was comparable to that of the standard drug loperamide (2mg/kg). The extract significantly reduced intestinal transit as observed by marked reduction in volume of intestinal contents (16-44%) of castor oil infected rats. One of the probable mechanisms of mode of action of A. nilotica may be its ability to enhance fluid and electrolyte absorption through the gastrointestinal tract or GI motility. Besides these possibilities, the extract under investigation may contain certain components having affinity with micro receptor, which is an opioid receptor located on GI mucosa and relieves diarrhoea when activated by an agonist (Mohammed et al., 2008).

Conclusion
The result of this investigation revealed that the seeds extract of the A. nilotica exhibited significant in-vitro and in-vivo antidiarrhoea activity. These results therefore support the ethno-botanical uses of A. nilotica as anti-diarrhoea herb.