Enabulele, O.I.
Faculty of Life Sciences,Department of Microbiology, University of Benin Edo State, Nigeria.

Tchounga, K.S., Ukaji, D.C., Ajugwo’ A.O.
Department of Medical Microbiology/ Medical Mycology, Faculty of Medical Laboratory Science,
Madonna University Elele Campus, Rivers State,Nigeria
All correspondence to: ukajidamian@yahoo.com

ABSTRACT
Serotype and biotype of Vibrio cholerae isolated from stool samples collected from patients with acute diarrhea and water samples were determined during September to November 2004 cholera outbreak in Uzebba in Edo–State, Nigeria. A total of 137 stool samples and 50 water samples were investigated. The samples were subjected to standard recommended microbiological techniques and confirmation of isolates by seroagglutination using Vibrio cholerae polyvalent O1 and O139 antisera and monovalent Ogawa and Inaba antisera, while biotyping was carried out by agglutination test and sensitivity to polymyxin B. Out of 137 stool samples, 63((45.98%) were found to be positive for Vibrio cholerae serogroup O1, and of 63Vibrio cholerae O1, 60(95.24%) were Vibrio cholerae serogroups O1 serotype Inaba, biotype classical and 3(4.76%) were found to be Vibrio cholerae serogroup O1, serotype Ogawa, biotype classical. Only stream water samples yielded growth of Vibrio cholerae serogroup O1, with 3(23.7%) out of 13 stream water samples. Well and bore hole water samples yielded no growth of Vibrio cholerae. Out of three Vibrio cholerae O1 isolated from stream water samples, 2(66.67%) were found to be positive, for Vibrio cholerae serogroup O1, serotype Inaba and classical biotype, while 1(33.33%) was Vibrio cholerae serogroup O1, serotype Ogawa and El -Tor biotype. This study demonstrated that some Vibrio cholerae O1 serogroup O1 isolated from stream water and stool samples had the same serotypes and biotypes. Therefore, there is need to perform a strong epidemiological surveillance for emergence and distribution of Vibrio cholerae O1.

Key words: Vibrio cholerae O1, serotype, biotype, water supplies, stool samples.

INTRODUCTION
Cholera (frequently called Asiatic cholera or epidemic cholera) is an acute diarrheal illness caused by a Gram negative slightly curved rod Vibrio cholerae (Ryan and Ray, 2004). Cholera is a worldwide problem, especially in developing countries. It has been very rare in developed nations for hundred years; however the disease is common today in other parts of the world, including the India sub-Asian continent and sub-Sahara Africa. Cholera is a major health threat in poor nations that frequently results in mortality. Although more than 100 serogroups of Vibrio cholerae exist, only two cause human disease. Vibrio cholerae O1 of which there are two biotypes (Classical and EL Tor); classified into serotypes Ogawa and Inaba and rarely Hikojima and Vibrio cholerae O139, which emerged in 1992. Cholera world wide is usually characterized by painless diarrhea and vomitting.
In 1991, there were more than a million cases in Central and South Canada. Several new cases have since been reported in Louisiana and other Gulf Coast areas (Werdlow
et al., 2002). African countries have in recent years experienced more epidemics and (WHO, 1999). A total of 29321 cholera cases and 10586 deaths were reported to WHO in 1998, with Africa accounting for the largest part with 72% of the global total (WHO, 1999). An explosive outbreak of cholera occurred among Rwanda refugees in Goma. Democratic Republic of Congo involved about 70,000 cases and caused about 12,000 deaths in 1994 (Siddique et al., 1995). In 1995, West Africa reported 64% cholera cases and 61% cases of cholera deaths (WHO, 1999). Between July and November 2001, several outbreaks were reported in the region, including 897 cases with 47 deaths in Cote d’voire and 575 cases with 21 deaths in Kano (WHO, 2001). In 2004, another outbreak involving 1316 cases and 76 deaths were again reported in Kano, while Edo-State (Uzebba) witnessed an outbreak of cholera involving 300 reported cases with 50 deaths (WHO, 2004). The Onslaught of cholera in Africa continent continues unbeaten as another one was reported in Nigeria involving 400 cases in Jigawa State, resulting in 15 deaths in October 2009 (WHO, 2009).

Strategies for prevention and control of this infectious disease depend on understanding the origin, transmission and other characteristics associated with it epidemiology. Therefore, this study was carried out to determine the prevalent serogroup, serotypes and biotypes of Vibrio cholerae responsible for the cholera outbreak in Edo-State, (Uzebba) between September to November 2004.

Materials and Methods
Stool samples were collected from patients with acute diarrihea. During the outbreak, exclusion criteria included patients using antimicrobial agents within the previous two weeks to avoid cases of antibiotic associated diarrhea. Water samples were collected in clean containers containing alkaline peptone water (APW) from various sources of water supply (streams, boreholes and wells).

Sample Processing
Stool samples collected in plain containers were cultured directly onto Thiosulphate Citrate Bile Salt Sucrose (TCBS) agar plates and incubated at 37oc for 24 hours. Water samples inoculated in APW were incubated at 37oc for 4 to 6 hours, after the incubation period, from the inoculated APW, plating was done onto TCBS agar and the plates incubated at 37oc for 24 hours. The isolates were purified by sub-culturing single colonies on sterile Nutrient agar plates which were then incubated at 37oc for 24 hours. Following standard morphological and biochemical tests according to Buchanan and Gibbons (1974), characteristic colonies grown on the selective TCBS agar were then confirmed for identification. The series of biochemical tests commonly used to identify V. cholerae (Baumann and Schubert, 1984; West and Cowell, 1984) were originally designed for clinical samples in order to specifically detect pathogenic vibrios. The series of biochemical tests include: Gram staining, oxidase , glucose and L-arabinose , methyl red , voges-proskauer and ornithine tests were performed.

Serological Test
Serological identification of isolates was done using slide agglutination test (Sakazaki and Donovan, 1984; Shimada et al., 1994), using polyvalent Vibrio cholerae O1 and Vibrio cholerae O139 antisera and monovalent Ogawa and Inaba antisera.

Biotyping
Biotyping of Vibrio cholerae isolates was done by using agglutination with chicken red blood cells and polymixin in B (50 units) sensitivity test (WHO, 1987).

RESULTS
A total of 137 stool samples collected from hospitalized patients during cholera outbreak from September to November 2004 in Uzebba (Edo – State), and water samples 50(13 stream water, 20 borehole water and 17 well water samples) were studied.

Table 1 shows the frequency of isolation, serotyping and distribution of Vibrio cholerae. Isolated from stool and water samples in Uzebba. Out of 137 stool samples, 63(45.98%) were found positive for Vibrio cholerae O1 and 60(95, 24%) out of 63, belonged to Inaba serotype, while 3(4.76%) were Ogawa serotype. None of the samples yielded growth of Vibrio cholerae O139. Water samples were also analyzed bacteriologically, 3((23.07%) out of 13 water samples collected from streams yielded growth of Vibrio cholerae O1 and borehole and well water samples yielded no growth. Table 2 shows the distribution of Vibrio cholerae O1 biotype isolated from stool and water samples. All vibrio cholerae O1 from stool samples both Inaba and Ogawa serotypes were Classical biotype, two Vibrio cholerae O1 Inaba serotype isolated from stream water samples were Classical biotype, while only Vibrio cholerae O1 Ogawa isolated from stream water sample was EL Tor biotype.
DISCUSSION
Cholera continues to be an important pubic Health problem among many poorer and vulnerable communities despite the fact that bacteriology, epidemiology and public health aspects of the disease were described in detail over a centuary (Shears, 2001). In the study, we isolated and identified 66 Vibrio cholerae out of which 63 isolates were from stool samples and 3 isolates from water samples.It was observed after serological test that the isolates were Vibrio cholerae O1 and that there was no serogroup O139 isolates. These findings are in agreement with the report of Tamang et al.,( 2005) reported that Vibrio cholerae O1 were predominant in Nepal, also similar reports were presented by Urassa et al.,( 2000);Inaet al.,(2007) reported that during his study in Manhica District hospital Southern Mozambique, all isolates were Vibrio cholerae O1. During this study, it was noticed that two serotypes Inaba and Ogawa co-existed with Vibrio cholerae O1 serotype Inaba being the most predominant with 62(93.94%) while 4(6.06%) were Vibrio cholerae O1 Ogawa serotype; and these isolates were both from stool and water samples. Our findings are in accordance with reports of Mercy et al.,( 2004) who isolated Vibrio cholerae O1 in Ghana with Inaba serotype having the highest occurrence over Ogawa serotype. Similar report was presented by Shukla et al., ( 2006 ) who recorded an emerged predominance of Vibrio cholerae O1 serotype Inaba over Ogawa between 2004 – 2006 in East Delhi.

The co-existence of the two serotypes recorded during our study was not in conformity with Inacio et al., (2007) who reported that only Vibrio cholerae O1 serotype Ogawa was isolated in Manhica District hospital Southern Mozambique. Our findings also contradicted claims by Hossein et al., ( 2005) that after a six year study on Vibrio cholerae in South Eastern Tran, all Vibrio cholerae O1 isolated were Ogawa serotype. EL Tor was the predominant biotype causing outbreak up till 1998. But during our study in Uzebba, the Classical biotype emerged predominant that although that one of the isolates from water sample was EL Tor biotype. The high emergence of Classical biotype in Uzebba could be justified by the
reports of Rafi et al., ( 2004) also isolated Vibrio cholerae O1 Classical biotype between 2000 – 2001 in Rawalpindi. This high occurrence of Vibrio cholerae O1 Inaba and Classical biotype in our study is not in accordance with the reports of Bradley et al.,(1997), Jacques et al.,( 2002) and Iwanaga et al.,( 2004), who reported that most of the strains of Vibrio cholerae O1 isolated from Madagascar, Bangladesh, Tanzania, Zaire, Latin America, Southern and Eastern regions of India were Ogawa serotype and EL Tor biotype. Our study revealed that high percentage of Vibrio cholerae isolated from both stool and water samples were Vibrio cholerae O1 Inaba and Classical biotype, which is also contrary to the findings of Hossein, et al.,(2005) that all strains of Vibrio cholerae O1 isolated between July and September 1998 in Goa belonged to EL Tor biotype, 53(66%) of them being Ogawa serotype, while 21(26%) were Inaba serotype.

Some isolates obtained from stream water samples when serotype and biotype were reported as Vibrio cholerae O1 Inaba serotype Classical biotype same as some of the isolates obtained from patient’s stool samples during the outbreak. One could be tempted to say that by referring to the phenotypic and genotypic characteristics of the isolates, this stream water that has been one of the sources of water supply in Uzebba community must have played an important role in the spread of cholera outbreak in that region. We can also say that outbreak in Uzebba is defined by social and environmental factors.The importance of aquatic reservoir depends on sanitary conditions of the community(CEDECO,2010. Cholera is spread mainly through drinking fecal – contaminated water. . While contaminated water remains the major route for cholera transmission (Shapiro, et al., 1999) food and utensils are also important (Rabbani and Greenough, 1999), emphasizing the importance of hygiene within the house hold (Fotedal, 2001).

Effective food hygiene measures include cooking food thoroughly and eating it while still hot; preventing cooked foods from being contaminated through contact with raw food or drinking water is important (Seas and Gotuzzo, ensuring proper management of excreta to avoid contamination of other water sources were important measures to reduce cholera transmission.   Education of the population at risk regarding appropriate hygienic practice is always recommended. Identification of local customs that place people at risk is also important in order to eliminate such practices (Sears and Gotuzzo, 2000). A greater understanding of the pathogen, its biology, ecology, epidemiology and strategies for treatment and prevention are essential to guide policies and programmes for the control of cholera. . Adequate measures to improve hygiene and sanitation and supply of safe potable water are needed to prevent any future outbreak of cholera in Uzebba.

The post Serotypes and Biotypes of Vibrio Cholerae O1 Isolated from Stool and Water Samples in Uzebba (Edo – State). appeared first on Nigerian Biomedical Science Journal.

Atiegha C. Igoni M , Victor I.,
Department Of Medical Laboratory Sciences, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Ayaowei I.T.
Department Of Health Information Management, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Eseimokumo Me.
Department Of Pharmarcy Technician Studies, College Of Health Technology Otuogidi-ogbia , Bayelsa State.

All correspondence to: IGONI M ; Bayelsa State College of Health Technology: e mail; achristo40@gmail.com.

ABSTRACT
The treatment of tuberculosis has evolved from streptomycin to the DOTS strategy .Even as at that it is a serious challenge. This retrospective study was carried out to evaluate the effectiveness of the method. A total of sixty seven(67) Tuberculosis cases were evaluated at the Tuberculosis Treatment centre Nembe comprehensive Health Centre, Bayelsa state , it covered a period of four (4) years, from 2009 to 2012. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. It is therefore recommended for community and family based treatment.

Key words: DOTS, Cured, treatment completed and treatment failure.

INTRODUCTION
Tuberculosis, a major health challenge and leading infectious killer is caused mainly in man by Mycobacterium tuberculosis. The bacilli enters the body by inhalation of droplets or dust particles containing it. It occurs as pulmonary tuberculosis or non- pulmonary tuberculosis. In pulmonary tuberculosis the pulmonary aveoli and surrounding lymph glands are lodged by the bacilli resulting in lesion characterized by acute inflammatory reactions with accumulation of fluid and white blood cells around the aveoli, while the non pulmonary tuberculosis includes the renal and urogenital tuberculosis, military tuberculosis and tuberculosis meningitis with varying symptoms1.
In sub-saharan Africa, cases of tuberculosis have increased dramatically, overwhelming control programs 2. This could be as a result of the fact that tuberculosis infection control interventions are not routinely implemented in contrast to what happens in high income countries with low prevalence of tuberculosis where infection control policy is routinely observed3. However the launch of directly observed treatment shortcourse (DOTS) in 1995 by the world health organization (WHO) has shown to be an effective intervention that will lead to reduced tuberculosis transmission and decreasing number of tuberculosis cases4,5. Also it is among the most cost-effective global health interventions available today6,7.
The DOTS strategy is based around a short-course treatment regimen for a minimum of six months of four drugs in combination (2 months of Isoniazide, Rifampicin ,Pyrazinamide and Ethambutol), a good management practice, sputum smear microscopy for diagnosis and the direct observation of doses to ensure adherence. Treatment success under DOTS concerns two outcomes- cured and treatment completed: “cured” if patient finish the treatment regimen with negative bacteriology at the end of the treatment whereas “treatment completed” refers to patients who have finished the regiment in full without showing evidence of treatment failure and without negative bacteriology. This treatment regiment however use to suffer from a number of drawbacks, with the combination of available drugs, the duration of treatment cannot be reduced below six months without a significance in relapses although re-infection with a different strain of mycobacterium tuberculosis can cause tuberculosis recurrence, which is considered to be an important measure of efficacy of tuberculosis treatment8,9. Also when treatment is given under sub-optimal conditions, regiments are associated with high rate of patients non-adherence specially in low income countries which harbor high burden of diseases10. The consequence of this is increased mortality and creation of clinic infections, drug resistant cases. The World Health Organization estimates in 2004, 424,203 cases of multidrug resistant tuberculosis globally among which 181,408 cases occurred in patients who had already been treated with standard (first line) therapy11.
This study was therefore carried out to determine the effectiveness of DOTS (Directly Observed Treatment Short course) on tuberculosis in the Nembe Local Government Area of Bayelsa state south-south Nigeria.

MATERIALS AND METHODS
Setting Comprehensive Health Centre Nembe, is situated in Nembe, the headquarters of Nembe local government area, about eighty kilometers south-east of Yenagoa the capital of Bayelsa State, Nigeria.

Subjects
The subjects of this report are those patients that reported to the TB treatment centre and those patients who were got through surveillance in the surrounding communities. A total of sixty seven positive cases were involved in the study. These cases were got over a four year period (from 2009 – 2012)

Laboratory analysis
Patients were given sterile wide mouthed plastic containers which they voided sputum into. Three specimens were collected from them over a two days period (spot, early morning and spot). Sputum analysis was done using the Ziehl-Neelson technique as described by cheesbrough12.

Method of drug administration
The strategy adopted here is the short course regiment that lasts for six months. Upon visit to the treatment center, Zeihl-Neelson(AFB) test is carried out. Treatment commences on patients who are newly diagnosed i.e. their sputum show smear positive. After the initial test, a second test (Zeihl- Neelson) is conducted upon revisit at the end of the second month of chemotherapy. The same thing applies
upon a second and third revisit which is at the end of the fifth and sixth months respectively.
For category 2 patients i.e. those who defaulted treatment or had a relapse, treatment is for seven months. Treatment on the intensive phase is for three months after which the second AFB test is done. The third and fourth AFB tests are done at the end of the fifth and seventh month of chemotherapy. Patients that absconded during treatment were not included in this work.

In 2009 eighteen new cases of sputum positives were recorded (six scanty and twelve +s). Upon first revisit i.e. after intensive phase, all eighteen persons were tested smear (AFB) negative. On second revisit the same feet was recorded. In the last revisit seventeen tested negative while one patient was AFB positive: table 1.

In 2010, seventeen persons were tested positive with varying degree of intensity of infection ( one scanty, eighty +s, four ++s and four +++s). sixteen persons tested smear negative at the end of the first revisit while person was tested positive. At the end of the fifth month of chemotherapy all fifteen persons tested smear negative (second revisit). After six months of chemotherapy one patient was tested smear positive while the other sixteen persons that continued their treatment to the end were tested negative. Conclusively, eleven persons were cured while one person was termed a “Treatment Failure”: table 2
Table 3 represents the summary of the 2011 treatment outcome. There were ten sputum smear positive cases in
2011. Eight of them were graded +, while one patient had 2+ and another 3+. Seven persons tested AFB negative upon the first revisit and one patients sputum was positive. In the second and third revisits, all eight patients had there AFB test negative. i.e. in 2011 eight persons were cured of tuberculosis in the treatment center.
Two scanty cases, twelve +s and eight cases of 2+s were recorded in the year 2012, twenty two cases in all. Three scanty cases were recorded in the first revisit stage, while nineteen tests were negative. In the second and third revisit all twenty two tests were smear negative. in 2012 twenty two persons were cured of tuberculosis in the treatment center. Table 4

Discussion
In this four years under study, the success of this method of treatment has been so tremendous. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. When the result was subjected to the chi-square test, it shows that there is a significant difference between those that are cured of TB by the dots strategy in the five years under study and those that were not cured by the strategy p<0.05, (234.845, df 3, assymp. Sig (2-sided) 0.000

Conclusion:
Since with the DOTS strategy it is very difficult for patients to skip treatment, (which in most times is the cause of resistance), the researcher recommends that this strategy should be adopted, be it community based or family based)

Acknowledgment
We wish to express our heartfelt thanks to God almighty for his abundance grace to carry out this research and the strength to stand it all, without him nothing would be done. We also greatly appreciate the provost of the Bayelsa State College of Health technology, Dr Adias T. Charles for his inspiration and motivation. Also, the research and manpower development department of the College was involved with the funding. Finally we thank the staffs of the comprehensive health centre Nembe, Bayelsa State.

The post Diagnosis And Treatment Of Tuberculosis By The Directely Observsed Treatment Shortcourse (DOTS): A Case Study Of Nembe Comprehensive Health Centre TB Treatment Unit, South-South Nigeria. appeared first on Nigerian Biomedical Science Journal.

Saleh, M.I.A., Mabrouk, M.A., Mohammed, A., Isa., A.I., Alhassan, A.W.

1. Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria.
Musa, K.Y.,

1. Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria.

Ayaowei IT
Department Of Health Information Management, College of Health Technology Otuogidi-ogbia , Bayelsa State.
Helmy, A.
1. Biotechnology Centre, Misr University for Science and Technology, Cairo, Egypt.

All correspondents to: M.I.A. Saleh, Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University,
Zaria, Nigeria. E-mail: alhajisaleh@yahoo.com

ABSTRACT
Nigella sativahas been used for medicinal purposes for centuries, both as a herb and when the seeds are powdered or pressed into oil in Asia, Middle East and Africa. It has been traditionally used for a variety of conditions and treatments related to respiratory health, stomach, intestinal, kidney, liver, circulatory and immune system support, and for general well-being .This study was aimed at investigating the effect of the n-butanol seed fraction of this plant as it affects gastric mucosal integrity and basal gastric secretions using the indomethacin –induced model. Phytochemical screening revealed the presence of flavonoids, alkaloids,, saponins, glucocinolates amongst others, whereas acute toxicity studies revealed a median lethal dose above 5000mg/kg. The rats were grouped into 6 (n = 5), with the extract fraction administered at 50, 100 and 200mg/kg subcutaneously, followed by pyloric ligation with indomethacin and cimetidine used as the standard drug. For the mucosal integrity study, ulcer and preventive indices were analysed, while volume of gastric juice, titratable acidity,acid output and pepsin concentration were assessed for basal gastric secretions. The three experimental doses of the extract at 50,100 and 200mg/kg showed a dose –dependent decrease in both ulcer and preventive indices. It also showed a significant (p<0.05) decrease in volume of gastric juice, titratable acidity, acid output and pepsin concentration in dose-dependent manner with the three experimental doses administered with the highest reduction at the 200mg/kg dose. The results obtained suggest that this fraction down regulated all those parameters which might be attributed to the presence of the phytoconstituents present in this fraction. Therefore, the extract fraction of this plant possesses gastroprotective and antisecretory effects further explaining the folkloric use of this plant in the therapy of peptic ulcer disease.
KEYWORDS: Nigella sativa, gastroprotection, basal secretions, NSAIDs

Introduction:

Peptic ulcers are breakages or discontinuities that can occur in the mucosal epithelial lining of either the stomach, small intestine, large intestine or the Merkel’s diverticulum along the gastrointestinal tract (Falase and Akinkugbe, 2010). They are known to occur world wide (Cemek et al., 2010) and are major causes of morbidity and mortality (Chaturvedi et al., 2007). The pathophysiology of peptic ulcer has been centralized on an imbalance between aggressive and protective factors. Factors such as stress, cigarette smoking, nutritional deficiencies, inadequate dietary habits, hereditary predisposition and frequent ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) all are known to increase
the gastric ulcer incidences (Klein et al.,2010).In developing countries, usually 50-90% of the populations are infected with Helicobacter pylori, which is the main organism responsible for majority of peptic ulcer cases, and children acquire the infection soon after being weaned. Many natural products and modern synthetic drugs have been used to treat the peptic ulcer disease, but so far a complete cure has not been achieved or discovered, and exploration of new anti-ulcer drugs has remained a field of active research (Bandyopadhyay et al.,2001).Although there are many products in the market for the treatment of gastric ulcers, including antacids, proton-pump inhibitors, anticholinergics and H2-receptor antagonists, most of these drugs produce several adverse reactions,such as hypersensitivity reactions, arrythmias, impotence, gynaecomastia, nephrotoxicity, and haemopoetic changes(Chang and Leung.,2002; Scholl et al.,2005).Development of tolerance and incidence of relapses and side-effects on clinical evaluation make their efficacy arguable, further promoting non-drug compliance to therapy (Santin et al., 2011). In addition, most of these medications are expensive, which further restricts their use (Santin et al., 2010).
This has been the basis for the development of new antiulcer drugs, which include herbal drugs (Altinkaynak et al., 2003). Herbs are used in many domains including medicine, nutrition, flavouring, beverages, dyeing, repellants, fragrances and cosmetics (Djeridane et al., 2006). The plant Nigella sativa has been used for medicinal purposes for centuries, both as a herb when pressed into oil in Asia, Middle East and Africa. It has been traditionally used for a variety of conditions and treatments related to respiratory health, stomach, intestinal, kidney, liver, circulatory and immune system support, and for general well-being. The seeds are used as carminative, aromatic, stimulant, diuretic, antihelminthic, galactagogue and diaphoretic(Gupta et al., 2009).The aim of the present study was to evaluate the effect of the n-butanol fraction of this plant seeds on gastric mucosal damage and secretions.
2.0 Materials and Methods:
2.1 Plant material:
Nigella sativadried seeds were obtained during the month of July, 2011 from Sabon-Gari market in Zaria. Botanical identification and authentication was done by Mr. U.A Gallah at the Herbarium section of the Department of Biological Sciences, Ahmadu Bello University, Zaria. A voucher herbarium specimen (No: 101201) was deposited at the herbarium for future references.

2.2 Extraction of the plant material:
Nigella sativadried seeds weighing about 2kg were crushed and pounded with pestle and mortar. The powder was extracted with aqueous ethanol (70%) in a Soxhlet Extractor, concentrated using rotaryevaporator at reduced pressure, suspended in methanol and partitioned with n-butanol to obtain the n-butanol (n-BuOH) fraction. The fraction was further concentrated in-vacuo and the residue obtained. The extract yielded about 80% of the residue.

2.3 Phytochemical screening of the fractions:
The preliminary analysis for the extract was conducted for the presence of flavonoids, alkaloids, saponins, steroids, glycosides, anthraquinones, resins, reducing sugars and other phytochemicals using standard procedures for analysis (Evans, 2002 and Harborne, 2007).

2.4 Acute toxicity studies:
Lethal Dose (LD50) determination was conducted using the method of Lorke (1983). In the initial phase, male rats were divided into three groups of 3 rats each, making a total of 9 rats. The rats were treated with the n-butanol fraction of the extract at doses of 10, 100 and 1000mg/kg subcutaneosly. Animals were observed for 24 hours and the number of death(s) or those that showed neurological signs were recorded. In the second phase, the animals were grouped into 4 groups of one rat each and treated with the fraction at appropriate doses subcutaneosly. The rats were observed for 4 h for deaths or neurological signs, and the final LD50 was calculated as the square root of the highest non-lethal
dose in which the animal survived multiplied by the lowest lethal dose in which the animal died.

2.5 Drugs and chemicals/reagents:
Cimetidine (Lek Pharma, Slovenia), Indomethacin (Liomethacin(R))(Cheisi, Egypt),
Thiopental Sodium (Abbott Laboratories, UK), Phenol Red (BDH Poole, England), Sodium Hydroxide (NaOH) (BDH Poole, England) for the preparation of 0.01N NaOH solution, Phosphate Buffered Saline (PBS),Casein Substrate Solution 1% (w/v) (Sigma-Aldrich, USA), Hydrochloric Acid (HCl) 0.1N(Sigma-Aldrich, USA), Trichloroacetic Acid Solution 6% w/v (Sigma-Aldrich, USA). All other chemicals and reagents were analytical grade.
2.6 Experimental animals:
A total of ninety adult male albino Wistar rats were used in this study. The animals were obtained from the Animal House, Faculty of Medicine, El-Kasr el-Ain, Cairo University, Egypt. Their weights ranged from 180 – 240g. They were maintainedunder a similar conditions of humidity, temperature and light/dark cycle respectively and each of the animal was kept in a single individual cage, with wide-meshed galvanized wire bottoms to decrease coprophagy as much as possible. The rats were given access to food and water ad libitum for two weeks to acclimatize, prior to the commencement of the experiment. The rats were treated in accordance to the internationally accepted principles of laboratory animal use and care. At the time of the experiment, all treatments were conducted between 9:00 and 10:00 (GMT+1) h to minimize variations in animal response due to circadian rhythm. The animals were divided into the following groups and subgroups for gastric mucosal damage and gastric secretion studies respectively.

2.7 Experimental design:
Group I: Study of gastric mucosal damage
Group IA; Normal saline (Negative Control)Five rats received normal saline (1ml/kg/rat S.C).
Group IB: Indomethacin-treated (Positive Control) Five rats received indomethacin (20mg/kg S.C) for the study of gastric mucosal damage.
Group IC: Cimetidine-treated Ten rats for the study of the effect of two different doses of cimetidine 50 and 100mg/kg S.C on gastric mucosal damage (5 rats for each dose)
.Ten rats for the study of effect of two different doses of cimetidine (50mg and 100mg/kg) S.C, given 30 minutes prior to indomethacin administration on gastric mucosal damage (5 rats for each dose).
Group ID: Nigella sativa extract treated Fifteen rats for the study of the effect of n-Butanol (BuOH) fraction, each at three different doses (50, 100 and 200mg/kg S.C),when given 30 minutes prior to indomethacin on gastric mucosal damage (5 rats for each dose).
Group II: Study of basal gastric secretion
Group IIA: Normal saline (Negative Control) Five rats received normal saline (2 ml/rat S.C).
Group IIB: Indomethacin-treated (Positive Control)Five rats received indomethacin (20 mg/kg S.C), followed by pyloric ligation for the study of basal gastric secretion.
Group IIC: Cimetidine-treated Ten rats for the study of the effect of two different doses of cimetidine, 50 and

100mg/kg S.C on basal gastric secretion (5 rats for each dose).
Ten rats for the study of the effect of cimetidine, 50 and 100mg/kg S.C, given 30 mins prior to indomethacin administration on basal gastric secretion (5 rats for each dose).Group IID: Nigella sativa-treated Fifteen rats for the study of the effect of n-Butanol (BuOH) fraction, each at three different doses (50, 100 and 200mg/kg S.C),when given 30 minutes prior to indomethacin on basal gastric secretion (5 rats for each dose).

2.8 Induction of gastric ulceration
After 48 hours of starvation, the animals were weighed and maintained in their individual cages. Then, indomethacin 20mg/kg was injected subcutaneously and the animals were then deprived of both food and water for 7 h (Urushidani et al., 1979). The animals were later sacrificed by decapitation (Satoh et al., 1983). Their stomachs were opened along the greater curvature, rinsed slowly with running water, then stretched out as much as possible by the use of pins on No.1 Whatman’s filter paper on a ceiling board.

2.9 Quantitative assessment of mucosal damage
The ulcerated areas in each stomach were measured with a transparent (mm) ruler scale, the result of each group were expressed as ulcer index (U.I) in mm of mean ulcer ± standard error of mean (Scepovic and Radmanovic, 1984).
The degree of ulceration was expressed as ulcerindex (U.I). It was calculated by multiplying ulcer score by 100 (Robert et al., 1968). Ulcer score for each group was calculated by dividing the total number of ulcers in each group by the total number of rats in that group (Robert et al., 1968).The percentage preventive index was calculated according to the method of Hano et al.(1976), which is expressed as:
Preventive Index (%) = (U.I. Indomethacin – U.I. Extract/drug plus) indomethacin x 100
U.I. Indomethacin

2.10 Collection of gastric secretion
The gastric juice was collected according to the technique of Shay et al. (1954) as modified by Levine (1965), where oesophageal ligation was avoided. The animals were fasted for 48 hrs to ensure complete emptying of the stomach, but allowed water ad libitum. Each animal was weighed at the end of the fasting period. Light anaesthesia was sodium thiopental 10 mg/kg intraperitoneal (Juliane et al., 2009), abdomen of each rat was opened via a midline incision and the stomach exteriorated. A pyloric ligature was made using a thread with care to avoid damage to the blood vessels or traction to the stomach. The abdomen was then closed by suture, cleaned thoroughly with distilled water or saline, and the animal was allowed to recover.
After 3 h, the rats were sacrificed by decapitation, abdomen of each of the animals were opened. The oesophagus was ligated, and the stomachs removed and washed with distilled water or saline. An opening along the greater curvature was made (Nwafor et al., 2000) and the gastric content drained into a graduated centrifuge tube, the volume noted, then centrifuged at 1,006 x g for 15 minutes.

2.11 Analysis of the gastric juice
Determination of each sample volume after centrifugation
The volume of 3 hours gastric secretion was measured after being subjected to centrifugation at 1,006 x g for 15 mins.
Determination of titratable acidity: A given volume of the gastric juice (1ml) was titrated against 0.01N NaOH. An end point of pH 7.0 as determined colorimetrically at 280nm by phenol red was used (Grossman, 1973; Davenport, 1977).The values were calculated as micro-equivalents per litre (Meq/L), which is equal to the number of millitres (ml) of 0.01N NaOH required to neutralize 1ml of gastric juice.
Titritable Acidity =
Volume of 0.01 N NaOH (mol) which neutralized 1ml of gastric juice
10
Determination of acid output: This was calculated by multiplying the volume (ml) of the gastric juice of each animal by the titritable acidity in that animal.
Determination of pepsin concentration: Pepsin concentration which is the major factor involved in the proteolytic activity of gastric secretion was determined in terms of the amount of protease enzymes produced after incubation of the substrate for 30 minutes with pepsin. It was determined by the spectrophotometric method devised by Jongensen (1954) and Hawk et al. (1960).
2.13 Statistical analysis
All data were expressed as Mean ± S.E.M (standard error of the mean) using SPSS Version 20. Statistical evaluation was done by analysis of variance (ANOVA) followed by post-hoc analysis by Duncan and Scheffe. Values of p<0.05 were considered significant (Microcal Software Inc., Northampton, USA).
3.0 Results:
The phytochemical screening revealed the presence of the following phytoconstituents as depicted in table I below:

The Phytochemical Analysis of N-Butanol Fraction of Nigella sativa L. seed extracts. Acute Toxicity Studies
The toxicity studies of the n-butanol seed extract of Nigella sativa in the first phase after being observed for 24hr, the rats did not show any signs and symptoms of toxicity or death. In the second phase, none of the rats produced any toxic symptoms or mortality up to the dose level of 5000mg/kg body weight, hence, they were considered safe for further pharmacological screening.

DISCUSSION AND CONCLUSION
To evaluate the gastroprotective effect of N-butanol fraction of Nigella sativa, the model of acute ulcer induced by a non-steroidal anti-inflammatory compound indomethacin was performed.
In this model, it was found that treatment with normal saline (2ml/rat) which was isotonic with the plasma as a control, showed no significant ulcer or lesion index with a preventive index of 97%. The group that received indomethacin 20mg/kg alone produced the highest lesion index when compared with all the treatment groups. There were gastric mucosal congestion, oedema, haemorrhage, lamina epithelial necrosis, leucocytic infilteration, blood vessels congestion with foci of necrotic tissues in the lesions. These results are consistent with previous studies that reported similar histopathological derangements and mucosal oxidative stress effects that involves weakening of gastric mucous, leading to formation of lesions in the gastric epithelium (Cyer, 2000; Chang and Leung, 2002; Ryo et al., 2006; Valcheva-Kuzmanova et al., 2007; El-Moselhy et al., 2009).
It was observed that the group that received 50 and 100mg/kg of the standard drug cimetidine alone, showed a significant decrease in gastric lesions (p<0.05) compared to the indomethacin 20mg/kg, cimetidine plus indomethacin group in the ulcer index with preventive indices of 73 and 79%, respectively. This corroborated with the studies of Marivane et al. (2011) who reported a decrease in lesion index on the gastric mucosa of rats treated with the H2-receptor antagonist cimetidine compared to indomethacin.
With regard to the effects of three different graded doses of the N-butanol fraction of Nigella sativa, it was found that treatment with the 50, 100 and 200mg/kg significantly reduced the lesion index compared with the control group (p<0.05) in a dose-dependent manner in this indomethacin-induced model. The highest dose of 200mg/kg N-butanol fraction produced the highest preventive index of 96% above that of the standard drug 50 and 100mg/kg cimetidine. A similar finding was reported by Marivane et al. (2011) that reported a significant reduction in lesion index, total injured area and the percentage injured area when extract of Brassica oleracea was used on indomethacin ulcer model.
Both Brassica oleracea and Nigella sativa are found to contain some flavonoids, especially quecertin and kacempferol mainly in glycosodic form and these are secondary metabolites that are widely distributed in nature with several biological activities including gastroprotective potentials (Martin et al., 1998). Croton urucurana with high flavonoid content also exhibited same mucosal cytoprotective potentials (Esmeraldino et al., 2005; Alves et al., 2008). The flavonoid content of this fraction had prevented and exerted a protective effect possibly by its inherent ability to scavenge free radicals, inhibit lipid peroxidation, increase mucous and prostaglandin contents of the gastric mucosa (Alanko et al., 1999).
Through phytochemical analysis of the fraction of N-butanol, apart from flavonoids, the presence of terpenoids, tannins, cardiac glycosides, steroids, saponins were detected amongst others. To further support and corroborate the possible fractions for the mucosal cytoprotection exhibited by this fraction of N. sativa containing flavonoids are the studies by Alcaraz and Hoult, 1985 that flavonoids increase mucosal prostaglandin content, inhibit histidine decarboxylase thereby decreasing histamine secretion (Bromer and Landry, 1985).
The presence of saponins in the fractions of N. sativa to improve on mucosal integrity has been reported in several other studies where plants containing saponins have been shown to possess antiulcer activity in several experimental ulcer models. Among these, saponins isolated from the rhizome of Panax japonicus and the fruit of Kochia scoparia (which contain approximately 20% of saponins) have been demonstrated to possess gastroprotective properties (Matsuda et al., 2003) in conformity with this study. The protective activities of all these saponins are not due to inhibition of gastric acid secretion, but probably due to activation of mucous membrane protective factors (Borreli and Izzo, 2000). Moreover, several plants containing high amount of saponins have been shown to possess antiulcer activity in several experimental bioassays, probably acting as an activator of mucus membrane stabilizing factors (Morikawa et al., 2006). Similarly, presence of tannins, terpenoids in N. sativa fractions further validate the cytoprotective property in the gastric mucosa observed in our study as reported by Al-Rehaily et al. (2002), where several saponins, tannins, terpenoids were found to possess gastroprotective properties. Additionally, Terpenoids are a widespread class of secondary compounds with several pharmacological activities, including anti-inflammatory and antiulcer activities (Arrietta et al., 2003). Plant extracts of Eleagnus angutifolia, Hibiscus esculentus, Papaver rhoeas, Phlomis grandiflora, Rosa canina all with a high flavonoid and saponin contents as reported by Ilhan et al. (2003) showed potent in vivo gastroprotective activity similar to that of Nigella sativa.
The interference of the N-butanol fraction of the extract using same model was also evaluated on parameters of basal gastric secretion. This method is an important procedure that reveals the possible changes of gastric secretory physiological parameters relating to volume of gastric secretion, titratable acidity, acid output relating to pH and the most important proteolytic enzyme in the stomach pepsin. The findings suggest that this fraction interfered with these major basal secretory indices of gastric juice.
Considering that the volume of gastric juice which is mainly acidic encompasses mucus, hydrochloric acid, pepsinogen, bicarbonates, intrinsic factor and protein plays a vital role in the aetiopathogenesis of gastric mucosal integrity, it is plausible to consider this fraction as a putative cytoprotective agent. This assumption was made based on the observation that the different quantities or volume of gastric juice obtained in this study showed a general inhibitory pattern with regard to its production in the stomach in this fraction evaluated. There was a significant reduction in the volume of gastric juice at the 100 and 200mg/kg extract treated groups when compared with the control. These results are in agreement with the studies of Muriel et al., (2008), who reported a significant decrease in volume of gastric juice on a similar ulcer model after using Green propolis.
With regard to the titratable acidity, there was no significant difference in all the three doses of N-butanol evaluated when compared with the control. Acid output significantly decreased in a dose related manner when compared with control. This decrease in stomach acidity facilitated the healing of gastric ulcers, because exposing the mucosa to high concentrations of acid favours mucosal epithelial damage (Laine et al., 2008). Contact between stomach acid and the mast cells of the submucosa and lamina propria causes mast cell degranulation and the release of histamine, which stimulates hydrochloric acid secretion by parietal cells and produces inflammation and acute oedema at the site of contact (Rodrigues et al., 2008). Overall, concentration of hydrogen ions in the gastric juice decreases reflective of high pH, further aggravating the aggressive factors (Lullmann et al., 2000). Consistently, hyperacidity is known to result due to uncontrolled hypersecretion of hydrochloric acid from parietal cells of gastric mucosa through the proton pump H+-K+ ATPase (Kishor et al., 2007). The results of 50mg/kg N-butanol was insignificant. Proteolytic activity as pepsin concentration significantly decreases (p<0.05) in a dose-dependent manner compared to control. A similar finding was reported by Halter et al. (1988) and Hatazawa et al. (2006).
In all the cimetidine treated groups, the volume of gastric juice, titratable acidity and pepsin concentration all significantly reduced compared to control. Gastric acid decimation by cimetidine has been attributed to its ability to antagonize the binding of histamine to the H2 receptor on the parietal cell membrane (Banji et al., 2010).The reported results have validated the folkloric use of N. sativa in the therapy of peptic ulcer disease. N. sativa offers protection against NSAIDs-induced gastric ulceration and down-regulate basal acid secretions to promote mucosal cytoprotection. The presence of phytoconstituents in this medicinal plant might be responsible for those pharmacological actions. In this context, extracts and active principles from plants could serve as leads for the development of new drugs. Therefore, this plant specie(s) have a great potential to be used as a gastroprotective agent in combination with others or alone.

The post Gastroprotective Effect of N-Butanol Fraction of Nigella sativa (L.) Seed Extract on Nsaid-induced Gastric Mucosal Ulceration and Secretions. appeared first on Nigerian Biomedical Science Journal.

Suleiman Ibrahim Eleha

Department of Chemical Pathology, University of Ilorin Teaching Hospital, Ilorin, Nigeria.
Oyedeji Samuel Oyewole
School of Medical Laboratory Science, Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria.
Muhammed Abdurrasheed Ola
Department of Histopathology, Faculty of Medical Laboratory Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria.
Adesina Adeyemi Adeleke
Department of Chemical Pathology, Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria
Afolabi O.O
Pathology Department, College of Health Sciences, University of Ilorin, Ilorin, Nigeria.

All correspondences to: Ibegbu A.O, aoibegbu@yahoo.com.

ABSTRACT
Pseudocedrela kotschyi is a medicinally important tree, belongs to the family Maliaceae, and have been widely used by traditional healers for treatment of various illnesses, many of which have not been scientifically scrutinized. Phytochemical screening and acute toxicity study (LD50) arethe two necessary preliminary investigations toward establishing therapeutic usefulness of any plant. The current study was carried out toprovide comparative phytochemical detail of methanol bark extract (BEPK) and aqueous leaves extract (LEPK) of P. kotschyi with their LD50 value, using standard procedure.Results showed that both extracts contained variedphytochemical constituents such as tannins, cardiac glycosides, steroids, and reducing sugars. Maximum phytochemicals was observed in aqueous extract. LD50 value was found to be 1,250 and 1,750 mg extract/kg body weightintraperitoneally respectively in rat, thus BEPK was found to be more toxic than LEPK.

Keyword: Pseudocedrela kotschyi, Cardiac glycosides, LD50, Tanins, Steroids)

INTRODUCTION

Pseudocedrela kotschyi (Schweinf) Harms belongs to the Maliaceae family and comprises a single species, a deciduous, monoecious, medium – sized tree up to 12–20 m tall (Ahua et al., 2007: Oliver-Bever, 1986: Salvina, 1989). P. kotschyi has numerous uses in traditional medicine, particularly its bark, roots and leaves, thus it is an important source of ingredient for local medicine (Kassim et al., 2009). The decoction is used as a wash for ulcers and in Northern Nigeria; the plant also serves as an occasional ingredient for use in arrow poison (Oliver-Bever, 1986). Roots of P. kotschyi are commonly used as chewing sticks in West Africa and it extract have been shown to inhibit the in-vitrogrowth and development of the schizont stage of Plasmodium falciparium, thus the root may provide affordable means of treating malaria (Kassim et al., 2009).Derived natural products such as flavonoids, terpenoids and steroids etc. have received considerable attention in recent years due to their diverse pharmacological properties including antioxidant and hepatoprotective activity (DeFeudis et al., 2003). The quantity and quality of phytochemicals present in plant
parts may differ from one part to another. In fact, there is lack of information on the distribution of the biological activity in different plant parts essentially related to the difference in distribution of active compounds (or active principles) which are more frequent in some plant parts than in others (Solomon et al., 2013). Successful determination of biologically active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure (Solomon et al., 2013).

Acute toxicity refers to those adverse effects occurring following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours (Muhammad et al., 2000). LD50 value (median lethal dose) is one way to measure the short term poisoning potential (acute toxicity) of chemical and in general, the smaller the LD50 value, the more toxic the chemical and the larger the LD50 value, the lower the toxicity (Gadanya, 2011). It is important to know that the actual LD50 value may be different for a given chemical depending on the route of exposure (Oral, dermal, inhalation) (Senin, 2006). It is usually expressed as the amount of chemical administered (e.g. milligrams) per 100grams (for smaller animals) or per kilogram (for bigger subjects) of the body weight of the test animal (Gadanya et al., 2011).

MATERIALS AND METHODS
Plant Materials
Different plant parts; leaves and bark of P. kotschyi were collected from Kisi, Oyo State, South West Nigeria (woody Savannah vegetation). The plant was identified by a plant Taxonomist. The leaves and stem bark were separated from the plants and washed in tap water, rinsed with distilled water. Then the plant parts were shade dried in room temperature till there is no loss of weight. They were ground into coarse powder and stored in room temperature for further study.

Extraction
The methanol and aqueous extracts were prepared by soaking 100 g each of the dry powdered plant materials in 1 L of methanol and water at room temperature for 72 h with occasionalshaking for effective extraction. The extracts were filtered after 72 h, first through a Whatmann filter paper No. 42 (125mm) and then through cotton wool. The extracts were concentrated using a rotary evaporator with the water bath set at 40°C. The percentage yield of extracts ranged from 7–19%w/w.

Animals
Twenty seven (27) healthy Wistar strain albino rats weighing between 150-200 g were obtained from the Laboratory Animal Centre of Osun State University, Osogbo, Osun State Nigeria. The rats were housed in clean metallic cages and kept in a well-ventilated room and allowed to acclimatize to the laboratory environmentalcondition for one week, prior to the commencement of the experiment. They were fed with standard animal pellet and water ad libitum.

Drugs and Chemicals
Hydrochloric acid, Acetic acid, Hydrogen Peroxide, Ferric chloride and the rest of the chemicals utilized were of analytical grade and were purchased from ROVERT Scientific Limited, Edo, Nigeria.

Phytochemical Study
Various phytochemical constituents were tested for,using different chemical tests; Alkaloids (Wagner’s reagent test), Cardiac glycosides (Keller-Killani test), Flavonoids (Alkaline reagent test), Sterols (Liebermann-Burchad’s reaction), Tannins (Ferric chloride test/ Braymer’s test), Phenol (Ferric chloride test), Quinones (HCl test), Saponins (Foam test) and Terpenoids (Salkoki’s test) as described by loan (1994).

Acute Toxicity Study
The method of Fixed Dose Procedure (FDP) (OECD, 1981) was used to establish LD50 value.
This method does not use death as an end point; instead it uses the observation of clear signs of
toxicity developed at one of a series of fixed dose levels to estimate the LD50 (Stallard and Whitehead, 1995).Animals
were fasted over-night prior to dosing (they were allowed access to water).
Following the period of fasting, they were weighed and the test substance administeredintraperitoneally as shown below.

In the 1st phase:There were 2 groups of 3 rats per group. Group 1 was treated with the BEPK extract at doses of300mg/kgand Group 2 was treated with1000 mg/kgof LEPK .

In the 2nd phase: There were 2 groups of 3 rats per group. Group 1 was treated with the BEPK extract at doses of750 mg/kg and Group 2 was treated with1,500mg/kg of LEPK
In the 3rd phase: There were 2 groups of 3 rats per group. Group 1 was treated with the BEPK extract at doses of1000 mg/kg and Group 2 was treated with 1,750mg/kg of LEPK
In the 4th phase: a group of 3 ratswere further treated with BEPK at dose of 1,250 mg/kg.

After the substance has been administered, food was withheld for a further 4 hour. They were closelyobserved in the first 4 hours and then hourly for the next 12 hours followed by hourly intervals for the next 56 hours (overall 72hrs) after the administration to observe any death or changes in general behaviours and other physiological activities (OECD, 2001).

DISCUSSION
The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body (Montilla et al., 2005). The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids, and phenolic compounds (Mir et al., 2013).
Phytochemical are naturally synthesized in all parts of the plant body; bark, leaves, stem, root, flower, fruits, seeds, etc. i.e. any part of the plant body may contain active components.Results of comparative study obtained for qualitative phytochemicals screening of BEPK and LEPKare presented in table 1. Ten phytochemicals werescreened for; alkaloid, cardiac glycosides, carbohydrate, flavonoids, phenols, saponins, sterols, tannins, terpenoids and quinines. All were found present in aqueous leaf extracts, while quinine and terpenoids were absent in methanol stem bark extract. This finding corroborates the reports of Asase et al.,(2008); Musa et al.,(2008) and Ojewale et al.,(2013).
Remarkably, the concentrations of flavonoids, alkaloid and tannins were higher in the leaf extract than the methanol stem bark extract. This suggests that the leaf offers a wider array of phytochemicals than the bark.
The presence of those selected phytochemical constituents in the plant made the plant to be a potential source of crude drug that can positively serve as source of modern drugs. This view was supported by Ahmadiani et al., (2000), who reported that tannins and/or flavonoids of medicinal plant origin were found to possessed significant
pharmacological activities: antidiarrheal, analgesic and anti-inflammatory among others in the animal body systems.Diets containing tannins at low dosages (0.15 – 0.20%), have been shown to improve wellbeing of the human body (Shiavone et al., 2008). Saponins enhance nutrient absorption and aid in animal digestion. Cardiac glycosides could improve circulation and heart function in congestive heart failure (El-olemy et al., 1994).

Acute Toxicity (LD50 Estimation)
The LD50 for BEPKwas estimated as1,250 mg/kg i.p. in rat (Table 2). While LEPK was found to have median lethal doses of 1,750 mg/kg i.p. in rat (Table 3). These values indicated that BEPK was slightly toxic to the test rats.Though the stating dose for LEPK was 1000 mg/kg, this isaccording to (OECD, 2000).
However, both the LD50 values were significantly lower than earlier value gotten by Ojewale et al., (2014) the reason for this might be due to difference in the solvents used for the extractions.
The World Health Organization bases its ranking of hazards chemicals on the lowest published rat oral LD50 in mg/kg body weight and in it ranking, any substance with oral LD50 values in the range of 200 – 2000mg/kg body weight oral falls into category II and classify as moderately toxic substances.
Thus, the indication is that both BEPK and LEPK are slightly toxics but could be used safely on human at lower doses (Adoun et al., 2012). The result also indicated that LEPK is less toxic in relation to BEPK, though both are relatively safe to the experimental model used, at the doses indicated.

Conclusion
Both methanol bark and aqueous leave extracts of Pseudocedrela kotschyishowed some of their chemical componentssuch as flavonoids, alkaloids, saponins, tannins, glycosides, phenol and sterols. There is no doubt thatthese plants are reservoir of potentially useful chemical compounds which serve as drugs, provide newer leads andclues for modern drug design. The LD50 of both the Leaves and Bark has been established in this study. So, its slighttoxicity at the higher doses should be taken into consideration during usage. Also, further clinical and pharmacological study should be conducted using lower dosage to investigate the unexploited potentials of this plant.

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