TOXICOLOGICAL AND ANTIDIARRHEAL STUDIES OF AQUEOUS EXTRACTS OF TWO MEDICINAL PLANTS: SCLEROCARYA BIRREA(A. RICH) HOCHST (ANACARDIACEAE) AND PSIDIUM GUAJAVALINN (MYRTACEAE) IN NMRI MICE Sawadogo Touwindséda Aimée, Tougouma Larba Colette, Ouedraogo Youssoufou, Da Filkpièrè Léonard, Bayala Balé and Belemtougri G. Raymond

Sawadogo Touwindséda Aimée, Tougouma Larba Colette, Ouedraogo Youssoufou, Da Filkpièrè Léonard, Bayala Balé and Belemtougri G. Raymond Laboratory of Animal Physiology, UFR of Life and Earth Sciences, University Joseph KI-ZERBO, 03 BP 7021, Ouagadougou 03, Burkina Faso. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 14 March 2020 Final Accepted: 16 April 2020 Published: May 2020

Sclerocaryabirrea and Psidiumguajava are two medicinal plants traditionally used in rural areas for the treatment of many diseases, including diarrhea. In our experiments, we used plants leaves aqueous extract for toxicity evaluation and preventive antidiarrheal effect study in NMRI mice. Sclerocaryabirrea and Psidiumguajava caused no mortality and no behavioral changes up to a dose of 5000 mg/kg body weight. The extracts of both plants were reported to be practically nontoxic. Diarrhea was censed induced by castor oil consumption, about 4 hours after oral administration in NMRI mice. Then 300, 500 and 800 mg/kg body weight of plant extract was given to animals. Sclerocaryabirrea extract induced an antidiarrheal protection of 40, 80 and 100% according to above respective used doses; these values are 60, 80 and 100% respectively with Psidiumguajava extract, in same conditions. A one dose test of 300 mg/kg body weight, Psidiumguajavaextract showed more antidiarrheal effect compared to Sclerocaryabirrea. Our work confirms the relevance in the traditional use of both plants in the treatment of diarrhea. A study has shown that in Burkina Faso, precisely in Ouagadougou, diarrheal diseases affect much more young children, with 55.7% of cases among infants (Sidibé, 2014). Because of difficulty to prevent diarrheal diseases (insufficient hygienic conditions in developing countries) curative treatment is prioritized but are too late, many times.
Present study consists in toxicity and preventive antidiarrheal activity of Psidiumguajava and Sclerocaryabirrea extracts evaluation in NMRI mice.

Sclerocaryabirrea:
The fresh leaves of Sclerocaryabirrea were harvested in Gampéla village, 25km from Ouagadougou, in July 2016. The identification was made from the herbarium of the University Joseph KI-ZERBO where a specimen was kept under the number ID 16959 and the sample number 6836. They were dried in the shade under ventilation in the laboratory at room temperature. Once dried, these leaves were ground using an electric grinder to obtain a fine powder which was used for extractions.

Psidiumguajava:
Fresh leaves of Psidiumguajava, were collected in the vicinity of University Joseph KI-ZERBO and were treated as those of Sclerocaryabirrea. One specimen was kept under ID number 17909 and sample number 6911. 948 Laboratory Animals: Naval Medical Research Institute (NMRI) mice was used. The mice were from 8 to 9 weeks-olds and weighing 20-35g respectively. The animals were kept in polycarbonate cages and housed under standard conditions of temperature (22 ± 3˚C), relative humidity (50 ± 10%) and dark/light cycle (12 h/12 h). The animals were given pelleted food containing an average of 29% protein and drinking water ad libitum. Study protocols and ethical issues were approved by Ethical committee of Faculty of University Joseph KI-ZERBO.

Aqueous extraction:
The leaves were stored in the Laboratory, protected from dust and humidity, under artificial ventilation and at room temperature. Once dried, the leaves were powdered for the preparation of the various extracts. Test samples of 100 g of the vegetable powder were placed in a 2000 mL stainless steel Erlenmeyer flask. For the aqueous maceration, a volume of 1000 mL of distilled water was added to each test sample and homogenized with a glass rod. After homogenization, the mixture was kept under constant mechanical stirring for 24 hours at laboratory room temperature. After this, the mixture was filtered twice, successively on a fine nylon fabric and then on cotton wool. The filtrate (aqueous macerate) obtained was distributed in 500 mL crystallizers and placed in a ventilated oven (MEMERT) set at 45°C for 24 hours. The concentrated extracts obtained were transferred to freezing bottles and used for the various tests. The yield of the aqueous extraction was 12.14% Sclerocaryabirrea and 12.26% Psidiumguajava.

Toxicity study: Acute toxicity:
The test was conducted according to OCDE (2001) guideline 423. It was performed with nine female mice, weighed and labelled and then divided into three groups of three mice. The control group received distilled water. Groups two and three received Sclerocaryabirrea and Psidiumguajava extract at 5000 mg/kg of body weight (BW), respectively. Twenty-four (24) hours prior administration of the extract, the mice were fasted and four (04) hours prior administration, they were deprived of drinking water. Administration is made in a single dose by oral route. Observations are made after 1 hour, 24 hours, 48 hours, 72 hours and for 14 days after extract administration. One hour after extract administration, mice are re-supplied with water and food. During this period, changes in behavior are noted as well as the number of dead animals.

Subacute toxicity:
Subacute toxicity was conducted according to the OCDE (2008) guideline 407. Female mice were used during subacute toxicity. They were divided into four (04) groups of six (06) mice each. Doses of 300, 400 and 800 mg/kg body weight of Sclerocaryabirrea and Psidiumguajava were administered. NaCl 0.9% was administered to the control groups. The test substance was daily administered, at the same time, over a period of 28 days, by single dose gavage in 1 mL/100g of body weight. Behavioral changes of the mice were noted during the experiment. Animals' weight and food consumption were weekly recorded during the test.

Antidiarrheal test:
The antidiarrheal test was performed according to the method of Shah et al. (2011a) with slight modifications. Twenty-five (25) female mice of eight to ten (8)(9)(10) week-old were used. The animals were fed 24 hours before the experiment and deprived of water 10 hours before.

Preliminary studies:
During the preliminary study the mice were divided into 5 groups of 5 mice each. All experimental mice received castor oil (10 ml/kg of BW) orally. Four hours (4 h) after castor oil administration, each mouse is placed in an individual cage containing blotting paper, and presence or absence of diarrheal faeces was observed.
All mice that did not have diarrhea were removed and those that did have diarrhea were collected and put in standard conditions for one week.

Antidiarrheal studies:
The mice were divided into 5 groups of 5 mice each: Group 1 received NaCl (0.9%) (negative control); Group 2 received 300 mg/kg BW of the extract; 949 Group 3 received 500 mg/kg BW of the extract; Group 4 received 800 mg/kg BW of the extract; Group 5 received 10 mg/kg BW of loperamide (positive control). One hour (1 h) after treatment, each mouse received 10 mg/kg BW of castor oil and 4 h after, observations were done as in above preliminary studies. The substances were administered orally.

Results: Aqueous extract:
The aqueous extract obtained was in brown crystalline form and the yield was 12. 14 % for Sclerocaryabirrea and 12. 26 % for Psidiumguajava.

Acute toxicity:
The dose of 5000 mg/kg body weight was used. Three female animals were used at each dose level. No mortality was observed in either plant.

Subacute toxicity:
Results are plotted in figure 3. Doses of 200, 400 and 800 mg/kg BW of Psidiumguajava administered resulted in a slight non-significant weight loss over the four weeks of the study. The same doses of Sclerocaryabirrea administered resulted in non-significant weight loss during the first week. However, a change in weight was recorded during the last three weeks.

Antidiarrheal test:
Sclerocaryabirrea extract and Psidiumguajava extract at doses of 300, 500 and 800 mg/kg significantly reduced castor oil induced diarrhea compared to the negative control. Sclerocaryabirrea extract administered at doses of 300, 400 and 800 mg/kg BW has a percentage of protection of 40, 80 and 100% respectively against castor oil induced diarrhea. The extract of Psidiumguajava administered at the same doses has a protection percentage of 60, 80 and 100% respectively. The loperamide used as a positive control has a protection percentage of 100%. The results obtained showed that the aqueous extracts of Sclerocaryabirrea and Psidiumguajava significantly reduced castor oil induced diarrhea. However, the 0.9% NaCl used had no effect on diarrhea. Castor oil is a purgative substance. It modifies the intestinal hydro-electrolytic exchanges and stimulates intestinal motility. Indeed, castor oil in the lumen of the small intestine is metabolized into ricinoleic acid which stimulates the secretion of prostaglandins and then reverses the results in stimulating secretion. On the other side this ricinoleic acid induces the release of certain mediators from the intestinal tract such as histamine, nitric oxide and prostaglandins which, in turn, stimulate intestinal secretion, motility, permeability, and prevent the reabsorption of sodium, potassium, and water (Belemtougri et al., 2016). By comparing our results with those obtained previously, we can confirm the antidiarrheal effect of aqueous extracts of Sclerocaryabirrea and Psidiumguajava leaves. Our work shows that Psidiumguajava leaves extracts are more active than Sclerocaryabirrea extracts at a dose of 300 mg/kg BW.

Conclusion and Perspectives:-
This study assessed the toxicity of two medicinal plants used in the traditional antidiarrheal treatment. The method of preparation used is maceration and the administration was done orally. The effects induced by Sclerocaryabirrea and Psidiumguajava, to treat diarrhea, seem to be due to various chemical groups: alkaloids, flavonoids, tannins and polyphenols that form the scientific basis for the traditional therapeutic use of these plants. The study of the acute toxicity of Sclerocaryabirrea and Psidiumguajava indicates that the extracts would be practically nontoxic at the doses used. On the other hand, the use of Sclerocaryabirrea over a long period of time should be done with caution.
From these results the following perspectives are considered: 1. Sub-chronic oral toxicity of Psidiumguajava and Sclerocaryabirrea followed by the determination of hematological and biochemical parameters; 2. Phytochemical screening of Sclerocaryabirrea and Psidiumguajava for the detection of chemical compounds such as flavonoids, tannins, alkaloids by thin layer chromatography; 3. Studies on the isolated intestine to determine the mechanisms of action of Sclerocaryabirrea and Psidiumguajava extracts; 4. Testing for antimicrobial activity with extracts of Sclerocaryabirrea and Psidiumguajava; 5. Studies on other models of diarrhea such as fluid accumulation and electrolyte secretion and gastrointestinal transit assay.