Phytochemical Screening and Cytotoxic Analysis of Three Local Vegetables Used in the Treatment of Bacterial Diarrhoea in Southern Benin (West Africa) : A Comparative Study

Research Laboratory in Applied Biology, Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, 01 P.O.Box 2009 Cotonou, Benin. Laboratory of Pharmacognosy and Essentials Oils, Institute of Applied Biomedical Sciences, Champ de Foire, Cotonou, Benin. Laboratory of Training and Research in Applied Chemistry, Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, 01 P.O.Box 2009 Cotonou, Benin. Laboratory of Biology and Molecular Typing in Microbiology, Faculty of Sciences and Techniques, University of Abomey-Calavi, 05 P.O.Box, 1604 Cotonou, Benin.


INTRODUCTION
Local vegetables are either wild or semicultivated species and important in both rural and urban areas of the world [1]. They are plants which leaves or aerial parts are integrated in the food habits of communities [2] and are strongly recommended because of their high nutritive values against the exotic varieties [3]. According to [4], most of local vegetables have medicinal properties and are used as medicinal foods. Although they are constituted mostly of water, they are valuable reserves of minerals, vitamins and phytochemical compounds such as alkaloids, flavonoids, glycosides and tannins that are very beneficial for health maintenance and the prevention of some illnesses [5][6][7][8][9][10]. Furthermore, some vegetables serve in phytomedicine for the treatment of various affections including stomach aches [11,12]. However, several studies established that some plant species are potentially toxic for human and animals [13]. In fact, phytochemical compounds are produced within the plants as poisons to fight pests and herbivores. Such poisons are very active substances that can cause acute effects in human and animals when they are ingested at high concentrations and chronic effects when they are accumulated [13]. Under stressful conditions caused by food scarcity, the consumption of high amounts of plant toxins can have serious consequences [14].
In Benin, 14.44% of the 27 local vegetable species recorded by [4] , are traditionally used as nutritious food by populations and would serve in phytomedicine by traditional healers to treat bacterial diarrhoea [15]. Among these vegetable species, the most cited and used by traditional healers in the treatment of bacterial diarrhoea are Ocimum gratissimum (29.05%), Vernonia amygdalina (16.80%), Crateva adansonii (13.49%) and Sesamum radiatum (11.41%). These vegetables leaves are not only used to treat bacterial diarrhoea, but they are also used in treatment of high blood pressure, constipation, malaria, dysentery, candidoses and the delivery at pregnant woman [15][16][17]. Nevertheless, in these plants, the chemical compounds responsible for biological activities as well as their toxicity remain unknown. This could cause enormous prejudices to prescribers, leading to issues like overdose, under-dose and poisonings and even death. The used doses remain inaccurate [18]. This imprecision constitutes a serious problem to traditional medicine improvement. Therefore, a surveillance of the toxicity and posology of plant extracts that are empirically administered is needed in order to avoid adverse therapeutic risks that can sometimes be tragic [19,20].
Regarding the fact that these local vegetables are widely consumed as food and used as medicine by local populations in Benin, their phytochemical characteristics and cytotoxicity need to be investigated. Results of their phytochemical screening and cytotoxicity in addition to the nutritional value of these three vegetables (V. amygdalina is very rich in magnesium (9000±1 mg/kg), sodium (2870±2 mg/kg), potassium (48200±1 mg/kg) and phosphorus (8738±2 mg/kg); C. adansonii has high calcium (24000±1 mg/kg), proteins (25248±1 mg/kg) and zinc (22±1 mg/kg) contents; S. radiatum, it is rich in iron (1360±1 mg/kg) and copper (38±1 mg/kg) [21]) will be used to impart knowledge on their nutritional and sanitary importance and promote their use. The objectives of the present study were to conduct a qualitative phytochemical screening of the leaves of V. amygdalina, C. adansonii and S. radiatum and to assess the cytotoxic activity of the leave extracts of V. amygdalina, C. adansonii and S. radiatum on shrimp larvae.

Materials
The material was basically plant material constituted of powders of the leaves of Vernonia amygdalina, Crateva adansonii and Sesamum radiatum. During two weeks, these leaves were collected from Ouèdo, Abomey Calavi Municipality, Pahou, Ouidah Municipality and Savalou Municipality respectively and identified at the National herbarium of Benin.

Preparation of the leave powders
Leaves of the three vegetables were thoroughly washed with distilled water containing Bleach (1/100) then dried at laboratory temperature for 20 days. The dried leaves were ground using mechanical grinder. The obtained powders were sieved using a 0.2 mm stitch sifter. They were then stored in sterile containers at laboratory temperature until use.

Phytochemical analysis
The chemical analyses were carried out at the laboratory of Pharmacognosy and essential oils of the institute of Applied Biomedical Sciences based on a phytochemical screening. It is a qualitative analysis based on coloration and precipitation reactions. This was performed on the crude powders of the three vegetables following the methodology described by [22] and repeated by [23]. This method permits to determine some constituents such as Alkaloids, Polyphenol, Triterpenoids and Reducing compounds.

Aqueous extract
100 g of plant powder were boiled for 30 minutes in 1000 ml distilled water. The decoction, after cooling was filtered once on absorbent cotton and once on filter paper Whatman N°1. The obtained filtrate was then dried in an oven at 50°C, to produce the aqueous extract (AE).

Hydro-ethanolic extract
100 g of plant powder were agitated for 72 hours in 1000 ml 70% ethanol then filtered once on absorbent cotton and once on filter paper Whatman N°1. The hydro ethanolic phase was dried in an oven at 50°C, to give the hydro ethanolic extract (HE).
The output (R) every extract of each vegetables leaves studied was calculated and was expressed in relation to plant material extracts (powder). Its calculation makes itself of following manner:

Statistical Analyses
To assess the toxicity of the extracts on A. salina larvae, a probit analysis was performed. The LC 50 values of the tested plant extracts were then calculated, using Microsoft Excel 2010 software. A confidence interval of 95% was defined to conclude statistical differences. 4. RESULTS

Phytochemical Screening
The major chemical groups present in the leaves of the three tested plant species are displayed in    Alkaloids are the most effective phytochemical compounds in therapeutic uses [5,42]. The presence of alkaloids in the studied vegetables is an advantage to consumers who can benefit from the analgesic, anti-inflammatory, antibacterial and resistance to illnesses and stress properties of these compounds Particularly Vernonia amygdalina contains high amounts of saponins traces of steroids and cyanogenic derivatives. Saponins are important nutritional sources [52]. They are glycosidic molecules [53] and have a useful expectorant action in curing infections of the superior respiratory tract. Saponins are implicated in the treatment of hypercholesterolemia and possess antibacterial and anti-diarrheal properties [18,54]. Steroids are known for their analgesic and cardio-tonic properties [55]. They regularize the metabolism of proteins and carbohydrates, increase muscles and bones synthesis and are also associated with hormonal control in women [55]. Cyanogenic derivatives are serious metabolite poisons. However, the very low presence of this anti-nutritional substance in leaves of V. amygdalina by the current study can allow its consumption without harm.

Cytotoxicity of the Studied Local Vegetables
The output (R) of hydro-ethanolic extracts of vegetables leaves is raised more than the one of aqueous extracts of this same vegetables. The ethanol diluted to 70% could be a good solvent of extracting of active principles of V. amygdalina, C. adansonii and S. radiatum leaves.
Several studies proved the relevance of larval toxicity test in exploratory studies of toxicity [56]. A positive correlation was recorded between larval toxicity test and lethal oral dose of medicinal plants in mice [57].
Additionally, with respect to the correlation that exists between larval toxicity and toxicity on human cells, notably the 9 PS and 9 KB cells of human nasopharyngeal carcinoma on one hand [58], the A-549 cells of the pulmonary carcinoma and the HT-29 cells of the colon carcinoma on other hand [59], V. amygdalina, C. adansonii and S. radiatum can be considered as harmless to human cells. In short, these vegetables can be used daily as a basic food, and in traditional medicine as well without any cytotoxic risk. This justifies their excessive consumption by population and by their use by Beninese traditional healers [15].
The Lethal Concentration 50 (LC 50 ) of the aqueous extracts (AE) of each of studied vegetable was lower than the one of their hydroethanolic extracts (HE). Such situation shows that the AE present more effect (lethally) on the larvae of (A. salina) than their HE. This could be explained by the strong polarity of the AE against the HE. These results are similar to those reported by [60] who demonstrated that the LC 50 of methanolic extracts of Fagonia olivieri was higher than the one of its aqueous extract. Therefore, variations of toxicity can be observed within different extracts of the same plant. The results also revealed that out of the three studied vegetables, V. amygdalina showed the strongest larval lethally (LC 50 = 1.796 mg/ml) followed by C. adansonii (LC 50 = 2.261 mg/ml) and S. radiatum with a poor larval lethally (LC 50 = 2.629 mg/ml).
V. amygdalina, C. adansonii and S. radiatum are more harmless than Tamarindus indica, a plant used as well in nutrition and in traditional medicine [61,62]. In fact, when it is used at high dose, this vegetable presents a cellular toxicity [63]. The harmlessness of each of the three studied vegetables is also better than the one of many other vegetables such as Boerhavia diffusa [64], Moringa oleifera [8], Solanum macrocarpon [65], Crassocephalum crepidioides and Crassocephalum rubens [66] and Manihot esculenta [67]. This study allowed to classify V. amygdalina, C. adansonii and S. radiatum in the group of non-cytotoxic vegetables where are found Solanum macrocarpon [65], Crassocephalum crepidioides and Crassocephalum rubens [66] and Amaranthus hybridus, a vegetable mostly consumed in Kenya [14].

CONCLUSION
The phytochemical screening of the leaves of V. amygdalina, C. adansonii and S. radiatum revealed the presence of tannins, alkaloids, flavonoids, steroids, mucilages, coumarins, anthocyanins, leucoanthocyanes, quinone derivatives, cyanogenic derivatives and reducing compounds indicating that they have strong medicinal properties. This confirms their use in the treatment of various affections in Benin. Furthermore, the non-toxicity of these vegetables will allow to sensitize populations on their values and to promote their use in food for nutritional purposes and in traditional medicine for the therapeutic properties. However other studies are necessary to assess the antibacterial, antidiarrheal, anti-diabetic, anti-inflammatory and the blood pressure regulation potentials assigned by the local communities to these vegetables. However, the use of sophisticated and more advanced methods for the phytochemical screening and the cytotoxicity tests would be paramount to improve this study.