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S. Moore and Crassocephalum Crepidioides (Benth.) S Int.J.Curr.Microbiol.App.Sci (2013) 2(8): 1-13 ISSN: 2319-7706 Volume 2 Number 8 (2013) pp. 1-13 http://www.ijcmas.com Original Research Article Phytochemical screening and toxicity studies of Crassocephalum rubens (Juss. ex Jacq.) S. Moore and Crassocephalum crepidioides (Benth.) S. Moore consumed as vegetable in Benin A.Adjatin1, A.Dansi1*, E.Badoussi2, Y.L.Loko1, M.Dansi1, P.Azokpota2, F.Gbaguidi2, H.Ahissou3, A.Akoègninou4, K.Akpagana5 and A.Sanni3 1Laboratory of Biotechnology, Genetic Resources and Plant and Animal Breeding (BIORAVE), Faculty of Sciences and Technology of Dassa, University of Abomey-Calavi, 071BP28, Cotonou, Benin 2Department of Nutrition and Food Technology, Faculty of Agriculture (FSA), University of Abomey-Calavi, BP 526, Cotonou, Benin 3Laboratory of Biochemistry and Molecular Biology, Faculty of Sciences and Technology (FAST), University of Abomey-Calavi (UAC), P.O. Box 526 Cotonou, Benin 4National Herbarium, Department of Botany and Plant Biology, Faculty of Sciences and Technology (FAST), University of Abomey-Calavi (UAC), BP 526, Cotonou, Benin 5Laboratoire de Botanique, Faculté des sciences, Université de Lome, BP 1515, Lome, Togo *Corresponding author e-mail: [email protected] A B S T R A C T Gbolo (C. crepidioides and C. rubens) is a wild or semi-domesticated aromatic traditional leafy vegetable highly consumed in Benin. The vegetable was said to have medicinal values and locally used as nutraceutical. Considering the scientifically K e y w o r d s established potential toxicity of some vegetable species to humans and animals, leaves extracts of C. crepidioides and C. rubens were subjected to qualitative phytochemical Crassocephalum screening and testing for cytotoxicity using the brine shrimp lethality bioassay. The crepidioides; phytochemical screening recorded the presence of pharmacologically important Crassocephalum substances such as tannins, coumarins, combined anthracene derivatives C-heterosides, rubens; flavonoids, mucilage, reducing compounds and Steroids. Alkaloids, anthocyanins, leafy vegetable; quinone derivatives, saponins, triterpenoids, cyanogenic derivatives, cardiac glycosides nutraceutical; and anthracene derivatives (except anthracene derivatives C-heterosides) were not phytochemical detected. The LC50 values of the leaves extracts were found to be 0.901 mg/ml for C. screening; crepidioides and 0.374 mg/ml for C. rubens hence indicating the non-toxicity of both toxicity; species. The outcome of this study lends support to the trado-medicinal uses of Benin. vegetable Gbolo in the treatment of various ailments in Benin and indicates the potential usefulness of this vegetable as nutraceutical to prevent or treat various diseases. However more studies are needed for the validation of the antimicrobial, anti- diabetic, anti-inflammatory and blood pressure regulation properties attributed by the local communities to this vegetable Gbolo Introduction Traditional vegetables from the wild or humans both in rural and urban set ups home gardens are mutually important for (Dansi et al., 2008). Traditional leafy 1 Int.J.Curr.Microbiol.App.Sci (2013) 2(8): 1-13 vegetables (TLVs) are those plants whose the vegetable locally known as Gbolo was leaves or aerial parts have been integrated found to be of paramount interest (Dansi et in a community s culture for use as food al.,2008). Gbolo comprising two species over a large span of time (Adeoti et al., namely Crassocephalum rubens (Juss. ex 2012). TLVs are highly recommended Jacq.) S. Moore and C. crepidioides because they have a relatively high (Benth.) S. Moore (Figure 1a, b), is highly nutritional value compared to the consumed throughout Benin. According to introduced varieties and their consumption Adjatin et al., (2013), the nutritional gives diversity to daily food intake, adding values of these two species although flavour and zest to the diet (Ahohuendo et slightly different are of high importance. al., 2012). According to Dansi et al., The contents of the leaves in raw proteins (2008) many traditional leafy vegetables and in crude lipids expressed in % of dry has some curative, regulative and matter are, respectively, 27.13%, 3.45 for stimulative properties besides food C. crepidioides; 26.43% and 2.75% for C. qualities and are used as nutraceutical. rubens. The content of vitamin C for 100 g Though the bulk of the weight of of fresh leaf is of 9.17 mg for C. traditional leafy vegetables is water, they crepidioides and 3.60 mg for C. rubens. represent sometimes a veritable natural The content of ash is of 19.76% and pharmacy of minerals, vitamin and 19.02% for C. rubens and C. crepidioides, phytochemical compounds such as respectively. The contents for sodium alkaloids, flavonoids, glycosides and (Na), potassium (K), magnesium (Mg), tannins. However several studies have calcium (Ca), iron (Fe), Manganese (Mn), established that some vegetable species are and copper (Cu) are 2129.04 mg, 4469.91 potentially toxic to humans and animals mg, 434.13 mg, 3845.88 mg, 1.6 mg, 8.22 (Agbaire et al., 2013). Plant chemical mg, and 2.6mg, respectively, for C. rubens compounds, toxic to humans and and these are higher than those in C. livestock, are produced as part of the crepidioides (Adjatin et al.,2013). plant s defence against being eaten by Traditionally, Gbolo is used as a pests and herbivores or to gain an nutraceutical and believed to have advantage over competing plants (Bharathi antibiotic, anti-helminthic, anti- 2008; Asaolu et al., 2009; Andzouana and inflammatory, anti-diabetic, anti-malaria Mombouli 2012; Agbaire et al.,2013). and blood regulation properties and also Plant poisons are highly active substances treats indigestion, liver complaints, colds, that may cause acute effects when ingested intestinal worms, and hepatic insufficiency in high concentrations and chronic effects in addition to its nutritional value (Adjatin when accumulated (Agbaire et al., 2013). et al.,2012). Since Gbolo is highly Under stress conditions, brought on by consumed (leaves eaten raw or cooked in food shortage, consumption of large sauce) by Benin local populations, its amounts of vegetable toxins can have phytochemical screening need to be negative consequences (Orech et al., carried out and its toxicity examined. The 2005). phytochemical screening and the toxicity results in addition to the already In Benin, a biodiversity inventory and established nutritional value of the two documentation survey recently conducted species of Gbolo will be used to create on TLVs throughout the country revealed awareness of their values and, when a total of 187 plant species among which possible (i.e. if not toxic and trado- 2 Int.J.Curr.Microbiol.App.Sci (2013) 2(8): 1-13 medicinal uses supported), popularize their tannins (ferric chloride test after usage in diets as nutraceutical. saturation with sodium acetate), Flavonoids (shinoda test and magnesium The objectives of this study were two powder), Cyanogenic derivatives (picric folds: Carry out a qualitative acid test), Triterpenoids (acetic acid test + phytochemical screening of the leaves mixture of acetic an hydride and sulfuric extract of C. crepidioides and C. rubens acid), Steroids (kedde reaction), Saponins and investigate the cytotoxic activity of C. (test index foam), Cardiac glycosides crepidioides and C. rubens leaves using (Raymond Marthoud reaction), brine shrimp assay Anthocyanins (test with hydrochloric acid and ammonia diluted to half), Materials and Methods Leucoanthocyanes (shinoda test), Mucilage (test of absolute alcohol), Source and Preparation of vegetable Reducing compounds (test with fehling s Gbolo samples solution), Coumarins (test with ether andammonia), Free anthracene Samples (stems and leaves) of Gbolo derivatives (test with chloroform (Crassocephalum rubens and andammonia), Combined anthracene Crassocephalum crepidioides) were derivatives (test with chloroform obtained from the germplasm maintained andammonia). at the Faculty of Science and Technology of Dassa (FAST Dassa) in Benin. The Brine Shrimp Lethality Assay stems and leaves of Gbolo were washed thoroughly under running tap water The cytotoxic activity of the extracts of the followed by sterile distilled water, cut two species of Gbolo was evaluated using into smaller pieces and dried under shade Brine shrimp lethality bioassay. Brine during for 9 days. The dried plant parts shrimp (Artemia salina Leach) also known were ground using electric blending as sea monkey are marine invertebrates of machine and the powdery samples about 1mm in size. The test is based on obtained were sieved using two sieves of the survival of shrimp larvae in sea water 0.2 mm (mesh size) and stored in air tight in the presence of the test solution. Its sterile containers until needed. interest lies in understanding the possible side effects that would result from Phytochemical analysis consumption of leaves of vegetable Gbolo on the body. A solution was prepared by Qualitative phytochemical screening of moderate heating for 20 minutes, the Gbolo species was carried out on the mixture of 1 g of powdered leaves of C. powdery samples, after extraction with rubens or C. crepidioides in 20 ml of aqueous solvent, using the standardly distilled water following Dougnon et employed precipitation and coloration al.,(2013). The concentration of 50 reactions as described by Houghton and mg/ml was obtained and a range of ten Raman (1998) and Dougnon et al.,(2013). successive dilutions (49 µg/ml, 98 µg/ml, Major secondary metabolites essayed and 195 µg/ml, 391 µg/ml, 781 µg/ml, 1582 the methods used were as follow: µg/ml, 3125 µg/ml, 6250 µg/ml, 12500 Alkaloids (Mayer s test), Quinone µg/ml, 25000 µg/ml) were made with sea derivatives (born-trager reaction), water from decoction. Eggs of A. salina Cathetic tannins (stiasny test), Gallic 3 Int.J.Curr.Microbiol.App.Sci (2013) 2(8): 1-13 were grown in an erlenmeyer containing beneficial for the maintenance of good sea water taken from the Atlantic Ocean health and prevention of some diseases and filtered before use. The mixture (eggs (Dansi et al., 2013). The qualitative and sea water) was left under stirring for phytochemical screening of the powdery 48 hours.
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