Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 2(5), 6-9, May (2013) Res. J. Recent Sci. International Science Congress Association        
6
 
Cytotoxicity of Leaves and Fruits of Solanum macrocarpon Linn (Solanaceae) against shrimp larvae (Artemia salina Leach)Dougnon T.V.1,2*, Bankolé H.S., Edorh A.P.1,3, Dougnon T.J., Klotoé J.R., Loko F., and Boko M.Laboratory of Toxicology and Environmental Health, Interfaculty Center of Formation and Research in Environment for the Sustainable Development, University of Abomey-Calavi (UAC), 01 BP 1463 Cotonou, BENIN Laboratory of Research in Applied Biology, Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, 01 BP 2009 Cotonou, BENIN Department of Biochemistry and Cellular Biology, Faculty of Science and Technology, University of Abomey-Calavi (UAC), 01 BP 526 Cotonou, BENINAvailable online at: 
www.isca.in
Received 3th October 2012, revised 5th November 2012, accepted 7th February 2013 Abstract  Traditional leafy vegetables are these plants whose leaves or aerial parts have been integrated in a community’s culture for use as food over a long span of time. These vegetables are highly recommended due to their relatively high nutritional value compared to the introduced varieties, and are also important in food security. They are also used as medicines. This study 
aims to assess the potential cytotoxicity of S. macrocarpon, a vegetable highly used in Africa.After hatching shrimp larvae 
for 48 hours, they were brought into contact with aqueous dilutions of the leaves as well as fruit for 24 hours. The variation 
in larval mortality as a function of concentration has been translated by a curve and semi-lethal concentrations were 
determined. In addition, the values of the half-lethal concentration (LC50 = 1.33 mg / ml for leaves and 1.51 mg / ml for fruit) were all greater than 0.1 mg / ml, the upper limit of toxicity.It follows then that the leaves and fruits were not toxic on shrimp larvae for the range of explored concentrations. These parts of vegetable can therefore be used both in traditional medicine and nutrition without immediate or medium-term major risks. Keywords: S. macrocarpon, cytotoxicity, LC50, vegetable. Introduction Vegetables are excellent contributions enrichment and diversification of food. They are the primary source of mineral nutrients, vitamins and other compounds involved in the health and human nutrition. In fact, vegetables are important in food security during periods of drought or crop failure and are also important sources of income. In addition to their nutritional value, vegetables are also sources of drugs2-4. In Benin, S. macrocarpon called "Gboma" in the local language Fon is grown by nearly of 95% of market gardeners. This high production is justified by the high consumption of this vegetable by people. Thus, the leaves and young fruits are prepared and eaten as a vegetable in several African countries. In addition, this plant appears to have an interest in traditional medicine in the treatment of various disorders. Despite the importance of the nutritional and medicinal vegetable, no scientific work to date, has provided scientific evidence that consumption of S. macrocarpon would have no effect on human cells. This is why the present study evaluated the cytotoxicity of leaves and fruits of S. macrocarpon against larvae of shrimp. It is justified by the fact that previous studies have revealed that the fruits of S. macrocarpon especially should be consumed with caution because of some chemical groups that phytochemical screening revealed8-9. This study is a contribution to a better understanding of this vegetable and to control possible risks associated with its use. Material and Methods Material: The leaves and mature fruit of S. macrocarpon (figure 1) were the plant material used in this study. They were purchased in July 2012 on the gardener site of Houéyiho at 6° 21' 20'' North latitude and 2° 21' 35'' East longitude in Benin. The plant was identified and authenticated by the National Herbarium of Benin under N° AA 6423/ HNB of 31 July 2012. Toxicity was evaluated at Laboratory of Pharmacognosy and Essential Oils with shrimp larvae whose eggs are marketed by the German Company JBL Gmbh & Co.KG. Plant preparation: The leaves were dried at room temperature of 16°C in Research Laboratory in Applied Biology (University of Abomey-Calavi) for 17 days while fruits were finely cut into small pieces and dried for 09 days. Dried leaves and fruits were ground using a SAYONA Moulinex commercially purchased. The powders obtained were sieved using sieves of 0.2 mm and stored in sterile containers until needed. Cytotoxicity test on shrimp larvae: This test is based on the survival of shrimp larvae in sea water in the presence of the test solution. Its interest lies in understanding the possible side effects that would result from consumption of fruits and leaves of S. macrocarpon on the body. It is a test of primary non-clinical toxicity proposed by Michael et al.10 and later developed by Vanhaecke et al.11, Sleet and Brendel12. This test is proposed 
Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(5), 6-9, May (2013)                              Res. J. Recent Sci.   International Science Congress Association 
7
as a simple method of bioassay for preliminary assessment of toxicity in search of natural active products13. Eggs of A. salinawere grown in an erlenmeyer containing sea water taken from the Atlantic Ocean and filtered before use. The mixture was left under stirring for 48 hours. Meanwhile, the eggs hatched to give birth to young larvae. Using a pipette, a colony of 16 live larvae was placed in contact with a series of solutions of graded concentrations of decoction of S. macrocarpon. These solutions and controls containing no solutions of S. macrocarpon were left stirring and read twenty-four hours later. In case of death in the control medium, the data are corrected by Abbott's formula14:  % Death = [(test-control) / control] × 100 The dose-response data were log-transformed and the Lethal Concentration 50 (LC50) determined by a linear regression study15. To assess the degree of toxicity from LC50 values, the correlation table (table 1) established by Mousseux16 was used. Preparation of test solutions: A solution was prepared by moderate heating for 20 minutes the mixture of 1 g of powdered leaves and fruits of S. macrocarpon in 20 ml of distilled water. The concentration of 50 mg/ml was obtained. A range of ten successive dilutions were made with sea water from decoction. Concentration values expressed in mg/ml of the dilutions contained in test tubes were 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, 0.09, and 0.04.  Table-1 Correspondence between LC50 and toxicity 
LC
50
 
Toxicité 
LC
50 
 0.1 mg/ ml 
(Non-toxic) 
0.1 mg/ ml �LC50  0.050 
mg/ ml 
+ (Low toxicity) 
0.050 mg/ ml �LC50
0.01 mg/ ml 
++ (Moderate toxicity) 
LC
50 
 0.01 mg/ ml 
+ + + (High toxicity) 
Statistical analysis: For the toxicity study, A. salina larvae were exposed to different concentrations of powdered decoction of leaves and fruits to determine the concentration that will kill 50% of larvae in 24 hours. Probit analysis was used to analyze data from toxicity tests. LC50 values of  tested powders were calculated with Microsoft Excel 2010. A confidence interval of 95% has been set.  
Figure-1 Leaves and fruits of S. macrocarpon
Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(5), 6-9, May (2013)                              Res. J. Recent Sci.   International Science Congress Association 
8
Results and DiscussionSeveral studies have shown that larval toxicity test is an excellent method for preliminary investigations of toxicity17. Para et al.18 have also demonstrated a positive correlation between larval toxicity test and the oral lethal dose of medicinal plants in mice. Shrimp larvae remained sensitive to leaves and fruits of S. macrocarpon with a threshold dose of mortality equal to 0.39 mg / ml (figures 2 and 3). Sensitivity curves showed that the death of the larval population increases with the concentration. This sensitivity thus followed a dose-response relationship.  In addition, the values of the half-lethal concentration (LC50 = 1.33 mg / ml for leaves and 1.51 mg / ml for fruit) were all greater than 0.1 mg / ml, the upper limit of toxicity16. We then deduce that the leaves and fruit are not safety on shrimp larvae for the range of concentrations explored. In addition, taking into account the correlation between larval toxicity and toxicity to cells, leaves and fruits of S. macrocarpon does not have harmful effects on human cells namely PS 9 and 9 KB (human carcinoma) on one hand19; A-549 cells of lung carcinoma and HT-29 cells of colon carcinoma on the other part20. In sum, the organs studied for this vegetable can be used for daily basic diet without exposure to adverse effects in short and medium term. This work reinforces the phytochemical study recently conducted by Dougnon et al9 who showed the richness of fruits in secondary metabolites. The non-cytotoxicity of S. macrocarpon justifies that the leaves and fruits are widely consumed as a vegetable in Africa.  In addition, the medicinal properties of this plant recognized in Africa can be seriously explored, since there is no cellular toxicity associated with its use. S. macrocarpon has better harmlessness compared to Tamarindus indica, a plant of the family Leguminoseae, which is also used as food and in traditional medicine21-23. Indeed, T. indica used in high doses, seems to have a certain cell toxicity24. It is the same with plants as Lannea barteri (K & Gillet), Sclerocaria birrea A. Rich, Annona senegalensis Pers., Kigelia africana (Lam). Benth, Ananas comosus L. Merr., etc. used in the treatment of malaria and various cancers in Nigeria25, Muntingia calabura used for its antibacterial properties in Brazil26, Abroma augusta, Acanthus ilicifolius used in traditional medicine in Bangladesh27. This study classified S. macrocarpon among the non-cytotoxic vegetables such as Amaranthus hybridus, a highly consumed vegetable in Kenya28. ConclusionThe present study scientifically demonstrated that both leaves and fruits of S. macrocarpon grown in Benin can be eaten and used in traditional medicine in short and medium term without major risk. However, long-term use should be done with caution, since no scientific study has explored this aspect. Conflict of interest: Authors declare no conflict of interests. 
Figure-2 Sensivity curve of the decoction of leaves against shrimp larvae 
Figure-3 Sensivity curve of the decoction of fruits against shrimp larvaeAcknowledgment We sincerely thank all the technicians of the Laboratory of Pharmacognosy and Essential Oils at Benin Center of Scientific and Technical Research for their various contributions. Authors also thank the National Program of Traditional Medicine and Pharmacopoeia of Benin for its support. References 1.Sinha N.K., Handbook of vegetables and vegetables processing, Blackwell Publishing Ltd, England, 788,(2011)2.Abbiw D.K., Diversity and traditional uses of African vegetables. In: Guarino L (Ed). Traditional African Vegetables: Proceedings of the IPGRI International Workshop on Genetic Resources of Traditional Vegetables in Africa, Conservation and Use. ICRAFHQ, Nairobi, Rome: Institute of Plant Genetic and Crop Plant Research: 29-30 (1997)3.Okafor J.C., Conservation and use of traditional vegetables from woody forest species in southeastern Nigeria. In: Guarino L (Ed), Traditional African Vegetables: 
Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(5), 6-9, May (2013)                              Res. J. Recent Sci.   International Science Congress Association 
9
Proceedings of the IPGRI International Workshop on Genetic Resources of Traditional Vegetables in Africa, Conservation and Use, ICRAF-HQ, Nairobi, Institute of Plant Genetic and Crop Plant Research, Rome, 31-38 (1997)4.Geissler P.W., Harris S.A., Prince R.J., Olsen A., Odhiambo R.A. and Oketch-Rabah H., Medicinal plants used by Luo mothers and children in Bondo District, Kenya, J. Ethnopharma,83, 39-54 (2002)5.Assogba-Komlan F. and Anihouvi P., Pratiques culturales et teneur en éléments anti nutritionnels (nitrates et pesticides) du Solanum macrocarpum au sud du Bénin, AFJAND, 7(4), 01 – 21 (2007)6.Dougnon T.V., Edorh P.A., Bankolé H.S., Dougnon T.J., Montcho S.A., Hounkpatin A., Gouissi M., Sossou B., Boko M. and Creppy E.E., Evaluation of the toxicological quality of the leaves of Solanum macrocarpum L. cultivated with the chicken’s droppings and water of marsh at Cotonou (Benin), Journal of Research in Environmental Science and Toxicology, 1(1), 001-006 (2012a)7.Sodipo O.A., Abdulrahman F.I., Alemika T.E. and Gulani I.A., Chemical composition and biological properties of the petroleum ether extract of Solanum macrocarpum L. (Local Name: Gorongo), British Journal of Pharmaceutical Research, 2(2), 108-128 (2012)8.Olaniyi A.A., Ayim J.S.K., Ogundaini A.O. and Olugbade T.A., Essential inorganic and organic chemistry, Omoade Printing Press, Ibadan, Nigeria, 582 (1998)9.Dougnon T.V., BankoléH.S., KlotoéJ.R., DougnonG., Gbaguidi F., Assogba F., Gbénou J., SahidouS., AtègboJ-M., Rhin B., Loko F. and EdorhA.P., Phytochemical, nutritional and toxicological analyses of leaves and fruits of Solanum macrocarpon Linn (Solanaceae) in Cotonou (Benin), Food and Nutrition Sciences, 3(12), 1595-1603 (2012b)10.Michael A.S., Thompson C.G. and Abramovitz M., Artemia salina as a test organism for a bioassay, Science, 123,  464 (1956)11.Vanhaecke P., Persoone G., Claus C. and Sorgeloos P., Proposal for a short-term toxicity test with  Artemia nauplii, Ecotoxicol Env Safety,5, 382-387 (1981)12.Sleet R.B. and Brendel K., Improved methods for harvesting and Counting synchronous populations of Artemia nauplii for use in developmental toxicology, Ecotoxicol Env Safety, 7, 435- 446 (1983)13.Solis P.N., Wright C.W., Anderson M.M., Gupta M.P., Phillipson J.D., A microwell cytotoxicity assay using Artemia salina. Plant Med., 59, 250-252 (1993)14.Abbott W.S., A method of computing the effectiveness of an insecticide, J. Econ. Entomol.,18, 265 (1925)15.Hafner E., Heiner E., Noack E. Arzn eim-Forsch., 27, 1871 (1977)16.Mousseux M., Test de toxicité sur les larves de Artemia salina et entretien d'un élevage de balanes, Rapport de stage de 2ème année. DEUST Aquaculture; Centre Universitaire de Nouvelle-Calédonie, France (1995)17.Quignard E.L., Pohlit A.M., Nunomura S.M., Pinto A.C., Santos E.V. and Morais S.K., Screening of plants found in Amazonas state for lethality towards brine shrimp, Acta Amazon., 33, 93-104 (2003)18.Parra A.L., Yhebra R.S., Sardinas I.G. and Buela L.I., Comparative study of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts,Phytomedicine, 8, 395-400 (2001)19.Pelka M., Distler W., Petschelt A. and Dent J., A new screening test toxicity testing of dental materials, 28, 341-345 (2000)20.Carballo J.L., Hernández-Inda Z.L., Pérez P., García-Grávalos M.D., A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products, BMC Biotechnology,  , 17 (2002)21.Anon B., The Useful Plants of India; CSIR: New Delhi, India (1986)22.Morton J., Tamarind In: Fruits of Warm Climates; Creative Resources Systems, Inc.: Miami, FL, USA, 115-121 (1987)  23.Pamploma-Roger G.D., Encyclopaedia of Medicinal Plants; Education and Health Library: Madrid, Spain, 2, 536 (1999)24.Nwodo U.U., Ngene A.A., Anaga A.O., Chigor V.N., Henrietta I.I., Okoh A.I. Acute toxicity and hepatotoxicokinetic studies of Tamarindus indica Extract, Molecules,16, 7415-7427 (2011) 25.Adoum O.A., Determination of toxicity levels of some savannah plants using Brine Shrimp Test (BST) Bajopas, 2(1), 135-138 (2009)26.Ramos S.C.S., de Oliveira J.C.S., da Câmara C.A.G., Castelar I., Carvalho A.F.F.U, Lima-Filho J.V. Antibacterial and cytotoxic properties of some plant crude extracts used in Northeastern folk medicine, Brazilian Journal of Pharmacognosy,19(2A), 376-381 (2009)27.Rahmatullah M., Sadeak S.M.I., Bachar S.C., Hossain M.T., Mamun A. and Jahan N., Brine Shrimp Toxicity Study of Different Bangladeshi Medicinal Plants, Advances in Natural and Applied Sciences, 4(2), 163-173 (2010)28.Orech F.O., Akenga T., Ochora J., Friis H., AagaardHansen J., Potential toxicity of some traditional leafy vegetables consumed in Nyang’oma division, Western Kenya, African Journal of Food and Nutritional Sciences, 5(1), 1-14 (2005)
<end>