Phytochemical screening , antioxidant and cytotoxic activity of different morphotypes of Corchorus olitorius L . leaves in the central region of Benin Republic ( West Africa )

1 Faculté des Sciences et Techniques de Dassa (FAST/Dassa), Université Nationale des Sciences, Technologies, Ingénierie et Mathématiques (UNSTIM), BP 14, Dassa-Zoumè, Bénin. 2 Ecole Normale Supérieure d’Enseignement Technique (ENSET), Université Nationale des Sciences Technologies, Ingenierie et Mathematiques (UNSTIM), BP 133 Lokossa, Benin. 3 Laboratoire de Pharmacognosie et des Huiles Essentielles, Faculté des Sciences et Techniques (FAST), Université d’Abomey-Calavi (UAC), BP 526, Cotonou, Bénin. 4 Laboratoire de Biochimie et Nutrition. Faculte des Sciences. Universite de Lome. B.P 1515. Lome, Togo.


INTRODUCTION
Human diets are becoming more varied and allow to cover the energy needs of human organism and the basal metabolism but without taking into account the nutritional and health security aspects (Mulungulungu et al., 2015).Nowadays, the light is focused on foods rich in nutraceutical and functional properties.Nutraceutical combining the words "nutrition and pharmaceutical" is a food or food product that provides health and medicinal benefits including the prevention and disease's treatment (Pepsi et al., 2012).The nutraceutical is used to accomplish desirable therapeutic outcomes with reduced side effects as compared with other therapeutic agents with great monetary success (Pepsi et al., 2012).
Edible plants have been used as nutraceutical in Benin and contributed to reduce health risks (Djengue et al., 2017a;Koukoui et al., 2015;Adjatin et al., 2013).The consumers' interest has been the use of foods with more natural antioxidants, dietary fibers, natural colorants, minerals, vitamins and synthetic additives free, etc.Thus, the consumption of fruits, vegetables and other nutraceutical foods is increasing.High consumption of fruits and vegetables is associated to the reduction of cardiovascular disease and some cancers risks (Lazou and Kronida, 2010).Generally, leafy vegetables are prominently used in the diets of people in the world particularly in Africa, Asia and Oceania, where they provide an essential part of nutritional and medicinal purposes (Dansi et al., 2008).Corchorus olitorius L. is traditional leafy vegetable belongs to the family of Malvaceae (Whitlock et al., 2003).Recently, Heywood et al. (2007) split the large Malvaceae and classified Corchorus within the family Sparrmanniaceae.Genus Corchorus consists of annual or short-lived perennial herbs and shrubs with many agriculturally important species.It consists of some forty species of which about thirty are found in Africa (Mbaye et al., 2001).In Benin, it is represented by five species Corchorus aestuans L., Corchorus fascicularis Lam., C. olitorius L., Corchorus tridens L. and Corchorus trilocularis L. (Akoegninou et al., 2006).C. olitorius L. is the most popular in Benin because it is cultivated countrywide as vegetable and its local name varies according to the regions (Dansi et al., 2008;Adjatin et al., 2017).It is recognized for its high intra-specific diversity and a wide distribution (Benor et al., 2011).In fact, the leaves are alternate, oval, lanceshade and toothed, allowed to identify different morphotypes (Adebo et al., 2015;Adjatin et al., 2017).The leaves of C. olitorius can be eaten fresh like spinach, cooked or stored as a dried powder for at least six months without any major loss of its nutritional value (Choudhary et al., 2013).In Benin, its leaves are used to make a sticky cooked sauce that accompanies the main courses (Adjatin et al., 2017).It is of high social economic importance especially for the local farmers for which, the livelihoods depend essentially on C. olitorius production and utilization (Komlan et al., 2013).C. olitorius is rich in protein, iron, calcium, vitamin (B, C), Beta-caroten and folic acid and constitute good nutritional supplements (Choudhary et al., 2013).Its leaves, roots and fruits are used in traditional medicine to treat various diseases such as gonorrhea, chronic cystitis, pain, fever and tumors (Kumawat et al., 2012).A regularly consumption of C. olitorius leaves helps to control blood pressure, cholesterol and lowers the risks of asthma, cancer, diabetes and heart disease (Handoussa et al., 2013).Also, decoctions of these leaves would treat many diseases such as typhoid fever, anemia, malaria and ulcer (Adjatin et al., 2017).Consequently, this vegetable is considered as an important medicinal taxon because of its several medicinal properties.Based on the aforementioned uses of this plant to cure many diseases, a qualitative phytochemical screening of leaves' extracts from different morphotypes of C. olitorius was carried out in order to identify phytochemical elements which confer to species, its several medicinal properties and also to investigate their antioxidant and cytotoxic activities.

Collection and extraction of plant material
A total of twelve accessions from seven morphotypes C. olitorius were collected from six districts of Central Benin (Table 1).Morphotypes included in the study were identified from study conducted by Adjatin et al. (2017) on phenotypic diversity of C. olitorius in the same region.
Fresh leaves samples of each accession were washed thoroughly under running tap water followed by sterile distilled water.Each sample was then cut into smaller pieces and dried under shade for two weeks.The dried leaves were ground with electric blending machine and the obtained powdery samples were sieved using a sieve of 0.2 mm (mesh size) and stored in air tight sterile containers for the analysis.

Phytochemical analysis
Qualitative phytochemical screening of C. olitorius was carried out on the powdery samples, after extraction with aqueous solvent (distilled water), using the standardly employed precipitation and coloration reactions as described by Houghton and Raman (1998) and used by Djengue et al. (2017b)

Total antioxidant capacity (TAC) assay
Antioxidant activity was related to the capacity of plant extract to trap the free radical molecules.The technique applied for this determination used the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method described by Velazquez et al. (2003).A volume of 1.5 mL of DPPH solution (4mg of DPPH dissolved in 10 mL of methanol) was mixed with 0.75 mL of leaves extract of each accession at different concentration (0.5 -3.5 mg/mL).After 15 min of incubation in the dark, at the room temperature, absorbance of the mixture was read at the wavelength of 517 nm with a spectrophotometer using ascorbic acid as blank and standard.The total antioxidant activity was expressed as Equivalent of Ascorbic acid per gram of aqueous extract (mmol EqAA/g).

Brine shrimp lethality assay
The cytotoxic activity of the extracts from the twelve accessions was assessed using Brine shrimp lethality bioassay.Brine shrimp (Artemia salina Leach) also known as sea monkey are marine invertebrates of about 1 mm in size.The test is based on the survival of shrimp larvae in sea water in the presence of the test solution.Its interest is linked in the understanding of the possible side effects that would result in consumption of C. olitorius leaves on the organism.A solution was prepared by moderate heating of the mixture of 1 g of powdered leaves in 20 ml of distilled water according to Agbankpé et al. (2015).The concentration of 50 mg/ml was obtained and from this decoction, a range of ten successive dilutions (49,98,195,391,781,1582,3125,6250,12500, and 25000 µg/ml) were made with sea water from decoction.Eggs of A. salina were grown in an erlenmeyer containing sea water taken from the Atlantic Ocean and filtered before use.The mixture (eggs and sea water) was incubated during 48 h.Meanwhile, the eggs were hatched to give birth to young larvae (nauplii).When using a pipette, a colony of 16 living larvae was placed in contact with the series of solutions of graded concentrations of C. olitorius decoction.These solutions and the control containing no extract of C. olitorius were incubated and read after 24 h.The total death and percentage of mortality at each dose level and control were determined.

Statistical analysis
Descriptive statistic of data from screening phytochemical was presented in the form of figures and tables.For the toxicity assessment, counting the number of survival larvae in each solution was allowed to evaluate the toxicity of the solution.The percentage of death was then calculated according to the following formula: For each sample, the lethal concentration that causes 50% death (LC50) was calculated at 95% confidence interval by linear regression analysis and also by using the probit analysis method following Djengue et al. (2017b).A regression line equation was derived for each extract with the mortality data obtained and, it was then used to calculate the LC50.The detailed mathematical steps used to derive the regression line equation are reported in the literature (Vincent, 2012).The correlation table established by Mousseux (1995) was used to assess the degree of toxicity of the different samples from LC50 values (Table 2).

Phytochemical screening
Phytochemical screening of the leaves extracts of the twelve samples of C. olitorius revealed the presence of several phytochemical compounds (Table 3).Flavonoids, leuco-anthocyanes, cathetic and gallic tannins, mucilage, quinone derivatives and reducing compound were present in all the studied extracts whereas saponins, triterpenoids, cardenolid derivatives, cyanogenic derivatives, combined anthracene O-heterosides and glycosides cardiotonic were not detected in any of the analysed extracts (Table 3).In addition, the phytochemical compounds such as alkaloids, steroids, coumarins, free anthracene derivative and anthracene Cheterosides were present in some morphotypes and

Anthracene derivatives
Free anthracene derivatives absents in others (Figure 1).The most of phytochemical elements found in all morphotypes are phenolic compounds with high antioxidant proprieties (Kumar et al., 2018).Moreover, it could be observed that the morphotypes Cor1, Cor2, Cor5 and Cor10 have more phytochemicals (58%) than the others (Figure 2).Both the percentage and the qualitative composition of phytochemical vary from one morphotype to another.These results are similar to those obtained by Bouabid et al. (2016) who notified a variability of phytochemical composition between two grapefruit varieties (Citrus paradisi).Considering the characteristics of the different morphotypes collected in the study area (Adjatin et al., 2017), the variations observed on the leaves are related neither to the shape and appearance of the leaves nor to the stems colour.These variations could be explained by the physiological stage of the plant during leaf harvesting.In fact, the bioactive constituents or secondary metabolites synthesized by the plants vary according to their different stages of growth (Okunlola et al., 2017).

Results (%)
However, some elements are found in the leaves at all stages of species growth and development and could be specific to certain varieties.For example, flavonoids are present in all samples of studies morphotypes.According to Okunlola et al. (2017), the presence of a phytochemical in all the stages of C. olitorius growth, showed that this phytochemical is synthetized in the plant at the vegetative, flowering and fruiting stages and justified then the presence of flavonoids in all samples of these morphotypes.In the current study, saponosides are absent in all studied morphotypes.These results are similar to those of Mohammed (2016).However, the results of Okunlola et al. (2017) showed the presence of saponosides in the leaves of C. olitorius during vegetative and flowering stages but indicated their absence at fruiting stage.Therefore, the phytochemical screening of the studies' morphotypes at different development stages of the plant is required to confirm or not the production phytochemicals which are secondary metabolic involved in the plant defence system according to Okunlola et al. (2017).

Total antioxidant capacity (TAC)
Total antioxidant capacity represents both oil soluble and water soluble antioxidants that are capable of scavenging reactive oxygen species (ROS) and protects from chronic diseases such as cancer, diabetics and arthritics.ROS that are produced as a result of cellular metabolism are highly toxic and are involved in the etiology of many chronic diseases due to oxidative damage to lipids, nucleic acids and proteins (Yanishlieva et al., 2006).The results of the antiradical activity of the aqueous extracts of the various C. olitorius morphotypes are as shown in Figure 3.Samples exhibited wide range of TAC values, expressed as the number of equivalents of ascorbic acid, from 17,527 mmol EqAA/g for Cor9 which is a variety with cordate and shiny leaves to 32,985 mmol EqAA/g for Cor1 variety with cordate and non-shiny leaves.All studied morphotypes possess an antioxidant potential on  (Oboh et al., 2009;Handoussa et al., 2013;Youssef et al., 2014).The presence of phytochemicals in extracts of all these morphotypes is in agreement with their antioxidant capacity.Among the phytochemicals, phenolic compounds are reported to be the main contributor of antioxidant activity in plant extracts (Dai and Mumper, 2010).Polyphenolic antioxidants from dietary sources are frequently a topic of interest due to widespread scientific agreement that they may help lower the incidence of certain cancers, cardiovascular and neurodegenerative diseases, and DNA damage and even may have antiaging properties (Obrenovich et al., 2010).Consumption of polyphenols has been linked to lowered risks of many chronic diseases including cancer, cardiovascular diseases, chronic inflammation and many degeneration diseases (Scalbert et al., 2005;Tsao, 2010).
Moreover, analysis of Figure 3 indicated that all samples have not the same antioxidant capacity and showed two categories of morphotypes.In the first groups, the morphotypes with a better radical scavenging activity whose antioxidant capacities are between 27.859 and 32.985 mM EAA/g of extract.These are Cor1, Cor2, Cor3, Cor5, Cor6 and Cor10 varieties.The second category constituted Cor4, Cor7, Cor8, Cor9 Cor11 and Cor12 varieties, is the one of morphotypes with low antiradical activity whose antioxidant capacities varies from 17.527 to 23.098 mM EAA/g.Among phytochemicals, only free anthracene derivatives are detected in the morphotypes of C. olitorius with high potential antioxidant.Then free anthracene derivatives found only in these morphotypes would act with another phytochemicals and might certainly explain their strongest radical scavenging activity of DPPH.In fact, levels of individual antioxidants in food do not necessarily reflect their total antioxidant capacity, which could also depend on synergic and redox interactions between the different antioxidant molecules (Hodzic et al., 2009).According Yanishlieva et al. (2006), free radicals and reactive oxygen species are involved in a variety of pathological events such as aging, inflammation, cancer, atherosclerosis, and diabetes.Then, regular consumption of C. olitorius, especially varieties having rounded and non-shiny leaves, elongated and shiny leaves, red stems or elongated and non-shiny leaves with lobed lateral, would be useful for the treatment of various diseases induced by free radicals as others traditional leafy vegetables such as Launeae taraxacifolia (Koukoui et al., 2015), Hibiscus sabdariffa, Solanum scabrum and Basella alba (Konan et al., 2016).
Besides, these values were greater than 0.1 mg/ml, which is the limit of toxicity (Mousseux, 1995).Then, in this study, that none of the morphotypes of C. olitorius investigated was harmless to shrimp's larvae.Taking into account the established correlation between the toxicity of shrimp larvae and that of human cells, all the morphotypes of C. olitorius involved in this study can be used daily both in food and in traditional medicine without risk of toxicity.C. olitorius can therefore be considered as a traditional nutraceutical leafy vegetable such as Lippia multiflora (Djengue et al., 2017a), Launea taraxacifolia (Koukoui et al., 2015), Crassocephalum crepidioides and Crassocephalum rubens (Adjatin et al., 2013).

Conclusion
The current study offers supporting evidence for medicinal effective use of C. olitorius.It revealed the presence of many phytochemical compounds in the studied morphotypes extracts.The number and the nature of these phytochemicals vary from a sample to another.All the morphotypes contained phenolic compounds whose biological activities are well established and possessed each one a total antioxidant capacity on DPPH.The potential antioxidant of the extract of morphotypes of C. olitorius is in relation with phenolic compounds that is responsible for their activity.There is a relationship between the antioxidant capacities and phenolic compounds implied that are the major contributors of antioxidant capacities of these plants.Plants naturally possess various therapeutic agents which properties depend on the nature and the variability of the plant.In sum, the morphotype with rounded and non-shiny leaves and the one with elongated and shiny leaves are the richest in phytochemicals and having potential antioxidant; these may be used efficiently in traditional medicine.Furthermore, the non-toxicity of these morphotypes will allow to promote their use in nutraceutical preparations.

Figure 2 .
Figure 2. Frequency of phytochemicals compounds identified in morphotypes of C. olitorius.

Table 1 .
List of morphotypes, samples and collection areas.
*Corresponding author.E-mail: aarlette2000@yahoo.fr.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License compounds (test with fehling solution), Coumarins (test with ether and ammonia), Free anthracene derivatives (test with chloroform and ammonia), and Combined anthracene derivatives (test with chloroform and ammonia).

Table 3 .
Qualitative assessment of phytochemical compounds in the extracts of C. olitorius.

Table 4 .
LC50 values of the different morphotypes of C. olitorius.