PHYTOCHEMISTRY AND IN VITRO ANTIOXIDANT ACTIVITIES OF FOUR CONSUMED PICKING PRODUCTS IN SIKASSO, MALI

The aim of this work was to study the phytochemistry and to evaluate the in vitro antioxidant activities of four fruits harvested consumed in Sikasso, Mali: Balanites aegyptiaca, Zizyphus mauritiana, Raphia sudanica and Saba senegalensis. A phytochemical screening has been performed using tube reactions. Total polyphenol and flavonoid were determined by the spectrophotometric method and in vitro antioxidant activities by DPPH test. The tests revealed in all the fruits the presence of many phytochemical groups. The mucilages have been found in the fruits of Raphia and Zizyphus. Fruits of Zizyphus had the highest polyphenolic content (1.72±0.07 mg GAE/100 mg) and Saba the lowest (0.99±0.07 mg GAE / 100 mg). The highest flavonoid content has been recorded with Raphia fruit (0.35±0.04 mg CE/100 mg) while Balanites fruit had the lowest content (0.16±0.034 mg CE/100 mg). As well the contents of polyphenols and flavonoids, the highest in vitro antioxidant activity has varied 30.66±2.16 % for Saba to 70.26±2.11 % for Raphia. These results have shown that these fruits are sources of many metabolites especially polyphenols and flavonoids and are endowed with antioxidant activities, therefore they could be used more in food to improve food ration and to fight against oxidative stress.

The aim of this work was to study the phytochemistry and to evaluate the in vitro antioxidant activities of four fruits harvested consumed in Sikasso, Mali: Balanites aegyptiaca, Zizyphus mauritiana, Raphia sudanica and Saba senegalensis. A phytochemical screening has been performed using tube reactions. Total polyphenol and flavonoid were determined by the spectrophotometric method and in vitro antioxidant activities by DPPH test. The tests revealed in all the fruits the presence of many phytochemical groups. The mucilages have been found in the fruits of Raphia and Zizyphus. Fruits of Zizyphus had the highest polyphenolic content (1.72±0.07 mg GAE/100 mg) and Saba the lowest (0.99±0.07 mg GAE / 100 mg). The highest flavonoid content has been recorded with Raphia fruit (0.35±0.04 mg CE/100 mg) while Balanites fruit had the lowest content (0.16±0.034 mg CE/100 mg). As well the contents of polyphenols and flavonoids, the highest in vitro antioxidant activity has varied 30.66±2.16 % for Saba to 70.26±2.11 % for Raphia. These results have shown that these fruits are sources of many metabolites especially polyphenols and flavonoids and are endowed with antioxidant activities, therefore they could be used more in food to improve food ration and to fight against oxidative stress.

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In Uganda, a study has been shown that wild edible plants can significantly complement cultivated exotic plants to reduce vitamin A deficiency in populations (Musinguzi et al., 2007). In Sahelian countries such as Mali, Burkina Faso and Senegal, edible wild plants are used as soldering food (Koné et al., 2009;Kouyaté et al., 2009;Thiombiano et al., 2014). Widely consumed, they improve the daily food intake as an energy source and through their micronutrient content (Makalao et al., 2015;Diarra et al., 2019). In 2016, Diarra et al. showed that the Malian flora is very rich in wild food plants and that fruits were the most consumed organs (62.2 %) by the population. Despite this floristic richness, statistics on malnutrition are worrying in Mali. The results of the ENSAN surveys in Mali showed that chronic food insecurity affects nearly a quarter (24%) of Malian households (ENSAN, 2016). As for the results of SMART Mali, the nutritional situation is considered precarious in several regions and particularly in Sikasso with prevalence's ranging from 5% to 10% of global acute malnutrition (SMART, 2016). Within the strategic framework of sustainable food security and in the context of poverty alleviation, the ways in which local populations exploit natural resources do not allow them to make the most benefits of these resources (Sène et al., 2018).
Studies have shown that many edible wild plants have a high level of antioxidant activity (Adjdir et al., 2018). Several metabolites are involved in the antioxidant and antimicrobial activities of plant extracts (Fatima et al., 2015;Bagewadi et al., 2019;Togola et al., 2019a). Among these metabolites, total polyphenol and flavonoid are predominant. It is why wild fruits are currently of great interest not only for the fight against malnutrition but also and above all for the treatment and/or prevention of some chronic diseases such as diabetes, cancer and high blood pressure. This craze for wild fruits began especially after several epidemiological studies showed that cancer rates were low among people who consume a lot of fruit rich in polyphenols and flavonoids and therefore in antioxidants (Riel, 1999); (Kabine et al., 2015).
Regarding this situation, it is necessary to optimise the level of knowledge on wild food plants in order to contribute to rational exploitation. This study was initiated within the overall framework of the valorisation of wild woody species of food interest. The aim is to contribute to the phytochemical study and evaluation of in vitro antioxidant activities of fruit extracts of four (04) harvested species consumed in Sikasso, Mali: Balanites aegyptiaca L.

Total polyphenol and flavonoid extraction:-
The extraction of polyphenols and flavonoids from the samples has been assisted by an ultrasonic bath. Ten grams (10 g) of vegetable pulp (weighed using a nahita Blue analytical balance series 5134 EX) have been extracted with 100 mL of methanol (AnalaR NORMAPUR UN1230 from VWR) diluted to 80 % with distilled water (80: 20; v/v) for 20 min. After filtration, the residue has been rinsed with 100 mL of methanol. This residue has been extracted again under the same conditions and filtered. The two filtrates were added and dry evaporated using a rotary evaporator (BUCHI). The resulting residue has been dissolved in 50 mL of methanol and the volume has been completed to 100 mL with distilled water. The resulting mixture was centrifuged using a centrifuge (SIGMA 1-6p), then made up to 100 mL with 50 % methanol and stored cold (0° C) before analysis (Koné et al., 2012).

Total polyphenol and flavonoid assays:-Total polyphenol:-
The total polyphenol contents were determined by the Folin-Ciocalteu method described by Fofié et al. (2017). One hundred microliters (100 µL) of properly diluted extract were introduced into a test tube. Then 100 µL of Folin Ciocalteu reagent were added to the mixture and stirred. After 5 min, 1 mL of a 7 % sodium bicarbonate (Na 2 CO 3 ) solution was added with stirring and the final volume was immediately increased to 2.5 mL with distilled water and vigorously stirred. After a 90 minute incubation at room temperature (30-35° C), the absorbances were read at 760 nm against a blank prepared with distilled water with a spectrophotometer (Zuzi 4010/1). The polyphenol levels were determined using a gallic acid standard curve Ci: Initial concentration of the mother solution (mg/L); D: Dilution factor. NB: All measurements have been repeated three times.

Flavonoids:-
The quantification of flavonoids was carried out by the colorimetric method described by Koné et al. (2012). This method uses aluminium trichloride which forms a yellow complex with flavonoids and sodium nitrite which forms a pink complex which absorbs in the visible at 510 nm. 100 µL of each properly diluted extract and 30 µL of a 5 % sodium nitrite (NaNO 2 ) solution were introduced into a test tube. After 5 minutes, 30 µL of 10 % aluminium chloride were added to the mixture. And 6 minutes later, 2 mL of 1 M soda were added and the mixture was stirred. The final volume was filled up to 700 µL with distilled water and vigorously stirred. The absorbances were read at 510 nm with a spectrophotometer (Zuzi 4010/1) against a blank prepared under the same conditions with distilled water. A standard curve has been established using a catechin solution (C1251-5G), powder; Sigma Aldrich) at different concentrations (     The figure 4 showed that the total polyphenol (p-value of 0.00031 < 0.05) and flavonoid (p-value of 0.0032 < 0.05) contents have been varied from one fruit to another.  (CIRAD, 2007). The maximum contents that they have recorded (in % GAE) in strawberries (0.26 %), lychee (0.22 %) and grapes (0.20 %); were lower than polyphenol contents that we have observed with our results. Studies have shown that the preventive properties of food and medicinal plants are due to the presence of polyphenols and flavonoids, among others, which are molecules with antioxidant power, hence their importance in regulating oxidative stress (Chaouche, 2014;Fagbohoun, 2014).
In scientific literature, it has been reported that the consumption of fruits rich in flavonoids reduces the risk of coronary thrombosis and myocardial infarction through their platelet inhibitory effect (Chira et al., 2008). The same study showed that phenolic compounds in red grapes (20-170 mg/kg at the pulp level) prevented the development of early atherosclerosis induced by atherogenic feeding. In these studies, the consumption of grapes (Hamburg Muscat variety at a dose equivalent to 600 g/day for a 70 kg man) or their juice (dose equivalent to 500 mL/day for a 70 kg man) reduced the surface area of lipid deposits on the aortic stick by 78 % and 93 % respectively. The physiological effects obtained for the nutritional consumption of grape polyphenol extract on atherosclerosis, diabetes or hypertension show an in vivo prevention of these pathologies. Grape polyphenols can therefore play a preventive nutritional role ( So our fruits with their polyphenol and flavonoid contents could serve as potential candidates in the fight against these diseases and malnutrition.

Screening for in vitro antioxidant activity:-
The figure 5 illustrates the chromatogram of the in vitro antioxidant activity of fruit extracts revealed by DPPH.

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This chromatogram showed that all extracts have revealed yellow patches on a purple background, indicating that the extracts have reduced the free radical DPPH. This activity could be due to the polyphenolic constituents present in these extracts. Many studies have attributed the antioxidant effect of plant extracts to polyphenols and flavonoids (Nesrine, 2014;Bruneton, 2016;Seggani & Boukehil, 2017). Therefore, the relatively high levels of polyphenols and flavonoids show that our fruits are said to be rich in antioxidants, which are currently the subject of many studies.

Evaluation of in vitro potential antioxidant:-
The antiradical potential of the extracts was evaluated by the DPPH method. The results obtained were expressed as percentages of inhibition of free radicals in DPPH (table 3). At the concentration of 100 µg/mL of extracts, the free radical inhibition rate has ranged from 16.07 ± 2.08% in S. senegalensis to 57.82 ± 2.41 in R. sudanica. At 125 µg/mL, this rate has varied in the same order and at the same extract level from 21.38 ± 0.26% to 70.26 ± 2.11%. This classification is consistent with the polyphenol and flavonoid levels obtained at the level of extracts, the highest of which have been recorded in R. sudanica extracts with 1.57 ± 0.04 mg GAE/100mg for polyphenols and 0.35 ± 0.04 mg EC/100mg for flavonoids and the lowest contents have been observed in S. senegalensis extracts with 0.99 ± 0.04 mg GAE/100mg for polyphenols and 0.17 ± 0.01 mg EC/100mg for flavonoids ( Figure 4). These results have revealed a strong capacity to reduce free radicals by extracts, which shows a strong antioxidant activity. However, all our extracts are less active than our positive control: ascorbic acid which at the concentration of 10 µg/mL induces a 94.45 % reduction.
Our results are similars to those reported by Abdoune & Yahiaoui (2015) which were 50.99 % inhibition rate with Zizyphus mauritiana fruit extracts. With different extracts (aqueous, methanolic and acetate) of Zizyphus fruit, Adjdir et al (2018) have also reported similar reduction capacities to ours with IC 50 ranging from 12 to 503.6 µg/mL. On the other hand, the inhibition rates (93.98 %) have been reported by Kabine et al. (2015) with Raphia sudanica fruit extracts are higher than ours.
It appears from this study that the extracts of our different fruits have a good antiradical activity. Antiradical activity being a good indicator of antioxidant potential, our extracts could be a potential source of natural antioxidants, which are currently in high demand. Because in addition to a definite interest in the conservation of edible foodstuffs and against photophobia-oxidation or photodegradation, they could prove useful in the prophylaxis and treatment of diseases in which oxidative stress would be incriminated (Kouamé et al., 2009;Tounkara et al., 2014). For this reason, wild fruits are currently of great interest not only for the fight against malnutrition but also and above all for the treatment and/or prevention of some chronic diseases such as diabetes, cancer and high blood pressure, as shown by epidemiological studies that have shown that cancer rates are low among people who consume a lot of antioxidant-rich fruits (Riel, 1999;Kabine et al., 2015). Therefore, in addition to their nutritional potential, the fruits of B. aegyptiaca, Z. mauritiana, R. sudanica and S. senegalensis would be promising in the fight against antioxidant stress.

Conclusion:-
This study showed that the fruit pulp extracts from the four wild food species studied contain many metabolites, which would justify their food and medicinal uses. The results of the assay showed that these fruits would be a potential source of polyphenols, flavonoids and natural antioxidants. Therefore, in addition to their nutritional potential, the fruits of B. aegyptiaca, Z. mauritiana, R. sudanica and S. senegalensis would be promising in the fight against antioxidant stress.