Evaluation of the nutritional and antioxidant potentialities of Capparis corymbosa

The study was undertaken with the objective of contributing to the search for new sources of dietary antioxidants. It aimed to assess the dietary potential, bioactive compound levels and antioxidant activities of the leaves and fruits of Capparis corymbosa, a plant food from Burkina Faso. Standard methods were used to assess the nutritional potential of leaves and fruits as well as the levels of bioactive compounds and antioxidant activities of leaf and fruit fractions. Levels of total sugar were 765.40 ± 134.67 mg/100 g for leaves and 2208.65 ± 194.55 mg/100 g for fruits. Protein levels were 22.722 ± 0.95 mg/100 g for leaves and 13.442 ± 0.91 mg/100 g for fruits. Both organs also had interesting contents of minerals and essential amino acids. Total phenolic levels ranged from 18.36 ± 0.90 g GAE/100 g to 16.46 ± 1.15 g GAE/100 g for leaves and from 4.58 ± 0.15 g GAE/100 g to 1.03 ± 0.31 g GAE/100 g for fruits while those of flavonoids ranged from 2.83 ± 0.23 g EQ/100 g to 2.08 ± 0.25 g EQ/100 g and from 1.78 ± 0.25 g EQ/100 g to 0.93 ± 0.41 mg EQ/100 mg, respectively for leaves and fruits. The butanol extraction fractions had the highest levels of polyphenols of both organs. Monitoring antioxidant activities using DPPH, FRAP and ABTS models showed that leaves and fruits displayed high antioxidant activities but leaves had higher antioxidant activities than the fruits. Antioxidant activity was correlated with total phenolic levels. These results reveal that C. corymbosa is an important source of phytonutrients, bioactive compounds and has good antioxidant activity and is therefore an alternative for the fight against diseases linked to oxidative stress and as a food supplement.


INTRODUCTION
In Burkina Faso, Capparis corymbosa (Capparidaceae) is a plant with great nutritional importance. The species is used as a dietary supplement, gathering or as a food supplement (Ayessou et al., 2018). In addition, many health benefits such as child care (colic and general tiredness) related to the consumption of C. corymbosa leaves and fruits have been reported (Busson, 1965;Zerbo et al., 2007). This species is used as a dietary *Corresponding author. E-mail: dibalacrepin@gmail.com. Tel: 0022670141596.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License supplement in children and type-II diabetic patients (Busson, 1965). Leaf extracts are suggested to be effective against kidney disease, biliary inflammation, rheumatism, etc., and good antioxidants (Zerbo et al., 2007). These different properties of the plant are certainly linked to its chemical composition. Indeed, Nacoulma (1996) and Sereme et al. (2001) reported that the richness of plants in secondary metabolites (tannins, flavonoids, saponosides) and active ingredients would be at the origin of their different properties. Despite all this high potential, little data exist on the phytochemistry, nutritional and antioxidant potential of C. corymbosa. Knowledge on antioxidant activities of the plant tissues could allow a better food valorization of the species.
The metabolism of aerobic organisms generates reactive oxygen species (ROS). The main ROS are hydroxyl radical, superoxide radical, and hydrogen peroxide. These ROS can also be generated by exposure to certain exogenous factors such as ionizing radiation, certain drugs, environmental pollutants and some synthetic food additives. ROS impair cellular function, particularly in the inflammatory process, phagocytosis and cellular signal transduction (Garait, 2006). The consequences of chronic exposure to high ROS levels are generally undesirable for the cells. The studies have reported that the accumulation of free radicals is the fundamental root cause of many age-related diseases (tumor proliferation, cataract, pulmonary edema) and affects several organs (muscles, heart, blood vessels, skin, etc). ROS are also factoring which potentiate the development of multifactorial conditions such as degenerative diseases (Parkinson's, Alzheimer's), type-II diabetes, osteoarthritis phenomena, cardiovascular disease, etc (Favier, 2003).
Fortunately, fruits and vegetables are a good source of dietary antioxidants (Valko et al., 2006). Well known natural antioxidants include phenolic compounds, glutathione, nicotinamides, ubiquinone (coenzyme Q 10 ), vitamins (C, E, A and K) and some enzymes (copper, zinc and manganese-dependent superoxide dismutase, catalase, selenium-dependent glutathione peroxidase, etc). Plants (leaves and fruits) are sources of phenolic compounds, vitamins, trace elements and especially beneficial natural antioxidants. Epidemiological studies have shown a positive correlation between the consumption of foods rich in polyphenols and lower risk of developing diseases like age-related neurodegenerative diseases (Mohamed and Darbar, 2010). This is often attributed to the antioxidant potential of phenolic compounds, those containing ortho-dihydrobenzene (catechols) due to their redox properties for eliminating reactive oxygen species or to chelate divalent metals (Atanasova, 2009). In addition, flavonoids and other phenolic compounds may also induce the expression of genes encoding antioxidant enzymes and proteins (superoxide dismutase, glutathione peroxidase, catalase, glutathione, etc). In research perspectives of dietary antioxidants, plant species still underexploited but accessible to rural populations constitute an interesting line of investigation (Garait, 2006). Therefore, an investigation was conducted on the chemical composition and bioactivities of C. corymbosa leaves and fruits.

Plant
Biological material consisted of leaves and ripe fruits of C. corymbosa. Sample collection was conducted during the months of June and July 2018 in the city park Bangréweogo (Ouagadougou). The species has been identified in plant biology laboratory of the University Joseph Ki-Zerbo. The leaves and fruits were dried by ventilation at room temperature (20-25°C) and ground into flour in a microanalytical mill to pass a screen of 0.5 mm. Milling was done at 4°C, and was performed carefully to avoid overheating. The flours were stored at -20°C prior to analysis to prevent endogenous enzymatic or nonenzymatic reactions, after the powders were subjected to different extraction solvents.

Preparation of extracts
Fifty grams (50 g) of powdered plant material as described earlier were extracted with 80% aqueous ethanol (500 ml) in ratio 1/10 (w/v) for 48 h under mechanic agitation (SM 25 shaker, Edmund BÜHLER, Germany) at room temperature (20-22°C). After filtration, ethanol was removed under reduced pressure in a rotary evaporator (BÜCHI, Rotavopor R-200, Switzeland) at approximately 40°C and freeze-dried with Telstar Cryodos 50 freeze-dryer. These ones were filtered and freeze-dried. The extract residues were weighed before packed in waterproof plastic flasks and stored at 4°C until use. The yields of different crude extract were calculated and expressed as grams of extract residues/100 g of dried plant materials.

Fractionation
The aqueous extracts were subjected to sequential liquid-liquid extraction with petroleum ether, dichloromethane, ethyl acetate and n-butanol. Each fraction was then collected and concentrated to dryness under reduced pressure to obtain hexan fraction, dichloromethane fraction, ethyl acetate fraction and n-butanol fraction. The fractions were freeze-dried. The fraction residues were packed in waterproof plastic flasks and stored at 4°C until use.

Determination of carbohydrate content
500 mg of sample were homogenized in 5 ml of 80% hot ethanol. After cooling, the homogenate was centrifuged at 4000 rpm for 10 min. The supernatant was used to estimate the soluble sugar content of fresh leaves. The soluble sugar content was determined using the phenol-sulfuric acid method as described by Dubois et al. (1965) and the absorbance was red at 490 nm. The total sugar content was expressed as μg glucose equivalent/100 gram of sample (μg GE/ 100 g of sample).

Determination of total protein
For proteins extraction, 500 mg of powder of leaves or fruits were homogenized in 5 ml of 0.1 M NaCl for 5 h at 150 rpm/min at room temperature. The samples were centrifuged at 10000 g during 30 min and the supernatant were collected to determine the protein content. The estimation of water-soluble protein content was performed according to the assay method of Bradford (1976). A volume of 50 µL of sample is added to 250 µL of Bradford reagent (Coomassie Brillant Blue G250). The reading is made using a spectrometer at 595 nm against a blank consisting of 50 µL of sample and 250 µL of buffer solution. BSA served as the standard for the preparation of the calibration curve (0-250 µg/mL (y = 0.0537x + 0.6136; R 2 = 0.9997). Results were expressed in mg per 100 mg of sample.

Determination of minerals and oligo-elements content
The minerals and oligo-elements determination were carried out according to a classical method (Norme international ISO 2171ISO , 2007. A mass of 3 g of the sample was introduced into crucibles before being calcined at 550°C in the muffle furnace for 24 h. The ash obtained was recovered in beakers and dissolved in 5 ml of concentrated hydrochloric acid, add 3 drops of hydrogen peroxide (H2O2). The whole was transferred to 100 mL volumetric flasks and made up to the mark with distilled water. Centrifugation was then carried out and the supernatant was recovered in sterile sampling tubes for spectrometric reading. The reading was taken with a Flame Atomic Absorption Spectrophotometer connected to a computer. The analyses permitted the determination of minerals such as calcium, magnesium, potassium and zinc.

Determination of amino acid content
The determination of amino acids was carried out by the PICO-TAG method (Thordser, 2011). For the determination of the complete amino acid profile, 500 mg sample was weighed and placed in a 20 mL volumetric flask. The volumetric flask is filled up to the mark with 0.1 M hydrochloric acid. Approximately 1 mL of the diluted sample is filtered through the 0.45 µm filters and then derivatized with phenylisothiocyanate (PITC) to produce the amino acids phenylthiocarbamyl (PTC). The derivatized sample were added 200 µl of PICO-TAG dilution solution (0.38 µg/µL), the amino acid derivatives were then separated by HPLC (2.3 µg sample/µL and detected by absorption spectrophotometry at values as low as 1 pmol using a wavelength at 254 nm.

Total phenolic content
Total polyphenols were determined by Folin-Ciocalteu method (Lamien-Meda et al., 2008). Aliquots (125 µL) of solution from extract or each fraction in methanol (10 mg/mL) were mixed with 62.5 µl Folin-Ciocalteu reagent (0.2 N). After 5 min, 500 µL of aqueous Na2CO3 (75 g/L) were added and the mixture was vortexed. After 2 h of incubation in the dark at room temperature, the absorbencies were measured at 760 nm against a blank (0.5 ml Folin-Ciocalteu reagent + 1 ml Na2CO3) on a UV/visible light spectrophotometer (CECIL CE 2041, CECIL Instruments, England). The experiments were carried out in triplicate. A standard calibration curve was plotted using gallic acid (Y= 0.0289x -0.0036; R 2 = 0.9998). The results were expressed as g of gallic acid equivalents (GAE)/100 g of extract or fractions.

Determination of flavonoid content
The total flavonoids were estimated according to the Dowd method, slightly modified (Hinnebourg et al., 2006). To an aliquot of 0.5 ml of Roamba et al. 203 methanol/AlCl3 (2%, w/v) were mixed 0.5 ml of extract or each fraction solution (0.1 mg/mL). After 10 min, the absorbencies were measured at 415 nm against a blank (mixture of 0.5 ml extract solutions and 0.5 mL methanol) on a UV/visible spectrophotometer (CECIL CE 2041, CECIL Instruments, England) and compared to a quercetin calibration curve (Y= 0.0289x-0.0036; R 2 = 0.9998). The data obtained were the means of three determinations. The amounts of flavonoids in plant extracts were expressed as g of quercetin equivalents (QE)/100 g of extract or fractions.

DPPH radical method
Radical scavenging activity of extract or each fraction against stable DPPH (2, 2'-diphenyl-1-picrylhydrazyl, Fluka) was determined with a UV/visible spectrophotometer (CECIL CE 2041, CECIL Instruments, England) at 517 nm as described by Hinnebourg et al., (2006). Extract solutions were prepared by dissolving 10 mg of dry extract in 10 mL of methanol. The samples were homogenized in an ultrasonic bath. Afterwards, 0.5 mL of aliquots which were prepared at different concentrations from each sample of extract were mixed with 1 mL of methanolic DPPH solution (20 mg/mL).
After 15 min of incubation in the dark at room temperature, the decrease in absorption was red. All experiments were performed in triplicate and expressed in mmol of ascorbic acid equivalent per mass of extract or fraction.

ABTS radical cation decolorization assay
For the assay (Hinnebourg et al., 2006), ABTS was dissolved in water to a 7 mM concentration. ABTS radical cation (ABTS ·+ ) was produced by reacting ABTS with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12 h before use. This mixture was diluted with ethanol to give an absorbance of 0.7 ± 0.02 at 734 nm using a UV/visible spectrophotometer (CECIL CE 2041, CECIL Instruments, England). Afterwards, 10 µL of the diluted sample (1 mg/mL -1 in methanol) which was allowed to react with 990 µl of fresh ABTS ·+ solution and the absorbance was taken 6 min exactly after initial mixing. Ascorbic acid was used as standard. Quercetin was used as positive control.

Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was performed as previously described (Velazquez et al., 2003). To an aliquot of 0.5 mL of extract or each fraction (1 mg mL -1 ) was mixed with 1.25 mL of phosphate buffer (0.2 M, pH 6.6) and 1.25 mL of aqueous potassium hexacyanoferrate [K3Fe(CN)6] solution (1%). After 30 min of incubation at 50°C, 1.25 mL of trichloroacetic acid (10%) was added and the mixture was centrifuged at 2000 × g for 10 min. Then, the upper layer solution (0.625 mL) was mixed with distilled water (0.625 mL) and a freshly prepared FeCl3 solution (0.125 mL, 0.1%). Absorbances were red at 700 nm on a UV/visible spectrophotometer (CECIL CE 2041, CECIL Instruments, England) and ascorbic acid was used to produce the calibration curve (Y= 0.008x-0.0081; R 2 = 0.9999). The iron (III) reducing activity determination was performed in triplicate and expressed in mmol Ascorbic Acid Equivalent per g of extract or fractions. Trolox, a reference compound was used as positive control.

Statistical analysis
The analysis of variance (ANOVA) was performed using XL-STAT The results shown in the previous figure by different letters are statistically distinct (p<0.0001). 2016 software to determine the variability of the parameters studied. Tukey's test at the 5% threshold was performed for means comparison.

Carbohydrates and protein content
The carbohydrates were determined using glucose as standard. Table 1 presents the carbohydrate and total protein contents expressed, respectively in mg GE/100 g fruit extract and mg GE/100 g leaf or fruit extract. The different contents varied significantly depending on the organs. Thus, in terms of total sugars, the fruits presented the best value (2304.144 mg GE/100 g) while in terms of total protein, the leaves presented the best content (22.722 ± 0.95 mg GE/100 g). The lowest values for total sugars (765.405 ± 19.54 mg GE/100 g) and protein (13.442 ± 0.91 mg GE/100 g) were recorded for leaves and fruit, respectively.

Mineral content of leaves and fruit
The mineral contents (Zn, Ca, Mg and K) expressed in mg/100 g of leaf and fruit powder are as shown in Figure  1. The mineral contents (Zn, Ca, Mg and K) expressed in mg/100 g of leaf and fruit powder are as shown in Figure  1. The results (Figure 1a and b) showed on one hand significant differences between the contents as a function of the minerals and on the other hand as a function of the organs. Calcium had the highest content while Zn and K had the lowest mineral content. In addition, the leaves had the highest Ca content compared to the fruit, which had the highest K content. No significant difference was recorded between the Zn and Mg contents of the leaf and fruit extracts.

Amino acid composition of leaves
The quantification of amino acids was limited to leaf samples only; fruits were not included in this analysis. Amino acid levels in the leaves are as shown in Figure 2. The analyses detected and quantified 17 amino acids with levels ranging from 0.30 to 7.24%. The highest content was recorded by aspartic acid (7.24%) followed by glutamic acid (3.87%), arginine (1.71%), lysine (1.02%) while the lowest content was observed in histidine (0.29%). The high levels of glutamic acid and aspartic acid could be explained by the conversion of glutamine and aspartate into their respective acids during the derivation process. In addition, apart from Tryptophan, all essential amino acids were detected and quantified in the leaves of Capparis spinosa. Their contents ranged from 0.43% for Isoleucine to 1.02% for Lysine.

Total phenolics and flavonoids contents of leaves and fruits
The total phenolic contents of the fractions of the different organs (leaves and fruits) expressed in g AG/ 100 g fraction are as shown in Figure 3. The total phenolic contents varied significantly by organ and also by fraction. In general, the different leaf fractions showed higher levels than the fruit fractions. The butanolic fraction had the highest content of phenolic compounds (181.962 ± 3.62 g EAG/100 g for the leaf fraction and 96.947 ± 2.57 g EAG/100 g for the fruit fraction) while the lowest value was recorded in the DCM fraction. (165.316 ± 2.76 g EAG/100 g for the leaf fraction and 37.414 ± 5.28 g EAG/100 g for the fruit fraction). The total flavonoid content expressed in mg EQ/100 mg of fraction of different fractions of leaves and fruits are as shown in Figure 4. The results show that the total flavonoid content of the leaves and fruit varied significantly in organ function and fractions. The butanol fraction of the leaves (2.83 ± 0.23 g EQ/100 g fraction) presented the best flavonoid content while the lowest concentration was recorded at the fraction DCM fruit (0.93 ± 0.41 g EQ/100 g fraction). In addition, the contents of total flavonoids in leaves were relatively higher than those fruit for all fractions.

Antioxidant activities
In the body, various types of oxidation occur and result in the accumulation of free radicals in the body. Also, the diversity and specificity of antioxidants (radical scavengers, electron donor or hydrogen) necessitate the evaluation of the antioxidant activity by various methods. Thus, three methods (DPPH, FRAP and ABTS) were used to evaluate the antiradical activity of the various fractions. The antioxidant activity of the different fractions is shown in Table 2. Except for the FRAP method, the variation of the various antioxidant activities is not a function of the organ. At the anti-radical activity DPPH (mg EAA/g) while the lowest activity was observed with the DCM fraction (9.54 ± 0.33 mg EAA/g) leaves. At the reducing power of the ABTS radical cation + , the best activity has been recorded with the fraction of the butanol fraction (17.03 ± 0.03 mg EAA/g) fruit while the lowest activity was observed with the DCM fraction (8.01 ± 0.56 mg EAA/g) of the body. For the reduction of Fe 3+ , the acetate fraction (0.48 ± 0.19 mg EAA/g) of the sheets presented the lowest activity whereas the highest activity with the butanol fraction (1.73 ± 0.18 mg EAA/g) leaves.
The Pearson correlation matrix between the amounts of phenolic compounds, flavonoids and antioxidant activities of the three methods is shown in Table 3. The results showed that the levels of phenolic compounds are negatively correlated respectively to levels of flavonoids (r = -0.642) and positively to iron reducer FRAP (r = 0.643). In addition, the flavonoid content is negatively and significantly (r = -0.528) correlated with iron reducing power while anti-radical activities determined by DPPH and ABTS methods are strongly (r = 0.811) correlated positively with each other.

DISCUSSION
This study focused on the evaluation of the nutritional and antioxidant potential of the leaves and fruits of C. corymbosa, a food plant of Burkina Faso flora. The study revealed protein and carbohydrate contents ranging from 13.44 to 22.72 mg/100 g and 765 to 20304.144 mg/100 g, respectively. The levels of total phenolics and  flavonoids ranged from 37.414 ± 5.28 to 181.962 g EAG/100 g and 0.93 ± 0.41 to 2.83 g EQ/100 g for total phenolics and flavonoids, respectively. Compared to the values reported on cowpea and Moringa oleifera, two plants with a recognized high nutritional potential, C. corymbosa appears to be a good source of nutrients, especially proteins and carbohydrates. Indeed, Sombie et al. (2019) reported protein contents ranging from 25.63 to 5.41 mg/100 g and 111 to 551 mg/100 g of fresh cowpea leaves, respectively. In addition, the results generally showed a significant variation in protein, carbohydrate, total phenolic and flavonoid contents between the leaves and fruits. The leaves had higher levels of protein, total phenolics and flavonoids than the fruits, which had higher levels of carbohydrates compared to the leaves. It appeared that leaves and fruit of C. corymbosa are also a good source of micronutrients, essential amino acids and mineral elements such as calcium, zinc, magnesium and potassium that highlight obviously its nutritional potential. This variation in nutrient concentrations in organ function may be due to various factors including genotype and ecotype orders. The found high macronutrient and micronutrient contents in both leaves and fruits of the plant may justify the use of the species as a food. The particularly high concentrations of lysine, histidine, phenylalanine and methionine pave the way for recovery of the species in the human diet given the important physiological roles of these amino acids in the body. These minerals are also essential for the proper functioning of our body. In fact, they contribute to the structure of bones and teeth and are associated with heart rate, muscle contraction, nerve conduction and in the water balance and acid-base of the body (Stadtman, 1992).
The fractions of leaves and C. corymbosa fruit presented some interesting levels of total polyphenols and total flavonoids. The presence of bioactive compounds in interesting amounts in fruits and leaves of C. corymbosa could be beneficial to health. Indeed, polyphenols and flavonoids are able to counteract the production of reactive oxygen species by neutrophils and therefore reduce the risk of disease (Manallah, 2012). They may inhibit the enzymes involved in the activation of cancer cells (Crozier et al., 2007). In addition, the leaves of fractions exhibited higher levels of polyphenols and flavonoids both in sheets at the level of fruit. The levels of bioactive compounds in organ function have been reported by many authors. Indeed, many secondary metabolites have a protective role for antioxidant plants against environmental aggressors or those pathogens (Garait, 2006). So they were found often in greater concentrations in the skin of the fruit or the outer leaves of leafy vegetables (Al-Snafi, 2015). This variability in levels of compound interest, variable therefore recommends a diet based on the two bodies to benefit both compounds in the leaves than in fruits.
The different fractions of leaves and C. corymbosa fruit showed good antioxidant activities. In addition, our results showed that the antioxidant activity evaluated by the FRAP method was strongly correlated with the polyphenol content. Similar results have been reported. Indeed, numerous studies have shown that the antioxidant properties of a plant product were strongly correlated with their content of phenolic compounds (Cai et al., 2004). The antioxidant potential of the latter is due ALUES redox properties that allow them to act as reducing agents hydrogen donors or electron and metal chelators (Meziti, 2009). The Capparis genus species is rich in phenolic compounds which are responsible for many biological activities including antioxidant activity, anticancer and antimicrobial (Turgut et al., 2015). The promising effect of antioxidants from plant health may result from their protective effect based on their ability to eliminate ROS. Some earlier reports C. corymbosa of antioxidant activity are very rare in the literature. Therefore, it is very difficult to compare our results with those of previous studies.

Conclusion
C. corymbosa may be good source of essentials aminoacids and also phenolic compounds that display in vitro high antioxidant activities. While leaves are rich in proteins, calcium and magnesium, fruits have high level of carbohydrates and minerals (calcium and magnesium). Additionally, butanol fractions of the two bodies have presented the best levels of total flavonoids and total polyphenols. The study also showed a significant variation in nutrient compounds concentrations in organ function which suggests a simultaneous consumption of both bodies.