Folin ciolcateu reagent, quercetin, sodium carbonate, aluminum trichloride, iron III chloride, potassium hexacyanoferrate [K3Fe(CN)6], gallic acid, quercetin, ethanol, vanillin, Na₂HPO₄, NaH₂PO₄, 2,2-diphenylpicrylhydrazyl (DPPH), ABTS [(2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonique)], trichloroacetic, acetic and ascorbic acid, hexane, dichloromethane, ethyl acetate, n butanol. All chemicals and solvents used were of analytical grad.

Whole plants of C. sesamoides and S. hermonthica were collected in the villages of Katchari and Mamasiol (urban commune of Dori) in September and October 2019. The whole plant samples were then washed with water and dried at room temperature away from sunlight and dust, before being used as whole plant powder for the experiments. The plants were identified at the National Herbarium of Burkina Faso (HNBF) under numbers 8758 and 8759 respectively.

Aqueous extracts were made by macerating 100 g of powder of each plant in 900 mL of distilled water for 24 hours under magnetic stirring. The macerate was then filtered twice using absorbent cotton and the filtrate was frozen at −4˚C. The frozen filtrates were then lyophilized using ALPHA 1 - 2 LD Plus freeze dryer and the extracts obtained were placed in a desiccator until their use.

Each plants powder (25 g) was extracted with 250 ml of acetone-water mixture (80:20) for 24 hours using an electric mixer. After filtration acetone was evaporated at 60˚C using heidolph brand rotavapor. The resulting aqueous extract (50 ml) was fractionated by successive liquid-liquid partitioning with an equal volume of n-hexane, dichloromethane, ethyl acetate and n-butanol. The organic solutions from the partitioning were then separately pooled and concentrated to dryness to form hexane, dichloromethane, butanol and ethyl acetate fractions. The final aqueous phase was lyophilized. The dichloromethane extract of interest in our present study was stored at 4˚C until use.

Fractionation allows the secondary metabolites to be distributed between the different solvents used, which could allow a concentration of specific molecules in the fraction, avoiding the effect of congestion and thereby optimizing their biological properties. This process would contribute to facilitate the isolation and characterization of interest molecule.

The total polyphenols content was performed by Folin-Ciocalteu method described by [13]. A volume of 125 µL of extract (at 0.1 mg/mL) was mixed with 625 µL of Folin-ciolcalteu reagent (0.2N). After 5 min of incubation in the dark, 500 µL of sodium carbonate (Na₂CO₃, 75 g/L) was added to the mixture. The mixture was then incubated for 2 h in dark before determination of total polyphenol contents at 760 nm against a gallic acid calibration curve (y = 4668e-3x − 0.034, r² = 0.9991). Each test was repeated 3 times and the results were expressed as mg of Gallic Acid Equivalent per 100 mg of extract (mg GAE/100 mg extract).

The total flavonoids content was determined according to the colometric method [13]. To 625 µL plant of plant solution (at 0.1 mg/mL), 625 µL of aluminum trichloride (AlCl3, 2%) were added. After 10 min of dark incubation, the flavonoids content was evaluated at 415 nm, using a quercetin calibration curve (Y = 1.259e-2x; r² = 0.9990). The tests were assessed in triplicate 3 times and the results were expressed as mg Quercetin Equivalent per 100 mg of extract (mg QE/100mg extract).

The determination of condensed tannins content was performed using the vanillin acid method [14].

0.5 mL of plant sample diluted in ethanol (1/100) was mixed with 1 mL of sulfuric vanillin solution (70%). After 15 min of incubation of the mixture in the dark at 30˚C, the condensed tannins content was measured at 500 nm against a tannic acid calibration curve (y = 0.0353x + 0.0016; r² = 0.9988).

The tests were repeated thrice and the results were expressed in mg Tannic Acid Equivalent per 100 mg of extract (mgEAT/g extract). Only the condensed tannins content of aqueous end dichloromethane extracts have been done.

This method evaluates the scavenging capacity of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical. 375 µL of extract of each plant (0.1 mg/mL) were mixed with 750 µL of DPPH solution (20 mg/L). After 15 min of incubation in the dark, the absorbance of the mixture was read at 517 nm on a GENESYS 30 spectrophotometer. The DPPH radical scavenging activity of each plant extract was measured using an ascorbic acid calibration curve (y = −2.224e-2x + 0.348, r² = 0.9966). The test was repeated 3 times and the results were expressed as µmol Ascorbic Acid Equivalent per gram of extract (µmol EAA/g).

The ability of plant extracts to reduce ferric iron to iron salts was evaluated by mixing 250 µL of plant extract (0.1 mg/mL) with 625 µL of phosphate buffer (0.2 M, pH 6.6) and 625 µL of potassium hexacyanoferrate [K3Fe(CN)6] (1%), then the mixture was incubated at 50˚C in a water bath. After 30 min, 625 µL of trichloroacetic acid (10%) was added to the mixture and the whole was centrifuged at 3000 rpm for 10 min. 0.3125 mL of the supernatant was then taken and mixed with 0.3125 mL of distilled water followed by 0.0625 mL of fresh iron chloride FeCl3 solution (0.1%). The absorbance of the reaction medium was measured at 700 nm. Each test was repeated 3 times and the reduction potential of ferric chloride was expressed as µmol Ascorbic Acid Equivalent per gram of extract (µmol EAA/g).

ABTS●+ cationic radicals were generated by mixing ABTS solution (7 mM) with 2.5 mM potassium persulfate. The mixture was then kept in the dark at room temperature for 12 hours before adjusting its absorbance with ethanol to 0.70 ± 0.02 at 734 nm.

The reduction potential of the ABTS●+ cationic radical by the extracts was determined by mixing 10 µL of extract (at 0.1 mg/mL) with 990 µL of the fresh ABTS solution. After 15 min of incubation in the dark, the absorbance of the mixture was read at 734 nm. The measurements were repeated 3 times and the results were expressed as µmol Ascorbic Acid Equivalent per gram of extract (µmol EAA/g) using the ascorbic acid calibration curve.

Table 1 shows Total Polyphenols (TP) as well as of Total Favonoids (TF) and Condensed tannins (TC) contents. The TF and TP content in the different extracts of both plants was varied. TF content ranged from 2.144 ± 1.66 to 3.814 ± 0.07 mgEQ/100mg for the different extracts of S. hermonthica while the TF content of the different extracts of C. sesamoides ranged from 2.406 ± 0.23 to 5.4273 ± 0.35 mgEQ/100mg have been registered. However, the ethyl acetate fraction obtained the highest TF content for both plants with 3.814 ± 0.07 mgEQ/100mg 5.4273 ± 0.35 mgEQ/100mg for s. hermonthica and C. sesamoides respectively.

The TP content was also varied for the extracts of both plants. A TP content ranging from 11.568 ± 0.30 to 74.871 ± 2.57 mgEAG/100mg was obtained for the extracts of S. hermonthica with 74.871 ± 2.57 mgEAG/100mg as the highest value obtained with the n-butanol fraction. the total polyphenol contents of C. sesamoides was ranged from 11.425 ± 0.539 to 47.236 ± 2.57 mgEAG/100mg. the highest value of polyphenols in C. sesamoides extracts was 47.236 ± 2.57 mgEAG/100mg obtained with the ethyl acetate fraction. Statistical analysis showed high significance (p < 0.05) between both plants extracts and fractions for TP and TF contents.

Aqueous extracts and dichloromethane fractions of both plants, of particular interest for our work, were quantified for condensed tannin content. The contents obtained were varied but the aqueous extract of C. sesamoides obtained the highest content with 3.028 ± 0.13 mgEAT/100mg.

The administration of a single dose of 2000 mg/kg of aqueous extract of C. sesamoides and S. hermonthica did not induce any signs of acute toxicity or mortality in the mice during the 72 hours of observation and after 14 days of follow-up.

ND: Not Determined.

Table 2 summarizes the free radical scavenging capacity by the DPPH, method reducing potential of ferric ions by the FRAP, as well as the ABTS cationic radical reduction capacity methods. Both plants extracts, showed higher antioxidant capacity with all the methods. The statistical analysis showed high significance (p < 0.05) between both plants extracts and fraction for the antioxidant capacities.