**2.5 Determination of the total phenolic and of the total flavonoid contents**

The concentration of total phenolics was measured by the method described by (Kim et al., 2003). Briefly, an aliquot (1 mL) of appropriately diluted extracts or standard solutions of gallic acid was added to a 25 mL volumetric flask containing 9 mL of ddH2O. A reagent blank was prepared using ddH2O. One mililiter of Folin & Ciocalteu's phenol reagent was added to the mixture and shaken. After 5 min, 10 mL of 7% Na2CO3 solution were added and the solution was then immediately diluted to volume (25 mL) with ddH2O and mixed thoroughly. After an incubation of 90 min at 23 °C, the absorbance versus prepared blank was read at 750 nm (Cary 50 Scan UV-Visible apparatus). Total phenolic contents of plant parts are expressed as mg of gallic acid equivalents (GAE) / g dry weight. All samples were analyzed at least in triplicate.

Total flavonoids were measured by a colorimetric assay that was developed by Zhishen et al. (1999). We can add to a 10 mL volumetric flask containing 4 mL ddH2O either 1 mL of aliquot of appropriately diluted sample or 1 mL of a standard solution of catechin. At zero time, 0.3 mL 5% NaNO2 was added to the flask. After 5 min, 0.3 mL 10% AlCl3 was added.

Identification, Quantitative Determination, and Antioxidant Properties

composition and the gradient elution program are reported in the table 1.

**2.9 HPLC analysis** 

O-phosphoric acid at pH 1.50

Table 1. HPLC solvent gradient elution program

made at least for each standard and plant extract.

Fig. 1. Chromatogram of standards (1mg/ml)

of Polyphenols of Some Malian Medicinal Plant Parts Used in Folk Medicine 135

The HPLC analyses were conducted with a Water 600 Pump apparatus. This apparatus was equipped with a quaternary solvent delivery system, a Rheodyne injector with 20µL sample loop and a UV detector Waters 486 Tunable which was fixed at 280 nm. Throughout this study, Alltech Intertsil ODS-5 C18 reversed phase column (150 mm, 4.6 mm, 5µm particle size) was used. The flow rate of the mobile phase was of 1 mL / min and the gradient elution was adapted from (Nakatani et al., 2000; Bouayed et al., 2007). The solvent

> Times (min) %A %B %C 0 100 0 0 5 65 12 23 11 0 15 85 29 0 22 78 36 0 25 75 42 0 25 75 55 0 35 65 60 0 50 50 70 0 100 0 75 100 0 0

Solvent composition: A =50 mM NH4H2PO4 at pH 2.60; B = 80% acetonitrile, 20 %A and C = 200 mM

Standards of five phenolic acids and two flavonoids were dissolved in 50% MeOH to make a concentration of 0.5; 0.25; 0.125 and 0.10 mg/mL. The plant part extracts and standards solutions were filtered through 0.45-µm olefin polymer (OP) syringe-tip filters. Then, phenolic acids and flavonoids present in the extracts were identified by matching the retention time against their corresponding standard. In this study, the standards used for comparison were gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, *p*-coumaric acid, isovitexin and quercetin-3-β-D-glucoside (Figure 1). Quantitative analysis was made according to the linear calibration curves of standards compounds. Three replications were

After 6 min, 2 mL of 1 M NaOH was added to the mixture. Immediately, the reaction flask had to be diluted to volume by the addition of 2.4 mL of ddH2O and thoroughly mixed. Absorbance of the mixture was determined at 510 nm (Cary 50 Scan UV-Visible apparatus) versus prepared water blank. Total flavonoids of plant parts are expressed as mg / g dry weight of catechin equivalents (CE). Samples were analyzed at least in triplicate.
