**2.9 HPLC analysis**

134 Macro to Nano Spectroscopy

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

Various methods have been introduced for the measurement of the total antioxidant capacity (Delgado-Andrade et al., 2005; Gülçin et al., 2006). In this study antioxidant activity was estimated by the method previously described (Kim et al., 2002, 2003). The *in vitro* antioxidant activities have been determined in two antioxidant tests. Among the different methods permitting to evaluate the antioxidant activities, these two simple stable radical chromogens have a high level of sensitivity and allow the analysis of a large number of samples in a timely fashion (Kim et al., 2002). It was reported that a single method is not enough to evaluate the antioxidant capacity of most of the complex natural products (Ozgen et al., 2006). Antioxidant capacity is expressed as mg of vitamin C equivalent (mg VCE) per

ABTS radical anions were used according to the method of (Kim et al., 2003). In brief, 1.0 mM of 2, 2'-azobis (2-amidino-propane) dihydrochloride (AAPH), a radical initiator, was mixed with 2.5 mM ABTS in phosphate-buffered saline (pH 7.4) and the mixed solution was heated in a water bath at 68 °C for 13 min. The resulting blue-green ABTS solution was adjusted to the absorbance of 0.650 ± 0.020 at 734 nm with additional phosphate-buffered saline. 20 µl of sample were added to 980 µL of the ABTS radical solution. The mixture incubated in a 37°C water bath under restricted light for 10 min. A control (20 µL 50% methanol and 980 mL of ABTS radical solution) was run with each series of samples. The decrease of the absorbance at 734 nm was measured (Cary 50 Scan UV-Visible apparatus) at an endpoint after 10 min. Total antioxidant capacity of plant parts is expressed as mg / g of dry weight of vitamin C equivalents (VCEAC). The radical stock solution had to be freshly prepared and all measurements of the tested samples were repeated at least three times.

The DPPH radical scavenging activity was determined according to the method of Kim et *al.*, (2002). The DPPH radical (100 µM) was dissolved in 80% of aqueous methanol. The plant extract solutions, 0.1 mL, were added to 2.9 mL of the methanolic DPPH solution and the mixture was vigorously shaken and was kept at 23 °C in the dark for 30 min. The decrease of the absorbance of the resulting solution was monitored at 517 nm (Cary 50 Scan UV-Visible apparatus) after 30 min. A control, which consists of 0.1 mL of 50% aqueous methanol and 2.9 mL of DPPH solution, was prepared. The DPPH radical scavenging activity of plant extracts is expressed as mg/g of dry weight of vitamin C equivalents (VCEAC). This measure was taken after 30 min reaction time. The radical stock solution had

to be daily prepared and the tests were repeated at least three times.

weight of catechin equivalents (CE). Samples were analyzed at least in triplicate.

**2.6 Determination of total antioxidant activity** 

**2.7 ABTS radical anion scavenging activity** 

**2.8 DPPH radical scavenging activity** 

g dry weight.

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


Solvent composition: A =50 mM NH4H2PO4 at pH 2.60; B = 80% acetonitrile, 20 %A and C = 200 mM O-phosphoric acid at pH 1.50

Table 1. HPLC solvent gradient elution program

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 made at least for each standard and plant extract.

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

Identification, Quantitative Determination, and Antioxidant Properties

L= leaves; TB= trunk barks; RB= root barks

**4. Discussion** 

*mucronata*.

Values are means of triplicate determination ± standard deviation.

Fig. 2. Vitamin C equivalent antioxidant capacity (VCE mg/g dry weight).

**3.3 Analysis of polyphenolic composition in plant part extracts using HPLC** 

The RP-HPLC results which are summarised in table 3, show that the protocatechuic acid (792.00 - 7.93 mg / 100g dry weight), the *p*-coumaric acid (1833.56 - 4.2 mg / 100g dry weight), the gallic acid (69.00 - 5.14 mg / 100g dry weight) and the chlorogenic acid (2286.08 - 62.09 mg / 100g dry weight) were encountered in high concentration, whereas the caffeic acid (149.86 -43.46 mg / 100g dry weight), the isovitexin (182.22 - 31.46 mg / 100g dry weight) and the quercetin-3-β-D-glucoside (83.53 - 70.89 mg /100g dry weight) were found in low concentration. It should be noted that the trunk and the root barks of *T macroptera* and the leaves of *V. heterophylla* contain the greatest number of compounds similar to the standards, whereas none of the standards was detected for the root barks of *Z. mucronat*a.

The leaves of *A. leiocarpus* had the highest total phenolic contents, which was 4- fold higher than those of the leaves of *V. heterophylla.* The lowest total phenolic and total flavonoid contents were found in the leaves of *V. heterophylla*. In the trunk barks of *T. macroptera*, the total phenolics and the total flavonoids were ranked first, followed by *A*. *leiocarpus* and then the *M. inermis* ones*.* The phenolic and flavonoid compounds were found in highest concentration first in the root barks of *T. macroptera* followed by the root barks of *C. populnea.* In contrast, it appeared that the lowest amount of flavonoids was found for the root barks of *Z. mucronata*. The total phenolic and the total flavonoid contents of the root barks of *T. macroptera* were respectively 11-fold and 4-fold greater than those of *Z.* 

of Polyphenols of Some Malian Medicinal Plant Parts Used in Folk Medicine 137
