**1. Introduction**

130 Macro to Nano Spectroscopy

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In biological systems, during the cellular respiration, reactive oxygen species (ROS) like hydroxyl radical (•OH), superoxide anion (•O2 <sup>−</sup>) and hydrogen peroxide (H2O2) are generated, as the natural consequence of oxidation reactions (Tarnawski et al., 2005). Reactive oxygen species (ROS) damage living cells causing lipid, protein, and DNA oxidation (Shukla et al., 2010). They are involved in the development of various diseases such as diabetes, rheumatic disorders (Luximon- Ramma et al., 2002), aging, cancer, cardiovascular or neurodegenerative disorders (Ju et al., 2004; Tarnawski et al., 2005), malaria and gastric ulcer (Gülçin et al., 2006).

The interest in searching natural antioxidants has recently increased. These natural products could be used in food or in medicinal materials to replace synthetic antioxidants which are about to be restricted owing to their side effects such as carcinogenesis (Gülçin et al., 2006). Many medicinal plants contain large amounts of antioxidants, such as polyphenols, which can play an important role in adsorbing and neutralizing free radicals, in quenching singlet and triplet oxygen, or in decomposing peroxides. The compounds that are responsible of antioxidant activity could be used for the prevention and treatment of free radical-related disorders (Gomez-Caravaca et al., 2006). Indeed, the consumption of antioxidants prevents different diseases such as neurological degeneration, inflammatory disorders, coronary diseases, aging and cancers (Djeridane et al., 2006). Hence, the studies on natural antioxidants have gained increasingly greater importance.

A large number of different plants have been studied as new sources of natural antioxidants (Cakir et al., 2003; Lee et al., 2000; Kumaran & Karunakaran, 2007; Muanda et al., 2009). For the first time, we report here the antioxidant properties of extracts from the following six Malian folk medicine plants, which were previously studied for their biological activities:

Identification, Quantitative Determination, and Antioxidant Properties

**2.3 Chemical reagents** 

(France).

analytical grade.

(0.750µm).

**2.4 Preparation of extracts** 

stored at -4°C until analyses were performed.

analyzed at least in triplicate.

of Polyphenols of Some Malian Medicinal Plant Parts Used in Folk Medicine 133

Standards: catechin and gallic acid, 3,4 dihydroxybenzoic acid (protocatechuic acid), chlorogenic acid, rutin were purchased from Across Organics (France). *p*-coumaric acid, isovitexin and quercetin 3-ß-D-glucoside were obtained from Fluka Chemical Company

Aluminium chloride (AlCl3), ascorbic acid, 2-2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), PBS buffer, 2-2'-azobis (2-methylpropionamidine) dichloride (AAPH), Folin-Ciocalteu's phenol reagent, sodium carbonate (Na2CO3), caffeic acid and sodium nitrite (NaNO2), stable free radical DPPH were purchased from Sigma Chemical Company (France). All commercial standards and reagents were of the highest

Each plant material was dried in a dark ventilated room for 5–7 days. The different parts of the plants (leaves, root barks, and stem barks) were ground to powder and sifted in a sieve

The extraction of the samples was performed by the ultrasound-assisted method (Kim et al., 2002, 2003). The air-dried plant material (10 g) was extracted using 100 mL of 80% aqueous methanol, the mixture of freeze-dried powder and 80% aqueous methanol was sonicated for 20 min with continual nitrogen gas purging. The mixture was filtered through Whatman N°2 filter paper and rinsing with 50 mL of 100% methanol. The extraction of the residue was repeated using the same conditions and the two filtrates were combined and transferred into a 1 L evaporating flask with an additional 50 mL of 80% aqueous methanol. The solvent was evaporated using a rotary evaporator at 40 °C. The remaining extract concentrate was first dissolved in 50 mL of 100% methanol and diluted to a final volume of 100 mL using distilled deionized water (ddH2O). The mixture was centrifuged at 1500g for 20 min and

**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

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.

*Anogeissus leiocarpus* (DC.) Guill. et Perrot (Combretaceae), *Cissus populnea* Guill. et Perr. (Vitidaceae), *Mitragyna inermis* (Willd.) O. Ktze. (Rubiaceae), *Terminalia macroptera* Guill. et Perrott (Combretaceae), *Vepris heterophylla* R. Let. (Rutaceae) and *Zizyphus mucronata* Willd. (Rhamnaceae). These plants were selected for their traditional used in the treatment of inflammatory diseases such as: malaria, oedema, arthritis, rheumatism, ulcer, gingivitis, conjunctivitis (Burkill, 2000; Malgras, 1992; Arbonnier, 2002; Inngjerdingen et al., 2004). Vonthron-Sénécheau et *al*., (2003) reported the *in vitro* antiplasmodial activity of the extracts of the leaves of *Anogeissus leiocarpus*. Geidam et al., (2004) reported evidence-proved similar effects of the aqueous stem bark extract from *Cissus populnea* on some serum enzymes in alloxane induced diabetic rats. They have attributed hypoglycaemic properties to these extracts. Aqueous extract from *Mitragyna inermis* has been used by traditional healers for the treatment of various diseases, particularly for hepatic illness, malaria and hypertention. Recently, studies by Ouédraogo et *al*., (2004) demonstrated the hypotensive, cardiotropic and vasodilatory properties of bark aqueous extract from *Mitragyna inermis*. To identify new antimalarial compounds, Conrad et *al*. (1998) selected *Terminalia macroptera* for an antiplasmodial screening. Moulis et al., (1994) studied the volatile constituents of the leaves of *Vepris heterophylla*. They found that among thirty-three compounds – that were identified by capillary GC – the main constituents were geijerene and pregeijerene. Recently, Mølgaard et *al.,* (2001) reported good activity of the extracts of root bark from *Zyzyphus mucronata* which were tested *in vitro* against tapeworms and schistosomules.

To our knowledge, there are no previous reports concerning *in vitro* antioxidant activities of these plant part extracts. The purposes of this study were to determine the total phenolic and the total flavonoid contents, to evaluate their antioxidant activities using 2, 2'-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) tests, and finally to identify and to quantify some polyphenolic compounds by using a RP-HPLC coupled to an UV detector.
