Phytochemical Composition and Antioxidant Capacity of Three Malian Medicinal Plant Parts

This study evaluates the levels of total polyphenolic compounds in three Malian medicinal plants and determines their antioxidant potential. Quantitative and qualitative analysis of polyphenolics contained in plants extracts were carried out by RP-C18 RP–HPLC using UV detector. The antioxidant activity was determined by three tests. They are phosphomolybdenum, DPPH (2,2-diphenyl-1 picrylhydrazyl) and ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic)] tests. The total phenolic and the total flavonoid contents varied from 200 to 7600 mg 100 g−1 dry weight (dw), expressed as gallic acid equivalents and from 680 to 12 300 mg 100 g−1 dw expressed as catechin equivalents, respectively. The total anthocyanin concentrations expressed as cyanin-3-glycoside equivalent varied from 1670 to 28 388 mg 100 g−1 dw. The antioxidant capacity was measured by determining concentration of a polyphenolic (in mg ml−1) required to quench the free radicals by 50% (IC50) and expressed as vitamin C equivalent antioxidant capacity. The IC50 values were ranked between 2.68 and 8.80 μg ml−1 of a solution of 50% (v/v) methanol in water. The uses of plants are rationalized on the basis of their antioxidant capacity.


Introduction
Several epidemiological studies suggest that plants rich in antioxidants play a protective role in health and against diseases [1], and their consumption lowered risk of cancer, heart disease, hypertension and stroke [2][3][4]. The major groups of phytochemicals that may contribute to the total antioxidant capacity of plant include polyphenols and vitamins (C and E). Phenolic compounds can be nonnutrients [5]. Phenolic compounds of plants are hydroxylated derivatives of benzoic acid and cinnamic acids and have been reported to possess antioxidative and anticarcinogenic effects. Phenolic compounds including flavonoids are important in plant defense mechanisms against invading bacteria and other types of environmental stress [5,6]. Flavonoids have long been recognized to possess anti-inflammatory, anti-allergic, antiviral and antiproliferative activities [5][6][7][8][9]. Several reports indicate that the antioxidant potential of medicinal plants may be related to the concentration of their phenolic compounds which include phenolic acids, flavonoids, anthocyanins and tannins [10,11]. These compounds are of great value in preventing the onset and/or progression of many human diseases [12]. The health-promoting effect of antioxidants from plants is thought to arise from their protective effects by counteracting reactive oxygen species [11]. Antioxidants are compounds that help delay and inhibit lipid oxidation and when added to foods tend to minimize rancidity, retard the formation of toxic oxidation products, help maintain the nutritional quality and increase their shelf life [13].
We have recently reported the evaluation of the antioxidant potential of some medicinal and dietary plants [14,15] and the positive correlation between peripheral blood granulocyte oxidative status and level of anxiety in mice [15][16][17].
The objectives of this investigation are (i) to evaluate the level of total phenolics, flavonoids and anthocyanins in three sub-Saharian medicinal plants (Daniella oliveri, Ficus capensis and Vitex doniana) used for treating hypertension and considered as diuretic, anti-inflammatory, antipyretic and antipurulent agents (Table 1) and (ii) to evaluate total antioxidant potential by using vitamin C equivalent antioxidant capacity (VCEAC) tests.   [27] slightly modified. The powder sample (2 g) was extracted twice with 20 ml of cold aqueous methanol solution (50%). The two volumes were combined, made up to 40 ml, centrifuged at 1238 g for 20 min and transferred in small sample bottles and stored at +4 • C in the dark until analysis.

Extraction of Polyphenol Compounds for RP-HPLC
Analysis. Polyphenols were extracted following the method described by Muchuweti et al. [28] slightly modified. Fresh samples (5 g) of plants portions were extracted twice with ethyl acetate (20 ml) and organic fractions were combined. After 30 min of drying with anhydrous NaSO 4 , the extract was evaporated to dryness at 40 • C. Then, the residue was dissolved in methanol/water [2 ml 1 : 1(v/v)] before analysis by RP-HPLC. The standard solutions were prepared by dissolving 1 mg ml −1 (m/v).

Spectrophotometer Analysis
Dosage of TPC. TPC were determined following Muchuweti et al. [28] method which was slightly modified. To a sample of 100 μl, distilled water was added to make the quantity 2 ml (Eppendorff tube), followed by addition of 1 ml of Folin-Ciocalteu reagent (1N) and sodium carbonate (20%). After 40 min at room temperature, absorbance at 725 nm was read on a spectrophotometer against a blank that contained methanol instead of sample. TPC were expressed in terms of equivalent amounts of gallic acid (GAE).
Determination of TFC. TFCs were measured according to a colorimetric assay slightly modified [12,29]. A 250 μl of standard solution of catechin at different concentrations or appropriately diluted samples was added to 10 ml volumetric flask containing 1 ml of didistillate waters (ddH 2 O). At time 0 min, 75 μl of NaNO 2 (5%) was added to the flask. After 5 min, 75 μl of AlCl 3 (10%) was added. At 6 min, 500 μl of NaOH (1N) was added to the mixture. Immediately, the solution was diluted by adding 2.5 ml ddH 2 O and mixed thoroughly. Absorbance of the mixture, pink in color, was determined at 510 nm versus the prepared blank. TFCs in medicinal plants were expressed as microgramcatechin equivalents (CE)/gram dry weight (dw). Samples were analyzed in three replications.
Evaluation of TAC. The anthocyanin contents of samples was estimated by a UV-spectrophotometer with the pHdifferential method [30,31] [36]. After each run, the system was reconditioned for 10 min before analysis of next sample. Under these conditions, 20 μl of sample were injected. All sample analysis was done in triplicate. Polyphenolic standards prepared by dissolving 1 mg ml −1 were used to generate characteristic UV spectra and calibration curves. The individual polyphenolic compounds in the sample were identified by comparison of their UV-visible spectra and their retention times with the spike of the corresponding polyphenolic standards.
The detection was carried out at 280 and 320 nm and their quantification was obtained by the comparison of the peaks area with the corresponding standards calibration curves. Collected results were reported as equivalent amount of commercial standard.
2.6. Antioxidant Activity. Three different tests have been used to determine the total antioxidant capacity: the phosphomolybdenum (PPM) test, the ABTS test and the DPPH test [37,38].

PPM Test.
The PPM assay is a DPPH scavenging method in which, hydrogen and electron transfer from antioxidant analytes to DPPH and Molybdenum(VI) complex occur in the DPPH and PPM. The transfers occur at different redox potentials in the two assays and also depend on the structure of antioxidant. Several flavonoids and phenols have been isolated from plant parts with potent DPPH scavenging activities [39], whereas the PPM method usually detects antioxidants such as vitamins C, E and some specific phenol [37]. In general, the extraction solvent affects the antioxidant capacity, the aqueous methanol extract showed better antioxidant activities than the organic extract, aqueous alcohol is considered to be the best solvent for the extraction of phenolic compounds from plant materials [40,41].
The total antioxidant capacity of the plant extracts was measured by the method described by Prieto et al. [37]; 100 μl of the sample solution was mixed with 900 μl of the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) against a blank containing 100 μl of methanol mixed with 900 μl of reagent solution. The absorbance of the test sample was measured at 695 nm. The antioxidant activity was expressed as vitamin C equivalent (mg 100 g −1 dry matter).

ABTS Test.
The method used in this test is the one developed by Vanden Berg et al. [38], slightly modified. One millimolar of AAPH solution was mixed with 2.5 mM ABTS as diammonium salt in phosphate buffered saline (PBS) solution 100 M potassium phosphate buffered (pH 7.4) containing 150 mM NaCl. The mixture was heated in a water bath at 68 • C for 20 min. The concentration of the resulting blue-green ABTS radical anion solution was adjusted to an absorbance of 0.65 ± 0.02 at 734 nm. The sample solution (60 μl) was added to 2.94 ml of the resulting blue-green ABTS radical solution. The mixture, protected from light, was incubated in a water bath at 37 • C for 20 min. Then the decrease of absorbance was measured at 734 nm. The control solution was consisted by 60 μl of methanol and 2.94 ml of ABTS radical anion solution. The stable ABTS radical anion scavenging activity of the plants phenolic compounds in the extracts was expressed as mg 100 g −1 dry plants powders and as mg 100 ml −1 standards compounds of VCEAC in 20 min. All radical stock solutions were prepared fresh daily.

DPPH Test
DPPH Evaluation. The antioxidant activity of plant extract was estimated using a slight modification of the DPPH radical scavenging protocol reported by Chen et al. [42]; 1 ml of 100 μM DPPH solution in methanol was mixed with 0.1 ml of plant extract. The reaction mixture was incubated in the dark for 20 min and thereafter the optical density was recorded at 517 nm against the blank.  [12,43] where At 0 : absorbance of the sample test after 0 min and At 20 : absorbance of the control after 20 min. Each assay was carried out in triplicate.
From a plot of concentration against %IP, a linear regression analysis was performed to determine the IC 50 value (concentration of a polyphenolic (in mg ml −1 ) required to quench the free radicals by 50%) for each plant extract. The DPPH radical scavenging activity of phenolic compounds was expressed as IC 50 value in micrograms per milliliter of fresh weight. A low IC 50 value represents a high antioxidant activity.
DPPH Determination. The DPPH scavenging activity was determined using a modified method of Kim et al. [35]. To 2.90 ml of an aqueous methanol solution (50%) of 100 μM of DPPH, 100 μl of the plant extracts solution was added. The mixture was shaken and allowed to stand at 20 • C in dark for 30 min. After the decrease in absorbance, the resulting solution was monitored at 517 nm. The DPPH radical scavenging activity of phenolic compounds was expressed as mg 100 g −1 of dry matter and as mg 100 ml −1 of VCEAC in 30 min. The control solution was consisted by 100 μl of methanol and 2.90 ml of DPPH solution. The radical solution was prepared daily.

Statistical Analysis.
Results are presented as mean ± standard error; statistical analysis of experimental result was based on analysis of variance. Significant difference was statistically considered at the level of P < .001.

TPCs, TFCs and TACs.
TPCs, TFCs and TACs were quantified using a UV-vis spectrophometric apparatus. The results of analysis are showed in Figure 1. No data were recorded for F. capensis leaves due to lack of sample.

RP-HPLC Analysis.
Quantitative and qualitative comparison of polyphenolic compounds (TPC, TFC, TAC) were conducted using RP-HPLC.
The retention time of standards and their corresponding concentration in the samples were collected in Table 2. The experimentation has been done in four replicates. However, it is important to note that numerous peaks were not identified owing to the absence of suitable standards.

Antioxidant Activity.
On the three plants screened, the extracts revealed good scavenging antioxidant activities as well as by PPM, ABTS or DPPH tests. The scavenging antioxidant activities of the different samples were reported in Table 3. Figure 2 showed the relationship between the antioxidant activities and the polyphenolic compounds (TPC, TFC, TAC) in the samples.

Discussion
The distribution of TPC in D. oliveri and V. doniana differs. The content of TPC are higher in leaves than in stem barks in V. doniana, whereas in D. oliveri TPC is more concentrated in the stem barks ( Figure 1). The concentration of TFC is very low in the root barks of F. capensis. The stem bark extracts of D. oliveri and F. capensis contain almost the same levels of TFC. Daniella oliveri plant parts, stem barks, root barks and leaves exhibit a similar TFC (Figure 1). For all the three plants, the concentration of TAC is lowest in the root barks.
RP-HPLC analysis revealed that the caffeic acid in the stem barks of D. oliveri is the most important phenolic compound (2410.4 μg ml −1 ), whereas its levels are too low in the other two plants (V. doniana, 8.2 μg ml −1 and F. capensis, 12.7μg ml −1 ). Moreover, it appears that rutin is in very high concentration (6363.0 μg ml −1 ) in the root barks of V. doniana and almost absent in the root barks of D. oliveri and F. capensis.   Rutin is the most important phenolic compound (11943.0 μg ml −1 ) in the leaves of V. doniana, while it is not detected in the leaves of D. oliveri (Table 2).
Antioxidant activity has been evaluated by three tests: PPM, ABTS and DPPH. The PPM assay showed that the highest value was 606.0 mg 100 g −1 dw (VCEAC) for the root barks of D. oliveri; in contrast, the lowest one was 60.0 mg 100 g −1 dw for the root barks of F. capensis (Table 3). The great variations observed between the different plants and plant parts could be explained by the fact that PPM essay evaluates the antioxidant activity of polyphenols, and others antioxidant agents which are not phenolic compounds [43]. To be more accurate about phenolic compounds, ABTS and DPPH tests have been done. ABTS tests showed that the antioxidant activity of different plants was almost the same. DPPH tests expressed as VCEAC varied from 91.3 mg 100 g −1 dw for the root barks of F. capensis to 205.5 mg 100 g −1 dw for the stem barks of V. doniana. In addition, the antioxidant activity evaluated as %IP revealed a similar behavior. The highest IP value was 93.3% for the stem barks of V. doniana and the lowest one was 28.4% for the root barks of F. capensis. The %IP and IC50 (μg ml −1 ) have been calculated to compare the antioxidant capacity of the studied plant parts extracts with those described by other authors in literature such as Adesegun et al. [44] and Ruchi et al. [43]. %IP values were relatively high (28.41-93.3%) and IC 50 relatively weak (2.7-8.8 μg ml −1 ). This revealed that these three Malian plants have very good antioxidant activities. Each plant contains generally different phenolic compounds with different amount of antioxidant activity.
Many studies indicate linear relationship between total phenolics and antioxidant activity [10,12,45]. In this study we found that polyphenolic compounds were not major contributors to antioxidant activity, since for TPCs, TFCs and TACs versus antioxidant activity, the correlation coefficients R 2 = 0.0998, 0.1641, 0.1135, respectively, were weak ( Figure 2). These correlations have been established using all plant parts (stem barks, root barks, leaves). In conclusion, our results suggest that these plants are strong radical scavengers and can be seen as potential source of natural antioxidants for medicinal and commercial uses.

Funding
Ministry of Scientific Research of the Republic Democratic of Congo grant (No. 132.49/060/KMB/07).