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Radical Scavenging Activity, Phenolic Content and Cytotoxicity of Bark and Leaves Extracts of Entada africana Guill. and Perr. (Mimosaceae)



A. Tibiri, O. Rakotonandrasana , G.O. Nacoulma and J.T. Banzouzi
 
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ABSTRACT

Dried ground leaves and barks of Entada africana were extracted by maceration in methanol and fractioned with chloroform, ethyle acetate and water. The total phenolic content of each fraction was determined spectrophotometrically according to Folin-Ciocalteu`s method and calculated as Tannic Acid Equivalent (TAE). Tannins and flavonoids were also determined. The total phenolic content was quite high, especially in the aqueous fraction (up to 39.7% TAE in the barks and 39.9% in the leaves). The antioxidant activity of lyophilized extracts was determined at room temperature by the means of the 2,2-diphenyl-1-picrylhydrazyl (DPPH!) colorimetric method with a detection scheme at 517 nm and expressed as EC50. The radical scavenging activity was evaluated as the difference in absorbance between a test sample and the control (methanol). Bark extracts had the best EC50, similar to those of rutoside and ascorbic acid for the aqueous and methanol fraction (5.7 and 5.3 μg mLG1, respectively). All the other extracts were moderately active (EC50 ranging from 6.9 to 20.0 μg mLG1), except the chloroform extracts (EC50 > 69 μg mLG1). Except for the crude and aqueous bark extracts, no extract was cytotoxic on KB or Vero cells.

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A. Tibiri, O. Rakotonandrasana , G.O. Nacoulma and J.T. Banzouzi , 2007. Radical Scavenging Activity, Phenolic Content and Cytotoxicity of Bark and Leaves Extracts of Entada africana Guill. and Perr. (Mimosaceae). Journal of Biological Sciences, 7: 959-963.

DOI: 10.3923/jbs.2007.959.963

URL: https://scialert.net/abstract/?doi=jbs.2007.959.963

INTRODUCTION

Entada africana is a leguminous tree used in traditional medicine throughout all sub-Saharan Africa, for the treatment of numerous diseases, such as fever (Bah, 1998), respiratory tract complaints (Occhiuto et al., 1999), diabetes, hypertension and diarrheas (Nacoulma-Ouedraogo, 1996). This tree is indicated in the traditional pharmacopoeias for its wound-healing, haemostatic, anti-rheumatism, anti-inflammatory and hepatoprotective properties (Diallo et al., 2001; Burkill, 1995; Oliver-Bever, 1986; Kerharo, 1974). Assays for the hepatoprotective activity were conclusive (Douaré, 1991; Sanogo et al., 1998). All plant parts are used, especially bark and leave.

These traditional indications led us to assess the antioxidant activity of Entada africana. In fact, very often in the relevant literature, a relationship is observed between hepatoprotective or anti-inflammatory properties and the antioxidant potential of the plant. Many hepatoprotective plants act by relieving liver cells from oxidative stress (Maxwell et al., 1999; Tilak et al., 2004; Bhattacharjee et al., 2006) and free radicals play a role in inflammation (Winrow et al., 1993). We therefore decided to assess the free radical scavenging activity of Entada africana leaves and bark extracts, using the DPPH colorimetric method, along with their phenolic content, since antioxidant activity in plants is often due to polyphenols and especially flavonoids and tannins (Yokozawa, 1998).

Indeed, a positive correlation between the antioxidant activity and the total phenolic content can be observed in a number of papers (Kumaran et al., 2007; Tawaha et al., 2007). Some data are already available on Entada africana phenolics, but they had to be completed. Bako et al. (2005) determined the phytochemical profile of the leaves and found tannins and flavonoids, without isolating them. Montoro et al. (2005) on the other hand isolated flavonoids in the roots, but without looking for the other phenolics. So, in order to look for a correlation between phenolics and radical scavenging activity, we had to get quantitative data and therefore to determine the amount of phenolics in both leaves and barks.

MATERIALS AND METHODS

Plant material: Fresh Entada africana was collected in Gampéla, 15 km east of Ouagadougou (Burkina Faso) in September 2005. A voucher specimen, identified at the department of Biology and Ecology of the University of Ouagadougou, was deposited under the registration number TA05-1 in the herbarium of the University. Leaves and barks were separately dried under ventilation at room temperature then finely ground on an electrical grinder.

Preparation of leaves and bark extracts: Leaves and barks (100 g of each) were extracted by methanol maceration at room temperature. The filtrates of the obtained extracts were concentrated under reduced pressure until all the methanol had evaporated. The concentrates were redissolved in distilled water and lyophilized. One gram of each lyophilized concentrate was dissolved in water and then sequentially extracted with chloroform and ethyl acetate (Ac-OEt). The water residue was then lyophilized. For each fraction and for the crude methanol extract, scavenging activity against DPPH was determined and the content of total phenolics, tannins and flavonoids was measured.

Free radical scavenging activity: The antioxidant activity of Entada africana leaves and barks was determined by the mean of DPPH colorimetric method as described by Velázquez et al. (2003), slightly modified. Three milliliters of a 20 mg mL-1 DPPH methanolic solution were added to 1.5 mL of a methanolic solution of each extract (1-100 μg mL-1). The mixture was shaken vigorously then incubated for 15 min in darkness at room temperature. Absorbance was measured at 517 nm. Methanol was used as control to zero the spectrophotometer (Agilent 8453E). Rutoside, ascorbic acid and quercetin were used as positive controls. The loss of free radical activity of the DPPH_ radical stock solution was reduced as much as possible as recommended by Blois (1958). All measurements were performed five times. The difference in absorbance between a test sample and a control (methanol) was taken as the activity and expressed as EC50, that is to say the concentration of antioxidant needed to decrease by 50% the initial concentration. This EC50 was calculated from the equation determined by linear regression for the dose/effect results.

Determination of the extracts cytotoxicity: The cytotoxicity of all the extracts of leaves and barks was tested on KB (Human epidermoid carcinoma) and Vero (African green monkey kidney) cells, according to the method described by Mbatchi et al. (2006). The reference used was taxotere.

Determination of the plant content in phenolic compounds
Total phenolic content:
The amount of total phenolic content in each leaves and barks extract was assessed according to the method of Singleton et Rossi (1965) using Folin-Ciocalteu reagent. For the preparation of calibration curve, 1 mL aliquots of 0.10 to 0.60 mg mL-1 ethanolic tannic acid solutions were mixed with 1 mL Folin-Ciocalteu reagent (2N) and 3 mL sodium carbonate (20%), then left 40 min at room temperature. The absorbance was read at 760 nm and the calibration curve was drawn. Zero point twenty-five milliliters of each extract (0.5 g L-1) were completed to 1 mL with distilled water and then mixed with the same reagents as described above. The resulting solution was vortexed and left for 40 min before measuring absorbance. Results were expressed as Tannic Acid Equivalent (TAE). All determinations were performed 5 times.

Non-tannic phenolics: Tannins were precipitated from each extract by polyvinyle polypyrrolidone (PVPP) during their standing at 4°C overnight. The precipitated tannins were removed by centrifugation (3000 rpm, 10 min). The supernatant, containing all phenolic compounds except tannins, was collected. Its absorbance was measured in the same way as for the total phenolics.

Non-flavonoidic phenolics: Flavonoids were precipitated by formaldehyde at pH < 0.8. Five milliliters of a concentrated HCl solution (50/50 v/v) and 5 mL of a formaldehyde solution (8 mg L-1, in distilled water) were added to 10 mL of each extract. The mixture was vortexed, then left 24 h at room temperature. Flavonoids were separated by centrifugation (3000 rmp, 10 min) and the supernatant, containing all phenolic compounds except flavonoids, was collected and filtrated. Its absorbance was measured in the same way as for the total phenolics.

Statistical analysis: Experimental values are means +/- SEM of the number of experiments indicated in the legends. Data were evaluated for statistical significance with one-way ANOVA followed by Dunnett’s multiple-range tests when appropriate (GraphPad Prism version 4.03 pour Windows, GraphPad Software, San Diego California USA). A p value of 0.05 or less was considered as statistically significant.

RESULTS

Scavenging activity against DPPH radicals: The aqueous and methanol extracts of barks were found to have the highest antioxidant activity (EC50 values of 5.7 and 5.3 μg mL-1, respectively), of the same order as those of rutoside and ascorbic acid. The Ac-OEt extract of bark is not far behind, with an EC50 value of 6.9 μg mL-1. However, their scavenging activity was twice inferior to the activity of quercetin. Both chloroform extracts (for barks and leaves) were inactive (EC50: 69 and 226 μg mL-1 respectively). All the other fractions (leaves extract) were moderately active, with EC50 values ranging from 9.5 μg mL-1 (methanol extract) to 20 μg mL-1 (aqueous extract; Table 1).

Cytotoxicity: Nearly all the barks and leaves extracts of Entada africana have a very low cytotoxic activity against Vero and KB cells at the concentration of 10 μg mL-1. Only two of the bark extracts (the crude extract and the aqueous extract) showed a measurable cytotoxicity against KB cells (respectively 53 and 57% of cell growth inhibition) (Table 2).

Phenolic content of Entada africana leaves and barks: Tannins and flavonoids concentrations were calculated from the values obtained for the non-tannic and non-flavonoidic phenolic compounds, respectively. The methanolic crude extract has a quite high content in total phenolics, especially in the aqueous fraction (up to 39.7% TAE in the barks and 39.9% in the leaves). Tanins and flavonoids are more abundant in the barks than in the leaves. Same proportions were observed for the aqueous fraction, as could be expected (Table 3).


Table 1: Extraction yield and EC50 values against DPPH of Entada africana leaves and bark extracts
Rutoside, 5.2 (5.0; 5.5); Ascorbic acid, 5.3 (5.1; 5.5); Quercetine, 2.5 (2.4; 2.6) Data in parenthesis represent the confidence interval at 95% (n = 5) a,b p>0,05 compared to rutoside and ascorbic acid

Table 2: Cytotoxicity of Entada africana leaves and bark extracts (Percentage of cell growth inhibition)
Taxotere (2.5 10-10 M): 65 (KB)/0 (Vero)

Table 3: Phenolic content of Entada africana leaves and barks, expressed in TAE (Tannic Acid Equivalent)
Data in parenthesis represent the confidence interval at 95% (n = 5), *Calculated from the values of non-tannic phenolics, **Calculated from the values of non-flavonoidic phenolics

DISCUSSION

As we had expected after having reviewed the traditional uses of the plant, Entada africana has a high antioxidant power, of the same level as ascorbic acid and rutoside, well-known free-radical scavengers. Our results confirm the previous study from Cook et al. (1998), who obtained with the trolox assay a relatively high antioxidant activity for Entada africana leaves (more active than potato and spinach). In our assay, the barks were more active than the leaves. The EAT values obtained for the total phenolic content, the tannins and the flavonoids are consistent with the results for antioxidant activity, since they are higher in the barks than in the leaves. We therefore think that the compounds responsible for the radical scavenging activity of the plant are likeliest to be found among the flavonoids or the tannins of the plant. This is supported by the results of Montoro et al. (2005), who isolated in the roots of Entada africana three myricetin-derived flavonols. These molecules had an interesting antioxidant activity, though lower than the standard, quercetin. Since this team used TEAC (Trolox Equivalent Antioxidant Capacity) and not DPPH as we did, results are not easy to compare. However, they seem coherent with the EC50 we obtained for leaves and bark extracts, which are lower than EC50 of quercetin but near EC50 of rutoside and ascorbic acid.

The cardio-protective and anti-tumor properties attributed to some flavonoids (Cook et al., 1998) make them useful compounds for medicine. Indeed, free radicals or oxidative injury underlay quite a number of human neurological and immune disorders and diseases such as diabetes, through mechanisms beginning to be well known. They also play a role in carcinogenesis and their level increase with exercise, fever, infection, etc. (Atawodi, 2004). Therefore, finding new radical scavenging compounds with a low cytotoxicity would be very interesting. The low cytotoxicity and the good antioxidant activity of Entada africana leaves and barks extracts give us good reasons to continue with our studies of this plant. Our next step will be to isolate this or these compounds through a bio-guided fractionation, in order to determine whether they are already known compounds or new ones. We will also check if there is a synergistic between the different phenolic compounds contained in the barks and leaves of Entada africana.

ACKNOWLEDGMENTS

We thank the researchers from CERMA, especially Ms Aline Prost, who helped in the writing and the translation of this study.

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