Antioxidant Activity of Hydro-Acetonic, Hydro-Methanolic and Aqueous Leaf and Bark Extracts of Sclerocaria birrea (A. Rich.) Hochst

Bioactive compounds in plants are associated with the reduction of chronic diseases. The free radical scavenging activity of different extracts of a medicinal plant, Sclerocarya birrea, has been investigated using the DPPH test, ABTS test and FRAP. Three extracts were prepared from the leaves and bark: hydro-methanolic, hydro-acetonic and aqueous. Phytochemical screening was carried out the standard methods followed by the determination of the polyphenol by Folin-Ciocalteu method. The analysis of variance (ANOVA) with the STATISTICA 7.1 and statistical significance was set at p < 0.05. Evolution of percent inhibition (PI) as well as the IC 50 of the extracts was obtained using the Origin Pro 8.5 software and Microsoft Excel. The results show that the bark extracts are about twice as rich in polyphenols as the leaves. With DPPH at 1.25 mg/mL, the bark has a PI of 91.04% ± 0.001% while leaves, reach 99.80% ± 0.021%. As for the ABTS test, the bark extract reached its maximum activity at 1.25 mg/mL with a PI the leaves. We can say that the bark has better activity than the leaves and the alcoholic extracts have given better results than the aqueous extract.


Introduction
The use of plants (fruits, leaves, etc.) by traditional medicine to fight against diseases linked to free radicals such as asthma, cancer and diabetes is well known [1]. Some vegetable phytochemicals (glucosinolates, thiosulfates, polyphenols, bioactive peptides, etc.) have positive effects on health [2]. They are strong antioxidants and they reduce the risk of chronic diseases by protecting against free radical damage, by modifying metabolic activation and detoxification of carcinogens [3]. Among all the chemical constituents of plants, phenolic compounds occupy an important place with several properties sought by man in particular in the analytical and biotechnological fields. Some plants such as green and black tea have strong antioxidant activity, which is due to their high polyphenol content [4]. Free radicals contribute to the occurrence of serious diseases such as cancer, diabetes and cardiovascular diseases [5]. Other phytochemical constituents (terpenoids, carotenoids, vitamins C and E) are also known for their antioxidant capacity [6]. The high cost of drugs, the inaccessibility of health care and the effectiveness and availability of medicinal plants are pushing populations towards herbal medicine. Many diseases including diabetes are treated satisfactorily and affordably with medicinal plants due to their active substances. In Senegal, leaves and bark of Sclerocarya birrea are also used for certain diseases, such as diabetes by population and the plant is very accessible [7] [8] [9]. According to that, investigations are conducted to assess their antioxidant activity through three methods and three extracts solutions like aqueous, hydro-methanolic and acetonic. This has been achieved in order to get complete information and to project on possible applications.

Collection and Identification of Plant
Leaves and bark of Sclerocarya birrea were collected in May 2019, the flowering period, at Diourbel region (Senegal). The plant was identified and authenticated in botany-biodiversité laboratory, plant biology department of Cheikh Anta Diop University in Dakar. Plant leaves and bark were washed with distilled water and air-dried at room temperature in the laboratory. Dried leaves and bark were ground to a fine powder using an electric grinder (Kenwood, France). The sifted powder with 1 mm mesh is stored at 4˚C in airtight jars. Thus, the powders are then subjected to two extraction methods: by infusion and by Soxhlet.

Determination of Total Phenolic Content
Total phenolic content (TPC) of the sample was estimated by the Folin-Ciocalteu method as described elsewhere slightly modified [13]. In brief, 50 µL of sample diluted to 450 µL with distilled water and standard was added to 2500 µL of ten times diluted Folin-Ciocalteu reagent. Then, after adding 2.5 mL of 0.75% sodium carbonate, (all from Sigma-Aldrich Chemie, Germany), the mixtures were vortexed and incubated at 50˚C in a water bath for 15 minutes. The samples prepared above were kept the samples pin the dark at room temperature for 30 minutes. Thereafter, the absorbance of each sample was read at 760 nm against the blank. Gallic acid was used for the calibration of a standard curve. Gallic acid dilution series at different concentration (0.02 -0.04 -0.06 -0.08 -0.1 -0.12 -0.14 mg/mL) was used for calibration curve in the same way as the extracts. TPC was determined as gallic acid equivalents (GAE) and values were expressed as mg GAE/g dry matter (DM) of plant extract.

Evaluation of Antioxidant
I (%): Antioxidant activity as a percentage of inhibition of DPPH. A co : absorbance of blank sample (t = 0 min); A Ex : absorbance of tested extract solution (t = 15 min).

Ferric Reducing Antioxidant Power assay (FRAP)
The ferric reducing power was determined according to the described method [16]. The antioxidant activity linked to the ferric reducing power of the extracts is expressed in Reducing Power (RP) using the following formula: A Ex : absorbance of tested extract solution A Co : absorbance of blank sample (t = 0 min).

Statistical Analysis
The analytical results obtained from tree trials were processed using analysis of variance (ANOVA) with STATISTICA 7.1. The results were considered statistically significant if P value was < 0.05. The IC 50 was obtained using the Origine Pro 8.5 software and Microsoft Excel.  [19] which detected in addition to these metabolites, the presence of saponosides. These compounds are frequently encountered in plant extracts such as Ziziphus mauritiana Lam [20].

Total Phenolic Content
Total phenolics constitute one of the major groups of compounds acting as primary antioxidants, it was reasonable to determine their total amount in leaves and bark extracts [11]. The content of phenolic compounds (mg GAE/g DM) in all extracts, determined from regression equation of calibration curve (y = 3.120x + 0.069, R 2 = 0.98) and expressed in gallic acid equivalents (GAE). The results show that the total phenolic contents vary significantly between the extracts of the plant parts studied but also the extraction solvents (

Antioxidant Activity
The results of the DPPH and ABTS radical tests were obtained by following the evolution of the percentage inhibition as a function of the variation in the concentration of the extracts.

DPPH Radical Scavenging Assay
The inhibition percentages show that at all the concentrations tested, our extracts significantly inhibit the DPPH radical as in the case of hydro-ethanolic extracts from the leaves and bark of Piliostigma thonningii Schumach [16]. In all the extracts tested, the barks exhibit greater activity than that of the leaves. Indeed, the greatest activity obtained at the concentration of 2.5 mg/mL, the bark has a percentage inhibition of 91.04% ± 0.001% compared to the leaves, the most important of which is 99.80% ± 0.021%. As for the ascorbic acid used as a reference has a PI with the DPPH radical of 94.86% ± 0.008% at 1.25 mg/mL then its activity evolved to a plateau up to 2.5 mg/mL (Figure 1). The percentages of inhibition obtained on the latter are greater than those of the acetone extract (71.76% at 0.825 mg/mL) on Foeniculum vulgare [22]. Thus, in order to better compare the anti-free radical activity of the various plant extracts, the IC 50 were determined. The extracts of the leaves (hydromethanolic and infused) have higher IC 50 than those of the bark with respective values of 0.480 ± 0.00014 mg/mL and 0.894 ± 00008 mg/mL against 0.156 ± 0.0007 mg/mL and 0.407 ±0.0001 mg/mL. However, the hydro-acetone extract of the bark has an IC 50 greater than that of the leaves with respective values of 0.361 ± 0.000028 mg/mL against 0.301 ± 0.00006 mg/mL. At the same time, ascorbic acid (vitamin C) exhibits an IC 50 of 0.051 ± 0.0000 mg/mL.

ABTS Radical Cation Decolourisation Assay
From Figure 2, it can be seen that whatever the extraction solvent, the extracts from the leaves and bark significantly inhibited the ABTS cation radical. As in the case of the DPPH test, in all the extracts tested, the barks exhibit greater activity than that of the leaves. Indeed, the bark extract reached its maximum activity at 1.25 mg/mL with a PI of 99.80% ± 0.003% compared to the extract of the   leaves which has a greater PI of 99.75 ± 0.003 at 2.5 mg/mL. At the weakest concentrate tested (0.039 mg/mL), the extracts inhibited 10.63% ± 0.007% the ABTS cation radical and 6.25% ± 0.003% the DPPH radical against ascorbic acid which inhibits 10.25% ± 0.002% the ABTS cation radical and 31.59% ± 0.012% the DPPH cation radical. At the highest concentration tested (2.5 mg/mL) and whatever the extraction solvent, the bark extract has the greatest PI with 91.04% ± 0.001% on the DPPH radical and 99.80% ± 0.021% on the radical ABTS against ascorbic acid 94.86% ± 0.008% on the DPPH radical and 99.80% ± 0.021% on the ABTS cation radical. By comparing the two methods, the PI shows that the bark extracts are more active than the leaf extract. However, ascorbic acid inhibits the ABTS radical (99.81% ± 0.014%) more than the DPPH radical (94.86% ± 0.008%). These results agree with those found on hydro-ethanolic extracts from the leaves and bark of Piliostigma thonningii Schumach [16].

FRAP Test
In analogy with the inhibitory power, the evolution of the reducing power as a function of the concentration of the extracts (Figure 3)    lower than those of the ABTS method. This could be explained by the presence of active substances which exhibit absorption bands at the same wavelength as the DPPH radical, thus leading to an increase in absorbance [6]. The FRAP test shows a better reducing power of the ferric ion of the bark compared to the leaves. Thus, by these 3 tests, we see that the antioxidant activity of the bark is stronger than that of the leaves. This could be explained by the polyphenol content of the bark which is about twice as high compared to the leaves.

Conclusion
This study has demonstrated the antioxidant activity of aqueous and hydro-al-