Phytochemical Assessment and Antioxidant Activities of Alcoholic and Aqueous Extracts of Acacia mearnsii De Wild

MATERIALS AND METHODS

Collection of plant material: The bark materials of A. mearnsii were collected from the plant growing within the University of Fort Hare campus in Alice, South Africa in September, 2010 while the study was carried out immediately after the plant material was dried. The plant was authenticated in the Department of Botany and a voucher specimen was prepared and deposited in the Griffen Herbarium of the University.

The bark samples were air-dried at room temperature and pulverized using a milling machine. Portions of about 100 g each of the pulverized samples were extracted separately with acetone, methanol, ethanol and water for 48 h. The extracts were filtered through Whatman No. 1 filter paper and evaporated to dryness under reduced pressure at 40°C using a rotary evaporator. The extracts were redissolved in their respective solvents to the required concentrations for the bioassay analysis.

Chemicals and reagents used: 2,2-azinobis-3-ethyl benzothiazoline-6-sulfonic acid (ABTS) diammonium salt, 1,1-diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), gallic acid, rutin, ascorbic acid (VC), quercetin and FeCl₃, were purchased from Sigma Chemical Co. (St. Louis, MO. USA) vanillin was from BDH Chemicals Ltd. (Poole, England) and Folin-Ciocalteu phenol reagent and sodium carbonate were from Merck Chemical Supplies (Darmstadt, Germany). All other chemicals used, including the solvents, were of analytical grade.

Determination of total flavonoids: Total flavonoids were estimated using the method of Ordonez et al. (2006).

Determination of ferric reducing power: The ferric reducing potential of the extract was determined according to the method of Kumar et al. (2005).

Determination of total phenol: The total phenolic content of the extract was determined by the modified Folin-Ciocalteu method (Wolfe et al., 2003).

Determination of total proanthocyanidins: The total proanthocyanidins were determined by using the procedure reported by Sun et al. (1998).

DPPH radical scavenging assay: For DPPH assay, the method of Liyana-Pathirana and Shahidi (2005) was adopted.

ABTS radical scavenging assay: The total antioxidant activity of the samples was measured by ABTS radical cation decolorization assay according to the method of Siddhuraju and Manian (2007).

Statistical analysis: The experimental results were expressed as the Mean±Standard Deviation. All assays were performed in triplicates. The data were subjected to one way analysis of variance using SPSS version 14.0. Differences between means at the 5% level were considered significant.

RESULTS AND DISCUSSION

Phytochemical compositions: The result showed that A. mearnsii has a considerable amount of total phenolic and flavonoids contents while the proanthocyanidins were very low in all the extracts. The quantities of the phenolic contents, total flavonoids and total proanthocyanidins were in descending order depending on the extract as shown in Fig. 1. The results indicated that acetone extract had the highest total phenolic content (47.88 mg g^-1) while ethanol extracts had the highest total flavonoid contents (7.98 mg g^-1).

Fig. 1:	Polyphenolic contents (mg g-1) of A. mearnsii. Data are presented as Means±Standard Deviation of three replicate with significant increases from all samples tested

While total proanthocyanidin content (0.51 mg g^-1) was the highest in methanol extract, the polyphenols contents of this plant were least obtained in the aqueous extract. This result is similar to earlier report of Zongo et al. (2010) indicating that alcoholic extract exhibited higher level of total polyphenol contents than water extracts. Quantitatively, in A. mearnsii, total phenolic contents were more than the flavonoids while the proanthocyanidins were the least. Many related polyphenols, commonly found in plants, have been reported to have several different biological activities, including antioxidant property (Luo et al., 2002; Afolayan et al., 2008; Krishna et al., 2010). According to Jayaprakasha and Patil (2007) and Hussein et al. (2010), there is a relationship between total phenolic content and antioxidant activity of plants. This is believed to be mainly due to redox properties of the phenolic compounds (Zheng and Wang, 2001) adsorbing and neutralizing free radicals, quenching active oxygen species as well as decomposing superoxide hydroxyl radicals. These phenolic compounds act as free radical terminators (Galvez et al., 2003) while flavonoids show antioxidant activity through scavenging or chelating process (Torane et al., 2011). El-Hela and Abdullah (2010) and Saikia and Adhyaya (2011) also noted a significant relationship between the free radical scavenging potency, the total phenolic and flavonoids contents of plant extracts. In this study, the extract having highest amount of flavonoids and phenolic compounds exhibited the highest antioxidant activity.

Total antioxidant power of extracts from A. mearnsii by the FRAP assay: The ferric reducing power of the different extracts of A. mearnsii was presented in Fig. 2. In both extracts and the standards studied, the reducing power or reductive capability of each of the extracts and the standards increased with increasing concentration. There are significant differences between the reductive capabilities of the extracts and those obtained for the standards such as Butylated Hydroxytoluene (BHT), Rutin and ascorbic acid. The reductive capabilities recorded was in the following order, Vitamin C>Rutin>BHT>Methanol>Acetone>Ethanol>Aqueous which showed that vitamin C exhibiting the highest reductive capability. At the highest concentration of 0.1 mg mL^-1, the reductive capability of each of the extract was 0.402 (aqueous), 0.421 (ethanol), 0.453 (acetone), 0.473 (methanol extracts), 0.633 (BHT), 0.706 (Rutin) and 1.218 (vitamin C) based on their spectrophotometric absorbance at 700 nm.

According to Chang et al. (2002) the observed reducing power is associated with antioxidant activity and may serve as a significant reflection of the antioxidant property of all the plant extracts.

Fig. 2:	Ferric reducing power determinations for the alcoholic and aqueous extracts of A. mearnsii. Data are presented as Means±Standard Deviation of three replicate with significant increases from all samples tested

Fig. 3:	ABTS radical scavenging activity of the alcoholic and aqueous extracts of A. mearnsii. Data are presented as Means±Standard Deviation of three replicate with significant increases from all samples tested

Furthermore, there is a linear relationship between concentrations and reducing power of the different extracts. This relationship is concentration dependent and may be associated with the presence of reductones (Duh, 1998) known to exert antioxidant activity by breaking the free radical chain via donating a hydrogen atom. These findings suggest that the A. mearnsii extracts are capable of donating electrons, and could, therefore, react with free radicals or terminate chain reactions.

ABTS radical scavenging activity of the extract from A. mearnsii: The results of the free radical scavenging activity of the different extracts of A. mearnsii determined by ABTS assay are shown in Fig. 3. Almost all the extracts had strong antioxidant abilities that exceeded that of BHT at varying concentrations. There was a steady increase in the ABTS radical scavenging capacity of all the extracts of A. mearnsii employed in present study. At the highest concentration of the extracts, the highest percentage inhibitions were recorded for the extracts and the standards.

The ABTS assay is based on the inhibition of the absorbance of the radical cation, ABTS, which has a characteristic long wavelength absorption spectrum (Sanchez-Moreno, 2002). While phenolic compounds scavenge radicals by forming a stable ABTS-H, the explicit method to measure the antioxidant activity of phenolic compounds is the decolorization of ABTS+radical. Decolorization of ABTS+,in present study, reflects the capacity of an antioxidant species to donate electrons or hydrogen atoms to inactivate this radical cation in a concentration dependent manner. The scavenging activity of these extracts towards ABTS radicals is similar to those earlier reported by Miller and Rice-Evans (1997). These results showed that polyphenols from bark of A. mearnsii had high antioxidant activities and that the activity of ABTS⁺ radical by the extracts was significant.

DPPH radical scavenging activity of extracts from A. mearnsii: In this study, the extracts of A. mearnsii exhibited varying degree of radical scavenging activity against the DPPH. At the lowest concentration of the extracts, high level of activity was observed in acetone, ethanol, methanol and aqueous extract. The activity of BHT was observed to be greater than activities of all the extracts at the lowest concentrations. This result showed that ethanol extract of the plant had the highest activity at the lowest concentration (Fig. 4). Their scavenging activities decreased with increase in the concentration of all the extracts as characterized by a rapid decline in the absorbance at 517 nm.

The ability of the extracts of A. mearnsii to act as a free radical scavenger or hydrogen donor was revealed by DPPH. This method is a rapid and sensitive way to survey the antioxidant activity of specific compounds or plant extracts. In this method, the antioxidant scavenges the DPPH radicals to form stable reduced DPPH molecules. The radicals formed are stabilized through the formation of non-radical products (Argolo et al., 2004). When the stable reduced DPPH molecules are formed in the presence of a free radical scavenger, the absorbance reduces and the DPPH solution is decolourised as the colour changes from deep violet to light yellow. The degree of reduction in absorbance value is indicative of the antioxidant power of the extract.

Fig. 4:	DPPH radical scavenging activity of the alcoholic and aqueous extracts of A. mearnsii. Data are presented as Means±Standard Deviation of three replicate

More yellowish colour of DPPH shows more antioxidant activity of the compounds or extracts tested (Moein et al., 2008). The observed decreases in free-radical scavenging activities with increase in concentrations of extracts are statistically significant from one extract to another. While Yu et al. (2002) reported that higher concentrations of extracts are more effective in quenching free radicals in the systems, Goupy et al. (2003) observed that the rapid reaction between antioxidants and DPPH occurs with the transfer of the most labile hydrogen atoms to the radical, while the subsequent slow steps depends on the residual H-donating capacity of antioxidant degradation products.

CONCLUSIONS

In conclusion, it is well known that free radicals are one of the causes of several diseases. The present study demonstrated that Acacia mearnsii had significant antioxidant and radical scavenging activities. Irrespective of the method used for the analysis, all the extracts showed antioxidant activity and free radical scavenging capability. While their activities are less than those of the commercially available synthetic antioxidants, phenolic content of this plant could be a good source of natural antioxidant substances which could help to neutralize free radicals and play a beneficial role in oxidative stress prevention.

ACKNOWLEDGMENT

The authors are grateful to the National Research Foundation (NRF) of South Africa for supporting this research.