6 ml/min. Separation of polyphenols was achieved using the following linear gradient system: 5–15% B in 6 min; 15–25% B in 3 min; 25–60% B in 3 min; 60–80% B in 0.6 min; 80–100% B in 0.8 min. The polyphenols were detected at a wavelength of 254 nm on a diode array detector. The polyphenol standards, consisting of gallic acid, protocatechuic acid, ellagic acid, quercetin and
kaempferol, were prepared in 50% methanol containing 20 mM DETC sodium salt and 5 μl volumes were injected into the UHPLC system and ran under the same conditions as described above. Akt signaling pathway All analyses were done in triplicate. Results were expressed as means ± standard deviations. The data were statistically analysed using the SPSS statistical software, version 15 (SPSS Inc, Chicago, Illinois, USA). An independent t-test was used for comparison of means between groups. One-way analysis of variance (ANOVA) and Tukey’s Honestly
Significant Difference test were used to compare means among groups. A Pearson correlation test was utilised to study the relationship between the antioxidant components and the antioxidant activities. The level of significance was set at p < 0.05. In the present study, B. racemosa from two different states, Kelantan on the east coast and Kedah on the UMI-77 west coast of Peninsular Malaysia were used for comparison purposes. In an attempt to find the optimal solvent for extraction of antioxidants from B. racemosa, four solvents of varying polarities were selected, namely water, ethanol, ethyl acetate and hexane ( Awah et al., 2010 and Khoo et al., 2008). The extraction yield and antioxidant components in the leaves and stems extracted with the four solvents are shown in Table 1. Among the different solvent extracts of the leaves collected from Kelantan and Kedah, the water extracts had the highest yield, followed, in descending
order, by the ethanol > ethyl acetate > hexane extracts. The aqueous extracts of plants are commonly shown to give higher yields than other solvent extracts ( Peschel et al., 2006). Extraction was performed at room temperature to prevent potential deterioration of antioxidant compounds as a result of heating or boiling ( Nurul Mariam et al., 2008). Polyphenol and ascorbic acid contents were highest in the water extracts, implying that most polyphenols in B. racemosa are Cell press polar. When the values were converted to freeze-dried weight, the polyphenol contents in the water extracts in this study (16.3–20.3 mg GAE/g freeze-dried tissue) were comparable to those of a previous study which reported that the methanol leaf extract of B. racemosa had 16.2 mg GAE/g of polyphenols ( Nurul Mariam et al., 2008). The polyphenol contents of the leaf water extracts of B. racemosa were comparable to, if not higher than several Chinese medicinal herbs (0.57–281 mg GAE/g of freeze-dried tissue) ( Liu et al., 2008) and Algerian medicinal plants (3.31–32.3 mg GAE/g of air-dried tissue) ( Djeridane et al., 2006).