Determination of antioxidant activity

The DPPH assay was carried out according to the method described by Wang et al. (2014). DPPH solution (6 M×10-5) in methanol was prepared and 2.7 ml of the solution was mixed with 300 lμ of extract. The solution was mixed vigorously, left in dark for one hour at room temperature and measured at 517 nm by using UV spectrophotometer (Lamda 35, Perkin Elmer, USA). Methanol was used as a blank, while 80% methanol with DPPH solution was used as the negative control. The butylated hydroxyanisole (BHA) was used as the positive control for comparison. The inhibitory percentage of the extract was calculated according to the following equation:

The concentration of extract providing 50% of radical scavenging activity (EC₅₀) was calculated from the linear regression of radical scavenging activity (RSA) against the extract concentration. A synthetic antioxidant Butylated hydroxyanisole (BHA) was used as reference standard for comparison.

For FRAP assay, the antioxidant capacity of the extracts was determined by the method developed by Xiao et al. (2015). Freshly prepared FRAP reagent was warmed at 37 °C in a water bath and the blank reading was taken at 593 nm using UV spectrophotometer (Lamda 35, Perkin Elmer, USA). One hundred microliter of sample extract was added with 300 lμ of distilled water and 3 ml FRAP reagent. The absorbance was measured after 30 min left in the dark. The absorbance of the reaction mixture was measured against initial blank reading and compared with the standard curve prepared by using several concentrations ranging from 0 to 2.4 mM of Fe (II) concentration. Results were expressed as m M Fe (II) equivalents/g dried weight (DW) extract.

The method for determination of ABTS radical cation assay was carried out according to the method described by Re et al., (1999) and adopted by Kong et al. (2013). ABTS salt with the concentration of 7 mM was dissolved in distilled water and mixed with 2.45 mM potassium persulfate. The reaction mixture was incubated at room temperature for 12 – 16 h in dark to generate free radicals. Thirty microliter of sample extract was mixed with 3 ml of diluted ABTS solution until the absorbance reached 0.70 ± 0.02. The absorbance was read at 734 nm after 6 min of reaction. Distilled water was used as the blank. Percentage of antioxidant capacity was calculated according to the formula below:

where AABTS was the absorbance of ABTS radical cation without sample or standard; Asampleofstandard was the absorbance of ABTS radical cation with sample or standard.

ORAC assay was used to measure the antioxidant scavenging activity against peroxyl radical generated by thermal decomposition of 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH) (Liu et al. 2016). AAPH solution, fluorescein and Trolox were prepared in a phosphate buffer (75 mmol/l, pH 7.4). The total mixture would be 20 lμ of AAPH, 170 lμ Fluorescein and 10 lμ of sample. Firstly, fluorescein and sample were mixed and incubated at 37 °C for 20 min in a microplate reader (Fluoroskan Ascent Microplate Fluorometer) and AAPH was added. After 30 s of AAPH added, the readings were taken at every cycle of 1 min in shaking mode using microplate reader. Ten microlitre of phosphate buffer without extract was used as blank. The antioxidant activity is expressed in micromole Trolox equivalents per gram of dry weight (DW) extract.

The total phenolic content in the extract was determined by using Folin-Ciocalteu assay reported by Deʇirmencioʇlu et al. (2016) with slight modifications. The sample extract (500 lμ) was mixed with 2.50 ml of freshly prepared Folin – Ciocalteu reagent, which had been previously diluted with distilled water (1:10 v/v). The mixture was incubated under room temperature for four minutes prior to its use. Then, 2 ml of 75 g/l sodium carbonate was added into the mixture before incubated at room temperature for 120 min. The absorbance of the mixture was measured at 760 nm by using UV spectrophotometer (Lamda 35, Perkin Elmer, USA).

Quantification of the phenolic content was based on the standard curve established (range of 0.05 – 0.30 mg/ml) and the content was determined as microgram of gallic acid equivalent (GAE) per g sample.

Phenolic compound standards were prepared by dissolving them with methanol into 10–100 μg/g. The standards used included gallic acid, ( +)-catechin, chlorogenic acid, ρ-coumaric acid, ferulic acid, vanillin, rutin, kaempferol, quercetin and mangiferin. On the other hand, the extracts were dissolved by methanol and filtered through 0.22 μm nylon membrane filter (Whatman, UK).

High Performance Liquid Chromatography (HPLC) was used to determine the phenolic compounds present in the sample extract according to the method described by Hassan et al., (2011) with slight modifications. Twenty microlitre of extract was injected automatically into Agilent 1200 series HPLC system equipped with a diode array detector (DAD). A 150 × 4.6 mm, 4 μm particle size reversed phase column (Phenomenex, USA) was used in this analysis. The column thermostat had been set at 25 °C. The mobile phase comprises of mixture of water/acetic acid (99.5:0.5 v/v) for solvent A and 100% of methanol for solvent B at a flow rate of 0.6 ml/min and measured at wavelength of 265 nm with a gradient elution program within 35 min of run time. The gradient elution began at 100% phase A, linearly decrease to 10% in 30 min. In the next 5 min, phase A increased to 100%. Quantification of phenolic compounds was determined by comparing the retention times and absorbance recorded in the chromatograms with the reference standards from the calibration curves constructed with the known standards. The amount of phenolic compounds was calculated and expressed as microgram per gram of dry weight (μ g/g DW).

Statistical Package for Social Science (SPSS) version 21.0 was used for data analysis. All of the triplicate results were expressed as mean ± standard deviation (SD). A significant difference between the variables was determined by one-way analysis of variance (ANOVA) with post hoc Tukey’s test at p < 0.05.