2.4.5. Phenolic Content and Composition
The total phenolic content was determined using Folin Ciocalteu method (Lamuela-Raventós, 2017). The calibration curve was plotted by mixing aliquots (1 mL) of Gallic acid solutions over a range of concentrations (800, 400, 200, 100, 50, 25, 12.5 and 6.25 μg/mL) with Folin Ciocalteu reagent (5 mL) and anhydrous sodium carbonate solution (4 mL). The phenolic content of the oleoresin was measured by mixing the sample (2.5 mL) and Folin Ciocalteu- anhydrous sodium carbonate reagents at the 1:1 (v/v) ratio. The solution was kept for 1 h at 25oC. The absorbance was measured at 725 nm using a spectrophotometer. Total phenolic content was expressed as mg of gallic acid equivalents (GAE) per gram of oleoresin (mg GAE/g oleoresin) using Equation 3:
C = C1 x \(\frac{V}{m}\) (3)
where C is the total phenolic content (mg/g) in GAE, C1is the concentration of gallic acid established from the calibration curve in mg/mL, V and m are the volume and weight of oleoresin, respectively.
The phenolic compounds were determined using the protocol described by (Tohma et al., 2017). The samples were prepared by mixing oleoresin (20 mg) with a mixture (2mL) of methanol:ethanol (90:10 v/v) in a glass tube with screw top (15 mL) and shaken in a wrist shaker for 1 h. Then, the solution was filtered with a PTFE filter (0.45 μm) and transferred to a 1 mL polypropylene vial (12 mm x 32 mm). The analysis was conducted using an HPLC system (Agilent Technologies, 1100 series, Santa Clara, CA, USA) equipped with a C18 column (TOSOH Bioscience LC, TSKgel ods-80, 4.6 mm ID×25 cm, 5μm Millipore Sigma, MA, USA) and a diode-array detector (DAD) (Hewlett-Packard, Palo Alto, CA, USA) operated at 260-430 nm. The mobile phase (elution rate of 1.0 ml/min) was composed of acetonitrile (solvent A), methanol (solvent B) and water with acetic acid (solvent C) with a gradient system as follows: 0-5 min 5-8% A, 5-8% B and 90-84% C; 5-15 min 8-10% A, 8-10% B and 84-80% C; 15-25 min 10-25% A, 10-0% B and 80-75% C; 25-35 min 25-30% A, 75-70% C; 35-45 min 30-60% A and 70-40% C. The injection volume was 20 μL and the column temperature was 30oC. Each phenolic compound was identified by their retention time in comparison with standard compounds. The peak areas were quantified using Chem Station software as areas under the curve and calibration curve prepared by their analytical standards. The concentration of each phenolic compound in oleoresin samples was determined using Equation 4:
Concentration of compound =\(\frac{\text{Area\ of\ sample}}{\text{Area\ of\ standard}}\) x Concentration of standard (4)