2.2. Quantitative Analysis of Active Compounds in the Extracts

High-performance liquid chromatography (HPLC) was performed to quantify major curcuminoids, including curcumin (CUR), demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC), as well as their degradation products such as ferulic acid (FA), 4-vinylguaiacol (4VG), vanillin (VN) and vanillic acid (VA) in extracts. Briefly, extracts were filtrated using a Millipore filter (pore size 0.45 μm) prior to HPLC analysis. The HPLC system (Agilent 1260 Infinity II, Agilent Technologies, Santa Clara, CA, USA) was equipped with Zorbax SB-C18 column (4.6 × 250 mm, 5 μm) at a flow rate of 1.0 mL/min. The mobile phase consisted of distilled water containing 0.4% acetic acid (A) and acetonitrile (B) in the gradient system using the following conditions: 0–30 min, 8–91% B; 30–39 min, 91–100% B; 39–44 min, 100% B; 44–47 min, 100-8% B; 47-50 min, 8% B. Sample detection was acquired at 260 nm and the injection volume of samples was 10 μL. The seven reference compounds of the curcuminoids and their degradation products with above 98% purity or suitable for HPLC analysis were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All the compounds in the samples were identified by comparison of their retention time and spectra with the reference compounds. Quantification was performed using calibration curves of each reference substance.