Online degasser

Composition of TFCH was analyzed by High-performance liquid chromatography (HPLC) system, mainly including quaternary gradient pump, online degasser, UV detector, and column thermostat was from Shimadzu Corporation. Chromatographic separation was attained on a Hypersil SB C18 column (Thermo Fisher Scientific, MA, USA) at 25°C. The mobile phase consisted of water-acetonitrile and is eluted with a gradient method (elution gradient: 0–15 min, 20% acetonitrile; 15–35 min, 60%–100% acetonitrile; 42–45 min, 100%–20% acetonitrile; 45–50 min, 20% acetonitrile). The flow rate of mobile phase is 1.0 ml/min, wavelength was 283 nm, and samples injection volume was 10 μl. The flavonoids in the samples were depending on the chromatographic peaks of the standard substances of composition of TFCH (neohesperidin, naringin, narirutin, and hesperidin).
The content of total flavonoids of TFCH was referred to previous method. Briefly, each flavonoid extract was dissolved in methanol (1:1, w/v) and consecutively added with 0.5 ml of 10% Al (NO₃)₃ solution, handed with 0.5 ml of 5% NaNO2 solution. Then, the absorbance of the samples was measured at 0, 5, and 10 min (wavelength was 510 nm). The content of total flavonoids substance in TFCH was calculated using a standard curve.