The PTFC composition was analyzed by high-performance liquid chromatography (HPLC) using a system that included a quaternary gradient pump, an online degasser, a UV detector, and a column thermostat produced by Shimadzu Corporation. Chromatographic separation was performed at 25°C on a Hypersil SB C18 column (Thermo Fisher Scientific, MA, United States). The mobile phase was water-acetonitrile and the samples were eluted using a gradient method consisting of the following 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 the mobile phase was 1.0 ml/min, the wavelength was 283 nm, and the injection volume was 10 μl. The flavonoids in the samples were identified based on the chromatographic peaks of the standard substances that constitute PTFC (neohesperidin, naringin, narirutin, and hesperidin).
The total flavonoid content of PTFC was determined as previously described (Jiang et al., 2019 (link)). Briefly, each flavonoid extract was dissolved in methanol (1:1, w/v). Then, 0.5 ml of 10% Al(NO3)3 solution was added followed by 0.5 ml of 5% NaNO2 solution. The absorbance of the samples was measured at 0, 5, and 10 min at a wavelength of 510 nm. The total flavonoid content in the PTFC was calculated using a standard curve.
The total flavonoid content of PTFC was determined as previously described (Jiang et al., 2019 (link)). Briefly, each flavonoid extract was dissolved in methanol (1:1, w/v). Then, 0.5 ml of 10% Al(NO3)3 solution was added followed by 0.5 ml of 5% NaNO2 solution. The absorbance of the samples was measured at 0, 5, and 10 min at a wavelength of 510 nm. The total flavonoid content in the PTFC was calculated using a standard curve.