Sting for six min, 4 mL of 4 NaOH was added. Immediately after shaking properly
Sting for 6 min, four mL of four NaOH was added. After shaking nicely and resting for 12 min, absorbance was measured at 510 nm. Total flavonoid content material was calculated using calibration curve (Y = ten.859X – 0.0617, R2 = 0.999) of rutin normal (HPLC grade, 98 purity, Solarbio Life Sciences, Beijing, China). Total anthocyanins were extracted following the protocol earlier described by Kim and Lee [62]. The 0.2 g plant material was added into 10 mL of 1 hydrochloric acid methanol option and kept for five h. Just after centrifugation at 1000 rpm for 20 min, ten mL supernatant was applied to measure the OD value with the sample at 530 nm and 560 nm. Equation (1) was made use of to calculate total anthocyanins.Plants 2021, ten,13 ofTotal anthocyanins mg -1 =(OD530 – 0.25 OD650) volume of extraction liquid (mL) . 4.62 104 fresh weight of passion fruit (g)(1)Procyanidin content was determined employing the method of Hellstrom and Mattila [63], with slight modifications. The 0.five g sample was accurately weighed inside a 10 mL centrifuge tube, 6 mL methanol was added, and ultrasonic remedy (power = 250, yield = 50 kHz) was performed for 20 min. Following becoming placed at area temperature, the supernatant was centrifuged to measure the absorbance at 546 nm. The procyanidin content material was calculated making use of calibration curve (Y = 0.0038X + 0.0202, R2 = 0.999) of procyanidin normal (HPLC grade, 95 purity, Solarbio Life Sciences, Beijing, China). 4.3. Determination of Flavonoid and Anthocyanin Metabolites For sample preparation, the strategy earlier described by Henry-Kirk et al. [64] was applied with some modifications. The 1 g plant material was ground along with liquid nitrogen, and 5 mL of methanol/formic acid/water (80:1:19, v/v/v) was added. Ultrasonic extraction was performed at 45 C for 60 min, and centrifugation was performed at 12,000 rpm for 10 min, and also the supernatant was filtered via MFMilliporeTM Membrane Filter (Cat. No. GSWP04700, 0.22 pore size) into an Agilent sample bottle for testing. 5 typical flavonoids of rutin, quercetin, luteolin, apigenin, and kaempferol (98 purity, Solarbio Life Sciences, Beijing, China) were ready together with the concentration of 0.1 mg L-1 , and 3 standard anthocyanins of cyanidin-3-O-glucoside chloride (98 purity, Solarbio Life Sciences, Beijing, China), peonidin-3-O-glucoside (95 purity, Solarbio Life Sciences, Beijing, China), pelargonidin-3-O-glucoside (95 purity, Solarbio Life Sciences, Beijing, China) were prepared. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was performed with Waters I-CLASS /XEVO TQS liquid mass spectrometer (Waters Corporation, Milford, MA, USA) for evaluation. The determination was performed on the Agilent-ZORBAX SB-C18 column in the flow price of 0.three mL in-1 as well as the column temperature was 40 C. The flavonoid and anthocyanin components were detected at 210 nm. A Waters 2996 diode array detector (Waters Corporation, Milford, MA, USA) was utilised to detect the eluted peaks. The contents of Propargite Epigenetic Reader Domain individual flavonoid or anthocyanin metabolites were calculated making use of calibration curve in the corresponding standard. All measurements had been performed with 3 replicates. The validation parameters consisted of linearity variety, limits of detection, and quantification [65]. The peaks had been identified by their retention occasions, comparing the UV isible spectra and spiking with requirements. Quantification has been carried out working with an external regular curve with 5 points (Table 2).Table two. Validation parameters for.
