Extraction of anthocyanidins was performed as previously described with a slight modification [28 (link)]. The fresh petals were first freeze-dried and ground into fine powder. Then, the resulted flour was macerated with methanol:water:formic acid:TFA (70:27:2:1, v/v) and left to rest in the dark at 4 °C for 24 h. The extracted fluid was filtered with 0.22 μm membrane filter and used in subsequent experiments. Hydrolysis analysis of anthocyanins was performed according to Huang [29 (link)] and Morit [30 (link)]. An aliquot of 300 μL sample concentrations mentioned in HPLC analyses was transferred to a fresh tube, acid-hydrolyzed by adding 300 µL of 6 M HCL, incubated at 90 °C for 1 h. Hydrolyzation solution was immediately cooled to room temperature, and then was filtered prior to injection for analysis.
HPLC analysis used a Waters 600 series high-performance liquid chromatograph (Waters, Milford, MA, USA), a Waters 2487 UV detector (Waters, Milford, MA, USA), an Azbil ADC15 column oven (Azbil, Sanbu, Japan), a Rheodyne 7725i manual injector (Rheodyne of IDEX, Chicago, IL, USA), and a Waters Empower Build 1154-C software (Waters, Milford, MA, USA). An aliquot of 300 µL sample concentrations mentioned in HPLC analyses was transferred to a fresh tube, acid-hydrolyzed by adding 300 μL of 6 M HCL, incubated at 90 °C for 1 h. Hydrolyzation solution was immediately cooled to room temperature, and then was filtered through a 0.22 μm filter membrane prior to injection for analysis. The chromatographic column was a C18 TSK gel ODS-80Ts QA (250 mm × 4.6 mm i.d., 5 μm) (Tosoh, Yokkaichi, Japan), with a flow rate of 0.8 mL/min and injection amount of 10 μL. Mobile phase A employed a 0.1% formic acid solution and phase B an 80% acetonitrile solution. The elution programme was as follows: 0 min, 88% A, 12% B; 15 min, 75% A, 25% B; 32 min, 62% A, 38% B; 40 min, 62% A, 38% B; 45 min, 88% A, 12% B; 50 min, 88% A, 12% B. The detection wavelength was 530 nm. Results of HPLC were verified by at least three independent experiments. Cy, Dp, delphinidin-3-O-glucoside (Dp3G), malvidin-3-O-glucoside (Mv3G), Pg, petunidin-3-O-glucoside (Pt3G) were purchased from ChromaDex (Santa Ana, CA, USA). The concentration of anthocyanidins was quantified by external reference methods using Dp-chloride as standards.
HPLC analysis used a Waters 600 series high-performance liquid chromatograph (Waters, Milford, MA, USA), a Waters 2487 UV detector (Waters, Milford, MA, USA), an Azbil ADC15 column oven (Azbil, Sanbu, Japan), a Rheodyne 7725i manual injector (Rheodyne of IDEX, Chicago, IL, USA), and a Waters Empower Build 1154-C software (Waters, Milford, MA, USA). An aliquot of 300 µL sample concentrations mentioned in HPLC analyses was transferred to a fresh tube, acid-hydrolyzed by adding 300 μL of 6 M HCL, incubated at 90 °C for 1 h. Hydrolyzation solution was immediately cooled to room temperature, and then was filtered through a 0.22 μm filter membrane prior to injection for analysis. The chromatographic column was a C18 TSK gel ODS-80Ts QA (250 mm × 4.6 mm i.d., 5 μm) (Tosoh, Yokkaichi, Japan), with a flow rate of 0.8 mL/min and injection amount of 10 μL. Mobile phase A employed a 0.1% formic acid solution and phase B an 80% acetonitrile solution. The elution programme was as follows: 0 min, 88% A, 12% B; 15 min, 75% A, 25% B; 32 min, 62% A, 38% B; 40 min, 62% A, 38% B; 45 min, 88% A, 12% B; 50 min, 88% A, 12% B. The detection wavelength was 530 nm. Results of HPLC were verified by at least three independent experiments. Cy, Dp, delphinidin-3-O-glucoside (Dp3G), malvidin-3-O-glucoside (Mv3G), Pg, petunidin-3-O-glucoside (Pt3G) were purchased from ChromaDex (Santa Ana, CA, USA). The concentration of anthocyanidins was quantified by external reference methods using Dp-chloride as standards.
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