Dismic 25 jp membrane filter

An aliquot of 10 mg of the 70% ethanol extract of safflower seed was dissolved in 10 mL of 50% methanol by repeated vortexing, and passed through a Dismic-25 JP membrane filter (Advantec Toyo, Tokyo, Japan; pore diameter: 5 µm). Next, 5 µL of the sample was injected into a reverse-phase HPLC system with an INNO C18 column (4.6 × 250 mm, 5 µm pore size, YoungJin Biochrom, Korea) at 35°C. The mobile phase comprised (A) a 0.1% aqueous solution of formic acid and (B) acetonitrile, and the following gradient conditions were used: 0 min, 0% B; 5 min, 6% B; 10 min, 22% B; 50 min, 28% B; 66 min, 50% B; and 70 min, 100% B. The flow rate was 0.8 mL/min. The UV absorbance at 254 nm was monitored by using an Agilent 1200 series diode array detector (Agilent Technologies, Waldbronn, Germany). All peaks were assigned through co-injection tests with authentic samples and comparison of the UV spectral data; through this process, compounds including serotonin and its derivatives were detected from safflower seed extract. All measurements were repeated in triplicate. Representative HPLC results are illustrated in Figure 1. The quantification of serotonin, N-(p-coumaroyl)serotonin, N-feruloyl serotonin, luteolin, and luteolin-7-O-glucoside was achieved by peak area measurement. The quantity of each identified compound is shown in Table 1.