Hypersil ods c18

The HPLC analysis was performed on an Agilent 1200 system equipped with a G1311A QuatPump (Agilent Technologies, Inc., Santa Clara, CA, USA), a G1322A degasser, a G1315D diode array detector (DAD), and a G1329A ALS with a 20 μL loop. An Hypersil ODS-C18 (250 mm × 4.6 mm i.d., 5 μm, Agilent Technologies, Inc., Santa Clara, CA, USA) column was used. The mobile phase (phase A) was 0.1% formic acid solution (v/v)—phase B was methanol. A gradient program was used as follows: 0–10 min, 20–30% B; 10–15 min, 30% B; 15–35 min, 30–95% B; 35–45 min, 95% B. The flow rate was 0.8 mL/min, and the injection volume was 10 μL. The chromatograms were recorded at 254 nm.
In addition, stock standard solutions of PUE, 3-MPR and PRX were prepared by dissolving in the analytical grade methanol for quantification.

HPLC analysis was performed on an Agilent 1200 system including a G1311A QuatPump, a G1322A degasser, a G1315D diode array detector (DAD), a G1329A ALS with a 20 μL loop. In addition, the HPLC column used was Hypersil ODS-C18 (250mm × 4.6mm i.d., 5μm, Agilent). The mobile phase consisted of water containing 0.1% formic acid (phase A) and acetonitrile (phase B). The gradient program was as follows: 0–12 min, 10–22% B, 12–38 min, 22–30% B, 38–50 min, 30–45% B, 50–55 min, 45–90% B, 55–65min, 90% B. The flow rate was 0.8 mL/min, and the wavelengths were 254 nm (for TIIA and CYT) and 280 nm (for SAB).
Calibration curves were established for SAB, TIIA and CYT by plotting the nominal concentrations of standard solutions versus peak areas. The linear ranges, linear regression equations and related details are listed in Table S1 in Supporting Information.

The amount of corosolic acid from L. speciosa (1 mg, Sigma Aldrich) was weighed and dissolved in 1 mL of ethanol for standard solution. Contents of corosolic acid from crude extracts were determined by HPLC, using Agilent Technologies 1260 Infinity, compared to the standard. The column Hypersil ODS C18, 4.0 × 250 mm, 5 Micron (Agilent) was used. The detection wavelength was 210 nm. The mobile phase consisted of two solvents: 0.1% phosphoric acid (A) and acetonitrile (B). The gradient elution was carried out by acetonitrile 55% to 100% (0–35 min). The flow rate was 1 mL/min, and 10 μL of the sample was injected.