Prep degasser

Preparative HPLC was generally carried out using Quaternary Gradient Module 2545, Photodiode Array Detector 2998, Autosampler 2707, Waters Prep Degasser and Waters Fraction Collector III. Software: Waters ChromScope v1.40 Beta (Waters, Milford, MA, USA). Stationary phase: Nucleodur^® C18 HTec, 5 µm, 250 × 21 mm, mobile phase: binary gradient of water (A) and acetonitrile (B) at a flow rate of approx. 15.5 mL/min.
Separation of fraction SE6 yielded 1 (118 mg) and 2 (1.2 mg). 3 (11 mg) was obtained from SE7, 4 (5.3 mg), 5 (13 mg) and 6 (0.65 mg) from fraction SE8. Subfractionation of MPLC fraction M2 and M3 led to 15 (39 mg). 16 (0.5 mg) was obtained from M4, and M5 yielded 17 (0.6 mg), 18 (0.9 mg) and 19 (0.3 mg).
Fractions N3 to N5 obtained from FCPC and SE3 were further purified using the following system: Two Waters 515 HPLC Pumps, Waters Pump Control Module II, Degasys DG-2410 (Uniflows, Tokyo, Japan), Waters 996 PDA Detector, software: Waters ChromScope v1.40 Beta Software (Waters, Milford, MA, USA), stationary phase: Eurospher 100 C18, 250 × 21 mm; 7 µm (VDS Optilab, Germany), mobile phase gradient: water (A), acetonitrile (B), flow: 10 mL/min, injection volume: 1 mL. 7 (0.12 mg), 8 (0.9 mg) and 9 (1.7 mg) were isolated from N3, N5 and N4 resp. Compound 14 (11 mg) was isolated from SE3 along with 10 (3.9 mg), 11 and 12 as a mixture (4.3 mg) and 13 (2.7 mg).

The stock solutions of 80% ethanol extracts of G. procumbens (GP1, GP2, and GP3) were each prepared in methanol at 20 mg/ml, and the reference standard, chlorogenic acid solution, was prepared at 1 mg/ml. Each stock solution was then diluted (two-fold dilution) into a series of concentrations. The diluted solutions of the extracts and the reference standards was analyzed separately by HPLC using the following conditions: analytical column XBridge™ C18 (4.6 × 250 mm, 5 mm) was used on Waters 2535 Quartenary Gradient Module with a photodiode array detector (Waters 2998) of wavelength ranging from 210 to 350 nm and Waters Prep Degasser. The mobile phase consisted of acetonitrile (A) and 0.1% orthophosphoric acid (B), with a stepwise gradient system (10% A–90% B at 0 min, 30% A–70% B at 30 min, 100% A at 40 min, and 100% A at 70 min) with a flow rate of 1 ml/min, and injection volume was 10 μL. The identification and quantification of chlorogenic acid in the extracts was performed by comparing the retention time and UV-Vis spectra of the sample peaks with the standard peak (chlorogenic acid) and by plotting a calibration curve of chlorogenic acid, respectively.