Quantitative Analysis of Ursolic Acid and Euscaphic Acid in Chaenomelis Fructus by HPLC-Evaporative Light Scattering Detection

Experimental Section

Materials and Reagents. Dried Chaenomelis Fructus samples from ten different habitats were obtained from herbal medicine markets in Daegu and Seoul, Korea. A voucher specimen was deposited at the Laboratory of Natural Products Medicine, College of Pharmacy, Kyungpook National University. Ethanol, dichloromethane, ethyl acetate, n-butanol, and methanol were obtained from Duksan Chemical (Anseong, Korea). Acetonitrile (HPLC grade) was purchased from J.T. Baker (Phillipsburg, NJ, USA). TFA was supplied from Sigma-Aldrich (St. Louis, MO, USA). Column chromatography was carried out using Kieselgel 60 (Merck, NJ, USA) and C-18 (Yamazen, Ultra Pack ODS-S-50B, Japan). 1H- and 13C-NMR analyses were performed with a Brucker Avance Digital 400 NMR spectrometer (Karlsruhe, Germany) at 400 and 100MHz, respectively. Chemical shifts were given in δ (ppm) from tetramethylsilane. Purified water was obtained by the Millipore-Q Water system (Millipore, Bedford, MA, USA). All other chemicals were of analytical grade and used as obtained commercially.

Isolation of Marker Compounds. Dried Chaenomelis Fructus (2.7 kg) was extracted with 95% ethanol for 3 h in three replicates. After filtration, ethanolic extract (195.0 g) was dispersed into 2 L of water and extracted by dichloromethane. The dichloromethane-soluble fraction (29.2 g) was loaded onto silica gel column chromatography (5.5 × 45 cm) using a mixed solvent of dichloromethane-acetone-methanol (200:1:1→1:1:1, gradient elution) to afford fractions of I-IV. Fraction II was precipitated using a mixed solvent of dichloromethane- methanol to yield ursolic acid as a white powder (1.02 g). Fraction IV (2 g) was chromatographed on reversed-phase C-18 (2.6 × 30 cm) with 60-100% methanol and the obtained subfraction IV-II (195 mg) was chromatographed on silica gel column chromatography (3.5 × 50 cm) using a mixed solvent of dichloromethane and acetone (30:1→6:1, gradient elution) to afford euscaphic acid (72.8 mg). The structures of ursolic acid and euscaphic acid were identified by 1H- and 13C-NMR (Table S1).

Preparation of Standard Solutions. Each standard of ursolic acid and euscaphic acid was accurately weighed, dissolved in methanol, and diluted to the appropriate concentration. Stock solution of standard containing ursolic acid or euscaphic acid at 1 mg/mL was prepared. A set of each standard solution was prepared by diluting the stock solution with methanol to concentrations ranging from 2 to 20 μg/ mL. All solutions were filtered through a 0.45-μm syringe filter and stored in the refrigerator at 4 ℃ before analysis.

Preparation of Samples. Each 50 g sample of dried Chaenomelis Fructus cultivated in ten different locations in Korea was refluxed with 100 mL of 95% ethanol for 3 h. After filtration through a 0.45-μm membrane filter, the filtrate was injected onto the HPLC for the quantitative determination of ursolic acid or euscaphic acid in the extracts.

HPLC Conditions. HPLC analysis was performed using a HPLC system consisting of Dionex Ultimate 3000 binary pump, automated sample injector, thermostatted column compartment, and Agilent 380-ELSD with Alltech Prosphere 300 C-4 column (250 × 4.6 mm id, 5 μm). The mobile phase was composed of deionized water containing 0.1% TFA (mobile phase A) and acetonitrile containing 0.1% TFA (mobile phase B). A linear gradient elution was performed from 40% to 70% B for 20 min at a flow rate of 1.0 mL/min. The evaporator temperature for the ELSD was set at 60 ℃ with the nebulizing gas flow-rate of 1.2 L/min. The sample injection volume was 20 μL.

Validation of HPLC Method. Calibration curves of ursolic acid and euscaphic acid were prepared with standards at concentrations ranging from 2 to 20 μg/mL. The regression equations were calculated in the form of y = ax + b, where y and x correspond to the peak area and concentration, respectively. The precision and accuracy were determined by analyzing each standard sample at a concentration of 10 μg/ mL. Intra-day precision was determined by repeating the analysis of each standard sample five times in a single day, and inter-day precision and accuracy were determined by repeating the analysis on three consecutive days. LOD and LOQ were defined as the minimum concentration at the signal-to-noise ratio (S/N) = 3 and 10, respectively.