Separation Of Ganoderic Acids From Ganoderma Lucidum By Preparative High-Performance Liquid Chromatography

This study separated and identified three major ganoderic acids from Ganoderma lucidum fruiting bodies by using Preparative High-performance Liquid Chromatography (Prep-HPLC).First, the crude extracts of ganoderic acids were separated from fruiting bodies of Ganoderma lucidum and purified on a silica gel column in advance to HPLC separation. By using the mobile phase which consisted of n-hexane-dichloromethane–chloroform-methanol(8:6:4:3) and further crystallization, three fractions named as F1, F2 and F3 were obtained.For HPLC separation, the conditions were studied on an analytical column ( SinoChrom ODS-BP, 5μm, 250mm×4.6mm). The temperature was set at 22℃, flow rate was 0.5 mL·min-1, and peaks were detected at 254nm. By manipulating mobile phase compositions, sample volumes and concentrations, and evaluating retention time and separation resolution as indexes, an optimized condition was found as methanol: water: acetic acid = 60:40:0.2, and 10 mL sample volume and 35 mg·mL-1 concentration, respectively.The conditions of flow rate and sample volume were linearly amplified to 70 times according to the ratio of the size of analytical and preparative HPLC column (300mm×40.0 mm, 15μm). The three fractions were purified at such conditions and the purified F1 and F3 were further recrystallized in methanol. After that their purity was detected as 93.3% (F1), 95.9% (F2) and 97.5% (F3) respectively and total recovery was 81.85%.Structural analysis showed that these three ganoderic acids all had a maximum absorbtion at 247nm. FT-IR results showed that they all had common absorption peaks of hydroxyl groups(3400 cm-1),carboxyl groups(3000 cm-1)and carbonyl groups(1710cm-1). The structure of F1 also had a penta-cyclic-ketone(1740cm-1) andα,β-unsaturated ketone (1650 cm-1). F2 and F3 all had a structure of hexa-cyclic-ketone (1708cm-1and 1666cm-1)MS analysis demonstrated that the molecular weights of the three compunds were 532D (F1), 514D (F2) and 516D (F3), respectively.The displacement values from 13C-NMR analysis revealed that F1 had three carbonyl groups (199.2ppm,217.6 ppm and 209.5 ppm), one carboxyl group (178.9 ppm), two carbons with double bonds (142.5 ppm and 157.0 ppm) and three hydroxyl groups (66.7 ppm,72.4 ppm and 77.6 ppm).F2 had three carbonyl groups (198.93 ppm,198.82 and 216.96 ppm), one carboxyl group (180.08 ppm), four double bond carbons (140.35 ppm,124.17ppm,157.13 ppm and 158.77 ppm) and two hydroxyl groups (68.95 ppm and 72.72ppm).F3 had three carbonyl groups (199.7ppm,208.8 ppm and 210.0ppm), one carboxyl group( 177.5ppm),two double bond carbons (161.8 ppm and 139.8ppm) and two hydroxyl groups (68.0 ppm and 71.5ppm). Based on these information, the structures of the compounds are identified as F1: ganoderic acid G (C30H44O8), F2: ganoderinic acid A (C30H42O7) and F3: ganoderic acid A(C30H44O7). This provides a theoretical base and practical information for ganoderic acids separation, purification and the standards preparation.