The Forsythia Chemical Separation Under The Guidance Of Chemical Fingerprint Analysis

Object:To investigate the chemical constitute of Forsythia suspense Vahl, high performance separation method directed by fingerprint chromatogram analysis was developed, series purity compounds, including pharmaceutical components, was separated with this method.Method:(1) The Ultra-pressure liquid chromatography-flight of time mass spectrum (UPLC-Q-Tof MS) analysis method was established. The crude extract of Forsythia suspense Vahl was analyzed, the main compounds was baseline separated and compounds was identified basing on the high resolution mass spectrum.(2) Forsythia suspense Vahl crude plant was extracted with 70% ethanol, crude extract was sequentially fractioned with ether ester and n-butanol, two fractions was obtained after vacuum evaporated. The butanol fraction was chromatographed with macroporous adsorption column and different concentrate ethanol eluted the column, 20%,40% and 60% ethanol was collected. High performance liquid chromatography method was developed and was used to analyze these three eluting fractions. Basing the HPLC analysis result, preparative method was optimized by analytical HPLC and three fractions were separated on HPLC preparative system. C18 column (20X250mm) was used and methanol-water was mobile phase, Ultraviolet detector was monitored at 220nm.(3) High resolution mass directed natural product chemical separation method was developed. The UPLC-Q-Tof provided the high efficiency separation ability and high resolution construct information. extract of Forsythia suspense Vahl was separated with UPLC and the mass spectrum of well separated compound was analyzed. The molecular formula was deduced from accurate molecular weight which was ensured basing on quasi-molecular ion and adducted ions information. By screening the library and literature, the chemical which had not been reported in Forsythia suspense Vahl was chosen as potential new compound and special separation process was developed.Results(1) Fingerprint chromatogram of Forsythia suspense Vahln was analysis with UPLC-Q-ToF method. 22 compound were baseline separated and their chemical construct was deduced basing on quasi-molecular ion information.(2) The butanol fraction was chromatography separated and three small fractions were obtained. HPLC analysis indicated pharmaceutical standard substance forsythoside A and phillyrinl were efficiently enriched in 40% and 60% ethanol elution fraction, respectively. By using preparative HPLC, rengyoside B, cornoside, were separated from 20% elution fraction. Acteoside, forsythoside A and forsythoside H were separated from 40% elution fraction. From the 60% elution fraction, (+)-pinoresinol-β-D-glueoside, (+)-epipinoresinol-D-glucoside,phillyrinl,phillygenin,pinoresinol,pinoresinol.monom ethyl ether, epipinoresinol, oleanolic acid, ursolic acid, sitosterol were obtained.(3) With the high resolution mass spectrum data analysis, two potential new compounds was chosen as target chemicals and separated by preparative HPLC method. 4-[2-(P-D-glucopyranosyloxy)ethyl]-4-hydroxy-2-cyclohexen-1-one and 4-[2-(β-D-glucopyranosyloxy) ethyl]-4,5-dihydroxy-2-cyclohexen-1-one were first time separated from Forsythia suspense Vahln.