Chemical Constituents From Rhizome Of Pulsatilla Koreana

Pulsatilla Koreana Naika belongs to the family Ranunculaceae. It is used as'Bai Tou Weng'in the northeast of china.'Bai Tou Weng'has been used to treat fungous infection by folks for a long time. Modern medicine study shows that many saponins from'Bai Tou Weng'have considerable antitumor activity. In our research, chemical constituents in pulsatilla Koreana Naika was extracted , isolated and identificated, to establish a foundation to find antifungous and antitumour drugs with high activity and low toxicity.Objective: chemical constituents in Pulsatilla koreana were extracted. Column chromatography (including silica gel, ODS, sephadex LH-20, macroporous resin etc.) and PHPLC were used to separate and purify the chemical constituents whose structures were elucidated by NMR (1D and 2D), ESI-MS,and hydrolysis methods.Methods: In our research, the rhizome of pulsatilla koreana was shattered. Then it was soaked and extracted with 70% alcohol. The extraction was condensed(d=1.18 g/ml,25℃) and evaporated to remove ethanol. Petroleum chloroform and acetoacetate was used individually to extract its constituents. Extracta sicca was obtained by reclaiming petroleum chloroform and acetoacetate respectively. the residual was diluted with purified water(1g crude herb in 5ml dilution). Macroporous resin AB-8 was used to adsorb the saponin in the dilution. It was eluted with 30%, 50%, 70% alcohol graduatedly after the saccharide adsorbed was washed out with Purified water. The elution was decoloured with macroporous resin D-280. Total saponin was obtained by reclaiming ethanol.TLC, column chromatography (gel silica, Sephadex-LH20, ODS) and PHPLC were used to separate the chemical constituents in total saponin and extractions of petroleum, chloroform and acetoacetate. In result, fiveteen compounds (I～ⅩⅤ) were isolated from the rhizome of pulsatilla koreana.Results: The structures of the constituents in pulsatilla koreana were elucidated by NMR(1D and 2D),ESI-MS,andhydrolysis methods. they are 3-O-β-D-glucopyrano syl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L- rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside(I), 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamno pyranosyl-(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl-hederagenin-28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside(II),3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl- (1→2)-α-L-arabinopyranosyl oleanolic acid-28-O-α-L-rhamno-pyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyrano-side(III),3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl -(1→2)-α-L-arabinopyranosyl hederagenin-28-O-α-L-rhamno- pyranosyl (1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyrano- side(IV), 3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyra-nosyl-(1→4)]-α-L-arabinopyranosyl oleanolic acid-28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside(V), 3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-α-L-arabinopyranosyl hederagenin-28- O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside(VI), 3-O-α-L-rhamnopyranosyl-(1→2)-α-L- arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)β-D-glucopyranoside(VII),3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosylhederagenin-28-O-α-L-rhamnopyranosyl-(1→4)β-D-gluco-pyranosyl-(1→6)β-D-glucopyranoside(VIII), oleanolic acid3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside(IX),hederagenin 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabino-pyranoside(X), hederagenin 3-O-α-L-arabinopyranoside(XI),β-sitosterol(XII),β-sitosterol-3-O-β-D-glucopyranoside(XIII),unsaturated fatty acid(XIV), oleanolic acid(XV).Conclusion: Fivteen constituents were isolated from pulsatilla koreana. Their structures were elucidated by the methods of spectroscopy and hydrolysis. Fourteen structures are established, the other is categoried to unsaturated fatty acid. Two compounds, 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl (1→2)-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl- (1→6)-β-D-glucopyranosylester(III) and hederagenin 3-O-α-L- arabinopyranoside (XI), were isolated from this plant for the first time.