Studies On Chemical Constituents And Bioactivities Of The Roots And Rhizomes Of Helleborus Thibetanus

The roots and rhizomes of Helleborus thibetanus Franch., locally known as'XiaoTaoErQi', is endemic to China and used for treatment of cystitis, urethritis, sores and traumatic injury in Chinese folk medicine. So far, there have been few reports on the chemical studies of H. thibetanus, and the chemical and active principles are not clear. Therefore, we have conducted studies on the chemical constituents of the title plant.Thirty-one compounds were isolated from the ethanol extract of the roots of H. thibetanus employing solvent extraction, various column chromatography (including silica gel, polyamide, Sephadex LH-20 and D101 macroporous resin), preparative high performance liquid chromatography (pre-HPLC), solvent washing and recrystallization. Two aglycones were obtained through enzymatic hydrolysis. On the basis of chemical and spectral methods including NMR, MS, IR, UV, ICP-MS, hydrolysis, and comparison with authentic samples, the structures of twenty-eight compounds were identified as sitosterol (HTP-C1), daucosterol (HTE-C1), deglucohellebrin (HTE-C2), polypodine B (HTE-C3), 20-hydroxyecdysone (HTE-C4), 14β,16β-dihydroxy-3β-[(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (HTE-C5), 14β-hydroxy-3β-[β-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (HTB50-C1), pregna-5,16-diene-20-one-3β-yl O-α-L-rhamnopyranosyl- (1→2)-O-[β-D-glucopyranosyl-(1→4)]-β-D-galactopyranoside(HTB50-C2), (23S,24S) -24-[(O-β-D-fucopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-diene-1β-yl O-(4- O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopy-ranoside (HTB50-C3), (25R)-26-[(O-β-D-glucopyranosyl)oxy]-furosta-5,20(22)- diene-3β-yl O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→4)]-β-D- galactopyranoside (HTB50-C5), 5β,14β-dihydroxy-19-oxo-3β-[β-D-glucopyranosyl)- oxy]bufa-20,22-dienolide (HTB30-C1), 5β,14β,16β-trihydroxy-19-oxo-3β-[(β-D- rhamnopyranosyl)oxy]bufa-20,22-dienolide (HTB30-C2), hellebrin (HTB30-C3), (25S)-22α,25-expoxy-26-[(O-β-D-glucopyranosyl)oxy]-3β-hydroxyfurosta-5-ene-1β- yl O-α-L-arabinopyranoside (HTB30-C4), 26-O-β-D-glucopyranosyl-3β-hydroxy-22α- methoxyfurosta-5,25(27)-diene-1β-yl sulfonate (HTB30-C5), adenoside (HTB30-C6), 3β-hydroxyspirost-5,25(27)-diene-1β-yl sulfonate (HTB30-C7), 3β-hydroxypregna- 5,16-diene-20-one-1β-yl sulfonate (HTB30-C8), 5β,14β,16β-trihydroxy-19-oxo-3β- [(β-D-glucopyranosyl)oxy]bufa-20,22-dienolide (HTB30-C9), 14β,16β-dihydroxy-3β- [β-D-glucopyranosyl-(1→4)-(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (HTB30-C11), (23S,24S)-24-{[O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl]oxy} -3β,23-dihydroxyspirosta-5,25(27)-diene-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)- (1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside (HTB30-C12a), (23S, 24S)-24-{[O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl]oxy}-3β,23-dihydroxy- spirosta-5,25(27)-diene-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamn- opyranosyl)-(1→2)-O-α-L-arabinopyranoside (HTB30-C12b), (23S,24S)-24-{[O-β-D- glucopyranosyl-(1→4)-β-D-fucopyranosyl]oxy}-3β,23-dihydroxyspirosta-5,25(27)-di-ene-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)- O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside (HTB30-C13a), (23S)-26-O-β-D-glucopyranosyl-3β,23-dihydroxyfurosta-5,20(22),25(27)-triene-1β-yl sulfonate (HTB30-C14), glucose (HTB30-C15), (23S,24S)-24-[(O-β-D-glucopyranosyl)oxy]- 3β,23-dihydroxyspirosta-5,25(27)-diene-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)- (1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside (HTB30-C17), (23S,24S) -21-hydroxymethyl-24-{[O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl]oxy}-3β, 23-dihydroxyspirosta-5,25(27)-diene-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O- acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyra-noside (HTB30-C19), 3β,14β,16β-trihydroxy-5α-bufa-20,22-dienolide (HTE-C5-S1) and 3β,14β-dihydroxy- 5α-bufa-20,22-dienolide (HTB50-C1-S1).Fourteen new compounds were obtained from the title plant. Among these new compounds, HTE-C5, HTE-C5-S1, HTB50-C1 and HTB30-C11 are 5α-bufadienolides, HTB50-C2 and HTB30-C8 are pregnanes, HTB30-C7, HTB30-C12a, HTB30-C12b, HTB30-C13a, HTB30-C17 and HTB30-C19 are spirostanes, HTB30-C5 and HTB30-C14 are furostanes.Effects on catecholamine secretion in cultured bovine adrenal medullary cells were conducted on some isolated compounds. HTE-C2 and HTE-C5 enhanced catecholamine secretion, and these results indicated that the two compounds show excitability effects on sympathetic activity.The studies on chemical components and bioactivities of the Helleborus genus in the past years were also summarized.