THE PROTECTIVE EFFECTS OF THE TOTAL SAPONIN OF DIPSACUS ASPEROIDES ON THE APOPTOSIS OF HIPPOCAMPAL NEURONS INDUCED BY β-AMYLOID PROTEIN

Objective To investigate the effects of the total saponin of Dipsacus asperoides (tSDA) and ginsenoside Rb1 (GRb1) on the apoptosis of primary cultured hippocampal neurons induced by β-amyloid protein (Aβ). Methods Primary cultured hippocampal neurons, the cultures were pretreated with tSDA and GRb1 on 10d for 24 hours respectively. Then the cultures were treated with 35 μmol·L -1 Aβ25-35 for 24 hours, observed the changing of survival rate of neurons and the apoptosis of neurons with biochemical analysis combining immunofluorescent cytochemical double-staining technique. Results Hippocampal neurons were treated with 35 μmol·L -1 Aβ for 24 hours, and survival rate of neurons downed to 52.6%. When neurons were pretreated by tSDA and GRb1, survival rate of neurons increased 11% to 15%. The findings of immunofluorescent cytochemical double-staining indicated that apoptotic neurons were obviously more than that of the blank group, reaching 43.9%.When neurons were pretreated by tSDA and GRb1, apoptotic neurons were downed to 16.6%, 10.8% respectively. Conclusion tSDA had the same effects as GRb1, protecting the neurons, antagonizing neurotoxicity of Aβ, increasing survival rate of neurons, and reducing apoptotic neurons induced by Aβ.

LC/MS/MS determination and pharmacokinetic studies of six compounds in rat plasma following oral administration of the single and combined extracts of Eucommia ulmoides and Dipsacus asperoides

AIM: To establish and apply a new LC/MS/MS method for the simultaneous, quantitative determination of six ingredients, aucubin(AU), geniposide(GP), geniposidic acid(GPA), pinoresinol diglucoside(PDG), secologanin(SLG), and loganin(LG) in single and combined extracts of Eucommia ulmoides and Dipsacus asperoides. METHOD: Using the LC/MS/MS-ESI--MRM mode to detect the six compounds, chromatographic separation was achieved on an Agilent Eclipse plus C18 column, and the mobile phase consisted of solvent A(CH3CN) and solvent B(H2O containing 0.01% CH3 COOH V/V). RESULTS: This method was successfully applied to quantify the six compounds in rat plasma after oral administration, and showed good precision, accuracy, reproducibility, and linear regression(r2 >0.99). CONCLUSION: The results showed that following the use of the two medicinal plants, for AU and GP, the values of tmax markedly increased, and the values of cmax markedly decreased. It was found that the compatibility of the medicinal plants might affect their pharmacokinetic properties of their constituents.

渝产续断多糖超声波辅助提取工艺的优化

为建立渝产续断(Dipsacus asperoids)多糖的最佳超声波辅助提取工艺,设计正交试验,以续断多糖的提取率为评价指标,对续断多糖的超声波辅助提取工艺参数进行考察。结果表明,最佳超声波辅助提取工艺为料液比1∶20(g∶m L),提取时间60 min,提取4次,测得续断多糖的平均提取率为7.472%,RSD为2.12%,表明续断多糖超声波辅助提取工艺稳定,质量可控。

川续断愈伤组织诱导及植株再生体系的建立

为解决川续断种苗快速繁殖的问题,需建立高效的组培苗再生体系。本研究利用植物组培技术,筛选川续断组培的最佳外植体,运用响应面优化法对川续断愈伤组织及丛生芽的诱导体系进行优化。结果显示,适合川续断组培的最佳外植体为幼苗叶片,最佳愈伤组织诱导培养基:MS+6-BA0.93 mg/L+NAA0.64 mg/L+KT 0.55 mg/L,诱导率98.62%,不同激素对愈伤组织诱导率大小的影响顺序:NAA>6-BA>KT,不同激素组合对愈伤组织诱导率大小的影响顺序为:NAA+KT>NAA+6-BA>6-BA+KT;丛生芽诱导的最佳培养基:MS+6-BA 0.76 mg/L+GA33.51 mg/L+IAA 0.93 mg/L,诱导率88.49%,不同激素对丛生芽诱导率大小的影响顺序为:GA3>6-BA>IAA,不同激素组合对丛生芽诱导率大小的影响顺序为:GA3+6-BA>GA3+IAA>6-BA+IAA;生根壮苗的最佳培养基:1/4MS+6-BA 0.1 mg/L+GA30.5 mg/L+0.5%活性炭,生根率81.92%。本研究建立的川续断组培苗高效再生体系,可为下一步研发川续断快繁优质种苗提供关键技术资料。

川续断皂苷合成路径关键基因的挖掘与生物信息学分析

川续断有滋补肝肾、强健筋骨、续接折伤、止崩漏的作用,主要有效成分为齐墩果烷型的三萜皂苷。为了探究川续断三萜皂苷生物合成途径及其关键酶的功能,本研究利用转录组测序对川续断三萜皂苷合成路径中的关键酶基因进行挖掘,并对相应核苷酸序列、氨基酸序列以及蛋白质结构特征等进行生物信息学分析。结果获得了鲨烯环氧酶3条候选基因、β-香树素合成酶1条候选基因、催化合成齐墩果酸的细胞色素氧化酶P4506条候选基因、催化合成常春藤皂苷元的细胞色素氧化酶P4508条候选基因以及催化合成崴岩仙皂苷A的糖基转移酶12条候选基因。本研究结果为川续断三萜皂苷合成通路的完全解析、川续断皂苷的生物合成以及川续断遗传改良和分子育种提供分子依据。