高速逆流色谱法分离纯化楤木总皂苷中的楤木皂苷A及活性研究

目的:采用高速逆流色谱法(HSCCC)对楤木大孔树脂纯化所得总皂苷中的楤木皂苷A进行分离纯化并做体外抗血小板研究。方法:采用75%乙醇渗滤提取楤木原药材,渗滤液经过大孔树脂纯化,所得总皂苷粉末作为高速逆流色谱分离的样品。采用TBE-300B型高速逆流色谱仪,以氯仿∶甲醇∶正丁醇∶水(4∶4∶1∶3.5)为溶剂体系进行分离纯化,采用两种逆流色谱操作模式(0~130 min为正接正转,体系下相流动;130~180 min为正接反转,体系上相流动,流动相流速为5.0 mL·min-1,仪器转速为850 r·min-1,温度为25℃)进行分离。采用二磷酸腺苷(adenosine diphosphate,ADP)诱导小鼠体外血小板聚集,分别采用不同浓度HSCCC分离后的四号馏分处理,检测空白对照管与不同浓度的4号馏分给药管富血小板血浆在5 min内的最大聚集率,计算血小板聚集的抑制率。结果:楤木大孔树脂提取物经过HSCCC一步纯化,得到的楤木皂苷A纯度为95.81%;分离所得楤木皂苷A呈浓度依赖的抑制ADP诱导的血小板聚集,最大抑制率达80%。结论:利用大孔树脂富集总皂苷,HSCCC分离纯化总皂苷中的楤木皂苷A,为楤木皂苷A的分离纯化提供了一种新的简便、高效的途径。抗血小板活性研究表明HSCCC法分离得到的楤木皂苷A具有初步的抗血小板凝聚作用。

Separation and purification of Aralia saponin A from Aralia saponin by high speed countercurrent chromatography

OBJECTIVE To separate and purify Aralia saponin A from total aralosides of aralia elata seem by high-speed counter current chromatography (HSCCC) and study its activity.METHODS The 75% ethanol percolate of Root bark of Aralia was purified by macroporous resin, then filtrated and dried under vacuum.The vacuum drying of total saponin powder was used as the sample separated by HSCCC.Two-phase solvent system containing chloroform:methanol:n-butanol:water (4:4:1:3.5) was run on a preparative scale by TBE-300B HSCCC instrument, and two countercurrent chromatographic operating modes (0-130 min as the forward rotation, phase flow under the system; 130-180 min as the forward inversion, phase flow on the system, mobile phase flow of 5.0 mL·min^-1, instrument rotation speed of 850 r·min^-1, and temperature of 25℃) were used for separation. Adenosine diphosphate (ADP) was used to induce platelet aggregation in mice in vitro, and fraction 4 after separation with different concentrations of HSCCC was used to treat them, respectively. The maximum aggregation rate of platelet-rich plasma in blank control tube and fraction 4 administration tube with different concentrations within 5 min was detected, and the inhibition rate of platelet aggregation was calculated.RESULTS The purity of saponin A was 95.81% after one-step purification of the aralia elata macropora resin extract, and showed concentration-dependent inhibition of platelet aggregation induced by ADP, with the maximum inhibition rate of 80%.CONCLUSION Aralia saponin A in aralosides of aralia elata is separated and purified by HSCCC with macroporous resin, which provides a new simple and efficient way for the separation and purification of Aralia saponin A.The antiplatelet activity study showed that Aralia saponin A isolated by HSCCC method had a preliminary antiplatelet aggregation effect.