知母皂苷抑制血小板聚集的活性成分筛选及构效关系分析

Screening of active ingredients of timosaponin for anti-platelet aggregation,and analysis of structure-activity relationship

目的筛选从知母中分离的甾体皂苷抑制血小板聚集的活性成分并对其进行构效关系分析。方法雄性Wistar大鼠心脏取血,离心分离获得富血小板血浆,分别加入呋甾烷型或螺甾烷型甾体皂苷化合物孵育3min后,以二磷酸腺苷(ADP)诱导大鼠血小板聚集,采用比浊法检测血小板聚集情况,通过比较不同甾体皂苷化合物对ADP诱导血小板聚集的抑制作用分析化合物的构效关系。结果 8个呋甾烷型甾体皂苷均无抑制血小板聚集的活性;4个螺甾烷型甾体皂苷在不同程度上抑制了血小板聚集,其中知母皂苷AⅢ作用最强,随后依次是知母皂苷AⅠ、知母皂苷AⅢ异构体和知母皂苷Ⅲ,而萨尔萨皂苷元没有活性;在苷元结构C-3位连接不同类型糖基的甾体皂苷抑制血小板聚集的作用不同,其中C-3位糖基为葡萄糖(D-Glc)时能完全抑制血小板聚集,而连接其他糖基如半乳糖(D-Gal)、阿拉伯糖(D-Ara)、核糖(D-Rib)、鼠李糖(L-Rha)或甘露糖(D-Man)时,抑制活性有不同程度的减弱。结论知母皂苷AⅢ是知母抑制血小板聚集的主要有效活性成分之一;甾体皂苷抑制血小板聚集的活性与其结构密切相关;第31、52、2位上的基团类型可影响甾体皂苷抑制血小板聚集的生物活性。

Objective To screen the active anti-platelet aggregation ingredients of steroidal saponins purified from Rhizoma anemarrhenae and analyze its structure-activity relationship. Methods Fresh blood from male Wistar rats was collected by cardiac puncture, and platelet-rich plasma was harvested by centrifugatioru Platelet suspension was incubated with furostanol saponins or spirostanol saponins for 3 minutes, and then the platelet aggregation was induced by ADP and monitored optically with turbidimetric method. Relationship between anti-platelet aggregation activity and structure characteristics of steroidal saponins was explored by comparing their inhibitory effect on ADP-induced platelet aggregation. Results AII the 8 furostanol saponins showed no inhibitory effect on platelet aggregation; 4 spirostanol saponins inhibited platelet aggregation in varying degrees, and the inhibitory effect of timosaponin AⅢ was the strongest, followed in order by Timosaponin AI, 22-α-β timosaponin AⅢ and timosaponin Ⅲ, while the sarsasapogenin has no anti-platelet aggregation activity. Platelet aggregation was inhibited in varying degrees by steroidal saponins with various monosaccharide chains at C-3 aglycone. Glycosylation of sarsasapogenin with glucopyranosyl donors completely inhibited the platelet aggregation, however, other sugar moieties, such as -Gal, -Ara, -Rib, -Rha or-Man, showed an attenuted inhibitory effects in varying degrees. Conclusions The timosaponin AⅢ is the main active ingredient of Rhizoma anemarrhenae for anti-platelet aggregation. The inhibitory activity of steroidal saponins is based on their structures, and the major structural requirements for the inhibition of platelet aggregation of steroidal saponins are the group situated at C-3, C-15 and C-22.