Evaluation Of The Anti-platelet Aggregation Activity Of Huo Xue Hua Yu Traditional Chinese Medicines And Food Materials

Thrombosis related cardiovascular disease(CVD) is a common ailment that seriously impacts on human’s health. The mechanism of cardiovascular disease is attributed to a very complicated pathogenesis including coagulation system dysfunction, hemorheology disorder, etc. And the platelet excessive activation and vascular endothelial injury are regarded as the most important factors. Under pathological condition, platelet activation will lead to the formation of insoluble thrombus, which finally induces blood clots and thrombotic diseases. Therefore, it is an effective way to treat CVD by development of anti platelet aggregation drugs. Traditional Chinese medicines(TCMs) and dietary food materials may be developed as anti platelet aggregation drugs because of abounding in natural resources, low side effects and obvious therapeutic effects. In this thesis, the in vitro anti platelet aggregation test model was developed and applied to evaluating the anti platelet aggregation activity of 31 TCMs and 14 food materials. In addition, the possible mechanism of anti platelet aggregation effect of Rhizoma Corydalis was also studied. The thesis consists of five main parts(chapters).In the first part, the mechanism of thrombosis formation and the role of platelet involved in the thrombosis formation process were introduced briefly. Then, the anti platelet aggregation effect of natural products was comprehensively summarized and their mechanisms could be classified into four approaches including inhibition of platelet membrane receptors, impacting on nucleotide system, inhibition of platelet granules secretion and impacting on arachidonic acid system. Finally, the research progress on chemical constituents and anti platelet effect of Rhizoma Corydalis were presented.In the second part, the in vitro anti platelet aggregation test model was established and the anti platelet aggregation effect of 31 TCMs was evaluated and compared. Firstly, three different polar extracts including aqueous, 90% ethanol and ethyl acetate extract of each TCM were prepared by reflux and liquid liquid extraction. Then, the platelet aggregation inhibition rate of 93 extracts were obtained by turbidimetry with aspirin as positive control, PBS and DMSO mixed solvent as blank control, and adenosine 5’ diphosphate(ADP), bovine thrombin(THR) and arachidonic acid(AA) as aggregation inducers. The results showed that Rhizoma Corydalis, Rhizoma Ligustici Chuanxiong, Radix Salviae Miltiorrhizae as well as Radix et Rhizoma Notoginseng possessed significant inhibitory effect, which provided experimental basis for developing them as clinical drugs.In the third part, the in vitro anti platelet aggregation test model described in previous part was applied to evaluate and compare the effects of 14 food materials. It can be concluded that concentrated juices of 14 foods had much stronger inhibitory activity than another three extraction parts(aqueous, 90% ethanol and ethyl acetate extract). In addition, Liliaceae family, Allium genus plants(leek, garlic, scallion and onion) and Citrus genus fruits(lemon and grapefruit) showed strong anti platelet aggregation effect induced by all three inducers(ADP, THR and AA). These results also provided scientific basis for reasonable selection of daily dietary with supplementary curative effects for the patients with CVD.In the fourth part, the possible anti platelet aggregation mechanism of Rhizoma Corydalis was studied by investigating its effect on platelet nucleotide system(c GMP) and platelet granules secretion(5 HT, P selectin and Ca2+). The results showed that the aqueous extract of Rhizoma Corydalis could inhibit 5 HT release induced by ADP, THR and AA, 90% ethanol extract had significant inhibitory effect on THR and AA induced 5 HT release, while ethyl acetate extract didn’t have obvious effect on ADP induced 5 HT release. All the three extracts of Rhizoma Corydalis were proved to have strong inhibitory effect on the level of Ca2+ both in resting or THR induced platelet. Ethyl acetate extract of Rhizoma Corydalis had much stronger inhibitory activity on THR induced P selectin secretion than that of aqueous and 90% ethanol extract. The level of c GMP in THR induced platelet was significantly increased by aqueous and 90% ethanol extract, but ethyl acetate extract didn’t have such effect. In conclusion, the possible inhibitory effect on THR induced platelet aggregation of aqueous and 90% ethanol extract of Rhizoma Corydalis may result from promoting the release of c GMP, inhibiting the level of 5 HT, P selectin as well as Ca2+. And the ethyl acetate extract may inhibit THR induced platelet aggregation through inhibition of the level of Ca2+ and P selectin.The fifth part is the conclusion and prospection of present study. Rhizoma Corydalis, Rhizoma Ligustici Chuanxiong, Radix Salviae Miltiorrhizae as well as Radix et Rhizoma Notoginseng, plus Liliaceae family, Allium genus plants and Citrus genus fruits could significantly inhibit platelet aggregation in vitro induced by ADP, THR and AA. The anti platelet aggregation mechanism of Rhizoma Corydalis was contributed to increasing the level of c GMP and decreasing the level of P selectin, Ca2+ as well as 5 HT. The anti platelet aggregation mechanism of active crude extracts as well as components from Rhizoma Corydalis could be further studied through platelet proteomics. On the other hand, the bioactive components and active mechanism of TCMs and food materials with higher anti platelet aggregation activity should be further studied.