Pharmacy Study On Natural Anticarcinogen Yuanhuacine And Its Injection

Daphne diterpene esters are principal activitive components of Daphne genkwa, which is an endemic traditional Chinese medicine. Yuanhuacine has potent inhibition against several cancer cell lines. Inhibition against DNA topoisomerase I (Topo I) is probably its anticancer mechanism. Yuanhuacine is another kind of Topo I inhibitor with completely different structure compared with known camptothecin and its analogs.In this thesis, standard fingerprint of daphne diterpene esters from the root extract was first established by HPLC-UV, which can be used for D. genkwa identification and quality control. The major peaks in the standard fingerprint were identified by LC-MS.Quantitative analysis methods of yuanhuacine and yuanhuadine were established herein. The root was refluxed with petroleum ether, isolated by silica gel column chromatography, then purified by preparative C18 HPLC to get yuanhuacine with a high purity. During this course, 6', 7'-dehydro-yuanhuadine was found first from D. genkwa and named yuanhuajine. Their structures were identified by a combination of UV, IR, MS, ¹H-NMR, ¹3C-NMR and 2D-NMR spectra.Water miscible injection of yuanhuacine (self-assembly nano micelle) was prepared and its quality analysis method was evaluated herein. Experiments first discovered that solution pH value played the most important role on the stability of yuanhuacine. When pH value during 7.0-9.0, yuanhuacine was very stable in water, while pH less than 6.0, its ortho-ester group would been opened and become inactivity against Topo I, which also explains the machanics that the traditional vinegar-processing of D. genkwa makes toxicity lower. Hemolysis test of yuanhuacine injection in vitro proved that it would not haemolyze erythrocyte and could be used for intravenous injection.Anticancer experiments proved that yuanhuacine and yuanhuajine had potent inhibition against A375 cells (IC₅₀ 5.50 and 6.90 μM respectively) and activity of Topo I (IC₅₀ 40.0 and 38.0 μM respectively). Rabbit's pharmacokinetics experiments of intravenous injection proved that relationship between plasma drug concentration and time accorded with two-compartment model in vivo. Biological half lives of a and β phase were 1.1 and 10.6 hours respectively. LC-MS experiments of metabolite proved that main metabolite was m/z 392 in urine, and there were no hydrolysis products of ortho-ester group (m/z 666) and 12-Hydroxy-daphnetoxin.