Study On The Effect Of Outer Capsule Of Ginkgo On Heptacarcinoma SMMC-7721

Cancer is one of the most dangerous diseases of human being.In nowadays,it has the highest mortality in place of blood vessel disease in china and is worthy of the name of "the first killer".But hepatocarcinoma is the second most ubiquitous malignance cancer,only inferior to gastric carcinoma,therefore it is called as "the second killer",because of high mortality.Many researches were reported that the anticancer component from natural plant could combine with special regulation target during the course of growth,develop and malignance of cancer.Screening out those active substance have been the hotspot in the work of finding new effective anticancer medicament,and it is also one of the most important aspects in research of biomedicine in china now.Ginkgo biloba L.is most precious plant.Its leaves,fruit and outer seed capsule et al has many medicinal properties.It creates the unexceptionable economical,ecological,social benefits.Ginkgo outer seed capsule is the fleshy section outside of Ginkgo,including many medicine properties.Many researchers have reported the extraction from ginkgo outer seed capsule including ginkgo biloba seed polysaccharide,ginkgol,ginkgoic acid,ginkgol flavonoids, ginkgolide and so on.Recently,researchers discovers that ginkgol,ginkgoic acid have many properties which include anti-bacterial,anti-anxiety,stabilization activity, however the most significant activity is ginkgol,ginkgoic acid have anti-cancer properties.Therefore,it is extremely potential anti-cancer resource.In this study,in order to purify the active component from ginkgol analogy, ginkgoic acid analogy,the purification protocol was optimized at first.Then,on this base,we used a series of methods including fragmentation,organic precipitation, column chromatogram Sephadex G-25,then in order to obtain rare ginkgol,ginkgoic acid,using ether,Na₂CO₃,NaOH,HCl extraction.After HPLC,six peaks were obtained.We named them GA₁,GA₂,GA₃,GA₄,GA₅ and GA₆ respectively.The effect of rare ginkgol analogy,ginkgoic acid analogy,GA₁,GA₂,GA₃,GA₄, GA₅ and GA₆ on the hepatocarcinoma cell SMMC-7721 proliferation and the inhibition rates were determined by MTT method respectively.The result shows that GA₁,GA₃ and GA₆ is effective inhibition activity.In these three samples,the most effective sample is GA₃,the IC₅₀ is 13μg/mL.The second one is GA₁,the IC₅₀ is 58μg/mL.GA₆ is the weakest inhibition activity.Comparison the cell cycle treated by GA₁,GA₆,GA₃ and cisplatin(positive control) was analyzed by flow cytometry.It was found that the SMMC-7721 cells grew slower than the control treated by all samples.However,only under GA₃ treatment,the apoptosis rate was higher than positive control(cisplatin).Therefore,we further investigate the inhibition activity of GA₃.We discovered that GA₃ not only effectively inhibit the cell proliferation,but also it can change cell morphologic characteristics.Annexin V-FITC/PI technology was used to study the potential molecular mechanism of inhibitory effect of GA₃ on the SMMC-7721.First,under GA₃ and cisplatin treatment,apoptosis rate and necrosis rate were obtained and analyzed by using flow cytometry.We discovered that under same concentration and time, SMMC-7721 treated by cisplatin mostly generated late apoptosis and cell necrosis, however,GA₃ mostly induces SMMC-7721 early apoptosis.In order to further confirmation,we apply fluorescence microscope to observe SMMC-7721 after the treatment of GA₃.The result shows that when the concentration of GA₃ is low, SMMC-7721 generated apoptosis,however,under the high concentration GA₃,it induces SMMC-7721 necrosis.Therefore,GA₃ has strong potential as effective anti-cancer drug under 10ug/mL.