Study On Anti-tumor And Anti-arrhythmic Activities,Structure-activity Relationships And Molecular Mechanisms Of Ginsneosides

Ginsenosides are a group of glycosides that contain a triterpene aglycone and sugar moieties,which are main active ingredients in ginseng.Most ginsenosides belong to dammarane-type tetracyclic triterpenoid saponins,and are classified into two types:protopanaxadiol and protopanaxatriol,according to their structural characteristics.The structure diversity of ginsenosides contribute to their multiple biological activities and pharmacological effects.Total ginsenosides and some monomers have been reported to have anti-tumor and anti-arrhythmic activites,however their structure-activity relationships and mechanisms are still not well understood.Further studies are required to discover ginsenosides with higher efficiency and less toxicity.This dissertation aims to study the structure-activity relationships of dammarane-type ginsenosides through comparing anti-tumor and anti-arrythmic activities of different protopanaxadiol and protopanaxatriol ginsenosides.The synergistic anti-tumor and anti-arrhythmia mechanisms of promising ginsenoside compositions or monomors were further investigated at the molecular level.The detail contents and results are as follows:1.Study on the relationship between structure of ginsenosides and their anti-tumor activityTo study the structure-activity relationship of ginsenosides,the inhibtory effect on cell proliferation of different dammarane-type ginsenoside monomers were evaluated by MTT assay on three colon cancer cell lines.The inhibitory effect of these ginsenosides are shown in the following descending order: PPD > Rh2 > GLXXV > Rg3 > F2 > GXVII >Rd > Rb1 > Rg2～Rh1～Re.The structure-activity relationship analysis indicates that anti-tumor activity of ginsenosides is associated with the type of aglycon and sugar residues.Protopanaxadiol ginsenosides generally shows stronger anti-tumor activity than protopanaxatriol ginsenosides.Moreover,the anti-tumor activity of protapanaxadiol ginsenosides exhibits a decreasing trend with increasing sugar moieties linked to aglycone skeletons.When containing the same number of sugar moieties,protapanaxadiol ginsenosides with sugar linkage at C-20 shows stronger anti-tumor activity.2.Synergistic anti-tumor activity and mechanism of ginsenoside compositionsTo study the synergistic anti-tumor acticity of ginsenoside compositions,ginsenoside monomers with excellent antitumor activity were combined with other ginsenosides to evaluate their inhibitory effects on cell proliferation by MTT assay.The results showed that Rg3(67 μM)and Rh2(33 μM)compositions could synergestically inhibit the proliferation of colon and breast cancer cells,but had no synergistic inhibitory effect on lung cancer and normal colon cancer cells.Synergistic anti-tumor effect of Rg3 and Rh2 compositions was further confirmed in CT-26 xenograft mice,and in vivo results showed that Rg3(10 mg/kg)and Rh2(5 mg/kg)compositions synergistically inhibited tumor growth and expression of the proliferation-associated protein Ki67 in tumor tissues.Results of synergistic anti-tumor mechanism study indicated that Rg3 and Rh2 could jointly promote glucocorticoid receptor activation.However,they inhibited the activation of different proteins in MAPK signaling pathway,and then synergistically induced caspase-dependent apoptosis.3.Study on the structure-activity relationship and mechanism of ginsenosides against arrhythmiaAnti-arrhythmic activity and cardiomyocyte toxicity of different dammarane-type ginsenosides were studied in barium chloride-induced arrhythmic rats and H9c2 cardiomyocyte respectively,in order to study their structure-activity relationship.Anti-arrhythmic activity of these ginsenosides are shown in the following descending order:Rg2～PPT > Rh1 > Re > Rd > Rg3～Rh2～PPD.Structure-activity relationship analysis indicates that protopanaxatriol ginsenosides generally have better anti-arrhythmic activity compared to protopanaxadiol ginsenosides.Cardiomyocyte toxicity of protopanaxatriol ginsenosides gradually decreases with increasing sugar moieties linked to aglycone skeletons.Based on the results of anti-arrhythmic activity and cytotoxicity studies,Rg2 was selected as a promising candidate for further pharmacodynamic evaluation and mechanism study.Anti-arrhythmic activity of Rg2 was evaluated on three types of drug-induced arrhythmia models.Results showed that Rg2 could significantly shorten the duration of arrhythmia and reduce the incidence of mortality and malignant arrhythmias,but had no obvious toxicity for normal rats,suggesting that Rg2 is an effective anti-arrhythmic candidate compound with minimal side effects.The anti-arrhythmic mechanism of Rg2 was studied in isolated myocardial tissues and cardiomyocytes,and it was found that Rg2 could inhibit calcium influx through inhibiting the activation of Ca MKII and Cav1.2,and thereby exerted anti-arrhythmic activity.4.Oral toxicity assessment of ginsenoside Rg2 with antiarrhythmic activityOral acute toxicity and subchronic toxicity of Rg2 were evaluated on animal models.Oral acute toxicity of Rg2 was evaluated by the maximum dose method.During the 14 days after intragastric administration of 10 g/kg Rg2,all the mice survived and behaved normally.There was no significant difference between control group and Rg2-treated group in body weight and food consumption,indicating that Rg2 has no obvious oral acute toxicity when the dose is less than 10 g/kg.To assess the subchrnic toxicity,rats were orally administrated with three doses of Rg2(1.75,3.5 and 5 g/kg/d)for 28 days.Rats in different groups were all survived and behaved normally during the 28-day administration period and the 14-day recovery period.No significant difference in body weight,food consumption and most hematological parameters was shown among groups.Morever,no obvious damage of major oragans could be observed in gross anatomy and pathological examination in high-dose group(5 g/kg).These results indicate that Rg2 has minimal sub-chronic oral toxicity when the dose is less than 5 g/kg/d.In summary,results of the structure-effect relationship study indicate that protopanaxadiol ginsenosides generally exhibit anti-tumor activity,while protopanaxatriol ginsenosides show anti-arrhythmic activity.Furthermore,Rg3 and Rh2 compositions have synergistic anti-tumor activity and Rg2 has significant anti-arrhythmic acticity with low toxicity.The synergistic anti-tumor and anti-arrhythmic mechanisms of these ginsenosides have been preliminarily illuminated.These results provide the theory basis for the development of safe and effective ginsenoside compositions and monomors for anti-tumor and anti-arrhythmic therapy.