| ObjectiveIn Traditional Chinese Medicine states,sargassum-licorice is an anti-drug couple which means it could be harmful to body once sargassum was used with licorice in one prescription.It is indicated in the 2015 "Pharmacopoeia of the People's Republic of China" that sargassum and licorice should not be used together.However,It is controversial that the toxicity of the compatibility application of sargassum and licorice.This study was to investigate the toxic mechanism of compatibility application of sargassum and licorice by look for toxic substances in heart,liver,and kidney toxicity respectively,then provide scientific basis for the clinical application.Method:There are five chapters in the study:The first chapter is literature study on the current research progressof the toxicity of compatibility of sargassum and licorice.The second chapter established a RP-HPLC method for simultaneous determination of liquirtin,isoliquiritin,liquiritigenin,isoliquiritigenin,glycyrrhizic acid(GL)in licorice.Determined the content of liquirtin and GL and then selected licorice herbs that met Pharmacopoeia standards.Compare changes in content of liquirtin,isoliquiritin,liquiritigenin,isoliquiritigenin and glycyrrhizic acid(GL),after sargassum and licorice were extracted together.The third chapter is a preliminary exploration of the toxicity of multiple administrations before and after sargassum-licorice compatibility.Rats were respectively exposed(gavage)to sargassum,licorice or licorice-sargassum extract with low,mid and high dose for 4 weeks.Drug toxicity was assessed according to organ weight coefficients,Serum levels of alanine aminotransferase(ALT),aspartate transaminase(AST),total bilirubin(T-BIT),alkaline phosphatase(ALP),creatine kinase(CK),a-hydroxybutyrate dehydrogenase(HBDH),lactate dehydrogenase(LDH),total cholesterol(T-CHO),triglyceride(TG),glucose(GLU),blood urea nitrogen(BUN),creatinine(CRE),and carries out heart,liver and kidney Histopathological examination.The fourth chapter was designed to investigate the effects of sargassum on the accumulation of the main active ingredients of licorice in normal rats.In section 1,a rapid and reliable UPLC-TQ/MS method was developed and validated to simultaneously determine the following six main components of licorice in rat plasma:liquirtin,isoliquiritin,liquiritigenin,isoliquiritigenin,GL,and glycyrrhetinic acid(GA).The effects of sargassum on the toxic-kinetic parameters of these six components of licorice following oral administration of single dose and multiple dose licorice extract and Sargassum-licoriceextract,respectively,were investigated.In section 2,to investigate the accumulation of these components in the heart,liver,and kidney tissues,A UPLC-TQ/MS method was established to simultaneously detect six main components of licorice in rat tissue.The fifth chapter was study that a preliminary exploration of the toxicity matrix of the compatibility application of Sargassum and licorice.In Section 1,the effects of drug-containing serum,extracts,and glycyrrhetinic acid on the survival rate of rat primary cardiomyocytes were compared by in vitro MTT assay.The changes of CK Myocardial enzymes in cell supernatants were measured using kits.In Section 2,to investigate the toxicological mechanisms of the compatibility application of Sargassum and licorice on liver,the expression of PPAR-?,PCK1,and G6PC,were analyzed by western blot.L02 cells viability were measured by MTT assay.Myocardial enzymes and lipid metabolism in cell supernatants were measured using kits.In Section 3,to study the toxicological mechanisms of the compatibility application of Sargassum and licorice on kidney,the levels of aldosterone,and cortisol in rat serum,and expression of HSD11B2 in kidney was detected,respectively.Results:1.After sargassum and licorice were combined,1 vs.1,the contents of the main active ingredients of the decoction of licorice changed,in which the content of liquirtin was reduced by 12.29%,while the content of isoliquiritigenin and GL increased by 13.60%and 10.53%,respectively.There was no significant change in the content of isoliquirtin and liquiritigenin.2.No significant toxicity was found in the control group and low-dose group(clinical dose).Following the 4-week treatment in rats,organ coefficients(heart,liver,kidney)and serum biochemical indicators(ALT,AST,ALP,CK,HBDH,LDH,TG,GLU,BUN)of the licorice-sargassum extract group(SL)were higher than those of the control group(P<0.01-0.05).Histological examination of the control and treatment groups confirmed that an inflammatory response was observed in organs(heart,liver,and kidney)of rats in the SL group.3.The combination of sargassum and licorice significantly increased the plasma concentration of GA,demonstrated by increased Cmax and AUC,in the single dose and multiple dose experiments(P<0.01-0.05).The accumulation of GA in heart,liver and kidney of HS-L group was significantly higher than that of HL group(P<0.01-0.05),which was about 114%,19%,and 190%higher than that of licorice group respectively.The exposure of liquirtin in the HS-L group was significantly lower than that in the HL group(P<0.05),and it was also decreased in the liver and kidney.4.In vitro experiments showed that GA can reduce the survival rate of rat primary cardiomyocytes and increase the level of CK in the supernatant of cells,indicating that glycyrrhetinic acid has obvious cardiomyocyte toxicity in vitro.After multiple doses of toxic doses,the expression of PPAR-a in the liver tissue of rats in the licorice group and sargassum-licorice group was down-regulated(P<0.05-0.01),and the expression levels of PCKl and G6PC protein were up-regulated(P<0.05-0.01).It was proved that GL and GA could reduce the survival rate of L02 cells,suggesting that GL and GA have obvious hepatotoxicity in vitro.After multiple doses of toxic doses,aldosterone levels in serum of high-dose licorice group andsargassum-licorice group decreased(P<0.01-0.05),and cortisol levels in high-dose sargassum-licorice group increased(P<0.05-0.01).The expression of HSD11B2 protein in kidney tissue of rats in the licorice group,sargassum-licorice group was significantly decreased(P<0.05-0.01),and the effect of the combined group was more significant.In vitro experiments demonstrated that GA can reduce the survival rate of rat HK-2 cells,indicating that glycyrrhetinic acid has obvious renal cell toxicity in vitro.Conclusion1.The increase of dissolution of GL may be related to the mechanism of its toxicity,after Sargassum and licorice were extracted together.2.The results of multiple-dose toxicity evaluation showed that the heart,liver and kidney may be the main toxic target organs of sargassum-licorice toxicity.3.After administration of high dosage sargassum-licorice extract,it was found that the increase of GA accumulation in plasma and the degree of accumulation in the heart,liver,and kidney,which suggested that,may be the main toxicant substance.4.The results of in vitro cell experiments showed that GA had obvious cytotoxicity to heart,liver and kidney cells in vitro,suggesting that glycyrrhetinic acid may be one of the main toxic substance bases of sargassum-licorice toxicity.The toxicity of the compatibility application of sargassum and licorice mainly was mainly result that increased accumulation of GA,and inhibited the expression of HSD11B2,resulting the aldosterone-cortisol system disorders.In vitro experiments also showed that GA can inhibit the proliferation of HK-2 cells,which may be one of the material bases of the combination of sargassum-licorice extract to produce nephrotoxicity. |
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