The Effect And Mechanism Research Of Liver Channel Tropism Of Vinegar-processing Rhizoma Curcumae Based On Microdialysis And Fluorescence Imaging

The concocted theory is an important part of Traditional Chinese Medicine(TCM),where the historic vinegar processing method has been widely used.Vinegar is warm and tastes sour.Herbal medicine processed with vinegar has the effect of liver target.It can also enhance the ability of activating blood,removing stasis and regulating qi to alleviate pain.Rhizoma Curcumae is the representative of Chinese medicine which is processed with vinegar and has the effect of liver channel tropism.The raw pieces are good at promoting qi circulation and eliminate toxins.The vinegar pieces have the hepatic target effect and are mainly used for the treatment of liver cancer,cholestasis and so on.Currently,studies related to VRC mostly focused on processing technology,chemical composition variation and anti-inflammatory and analgesis effect,while less is known about its processing mechanism.This subject comes from the National Natural Science Foundation of projects.Early studies had shown that VRC could improve the cell survival rate and increase the bile excretion.It could inhibit CYP1A2 and CYP2E1 and had an induction on the CYP3A4.Moreover,it could promote curdione in rat liver tissue distribution,and the concentrations of curcumin in liver tissue was significantly higher than other major organizations after giving rats curcumin turmeric extract.The above study could explain the mechanism of VRC hepatic target effect to some extent.The traditional processing and meridian tropism theory is an extremely complex and esoteric system,it should be researched in multi-level,multi-angle and with multi-methods.On the basis of preliminary studies,this subject closely linked to the overall concept of Chinese Medicine and tried to explore the vinegar-processing mechanism of Rhizoma Curcumae with modern technologies.The main contents of this paper are as follows:1.Through access to relevant ancient and modern literatures,we systematically collected the relevant researches of chemical composition,pharmacological effects,pharmacokinetics,processing history and mechanism of Rhizoma Curcumae.And at the same time,literatures about hepatic fibrosis,microdialysis and fluorescence imaging have also been studied.2.Curcuma wenyujin was chose in our experiment.It was prepared and identified in accordance with Chinese Pharmacopoeia.The results showed that the volatile oil content of RRC and VRC were 1.92±0.17%and 1.52±0.13%,respectively.All of them meet the requirement of Chinese Pharmacopoeia and can be used in the later experiments.3.UPLC-TOF/MS was used for the identification of RRC and VRC.The accurate molecular weight measurement of TOF/MS helped us to identify a total of 20 ingredients.The total intensity of RRC and VRC were calculated by PeakView software.The intensity of 17 kinds of ingredients which have stable response was calculated,too.The relative content of each compound was expressed by the ratio of intensiy.The results showed that the total intensity of RRC was 18638627.0±3888307.3,while the VRC was 14308138.3±2700374.7.Among the 17 kinds of ingredients,the intensity of dehydrocurdione,curcarabranol,bisacurone in VRC increased,while the others reduced.The relative contents of curcumol,ar-turmerone,procurcumenol,dehydrocurdione,curcarabranol,bisacurone in VRC were increased.Therefore,the chemical composition of Rhizoma Curcumae changed after been processed by vinegar.4.Rats models of hepatic fibrosis were made by subcutaneous injection of 40%CCl4 olive solution and fed with alcohol and high fat food.The automatic biochemical analyzer and pathological section were used to evaluate the anti-hepatic fibrosis effect of RRC and VRC.The results showed that both RRC and VRC can improve liver function parameters in different degrees.But the effect of VRC is superior to RRC.VRC can significantly reduce the level of MDA(P<0.01)and increased SOD activity(P<0.01),and the high dose group of VRC was better than colchicines group.Thus the results indicate that RRC may have significant impact on lipid metabolism of the body.5.The simultaneously microdialysis sampling system of blood and liver were established,and RRLC-MS was utilized in the determination of curdione and curcumol.The pharmacokinetics parameters were calculated by WinNonlin.The in vitro and in vivo recovery of curdione and curcumol were investigated first.And factors affecting the calibration of microdialysis probes were assessed.The revoery results indicated that the sites-microdialysis sampling could be used for the pharmacokinetic study of curdione and curcumol,and retrodialysis could be applied to assess the in vivo recovery of curdione and curcumol.The pharmacokinetic results showed that the liver distribution of curdione and curcumol were 0.3710.08 and 0.85±0.11,respectively,after oral administration of RRC.And the liver distribution of curdione and curcumol after oral administration of VRC were 0.55±0.09 and 0.86±0.09.respectively.Compared with RRC group,the liver distribution of curdione in VRC group increased significantly(P<0.05),which may help to explain the mechanism of liver channel tropism of VRC.6.The basic theories of Chinese medicine consider that bile derives from liver,so we chose bile as the object of this experiment.Under the guidance of metabonomics theory,UPLC-TOF/MS combined with PCA and T-test analysis were applied to compare the bile metabolites between RRC and VRC.With the boundaries of P<0.05,13 kinds of compounds with significant difference were found,and 9 of them were identified.Through the analysis of the biological founction and metabolic pathways,we found that all of them were associated with the body lipid metabolic pathways.Therefore we speculated that VRC had important influence on lipid metabolism process.7.The studies above showed that curcumol could not be detected in both blood and liver microdialysate at 5.5 h after oral administration of drug.So we tried to apply fluorescence imaging technique to visualize tracking of curcumol in the process of absorption and metabolism in nude mice.The results showed that after monomer curcumol injected into nude mice,fluorescent immediately distributed to the whole body.5 min later,the bladder significant enriched with fluorescence,and the intensity was higher than the other organs.Thus curcumol showed significant renal excretion characteristic.In addition,there were significant fluorescence intensity in both lungs and brain,which metabolic rates were slower than heart.Due to the large surface area of liver,the average intensity is weaker,but total signals were highest.In summary,this subject selected liver fibrosis model which is closely related to the clinical application of Rhizoma Curcumae to compare the therapeutic effect of RRC and VRC.Under the guidance of Chiniese medicine theories,we combined multiple modern methods in our research,such as UPLC-TOF/MS,microdialysis and fluorescence imaging.Both the research ideas and technologies were innovative.The results could further explain the mechanism of liver channel tropism of vinegar-processing Rhizoma Curcumae.It also provides new ideas for the modernization research of TCM.