| Cholestasis can be seen in clinical as primary biliary cirrhosis(PBC)and primary sclerosing cholangitis(PSC).Cholestasis always results from an impairment or disruption of bile production and causes intracellular retention of toxic bile constituents,including bile salts.If left untreated,cholestasis leads to liver fibrosis and cirrhosis,which eventually results in liver failure and the need for liver transplantation.Currently,the only therapeutic option available for these patients is ursodeoxycholic acid(UDCA),which slows the progression of PBC,particularly in stage Ⅰ and Ⅱ of the disease.For PSC,UDCA therapy does not improve survival,and recommendations for its use remains controversial.These considerations emphasize the need for alternative therapies.Hepatic transporters,located along basolateral(sinusoidal)and apical(canalicular)membranes of hepatocytes,are integral determinants of bile formation and secretion.Once entrance into hepatocytes,bile acids are synthesised by CYP7A1,CYP8B1,and CYP27A1.Phase Ⅱ enzymes like UGT1A1 are responsible for the formation of conjugated bile acid.Nuclear receptors are critically involved in the regulation of these hepatic transporters and are natural targets for therapy of cholestatic liver diseases.Chlorogenic acids are esters formed between caffeic and quinic acids,and represent an abundant group of plant polyphenols present in the human diet.Results of epidemiological studies suggest that the consumption of beverages such as coffee,tea,wine,different herbal infusions,and also some fruit juices are linked to reduced risks of developing different chronic diseases.These beverages contain Chlorogenic acids present in different concentrations and isomeric mixtures.The underlying mechanism(s)for specific health benefits attributed to Chlorogenic acids involves mitigating oxidative stress,and hence the related adverse effects associated with an unbalanced intracellular redox state.There is also evidence to show that Chlorogenic acids exhibit anti-inflammatory activities by modulating a number of important metabolic pathways.Other biological activities of chlorogenic acids have been reported includingantibacterial,anticarcinogenic,antihypertensive,regulation of glucose and lipid metabolism and antidepressant properties.Due to potent antioxidant and anti-inflammatory activities,chlorogenic acids was demonstrated to counteract liver injure induced by hepatotoxicant or ischemia/reperfusion.Artemisia capillaris(Yin-Chen)is another common used traditional herb rich in chlorogenic acids for treatment of liver diseases,in which chlorogenic acids was revealed as one of the hepatoprotective and choleretic components.As a well-known antioxidant,however,the mechanism involved in the choleretic effect of CHLOROGENIC ACIDS was not fully understood.Pharmacokinetics describes the course of a drug when it enters the body,including absorption,distribution,metabolism and excretion.Pharmacokinetics allows us to understand the profile of drug concentration over time and the links to clinical practice.It allows us to recommend drug-dosing regimens or for novel drug therapies,provides us with knowledge to prescribe safely and effectively.Pharmacokinetics describes drug disposition mediated by human body.Inevitably there are individual differences.Age,gender,disease and other factors can change drug pharmacokinetics,consequently change the therapeutic effects.Under disease conditions,the function of absorption,metabolism and excretion disturbs by pathological environment.Considering the pharmacokinetic changes,individual regimen is therefore necessary.Pharmacokinetics of chlorogenic acid has been fully understood under physiological conditions.However,effect of disease on the Pharmacokinetics of chlorogenic acid has not been investigated thoroughly.In present study,a rat model with bile duct ligation(BDL)was established to investigate the effect and mechanism of chlorogenic acid on the expression of hepatobiliary transport and metabolism system.Moreover.a comparative pharmacokinetic study in normal and BDL Rats was conducted to reveal changes in pharmacokinetic of chlorogenic acid induced by cholestasis.Part Ⅰ Effect of Chlorogenic Acid against hepatotoxity and cholestasis in bile duct ligation ratsObjective:To explore the effect of CA on the liver injure induced by bile duct ligation(BDL).Methods:Rats were randomly divided into six groups(n=6):sham+vehicle group,sham+100mg/kg CA group;BDL+vehicle group,BDL+20mg/kg CA group,BDL+50mg/kg CA group,BDL+100mg/kg CA group.Rats received vehicle(0.50%sodium carboxymethylcellulose solution)or CA(20,50 or 100 mg/kg/d)orally for 3 days.On the 4th day,rats were sham or BDL operated and orally administrated vehicle(0.50%sodium carboxymethylcellulose solution)or CA(20,50 or 100 mg/kg/d)for another 3 days or 7days.Then rats were sacrificed,and blood and liver were collected.A portion of liver was fixed in 10%(v/v)neutral formalin,processed by standard histological techniques,stained with haematoxylin and eosin(H&E).and examined for morphological evidence of liver injury.Results:After BDL,serum levels of ALT,AST,total bilirubin and total BAs were increased significantly and typical pathological changes were observed in liver morphology.CA treatment decreased the biochemical indicator levels and ameliorated the liver pathological changes.Conclusion:CA protected against liver injury and attenuated cholestasis induced by BDL in rats.Part Ⅱ Effect and mechanism of Chlorogenic Acid against hepatotoxity and cholestasis in bile duct ligation ratsObjective:To reveal the regulation of the hepatobiliary transport and metabolism system and upstream nuclear receptors FXR and CAR.Methods:After BDL and treatment of CA for 7d,the liver was used to determinate expression of mRNA and protein of transporters,enzymes,nuclear receptor and SIRT1 through qRT-PCR and western blot method.Results:After BDL,hepatic uptake transporters(Ntcp,Oatp 1a4,and Oatp 1b2)were down-regulated and efflux transporters(Bsep and Mrp 2/3/4)were up-regulated.Furthermore,expression of Cyp 7a1,Cyp 8b1,and Cyp 27a1 were inhibited while Ugt 1a1 was induced.Moreover,up-regulation of FXR,CAR and SIRT1 was observed in the liver of BDL rats.CA treatment enhanced the degree of expression changes in the transporters and enzymes.The expression of SIRT1 but not FXR or CAR was increased after administration of CA in BDL rats.Conclusion:These results suggested that CA attenuated cholestasis by decreasing uptake and synthesis and accelerating metabolism and efflux of bilirubin and BAs via regulation of SIRT1-FXR/CAR.Part Ⅲ Development of LC-MS/MS methods for determination of Chlorogenic acid in rat plasmaObjective:To develop a sensitive ans specific HPLC-MS/MS methods for determination of Chlorogenic acid in rat plasma.Methods:Chlorogenic acid were isolated from biological samples by protein precipitation with methanol,and then chromatographed on a Hypersil ODS-C18 column,using methanolwateracetic acid(20:80:0.097,v/v/v)as the mobile phase.The detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source(ESI).Detection was performed in multiple reaction monitoring(MRM)mode using the transitions m/z 353→m/z 191(Chlorogenic acid),m/z 179→m/z 135(Caffeic acid,internal standard).Results:A LC-MS/MS assay for determination of Chlorogenic acid in the rat plasma was developed and validated.Excellent linearity was obtained over the concentration range of 1.00 to 1000 ng/mL for Chlorogenic acid in the rat plasma.The Hypersil ODS-C18 column provided isocratic chromatographic separation of the analytes with a run time of 3 min.Conclusions:A sensitive,rapid and convenient LC-MS/MS methods were developed and validated for determination of Chlorogenic acid in biological samples.The methods were proved suitable for the pharmacokinetic study of Chlorogenic acid in rats. |
