Pharmacological Activity And Pharmacokinetics Investigation Of Crocins And Iridoid Glycosides From Gardenia

Objective: Investigate in vitro antioxidant activity of gardecin and several different crocins from gardenia;Investigate protective effects of crocins and iridoid glycosides extracted from gardenia and saffron extracts against liver injury induced by methionineand choline-deficient diet(MCD)in mice;Investigate pharmacokinetics of crocin-1 in rats and its distribution in the rat heart,liver,spleen,lung and kidney tissues,which provide the basis for clarifying the crocin-1 pharmacological activity in organ tissues of rats.Methods:(1)Determine in vitro antioxidant activity of novel compound gardecin obtained from gardenia compared with 3 different crocins by ·OH free radical scavenging activity experiment,reduction ability model experiment,chelating capacity of Fe2+ experiment and nitrite inhibition experiment.(2)Male C57BL/6 mice were fed either the MCS diet or the MCD diet and divided into 8 groups,8 rats in each group: MCS group,MCD group,MCD+TIG groups(100mg/kg,50mg/kg),MCD+TCG groups(100mg/kg,50mg/kg),MCD+SEA groups(25mg/kg,12.5mg/kg).The liver index was calculated by liver weight and body weight ratio.The livers morphology was photographed by a camera.The liver sections were observed and photographed under a light microscope after stained by HE,Sirius red and Masson.The serum levels of triglyceride(TG),total cholesterol(TC),alanine aminotransferase(ALT)and aspartate transaminase(AST)were determined by enzyme-linked immunosorbent assay(ELISA).(3)10 male SD rats were randomly divided into two groups,which are crocin-1 group and control group with 5 rats in each group.Rats were deprived of food for 12 hours before oral administration(but not water).A saline solution containing crocin-1(300mg/kg)was administered in treatment group by intragastric gavage,while a saline solution was administered in control group by the same way above.Then at 0.5h and 6h after the administration,each rat was anesthetized with Chloral hydrate,blood was extracted from the caudal vena cava with a heparinized syringe.Rats were sacrificed by tail vein embolization and hearts,livers,spleens,lungs and kidneys were removed at 0.5,1h,2h,4h and 6h.Plama and homogenate samples were subjected to LC-MS measurement after preparation.Results:(1)In four antioxidant models,including OH radicals scavenging,reducing power,Fe2+-chelating and nitrite scavenging,IC50(or EC50)of gardecin were significantly smaller than those of crocins.(2)After 8 weeks fed with MCD diet,the MCD mice with or without treatment displayed a significant reduction in liver index.Liver morphology,HE staining,Sirius red staining and Masson staining showed that MCD diet caused the steatosis,hepatocellular ballooning degeneration and fibrosis compared with MCD group;TCG,TIG and SEA can significantly attenuate hepatic lesions compared with MCS group.Serum biochemical analysis showed that TG levels and TC levels declined sharply while ALT and AST levels raised significantly in MCD diet group compared with the MCS group(P<0.05);TG levels and TC levels had raised while ALT and AST had decreased in MCD+TCG groups,MCD+TIG groups and MCD+SEA groups compared with the MCD group(P<0.05),furthermore,high dose groups showed more significant change than low dose groups.(3)LC-MS detection results showed that there are at least three main metabolites and crocin-1 in blood and tissues of rats after oral administration of crocin-1.For liquid chromatography-mass spectrometry(LC-MS)analysis,it can be inferred that these metabolites are crocetin,crocetin-monoglucuronide and-diglucuronide,which were widely distributed in blood,heart,liver,spleen,lung and kidney of rats.Crocin-1 reached its maximum relative amount respectively at 2h,1h,2h,2h and 2h in heart,liver,spleen,lung and kidney.While crocetin were at 4h,1h,0.5h,1h and 2h,crocetin-monoglucuronide were at 1h,4h,2h,2h and 6h,crocetin-diglucuronide were at 1h,0.5h,2h,1h and 1h.Conclusion:(1)Antioxidant activity of gardecin is significantly stronger than those of crocins.Sugar moiety is a beneficial factor for antioxidant activity of crocins.(2)TCG,TIG and SEA have a significant therapeutic effect on nonalcoholic steatohepatitis in mice.(3)For the analysis of LC-MS,the crocin-1 and its metabolites crocetin, crocecin-monoglucuronide and crocetin-diglucuronide had been found in blood,heart,liver,spleen,lung and kidney of rats after oral administration of crocin-1.According to this result,we can conclude that crocin-1 may be hydrolyzed into crocein before or during the intestinal absorption,thereafter crocetin would be partly metabolized into mono-or diglucuronide conjugates in liver,finally,the crocecin and its mono-or diglucuronide conjugates were incorporated into blood circulation,which were widely distributed in heart,liver,spleen,lung and kidney of rats by blood flow.