Preparation And Plasma Kinetics Study Of Cis/Trans Crocetin

The gardenia belongs to the Rubiaceae family,and its dried and ripe fruits are commonly used in clinical Chinese medicine.Our research group previously screened drugs with anti-high altitude hypoxia activity from a large number of natural plants,and found that gardenia yellow pigment extracted from gardenia had good pharmacological activities such as anti-oxidation,anti-hypoxia and anti-fatigue.The main active component of gardenia yellow pigment,crocin-1,was first metabolized into its aglycone crocin acid in vivo and then absorbed into the blood.Therefore,on the basis of the research group,a large number of high-purity crocus acid was prepared in this paper.The in vitro antioxidant activity and rat plasma kinetics of cis and trans crocetin were then studied to provide a theoretical basis for subsequent experiments.The research content of this paper includes the following four parts:1.Preparation and process optimization of trans crocetin.high-performance liquid chromatography（HPLC）was established for the determination of crocetin in the samples.The evaluation index was based on the purity and yield of crocetin.The solid-liquid ratio,Na OH concentration,hydrolysis temperature,and hydrolysis time were investigated.The optimal conditions for alkaline hydrolysis of gardenia yellow pigment were obtained as follows:solid-liquid ratio 1:6 g/m L,Na OH concentration 3mol/L,hydrolysis temperature 55°C,and hydrolysis time 60 min.Under this condition,the crocetin yield was 15.33%±1.25%and a content of 97.24%±0.78%.Finally,high-purity crocetin was prepared from the gardenia yellow pigment in a single step.This method is simple,environmentally friendly,and suitable for industrial applications.At the same time,in the research process,it was found that crocetin has two configurations of cis and trans.In order to further study the difference between cis and trans isomers,it is necessary to prepare high-purity cis crocetin.2.Preparation and characterization of cis crocetin.cis crocetin is obtained by two different methods.1)Taking the gardenia yellow pigment as raw material,on the basis of the technology for preparing trans-crocetin,cis-crocetin was obtained that further extracting and separating from the discarded filtrate;2)Therefore taking trans-crocetin as raw material,using iodine induction for the first time the method converts the trans-crocetin to the cis-structure.The crude cis-crocetin obtained by the two methods was purified by silica gel column chromatography.The elution solution was dichloromethane-methanol-acetic acid（35:1:0.01）.The structure was characterized by HPLC,NMR,IR and MS.The effects of reaction solvent,iodine concentration and drug concentration on the reaction were studied by high performance liquid chromatography（HPLC）,and the optimal conditions for configuration conversion were preliminarily screened.The optimal conditions obtained after screening were as follows:DMF was used as the drug solvent,the iodine concentration and drug concentration were 2.5 mg/m L,and irradiation time was appropriate under the visible light of 30 W power.3.In vitro antioxidant studies of trans and cis crocetin.The in vitro antioxidant activity of trans and cis crocetin was evaluated by the chemiluminescence method.Luminol was used as a chemiluminescence reagent to compare the changes of luminous intensity in the system after adding trans and cis crocetin.The inhibition rate and half-inhibitory concentration(IC₅₀)were calculated to evaluate the scavenging ability of·O₂^-,H₂O₂ and·OH-.The experiments found that the IC₅₀ of trans-crocetin on·O₂^-,H₂O₂ and·OH-were 6.39,4.67,8.63μg/m L,respectively,and the half-inhibitory concentrations of cis-crocetin on·O₂^-,H₂O₂ and·OH-were 13.29,8.47,6.41μg/m L,respectively.The results showed that both trans and cis crocetin had strong antioxidant activities,and compared with the control group ascorbic acid,there was a very significant difference.4.Plasma kinetics of trans and cis crocetin in rats.high-performance liquid chromatography（HPLC）was established for measuring trans and cis-crocetin in rat plasma.The results showed that the AUC（0-t）,AUC（0-∞）and C_max of cis-crocetin were significantly higher than those of trans-crocetin（P<0.05）,which are 1.50,1.24 and8.54 times of the trans,respectively.The half-life(t_1/2)of cis-form is significantly smaller,about 1/2 of that of trans,and T_max is 1.16 h shorter than that of trans.In addition,after administration of cis-crocetin,it was partially converted into trans-crocetin（CT-trans-crocetin）in vivo,and AUC（0-∞）and C_max of CT-trans-crocetin were 0.79 and 1.15 times of those of the direct administration of the trans-crocetin group.Therefore,the absorptivity and absorption rate of cis-crocetin is significantly higher than that of trans-crocetin.However,the half-life of cis-crocetin is short,but some of it will be converted into trans-crocetin,which exists in vivo in another configuration and extends its residence time in vivo.