| Galangal is the rhizome of Alpinia officinarum Hance,a genus of the genus Ginger,which was first published in named as“Ming Yi Bie Lu”.It was also included in the 2015 edition of the Chinese Pharmacopoeia and was included in Drug and Food Homologous Directory by the National Health and Wellness Committee.Galangin,chemically known as 3,5,7-trihydroxyflavone,is an active compound existing in galangin and honey.It has strong pharmacological effects such as anti-bacterial,hypoglycemic,anti-obesity,anti-cancer and anti-inflammatory.At present,Alpinia officinalis is widely used in food,medicine and health care products.Galangin,an active component of its flavonoids,has attracted much attention.However,the study of galangin is mostly focused on its pharmacological activity in vitro.The absorption and metabolism of galangin in vivo are not very clear,which affects the elucidation of its pharmacodynamic substances and mechanism.In order to further study the mechanism of action of galangin in vivo and provide reference for rational drug use in clinic,this thesis explored the metabolism,absorption and transport mechanism in vivo and in vitro in normal rats,and using type 2 diabetic rats for pharmacokinetics and absorption.The main findings are as follows.Part one Metabolic profiling of galangin in vivo and in vitro using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometryObjective:To develop a rapid method based on UHPLC-Q-TOF-MS/MS to separate and characterize metabolites in rat liver microsomes(RLMs),rat plasma,rat bile,rat urine,rat feces and rat tissues after oral administration of galangin,and identification of metabolites of galangin.Methods:This strategy was characterized by the following:a novel and unique multiple mass defect filter(MMDF)combined with dynamic background subtraction(DBS)dependent on-line data acquisition method was developed to trace all probable metabolites of galangin.MMDF and DBS methods could trigger information-dependent data acquisition(IDA)scan for the low-level metabolites masked by background noise and endogenous components.A combination of data processing methods including extracted ion chromatography(XIC),mass defect filter(MDF),product ion filter(PIF)and neutral loss filter(NLF)were employed to find the metabolites of galangin.Then,the structures of the metabolites were elucidated based on accurate mass measurement,fragmentation rule of parent drug and relevant drug bio-transformation knowledge.Finally,an important parameter Clog P was used to estimate the retention time of isomers.The analysis samples were taken from:(1)blood samples,bile,urine,feces and tissues of rats after gavage with galangin;and(2)mixture of galangin and liver microsomes in vitro.Results:Based on the proposed strategy,27 metabolites(including 8phase I and 19 phase II metabolites)and 13 metabolic pathways were identified in rat after oral administration of galangin.The results indicated that glucuronidation,sulfate conjugation,oxidation and methylation were the main metabolic pathway.Metabolites related to oxidation,reduction and hydrolysis of galangin were detected in plasma,urine,bile,feces and tissue samples.Only oxidation,methylation and glucuronidation reactions were found in the study of in vitro metabolism.Conclusions:UHPLC-Q-TOF-MS/MS technology is used to establish a method for the analysis of metabolites in galangin.This method is simple,rapid and accurate for the analysis of metabolites in different pathways and tissues.The metabolism of galangin in rats is a combination of phase I and phase II metabolism.These results provide a reference for further identification and analysis of metabolites for galangin in vivo.Part two Pharmacokinetics of galangin and its metabolites in healthy rat and diabetic rat models based on HPLC-MS/MSObjective:To establish a sensitive,specific and rapid high performance liquid chromatography-mass spectrometry(HPLC-MS/MS)was developed for determination of galangin and its metabolites at different groups(blank control group,high-fat control group,type 2 diabetes experimental group)rat plasma after oral administration or tail vein injection of galangin.Methods:The rat plasma samples were pretreated by simple protein precipitation with methanol,using sulfamethoxazole as internal standard(IS).Chromatographic separation was carried out on a Wonda Cract ODS-2 C18column at 25?.Linear gradient elution was performed using acetonitrile,methanol and water containing 0.1%(v/v)formic acid as mobile phase at a flow rate of 1.0 m L·min-1.The injected volume for all samples was 10?L.The detection of galangin and its metabolites were achieved using HPLC-MS/MS method under the positive and negative ionization mode via electrospray ionization(ESI)source.Quantitation analysis was accomplished by Multi Quant software.Results:There was a good linear relationship between galangin and its metabolites in rat plasma.The relative standard deviation(RSD)of intra-day and inter-day precision was less than 6.34%.The average recovery of galangin was in the range of 85.63%-93.47%.The absolute bioavailability of galangin in normal rats was 2.90%.The pharmacokinetic parameters of galangin in rats fed with high-fat diet had no significant effect.However,the absolute bioavailability of galangin in type 2 diabetic rats was as high as 12.70%.In the normal group,the metabolites of galangin appeared for 5 minutes in blood after gavage,including apigenin,kaempferol,quercetin and chrysin.Apigenin was the highest amount of metabolite formed after oral administration of galangin.Its AUC(0-)?value was 3.7 times that of galangin,while quercetin was 0.60 times,kaempferol was 0.28 times and chrysin was 0.23 times.Conclusions:A method for simultaneous determination of galangin and its metabolites in rat plasma by liquid chromatography-mass spectrometry is established.Compared with normal rats,type 2 diabetic rats significantly change the pharmacokinetic parameters of galangin after gavage,and improve the absolute bioavailability of galangin.Apigenin,quercetin,kaempferol and salicin are the main metabolites.The results can provide references for daily diet and rational drug use of galangin in clinic.Part three Effects of P-gp,MRP2 and BCRP inhibitor on intestinal absorption of galangin in rats by in-situ single-pass perfusion modelObjective:To study the absorption characteristics of galangin in different intestinal segments and the effects of efflux protein inhibitors on the absorption of galangin in combination with efflux protein inhibitors(Verapamil,MK571,KO143).The characteristics of intestinal absorption of galangin in type 2 diabetic rats(DM)and DM rats for long-term administration(galangin,8 mg·kg-1·day-1,ig,14 weeks)were investigated.Methods:The in-situ single-pass perfusion model was used in normal rat group,DM rat group,normal rat long-term administration group and DM rat long-term administration group.Phenol red was used as marker.The content of galangin in perfusate was determined by HPLC-UV,and the absorption constant(Ka)and effective permeability coefficient(Peff)of galangin were calculated.The experimental items included:different intestinal segments(duodenum,jejunum,ileum,colon)and the addition of P-gp inhibitors(verapamil,100?mol·L-1),MRP2 inhibitors(MK571,50?mol·L-1)and BCRP inhibitors(KO143,?mol·L-1).Results:The intestinal absorption of galangin in normal rats belonged to Fick diffusion principle in the range of 5-20?g·m L-1.The higher the concentration of galangin,the more it was absorbed,and there was no obvious active transport mode.After adding inhibitors(verapamil,MK571,KO143),the inhibition efflux effect of different intestinal segments was different in intensity,and the absorption characteristic parameters of galangin in duodenal and jejunal segments were significantly increased.The Peff values of inhibitors on duodenum and jejunum were up-regulated by 26.01%,41.30%(verapamil),40.01%,83.33%(MK571)and 28.05%,42.91%(KO143),respectively.The above values were not more than 50%,suggesting that galangin was not the substrate of P-gp,MRP2 and BCRP.Compared with normal rats,the absorption of galangin for DM in jejunal and ileal segments increased significantly,the difference of Ka and Peff values were statistically significant(P<0.05),and the cumulative absorption increased significantly.The trend was in good agreement with the pharmacokinetic curve and related parameters of galangin DM group.In the high-fat group,the absorption of duodenal and jejunal segments increased,and the values of Ka and Peff in jejunal segments increased significantly(P<0.05),suggesting that high fat diet contributes to the uptake of galangin in intestinal epithelial cells,which may be related to the better lipophilicity of galangin.After long-term administration,the cumulative absorption behavior of DM group was consistent with that of normal rats,indicating that the intestinal tract recovered to normal absorption level.Conclusions:Galangin is obviously absorbed in the duodenal and jejunal segments of normal rats.Affected by the efflux of P-gp,MRP2 and BCRP transporters,adding efflux inhibitors can increase the absorption rate and amount.P-gp affects the absorption of galangin in duodenal,jejunal and ileal segments;MRP2 affects absorption of galangin in duodenal and ileal segments;BCRP affects absorption of galangin in duodenal and jejunal segments;compared with the normal group,the absorption of galangin in DM group increases significantly,which is consistent with the pharmacokinetic test.The absorption characteristics of long-term administration group of DM are consistent with that of normal group,which indicate that galangin can improve intestinal absorption of DM rats.Part four Transport research on galangin across Caco-2 monolayer modelObjective:To evaluate the bidirectional transport of galangin in Caco-2cell monolayer model and the effect of galangin on P-gp,MRP2,BCRP and P-gp.In addition,the effect of galangin on P-gp was investigated.Methods:Caco-2 cell monolayer was used as an in vitro model for the absorption and transport of galangin in the small intestine.The bidirection transport of galangin was tested,and the transport rate constant(V),apparent permeability coefficient(Papp)and efflux ratio(ER)were calculated.The effect of galangin on the activity of P-gp on the monolayer membrane of Caco-2 cells was evaluated by the transportability of P-gp substrate Rhodamine 123(Rho123)on both sides of the cell monolayer.Results:The cumulative transport of galangin increased with the increase of concentration in Caco-2 single-layer model,and the absorptive capacity(AP?BL)was smaller than that of the efflux capacity(BL?AP).The value of Papp(AP?BL)was about 2.09×10-8 cm·s-1,much less than that of Papp(BL?AP)5.59×10-8 cm·s-1,both of which were less than 1×10-6 cm/s,,suggesting that the bioavailability of galangin after oral administration was poor.The V value was positively correlated with mass concentration,and ER value was greater than 2.There was significant efflux in the transport of galangin,which could be inhibited by P-gp inhibitor(verapamil),MRP2 inhibitor(MK-571),BCRP inhibitor(KO143).Preincubation of Caco-2 monolayer model with galangin could significantly increase the efflux of Rho123,suggesting that galangin could induce P-gp on Caco-2 monolayers,resulting in up-regulation of expression.Conclusions:The absorption of galangin on Caco-2 cell monolayer model is efflux and can be inhibited by inhibitors.These results indicate that P-gp,MRP2 and BCRP are involved in the absorption and efflux transport of galangin in Caco-2 cells respectively,suggesting that P-gp,MRP2 and BCRP may be one of the main physiological barriers restricting their oral bioavailability.Galangin can induce up-regulation of P-gp expression in Caco-2 cell monolayer model,resulting in evident efflux.Part five Effect of galangin on P450 enzyme activity and m RNA expression in ratsObjective:To establish a sensitive and rapid high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)and real time polymerase chain reaction(RT-PCR)were employed to evaluat the effects of galangin on cytochrome P450(CYP450)-mediated metabolism in rat liver,using two different approaches,to predict drug–drug interactions.Methods:A“cocktail-probes”approach was employed to evaluate the activities of different CYP450 enzymes.Blood samples of seven probe drugs were analysed using liquid chromatography-tandem mass spectrometry in positive and negative electrospray-ionisation modes.Seven probes were administered to rats received gavage of galangin(8 mg·kg-1,8 weeks).Blood samples were taken regularly for determination and pharmacokinetic parameters of seven probes were calculated.Meanwhile,real-time quantitative polymerase chain reaction(RT-PCR)was used to study m RNA expression level of CYP in the liver of the rats in the drug-administered group.Results:The AUC(0)–?</sub>and Cmax values of CYP1A2 and CYP2B3 were significantly lower in the galangin-administered group compared with the control group,and the AUC(0)–?</sub>and Cmax values of CYP2C13 and CYP3A1 in the galangin-administered group were significantly increased.No significant differences were observed in the pharmacokinetic curves of CYP2C11,CYP2D4 and CYP2E1.The RT-PCR test showed that the m RNA expression results of different CYP enzymes in the liver of the drug-administered group were consistent with the pharmacokinetic results.Conclusions:Long-term administration of galangin can change the activity of CYP450 in liver,suggesting that there may be interaction between galangin and drug. |
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