| Pinnatifolone A is a sesquiterpenoid with a wide range of pharmacological effects isolated from Syringa oblata Lindl.of the genus Lilac in the family Mignonaceae.Pi nnatifolone A has potential for further development as an active ingredient in tradition al Chinese medicine.Pharmacokinetics is critical in the development of new drugs.Ph armacokinetic studies aid in evaluating the clinical efficacy of TCM active ingredients,guiding their rational use,and promoting TCM development.Complete in vivo and e x vivo preclinical pharmacokinetic evaluation is a regulatory requirement to better und erstand the efficacy,safety and toxicity of new investigational drugs.Based on literatu re research,no metabolite analysis and pharmacokinetic studies of Pinnatifolone A in c omplex biological samples have been reported so far.Therefore,the following studies were conducted:1.Pinnatifolone A anti-inflammatory activity studies in vitroModern research have discovered that the sesquiterpenoids have potent anti-infla mmatory properties despite their unique structural features.Using the appropriate kits,it was determined how Pinnatifolone A affected the viability of cells and the productio n of NO,TNF-α,and IL-6 by the LPS-induced RAW264.7 inflammatory cell model.P innatifolone A was not hazardous to cells at concentrations of 6.25,25,50,and 100μM,according to the study.Compared with LPS model group,the release of NO from P innatifolone A was significantly reduced at 25,50,and 100μM（p<0.0001）.When Pin natifolone A concentration was 6.25,25,50,100μM,the secretion of TNF-αand IL-6decreased compared with that of the model group.This indicates that Pinnatifolone A can reduce the secretion of TNF-αand IL-6 to A certain extent,and Pinnatifolone A ha s good anti-inflammatory activity.2.Establishment of determination method for Pinnatifolone A LC-MS/MS in biological SamplesIn order to quantitative analysis of Pinnatifolone A in biological samples.Verapamil（m/z 455.1→165.0）was used as internal standard in MRM positive ion mode for simultaneous detection of Pinnatifolone A（m/z 249.1→181.1）.The samples were separated by a 0.1%formic acid-acetonitrile gradient run.The methodological validation showed good linearity with intra-day precision within±15%,inter-day precision between 5.29%～13.74%and accuracy between-9.05～13.52%;the extraction recoveries of the quality control samples were 105.37%,103.30%and102.44%,and the recoveries of IS and matrix effects were 110.23%and 91.26%,respectively.In addition,the matrix effect values for the QC samples were 97.84%,94.36%and 100.97%,respectively.Short-term,long-term,freeze-thaw,and automatic sampler stability RSD and RE（%）were within±15%.A rapid and sensitive LC-MS/MS method was developed for the analysis of Pinnatifolone A in biological samples.3.Pharmacokinetics of Pinnatifolone A in male and female ratsTo investigate the pharmacokinetic process of Pinnatifolone A after administration in male and female rats.The established LC-MS/MS method was used to determine the blood concentration of Pinnatifolone A at different time after oral administration（140mg/kg）and intravenous administration（6.3 mg/kg）in male and female rats.The pharmacokinetic results showed that the area under the drug-time curve（AUC0-t）after oral administration of Pinnatifolone A in female rats（6770.76±1193.32 ug/L*h）was14.6 times higher than that in male rats（462.35±342.26 ug/L*h）（p<0.0001）;the peak drug concentration(Cmax)in female rats（2150±168.88 ug/L）was 4.3 times higher（p=0.0496）than that of male rats（496.19±341.55 ug/L）.The apparent volume of distribution（Vd）was（36.18±10.68）L/kg in female rats and（630.39±577.99）L/kg in male rats.clearance（CL）was（20.99±3.33）L/h/kg in female rats and（472.37±437.31）L/h/kg in male rats.In female rats,the absolute bioavailability of Pinnatifolone A was about 30.36%in female rats and 2.91%in male rats,and the higher bioavailability in female rats may be related to the smaller hepatic first-pass extraction due to slow metabolism.The in vivo pharmacokinetic behavior of Pinnatifolone A in female and male rats was significantly different,providing a reference for the in vivo pharmacokinetic study and rational clinical application of Pinnatifolone A.4.Metabolic Differences of Pinnatifolone A in different species of liver microsomesBased on the results of the previous study,to further verify that the higher F value of Pinnatifolone A in female rats may be caused by slow liver metabolism.In this chapter,we investigated the metabolic stability of Pinnatifolone A in human,monkey,dog,rat and mouse liver microsomes by using the established LC-MS/MS method.The results show that The half-life t1/2 of Pinnatifolone A in the liver microsomes of the five female species increased in descending order:SD rat（18.13 min）,Beagle（49.05 min）,cynomolgus monkey（56.99 min）,human（58.30 min）,and mouse（281.79 min）.The half-life t1/2 of the liver microsomes of five male species was 8.73 min in SD rats,9.55min in beagle dogs,18.14 min in mice,29.86 min in cynophilic monkeys,and 57.17min in humans.Pinnatifolone A has moderate metabolic stability in female beagle,cynomolgus monkey and female liver microparticles,and stable metabolic stability in female mouse liver microparticles.The metabolism of liver microparticles in male SD rats,beagles,mice and cynomolgus monkeys was unstable,while that in male liver microparticles was moderate.The liver microsomal CL int（mic）of male SD rats was twice as high as that of female SD rats.The CL int（mic）of male beagles and mice was significantly different from that of female beagles and mice.It is easily metabolized in male beagles and SD rats.Pinnatifolone A has species differences in metabolism in vitro,which may affect metabolism in vivo simulation,so attention should be paid to the selection of experimental animals.5.Metabolites of Pinnatifolone A in male and female ratsTo evaluate the Pinnatifolone A metabolites in male and female rats’plasma,urine,and feces and to predict their metabolic pathways.Plasma,urine,and feces were collected after Pinnatifolone A140 mg/kg gavage in male and female rats,and the samples were detected by UPLC-Q-TOF-MS/MS technique in positive ion mode,and identified and analyzed by combining Peak View and Metabolite Pilot software.The structure of the metabolites was inferred from the extracted ion chromatograms and secondary cleavage patterns of mass spectra with blank biological samples and controls,and a total of prototypes and 18 metabolites were detected in fecal samples,and 13 and7 metabolites were detected in urine and blood samples,respectively.N-acetylcysteine binding metabolites were the major metabolites in the feces and urine of rats;moreover,the levels of N-acetylcysteine binding metabolites were significantly different between male and female rats.Hydrogenated reduced metabolites were specific to the feces and urine of female rats.Mono-hydroxylated,S-cysteine bound,Ketone formation,oxidized and sulfated,and di-hydroxylated metabolites were specific to the feces of male rats.Oxidation and glucuronide-binding,deoxygenation,internal hydrolysis,and dihydroxylation metabolites were specific to male rat urine.Glucuronide-binding metabolites were the major metabolites in the plasma of both male and female rats,with glucuronide-binding metabolites in females being twice as high as those in males.Oxidized glucuronide-binding and di-hydroxylation metabolites were identified only in male rat plasma,whereas deoxy-and desaturated metabolites were identified only in female rat plasma.Pinnatifolone A metabolic pathways included hydrogenation reduction,deoxygenation,desaturation,oxidation,dehydration,demethylation,internal hydrolysis,sulfation,S-cysteine binding,N-acetylcysteine,and glucuronidation.The results showed that the metabolic profiles of female rats differed from those of male rats.In addition,the ESI-MS cleavage patterns of sesquiterpenes were enriched,providing a reference for the mass spectrometric analysis of these compounds as well as a basis for an in-depth study of the pharmacological effects and toxicity of Pinnatifolone A.In this study,Pinnatifolone A has been verified that Pinnatifolone A can inhibit the production of NO,TNF-αand IL-6 in the model group at the concentration range of6.25～100μM,and has good anti-inflammatory activity.The LC-MS/MS analysis method for Pinnatifolone A in biological samples was established for the first time,and the method was applied to study the pharmacokinetics of Pinnatifolone A in male and female rats.It was found that the pharmacokinetic behavior of Pinnatifolone A is significantly different in male and female rats.The metabolic stability of Pinnatifolone A was studied through the in vitro liver microsomal incubation system,and the differences in metabolic rate of Pinnatifolone A between male and female species were compared,and the differences in metabolic rate of Pinnatifolone A between male and female species were initially verified,which may be the reason for the differences in pharmacokinetic properties of male and female rats.Finally,the metabolites in male and female rats were analyzed and the differences in metabolites between male and female rats were found,which further verified the results from the perspective of in vivo.The pharmacokinetic characteristics of Pinnatifolone A are elucidated in order to provide reference for mass spectrometry analysis of this kind of compounds and for further research and development of Pinnatifolone A. |
