Study On The In Vivo And In Vitro Metabolism Characteristics Of Isoquinoline Alkaloids From Linderae Radix And The Changes Of Bioactivity After Biotransformation

Recent studies have demonstrated that the isoquinoline alkaloids from Linderae Radix possessed obvious anti-inflammation and analgestic effects.And in vitro and in vivo drug metabolism and pharmacokinetic studies of norisoboldine,a major bioactive components of Linderae Radix have been reported.To further reveal the metabolism characteristics and dynamic profiles of isoquinoline alkaloids from Linderae Radix,and clarify the effective substances of Linderae Radix for anti-rheumatoid arthritis,an investigation of drug metabolism and pharmacokinetics of other alkaloids with similar structure is necessary.In the present study,we focused on the in vitro and in vivo metabolism and pharmacokinetic studies of boldine,isoboldine and reticuline and their in vitro bioactivity of reduced the production of nitric oxide were assessed with and without microsomal incubation by nitrate acid reductase method.Methods1 After incubation of boldine,isoboldine and reticuline using in vitro human live microsomal system,ultra-performance liquid chromatography coupled with triple-quadrupole mass spectrometry(UPLC-MS/MS)was utilized to identify their metabolites.2 The metabolites of boldine,isoboldine and reticuline in plasma,urine,feces and bile were identified using UPLC-MS/MS method after oral administration.3 Three rapid,selective and sensitive methods using UPLC-MS/MS were firstly developed for determination of boldine,isoboldine and reticuline in rat plasma,respectively.The detection was performed by MRM mode via electrospray ionization(ESI)source operating in the positive ionization mode.The precursor-to-product ion transitions were at m/z 328.22>264.91 for boldine,m/z 328.33>265.05 for isoboldine,m/z330.32>191.87 for reticuline and m/z 330.24>180.93 for sinomenine(IS),respectively.The in vivo pharmacokinetic analysis of boldine,isoboldine and reticuline were investigated by using UPLC-MS/MS method after oral administration and intravenous injection.4 A commercially-available cell viability assay was employed to evaluate their cytotoxic effects of boldine,isoboldine and reticuline in the presence or absence of LPS on RAW264.7 cells using the MTT-based colorimetric.Nitric oxide was measured in culture supernatant by nitrate reductase.Results1 Two phase Ⅱ metabolites were detected in both incubation systems,and identified as glucuronide conjugates(M1 and M2)of boldine,isoboldine and reticuline,which are isomers.However,no phase Ⅰ metabolite of boldine,isoboldine and reticuline were found.At the same time,these studies also showed that there were obvious differences between species.2 Five new metabolites were detected and identified in rats after oral administration of boldine and isoboldine,they were identified as single glucuronide conjugate(M1 and M2,m/z504),single sulfated conjugate(M3,m/z408),double sulfated conjugate(M4,m/z488),single glucuronide and single sulfated conjugate(M5,m/z584).Meanwhile,four new metabolites of reticuline in rat were elucidated and identified after oral administration,they were identified as single glucuronide conjugate(M1 and M2,m/z506),single sulfated conjugate(M3 and M4,m/z410).The results indicated that glucuronide and sulfated conjugate were the main metabolic pathways of boldine,isoboldine and reticuline in vivo.However,reticuline was also easily biotransformed to other metabolites in rats.Their m/z were 328(A1～A3),408(B1),and 504(C1～C5)in bile sample,respectively.3 The calibration curves were linear over the concentration ranges of 0.002555～2.555μg/mL for boldine,0.0048～2.4μg/mL for isoboldine and 0.00231～1.155 μg/mL for reticuline respectively.The methods was validated according to the bioanalysis requirements of FDA.Their absolute bioavailbilities of boldine,isoboldine and reticuline in rats were 6.3%,1.3%,and 5.5%,respectively.4 Boldine and isoboldine(10-100μM)in the presence or absence of LPS,except at the highest concentration(100μM),did not display remarkable cytotoxicity on RAW 264.7 cells.Reticuline showed no cytotoxicity on the growth of RAW264.7 cells at the concentration range from 10 to 100μM.The result of nitrate reduction test of boldine,isoboldine and reticuline showed that they both had anti-inflammatory efficacy,whether in presence of liver mirosome or not.Conclusion1 The results indicated that glucuronide and sulfate conjugate were the main metabolic pathways of boldine,isoboldine and reticuline.However,no phase Ⅰ metabolite was found,which suggests that boldine,isoboldine and reticuline can’t be metabolized by CYP450 enzyme.The in vitro metabolism study showed that isoquinoline alkaloid of Linderae Radix would be vulnerable to metabolized by UDP-glucuronosyltransferases,and the study also found that there are obvious differences between species.All these findings will contribute further understanding of the intermediate processes and metabolic mechanism.2 Poor bioavailability of the three isoquinoline alkaloid is primarily due to extensive first-pass effect.The results of anti-inflammatory activity in vitro showed that they both had anti-inflammatory efficacy,whether in presence of liver mirosomes or not,prompting that isoquinoline alkaloid of Linderae Radix not only prototype compound but also its metabolites may also be effective.