Studies On The Pharmacokinetics And Microbial Transformation Of Triterpenoids From Ilex Rotunda

Ilex rotunda Thunb.,an evergreen subtropical tree from genus Ilex,is widely distributed in southern China.The dried stem barks of I.rotunda,called Ilicis Rotundae Cortex or“Jiubiying”in China,have been officially listed in the Chinese Pharmacopeia 2015 Edition.Ilicis Rotundae Cortex has been commonly applied in traditional Chinese medicines to treat gastrointestinal and cardiovascular diseases.On the basis of previously phytochemical research,triterpenoids and phenylpropanoids are generally considered to be the main bioactive and characteristic constituents of I.rotunda.In this study,an investigation on the chemical constituents and in vivo process of I.rotunda was carried out.A rapid and sensitive ultra-high performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry（UPLC-TOF-MS/MS）was developed for the identification of chemical constituents in the 70%Et OH extract of I.rotunda.Based on the accurate mass measurement,the reference substances,the retention times and the mass spectra fragmental patterns,40 constituents were identified or tentatively characterized,including 36 triterpenoids and 4 phenylpropanoids.This study preliminarily illuminated the major chemical components of the Et OH extract of I.rotunda.Moreover,24,36 and 38 prototype compounds were determined in rat’s plasma,urine and feces,respectively,after oral administration of the 70%Et OH extract of I.rotunda by“in vitro to in vivo”translation approach.Then the LC-MS method for the simultaneous determination of plasma concentrations of six absorption bioactive triterpenoids were established and further applied to a pharmacokinetic study.Plasma was pretreated by protein precipitations with methanol and digoxin was used as the internal standard.All samples were separated by an ACQUITY UPLC BEH C₁₈column（100×2.1 mm,1.7μm）via a gradient elution using 0.1%formic acid in both Me CN（A）and H₂O（B）as mobile phases at the flow rate of 0.4 m L/min and matain at40°C.Processed samples in the autosampler were maintained at 4℃and injection volume was 5μL.The mass spectrometer was performed under the following optimal parameters:nebulizer gas pressure at 55 psi,heater gas pressure at 55 psi,curtain gas pressure at 35 psi,ion spray vltage at-4500 V,source temperature at 550℃,declustering potential at 100 V.The method was validated for sufficient specificity,precision,accuracy,and sensitivity.The intra-day precisions was less than 12.4%and the inter-day precisions was less than 13.5%.The extraction recoveries of the analyses were all ranging from 81.2%～98.2%and81.3%～112.5%,respectively.The validated method was successfully applied to a pharmacokinetic study of I.rotunda in rat plasma after oral administration of the 70%Et OH of I.rotunda.Thus,the presently developed methodology was simple,robust,accurate and precise would be useful for the pharmacokinetics study of I.rotunda.In addition,rotundic acid,a representive triterpenoid in I.rotunda,was selected as an example to understand the in vivo metabolic process of other triterpenoids and provide a basis for furture pharmacological research of this plant medicine.11 metabolites were detected in rat plasma,urine and feces after oral administration of rotundic acid.In order to confirm their structures,14 strains of different species including Mucor polymorphosporus,Mucor plumbeus,Syncephalastrum racemosum,Cunninghamella blakesleana,Penicillium melinii and Aspergillus niger were used to screen the best condition for biotransformation.All of them showed conversion ability,among which S.racemosum AS 3.264 was the strongest proportion strain,and this organism was used for identifying and preparing metabolites in vitro.As a result,6 metabolites were identified from biotransformation.By comparison of the metabolites in rats and those in microbial samples,three microbial metabolites were identical to those found in rats.Moreover,three metabolites were obtained by the means of amplifying incubation and their structures were determined by various spectroscopic analyses,hereby the structure of some metabolites in vivo were confirmed.Rotundien A was proved to be a previously undescribed compound,and rotundanonic acid and ilexolic acid A were isolated from microbial conversion for the first time.