Investigation On The Chemical Constituents Of Diterpenoids In The Isodon Rubescens (Hemsl.)hara For Quality Control And The Metabolism Of Oridonin In Different Liver Microsomes

Isodon rubescens(Hemsl.)Hara(IR),known as Dong-ling-cao in China,is a well-known Chinese medicinal herb.It has been listed in the Chinese Pharmacopoeia and used to treat inflammatory and pain related diseases,such as pharyngitis,sore throat,stomachache,and cough.IR widely distributed in China,and there are obvious differences in chemical components in different origin.At the same time,the Isodon plants were similar in appearance,and the origin of the medicinal plant was lack of regulation in the medicine market.Therefore,it is very significative for the quality of the medicine to develope a novel and effective method to identify and control the diterpenoid in IR samples.In recent years,a novel independent acquisition mode called SWATH(sequential window acquisition of all theoretical fragment-ion spectra),which sequentially acquires MS/MS of all precursor ions across a specified mass range,are used to collect the same spectrum precursor and fragment ions using a collision energy rang.SWATH method could give better quality of MS/MS spectra with an identical MS/MS acquisition hit rate.Therefore,this study coupled ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS/MS)with SWATH method was employed to quickly identification of diterpeoids ingredients in IR plants.This study could provide technical support for further development and utilization of the active diterpenoid ingredient.Meanwhile,principal component analysis(PCA)was employed for better evaluation the IR samples quality.These results would be used for evaluation the holistic quality control in IR herbs and other TCM(traditional Chinese medicine)plants.Oridonin(ORI)is an active natural ent-kaurane diterpenoid ingredient with notable anti-inflammation and anti-cancer activities.It has many pharmacological and biological activities such as anti-tumor,antagonistic activity,anti-inflammatory,anti-leukemia,anti-viral and anti-oxidation activities.Drug metabolism research is an indispensable part at various stages of drug discovery and development.And it is significative for illuminating the drug metabolic pathway and evaluating the drug safety.Therefore,a strategy was developed to identify metabolites and to assess the metabolic profiles of ORI in vitro using UHPLC-Q-TOF-MS/MS.Meanwhile,the metabolic patterns of ORI metabolites were summarized in vitro.And the metabolism differences of ORI in the liver microsomes of four different species were investigated and analyzed,and this research can reveal the pharmacological activities and toxicological implications of a drug to ensure that the drug can be safely used in humans.Part one Rapid identification of ent-kaurane diterpenoids in Isodon rubescens(Hemsl.)Hara extract with a practical technique of SWATH based on UHPLC-Q-TOF-MS/MSObjective: To establish a novel stragety based on UHPLC-Q-TOFMS/MS coupled with SWATH method for rapid identification the diterpenoid ingredient in IR herbs.PCA was used for better evaluating the IR samples quality.Methods: Firstly,on-line data were acquired using full-scan,and accurate MS/MS data were obtained using a novel SWATH acquisition strategy.We specified a mass range of 100~1000 Da for our SWATH MS/MS acquisition with 30 scan cycle.The application of SWATH indicated sequential window acquisition of all theoretical fragments according to multiple precursor ion windows.Secondly,Peak ViewTM software and Master ViewTM software were used to post-acquisition data mining techniques.Then,structures for the compounds were successfully identified based on accurate mass and the mass fragmentation pathways.Finally,the useful parameter Clog P,was introduced to distinguish the structural isomers.The established method was applied to analyze 24 samples from different areas.PCA was used to find main factors related to quality.The chromatographic separation was carried on a Phenomenex Kinetex C18(100 mm × 3.0 mm,2.6 ?m)with a guard column Phenomenex Kinetex C18(4.0 mm × 3.0 mm,2.6 ?m).The column temperature was set at 25 °C.The mobile phase consisted of water(A)and methanol(B)with a flow rate of 0.3 m L/min.All the analytes were detected within 37 min.The injection volume was 2 ?L.Results: Based on the proposed method,a total of 60 ent-kaurane diterpenoids,including 33 in 7,20-epoxy-ent-kauranes,7 in 6,7-seco-emein,3 in 7,20-diepoxy-ent-kauranes,2 in 6,7-seco-aldactone,5 in C-20-oxygenated-ent-kauranes,10 in C-20 non-oxygenated ent-kauranes,were identified out.And then,the mass spectrometry cracking rule of diterpenoids in IR herbs was tentatively summarized.PCA results revealed there were significant differences in different batches of samples.At the same time,PCA suggested that C2 and C56 could be potential chemical markers for the discrimination of different batches of IR herbs.Conclusions: This study coupled UHPLC-Q-TOF-MS-SWATH the qualitative analysis of ent-kaurane diterpenoids in IR extracts.This is the first report on the qualitative analysis of ent-kaurane diterpenoids based on SWATHTM method.The method is quick,simple,rapid,specific,sensitive,accurate and reliable for qualitative and quantitative analysis.This stragety provided a practical and feasible way to analyze of the chemical ingredients in IR herbs.And this study could also be used for evaluation the holistic quality control in IR herbs and other TCM plant.At the same time,PCA could provide not only the foundation for evaluation of the quality difference but also rational use of drug resources and save medicine resources.Part two Identification and comparative oridonin metabolism in different species liver microsomes by using UHPLC-QTOF-MS/MS and PCAObjective: To develop an UHPLC-Q-TOF-MS/MS method for identifying and comparing of ORI metabolites in human,monkey,rat,and mouse liver microsomes,as well as to explain the metabolic pathway and metabolic type of the ORI in vitro.Meanwhile,PCA was introduced for better evaluation the metabolism differences.Methods: The liver microsomes incubation system of ORI was developed and the blank group,control group and sample group were obtained by vortex and centrifugation.Then,detection was performed by UHPLC-Q-TOF-MS/MS system.Firstly,on-line data were acquired using full-scan,and accurate MS/MS data were obtained using an effective MMDF(multiple mass defect filter)and DBS(dynamic background substract)-dependent data acquisition method.Next,the post-acquisition data mining was performed using various data-mining tools such as XIC(extract ion chromatogram),MDF(mass defect filter),PIF(product ion filter),and NLF(neutral loss filter).Then,the structures of the ORI metabolites were clarified based on the accurate mass measurement,relevant drug bio-transformation knowledge,previously investigated fragmentation regulations of ORI and MS/MS spectrum of metabolites.Finally,the useful parameter Clog P,was introduced to distinguish the structural isomers.The chromatographic separation was performed a Phenomenex Kinetex C18 column(100 mm×3.0 mm,2.6 ?m)with acetonitrile and water as gradient eluents.The temperature was set at 25 oC.The mobile phase flow rate was set at 0.4 m L/min and the injection volume was 5 ?L.The elution programme was optimized for 15 min.At the same time,the metabolism differences of ORI in the liver microsomes of four different species were investigated using a PCA based on the metabolite absolute peak area values as the variables.Results: Based on the above method,27 metabolites,including 24 metabolites in human liver microsomes(HLMs),14 metabolites in monkey liver microsomes(Mon LMs),5 metabolites in rat liver microsomes(RLMs),and 2 metabolites in mouse liver microsomes(Mou LMs)were structurally characterized.Meanwhile,the postulated major metabolic pathway of incubated ORI with HLMs,Mon LMs,RLMs,and Mou LMs was inferred.ORI mainly undergoes dihydroxylation and dehydration reactions,particularly the dihydroxylation metabolism.The PCA results show that the metabolism of ORI in 4 different species has a remarkable interspecies difference,and monkeys should be selected as models of human in future pharmacology and toxicology.Conclusions: This is the first research of the identification and comparing of metabolites of ORI in vitro by UHPLC-Q-TOF-MS/MS,and then PCA was introduced to investigate metabolic similarities and differences among four species.This study should be helpful when selecting a suitable species for ORI toxicology and efficacy evaluation.Moreover,these proposed methods will provide references to study other ent-kauranes diterpenoids.