Investigation On The Chemical Constituents In The Isodon Serra(maxim.)hara And Metabolism Of Ponicidin In Vivo And In Vitro

Isodon serra is the dried entire plant of Isodon serra?Maxim.?Hara.it has been used as a local folk medicine in China for treatment of enteritis,jaundice,acute jaundice hepatitis,acute cholecystitis,laryngopharyngitis,lepromatous leprosy and ascariasis.Isodon serra widely distributed in China,and the diterpenoids are the most important active ingredients in Isodon serra.Therefore,it is very significative for the quality of the medicine to detect and identify of the diterpenoids.Since diterpenoids in medicinal materials are diverse and low in content,it is difficult to effectively identify the compounds using conventional techniques.Therefore,it is very necessary to develope a novel and effective detection technique to identify the diterpenoids in Isodon serra sample.Therefore,this study coupled ultra-high performance liquid chromatography-quadrupoletime-of-flightmassspectrometry?UHPLC-Q-TOF-MS/MS?with SWATH?sequential window acquisition of all theoretical fragment-ion spectra?method was employed to quickly identification of diterpeoids ingredients in Isodon serra plants.This study could provide technical support for further development and utilization of the active diterpenoid ingredient.This study could provide technical support for further development and utilization of the active diterpenoid ingredient.These results would be used for evaluation the holistic quality control in Isodon serra herbs.Ponicidin is an active natural ent-kaurane diterpenoid ingredient originating from many Isondon herbs.Currently,ponicidin has attracted increasing attention due to its immunoregulatory,anti-inflammatory and antiviral features especially for upper respiratory tract infections.Drug metabolism research is an indispensable part at various stages of drug discovery and development.Therefore,it is necessary to study the metabolites of ponicidin in vivo and in vitro,and it can provide necessary preliminary research data for the further development and utilization of ponicidin.In this paper,a technique was used to identify the metabolites of ponicidin in vivo and in vitro using UHPLC-Q-TOF-MS/MS.Simultaneously,The mass spectrometry cleavage of metabolites of ponicidin in vitro and in vivo was analyzed.The metabolic differences of different species of liver microsomes were compared and the possible metabolic pathways were speculated.Part one Rapid identification of ent-kaurane diterpenoids in Isodon serra ?Maxim.?HaraObjective:To establish a novel stragety based on UHPLC-Q-TOF-MS/MS for rapid identification the traces diterpenoid ingredient in Isodon serra herbs.Methods:First,the mass fragmentation and fragmentation patterns of nine major ent-kauranes diterpenoids were studied.Next,parent ions were obtained by electrospray ionization?ESI?negative ion full-scan mode,and accurate MS/MS data were obtained using SWATH technique.The TOF-MS full scan was performed in the SWATH mode of the methanol extract of Isodon serra to obtain a total ion chromatogram?TIC?.Then,the known molecular formulas in the database were input into the data processing MasterView software.Combined with the cleavage of the enantio-kautane diterpenoids and the reported cleavage pathways of the compounds in the literature,chromatographic peaks within±5 ppm error range were idented.The chromatographic separation was carried on a Phenomenex Kinetex C₁₈with a guard column Phenomenex Kinetex C₁₈.The column temperature was set at 25°C.The mobile phase consisted of water and methanol with a flow rate of 0.3 mL/min.All the analytes were detected within 37 min.The injection volume was 2?L.Results:A total of 48 ent-kauranes diterpenoids in Isodon serra were identified by the SWATH technique in UHPLC-Q-TOF-MS/MS for the first time.Compounds 6,9,10,11,12,27,22,24,25,37,38,40 have been reported in the literature,and the remaining 36 compounds have been identified for the first time.Simultaneously,the mass spectrometry cracking rule of diterpenoids in Isodon serra herbs was tentatively summarized.Conclusions:This study coupled UHPLC-Q-TOF-MS/MS combined with SWATH technology to rapidly identify 48 enantio-kaurene diterpenoids in Isodon serra for the first time.The method is quick,simple,rapid,specific,sensitive,accurate and reliable for qualitative and quantitative analysis.This study has important implications for understanding the pharmacodynamic material basis and quality control of Isodon serra.Part two Identification of ponicidin metabolites in vivo and in vitroObjective:The UHPLC-Q-TOF-MS/MS method was developed to identify metabolites of ponicidin in vivo and in vitro.Simultaneously,the metabolic differences were compared in vivo and in vitro and metabolic differences between different species,and the metabolic pathways of ponicidin in vivo and in vitro were elucidated.Methods:Urine and bile samples were collected after single oral administration of ponicidin to rats.Then the samples were pretreated by liquid-liquid extraction with ethyl acetate.Simultaneously,the liver microsomes incubation system of ponicidin 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 The chromatographic separation was performed a Phenomenex Kinetex C₁₈column?100 mm×3.0 mm,2.6?m?with acetonitrile and water as gradient eluents.The temperature was set at 25°C.The mobile phase flow rate was set at 0.3 mL/min and the injection volume was 5?L.The elution programme was optimized for 21 min.Firstly,on-line data were acquired using full-scan,and accurate MS/MS data were obtained using SWATH independent data acquisition method.Next,the post-acquisition data mining was performed using various data-mining tools such as MetabolitePilot and MasterView softwares to obtain accurate metabolite information.Then,the structures of the ponicidin metabolites were clarified based on the accurate mass measurement,relevant drug bio-transformation knowledge and MS/MS spectrum of metabolites.Finally,the useful parameter Clog P was introduced to distinguish the structural isomers.Conclusions:In this study,a practical UPLC-Triple-TOF-MS/MS method was developed for the successful characterization of 20 metabolites of ponicidin in vitro and in vivo using the novel SWATH data acquisition method.The major metabolic biotransformation is reduction.hydrolysis,oxidation,methylation and glucuronidation.The above studies provide references for the elucidation and pharmacological studies of ponicidin in vivo and in vitro.The method is rapid and sensitive,and it can be used for analysis of the metabolites of other monomer components in vivo and in vitro.