Studies On The Pharmacodynamic Substances Of Ginkgo Biloba Extract And Metabolites Identificaion Of Helicid In Vivo And In Vitro Based On Liquid Chromatography-mass Spectrometry Technique

Ginkgo biloba extract is the dry leaf of Ginkgo biloba L.It is mainly used for the treatment of blood stasis,chest pain,hemiplegia,lung deficiency,cough and hyperlipidemia.Ginkgolide B（GB）is one of the diterpenoid of Ginkgo biloba with a C20 cage skeleton consisting of six members rings,and is the important active ingredients of the traditional Ginkgo biloba extract.This experiment was the first study by using Ultra-highPerformanceLquidChromatography-Quadrupole Time-of-Fligh mass spectrometry（UHPLC-Q-TOF-MS）to to identify and analyze metabolites of GB in rat liver microsomes（RLMs）and in vivo（plasma,bile,unire and feces）,Fifty-three different metabolites of GB were detected by comparing with the blank samples,respectively.And this study developed a HPLC-QTRAP-MS/MS method for the content determination of 22 compounds in Ginkgo biloba extract and 4saponin components and 6 flavonoids components in Ginkgo Radix Notoginseng Fructus Crataegi Tea Polyphenol.The method was simple and sensitive.It can provide a scientific basis for the quality control of Ginkgo biloba extract and Ginkgo Radix Notoginseng Fructus Crataegi Tea Polyphenol.Helicid is an active natural aromatic phenolic glycoside ingredient originating and has the significant effects of sedative hypnosis,anti-inflammatory analgesia and antidepressant.In this study,we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter（MMDF）and dynamic background subtraction（DBS）in UHPLC-Q-TOF-MS.Twenty metabolites of Helicid were detected by comparing with the blank samples,respectively.This experiment not only proposed a method for rapidly detecting and identifying metabolites,but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo.Part one Identification of metabolites of Ginkgolide B in vivo and in vitro using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometryObjective:To build a method to detectand identify GB metabolism in vivo and in vitro by employing UHPLC-Q-TOF-MS technology simultaneously.Methods:Agilent Poroshell 120 SB-C18 column（2.1×100 mm,2.7μm）,gradient elution,A for aqueous phase（0.1%formic acid-acetonitrile）,B for organic phase（0.1%formic acid-water）,elution procedure 0%⁵%B,0 min¹ min;1 min⁵ min,5%²5%B;25%⁹5%B,5 min¹5min.source temperature is 650°C,column temperature is 40.0°C,electricity spray ionization source（ESI）,negative ion selective ion detection.Eighteen spraque-dawley rats were divided into three groups,six in the feces and urine group,six in the plasma and bile groups,extracted with acetonitrile,reconstituted with 95%acetonitrile,and the supernatant was injected.MetabolitePilot^TM 2.0 was analyzed for metabolites and analyzed using PeakView^?2.2 software and Masterview^?1.1 software chromatograms.Results:A total of 53 metabolites were identified in vitro and in vivo.Including of 17 metabolites in RLMs,18 metabolites in plasma,26metabolites in bile,30 metabolites in urine and 37 metabolites in feces.Conclutions:This study established a powerful and quick tool to potential metabolites identification.And the active metabolites will be greatly helpful in elucidating the potential biological mechanism of terpenoid compound derivatives in clinical application.Part two Quantification of 22 compositions in Ginkgo biloba extract by HPLC-QTRAP-MS/MSObjective:To Establish a HPLC-QTRAP-MS/MS method for the simultaneous determination of 22 components in Ginkgo biloba extract.Methods:The chromatographic column was Wonda Cract ODS-2C18 column（4.6 mm×150 mm,5.0μm）,the mobile phase was water（C,0.1%formic acid）-acetonitrile（D,0.1%formic acid）,gradient elution.The ion source was analyzed by negative ion detection mode and multi-reaction monitoring mode.Results:There was a good linear relationship between peak area and mass concentration of 22 components in 15 batches of Ginkgo biloba extract by HPLC-QTRAP-MS/MS（r≥0.9977）.The recovery was 89.25%±105.9%.The RSD values of precision,stability and repeatability were all less than 3.94%.Conclusions:This study provided a simple,rapid and sensitive method for the determination of 22 components in Ginkgo biloba extract.Part three Quantification of 10 components in Ginkgo Radix Notoginseng Fructus Crataegi Tea PolyphenolObjective:To develop a HPLC-QTRAP-MS/MS method for the content determination of apigenin,luteolin,kaempferol,quercetin,myricetin,isorhamnetin,notoginsenoside R1,ginsenoside Re,ginsenoside Rd and ginsenoside Rb1 in Ginkgo Radix Notoginseng Fructus Crataegi Tea Polyphenol.Methods:Analysis was performed on a Diamonsil C 18 column（150 mm×4.6 mm,5μm）eluted with 0.1%formic acid-water（A）and0.1%formic acid-acetonitrile（B）in a gradient program.The multiple-reaction monitoring scanning（MRM）was employed for the quantification with switching electrospray ion source polarity in negative ion mode.Results:The regression equations showing linear relationships between peak areas and contents of ten compound of in three batches of Ginkgo Radix Notoginseng Fructus Crataegi Tea Polyphenol were obtained by using HPLC-QTRAP-MS/MS method（r≥0.9985）.The average recoveries of the compounds ranged from 88.44%to the precision in terms of RSD was in the range from 0.58%to 1.82%.Conclutions:The method was simple and sensitive.It could be used as a quantitative determination method for the components including flavonoids and saponins in Ginkgo Radix Notoginseng Fructus Crataegi Tea Polyphenol.Part four Identification of metabolites of Helicid in vivo using ultra-highperformanceliquidchromatography quadrupole time-of-flight mass spectrometryObjective:To build a method to detect and identify Helicid metabolism in rats by employing UHPLC-Q-TOF-MS/MS technology simultaneously.Methods:Agilent Poroshell 120 EC-C18 column（2.1×100 mm,2.7μm）,gradient elution,A is organic phase（methanol）,B is aqueous phase（0.1%formic acid-water）,elution procedure is 0 min¹ min0%⁵%B;1min¹0 min 5%²1%B;10 min¹5 min 21%⁴5%B;15min²0 min 45%⁹5%B.source temperature is 650°C,column temperature is 40.0°C,electrospray ionization source（ESI）,negative ion selective ion detection.Twelve SD rats were divided into three groups,four in the feces and urine group,four in the plasma and bile groups,extracted with ethyl acetate,reconstituted with methanol,and the supernatant was injected.Results:A total of 25 metabolites were detected in this experiment,including 10 in feces,11 in urine,6 in bile,and 1 in plasma.The biotransformation route of Helicid was identified as demethylation,oxidation,dehydroxylation,hydrogenation,decarbonylation,glucuronide conjugation and methylation.Conclutions:This is the first study of simultaneously detecting and identifying Helicid metabolism in rats by employing UHPLC-Q-TOF-MS technology.It not only provided useful information for further study of the pharmacology and mechanism of Helicid in vivo,but also provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.