Qualitative Analysis And In Vivo Metabolic Profiling Of Farfarae Flos By UHPLC-Q-TOF-MS Technologies And Study On Characteristic Fingerprints Of Flos Genkwa And Wikstroemia Chamaedaphne

Farfarae flos,the dried flower buds of Tussilago farfara L.,also known as Donghua,is mainly distributed in Hebei,Henan,Shanxi,Hubei,Sichuan and other places.It has been listed in the Chinese Pharmacopoeia and is usually used to treat coughs,bronchitic and asthmatic conditions as an important traditional Chinese medicine.Several compounds have been isolated from this medicine,including sesquiterpenes,triterpenes,flavonoids and pyrrolizidine alkaloids etc.Several pharmacological investigations have demonstrated that sesquiterpenoids contained in the Farfarae flos have many biological activities,including the inhibition of platelet activity and hypertensive effect.Besides those therapeutic actions,the pyrrolizidine alkaloids（HPAs）can be hepatoxic.However,the in vivo metabolism study of Farfarae flos extract is still not reported.The present study aims to analyze and identify metabolites of major bioactive components and toxic ingredients by UHPLC-Q-TOF-MS/MS.Xuelizhike Syrupusb has been listed in Fifteenth volumes of Chinese herbal prescription of the Drug standard of ministry of health.It is constituted of Pear ointment,Folium eriobotryae,Radix asteris,Farfarae flos,Balloon flower,Semen armeniacae Amarum and Radix peucedani and usually used to treat gas inverse cough and lung asthenia cough conditions.However,the quality standard of Xuelizhike Syrupusb has no content control project,the present study aims to determine the content of multiple components by UHPLC-Q-TOF-MS/MS.Flos genkwa,the dried flower buds of Daphne genkwa sieb.et Zucc.,has been listed in the Chinese Pharmacopoeia and usually used to treat water swelling and phlegm retention syndrome as an important traditional Chinese medicine.Wikstroemia chamaedaphne,the dried flower buds of Wikstroemia chamaedaphne Meisn.,is usually used to treat water swelling and laxative.As a fake of Flos genkwa,Wikstroemia chamaedaphne was often regarded as Flos genkwa to treat disease in the market.Because the functions are not exactly same,they can not be used confusing.The study established the fingerprint of Flos genkwa and Wikstroemia chamaedaphne,which can be used for the identification between them.Part one The analysis of the chemical composition of the Farfarae flos by UHPLC-Q-TOF-MSObjective:The chemical constituents in the methanol extract from the Farfarae flos were rapidly identified using UHPLC-Q-TOF-MS in positive and negative ion modes.Methods:The analysis was performed on an Agilent Poroshell 120EC-C₁₈ chromatographic column（2.1 mm×100 mm,2.7μm）.The mobile phase consisted of acetonitrile and 0.1%formic acid.In positive ion mode,gradient elution:0¹ min,5%¹7%B;1³ min,17%¹9%B;3¹4 min,19%⁴4%B;14¹6 min,44%⁶6%B;16²6 min,66%⁸7%B;26²8 min,87%⁹5%B;28³3 min,95%B.In negative ion mode,gradient elution:0²min,5%¹4%B;2¹0min,14%³2%B;10¹5 min,32%B.The flow rate was 0.4 mL/min,the injection volume was 5μL.The information of the compounds was analyzed by positive and negative ion modes mass spectrum information,elements composition,reference substance retention time or mass spectrum parameters of compounds in literature.Results:Thirty-four compounds in Farfarae flos extracts were identified,combined with provided accurate molecular weight compounds by UHPLC-Q-TOF-MS,including twelve kinds of terpenoids,eight kinds of flavonoids,seven kinds of phenolic acids,two kinds of pyran compounds,one kind of phenolic ketones,one kind of fat ketones and three kinds of alkaloids.Conclusion:The method provides the theory basis for quality control and the clinical reasonable application and provides the reference for clarifying its efficacy material base.Part two Identification of metabolites of main ingredients in rat after lavage Farfarae flos extract by UHPLC-Q-TOF-MS/MSObjective:To identify the metabolites of two kinds of main active ingredients and hepatotoxic pyrrolizidine alkaloids（HPAs）in plasma,bile,urine and feces samples of the rats after oral administration of Farfarae flos extract in vivo.Methods:Different time points of plasma,bile,urine and feces of rats after oral administration of Farfarae flos extract at a dose of 50 g/kg were collected,then the metabolites were detected and analyzed by UHPLC-Q-TOF/MS.The chromatographic separation was carried on Poroshell 120 EC-C₁₈（2.1mm×100 mm,2.7μm）,The column temperature was maintained at 25℃.The mobile phase consisted of water containing 0.1%formic acid（A）and acetonitrile（B）.The gradient elution program was optimized for the separation,and the program was as follows:5²5%B from0 to10 min,25⁴5%B from 10 to 12 min,45⁹5%B from 12 to 40 min,95%B from 40 to 45 min.Elution was set to a flow rate of 0.4 mL/min and the injection volume was 5μL.Results:A total of 35,37,18 and 9 metabolites of tussilagone,methl butyric acid tussilagin ester,senkirkine and senecionine in rats were tentatively identified in plasma,bile,urine and feces.Conclusion:This study is the first reported analysis and characterization of the metabolites and the proposed metabolic pathways might provide further understanding of the pharmacokinetics and the pharmacological action mechanism of Farfarae flos extract in vivo.Part three Quantification of seven chemical compositions in Xuelizhike Syrupusb by UHPLC-Q-TOF-MS/MSObjective:To develop an UHPLC-Q-TOF-MS/MS method for the determination of marmesin,xanthotoxin,bergapten,isopimpinellin,tussilagone,praeruptorin E,shiononein Xuelizhike Syrupusb.Methods:Analysis was performed on an Agilent Poroshell 120 EC-C₁₈column（2.1 mm×100 mm,2.7μm）eluted with 0.1%formic acid 1mmol/L ammonium formate（A）and acetonitrile（B）in a gradient program.The flow rate was 0.4 mL/min,the injection volume was 5μL,and the column temperature was 40℃.Electro spray ionization（ESI）was in the positive ion mode.Results:The regression equations showing linear relationships between peak areas and contents of each compound were obtained（r²>0.9950）.The average recoveries of the compounds ranged from 97.13%to 103.1%.Conclusion:The method is rapid,simple and sensitive and can provide the scientific basis for the quality control of Xuelizhike Syrupusb.Part four Study on the HPLC characteristic fingerprints of Flos genkwaand Wikstroemia chamaedaphneObjective:To establish the characteristic fingerprints of Flos genkwa and Wikstroemia chamaedaphne.Methods:HPLC-UV was used to establish the characteristic fingerprint,The analysis was performed on an Diamonsil C₁₈ chromatographic column（4.6 mm×250 mm,5μm）.The mobile phase consisted of acetonitrile and0.1%formic acid,gradient elution,the flow rate was 0.7 mL/min,the detection wavelength was set at 350 nm,the injection volume was 20μL.Results:The characteristic fingerprints of Flos genkwa and Wikstroemia chamaedaphne were constructed,six characteristic peaks of Flos genkwa and four characteristic peaks of Wikstroemia chamaedaphne were established by HPLC-UV.Conclusion:The established method can provide the scientific basis for the identification and quality control of Flos genkwa and Wikstroemia chamaedaphne,and provide a guarantee for the safety of clinical medication.