| Wu Zi Yan Zong formula（WZYZ）is an ancient prescription which commonly used in clinical practice in China.It has proven clinical effects on supplementing kidney and improving spermatorrhea.It is mainly used to treat nocturnal emission,impotence,premature ejaculation,unclear after urination,and infertility caused by kidney deficiency.At present,the researches on the WZYZ have been mainly focused on qualitative and quantitative analysis and pharmacological study of one active constituent or more.So far the chemical composition of WZYZ has not been comprehensively explored,restricting further application and development.Therefore,this study is aimed at comprehensive analysis of the chemical constituents of WZYZ by high performance liquid chromatography（HPLC）and high performance liquid chromatography quadrupole time-of-flight mass spectrometry（UHPLC-Q-TOF-MS）.Schisandra chinensis is one of the herbs formulated in WZYZ.The Schisandra chinensis is mainly used to treat prolonged cough asthma,dream residual semen,recurrent urine,prolonged diarrhea,self-sweat and night sweat,internal heat and thirst and so forth.The lignans were found to be the main active compounds of Schisandra chinensis in the preliminary experiments and by literature retrieval.This kind of ingredients not only has certain effects on the kidney,liver and reproductive system,but also has pharmacological effects such as antibacterial,immune enhancement,anti-tumor and anti-human immunodeficiency virus（HIV）.Many researchers have studied the in vivo metabolic dynamics and metabolites of lignans,but the analysis of metabolites in vivo is still open for further study to clarify the components that play the efficacy of substances.In this research,UHPLC-Q-TOF-MS is to be used to study the in vivo metabolisms of the main lignans of Schisandra chinensis,for further illustration of the pharmacodynamics basis of WZYZ.Part one Study on fingerprints of Wu Zi Yan Zong by HPLCObjective:To establish the HPLC fingerprints of Wu Zi Yan Zong formula and to identity the characteristic peaks ion.Then the peaks of every single herb were matched with the peaks of WZYZ formula in order to study the chemical composition of the formula.Methods:The HPLC-DAD analysis was carried out using acetonitrile-0.4%phosphate acid aqueous solution at the flow out on a Waters Symmetry（?）C18（250 mm×4.6 mm,5μm）,using a mobile phase of rate of 1.0m L/min.The detection wavelength was set at 254 nm and column temperature was maintained at 35℃.Similarity on 12 batches of WZYZ was estimated,and peak pattern matching of the original medicine was conducted.Results:The results showed that 24 common peaks were defined as follows:The peaks No.1 was gallic acid,No.4 was geniposidic acid,No.8was chlorogenic acid,No.19 was hyperin,No.20 was isoquercitrin,No.21was acteoside,No.22 was kaempferol-3-O-rutinoside,and No.23 was isoacteoside.The peak pattern matching showed that WZYZ had 8（No.2,3,6,7,8,11,12 and 18）,10（No.6,7,8,9,10,15,16,19,20 and 24）,7（No.1,3,7,13,14,17 and 22）,3（No.4,21 and 23）,and 1（No.3）matching peaks with Lycium barbarum,Cuscuta chinensis,Rubus chingii,Plantago asiatica,and Schisandra chinensis,respectively.The fingerprint similarity among the samples were all over 0.959.The fingerprint similarity between the chromatographic control fingerprint and samples were all over 0.979.Conclusion:Similarity evaluation combined with the peak matching of fingerprint for WZYZ could provide the scientific and simplicity methods for its identification and quality control.Part two The analysis of 117 kinds of chemical compositions of the WZYZ by UHPLC-Q-TOF-MSObjective:The chemical constituents of WZYZ were rapidly identified using UHPLC-Q-TOF-MS in positive and negative ion modes.Methods:The analysis was preformed on a Thermo Accucore C18chromatographic column（150 mm×2.1 mm,2.6μm）.The mobile phase consisted of 0.1%acetic acid-2 mmo L/L ammonium acetate and acetonitrile.The gradient elution method was carried out respectively in positive and negative ion mode.The flow rate was 0.4 m L/min,the injection volume was 2μL.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.Then the identified chemical compositions were also traced back to its original herbs.Results:One hundred and seventeen compounds in WZYZ were identified,combined with provided accurate molecular weight compounds by UHPLC-Q-TOF-MS,including of twenty senen kinds of flavonoids,forty six kinds of phenylpropanoids,seventeen kinds of terpenoids,twenty two kinds of organic acids and five other ingredients.Conclusion:The method provides the theory basis for quality control and clinical reasonable application and provides the reference for clarifying its efficacy material base.Part three Identification of metabolites of main ingredients in rat after lavage Schisandra chinensis extract by UHPLC-Q-TOF-MSObjective:To identify the active ingredients and the metabolites of six kinds of the main compounds（Schizandrin A、Schisandrol A、Schizandrin B、Schisandrol B、Schizandrin C、Schisantherin A）in plasma,bile and urine samples of the rats after oral administration of Schisandra chinensis extract in vivo.Methods:Different time points of plasma,bile and urine of rats after oral administration of Schisandra chinensis extract at a dose of 12 g/kg were collected,the the metabolites were detected and analyzed by UHPLC-Q-TOF-MS.The chromatographic separation was carried on Thermo Accucore C18（150 mm×2.1 mm,2.6μm）,the column temperature was maintained at 40℃.The mobile phase consisted of 0.1%acetic acid-2mmo L/L ammonium acetate and acetonitrile.Elution was set to a flow rate of0.4 m L/min and the injection volume was 4μL.Results:A total of 33 kinds of prototype components and 149（130kinds of phase I and 19 kinds of phase II）metabolites of the six main active ingredients were identified in plasma,bile and urine.To the best of our knowledge,75 metabolites among the 149 were first reported.Conclusion:In this paper,the in vivo metabolites of Schisandra chinensis were deeply studied,and the proposed metabolic pathways might provide further understanding of the pharmacology and clinical application of Schisandra chinensis. |
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