Comparision Of Quality Between Hawthorn Leaves And Guang Hawthorn Leaves

Hawthorn leaves,known as a Chinese traditional medicine(CTM),and its preparation have been used in the treatment of cardiovascular and cerebrovascular diseases,which have good effects showed by modern pharmacological studies.Hawthorn was known as the Rosaceae Crataegus L.and Rosaceae Malus Mill.The origins of hawthorn were complex.Rosaceae Crataegus L.Crataegus pinnatifida Bge.,Crataegus pinnatifida Bge.Var.major N.E.Br.,Crataegus cnneaia Sieb.et Zucc.and Rosaceae Malus Mill.Crataegus scabrifolia(Fr)Rehd.,Malus doumeri(Bois)Chev,Malus leiocalyca S.Z.Huang were used for medical treatment.In addition to the Chinese pharmacopoeia records of hawthorn leaves(Crataegus pinnatifida Bge.Var.Major N.E.Br.or Crataegus pinnatifida Bge.),some provinces also conventional wide Yun hawthorn leaves and Guang hawthorn leaves.Review of the literature,flavonoids and organic acids were the same compositions in hawthorn leaves metioned in above.However the kinds and contents have bigger difference,which could cause the difference of the pharmacological and clinical curative effect.In order to make reasonable use of hawthorn leaves,33 hawthorn leaves contained in Chinese pharmacopoeia and 8 batches Gaung hawthorn leaves produced in guangdong guangxi province were compared in this study.In previous study,focus was put on the fingerprint and flavonoids content determination by high performance liquid chromatography(HPLC).However,these methods suffered from long run time,low sensitivity and resolution.UPLC has the advantage in increasing resolution and improving sensitivity.The combination of LC and MS can provide high separation efficiency and identification for Traditional Chinese Medicine(TCM).Especially effectivelydetect for the weak ultraviolet absorption,separation effect is poor and content in low levels,which can not applied to LC-DAD qualitative and quantitative analysis.In this study,an ultra performance liquid chromatography(UPLC)method was developed to achieve the reliable fingerprint of hawthorn leaves and Guang hawthorn leaves,which was obtained by hierarchical cluster analysis and principal component analysis and completed simultaneously determination of 5 components.Furthermore,the component in hawthorn leaves were identified by LC-MS/MS.Moreover,8 components,especially for the epicatechin,rutin,shanyenoside A,were deteminted simultaneously by LC-MS.Part one Study on the UPLC fingerprint and simultaneous determination ofhawthorn leaves and Guang hawthorn leavesObjective:To establish the common fingerprint and simultaneously determination 5components method of hawthorn leaves and Guang hawthorn leaves by UPLC,which could improve the quality control level of hawthorn leaves from the qualitative and quantitative view.Methods:Chromatographic separation was conducted on an Acquity UPLC BEH C18 column(50×1.7 mm,1.6 μm)from Waters(Milford,MA,USA)main tained at 30 ℃.The mobile phase made of ACE(A)and 0.1% formic acid in water(B)and the flow rate was 0.2 m L·min-1,The gradient program was as follows: T: 0~4~7~14~16~25~28~30 min,A(12%~12%~17%~17% ~25%~40%~80%~80%),B(88%~88%~83%~83%~75%~60%~20%~20%).The det ection wavelength was set at 320 nm.Evaluate the similarity of hawthorn leaves and Guang hawthorn leaves and mark common peaks with the software of Traditional Chinese medicine similarity evaluation system(2004A version).Then,hierarchical cluster analysis and principal component analysis were used to establish the UPLC common fingerprint model with the peak area ofcommon peaks.Results:1.The UPLC fingerprint of 33 hawthorn leaves were established and 8common peaks were marked.The similarities between the samples and the reference chromatogram differ from 0.441~0.993.2.33 batches samples were divided into three groups.Group Ⅰconsisted of NO.2-NO.21,NO.23-NO.31 and NO.33,group Ⅱ was made of NO.1 and NO.32,and group Ⅲ was NO.22.3.By eliminating group Ⅱ and group Ⅲ abnormal samples,selecting group Ⅰ to re-establish the more accurate fingerprint spectrum,10 common peaks were found and the similarity was between 0.808 and 0.977.4.The UPLC fingerprint of 8 Guang hawthorn leaves were established,and 20 common peaks were marked.The similarities between the samples and the reference chromatogram differ from 0.917~0.977.5.Simultaneously determination 5 components of 33 hawthorn leaves and 8 Guang hawthorn leaves by ESM in the same conditions.Conclusion:The UPLC fingerprint and simultaneously determination methods established in the study were accurate and reliable,which could compare between hawthorn leaves and Guang hawthorn leaves from the semi-quality ative and quantitative view.Part two Study on the chemical components in hawthorn leaves based onLC-MSObjective:Identify the components with the reference substance and literature in hawthorn leaves of different origins by LC-MS to improve its quality control level from the qualitative view.Methods:Chromatographic conditions: The same conditions with the Part one;Mass spectrometry conditions: The conditions of the MS spectrometerwere as follows: ion source gas 1 50 psi,ion source gas 2 50 psi,curtain gas35 psi,temperature 600 ℃,ISVF 5500 V(in positive modal)/-4500 V(in negative modal).Results:11 components were identified by Triple TOF 5600 MS spectrometer with the assist of references and standards.Conclusion:The chemical components were identified by a hybrid triple quadruple time-of-flight mass spectrometer,which could improve the quality control level of hawthorn leaves of different origins from the qualitative view.Part three Multi-constituent determination by HPLC-MS of hawthornleaves and Guang hawthorn leavesObjective:To establish a multi-constituent determination method and add quantit ative indicators of hawthorn leaves and Guang hawthorn leaves,which could improve the quality control level.Methods:Chromatographic separation was conducted on a Poroshell 120 SB-C18column(2.1×100 mm,2.7 μm)maintained at 30 ℃.The mobile phase consist of acetonitrile(A)and 0.1% formic acid in water(B),and the flow rate was at0.15 m L·min-1.The gradient program was as follows: T: 0~10~25~35~40~45~55 min,A(90%~88%~80%~80%~40%~20%~10%),B(10%~18% ~20%~20%~60%~80%~90%).The detection wavelength was also set at 320 nm and the injection volume was 1 μL.Mass spectrometric detection was carried out on an Agilent Technologies6310 mass spectrometer.The parameters of the mass spectrometer were optimized and set as follows: Nebulizer pressure at 20 psi,drying gas flow rate at 10 L·min-1 at 350 ℃,Auto Scan range set from 100 to 700 m/z.Results:8 ingredients of 33 hawthorn leaves and 8 guang hawthorn leaves ofdifferent origins were quantified by LC-MS method.Conclusion:The method established in the study added the quantitative detection index,which was accurate and realiable to evaluate the quality of hawthorn leaves and Guang hawthorn leaves.