| China is the place where Chinese herbal medicine originated,commonly used Chinese herbal medicine has about 5000 species,Chinese herbal medicine affected by various aspects in its growth process such as geography、environment、climate、planting methods and so on,simultaneously the uniqueness of the processing technology,formed the authentic medicinal materials with typical regional features.Polysaccharides widely exists in Chinese herbal medicine,is one of the important active substances which contribute playing particular efficacy of Chinese herbal medicine,and polysaccharides is also used for quality control of Chinese herbal medicine.As the development of Chinese medicine polysaccharide drugs and health products,it’s increasingly important to set up the impeccable and reliability quality control system of Chinese herbal medicine.This paper relies on high performance liquid chromatography-electrospray ionization time of flight mass spectrometry(HPLC-ESI-QTOF-MS)to establish a method to analyze the glycoside bond type of oligosaccharide,and classifying the raw material according to origins or grades in the meantime,to study the fingerprint spectrogram of polysaccharides hydrolysate from Panax ginseng C.A.Meyer、Prunella vulgaris L、Cordyceps sinensis(Berk.)Sacc.And analyze their oligosaccharide structure.Finally,this paper uses chemometrics method to analyze the difference of three kinds of Chinese herbal medicine in origins or grades and build the complete analysis method of Chinese herbal polysaccharides saccharides mapping relied on HPLC-ESI-QTOF-MS.This paper is divided into five parts:Chapter I:It introduces the structural and active research progress of PPG、PPVL and PCS,and summarizes various saccharides analysis methods.Then it describes the superiority of Liquid chromatography-mass spectrometry(LC-MS)technology in saccharides analysis and expounds the research progress of saccharides mapping.Chapter Ⅱ:The crude PPG、PPVL and PCS are extracted by hot water extracting and alcohol precipitation.The physico-chemical properties were determined by chemical method,and the results indicates that:the total sugar content of PPG originated from Heilongjiang(PGl)、Liaoning(PG2)and Jilin(PG3)are 95.61%、93.24%and 93.89%respectively,the uronic acids content are 10.16%、9.54%and 9.22%respectively,the protein contents are 2.51%、0.66%and 2.44%respectively;the total sugar content of three grades PPVL1、PPVL2 and PPVL3 originated from Zhangshu of Jiangxi are 46.17%、55.09%and 50.63%respectively,the uronic acids content are 21.11%、19.55%and 22.20%respectively,the protein contents are 34.06%、33.97%and 35.54%respectively.the total sugar content of four grades PCS1、PCS2、PCS3 and PCS4 originated from Naqu of Tibet are 63.48%、66.79%71.96%and 79.32%respectively,the uronic acids content are 7.69%、4.84%6.09%and 9.01%respectively,the protein contents are 14.13%、15.66%、16.69%and 20.11%respectively.The total sugar content of PCS5 originated from Yushu of Qinghai is 63.89%,the uronic acids content are7.74%,the protein contents are 19.21%.The relative molecular weight analysis relied on high performance gel permeation chromatography show that the relative molecular weight of PPG is 4.3×10~3 kDa,the relative molecular weight of PPVL is2.1×10~3 kDa,the relative molecular weight of PCS is 3.2×10~3kDa.Chapter Ⅲ:The MS/MS spectra of 6 disaccharides with different glycosidic linkage are studied by ESI-QTOF-MS without derivation in negative-ion mode.The different disaccharides with different glycosidic linkage fracture in different way.The glycosidic linkage can be determined by the mass loss of the characteristic fragment ions.It is concluded that the fragmentation regularity of different glycosidic linkage in MS/MS,and the glycosidic linkage of 5 linear oligosaccharides include 4 trisaccharides and 1 tetrasaccharides are determined successfully.At addition,the MS/MS spectra of Xylotriose is analyzed particularly.This method don’t need derivation and have high sensitivity,good accuracy,and only demand a small quantity of samples.Chapter Ⅳ:The analytical method of 3 Chinese herbal polysaccharides hydrolysate is builded relied on high performance liquid chromatography-electrospray ionization-time of flight mass spectrometry.The partial acid hydrolysis of polysaccharides samples without derivatives was analyzed in negative-ion mode with 0.1%aqueous solution of formic acid and acetonitrile as mobile phase.Combining with the fragmentation regularity of different glycosidic linkage in MS/MS.The results show that:PPG mainly contains 1,4-pyranohexosan and a few of 1,4-pyranohexosan,PPVL contains 1,4-pyranohexosan and may contains 1,4-pentosan,PCS contains 1,4-pyranohexosan and 1,4-pyranohexosan,and may also contains particular pyranohexosan with unit structure of-Hex-1,4-Hex-1,6-.Chapter V:Three Chinese herb medicine polysaccharides samples was analyzed and modeled used by significance analysis(ANOVA and Fold Change analysis)、clustering analysis、principal component analysis(PCA)and partial least square discriminant analysis(PLS-DA),each groups of three Chinese herb medicine samples obtain the expected distinguish according the difference of origins or grades,and several potential marker compounds which can distinguish each groups are found preliminary,include:9 potential marker compounds of distinguishing Panax ginseng C.A.Meyer from three origins(Heilongjiang、Liaoning、Jilin);11 potential marker compounds of distinguishing three grades Prunella vulgaris L origined from Zhangshu of Jiangxi;29 potential marker compounds of distinguishing Cordyceps sinensis(Berk.)Sacc.from two origins(Naqu of Tibet、Yushu of Qinghai)and 76 potential marker compounds of distinguishing four grades Cordyceps sinensis(Berk.)Sacc.origined from Naqu of Tibet.Based on HPLC-ESI-QTOF-MS detecting,and combined with multifarious chemometrics methods to analyze Chinese herb medicine,then to set up the fingerprint spectrogram and model of Chinese herb medicine,and to find the characteristic potential marker compounds finally.The method in this study is all-purpose and common approach used in fingerprint spectrogram analysis of Chinese herb medicine such as saccharide mapping、geographical origin traceability、quality monitoring and so on with its high reliability、high accuracy and extensive applicability. |
