Differential Profiling Of Chemical Constituents From Different Medicinal Parts Of Panax Notoginseng And Its Preparations

Notoginseng originates from the dry roots and rhizomes of Panax notoginseng(Burk.)F.H.Chen.It is widely used in the treatment of cardiovascular diseases and traumatic bleeding.Traditionally only the main roots were used as the medicinal organ of Notoginseng by removal of the rhizome and rootlets.In recent years,increasingly widespread use of Notoginseng and Notoginseng-containing medicinal and health products led to a quick shortage of Notognseng crude drug resource,and meanwhile,it was reported that the rhizomes contain not only similar chemical profile to those of roots,but also higher contents of saponins based on the conventional chromatographic analyses.In considering the effective utilization of medicinal resources and the convenience of origin-processing,the Notoginseng crude drug has been revised as the root and rhizome of Panax notoginseng(Burk.)F.H.Chen,since the 2005 edition of Chinese Pharmacopoeia.In addition,foreign pharmacopoeia and the Chinese medicinal material standards in Hong Kong also have different provisions on the medicinal part of panax notoginseng.The combination of the two medicinal parts(roots and rhizomes)of Notoginseng has been argued not only by TCM practitioners but also by manufacturers.Firstly,It was reported that the root and rhizome of notoginseng showed different bioactivity in anticoagulation,anti-inflammation and antioxidation.Secondly,some of the final products like Xueshuangtong tablets,injections are prepared from the main roots,while some others like Xuesaitong tablets,injections,are from the rhizomes.Furthermore,the final products,Xuesaitong injection(prepared from the rhizomes)and Xueshuangtong injection(prepared from the roots)indicated more or less adverse drug reactions(ADRs)in clinical.T Pharmacological study results showed that notoginsenosides achieved the purpose of treating diseases through the integration of multi-component,multi-signaling pathway regulation and multi-mechanism.Therefore,it can be inferred that the difference in the composition of saponins is bound to affect the safety and efficacy of drugs.Leaf and flower of notoginseng also were abundant in ginsensides.In order to further clarify the chemical differences from different medicinal parts of notoginseng in detail,especially for the confused two parts of root and rhizome,and standardize and guide usage of different medicinal part scientifically,then improve quality evaluating method,profiling of the differential analysis was carried out by using liquid chromatography coupled with mass spectrometry.Quality consistency evaluation between XST and XSh T injection has been completed subsequently.A strategy by combing characteristic fingerprinting based on precursor ions graph of UHPLC-MS/MS coupled with charged aerosol detection(CAD)and targeted separation using online multi heart-cutting 2DLC-CAD/MS method was been developed to screen,identify and confirm the intrinsical markers which can be utilized to discriminate intuitively the roots and rhizome of Notoginseng from each other in the first chapter of this thesis.The product ions m/z of 459,475 and 455 which were the characteristic MS~2ions to diagnose PPD,PPT and OA type of ginsenosides depending on their fragmenting pattern in MS/MS were chosen to acquire their precursor ions chromatograms to profile the chemical difference between root and rhizome of notoginseng intuitionally.The monitoring for other types of ginsensides which not belonged to the above three types could be covered by charged aerosol detection(CAD)in parallel with MS/MS.Serval potential markers to discriminate between two medicinal parts of notoginseng have been screened from their characteristic fingerprints with the help of partial least squares discriminant analysis(PLS-DA).Multi heart-cutting 2DLC(MHC-2DLC)method which select C30 as the second dimensional column by keeping first dimensional separation performance has been constructed to carry out targeted separation for these potential markers according to retention time.Five differential markers have been verified to be reliable through 33 batches of herbal material finally.Five markers were identified tentatively to chikusetsusaponin L5,stipuleanoside R2,ginsenoside Rb2 and malonyl-ginsenoside Rb1,Rd by comparing with the known references based on the retention time and high resolution MS and its MS~2 in MHC-2DLC-MS/MS.The first three non-mal-ginsenosides of these markers were confirmed to be as differential markers according to mass transferability from medicinal materials to preparations finally.The quantitative method for five active ginsenosides(notoginsenoside R1,ginsenoside Rg1,Re,Rb1,Rd)plus three discriminative markers was developed to improve the quality evaluating standard by fully automated 2DLC coupled with CAD.Method validation results showed that the linear correlation,reproducibility and recoveries for all the markers meet the requirements of content determination and also can be operated in practice.Therefore,the quality evaluation methods of medicinal materials were improved.Rapid Chemical profiling to ginsenosides in the leaves and flowers of notoginseng were carried out by constructing an online comprehensive 2DLC-Q-orbitrap HRMS system combined with UHPLC-Q-orbitrap-HRMS in the second chapter of this thesis.Sample was extracted by two-steps pressurized solvent extraction method.The first step was to remove of fat-soluble chlorophyll with chloroform and the second step was to extract the targeted analyte with methanol rapidly.Sample solution was separated into30%,50%,70%and 95%methanol eluate by Retain PEP column roughly for sample injection analysis.The online comprehensive 2DLC was constructed by hydrophilic interaction chromatography combined with reversed-phase chromatography.The solvent effect was effectively weakened by using 350μL static mixer as 2D interface and the sampling volume below 80μL.The results showed that ~1D-HILIC and ~2D-RP had good orthogonality for ginsenosides separation.The theoretical peak capacity reached 6879,and the effective peak capacity was 948,which was nearly 7 times higher by online HILIC×RP-Q-Orbitrap HRMS system than the first dimensional separation.It was beneficial to identify the compound accurately depending on high-quality MS~2 data by decreasing the overlapped peaks.A total of 111 ginsenosides were identified from leaves and flowers of notoginseng.Five compounds were not reported in the leaves and flowers of notoginseng before.It was confirmed that there was the special structural type of ginsenosides with malonyl-sapoginin in leaves and flowers of notoginseng.Solid foundation was laid to prepare this type of ginsensides by MS-guided separation method according to fragmentation pattern behaved in MS/MS.The pre-experiment showed that the relative amount of non-saponins in the aqueous extract of panax notoginseng was about 47.34%.Ion-pairing chromatography on porous graphitic carbon(PGC)column coupled with Charged Aerosol Detection(CAD)was developped to profiling to water-soluble non-saponins part from root and rhizome of notoginseng in the third chapter of thesis.The SPE column of Retain PEP was used to enrich and prepare the sample solution.Serval critical conditions,such as the types of ion pair reagents,concentration and gradient program were optimized.The chemical differences between root and rhizome were compared and evaluated by means of HCA,similarity analysis and PCA.Finally,we constructed the fingerprint analytical method successfully,which also could be used to quantify dencichine in notoginseng.Results showed that the chemical profile of water-soluble non-saponins part was similar between root and rhizome holistically.Nevertheless,the contents of 3 components and their relative proportions to internal reference were significantly different with the help of PLS-DA recognition.In addition,panax notoginseng,panax ginseng and panax quinquefolium were also compared from water-soluble non-saponins part by the constructed fingprint analytical method.The results showed that the chemical profiles of water-soluble and non-saponins part were similar holistically.However,the average content of dencichine in notoginseng was at least 4.3 times higher than that of the other two herbs,which led to a large difference in the proportion of GABA to dencichine.Because dencichine and GABA have the oppsite effects on central nervous system,content-effect relationship between them will also affect the orientation of the three traditional Chinese medicines on the central nervous system to some extent.In the fourth chapter,the chemical differences of xueshuantong(XSh T)and xuesaitong(XST)injection deriving from root and rhizome of panax notoginseng respectively were analyzed.The comprehensive chemical profiling to the differences between XST and XSh T was carried out by RPLC-CAD,HILIC-UV and 2DLC-CAD constructed in the above first chapter,and combined with similarity evaluation software,hierarchical cluster analysis(HCA)and principal component analysis(PCA).Qualitative and quantitative analysis for the differential components were accomplished by UHPLC-HRMS coupled with CAD.The influence of heating time on composition change was investigated through thermal damaging test in the process of evaluating the chemical consistency for XST injection.As a result,a total of 17 differential components were confirmed and identified by LC-HRMS,among which,except for 3 differential markers coming from different parts of medicinal materials,the other 14 components were all derived from different preparating processes of total saponins between XST and XSh T injection.The consistency evaluation of XST showed that the similarity between injection,intermediate and its freeze-dried powder was slightly lower.The results of thermal-damaging test confirmed that the ginsenoside compounds could transform each other and keep mass balance for the varied components calculated by relative area roughly.Total10 compounds with obvious changes were identified in the experiment preliminarily.It was inferred that the reactions of deglycosylation and dehydration of sapogenin in the process of sterilizing at high-temperature were the intrinsic reasons for the transformation of components and inducing the difference in injection.The conclusion of this thesis not only improves the quality evaluating standard of panax notoginseng but also provides a scientific basis for the rational usage of different parts and its total saponins extracts.Solid foundations have been laid for the comparative study on the Pharmacological effects from different parts of panax notoginseng and the discovery of potential active compounds in the future.