Studies On The Chemical Ingredients Of Uncaria Sessilifructus Roxb.Based On Three-dimensional Chromatography/High-resolution Mass Spectrometry Enhanced Scaning Technique

The study of the material basis of the traditional Chinese medicine system is the core of the modernization of traditional Chinese medicine and the basis of quality control and biological effect research.Many types of active ingredients coexist in traditional Chinese medicine,with large differences in acidity and alkalinity,and a wide range of molecular weight,polarity,and content.This brings great obstacles to the discovery of active ingredients derived from traditional Chinese medicine or natural medicine.Liquid chromatography-mass spectrometry is one of the most commonly used techniques for TCM analysis and rapid discovery of TCM active molecules.However,due to a single separation mechanism and low-specificity scanning methods,many trace active ingredients cannot be characterized and identified.Uncaria sessilifructus Roxb.is a vine belonging to the genus Rubia of the Rubiaceae family and is mainly distributed in Yunnan,Guangdong,and Guangxi in China.The Dai people in Xishuangbanna call it the"King of the King",which is a traditional medicinal material in Xishuangbanna and a Xishuangbanna Prescription medicinal materials in hospital preparations are widely used in the region.Modern pharmacological studies have shown that Uncaria sessilifructus has a variety of pharmacological activities such as anti-hypertensive,anti-cancer,anti-inflammatory,analgesic,enhance immunity,anti-convulsant,anti-epileptic,anti-oxidant and so on.Uncaria sessilifructus plants are typical of the coexistence of multiple types of active ingredients,of which alkaloids and triterpene acids are the two main functional components.However,the lack of powerful analytical techniques for the multi-component characterization of Uncaria sessilifructus has hindered the modernization of Uncaria sessilifructus research and further development and utilization.In this thesis,a offline three-dimensional liquid chromatography was constructed using ultra high performance liquid chromatography/quadrupole electrostatic field orbit trap mass spectrometry（UHPLC/Q-Orbitrap MS）with alkaloids and triterpenoids in Uncaria sessilifructus as the main target,by integrating highly orthogonal chromatograms of different mechanisms for full chromatographic separation;establishing improved data-dependent acquisition（DDA）technology including parent ion list（PIL）to sensitively monitor target components of alkaloids and triterpenoids,and collect the decomposition information of other unknown mass components;and then through the analysis of high-resolution mass spectrometry data and the study of the cracking rules,to achieve the systematic characterization and identification of the multi-components of the Uncaria sessilifructus,and systematically explain its material basis.Established a three-dimensional liquid chromatography separation system based on ion exchange chromatography（HILIC）-hydrophilic interaction chromatography（HILIC）and reverse-phase ultra-high performance liquid chromatography（RP-UHPLC）to achieve the classification and system characterization of alkaloids and triterpenoids.Taking into account the great differences in p H between alkaloids and triterpenoid acids,this paper designs a three-dimensional chromatography method based on the differences in p H（first dimension）,polarity（second dimension）,and distribution（third dimension）.First,with the goal of separating alkaloids from triterpene acids,the first-dimensional chromatography examined anion exchange columns and cation exchange columns,respectively.It was confirmed that Pheno Sphere^TM SCX cation exchange columns can completely retain alkaloids,and then achieve the same relationship with three separation of terpenic acid（non-retained）components.The optimal mobile phase is methanol:20 m M ammonium acetate/0.05%formic acid water=58:42.Next,a two-dimensional chromatographic separation system was established with the total alkaloids and total triterpene acids prepared by the IEC as targets.Screening through stationary phases（9 columns）and mobile phases,using CSH Phenyl-Hexyl columns,acetonitrile/0.1%formic acid water as mobile phase,achieving better chromatographic separation of total alkaloids and total triterpene acids under the same conditions（third dimension Chromatography）;Then use the separation of 16 components as an index,compare 6 HILIC columns,compare them for the separation of alkaloids and triterpene acids and CSH Phenyl-Hexyl columns,and select the best orthogonality Acchrom XAmide（R²=0.1619）chromatographic column,acetonitrile/0.1%formic acid water as mobile phase,as second-dimensional HILIC separation conditions.The established SCX×CSH Phenyl-Hexyl×XAmide offlinethree-dimensional liquid chromatography has realized the separation and separation of alkaloids and triterpenoids from the vine,with high selectivity and greatly increased peak capacity.On the Q Exactive Q-Orbitrap high-resolution mass spectrometer,establish a fast positive-negative mode switching PIL/Full MS/dd-MS²（top N）acquisition method,set dynamic exclusion and"If idle-pick others",respectively,at positive and negative ESI In this mode,sensitive characterization of alkaloids and triterpene acids in Uncaria sessilifructus is achieved.First,mass spectrometry filtering（MDF）algorithm was used to filter the first mass spectrometry data of Uncaria sessilifructus,and 17 masses of alkaloids and 9 masses of triterpene acid were obtained,and the respective parent ion lists were constructed.The main ion source parameters（spray voltage,capillary gas temperature,and auxiliary gas temperature）and cracking energy were optimized.As a result,multiple alkaloids and triterpene acids were observed at a spray voltage of 3.5 k V,capillary gas temperature of 400℃,and auxiliary gas temperature of 450℃.The component response is the best,with an optimal cleavage energy of 30 V/40 V/50 V.This mass spectrometry method,because of the introduction of PIL,can greatly improve the ability to identify target masses and collect secondary mass spectrometry information of unknown masses at the same time.It is a systematic characterization technology for targeted and non-targeted multi-component Chinese medicines.Simplified methodological verification shows that the established offline3D-LC/MS method has good precision（RSD<5%）and reproducibility（RSD<10%）.By analyzing the high resolution mass spectrometry data of 14 segmented samples（8alkaloid segments and 6 triterpene acid segments）obtained from the second-dimensional chromatographic separation,a total of 308 compounds were detected,of which 128 were target compounds.There are 180 non-target compounds.The identified compounds are mainly divided into two categories:85 alkaloids,29 triterpenoids,and 14 other compounds.Compared with the traditional one-dimensional chromatographic separation method,the theoretical chromatographic peak capacity of the method has been greatly improved,which enhances the ability to identify the multi-components of Uncaria sessilifructus.In this thesis,IEC×HILIC×RPLC offline three-dimensional chromatography was established for the first time to separate the traditional Chinese medicine components with acid and alkaline active ingredients,combined with an efficient and sensitive positive-negative switching improved DDA scan,to achieve the in-depth characterization of the traditional Chinese medicine Uncaria sessilifructus.It is beneficial to the quality control and development of Chinese medicine of Uncaria sessilifructus.At the same time,this paper also provides a methodological reference for the systematic characterization of the complex system of Chinese medicine.