Characterization Of Chemical Constituents Of Different Uncaria And Evaluation Of Their Effects On Mesenteric Relaxation In Vitro

Uncariae Ramulus Cum Unicis（Gou-Teng）has been commonly used in the treatment of cardiovascular and central nervous system diseases.According to the Chinese Pharmacopoeia（2020 edition）,the stems and hooks of Gou-Teng has five species,including Uncaria rhynchophylla（Miq.）Miq.ex Havil.（URM）,Uncaria hirsuta Havil.（UHH）,Uncaria sessilifructus Roxb.（USR）,Uncaria macrophylla Wall.（UMW）and Uncaria sinensis（Oliv.）Havil.（USH）.Currently,the research of Gou-Teng is mainly focus on one of the above-mentioned single varieties,and there is a lack of comprehensive and systematic analysis and activity evaluation of various chemical constituents.In this thesis,the chemical constituents of five original plants of Gou-Teng recorded in the Chinese Pharmacopoeia were characterized and their relaxation effects on isolated rat mesenteric arterial rings were studied,which provide reference for the quality control and rational application of Gou-Teng.In this thesis,the fingerprints of 30 batches of URM were determined by ultra-high performance liquid chromatography（UHPLC）,and the similarity was calculated to evaluate the quality of URM from the perspective of chemical profile spectrum.Then,the primary metabolites of different varieties of five original plants of Gou-Teng were identified by¹H-NMR spectra,and the secondary metabolites were identified by ultra-high performance liquid chromatography combined with quadrupole orbitrap mass spectrometry（UHPLC-Q-Orbitrap MS）.After multivariate statistical analysis,the different components of five original plants of Gou-Teng were screened out.Subsequently,the contents of 14 compounds in different batches of Gou-Teng were determined by ultra-performance liquid chromatography combined with triple quadrupole mass spectrometry（UPLC-MS/MS）.In addition,since the clinical application of Gou-Teng is mainly used for lower blood pressure,the relaxation effects on isolated rat mesenteric arterial rings of five original plants of Gou-Teng were also investigated,and the active compounds were determined by components-activities correlation analysis.The specific research contents and results are shown as follows:1.The fingerprints of 30 batches of URM were established by UHPLC,and the similarity evaluation was carried out by using"Chinese medicine chromatographic fingerprint Similarity evaluation software（2012 Edition）".The analysis results showed that there were 15common peaks in URM,including seven known peaks,which were chlorogenic acid,isomitraphylline,isorhynchophylline,rhynchophylline,geissoschizine methylether,hirsuteine and hirsutine.The similarity analysis results showed that the URM has good consistency,and the similarity of 30 batches of URM is between 0.850 and 0.995.2.Using ¹H-NMR spectra to identify primary metabolites of five original plants of Gou-Teng,and 31 primary metabolites were identified,including 10 organic acids,7carbohydrates,7 amino acids,3 cholines,1 kind of vitamins B and 3 kinds of other compounds.3.The UHPLC-Q-Orbitrap MS method was used to scan the primary precursor ion and secondary product ion in the ESI positive and negative ion mode to identify the secondary metabolites and several primary metabolites of five original plants of Gou-Teng.A total of123 compounds were separated and detected,including 76 alkaloids,11 flavonoids,9triterpene acids and 27 other compounds.At the same time,35 different metabolites were screened out by principal component analysis（PCA）and orthogonal partial least squares discriminant analysis（OPLS-DA）.4.A UPLC-MS/MS analysis method was further established to determine the content of isomitraphylline,yohimbine,scopoletin,mitraphylline,corynoxeine,isorhynchophylline,ajmalicine,rhynchophylline,uncarine E,isocorynoxeine,akuammigine,geissoschizine methylether,hirsuteine and hirsutine.The results showed that there were significant differences among the content of different varieties of Gou-Teng.5.Using the isolated rat mesenteric aortic ring method,dehydroacetylcholine was used to detect the endothelial function integrity of the pre-contracted vascular ring,and to evaluate the diastolic effect of the water extracts of five original plants of Gou-Teng.The results showed that the aqueous extracts of five original plants of Gou-Teng has vasodilatory efficacy in a dose-dependent manner,which indicated that the aqueous extracts of five original plants of Gou-Teng have the effect of vasodilation.As a comparison,USH had lowest ED₅₀ value and the overall relaxation effect was slightly stronger compared with others.Then,the potential relaxing active components in USH were screened through the correlation analysis of components and activities.