Isolation,Identification And Biological Activity Of Chemical Components From Hydrocotyle Sibthorpioides Lam.

Hydrocotyle sibthorpioides Lam.（HSL）,which is one of the traditional Chinese medicines of the genus Umbellifera,is a perennial herb.The main work of the thesis is as follows:1.Preliminary identification of the chemical constituents of EtOAc-Soluble fractions by electrospray ionization mass spectrometry（ESI-MS/MS）.2.Separation of chemical components（separation by column chromatography and high-speed countercurrent chromatography）.3.Spectral characterization and structural analysis.This academic dissertation mainly carried out electrospray ionization mass spectrometry（ESI-MS/MS）for the preliminary identification of the chemical constituents in the extraction of EtOAc-Soluble fractions from HSL,and high-speed counter-current chromatography（HSCCC） was used to separate and prepare its chemical constituents.Their chemical structure was identified by means of characterization such as nuclear magnetic resonance（NMR） spectroscopy.Finally,the anti-inflammatory activity of the isolated compounds was studied,and the following research results and conclusions were obtained.1 This chapter studied the chemical composition of EtOAc-soluble fractions of HSL by chromatography-mass spectrometry（HPLC-PAD-ESI-MS/MS）.The chromatographic peak information（ultraviolet spectroscopy,mass spectrometry data）combined with the information provided by the literature and mass spectrometry database（Natural Organic Chemical Mass Spectrometry Atlas）provides preliminary identification of chemical components and provides a better scientific research for HSL.2 After preliminary analysis by HPLC,the EtOAc-soluble fractions of HSL was initially separated by Sephadex LH-20.Next,the sample obtained by the initial separation was separated by a HSCCC separation and a n-hexane-n-butanol-water solvent system.Finally,14 kinds of monomer compounds were isolated from the EtOAc-soluble fractions.Their structure of 14 monomeric compounds was identified by ¹H NMR and ¹³C NMR:Vanillic acid（1）,p-hydroxybenzoic acid（2）,Protocatechuic acid（3）,Gallic acid（4）,Quercetin（5）,Kaempferol（6）,Luteolin（7）,Apigenin（8）,Myricitrin（9）,Quercitrin（10）,Astragalin（11）,Myricetin3-O-galactoside（12）,Isomyricitrin（Myricetin 3-O-glucoside）（13）and Quercetin3,3’-di-α-L-rhamnopyranoside（16）.3 Four monomeric compounds were isolated from the n-BuOH-soluble fractions by DM 301 macroporous resin coarse fraction,Sephadex LH-20 purification,reversed phase silica gel（ODS/RP-18）chromatography and combined gradient elution chromatography（gradient elution HSCCC）.Compound 15 and compound 17 with large partition coefficients were separated by gradient countercurrent chromatography,and glacial acetic acid was added to the solvent system to improve the partition coefficient of the material,which provided a novel methodological reference for the separation of flavonoid glycosides.Their structure of 14 monomeric compounds was identified by ¹H NMR and ¹³C NMR:Luteolin-7-O-β-D-glucuronide（14）,Apigenin7-O-β-D-glucuronide（15）,Quercetin 3,3’-di-α-L-rhamnopyranoside（16） and myricetin 3,3’-di-α-L-rhamnopyranoside（17）.4 Monoclonal macrophages（RAW 264.7）were induced by lipopolysaccharide（LPS）,and the inhibition of tumor necrosis factor（TNF-α） and interleukin（IL-6） in RAW 264.7 was tested using an ELISA kit.The results showed that Compound 2 had strong anti-inflammatory activity,and the activity of organic acid compounds was higher than that flavonoids.