| Callicarpa nudiflora Hook.et Arn is a plant of Callicarpa L.in Verbenacea,mainly produced in Hainan,Guangdong,Guangxi,Jiangxi,etc.It has been included in the Chinese Pharmacopoeia 2020 edition as a new medicinal material.Callicarpa nudiflora is widely used in clinical practice as a traditional Li medicine.Its roots and leaves can be used as medicine,with the effects of antibacterial and hemostatic,anti-inflammatory and detoxifying,dispersing blood stasis and swelling,dispelling wind and dispelling dampness.Modern pharmacological studies have shown that Callicarpa nudiflora’s main pharmacological components,phenylethanoid glycosides(Ph Gs),have the ability to inhibit neuroinflammation,improve cognitive dysfunction caused by neuroinflammation and antioxidant activity.Therefore,in order to further study Callicarpa nudiflora and find more Ph Gs with novel structures and good biological activities,this thesis was conducted to separate the components of Callicarpa nudiflora aqueous extracts from ethyl acetate extraction sites and to establish a cellular model of neuroinflammation using lipopolysaccharide(LPS).The cellular model of Callicarpa nudiflora was used to explore the alleviating effect of Ph Gs on LPS-induced neuroinflammation and the potential molecular mechanism,aiming to provide a scientific basis for the comprehensive utilization of Callicarpa nudiflora and the development of new anti-neuritis natural drugs.The main experimental results are as follows:(1)The ethyl acetate part of Callicarpa nudiflora was purified by normal-phase silica gel column chromatography,reversed-phase ODS column chromatography,Sephadex LH-20 gel column chromatography,MCI column chromatography and reversed-phase preparative liquid chromatography,and the structure was identified by a combination of spectroscopic techniques such as nuclear magnetic spectroscopy and high-resolution mass spectrometry.As a result,48 compounds were isolated and identified,including 18 phenylethanol glycosides,11 sesquiterpenoids,9 diterpenoids,8 flavonoids,1 lignan and 1 other class.And according to the results of numerous pharmacological studies,Ph Gs were found to modulate cognitive dysfunction caused by neuroinflammation and antioxidant activities.Therefore,in this thesis,18 Ph Gs were selected for an in-depth study to investigate their anti-neuroinflammatory activity and mechanism of action.Among the 18 Ph Gs identified were parvifloroside A(1),calceolarioside B(2),2′′-O-β-apiosylverbascoside(3),1’-O-β-(3,4-dihydroxyphenyl)-ethyl-[4’’’’-O-caffeoyl-(α-L-rhamnopyranosyl)]-(1→3’’)-D-galactopyranoside(4),parvifloroside B(5),desrhamnosylacteoside(6),torenoside B(7),myricoside(8),isomartynoside(9),cistanoside D(10),acteoside(11),isoverbascoside(12),campneoside I(13),martinoside(14),plantainoside B(15),plantainoside C(16),betonyoside B(17),epimeridinoside A(18).Among them,compounds 3,4,5,6,7,16-18 were isolated for the first time from the genus,and compounds 1,2,10,14,15 were isolated for the first time from the plant.The above results have enriched the study of chemical composition of Callicarpa nudiflora and laid a certain foundation for the study of Callicarpa nudiflora related medicinal substances.(2)This thesis provides a preliminary prediction of the targets and mechanisms of action of Callicarpa L.phenylethanolide against neuroinflammation by a network pharmacology approach.The final analysis showed that most Ph Gs targeted important targets corresponding to neuroinflammation,and further pathway enrichment analysis of potential target genes was performed.Finally,15 key target proteins were screened out of 67 neuroinflammation-related target proteins,and 20 potential pathways of action of phenylethanolide against neuroinflammation were screened out of 161 signaling pathways.Finally,IL-6,TNF-α,and P38 were screened as core targets,and MAPK signaling pathway played an important role in the therapeutic effects of Ph Gs on neuroinflammation.These results provide a theoretical framework for the use of phenylethanolide for the treatment of neuroinflammation.(3)In this thesis,interleukin 6(IL-6),a key target predicted by network pharmacology,was used as the active research target to construct a pharmacophore model based on common molecular features to further explore the structural commonality of active components in Ph Gs against neuroinflammation.In this thesis,26 experimentally validated IL-6 inhibitors were selected from published papers as a training set,and molecular overlap and common pharmacodynamic features were searched to construct a pharmacodynamic cluster model based on IL-6 receptor antagonists.After that,18 Ph Gs identified in Chapter 2 were screened for activity by the preferred pharmacophore models.It provides a theoretical basis for further study of the anti-neuroinflammatory mechanism of action of Ph Gs in Callicarpa nudiflora and provides a lead compound for the development of new drugs against neurodegenerative diseases.(4)In this thesis,the phenylethanol glycosides isolated from Callicarpa nudiflora were selected based on the 3D-QSAR pharmacophore model and the Griess method,and the molecular mechanism of their anti-neuritis effects was investigated in depth.Finally,based on the pharmacophore model and NO inhibition results,the active compound,cistanoside D(CD),with high efficiency and low toxicity,was selected and its molecular mechanism of anti-neuritis effect was investigated in depth.The results showed that CD could inhibit LPS-induced M1 polarization of BV2 microglia,promote M2 polarization of microglia,and inhibit early apoptosis by reducing ROS production in cells.It also attenuates the expression of microglia IBA-1and NLRP3 inflammatory mediators and blocks NF-κB entry into the nucleus.It also protects BV2 cells from LPS damage and further ameliorates neuroinflammation by affecting the expression of related proteins on the NF-κB/MAPK inflammatory pathway. |
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