Discovery Of Natural Products With Lipid-lowering Activity And Their Mechanism Of Action

Hyperlipidemia is a major risk factor of atherosclerosis which can lead to a series of cardiovascular and cerebrovascular diseases and greatly threaten human health.Even though statins have a good efficacy and safety,some medical need is still unmet.Proprotein convertase subtilisin/kexin type 9?PCSK9?is one of the most promising target by far,and its inhibitors are anticipated to be the new revolutionary lipid-lowering drugs.At present,the development of PCSK9 inhibitors is mainly focused on biological agents,whereas that of small molecule inhibitors is still in its initial stage,with both opportunities and challenges.Traditional Chinese medicine?TCM?often has its unique advantages in the treatment of complicated diseases,especially some cardiovascular and cerebrovascular chronic diseases.Morden Chinese medical research indicates that hyperlipidemia is closely related to stasis syndrome of TCM,thus there is the potential to discover hits or leads from TCM herbs with stasis-dissolving effects.In this dissertation,three medicinal plants with stasis-dissolving effects,including Euphorbia helioscopia,Celastrus orbiculatus and Trichosanthes cucumeroides were studied.Altogether,146 compounds,including 21 new ones were isolated from the extracts of the three plants by various column chromatography,such as Silica gel,Chromatorex C₁₈,Sephadex LH-20,TSK gel Toyopearl HW-40F and semi-preparative HPLC.Their structures were identified on the basis of extensive spectroscopic means,chemical communication,circular dichroism?CD?,X-ray crystallography and Mosher's method.In order to improve diversity of jatrophane categories,we prepared 21 derivatives through structural modification.We tested the in vitro lipid-lowering activity of the identified compounds,from which we selected 2active compounds to evaluate their in vivo activity and explore their mechanism of action.65 compounds including 35 jatrophane macrocyclic diterpenes?A1-A6,A15-A43?,2 lathyrane macrocyclic diterpenes?A7,A44?and 28 triterpenes and other types of compounds?A8-A14,A45-A65?were isolated from E.helioscopia.Among them,compounds A1-A14 were new and their absolute configurations were determined by chemical communications,X-ray crystallography,circular dichroism and Mosher's method.Alkaline hydrolysis of the abundant jatrophane A33 using potassium carbonate yielded the main product A66,whose structural modification at14-OH gave rise to 21 acylated derivatives?A67-A87?.71 compounds including 15 ent-kaurane diterpenes?B1,B2,B24-B36?,17abietane diterpenes?B3,B8-B23?,2 ent-pimarane diterpenes?B4,B5?,1 ent-labdane diterpene?B6?,16 oleanane triterpenes?B37-B52?,3 ursane triterpenes?B7,B53,B54?,12 friedeiane triterpenes?B55-B66?,3 steroids?B67-B69?and 2 flavonoids?B70,B71?were isolated from C.orbiculatus.Among them,compounds B1-B7 were new and their absolute configurations were determined by X-ray crystallography.7 known cucurbitane triterpenes or their saponins?C1-C7?were isolated from T.cucumeroides.The isolated and structurally modified compounds were subjected to DiI-LDL uptake assay to analyze their effects on LDL-Uptake.Compounds A22?A31?A36?A44?A56?A68?B43?B46?B58 and C4 exhibited significant effect of improving LDL-Uptake rates in HepG2?with LDL-Uptake rates over 1.3?.SAR studies for the jatrophanes suggested that substituted patterns of C-9,steric hindrance between C-14and C-15,and the long conjugated fragment from C-5 to the carbonyl?C-9?were essential for the activity.SAR studies for the cucurbitanes revealed that hydrogenation of the double bond between C-23 and C-24 increased the activity while subsititution of a hydroxyl group at C-24 decreased the activity.Western Blot assay was carried out to analyze whether these compounds had influence on the LDLR and PCSK9 protein level in HepG2 cells.Results showed that different types of compounds increased LDLR protein levels in HepG2 cells does-dependently,but their regulation effect of PCSK9 protein was different.Interestingly,triterpenes A56?B43?B46?B58 and C4 could reduce the expression of PCSK9 protein,and are potential natural source of small-molecule PCSK9 inhibitors.To evaluate compounds A31 and C4's activities in vivo,Golden Syrian Hamsters were used as evaluation models.Results showed that both of them could significantly reduce serum CHOL and LDL-C,moreover,C4 could even reduce serum TG.Mechanism research shown that compound A31 could stabilize LDLR mRNA through stimulating phosphorylation of ERK,and consequently increase LDLR protein level in HepG2 cells.Differently,compound C4 could improve the activity of LDLR promoter and reduce the activity of PCSK9 promoter,thereby affecting the level of each mRNA and protein.