【作者】 杨丽；

【导师】 张学武；

【作者基本信息】 华南理工大学 ， 食品科学与工程， 2024， 博士

【摘要】 墨旱莲（Eclipta prostrata L.）是菊科醴肠属植物,在中国因其具有较好的药用价值而广受关注,而有关其纯化组分药理活性研究报道少。本文对墨旱莲皂苷黄酮组分进行提取分离和成分分析,并利用多种细胞模型对其抗炎肝保护作用机制进行系统研究,以期为开发食品新资源及保健品提供研究基础。（1）从墨旱莲中用HPD300树脂经层析同时分离获得皂苷（MZ2）和黄酮（MF1）纯化组分,皂苷和黄酮总含量分别为89.73%和59.17%,利用傅里叶红外光谱分析官能团发现,MZ2中有皂苷类化合物且可能含呋喃型,MF1中有黄酮类化合物,进一步采用UPLC-MS/MS质谱技术检测并鉴定MZ2中主要含有3种皂苷、1种蒽醌类和2种黄酮等化合物,而MF1中主要含有2种黄酮、2种脂类和1种皂苷等化合物。（2）对MF1和MZ2纯化组分的抗氧化、抗炎及肝损伤保护作用进行初筛结果表明,不同组分对不同的自由基和氧化还原物质的作用能力不同,其中,对ABTS和DPPH自由基具有较好的清除能力,以及较强的铁离子还原能力,MF1抗氧化作用比MZ2会稍强。两者对LPS刺激RAW264.7细胞释放NO有较强的抑制效果,且接近阳性对照地塞米松（DXM）,MZ2相对比MF1抑制效果更好。此外,测定了酒精诱导的L02细胞肝损伤保护中存活率的情况,两者对缓解酒精诱导L02细胞肝损伤保护效果较好,MZ2相对比MF1保护效果强,且接近阳性对照水飞蓟素（Silymarin）干预的效果。（3）以LPS诱导的RAW264.7细胞为模型,对MF1和MZ2进行系统的抗炎活性及机制研究。结果显示两者能显著抑制促炎因子TNF-α,IL-1β和IL-6分泌量,且能不同程度的抑制TNF-α、IL-6、IL-1β、i NOS和COX-2的mRNA表达,同时,Western blot显示两者能抑制COX-2蛋白表达且能抑制p38和JNK磷酸化,而MZ2又能抑制p65、IκBα和IκKα/β磷酸化,进而通过调节NF-κB和MAPK信号通路来抑制炎症。此外,通过对蛋白（COX-2、p65、p38和JNK）和主要活性小分子物质半柔性对接,根据最低的结合能筛选的对象Cynaroside,Eclalbasaponin A和Deoxylimonin,利用三维可视化预测其是主要起抗炎作用的化合物。（4）以肝细胞L02为研究对象,分别通过酒精、化学物质（H₂O₂提供活性氧和LPS致炎）及肝脂肪变性（FFA）诱导建立了四种肝损伤模型,探究其氧化应激及抗炎机制。在酒精诱导损伤模型中,MZ2能极显著降低ALT、AST酶活力减少转氨酶的泄出;MF1和MZ2能分别极显著提高SOD活力和降低MDA含量而对抗氧化,同时,上调Nrf2和HO-1 mRNA和蛋白的表达水平。H₂O₂诱导L02化学损伤模型中,MZ2极显著降低ALT、AST酶活力对损伤有修复作用,MF1和MZ2能提高SOD和GSH-Px活力而对抗氧化,同时,上调Nrf2和HO-1蛋白使得Nrf2/HO-1介导的氧化应激被激活。在FFA诱导的L02肝脂肪变性的模型中,油红O染色与TG的含量测定显示MF1和MZ2可能是通过抑制L02对脂肪累积起到改善肝损伤,同时,二者极显著降低ALT、AST酶活力,不同程度提高SOD和GSH-Px活力对抗氧化应激。此外,MF1和MZ2（100μg/m L）对Nrf2、γ-GCL和HO-1蛋白上调,使得Nrf2/ARE介导的氧化应激被激活。LPS诱导L02肝炎损伤模型中,MF1和MZ2能显著抑制LPS诱导L02细胞释放TNF-α和IL-6,不同程度的显著下调TNF-α、i NOS和IL-6mRNA的表达,从而抑制促炎因子释放起到减缓肝损伤。（5）利用LC-MS/MS技术对所诱导的模型细胞胞内代谢物进行分析,探索诱导模型与皂苷纯化组分给药组组间代谢差异物区别与不同代谢通路,从代谢水平探究其肝损伤的保护机制,在酒精诱导模型中代谢物通路包含烟酸和烟酰胺代谢、甘油磷脂代谢、核苷酸代谢;FFA诱导模型中代谢物通路包含核苷酸代谢、精氨酸和脯氨酸代谢、甘油磷脂代谢、嘧啶代谢;LPS诱导模型中代谢物通路包含甘氨酸、丝氨酸和苏氨酸的代谢;由此MZ2可能通过影响以上代谢途径而发挥肝损伤保护作用,同时表明不同模型组会影响机体的代谢不同,而MZ2可能通过干预相关代谢逆转保护细胞免受损伤。更多还原

【Abstract】 Eclipta prostrata L.belong to the Eclipta prostrata in the asteraceae family.It has been widely concerned for its good medicinal values in China.However,little information is available about pharmacological activities of its components.In the present study,the saponin flavonoid fraction was extracted,isolated,and structurally characterized,and the the mechanism of anti-inflammation and liver injury protection of the saponin flavonoid fraction were evaluated using various cell models,in order to provide research basis for developing new food resources and health care products.Purified fractions of saponins（MZ2）and flavonoids（MF1）were obtained by simultaneous chromatography with HPD300 resin,and the total contents of saponins and flavonoids were 89.73%and 59.17%,respectively.Functional group analysis by fourier infrared spectroscopy showed that there were furan-type saponins in MZ2,and there were flavonoids in MF1.Further,by using UPLC-MS/MS mass spectrometry,MZ2 fraction contained mainly 3 saponins,1 anthraquinones and 2 flavonoids,while MF1 fraction contained mainly 2 flavonoids,2 lipids and saponins,etc.The results of the antioxidant,anti-inflammatory and liver injury protective effect of the purified fractions of MF1 and MZ2 showed that had good scavenging ability for ABTS and DPPH free radicals and strong iron ion reduction ability,and the antioxidant effect of MF1 was slightly stronger than that of MZ2.Both of them had a strong inhibitory effect on NO release fromRAW264.7 cells stimulated by LPS,and its result were close to dexamethasone（DXM）,and the inhibitory effect of MZ2 had a better than that of MF1.In addition,the survival rate of alcohol-induced liver injury in L02 cells was measured.MZ2had a good protective effect on alcohol-induced liver injury in L02 cells,and was close to the effect of silymarin as a positive control,and the protective effect MZ2 had a stronger than that of MF1.Based on LPS-induced RAW264.7 cells inflammatory model,the anti-inflammatory activity and mechanism of MF1 and MZ2 were studied systematically.The results showed that they could significantly inhibit the release of inflammatory factors TNF-α,IL-1βand IL-6,and inhibit the mRNA expression of TNF-α,IL-6,IL-1β,i NOS and COX-2.The results of western blot showed that MZ2 inhibited the expression of COX-2 protein and the phosphorylation of p38 and JNK,while MZ2 inhibited the phosphorylation of p65,IκBαand IκKα/β.Therefore,they inhibited inflammation by regulating NF-κB and MAPK signaling pathways.In addition,by the semi-flexible docking of proteins（COX-2,p65,p38,and JNK）with the main active small molecules,the target compounds（Cynaroside,Eclalbasaponin A,and Deoxylimonin）were screened according to the lowest binding energy.Through 3D visualization analysis,they were predicted to be the main compounds that play an anti-inflammatory role.To explore the oxidative stress and anti-inflammatory mechanism,four kinds of liver injury models were established,including alcohol-induced,chemical substances（H₂O₂-induced and LPS-induced）and hepatic steatosis（FFA-induced）liver injury,respectively.In alcohol-induced injury model,MZ2 significantly decreased the activity of ALT and AST enzymes for reducing the leakage of transaminase.MF1 and MZ2 can significantly increase SOD activity and decrease MDA content,and up-regulate Nrf2 and HO-1 mRNA and protein expression levels.In H₂O₂-induced injury model,MZ2 significantly reduced the activity of ALT and AST enzymes,indicating that MZ2 had a repairing effect on damaged cells.MF1 and MZ2 could increase the activity of SOD and GSH-Px to combat peroxide,meanwhile,Nrf2 and HO-1 proteins were up-regulated to activate Nrf2/HO-1 mediated oxidative stress.In FFA-induced injury model,the results of oil red O staining and TG content determination showed that MF1 and MZ2 may protect against liver damage by inhibiting accumulation of fat in the L02.Furthermore,they significantly reduced the activity of ALT and AST enzymes,and increased the activity of SOD and GSH-Px to combat oxidative stress.In addition,MF1 and MZ2（100μg/m L）up-regulated Nrf2,γ-GCL and HO-1 proteins,leading to activate the Nrf2/ARE-mediated oxidative stress.In LPS-induced injury model,MF1 and MZ2 can significantly inhibit the release of TNF-αand IL-6,and significantly down-regulate the expression of TNF-α,i NOS and IL-6 mRNA.The possible reason is to protect liver cells from damage by inhibiting the release of inflammatory factors.We used LC-MS/MS technology to analyze intracellular metabolites of different induced-models,aiming to compare the differences of metabolites and metabolic pathways between different groups,and to further explore the protective mechanism of liver injury from the metabolic level.In alcohol-induced model,the metabolite pathway includes niacin and nicotinamide metabolism,glycerophospholipid metabolism and nucleotide metabolism.In FFA induced model,the metabolite pathway includes nucleotide metabolism,arginine and proline metabolism,glycerophospholipid metabolism and pyrimidine metabolism.In LPS-induced model,the metabolite pathway includes glycine,serine and threonine metabolism.Therefore,MZ2 may play a protective role in liver injury by affecting the above metabolic pathways,and it also indicates that different models show different metabolism,and MZ2 may protect cells from damage by interfering with related metabolism.更多还原