Study On The Anti-inflammatory Material Basis And Mechanism Of Action Of The Effective Parts Of Morus Nigra L.fruit

The Xinjiang Medicinal Mulberry（Morus nigra L.）is a half-cultivated,half-wild species and is the only natural 44-ploid（n=7）mulberry germplasm resource in China.As a traditional medicinal fruit of the Xinjiang Uyghur people,the Morus nigra L.fruit contains a large number of bioactive components,such as phenols,flavonoids and anthocyanins,with biological activities such as antioxidant,anti-inflammatory,anti-tumour and anti-atherosclerosis.There have been many reports in the literature on the anti-inflammatory activity of extracts from the Morus nigra L.fruit,but the specific anti-inflammatory material basis and potential mechanisms have not yet been elucidated.Therefore,in this study,the effective site of the anti-inflammatory effect of Morus nigra L.fruit was identified through in vitro experiments,and its anti-inflammatory mechanism was further investigated.On this basis,the potential anti-inflammatory active components of the active site were explored by LC-MS analysis,network pharmacology and molecular docking,with the following main studies:1.Determination of the effective active part of Morus nigra L.fruitIn this study,the content of TPC and TFC in the methanol total extract,petroleum ether extract,ethyl acetate extract,n-butanol extract and water fraction of Morus nigra L.fruit were determined,and their antioxidant and anti-inflammatory activities were evaluated.The results showed that there were some differences in the contents of TPC and TFC in different parts,and the contents of TPC and TFC in the ethyl acetate part were the highest,which were 399.67±3.58 mg GAE/g and 155.79±5.17 mg RE/g,respectively.Similarly,the ethyl acetate fraction exhibited the strongest antioxidant activity in all three in vitro antioxidant models,with IC₅₀ values of 0.18 mg/m L and0.64 mg/m L for the radical scavenging abilities of DPPH and ABTS,respectively,and a FRAP equivalent of 2.69 mmol Fe²⁺/g.Compared with other sites,the ethyl acetate site exhibited stronger anti-inflammatory activity in vitro,which could significantly reduce the production of NO and inhibit the denaturation of bovine serum albumin in LPS-induced RAW264.7 cells in a dose-dependent manner.Therefore,it was determined that the ethyl acetate part of the Morus nigra L.fruit is the effective part of the anti-inflammatory effect.2.Study on the anti-inflammatory mechanism of the effective parts of Morus nigra L.fruitUsing LPS-induced RAW264.7 macrophages as an inflammatory model,the in vitro anti-inflammatory mechanism of the effective part of Morus nigra L.fruit was explored.The results showed that the effective parts of Morus nigra L.fruit significantly inhibited the release of ROS and the expression of inflammatory factors IL-6,IL-1βand TNF-α,as well as the expression of INOS,COX-2 and 5-LOX proteins and the activation of TLR4/NF-κB/MAPK pathway,and activated the Nrf2/HO-1 pathway.3.Study on the chemical constituents of the effective parts of the Morus nigra L.fruitThe chemical composition of the effective parts of the Morus nigra L.fruit was characterized by LC-MS and the main active ingredients were quantified by UPLC-PDA..The LC-MS results were analyzed by Xcalibur software,and a total of 56compounds were inferred and identified,including 27 flavonoids,19 organic acids,4fatty acids,3 coumarins,and 3 other compounds.The content of the main active components chlorogenic acid,caffeic acid,cryptochlorogenic acid,quercetin,neochlorogenic acid and ferulic acid were 8.69 mg/g,8.02 mg/g,6.81 mg/g,6.75 mg/g,5.97 mg/g,1.22 mg/g.4.Based on network pharmacology and molecular docking,the main anti-inflammatory active substances in the effective parts of Morus nigra L.fruitsThe potential anti-inflammatory active ingredients and targets of action of the active parts of Morus nigra L.fruit were predicted using network pharmacology,and the potential active ingredients were molecularly docked with the core targets for the treatment of inflammation,and those with strong binding ability were selected as main anti-inflammatory active ingredients.The results showed that 547 chemical composition-related targets and 931 inflammation-related targets were obtained from the online database,of which 137 were common targets.GO enrichment analysis involves biological processes including regulation of inflammatory responses,bacterial-derived molecular responses,lipopolysaccharide and reactive oxygen metabolism processes.KEGG pathway enrichment yielded a total of 150 pathways,five of which are closely related to inflammation:TNF,MAPKs,Toll-like receptors,NF-κB and arachidonic acid metabolic pathways.By constructing a"component-target-pathway"network,14 active components were screened based on Degree values,including mustard,ferulic acid,naringenin,caffeic acid,apigenin and lignan,and 13 key targets,including ALOX5,PTGS2,EGFR,ALOX5 and MMP9.The molecular docking results showed that the docking scores of the 14 active ingredients with the key targets ALOX5 and PTGS2 were above the threshold values.Lacinin,isorhamnetin,kaempferol and quercetin had higher docking scores with ALOX5,while isorhamnetin,lignan,quercetin and naringenin had higher docking scores with PTGS2.In summary,this study clarified the anti-inflammatory material basis and mechanism of action of the effective parts of Morus nigra L.fruit,and provided a theoretical basis for the further development and utilization of Morus nigra L.fruit.