Components Of Eucommia Ulmoides Oliver Leaves And Their Neuroprotective Mechanism Study

Alzheimer's disease(AD)and Parkinson's(PD),as the two most common neurodegenerative diseases,have gradually become one of the important threats to the health of the elderly in today's society with the aging of the global population.The pathogenesis of AD and PD has not been clarified exactly,and clinical medication can only relieve symptoms,and there is a lack of effective treatments.Current studies have found that oxidative stress and neuroinflammation may be the cause of AD and PD.Therefore,there is an urgent need to find neuroprotective compounds against neurodegenerative diseases from traditional Chinese medicines and natural medicines.This article made full use of the laboratory research platform,Eucommia ulmoides as the research object.The natural products were separated and purified by chromatography from the ethyl acetate phase of Eucommia ulmoides leaves.NMR,MS,ECD,and UV analysis methods were identified 38 compounds,including 36known compounds and two new compounds.At the same time,the biological activities of the compounds were screened and discovered compounds with neuroprotective activity.Key results are as follows.1.The compounds were separated from the ethyl acetate phase of Eucommia ulmoides leaves,including twelve lignans,three coumarins,six flavonoids,five phenylpropanoids,two iridoids,two triterpenoids,and eight other compounds.Among them,the two new compounds were lignan and fatty acid.The results of?-glucosidase inhibitory activity showed that the IC₅₀ value of the compoundsCinchonainIb(9),Pelargidenon(17),3',4',5,7-Tetrahydroxy-3-methylflavone(19)and Quercetin(20)were 19.1,18.9,13.4and 14.7?M.Thirteen compounds(14,15,17,19,27,28,30,31,32,33,35,and 38)showed NO inhibited activity.And using ELISA assay tested inflammatory factors(IL-1?,TNF-?,PGE2,and IL-6)in BV-2 cells,the results showed that 27,28,30,31,32,33,35,and 38,the compound Ulmoidol was found(30)had anti-neuroinflammation activity.Using H₂O₂ induced PC-12 cells as the cell model of oxidative stress,tested the cell viability,and the activities of LDH,ROS,GPx,and SOD under the action of 25?M flavonoids and lignan compounds,screening six bisdpoxylignans compounds(2-7)with the best neuroprotective activity.2.The ursane-type C₂₉-triterpenoid,Ulmoidol(ULM,30),significantly inhibited the production of proinflammatory mediators and reduced the expression of inducible nitric oxide synthase(i NOS)and cyclooxygenase-2(COX-2).Moreover,ULM inhibited the cluster of CD14/TLR4 signaling pathway and further limited the activation of NF-?B and MAPK signaling pathways.Notably,electrophoretic mobility shift assay(EMSA)and molecular docking analyses indicated that ULM could prevent PU box binding-1(PU.1)from binding to DNA,suggesting that PU.1might be a potential ULM target.In conclusion,ULM alleviates neuroinflammatory responses in microglia,which could be partly explained by its targeting of PU.1 and the resulting suppression of the TLR4/MAPK/NF-?B signaling pathways.These results suggested that ULM may have therapeutic potential as an agent for treating neuroinflammation-related neurodegenerative diseases.4.Using the Davaid 6.7 database,analyzed lignans with BP,CC,and MF,combined with the drug-target-disease network diagram,found that lignans were related to PD disease targets:GSK-3?,PI3K,SOD2,and MAPK.Different concentrations of 2-7 affected cells to test cell viability,LDH,ROS,GPx,and SOD viability,further selecting the three compounds(2,3,and 5)with the best activity for in-depth study.The results showed that the antioxidant protein(HO-1,NQO-1,and CAT)protein expression all be increased,the PI3K/AKT/GSK-3?/Nrf2 signaling pathways were activated in the cell,and the protein expression of Nrf2 of the cytoplasm and nucleus were showed an upward trend with the increase of compound concentration.In addition,LY294002,a specific inhibitor of PI3K,was added to the experimental group;the Western blot results further proved that compounds 2,3,and5 dose-dependent increased the protein expression of Nrf2 inside and outside the nucleus and antioxidant enzymes HO-1,NQO-1 and CAT by activating the PI3K/AKT/GSK-3?signaling pathway.