| Background:Euonymus alatus is a winged appendage of Celastraceae.It is a medicinal plant and is originally recorded in Shennong Bencao.In ancient,it was mainly used for gynecological diseases,the role of Euonymus alatus in vaginal bleeding,bloating and detoxification was recorded in the Compendium of Materia Medica;In modern times,Euonymus alatus is mainly used for diabetes and its complications and tumors,but the pharmacodynamic components and mechanism of action of Euonymus alatus are not clear.Therefore,this experiment systematically studied the chemical constituents of Euonymus alatus,and determined the content and the antioxidant activity in vitro;Selecting of compound EA-3 has a strong effect on DR through experiments such as transwell and tube formation of RVECs RF/6A cells;Then carry out pharmacodynamic experiments and mechanism exploration:In the CAM and OIR mouse models,EA-3 can significantly inhibit the formation of neovascularization in CAM and the formation of diseased vascular clusters in the retina of OIR mice,and promote the normalization of diseased vessels;The mechanism by which EA-3 inhibits angiogenesis may be the inhibition of high glucose-induced VEGF-VEGFR-mediated expression of the core protein of the Hippo pathway.Obiective:In order to lay the foundation for future study of Euonymus alatus,the chemical composition in Euonymus alatus have been studied through the extraction and separation technology and HPLC-MS technology;Quantitative analysis of monomer compounds isolated from Euonymus alatus have been studied,promoted the quality standard of Euonymus alatus;To investigate the pharmacological action and mechanism of the compound EA-3 in Euonymus alatus by in vivo and in vitro experiments,and provide theoretical basis for the development and utilization of Euonymus alatus.Methods:Part 1:The study of the chemical constituents from Euonymus alatus.The ethyl acetate extraction fraction of Euonymus alatus was isolated and purified by separation techniques such as silica gel,sephadex LH-20 and C18.The isolated compounds were identified by techniques such as LC/MS and nuclear magnetic resonance.By reviewing the literature,summarizing the relative molecular mass and cleavage law of known compounds in Euonymus alatus,a database of known compounds of Euonymus alatus was established.The mass spectrometry cleavage rules of flavonoids,aliphatic and steroids et al in the literature were summarized.The unknown compounds in Euonymus alatus were identified by the compound cleavage rules.The chemical composition of the Euonymus alatus methanol extract was quickly identified by the cleavage rules of the Euonymus alatus known compound database and various compounds.Part 2:Quantitative analysis of Euonymus alatus.An HPLC method was used for simultaneous determination of protocatechuic acid,quercetin and kaempferol.First,the instrument and experimental methods were examined,including standard curve,precision test,repeatability test,stability test and recovery test;After the methodological examination,the content of three polyphenols in the test solution was determined by HPLC method using the same experimental method.Part 3:Evaluation of antioxidant activity of polyphenols in Euonymus alatus.The DPPH method was used to determine the antioxidant activities of protocatechuic acid,quercetin and kaempferol,and the natural antioxidant ascorbic acid Vc was used as a control.Part 4:Study on the mechanism of action of EA-3 in inhibiting diabetic retinopathy.Transwell migration assay was used to detect the migration of RVECs RF/6A cells;The Matrigel assay was used to detect the formation of tube in RVECs RF/6A cells;The expression of VEGF-VEGFR and Hippo signaling pathway-related proteins was detected by Western Blot;The neovascularization was detected by CAM experiments;The OIR mouse model was used to investigate the pathological vascular clusters and avascular regions of the mouse retina.Results:Part 1:Twelve compounds were isolated from the ethyl acetate fraction of Euonymus alatus,including six flavonoids,two steroids,one phenolic,one triterpenoid,one aliphatic and one phenylpropanoid.The structures were identified as friedelin(1),stigmasterol(2),sitostenone(3),?-hydroxymethyl ester(4),3-(4-hydroxy-2-methoxyphenyl)-acrylic acid-4-hydroxy-3-methoxyphenyl ester(5),apigenin(6),naringen(7),kaempferol(8),quercetin(9),hesperidin(10),rutin(11),protocatechuic acid(12).Among them,the compound?-hydroxymethyl ester and 3-(4-hydroxy-2-methoxyphenyl)-acrylic acid-4-hydroxy-3-methoxyphenyl ester were isolated from Euonymus alatus for the first time.Using UPLC-LTQ-Orbitrap-MS technology,65 components were identified from the methanol extract of Euonymus alatus,including 36 flavonoids,4 phenolic acids,7 triterpenoids,12 aliphatics and 6 other of the class composition,29 of them were first discovered in the Euonymus alatus.Part 2:The extraction method,extraction solvent and detection wavelength were investigated.The method for simultaneous determination of three polyphenols in Euonymus alatus was established by HPLC.The results showed that the injection amount of protocatechuic acid,quercetin and kaempferol was linear with the integral value of chromatographic peak area in the range of 3.2-1208,3.6-1440,4.0-1600 ng,and the regression equation was y=3055.4x-5311.9,y=4377.6x-31603,y=3015.9x-49183;The methodological findings showed that the RSD was less than 3%,the recovery rate(n=9)was 100.09%-100.57%,and the RSD was less than 3%.The method is simple,accurate,rapid and highly reproducible and can be used for the determination of three polyphenolic compounds in Euonymus alatus.The content of protocatechuic acid,quercetin and kaempferol in the samples were calculated by regressionequation:14.80,19.09,33.29 ?g·g-1.Part 3:The DPPH method results showed that the three polyphenolic compounds in Euonymus alatus have DPPH free radical scavenging effect,and the ability to scavenge DPPH free radicals is quercetin>protocatechuic acid>Vc>kaempferol,suggesting that these three polyphenolic compounds have antioxidant activity.Part 4:The results of Transwell migration assay showed that EA-3 significantly reduced the migration of RVECs RF/6A cells induced by high glucose,with the maximum inhibition rate of 148.98%.The results of Matrigel experiment showed that the length of tube caused by high glucose after EA-3 treatment was significantly reduced,and the highest inhibition rate was as high as 231%.Western Blot analysis showed that EA-3 significantly inhibited the increase of core protein in the VEGF-VEGFR mediated Hippo signaling pathway caused by high glucose.The results of CAM experiments showed that after EA-3 treatment,the area of neovascularization in CAM was significantly reduced compared with the blank control group,and the highest inhibition rate was 41.17%.The results of the OIR mouse model showed that after the EA-3 treatment,the vascular clusters and avascular regions in the retina were reduced and the blood vessel growth was normalized compared with the model group.Conclusion:A systematic study on the chemical constituents of the Euonymus alatus was carried out to establish a method for the separation and analysis of compounds in Euonymus alatus,providing a material basis for the study of Euonymus alatus medicinal components;The method for determining the content of Euonymus alatus medicinal materials was established by using HPLC technology,which provided a theoretical basis for improving the quality standards of Euonymus alatus.EA-3 may have the effect of inhibiting angiogenesis that inhibits angiogenesis in vitro and in vivo by reducing VEGF-VEGFR mediated expression of core proteins in the Hippo pathway. |
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