Screening Of Active Components Of Lotus Seedpod And Preliminary Study On Its Anti-oxidation And Anti-inflammatory Mechanism

Objective:The preliminary study on the antioxidant activity and anti-inflammatory mechanism of the extracted compounds separated by ethyl acetate of lotus seedpod was conducted to provide theoretical basis for the development and utilization of lotus seedpod.Methods:The ethyl acetate extract of lotus seedpod was separated and extracted on silica gel column.The antioxidant capacity of its chemical constituents was investigated by scavenging DPPH free radical,ABTS⁺free radical and Fe³⁺reducing ability.Mice mononuclear macrophage RAW264.7 was stimulated to produce inflammation by lipopolysaccharide（LPS）.The anti-inflammatory activity of the compounds isolated by silica gel column chromatography from the rosary was detected by interfering with the inflammatory test.The anti-inflammatory activity of the compounds with obvious and stable anti-inflammatory effects was detected by gradient concentration anti-inflammatory test and cell viability test.The effect of ethyl retrooleate on the protein expression of nitric oxide synthase（iNOS）and cyctoxase（COX-2）was investigated by Western blot.Results:1.Through the analysis of physical and chemical properties and spectrum(¹³C NMR,¹H NMR,etc.),11 compounds were separated from the ethyl acetate extract of Chinese medicine lotus seedpod by silica gel column chromatography,including 2 fatty acid compounds and 4terpenes Class,5 flavonoids.The two fatty acid compounds are linoleic acid ethyl ester（Lin-oleic acid ethyl ester,1）and（e）-9-octadecenoic acid ethyl ester（（e）-9-octadecenoic acid ethyl ester,2）.The four terpenes compounds include:7β-Hydroxy betulinic acid（7β-hydroxy Betulinic acid,3）,betulinic acid（Betulinic acid,4）,α-amyrin alcohol（α-amyrin,5）,β-amyrin alcohol（β-amyrin,6）.The five flavonoids compounds include:kaempferol-3-rutino-side（Kaempferol-3-rutinoside,7）,Isorhamnetin-3-O-glucoside（1→6）-β-D-glucoside（Isorham-netin-3-O-glucoside（1→6）-β-D-glucoside,8）,Quercetin（Quercetin,9）,Rutin（Rutin,10）,Quer-cetin-3-O-β-D-galactopyranoside,（Quercetin-3-O-β-D-galactopyranoside,11）.Among them,compound 3 is a new compound,and compounds 1,2,4,5,6,7,8,10,and 11 were isolated from the lotus plant for the first time.2.Antioxidant experiments showed that kaempferol-3-O-rutoside,isorhamnetin 3-O-di-glucoside,quercetin,rutin and quercetin 3-O-β-D-galactoside all had strong scavenging ability of DPPH free radical,ABTS⁺free radical and reducing antioxidant ability of Fe³⁺.DPPH radical scavenging assay showed that the antioxidant capacity of 7β-hydroxy Betulinic acid,Betulinic acid,α-aromatic alcohol,β-aromatic alcohol,ethyl linoleate and oleic acid was almost zero.3.Lotus seed pot compounds of LPS induced NO have good inhibitory effect,compared with model group,quercetin,linoleic acid ethyl ester,ethyl oleate,7 beta hydroxy Betulinic acid and compound of alpha and beta fragrant incense resin resin alcohol,rutin and quercetin-3-O-beta-D-galactose NO significant reduction in the concentration of glycosides,statis-tically significant difference（P<0.01 or P<0.05）.The inflammatory gradient concentration inhibition test and cell viability test of three compounds,including ethyl linoleate,ethyl retrooleate and rutin,showed that ethyl retrooleate had the strongest anti-inflammatory activity and was relatively stable,and had no obvious toxic effect on cells.When the concentration of ethyl retrooleate was 20μg/mL,it had a slight inhibitory effect on the production of LPS-in-duced NO,and the inhibitory effect on NO increased with the increase of drug concentration.When the concentration of ethyl retrooleate was 100μg/mL,the inhibitory effect on NO could reach about 80%,and the inhibitory effect was significantly enhanced（P<0.01）.4.Western blot analysis showed that ethyl retrooleate could inhibit the expression of iNOS and COX-2 protein in RAW264.7 macrophages induced by LPS.Compared with blank group,the protein expressions of COX-2 and iNOS in RAW264.7 cells in two model groups were significantly increased,and the difference was statistically significant（P<0.01）.Compared with model group,the protein expression of iNOS did not decrease when the drug concentration was 5μg/mL,and the protein expression of iNOS was significantly decreased when the ethyl retrooleate concentration was 10μg/mL,with statistical significance（P<0.01）.When the drug concentration was 40μg/mL,the protein expression of COX-2 decreased significantly,and the difference was statistically significant（P<0.01）.When the drug concentration was lower than40μg/mL,the protein expression of COX-2 was not significantly decreased,indicating that the ethyl retrooleate could effectively inhibit the protein expression of iNOS and COX-2 in a certain dose dependent manner.In this study,ethyl retrooleate decreased the expression of iNOS and COX-2 protein induced by LPS,suggesting that ethyl retroate could exert anti-inflammatory effects by inhibiting the expression of iNOS and COX-2 protein in RAW264.7 macrophages induced by LPS.Conclusions:1.Flavonoids in lotus seed pot,namely kaempferol-3-O-rue glucoside,isorhamnetin 3-O-β-D glucosyl（1→6）-β-D-glucoside,quercetin,rutin and quercetin-3-O-β-D-galactoside,showed good antioxidant activity.2.The ethyl retrooleate compound in lotus seed can exert anti-inflammatory effects by inhibiting the expression of iNOS and COX-2 protein in RAW264.7 macrophages induced by LPS.