Structure Elucidation And Activity Evaluation Of Novel Glycoglycerolipids From Perilla Frutescens

Oxidative damage and chronic inflammation can cause serious harm to human health.The occurrence of aging,degenerative diseases,cardiovascular diseases and cancer are closely related to oxidative damage and chronic inflammation.As a new type of surfactant,glyceroglycolipid has a variety of physiologically active functions,such as antioxidant,anti-inflammatory,anti-cancer and antibacterial.Perilla,listed by the Chinese Pharmacopoeia as "medical and food use" is one of the important sources of glycoglycerolipids.This paper mainly uses biological separation technology to separate glycoglycerolipids from perilla leaves and elaborate on its molecular structure by combining mass spectrometry and NMR spectroscopy techniques.Anti-inflammatory activity,and its antioxidant activity in vitro was measured by various antioxidant detection methods,aiming to excavate new glycoglycerolipids,detect its physiological activity and explore the relationship between the molecular structure of glycoglycerolipids and physiological activity,as the application of food,medicine and health products provides a theoretical basis.1.Isolation and structure elucidation of glycoglycerolipids from Perilla frutescensThree kinds of glyceroglycolipid compounds were extracted,purified and identified from perilla leaves.Two DGDG-type glyceroglycolipid compounds were reported for the first time.The silica gel column chromatography gradient elution method,thin layer chromatography technology and semi-preparative high performance liquid chromatography were used to separate and purify the glycoglycerolipids in perilla leaves,and three glyceroglycolipid compounds with different retention times(compound 1,9.7min;Compound 2,10.7min;Compound 3,14.2min);Using MADILTOF/TOF-MS/MS technology to identify the molecular composition of the three compounds,the results showed that Compound 1 was MGDG type glyceroglycolipid compound,Compound 2 and Compound 3 were DGDG-type glyceroglycolipid compounds;Finally,1D NMR(1H NMR and 13C NMR)and 2D NMR(HMBC,HMQC and TCOSY)were used to determine the type of glycosyl group,fatty acyl and glycerol linkage sites in the three compounds.The position of double bond and its geometric configuration were also analyzed,and the three compounds were identified as 1,2-2-O-(9Z,12Z,15E-octadecatrienoyl)-3-O-(β-D-galactopyranosy)-sn-glycerol,1O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-(6Z,9Z,12Z-octadecatrienoyl)-3-O-[β-D-galact-opyranosyl-(1’→6")-α-D-galactopyranosyl]-sn-glycerol and 1-O-hexadecano yl-2O-(9Z,12Z,15-Z-octadecatrienoyl)-3-O-[β-D-galactopyranosyl-(1"→6")-α-D-galactop-yranosyl]-sn-glycerol.2.Evaluation of anti-inflammatory activity of glyceroglycolipid in perilla leavesThe MTT method was used to detect the toxic effects of LPS and glycoglycerolipids on macrophages.The test results showed that LPS has no toxic effects on macrophages.The maximum safe dose of glycoglycerolipids on macrophages was 200μg/ml,so the low dose of 50μg/ml,medium dose 100 μg/ml,high dose 200 μg/ml were selected as the final test concentration of glycoglycerolipids.The effects of three glyceroglyco lipid compounds on cellular NO secretion were detected by Griess method.The results showed that all three compounds could significantly reduce cellular NO secretion levels in a dose-dependent manner.The effect of three glyceroglycolipid compounds on the secretion of IL-1β,IL-6 and TNF-α in cells was detected by ELISA.The results showed that 200μg/ml glyceroglycolipid compounds could significantly reduce the secretion level of IL-1β、IL-6 and TNF-α.In summary,three glyceroglycolipid compounds isolated from perilla leaves can effectively inhibit the level of inflammatory factors secreted by LPS-induced rat macrophages RAW264.7.The preliminary structureactivity relationship showed that the anti-inflammatory capacity of glycoglycerolipids increased with the increase of fatty acyl unsaturation,and decreased with the increase of the number of sugar groups.3.Evaluation of antioxidant activity of glyceroglycolipid in perilla leavesThe antioxidant activity of glycoglycerolipids with three different molecular species in Perilla leaves was determined by chemical evaluation method and cell oxidative damage model.The relationship between the molecular structure of glycoglycerolipids and its antioxidant activity was also preliminarily discussed.The results indicated that antioxidant activities of glycoglycerolipids with three different molecular species were different.Antioxidant capacity of monogalactosyldiacylgycerol(MGDG)and digalactosyldiacylglycerol(DGDG)composed of fatty acyl CI 8:3/18:3 was higher than DGDG composed of fatty acyl C18:3/16:0,all glycoglycerolipids with strong reducing power can not only inhibit the production of 2,2’-azino-bis-3-ethylbenzthiazoline-6sulfonic acid(ABTS),1,1-Diphenyl-2-picrylhydrazyl(DPPH),hydroxyl radicals(·OH)and superoxide anion radicals(O2-·)in a dose-dependent manner,but also significantly improve the activity of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-PX).In summary,glycoglycerolipids with different fatty acyl composition have different antioxidant capacity,and the preliminary structureactivity relationship showed that the number of double bonds in the acyl group and the number of sugar groups have a greater influence on the antioxidant activity of the glycoglycerolipids.The antioxidant activity of glycoglycerolipids weakens as the number of sugar groups increases.When the number of sugar groups is constant,the antioxidant activity of the glycoglycerolipids with a fatty acyl group as bis-linolenoyl is greater than that of a glyceroglycolipid with a fatty acyl group as linoleyl/palmitoyl.