Studies On The Active Constituents Of Diaphragma Juglandis Fructus

Diaphragma juglandis Fructus,the wooden diaphragma of the kernel of Juglans regia L.,has long been used to Chinese folk medicine,are widely distributed in China.The chemical constituents of Diaphragma juglandis Fructus include flavonoids,cardiac glycosides,alkaloid,and polysaccharides.Diaphragma juglandis Fructus has the effect of strengthening the kidney to stop nocturnal emission,and is used in the treatment of kidney disease in clinical.In addition,its crude extract also has antibacterial,antioxidant activity.In the literature,there are mainly studies on the small molecular compounds,and little research has been done on polysaccharides.Polysaccharides have great value to research,and have become a hot topic in natural medicine research.Objective:Extraction,isolation and purification of polysaccharides from Diaphragma juglandis Fructus,and preliminarily explore the activities of polysaccharides;Studying the small molecular components of the Diaphragma juglandis Fructus systematically,and investigate the activities.Methods:Polysaccharides from Diaphragma juglandis Fructus were obtained through hot water extraction-ethanol precipitation-Sevag method.Through DEAE sephadex A-25 and sephadex G-75 column chromatography to isolate and purificate polysaccharides.On this basis,the structure of polysaccharides were characterized by infrared absorption spectroscopy?IR??ultraviolet absorption spectroscopy?UV??specific rotation?high performance liquid chromatography?HPLC?and gel permeation chromatography?GPC?.The inhibitory effects of polysaccharides on?-glucosidase and the formation of advanced glycation end products?AGEs?were studied.The chemical constituents were isolated from Diaphragma juglandis Fructus by Silica gel,Sephadex LH-20,ODS column chromatography,re-crystallization,preparative TLC and pre-HPLC.The structures of the compounds were identified on the basis of physical properties,chemical evidence and spectroscopic analysis including MS,NMR,UV,IR.Cupricions(Cu²⁺)reducing ability?anti linoleic acid oxidation ability?scavenging superoxide anion activity?the hydrogen peroxide scavenging assay and antibacterial activity were evaluated for the isolated compounds.Results:Two new polysaccharides P4a and P5a were obtained.The relative molecular weight of P4a and P5a was 7015 Da and 13057 Da.The molar ratio of monosaccharide composition of P4a was mannose:rhamnose:glucose:galactose:xylose:arabinose:fucose=1:23.1:11.4:72.2:19.9:25.7:2.6 and P5a was mannose:rhamnose:glucose:galactose:xylose:arabinose:fucose=1:36.2:26.7:82.7:12.3:21.9:2.5.Pharmacological studies showed that the IC₅₀0 of two polysaccharides on?-glucosidase were 192.1 and 55.7?g/mL,respectively,and the IC₅₀0 of inhibition on AGEs was 117.5 and 85.58?g/mL.A phytochemical investigation on the extract of Diaphragma juglandis Fructus resulted in the isolation of thirteen compounds,?-sitosterol?1??daucosterol?2??quercitrin?3??gallic acid?4??taxifolin-3-O-?-L-arabinofuranoside?5??protocatechuic acid?6??naringenin?7??texifolin?8??progallin A?9??juglanin D?10??isoquercitrin-6"-O-3'",4'",5'"-trihydroxybenzoyl?11??4,5,8-trihydroxy-?-tetralone-5-O-?-D-glucopyranoside?12??4,5-dihydroxy-?-tetralone-4-O-?-D-glucopyranoside?13?.The pharmacological results showed that most of the compounds had good antibacterial and antioxidant activities.Conclusions:Polysaccharides from Diaphragma juglandis Fructus showed good inhibitory activity on?-glucosidase and AGEs,which have great potential in the treatment of diabetes and cardiovascular disease;Thirteen compounds were isolated from Diaphragma juglandis Fructus.Among them,compounds 12?13 were isolated from the genus for the first time.The most compounds show good activities on antimicrobial and antioxidant,providing the material basis for exploitation of natural preservatives.