Separation And Identification Of Chemical Constituents From Diaphragma Juglandis Fructus And Study On The Anti-inflammatory Activity

Diaphragma juglandis Fructus,known as the walnut diaphragm,is the dry wood diaphragm tissue that grows inside the walnut.Diaphragma juglandis Fructus has been used for treating different illnesses,such as diuresis and clearing heat,spleen and kidney fixation,arresting seminal emission because it contains large amounts of bioactive compounds,including phenolic acids,flavonoids,alkaloid,saponins and polysaccharides.In recent years,reports of comprehensive study about chemical composition corresponding to Diaphragma juglandis Fructus are less.The separation and identification of chemical constituents is not comprehensive.Mostly,the material base of efficacy is not clear.Purified compounds need to be further research and development.In this study,modern chromatographic technique combination with the separation and purification methods was used to study systematically chemical constituents from Diaphragma juglandis Fructus.And advanced spectrum technology was used to identify and analyze the structure.On the basis of the research on the chemical composition,anti-inflammatory activity of isolated compounds from Diaphragma juglandis Fructus was screened for the first time which lays the foundation of further exploitation and utilization of Juglans regia L.genus plants and further understanding of medicinal active ingredients.The main research results and conclusions are as follows.1.The high-speed counter-current chromatography,reversed-phase C₁₈ column chromatography and preparative high performance liquid chromatography?pre-HPLC?were used for the separation and purification.Twenty-four compounds were isolated of the ethyl acetate fraction from Diaphragma juglandis Fructus.Their chemical structures were identified as gallic acid?1?,dihydrophaseic acid?2?,blumenol B?3?,quercitrin?4?,protocatechuic acid?5?,taxifolin-3-O-?-L-arabinofuranoside?6?,p-hydroxybenzoic acid?7?,vanillic acid?8?,ethyl gallate?9?,dihydroquercetin?10?,?4S?-4-hydroxy-1-tetralone?11?,?+?-dehydrovomifoliol?12?,?6R,9R?-9-hydroxymegastigman-4-en-3-one?13?,?6R,9S?-9-hydroxymegastigman-4-en-3-one?14?,isosclerone?15?,quercetin?16?,2,3-dihydro-3-hydroxy-2-oxo-1H-indole-3-aceticacid?17?,juglaninD?18?,2-hydroxycinchoninicacid?19?,C-veratroylglycol?20?,quercetin3-O-?6?-O-galloyl?-?-galactoside?21?,quercetin 3-O-?6?-O-galloyl?-?-glucoside?22?,hyperoside?23?,isoquercitrin?24?.Compounds 12,13,14,17,20 and 22 are found in Juglans regia L.genus for the first time and compounds 2,3,11,19,23 and 24 are isolated from Diaphragma juglandis Fructus for the first time.2.The alkaline silica gel column chromatography and pre-HPLC were used for the separation and purification.Four compounds were isolated of the petroleum ether fraction from Diaphragma juglandis Fructus.Their chemical structures were identified as2-ethoxyjuglone?25?,juglanin A?26?,isosclerone?9?,4-megastigmen-3,9-dione?27?.Compound 27 is found in Juglans regia L.genus for the first time.3.The macroporous resin?MCI?column chromatography,reversed-phase C₁₈ column chromatography and pre-HPLC were used for the separation and purification.Four compounds were isolated of the remaining aqueous fraction from Diaphragma juglandis Fructus.Their chemical structures were identified as 3?-O-?E-4-coumaroyl?-quinic acid?28?,5?-O-?E-4-coumaroyl?-quinic acid?29?,vanillic acid-4-O-?-D-glucopyranoside?30?and litchiol A?31?.The four compounds are found in Juglans regia L.genus for the first time.4.The RAW264.7 inflammatory model cell was established by LPS.The inflammatory cytokine NO expression was studied on each isolated compounds by this model.These results showed that compounds gallic acid?1?,isosclerone?9?,?+?-dehydrovomifoliol?12?,quercetin?16?,2-ethoxyjuglone?25?and juglanin A?26?can significantly inhibit the expression of inflammatory cell model of NO.It revealed that six compounds have potential anti-inflammatory activity which lays the foundation of medicinal development.