Diaphragma juglandis fructus contains various bioactive constituents. Fourteen compounds were isolated from Diaphragma juglandis fructus by preparative high performance liquid chromatography (pre-HPLC) and high-speed counter-current chromatography (HSCCC). Their structures were identified by nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS). Compounds (+)-dehydrovomifoliol (12), (6R,9R)-9-hydroxymegastigman-4-en-3-one (13) and (6R,9S)-9-hydroxymegastigman-4-en-3-one (14) are found from Juglans regia L. for the first time. Compounds dihydrophaseic acid (2), blumenol B (3) and (4S)-4-hydroxy-1-tetralone (11) are isolated from Diaphragma juglandis fructus for the first time. The anti-inflammatory effects of isolated compounds were evaluated by an in vitro model of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Compounds gallic acid (1), ethyl gallate (9) and (+)-dehydrovomifoliol (12) exhibited inhibitory activity on the nitric oxide production of RAW 264.7 at a concentration of 25 μM. The result indicated that the combination HSCCC with pre-HPLC is an effective way for compound separation and purification. And Diaphragma juglandis fructus constituents have the potential for the treatment of inflammatory-related diseases.
Keywords: Diaphragma juglandis fructus; anti-inflammatory activity; chemical constituents; high-speed counter-current chromatography; preparative separation.