Studies On The Constituents From The Shells Of Carya Cathayensis And Anti-colorectal Cancer Activities

Carya cathayensis is a deciduous tree, growing mainly in the south and north of China. The extracts of green-walnuts and shells have been used in folk medicine more than four hundred years, according to the theories of Traditional Chinese Medicine,for treatment of rheumatic arthritis, gynecological disorders, constipations and so on.In modern pharmacological studies, it has demonstrated that they also possess actions of anti-cancer,free radical scavenging, cardiotonic, anti-inflammation and anti-nociception.By means of many chromatographic methods, including the open column chromatography of silica gel, Sephadex LH-20, reversed phase ODS and semi-preparative HPLC, thirty two compounds were isolated from the bioactive part of anti-colorectal cancer, EtOAc partition, which was obtained from 75% ethanol extract of the shells of Carya cathayensis. On the basis of physico-chemical data and spectral methods (1H-NMR, 13C-NMR, 1H-1H COSY, HSQC, HMBC, NOESY and CD),twenty five compounds were identified as (7R, 8S,8'R)-4,4',9-trihydroxy-7,9'-epoxy-8,8'-lignan (1),(7R,8S,8'R)-4,4',9-trihydroxy-7'-keto-7, 9'-epoxy-8,8'-lignan (2), carayensin-A (3), carayensin-B (4), carayensin-C (5), (E)-3-[(2S, 3R)-2,3-Dihydro-2-(4'-hydroxy-3'-methoxyphenyl)-3-hydroxymethyl-7-methoxy-1-benzo[b]furan-5-yl]- 2-propenal (6), evofolin-B (7), ligballinol (8), vanillin (9), isovanillin(10), p-hydroxybenzoic acid (11), p-hydroxybenzaldehyde (12), 3-(4'-hydroxy-phenyl)-2-propenol (13), 3-(4'-hydroxy-3'-methoxy-phenyl)-2-propenol (14),5-hydroxy-3-methoxybenzaldhyde (15),1 -(4'-hydroxy-phenyl)-1 -propanone (16),2-formyl-3-hydroxyphenylpropanol (17), 4-(2, 3, 4-tri-hydroxybutoxy)- benzoic acid(18), 4-(2, 3, 4-tri-hydroxylbutoxy)-benzaldehyde (19), 4-(2-hydroxyenthoxy)-benzaldehyde (20), Quercitin (21), Quercitrin (22), Rutin (23), ?-sitosterol (24),daucosterol (25). Among them, compounds 1-5, 18-19 were new compounds,compounds 6, 20 were new natural products, and 7-17, 21-23 were obtained for the first time from the Carya sp. and from the C. cathayensis, respectively.In order to obtain the effective compounds of inhibiting HCT-116 and HT-29, the inhibitory effects of seventeen compounds isolated from the shells of C. cothayensis were determined by MTT assay in vitro. The results indicated that the compound 6,(E)-3-[(2S, 3R)-2, 3-Dihydro-2-(4'-hydroxy-3'-methoxyphenyl)-3-hydroxymethyl-7-methoxy-l-benzo[b]furan-5-yl]-2-propenal (E2S), was considered to be potential as anti-colorectal cancer agent, which more effective than the positive control Juglone.The inhibitory mechanism of E2S on HCT-116 and HT-29 were examined for the signaling pathways by Western blotting assay. The results demonstrated that E2S may have the effect on HCT-116 through inhibiting the protein expression of NFAT4 by the degradation of p-JNK and controlling gene transcription factor by the degradation of c-Myc and have the effect on HT-29 through the protein expression degradations of p-JNK and c-Myc.A reversed-phase high performance liquid chromatography (HPLC) method was developed for the quantitative determination and identification of E2S and Juglone,isolated from different parts of C. cathayensis. The validation tests indicate that the method is of good reproducibility and accuracy and practical for quantitative determination of bioactive constituents of C. cathayensis on anti-colorectal cancer. In result, E2S was detected in the shells of C. cathayensis with the content of 0.08%, and none in other parts.In summary, the bioactive parts of C. cathayensis on anti-colorectal cancer exists shells. The bioactive constituents in the shells were mainly in EtOAc extracts. E2S showed the most significant activities against HCT-116 and HT-29 in the pure compounds from the active parts, which were obtained from 75% ethanol extract of the shells of Carya cathayensis. E2S are speculated to be potential as anti-colorectal cancer agents, and would provide more potent derivative with a suitable modification.The present study provided some chemical and pharmacological information for the screening candidate drugs for the therapy of colorectal cancer from C. cathayensis.