|Budget Amount *help
¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 2002 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 2001 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Isolation of quinone reductase (QR)-inducible constituents from freeze-dried Ashitaba leaves (Angelica keiskei Koidz.) was performed. The dried leaves were extracted with 80 % methanol, and ethyl acetate-soluble fraction was separated therein. The fraction was then chromatographed on a silica gel column by eluting with mixtures of n-hexane and ethyl acetate, in which six major components (Pk1, Pk2, Pk3, Pk4, Pk5, and Pk6) were detected by HPLC analysis. Among the components, both Pk3 and Pk4 were found to induce QR. In addition, the component, Pk6 showed a strong cytotoxicity. The QR-inducible components, Pk3 and Pk4, were further purified using a C18 Sep-Pak cartridge, by eluting the cartridge with 50% and 70 % acetonitrile in water, respectively. Also, the cytotoxic component, Pk6 was isolated on the cartridge by eluting with 100% acetonitrile. These isolates were analyzed by HPLC-MS. Mass spectrum of the QR-inducer, Pk3 showed an apparent fragment ion at m/z 285 which may be due to a flavonol, luteolin, along with the molecular ion peak at 645 (M^+-H). It was presumed that this compound might be a flavonol glycoside having luteolin as aglycone. On the other hand, the cytotoxic component showed an ion peak at m/z 338 as the molecular ion, which was identical to that of a chalcone derivative, 4-hydroxy derrick (2',4-dihydroxy-3'-(γ,γ-dimethylallyl)-4'- methoxychalcone). This suggested that the cytotoxic component should be 4-hydroxy derricin, which has been known as a constituent of Ashitaba root. The component was found in other vegetables such as Mitsuba (Cryptotaenia japonica Hassk), Seri (Oenanthe javanica DC.), and Parsley (Petroselium crispum Nym.). Effect of the components, Pk3 and Pk4 on oxidative cellular damage was evaluated on the basis of amount of 8-OhdG formed by menadione-mediated oxidation of DNA, in which both components showed no suppressive effect. This should be further studied along with improvement of the evaluation method.