Angular Dihydropyranocoumarins from Peucedanum japonicum Roots

Abstract

Peucedanum japonicum Thunberg, belongs to Umbelliferae family, was distributed in southern and eastern Asian countries. Its roots were traditionally used as a medicine for cold and neuralgic diseases in Korea and Taiwan. It was reported that the roots of this plant contained coumarins, chromones, polyacetylenes, sugar alcohols, and steroid glycosides. Pharmacological researches revealed that P. japonicum roots showed antioxidative, anti-inflammatory, antifungal activity and cytotoxic effect against lymphocytic leukaemia. In this study, sixteen new angular dihydropyranocoumarins (1-16) and three new angular monohydromonohydroxyfuranocoumarins (41-43) were isolated along with forty-five known compounds from the n-hexane and CHCl3 fractions of the P. japonicum roots. The known compounds were characterized as angular dihydropyranocoumarins (17-40), linear furanocoumarins (44-47), an angular dihydrofuranocoumarin (48), a linear dihydropyranocoumarin (49), simple coumarins (50-58), a chromone (59), ferulic acid derivatives (60-61), a lignan (62), a phenylpropanoid (63), and an indole alkaloid (64). Isolated angular dihydropyranocoumarins (khellactones) included monoacyl- and diacyl-type khellactone esters. In the case of monoacylkhellactones, the absolute configuration was easily determined by the Mosher method. However, the absolute configuration of diacyl khellactone esters was difficult to assign due to the absence of free hydroxyl group. Therefore, partial alkaline hydrolysis prior to MTPA derivatization and X-ray diffraction analysis were applied to determine the absolute configurations at 3- and 4-positions. Because the success of partial hydrolysis and single crystallization was very difficult, ECD spectroscopy was suggested to confirm the absolute configuration of most of the compounds. Interestingly, a few enantiomers were discovered and isolated by enantio-selective column. Enantiomers were usually detected and isolated using chiral column. However, RP-HPLC analysis with MTPA reaction products could be alternative method to confirm enantiomer existence without testing a number of chiral-selective columns. In the case of cis-monoacylkhellactones, the interconversion of substituents at 3- and 4-position was observed. The major MS fragment peak of khellactone esters was detected without a C-4 substituent. Thus, the position of substituents at 3 and 4 could be determined by MS fragmentation analysis without HMBC measurement. The NO production inhibitory activity of isolated compounds were tested using Griess assay in LPS-induced RAW264.7 cells. As a result, 1, 3-6, 22, 31, and 36-38 showed significant activity without cytotoxicity. The isobutyryl, senecioyl, 2-methylbutyryl, and isovaleryl moieties at 3 position played more important role than the acetyl and angeloyl groups. In conclusion, the absolute configuration of isolated compounds were assigned by various methods. Those methods will become useful guide to solve similar structures. The isolated compounds which significantly inhibited NO production were suggested as anti-inflammatory candidates from natural resources.