|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1991 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1990 : ¥1,500,000 (Direct Cost : ¥1,500,000)
1. Isolation and identification of ten kinds of Oryzalides and related compounds.
A novel antibacterial diterpene substances, Oryzalide A and structurally related 9 substances, were isolated from healthy rice leaves and the structures were determined. An ethyl acetate-extractable acidic and neutral crude extracts were isolated from about 20 kg fresh weight of flag leaves of cv. Norin 27, and they were purified by silica gel and sephadex LH-20 column chromatography, HPLC, partition TLC, etc., and finally their structures were determined by ^1H-NMR, ^<13>C-NMR, HR-MS, IR, etc. As the results, four kinds of acidic substances of Oryzalides A, B, and Oryzalic acids A, B, and six kinds of neutral substances of Compounds A, B, C, D, E and F were newly found. The structural characteristics, yields and antibacterial activities of the above mentioned ten substances were clarified.
2. Establishment of a microanalytical method of Oryzalides and Oryzalic acids, and measurement of an endogenous level
in rice leaves. Oryzalides(A and B)and Oryzalic acids(A and B)were analyzed by means of GC-MS-SIM equipped with a capillary column after conversions to the Methyl ester derivative. Amounts of endogenous Oiyzalides and Oryzalic acids were determined by addition of deuterated labeled[17-D_2]Oryzalide B as an internal standard. Major components were found to be Oryzalide A and Oryzalic acid A in fresh leaves of rice plant when monitoring at m/z 305 fragment ion, which was a characteristic and common ion among the four substances.
As the results of preliminary measurement using the above microanalytical method, all Oryzalides and Oryzalic acids were detected from healthy leaves of 8 rice cultivars different in their resistance to bacterial leaf blight, but no correlation was observed between the amount and the degree of resistance. An endogenous four substances were almost detected from leaves but not from roots and ears, and their amount increased as a growth of rice plant proceeds. When the leaves were wounded by a needle bundle-prick, the amount increased to 3-4 times compared with that of not wounding healthy leaves(control). In the leaves inoculated with a compatible or an incompatible strain of pathogenic bacterium, the amount increased to 7.2 or 9.1 times of control, respectively. Less