Studies on Oryzalide A, a novel antibacterial diterpene, isolated from healthy leaves of rice plant

1. 十种稻谷苷类化合物及其相关化合物的分离鉴定。从健康水稻叶片中分离到一种新型抗菌二萜物质Oryzalide A及其结构相关物质，并对其结构进行了测定。从鲜重约20 kg的枸杞旗叶中分离出乙酸乙酯可提取的酸性和中性粗提取物。通过硅胶和sephadex LH-20柱层析、HPLC、分区TLC等方法纯化，最后通过^1H-NMR、^<13>C-NMR、HR-MS、IR等方法测定其结构。结果发现，化合物A、B、A、B中有4种酸性物质，化合物A、B、C、D、E、F中有6种中性物质。阐明了上述10种物质的结构特征、产率和抑菌活性。水稻叶片中米扎里酯和米扎里酸微量分析方法的建立及内源含量的测定。转化为甲酯衍生物后，采用气相色谱-质谱联用仪（GC-MS-SIM）对米扎里酯（A、B）和米扎里酸（A、B）进行分析。采用氘化标记的[17-D_2]米扎里酯B作为内标，测定内源性米扎里酯和米扎里酸的含量。在m/ z305片段离子监测下，发现水稻鲜叶中的主要成分为米扎利胺A和米扎利酸A，这是四种物质中常见的特征离子。采用上述微量分析方法进行初步测定，从8个抗白叶枯病能力不同的水稻品种健康叶片中检测到所有的米扎里酯和米扎里酸，但含量与抗白叶枯病程度无相关性。四种内源物质在水稻叶片中几乎检测到，而在根和穗中检测不到，并且随着植株的生长而增加。针束刺伤叶片后，其用量比未刺伤健康叶片（对照）增加3-4倍。病原菌亲和株和不亲和株接种叶片，其数量分别增加到对照的7.2倍和9.1倍。少

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 … More 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