Five minor ferulic acid esters (Ⅰ～Ⅴ) were identified in a petrol extract of Melia azedarach stem bark, one (Ⅰ) of which was isolated with no impurity for the first time. By chemical and spectral methods, their structures were elucidated as hexacosylferulate (Ⅰ); tetracosylferulate (Ⅱ); pentacosylferulate (Ⅲ); heptacosylferulate (Ⅳ) and octacosylferulate (Ⅴ), respectively.

AIM: To construct the plasmid vectors containing different regions of human eNOS promoter coupled to a red fluorescent protein reporter gene, which may express in mammalian cells. METHODS: Different regions of human eNOS promoter were subcloned respectively into a red fluorescent protein vector, pDsRed1-1. These recombinant vectors, pDsF1033Red, pDsF494Red and pDsF166Red, were then transfected into NIH3T3 cell lines, followed by the observation under a fluorescent microscope. RESULTS: After identified to be right by double restriction enzyme digestion, PCR and sequencing, the vectors might be effectively expressed in NIH3T3 cells. 95 % of the red fluorescent emitted by a red fluorescent protein dispersed all over the cells, appearing at 48-60 h after transfection, reaching peak at 96-144 h, becoming the strongest in light at 144 h, gradually disappearing after 168 h and remaining little red fluorescent in 21 days. The quantity and intensity in expressions of red fluorescent protein drived by different regions of human eNOS promoter were clearly lower than by a strong promoter, p CMVIE . CONCLUSION: The red fluorescent protein reporter gene vectors containing different regions of human eNOS promoter are successfully constructed and may efficaciously express in mammalian cells, appearing not strong transcriptional activities, which provide practical and feasible tools to study functions of different regions of human eNOS promoter and roles of cis-elements in it. [