Preparation Of Kaempferol Nitro Derivative And Study Of Anti-reproductive Function

Nitration is the process of introducing nitro（-NO₂）into the molecules of organic compounds.Heme proteins in organisms,such as myeloperoxidase,soybean peroxidase and horseradish peroxidase,can catalyze the nitration of some compounds,such as protein tyrosine,aromatic compounds or phenolic compounds,with the participation of NO₂^-and H₂O₂.3-nitrotyrosine can be generated from tyrosine in the HRP/H₂O₂/NO₂^-system,which is an important form of protein post-translational modification.Kaempferol is a flavonoid compound with similar structure to tyrosine,which can be obtained from many vegetables and fruits.It has the effects of anti-oxidation,antibacterial,anti-tumor,anti-diabetes and anti-reproduction effects.Therefore,kaempferol has a wide range of applications in food,health care products,cosmetics and biomedicine.In this study,HRP/H₂O₂/NO₂^-reaction system,which was constructed using horseradish peroxidase as a tool enzyme,was used to detect the production of nitro derivatives of kaempferol and thus determine the optimal reaction system.The physicochemical properties of kaempferol nitro derivatives were compared with those of kaempferol,and the effects of kaempferol nitro derivatives on reproduction and life span were examined using C.elegans as a model organism.The results showed that kaempferol nitro derivative could be detected using a biphasic immiscible system of ethyl acetate-buffer.The catalytic efficiency of horseradish peroxidase（HRP）was higher than millet peroxidase（Pm POD）.When the reaction buffer was p H6,the maximum product was produced.When the sodium nitrite content was more than 10 times higher than the amount of kaempferol,the amount of product was no longer significantly increased.Kaempferol nitro derivatives in aqueous phase were separated and purified by macroporous adsorption resin HPD 100.After extraction with ethyl acetate,pure kaempferol nitro derivatives were obtained.The maximum solubility of the nitro-derivative of kaempferol was 94.876μg/m L in water,which was about 48 times than kaempferol.The structure of the nitro derivative of kaempferol was determined by LC-MS and NMR spectroscopy.The nitro（-NO₂）was added to the adjacent position of the phenolic hydroxyl group of the B ring of kaempferol.Caenorhabditis elegans was used as a model organism to evaluate the anti-reproductive effects of kaempferol and its nitro derivatives.Kaempferol significantly inhibited egg laying and ovulation in a dose-dependent manner.Compared with kaempferol,kaempferol nitro derivatives have a more significant inhibitory effect on reproduction,which may be related to its good solubility.Lifespan experiments showed that kaempferol could prolong the lifespan of C.elegans,while equal concentrations of nitro-kaempferol could shorten the lifespan of C.elegans.The results of quantitative real-time PCR showed that the transcription levels of reproductive related genes were significantly decreased in the kaempferol nitro derivatives treatment group.Among them,vitellogenin gene vit-2,fertilization related gene spe-11 and germ cell proliferation and differentiation related gene glp-1 were extremely significant down-regulated.The gene cpb-3 involved in meiosis and the gene fem-3 involved in sex determination were significantly down-regulated.These results indicate that kaempferol and its nitro derivatives affect the process of sperm-egg formation,resulting in the block of sperm-egg conversion,the block of egg activation,and the defect of egg generation,thus playing an anti-reproductive role.