Biological Activity Studies Of Flavonoids In The Citrus Peel Slag

Citrus peel slag is the waste after rutaceae Citrus fruit and cultivated varieties ofthe fruit processed, which was mainly processed through the landfill at present in thedomestic. Citrus flavonoids are a series of significant biological activity of flavonoids,mainly in the rutaceae citrus species and cultivars of fruit or peel. Citrus peel is the rawmaterial of traditional Chinese medicine dried tangerine or orange peel, and citrusflavonoids is the main efficacy composition of dried tangerine or orange peel. Therefore,the study of citrus flavonoids extraction, purification and biological activity is greatsignificance to citrus peel residue scientific utilization and environmental protection.This topic used orange peel slag as raw material according to preliminary researchof Research Group, maked up the citrus compounds called flavonoids by ethanolrefluxing extraction, and determined the citrus flavonoids ingredients bySpectrophotometry.Then neuromedin U-2receptor agonist activity was measured by reporter genescreening model of neuromedin U-2receptor agonists on different citrus pomace extractand nine kinds of citrus flavonoids monomer and its derivatives, performance and oneof the active substance, half effective concentration and potency were analysed. At lasttrue positive test was done by siRNA interference experiments on the active substancein citrus bioflavonoids.1、The extraction, purification and detection of the total flavonoids of citrus peelThe flavonoids of orange peel were extracted from citrus peel by refluxingextraction. At first citrus peel was degreased and decolored with petroleum.Then, water,60%ethanol,75%ethanol, and95%ethanol were used respectively to refluxingextraction with material liquid rate l:20, extraction temperature80℃, reflux extraction3times and each extract for4h. At last, filtering, vacuum distillation, low-temperaturedrying were done to obtain the total flavonoids of citrus peel.Spectrophotometry was used with rutin as a standard product to detect the flavonoidextract species citrus content in510nm place. The results were that the flavonoidscontent by orange peel slag water,60%ethanol,75%ethanol,and95%ethanol waterextraction in the sample were61.143%,62.684%,72.116%and71.556%. Then theprecision experiments and the add kind of recovery experiments were done to assess the detection methods. The result indicated that this method could be used to detect orangepeel extract citrus flavonoids.2、The construction of the negative cell linesHEK293cells were transfected with NMU2R expressing plasmid (NMU2R/pCDNA3.1) and reporter plasmid (3×MRE/3×CRE/SRE-Luciferase/pGL3) with a1:5ratio. After including800ug/ml of G418medium screening, pick monoclonal cells.Of CRE (cAMP response element) activation agent forskolin role in cell, screening toget one pair of forskolin dose-dependent effect of endogenous ligands for NMU2Rresponse to the negative control cell lines.3、Study of Neuromedin U2receptor agonist activityUsing to stabilize to express NMU2R, and the expression of luciferase cell lines(NMU2R cell line) dose-dependent manner in after by NMU2R agonist stimulation andbuild to get the negative control cell line, citrus skin extract and nine kinds of citrusflavonoids its derivatives to the detection of the fluorescence response. Found in allcitrus skin extract and citrus bioflavonoids hesperidin and nobiletin the NMU2R celllines can activate the reporter gene is abundantly expressed in the negative control doesnot activate reporter gene expression in cell lines, so the orange skin extract citrusflavonoid hesperidin and nobiletin may activate the effect of NMU2R; factors on theactivation of the best citrus skin extract, hesperidin and nobiletin performance,half-effect concentration and potency testing find the lowest half-effect concentration ofnobiletin.4、siRNA interference analysisTwo siRNA of NMU2R was desig designed n and built, the western blot assayinterference of two siRNA efficiency interfere with efficient siRNA-NMU2R-1forNMU2R-1sequence siRNA interference NMU2R of cells, again citrus bark extract,hesperidin and nobiletin fluorescence response detected. Orange skin extract, hesperidinand nobiletin on the activation of the luciferase gene in NMU2R cells is mediated byNMU2R.