| The walnut (Juglans regia L.) is a member of the family Juglandaceae, widely distributed in China. The yield and planting area rank first in the world. Walnut shell is one of the byproducts of the walnut, which contains lignin, cellulose, polysaccharides, flavonoids, saponins, volatile oil, tannins, coumarins and trace elements etc. In recent yesrs, studies have been reported that flavonoids possess anti-inflammatory, antiviral, antioxidation, antitumor and other biological activity. Preliminary experiments showed that the walnut shell included more flavonoids. The paper exploited the walnut shell as raw materials, optimizing the extraction process of total flavonoids from walnut shell. The crude extract was preliminary purified by organic solvent extraction and macroporous adsorption resins, which were isolated and purified by repeated silica column chromatography and identified by their physical characteristics and nuclear magnetic resonance spectrum. Its lipid-lowering activity was detected on a cellular level, the main contents and results were as follows:1. In the present study, the optimum conditions for the extraction of total flavonoids from Walnut Shell were determined by using response surface methodology. On the basis of single-factor experiments, selecting extraction temperature, ethanol concentration and liquid-to-solid ratio as independent variables, response surface methodology was applied to investigate the effect of three variables on extraction yield of total flavonoids. A regression model describing extraction content of total flavonoids as a function of these three variables was established. The optimal conditions for achieving the high extraction rate of total flavonoids from Walnut Shell were ethanol concentration of59%, extraction temperature of78℃and liquid-to-solid ratio of33:1. The result of verification experiment was coincided with predictive value, the content of total flavonoids from Walnut Shell was3.61mg·g-1.2. The crude extract(779g) was obtained from7kg walnut shell powder usied by alcohol extraction, which was preliminary purified by organic solvent extraction and macroporous adsorption resins. Extracted with ethyl acetate and n-butanol three times, obtaining the extractl(131g) and extract2(463g). The lipid-lowering activity of the two extracts were preliminary determinated, the results showed that ethyl acetate phase greatly promoted the growth of fat cells, inconspicuously reduced lipid content of cells; While n-butanol phase was not significantly about promoting the growth of fat cells, lipid-lowering activity was better, so it can be isolated and purified for the further study.In this paper, five macroporous adsorption resins were selected to compare their adsorption and desorption performances for flavonoids from Walnut Shell by using static adsorption experiment. The best performance resin was NKA-9whose dynamic adsorption performance was further researched. The excellent technological parameter were:the pH value of sample5.0, current velocity1.0mL-min-1, concentration of sample1.0mg·mL-1and5BV95%ethanol. The purity of flavonids was3.58%in the crude extract from Walnut Shell, increasing to62.3%after purification with the NKA-9resin. The coefficient of recovery was88.9%.3. The Walnut shell extract which preliminarily purified separated on gel column,12crude components were selected with gradient elution by chloroform:methanol (100:1;80:1;60:1;40:1;30:1;20:1;:1;5:1;1:1). Choosing four crude components with colors from light yellow to yellow were further subdivided and separated on gel column, using different polarities of elution system obtained6compounds. According to the color and the yield, selecting four compounds which were identified by Thin-layer chromatography(TLC) and nuclear magnetic resonance (NMR). They were stearic acid, β-sitosterol,5-hydroxyl-4’-methoxy-7-methyl-flavone, daucosterol. The5-hydroxyl-4’-methoxy-7-methyl-flavone was newly discovered.4. In order to successfully constructe the model of fat cells, using insulin, dexamethasone and IBMX for inducting3T3-L1cells into fat cells, detecting the lipid-lowering activity of5-hydroxy-4’-methoxy-7-methyl-flavone. The proliferation activity of the5-hydroxy-4’-methoxy-7-Methylflavone for the3T3-L1cell was detected by determining cell survival rate with MTT. The effect of the5-hydroxy-4’-methoxy-7-Methylflavone to fat cell viability was detected by oil red O semi-quantitative methods, TG assay kit quantified the content of TG. The result indicated the lipid-lowering activity of5-hydroxy-4’-methoxy-7-methyl-flavone.The lower concentration of5-hydroxy-4’-methoxy-7-Methyl flavone promoted the growth of cells, the high concentration would directly inhibit the growth of cells within the entire concentration range. The cell survival rate was higher than50%, we could develop the further study about the effect of5-hydroxy-4’-methoxy-7-Methyl flavone to cell differentiation at1～100mg-L-15-hydroxy-4’-methoxy-7-methyl flavonoids inhibitied cell differentiation, the effect was directly proportional to the concentration, and lower concentrations had better lipid-lowering activity. The relative value of TG content reached the minimum (closing to50%) at the highest concentration of100mg·L-1, It indicated that the inhibition of5hydroxy-4’-methoxy-7-methyl-flavone to cell differentiation was very significant and lipid-lowering ability was up to50%. |
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