Study On Purification Process Of Dihydroquercetin From Larch And Its Comparison With The Biological Activity Of Dihydromyricetin

Dihydroquercetin, also known as taxifolin(TF), is a kind of natural active substance, which belongs to dihydro flavonols. Because of its antioxidant, anti-virus, sunscreen whitening, inhibit cancer cell growth, inhibit high glucose-induced myocardial apoptosis and other biological activity, and is widely used in food, health products, cosmetics, medicine, industry, agriculture and other areas. Purification process of dihydroquercetin from Larch was optimizated, and anti-inflammatory, anti-tumor, anti fatigue activity of dihydroquercetin and dihydromyricetin(DMY) was compared. It aimed that which was more effectively on enhance immunity and playing a better role in the prevention and treatment of disease of dihydro flavonols, provided a scientific basis for experimental research and clinical application of dihydro flavonols.Single factor method combined with Box-Behnken response surface methodology was used to optimize four factors including solid/liquid ratio, recrystallization temperature and time by establishing a model involving four independent variables at three levels each based on TF purity, and get the regression equation. Regression analysis combined with the actual conditions to determine the best purification process conditions were determined as recrystallized for 20 h at 5 ? at a liquid-to-solid ratio of 1:12(TF: water), the purity of dihydroquercetin is 96.20%. The method was low cost, large amount of sample treatment, and followed the principle of saving energy and green environmental protection, and can be used for mass industrial production.1 mg/L lipopolysaccharide(LPS)- induced RAW264.7 macrophages in vitro model of inflammation was established;Cell viability was detected by the cytotoxicity test(MTT) method;Griess reagent method was used to determine the amount of NO released from cell supernatant;The content of cytokine IL-1, IL-6, TNF-, and PGE2 in supernatant was detected by ELISA assay; Gene expression were detected with the methods of RT-PCR. The results show that TF and DMY 2.5-20?g / mL showed no cytotoxicity on macrophages of RAW264.7. Meanwhile, TF high, medium and low dose group and DMY high, medium and low dose group significantly inhibited LPS-induced cells to release of NO and PGE2(P<0.01), and showed a dose-dependent manner; TF high, medium and low dose group and DMY high, medium and low dose group could effectively reduce IL-1?, IL-6, TNF-? gene expression, while reducing LPS-induced cells to produce cytokines levels. These results showed that dihydroquercetin and DMY had good anti-inflammatory effects in vitro and anti-inflammatory effect of TF was better.To establish the H22 implanted hepatocellular carcinoma animal model which was used to analyze the effect of TF and DMY on the growth of transplanted tumor. Mice were divided into six groups 24 h after modeling: model, positive control(cytoxan, 25mg/kg), low-, high-dose(10,30mg/kg) TF and DMY groups. In addition to the positive control group were administered, the other groups were administered orally, the effects of H22 on tumor inhibition rate, immune organ index, biochemical index and factor in tumor bearing mice were determined. Observe the degree of necrosis in tumor tissue by Hematoxylin and Eosin(H&E). Results showed that both low(10mg/kg) and high(30mg/kg) doses of TF and DMY inhibited tumor growth, could increase the thymus index and spleen index of H22 tumor-beating mice. The inhibitory effect was most pronounced(58.8%) with high doses of TF. Both low and high doses of TF and DMY also increased interleukin-2(IL-2) and tumor necrosis factor-?(TNF-?) levels, elevated levels of SOD, reduced MDA level. In addition, no toxicological effects were observed on hepatic function and renal function in low and high doses of TF and DMY. In summary TF and DMY had antitumor effect on H22 transplanted tumor in mice, the inhibitory effect was most pronounced with of TF, and was dose dependent. The possible mechanisms may be related to the enhanced antioxidant capacity, and improved immune function and elevated cytokine levels. Its mechanism may be related to enhance antioxidant capacity, improve the body's immune function, and elevated cytokine levels.The 70 male mice were randomly divided into control group, TF dose group(10mg / kg, 20 mg.kg and 40 mg / kg) and DMY dose group(10mg / kg, 20 mg.kg and 40 mg / kg), with continual intragastric administration for 28 days.. All the mice were evaluated for their weight-loaded swimming duration, including blood lactic acid(BLA), blood urea nitrogen(BUN), liver glycogen(Glycogen), superoxide dismutase(SOD), glutathione peroxidase(GSH-Px) and malondialdehyde(MDA) and other biochemical indicators of the impact; Real-time quantitative RT-PCR was used to detect gene expression of PGC-1? and PPAR?, western blot assay protein expression of PGC-1?. As compared with control group, TF at the high?middle?low dosages and DMY at the high?middle?low dosages could prolong weight-loaded swimming duration, reduce the accumulation of BUN in the body after exercise-induced fatigue in mice, increase the reserves of glycogen in the body, reduce the level of BLA and MDA, increase the content of SOD and GSH-Px; Real-time fluorescence quantitative RT-PCR showed that Dihydrogenquercetin dosage and DMY group PGC-1? and PPAR-? mRNA expression levels significantly higher as compared with the control group, Western blotting results showed that drug treatment PGC-1? protein expression levels were significantly increased. The above results showed that, TF and DMY can accelerate clearance of free radical, enhance mice antioxidant capacity and improve exercise ability of mice, thus play the anti-fatigue role, and the effect was also related to the gene expression of PGC-1? and PPAR? in skeletal muscle.