| Pu-erh tea belongs to a post-fermented tea, which is one of the unique and famous dark tea in China. It is favored by the vast number of consumers because of its many special health functions. Tea pigments are regarded as one of the main compositions of the pharmacological action of pu-erh tea. At present, the research of chemical composition, nature and pharmacological mechanism of pu-erh tea pigments has become an inescapable and very important difficulty. The paper studied the pu-erh tea pigments extraction, fraction and bioactivity, hoping tea pigments, especially, including the water-soluble pigments which is the rest of the pu-erh tea pigments which were extracted by chloroform, ethyl acetate, n-butanol in turn, its complexity in composition and properties, could be better understood than ever before, and tea scientific theories will be enriched, part of pharmacological mechanism of pu-erh tea pigments is reasonably expounded. The main results are as follows:1 Response surface analysis of extraction technology of pu-erh tea pigments and its antioxidant activityColor difference value E with distilled water was regarded as evaluation index of pu-erh tea pigments extraction for the first time, which is a simple, safe and fast assessment for different effect of pu-erh tea pigment extraction under different conditions. Through the quadratic regression design, regression model was established between color difference value E of pu-erh tea pigment extraction and ratio of solvent to material, extraction temperature and time of extraction. 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH-)scavenging activity of the crude extract of pigment from pu-erh tea was also researched. If we only took into account color difference value E, the optimum conditions of pigment extraction were:ratio of solvent to material 45:1(v/w), extraction temperature 100℃and time of extraction 150 min. Under these conditions, color difference value E was up to 52.97, which is higher 4.73%than the highest one in both single-factor tests and Box-Behnken composition. And relative standard deviation of color difference value E was 1.37% and differential value was 1.3%compared to the forecast value. Pu-erh tea pigment has excellent scavenging ability of DPPH* and its IC50 value was 30.83μg·mL which suggested that pu-erh tea pigment can be used as a natural anti-oxidant for further development and utilization.2. The effects of different extracts of pigments crude extract of pu-erh tea on the activities of two kinds of digesting enzymes and their properties analysisPigments crude extract (water extracts) of pu-erh tea were extracted by chloroform, ethyl acetate, n-butanol in turn. The effects of different fractions were investigated on the activities of a-glucosidase and pancreatic lipase. The results showed that different fractions of pu-erh tea pigments had different effects on a-glucosidase activity. The sample of chloroform fraction could increase a-glucosidase activity a little and the relative activation rate was 8.33%. However, The water extracts, the ethyl acetate fraction and n-butanol fraction and water fraction showed obvious inhibition, inhibition rates being 52.29%,82.52%,65.84%,58.22% respectively. All fractions of pu-erh tea could inhibit pancreatic lipase activity. And inhibiting rates of chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction and the water extracts were 1.80%> 13.65%.4.57%.7.55%.9.00%. Through chromatism parameter determination, ultraviolet-visible spectroscopy scanning, Fourier infrared spectral scanning and ACQUITY UPLC-Xevo QTof MS analysis of different fractions in the same conditions, the results showed that differences of quality and quantity of their intrinsic chemical composition existed among different extraction fractions, which thus determined the varying effects of the different extraction fractions on activities of two types of enzymes.3 Study on fraction, correlative properties and activity screening of water-soluble pigments from pu-erh teaWater-soluble pigments was installed in hydroxypropyl glucan gel(Sephadex LH-20) column with acetone-aqueous solution for elution. The fractions of water-soluble pigments were collected respectively and their physico-chemical and spectral properties and the effects of different fractions on the activities of a-glucosidase and pancreatic lipase were futher studied. The results indicated that water-soluble pigments in pu-erh tea could be classified best, when 40% acetone-aqueous solution which is the optimum eluent was used, with a flow rate of 0.05 BV/h, for 6 obvious strips could be clearly seen in column. The differences exist in their chromatism parameter values, total reducing power, pH value, the content of conventional component, UV and IR spectroscopy scanning, stability and ACQUITY UPLC-Xevo QTof MS analysis. The study further demonstrated the complexity of compositions and properties of water-soluble pigments. Research also showed that all fractions of water-soluble pigments could inhibit the activities of a-glucosidase and pancreatic lipase, and inhibitory effect of fraction V among them is best.4 The effects of fraction V on proliferation and differentiation of 3T3-L1 preadipocytes and differentiation process related gene and protein expression10μg/mL,20μg/mL,30μg/mL,50p.g/mL and 100μg/mL fraction V respectively disposing 3T3-L1 preadipocytes for 24 hours could slightly inhibit cell proliferation which had no obvious difference with normal control group comparison. And then fraction V with different concentrations mentioned above disposing 3T3-L1 preadipocytes, cell proliferation was restrained after 48 hours and had significant difference (P<0.05) between normal control treatment group and 20μg/mL or 30μg/mL fraction V treatment group, and had very significant difference (P<0.01) between normal control treatment group and 50μg/mL or 100μg/mL fraction V treatment group; Under fraction V with various concentration disposing 3T3-L1 preadipocytes 72 hours, cell proliferation also was restrained, and had significant difference (P<0.05) between normal control treatment and 30μg/mL or 100μg/mL fraction V treatment group, and was not statistically significant between the other treatment group and normal control group.10μg/mL,20μg/mL,30μg/mL,50μg/mL and 100μg/mL fraction V respectively disposing 3T3-L1 preadipocytes (confluence about 70%) 48 hours, all can increase reactive oxygen content,30μg/mL fraction V treatment group among them had the highest level, and very significant difference (p<0.01VS.control group) exist in 20μg/mL,30μg/mL,50μg/mL and 100p,g/mL treatment group compared with control group.fraction V inhibiting cell proliferation and increasing reactive oxygen content might be one of its " weight loss " mechanism and the IR could improved through " weight loss ".lipid concentration was increased in the fraction V with various concentration (10μg/mL,20μg/mL,30μg/mL,50μg/mL and 100μg/mL) groups in the experiment respectively (p<0. 01VS. control group). However, lipid accumulation effect was lower than rosiglitazone group.50μg/mL fraction V significantly decreased the mRNA and protein expression of PPARy2, C/EBPa and PTP1B and GLUT4 protein expression, and increased GLUT4 mRNA expression. The fraction V may have function of maintaining reasonable lipid metabolism balance by decreasing mRNA and protein expression of PPARy2 and C/EBPa, and may decrease PTP1B protein expressions for activating the insulin receptor proteins to promote GLUT4 protein to the membrane transposition and glucose intake, which may improve insulin resistance and prevention of MS effect.
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