| In this study, grape seed procyanidins were separated by methanol-chloroform solvent two-phase precipitation method. The method of spectrophotography determined the average degree of polymerization of procyanidins. The aim of this study is conducting preliminary research about the interaction and inhibition ratio between proanthocyanidins and protein in vitro, the activity of digestive enzymes and nutrient uptake in normal rats. The process and results are as followed:1. Separation of grape seed proanthocyanidinsGrape seed proanthocyanidins were separated using methanol and chloroform. The grape seed proanthocyanidins powder was dissolved in aqueous solution to reach a certain concentration. This solution was extracted by ethyel acetate to get ethyl acetate phase and aqueous phase. The monomer were removed by a certain concentration of ethyel acetate and toluene and then got Fo. Aqueous phase was evaporated to dryness and then was redissolved in methanol. Isometric chloroform was adjusted to get F1-F5 responsively.2. Determination of the degree of proanthocyanidinsThe relative error was 7.8±1.28%between phloroglucinol and spectrophotometry, which was a smaller gap. Determine the degrees of proanthocyanidins fastly and efficiently. We used spectrophotometry to determine the 6 fragments by methanol-chloroform solvent two-phase precipitation method. The vanillin-glacial acetic acid method was used to determine the molar concentration of procyanidins. The vanillin-methanol method was used to determine the procyanidins content. By measuring, the average degree of F0、F1、F2、F3、F4、F5 was 2.36、 4.46、5.54、7.15、9.12、11.63 respectively.3. Interaction between proanthocyanidins and proteinDifferent concentration and degrees of proanthocyanidins were mixed with gelatin in volumetric flask. Study the complex effect and suppressing discipline between protein and proanthocyanidins. The study found that interaction between proanthocyanidins and protein were related to the content of proanthocyanidins. When the concentration of gelatin is 0.15mg/ml,28%、47%、80%、84%、86% of gelatin in solution was precipitated by 0.05mg/mL. O.lmg/mL、0.15mg/mL、0.2mg/mL、0.25mg/mL of proanthocyanidins. Additional, interaction between proanthocyanidins and protein were related to the degree of proanthocyanidins. When the concentration of gelatin was 0.15 mg/mL,0.05%、32%、49%、 61%、72%、82%、83% of gelatin in solution was precipitated by 1、2.36、4.46、5.54、7.15、 9.12、11.63 of the degree of procyanidin polymerization respectivly.4. The effects of different degrees of procyanidin polymerization on the nutrient absorption and digestive enzyme activity in rats.24 male Kunming white rats were randomly divided into three groups, that was control group, oligomeric procyanidin group and polymeric procyanidin group. Each group contained 8 replicates. The specimens were used for a series of gavage experiments lasting 56 d. The results found that the body weight gain of polymeric group was 8.52±1.59 g and the body weight gain of oligomeric group was 10.94±0.92 g. The final weight of rats from the polymeric group was significantly lower than the control group. The final weight of rats from the oligomeric group was also lower than the control group, but the difference was not significant. The research found that the total food intake of polymeric group was 242.22±15.04 g and the total food intake of oligomeric group was 255.89±13.29 g. The total food intake for experimental groups were significantly lower than the control group. The results found that the food conversion rate of polymeric group was 3.52±0.63% and the food conversion rate of oligomeric group was 4.28±0.33%. The polymer group had significantly lower food conversion rate than the control group, the oligomer group also had lower food conversion rate than the control group, but the difference was not significant. The α-amylase activity of polymeric group in intestinal and pancreatic was 0.20±0.03 U mgprot-1,0.26±0.05 U mgprot-1 respective. The α-amylase activity of oligomeric group in intestinal and pancreatic was 0.27±0.06 U mgprot-1,0.35±0.06 U mgprot-1 respective. The intestinal and pancreatic α-amylase activity from all the experimental groups were significantly lower than that from the control group, the intestinal and pancreatic a-amylase activity from the polymeric group was significantly lower than that from the oligomeric group. The trypsin activity of polymeric group in intestinal, pancreatic and stomach was 3200.25±422.29 U mgprot-1,3782.56±564.98 U mgprot-1,3200.25±422.29 U mgprot-1 respectively. The trypsin activity of oligomeric group in intestinal, pancreatic and stomach was 3900.31±386.49,4652.49±574.35,25.76±3.46 respectively. The activity of the intestinal, pancreatic and stomach proteases from the polymeric group were significantly lower than that from the control group, the activity of the oligomeric group was lower than that of the control group, but the difference was not significant. The apparent digestibility of protein and fat in polymeric group was 80.53±5.49%. 82.65±3.42%respectively. The apparent digestibility of protein and fat in polymeric group was 84.89±2.59%,86.08±1.68%respectively.The apparent digestibility of protein and fat from the polymeric group were significantly lower than that from the control group, the values of the oligomeric group were also lower than that of the control group, but the difference were not significant. In conclusion, the rats from the polymeric group had significantly less weight gain, total food intake, food conversion rate, amylase activity, protease activity and digestibility of major nutrients compared with the control group. Rats from the oligomeric group only had significantly reduced total food intake, a-amylase activity and apparent digestibility of calcium and zinc. The results showed that polymeric procyanidin solution had a larger influence on the nutrient absorption and digestive enzyme activity in rats. Therefore we can reduce polyphenol anti-nutritional by reducing the degree of polymerization of proanthocyanidins. |
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