Studies On Perparation And The Antitumor Activity Of Peptides From Rape Pollon Glutelins

Rapeseed is one of the most important oilseed crops cultivated in the world, thus rape pollen is the cheap high-yielding pollen. However, because the rape pollen has a special unpleasant flavor that can not be accepted by young people, it is not popular to consumers directly as a commodity for sale, leading to large bulks of storage. Protein content is as high as 20%-25% in Rape pollen, which is comparable to soybeans and peas, suitable proportion of its amino acid composition, with higher contents of essential amino acids, much peptides of higher quality physiological activity were expected to be obtained after the enzymatic modification. But no disquisition on preparation of rape pollen peptides with the proteinase has been reported as yet. Cell-fragmentated and defatted rape pollen was used as material in this study. A research on preparing rape pollen protein fractionation and part of their functional properties were performed for the first time. Based on this, rape pollen gluntelins was hydrolyzed with alcalase and the peptides of rape pollen gluntelins(PRPG) were obtained. The antitumor activity and mechanisms of PRPG were also studied here. The main results were shown as follows:1 Classification of rape pollen protein isolateAll the nutrient contents of fresh rape pollen is not much different from the stale one, and the stale rape pollen is much more conducive to breaking cell. The protein contents in rape pollen account for 22.4% after being defatted and cell-fragmentated. The results showed that glutelins and albumins were found to be the predominant proteins in rape pollen, comprising 55.7% and 39.0% of total proteins, While globulins and prolamins were 3.2% and 2.1% respectively.2 Nutritive value evaluation of pollen protein isolatesThe physico-chemical properties of pollen protein were investigated, including water absorption, oil absorption, emulsifying activity and emulsion stability. Albumins have better function, including water absorption, oil absorption, emulsifying activity and emulsion stability. So albumins can be widely applied in the food and beverage industry. The scavenging hydroxyl radical capability of pollen protein was studied, the inhibition rate .OH of glutelins was and of albumins was 54.35%. Amino acid profile of the isolates indicated that the total essential amino acid of each protein fraction was high. Amino acid ratio coefficient (SRC) is to evaluate the protein quality by dispersion that all essential amino acids diverge from the amino acid model, SRC is highly correlated with the biological value and also is closer to biological value in number. It also showed that the SRC of glutelins was the highest, therefore its nutritional value is the best. Glutelins in human gastric acid environment was of very low bioavailability, therefore, Glutelins was used as the experimental material for the follow-up experiments, and then enzymatic modification not only could increase its bioavailability, but also increase its biological activities.Through orthogonal test, the optimum conditions of extraction of glutelins were obtained on the basis of single factor test, as follow: ratio of solid-liquid 1:20, NaOH concentration 0.30%, pH value 11, time 75min, temperature 70℃.3 Preparation of peptides of rape pollen glutelinsDegree (DH) were used as response values in analysis of response surface regression (RSREG), the optimum conditions of alcalase enzymatic hydrolysis have been determined by mono-factor analysis and response surface methodology as follows: pH=9, hydrolyzing temperature 50℃, enzyme concentration 1460U per gram of substrate, concentration of substrate 6%, hydrolyzing time 2h. And now DH could reach 24.5%.PRPG was graded by Sephadex G-25 column with salt solution as eluant and fractions were pooled into three major groups(P1, P2 and P3). The overall recovery rate was 60.21% and the proportion of P1,P2,P3 were 15.7%,70.2%,14.1%. The antioxidant capability of the groups was different. Crude enzymolysis peptides(2mg/mL) and its three separate components (2mg/mL) all could scavenge hydroxyl free radicals effectively, and P3 which was 14.1% can scavenge hydroxyl free radicals more effectively than at the same concentration of the crude peptides. P1 even have lower antioxidant activity than the same concentration of albumins and globulins.4. Antioxidant activities of PRPGThe antioxidant effects of PRPG were studied in some modified chemical systems, with mice liver mitochondria and mice red blood cell (RBC), mice serum and liver homogenate in vitro and mice in vivo. The deoxidation was measured by K₃[Fe(CN)₆]system and the content of malondialdehyde(MDA) was analysed by the reagent kits. The results showed that PRPG had higher reductive activity and could scavenge hydroxyl radicals in some modified chemical systems. In vitro, PRPG could inhibit the swelling of mice liver mitochondria and the hemolysis of mice RBC induced by H₂O₂, also, could heighten the antioxidant capability of mice serum, and inhibit the formation of malondialdehyde(MDA) in mice liver mitochondria and mice liver homogenate. In vivo, there was a significant difference in MDA value of mice liver homogenate between the control and the groups treated with PRPG(150mg/kg·bw·d). All the above indicated that PRPG had evident antioxidant function.5. Study on the antitumor activity of PRPGThe results showed PRPG of different concentration could inhibit the growth of S180 and Hela cell in vitro, and the significant result appears from the eventually concentration of 1mg/mL, of which the inhibition rate on S180 and Hela is 35.72% and 34.70%.The results in vivo showed that PRPG could significantly inhibit the growth of S180 cell, the inhibition rate of 100 (mg/kg.d) dosage group was respectively 45.44%. PRPG could significantly increase the weight of immune organ, phagocytic function of monocytes, T lymphocyte transformation test, and NK cell activity, restore DTH response and antibody product, which showed that PRPG could increase both specific and non-specific immune function in tumor-bearing mice.The antioxidant capacity of mice greatly decline after they were inoculated with S180 cell. PRPG could remarkably increased SOD and GSH-Px activity and MDA content in serum, which indicated that PRPG could enhance its antioxidant capacity in tumor-bearing mice.Peripheral blood of tumor-bearing mice were greatly abnormal. white blood greatly increased, and red blood cell and hemoglobin famously deceased. PRPG could importantly make them regained.Cy had notable damage on immune organ. PRPG used in combination with Cy could display synergism. It could make antitumor activity of Cy raised to 55.06%%, and reduce toxicity of Cy on immune organ.The effect of PRPG on histomorphology of tumor, liver, kidney and spleen were tested through transplantable animal tumor, compared with Cy. The results of histomorphology showed the PRPG had no obvious toxicity on liver and kidney and spleen in tumor-bearing mice, and could protect them. The result also expressed that PRPG used in combination with Cy could display synergism and reduced Cy toxicity on immune organ. 6 Effect of PRPG on production and mRNA expression of cytokines in spleen cells of tumor-bearing miceThe results of two-antibody-sandwich ELISA indicated that PRPG could remarkably improve the production of TNF-a, IL-2 and IL-6 in spleen cells of tumor-bearing mice, and the results of reverse transcription-polymerase chain reaction showed that PRPG could great raised mRNA expression of TNF-a and IL-6, and induced dose-dependence, which announced that the function of anti-tumor of PRPG was due to enhance cytokine mMRA expression.