蜂毒活性肽的分离纯化及生理功能研究

发布时间：2019-06-24 17:44

【摘要】：蜂毒活性肽是蜂毒中的主要物质,具有抗炎、抗癌、抗辐射等功能。蜂胶含有丰富的黄酮类物质,具有抗菌、抗氧化、抗癌等多种功效。本文以蜂毒和蜂胶为原料,研究了蜂毒活性肽的分离纯化,以及蜂胶黄酮类物质的提取工艺,以抗氧化、抗痛风、抗风湿性关节炎生理活性为指标,经复配获得蜂毒蜂胶复配产品。主要研究结果如下:1.以蜜蜂蜂毒为实验材料,经Sephadex凝胶、醇沉、反相高效液相色谱(RT-HPLC)优化分离纯化条件。通过Sephadex G-25凝胶,以流速28 m L/h、上样量1.0 m L、上样浓度1.0 mg/m L,获得两个组分P1、P2。收集P1组分进行冷冻干燥,用3倍体积无水乙醇沉淀,3000 r/min离心5 min,沉淀即为蜂毒肽,测得蜂毒肽的相对分子量为2840 Da左右,提取率为96%,纯度为98%;上清经RT-HPLC,色谱柱Zorbax SB-C18(4.6×250μm)、进样量10μL、乙腈-0.1%三氟乙酸水溶液为流动相,得到三种组分Q1、Q2、Q3,分别为肥大细胞脱粒肽(MCD肽)、蜂毒明肽、镇静肽,相对分子量2592 Da、2035 Da、2700 Da左右,提取率分别为78.6%、89%、70%,其中MCD肽和蜂毒明肽纯度分别为91.28%、90.16%。2.以蜜蜂蜂胶为实验材料,经单因素与响应面研究,优化蜂胶黄酮类物质的提取条件为:乙醇浓度82%,提取时间3 h,提取温度83℃,料液比1:10,在此条件下蜂胶黄酮类物质的得率为46.30%。3.蜂毒活性肽各组分抗氧化、抗痛风、抗风湿性关节炎生理活性研究。经体外抗氧化实验,0.20 mg/m L蜂毒肽对羟基自由基清除率为15.32%,对超氧化物阴离子的清除率为16.04%;相同浓度的蜂毒明肽、MCD肽对羟基自由基清除率分别为3.56%、1.14%,对超氧化物阴离子的清除率分别为4.51%、1.34%。说明蜂毒肽是蜂毒中主要抗氧化物质。经小鼠痛风实验,蜂毒肽、蜂毒明肽、MCD肽高剂量组与模型组相比XOD活性降低了46.94%、42.00%、31.54%,UA含量降低了62.01%、57.38%、59.75%;与空白组相比CRE含量高了14.63%、19.34%、20.47%,BUN含量高了12.79%、19.16%、17.17%。说明蜂毒活性肽中抗痛风的主要物质为蜂毒肽。经小鼠风湿性关节炎实验,蜂毒肽、蜂毒明肽、MCD肽高剂量组与模型组相比TNF-α含量降低28.32%、24.68%、23.29%,IL-6含量降低54.52%、51.97%、50.99%,IL-1β含量降低26.60%、20.81%、18.94%。表明蜂毒活性肽中抗风湿性关节炎的主要物质为蜂毒肽。4.蜂胶黄酮类物质抗氧化、抗痛风、抗风湿性关节炎生理活性研究。经体外抗氧化实验,0.83 mg/mL的蜂胶黄酮类物质对羟基自由基的清除率为96.39%,对超氧化物阴离子的清除率为91.89%,说明蜂胶黄酮类物质具有抗氧化的功效。经小鼠痛风实验,蜂胶黄酮类物质高剂量组XOD活性比模型组降低45.58%,UA含量降低34.38%;与空白组相比CRE含量升高7.69%,BUN含量升高17.91%。可见蜂胶黄酮类物质具有对抗痛风的作用。经小鼠风湿性关节炎实验,蜂胶黄酮类物质高剂量组TNF-α含量比模型组降低28.08%,IL-6含量降低49.44%,IL-1β含量降低18.45%。表明蜂胶黄酮类物质具有对抗风湿性关节炎的功效。5.蜂毒蜂胶抗氧化、抗痛风、抗风湿性关节炎生理活性复配条件研究。经体外抗氧化实验,0.2 mg/m L的蜂毒肽与0.83 mg/m L的蜂胶黄酮类物质按照1:5配制,对·OH的清除率为96.40%,对超氧化物阴离子自由基清除率为91.90%。经小鼠痛风实验,0.05 mg/mL蜂毒肽与0.326 mg/m L蜂胶黄酮类物质按照1:8的比例进行调配。与模型组相比,1:8组XOD活性降低56.75%,UA含量降低46.81%;与空白组相比,CRE、BUN含量分别上升8.66%、25.78%。说明复配产品具有缓解痛风的效果,且不造成肾功能的损伤。经小鼠风湿性关节炎实验,0.05 mg/m L蜂毒肽与0.326 mg/mL蜂胶黄酮类物质按照1:7的比例进行调配。与模型组相比,1:7组血清TNF-α、IL-6、IL-1β含量分别降低33.28%、50.60%、15.81%。说明复配产品具有缓解风湿性关节炎的效果。目前,痛风、风湿性关节炎等慢性疾病的患病几率呈现逐年上升趋势,研究缓解痛风、风湿性关节炎的复配产品具有重要意义。对蜂毒、蜂胶产品的开发利用奠定了理论基础。
[Abstract]:The bee venom active peptide is the main substance in bee venom, and has the functions of resisting inflammation, resisting cancer, and resisting radiation. Propolis contains rich flavonoids, and has effects in resisting bacteria, resisting oxidation, and resisting cancer. In this paper, bee venom and propolis as raw materials, the separation and purification of bee venom active peptide and the extraction process of the propolis flavonoids were studied, and the compound product of bee venom was obtained by compounding the anti-oxidation, anti-gout and anti-rheumatoid arthritis physiological activities. The main results are as follows:1. The separation and purification conditions were optimized by Sephadex gel, alcohol precipitation, reverse phase high performance liquid chromatography (RT-HPLC) using bee venom as an experimental material. The two components P1 and P2 were obtained by a Sephadex G-25 gel at a flow rate of 28 m L/ h, an upper sample amount of 1.0 m L, and a loading concentration of 1.0 mg/ m L. collecting the P1 component for freeze drying, precipitating with 3 times volume of absolute ethanol, centrifuging at 3000r/ min for 5 min, and precipitating to obtain the bee venom peptide, the relative molecular weight of the bee venom peptide is 2840 Da, the extraction rate is 96%, the purity is 98%, the supernatant is clear through RT-HPLC, the column Zorbax SB-C18 (4.6 to 250.mu. m), The sample amount is 10 & mu; L, and the acetonitrile-0.1% trifluoroacetic acid aqueous solution is the mobile phase to obtain three components Q1, Q2 and Q3, respectively the mast cell degranulation peptide (MCD peptide), the bee venom peptide, the sedative peptide, the relative molecular weight of 2592 Da, the 2035 Da and the 2700 Da, the extraction rate is 78.6% and 89%, respectively, The purity of MCD peptide and melittin was 91.28% and 90.16%, respectively. Using bee glue as an experimental material, the extraction conditions of the propolis flavonoids were optimized by the single factor and the response surface. The extraction conditions of the propolis flavonoids were as follows: the ethanol concentration was 82%, the extraction time was 3 h, the extraction temperature was 83 鈩，

本文编号：2505242