【摘要】：蜂毒活性肽是蜂毒中的主要物質(zhì),具有抗炎、抗癌、抗輻射等功能。蜂膠含有豐富的黃酮類物質(zhì),具有抗菌、抗氧化、抗癌等多種功效。本文以蜂毒和蜂膠為原料,研究了蜂毒活性肽的分離純化,以及蜂膠黃酮類物質(zhì)的提取工藝,以抗氧化、抗痛風、抗風濕性關(guān)節(jié)炎生理活性為指標,經(jīng)復(fù)配獲得蜂毒蜂膠復(fù)配產(chǎn)品。主要研究結(jié)果如下:1.以蜜蜂蜂毒為實驗材料,經(jīng)Sephadex凝膠、醇沉、反相高效液相色譜(RT-HPLC)優(yōu)化分離純化條件。通過Sephadex G-25凝膠,以流速28 m L/h、上樣量1.0 m L、上樣濃度1.0 mg/m L,獲得兩個組分P1、P2。收集P1組分進行冷凍干燥,用3倍體積無水乙醇沉淀,3000 r/min離心5 min,沉淀即為蜂毒肽,測得蜂毒肽的相對分子量為2840 Da左右,提取率為96%,純度為98%;上清經(jīng)RT-HPLC,色譜柱Zorbax SB-C18(4.6×250μm)、進樣量10μL、乙腈-0.1%三氟乙酸水溶液為流動相,得到三種組分Q1、Q2、Q3,分別為肥大細胞脫粒肽(MCD肽)、蜂毒明肽、鎮(zhèn)靜肽,相對分子量2592 Da、2035 Da、2700 Da左右,提取率分別為78.6%、89%、70%,其中MCD肽和蜂毒明肽純度分別為91.28%、90.16%。2.以蜜蜂蜂膠為實驗材料,經(jīng)單因素與響應(yīng)面研究,優(yōu)化蜂膠黃酮類物質(zhì)的提取條件為:乙醇濃度82%,提取時間3 h,提取溫度83℃,料液比1:10,在此條件下蜂膠黃酮類物質(zhì)的得率為46.30%。3.蜂毒活性肽各組分抗氧化、抗痛風、抗風濕性關(guān)節(jié)炎生理活性研究。經(jīng)體外抗氧化實驗,0.20 mg/m L蜂毒肽對羥基自由基清除率為15.32%,對超氧化物陰離子的清除率為16.04%;相同濃度的蜂毒明肽、MCD肽對羥基自由基清除率分別為3.56%、1.14%,對超氧化物陰離子的清除率分別為4.51%、1.34%。說明蜂毒肽是蜂毒中主要抗氧化物質(zhì)。經(jīng)小鼠痛風實驗,蜂毒肽、蜂毒明肽、MCD肽高劑量組與模型組相比XOD活性降低了46.94%、42.00%、31.54%,UA含量降低了62.01%、57.38%、59.75%;與空白組相比CRE含量高了14.63%、19.34%、20.47%,BUN含量高了12.79%、19.16%、17.17%。說明蜂毒活性肽中抗痛風的主要物質(zhì)為蜂毒肽。經(jīng)小鼠風濕性關(guān)節(jié)炎實驗,蜂毒肽、蜂毒明肽、MCD肽高劑量組與模型組相比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風濕性關(guān)節(jié)炎的主要物質(zhì)為蜂毒肽。4.蜂膠黃酮類物質(zhì)抗氧化、抗痛風、抗風濕性關(guān)節(jié)炎生理活性研究。經(jīng)體外抗氧化實驗,0.83 mg/mL的蜂膠黃酮類物質(zhì)對羥基自由基的清除率為96.39%,對超氧化物陰離子的清除率為91.89%,說明蜂膠黃酮類物質(zhì)具有抗氧化的功效。經(jīng)小鼠痛風實驗,蜂膠黃酮類物質(zhì)高劑量組XOD活性比模型組降低45.58%,UA含量降低34.38%;與空白組相比CRE含量升高7.69%,BUN含量升高17.91%。可見蜂膠黃酮類物質(zhì)具有對抗痛風的作用。經(jīng)小鼠風濕性關(guān)節(jié)炎實驗,蜂膠黃酮類物質(zhì)高劑量組TNF-α含量比模型組降低28.08%,IL-6含量降低49.44%,IL-1β含量降低18.45%。表明蜂膠黃酮類物質(zhì)具有對抗風濕性關(guān)節(jié)炎的功效。5.蜂毒蜂膠抗氧化、抗痛風、抗風濕性關(guān)節(jié)炎生理活性復(fù)配條件研究。經(jīng)體外抗氧化實驗,0.2 mg/m L的蜂毒肽與0.83 mg/m L的蜂膠黃酮類物質(zhì)按照1:5配制,對·OH的清除率為96.40%,對超氧化物陰離子自由基清除率為91.90%。經(jīng)小鼠痛風實驗,0.05 mg/mL蜂毒肽與0.326 mg/m L蜂膠黃酮類物質(zhì)按照1:8的比例進行調(diào)配。與模型組相比,1:8組XOD活性降低56.75%,UA含量降低46.81%;與空白組相比,CRE、BUN含量分別上升8.66%、25.78%。說明復(fù)配產(chǎn)品具有緩解痛風的效果,且不造成腎功能的損傷。經(jīng)小鼠風濕性關(guān)節(jié)炎實驗,0.05 mg/m L蜂毒肽與0.326 mg/mL蜂膠黃酮類物質(zhì)按照1:7的比例進行調(diào)配。與模型組相比,1:7組血清TNF-α、IL-6、IL-1β含量分別降低33.28%、50.60%、15.81%。說明復(fù)配產(chǎn)品具有緩解風濕性關(guān)節(jié)炎的效果。目前,痛風、風濕性關(guān)節(jié)炎等慢性疾病的患病幾率呈現(xiàn)逐年上升趨勢,研究緩解痛風、風濕性關(guān)節(jié)炎的復(fù)配產(chǎn)品具有重要意義。對蜂毒、蜂膠產(chǎn)品的開發(fā)利用奠定了理論基礎(chǔ)。
[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，

本文編號：2505242