【作者】 姚瑞祺；

【导师】 仇农学；

【作者基本信息】 陕西师范大学 ， 农产品加工及贮藏工程， 2009， 硕士

【摘要】 多酚是广泛存在于植物中的一类多羟基酚及其衍生物的总称。多酚的邻位酚羟基极易被氧化,对活性氧等自由基也有较强的捕捉能力,因此具有很强的抗氧化活性和清除自由基的能力,能有效预防癌症、心血管疾病和神经组织退化等多种疾病。青果是我国特产,在南方多个省份均有广泛分布,对青果多酚的开发利用具有重要的现实意义。本文主要从以下几个方面对青果多酚进行试验研究：1.以多酚含量和抗氧化活性为指标对青果与其他85种药食两用植物进行比较,对青果多酚与人工合成抗氧化剂BHT的抗氧化活性进行比较。2.选择适合提取青果多酚的溶剂,利用Design-expert软件建立青果多酚的提取模型,并对模型进行验证；3.选择适合纯化青果多酚的大孔吸附树脂,比较不同浓度乙醇洗脱物的抗氧化活性；4.利用不同极性溶剂对青果多酚进行萃取,研究萃取物的纯度和抗氧化活性,确定青果多酚抗氧化活性成分所在的极性区间；5.利用葡聚糖凝胶Sephadex LH-20进一步分离青果多酚,采用高效液相色谱(HPLC)分析青果多酚的组成。6.采用抗氧化活性跟踪法对青果多酚进行研究,研究青果多酚抗氧化活性成分所在的区间及其物质组成。研究结果表明：(1)青果的多酚含量和抗氧化活性均远超其他药食两用植物。青果多酚抗氧化活性优于BHT,对DPPH·的半数清除浓度IC50为0.140mg/mg,为BHT的6.336倍。(2)甲醇、乙醇和丙酮三种溶剂在最适浓度下,对青果多酚的提取能力大小顺序为：甲醇>乙醇>丙酮,试验选择甲醇为提取溶剂。(3)基于星点设计(CCD)青果多酚提取试验结果表明：甲醇浓度(A)、提取温度(B)和提取时间(C)极显著；甲醇浓度平方项(A2)、提取温度(B2)、提取时间(C2)极显著；提取温度与提取时间的交互项(BC)夕极显著,甲醇浓度与提取时间的交互项(AC)显著,甲醇浓度与提取温度的交互项(AB)不显著,各影响因素对于青果多酚提取率的影响不是简单的线性关系。通过Design-Expert软件,建立了以甲醇浓度、提取温度、提取时间为自变量的青果多酚提取模型：Y=0.39+4.883*10-3A+2.514*10-3B+3.064*10-3C-7.919*10-4AB-1.368*10-3AC-1.944*10-3B C-2.419*10-3 A2-1.197*10-3B2-1.298*10-3C2根据此模型得到青果多酚的最佳提取工艺参数为：甲醇浓度为78.2%,提取温度64.4℃,提取时间7.4h；以此条件进行试验,得到提取量平均值为0.3926g/g DM,与模型预测值的提取量值0.3932g/g DM相比,相对误差为1.53%,证明该模型是合适有效的。在该提取模型下得到的青果多酚纯度为62.63%,总抗氧化能力FRAP值为16.15mmol/g,对DPPH·的半数清除浓度IC50为0.140mg/mg。(4)采用乙酸乙酯、正丁醇2种不同极性的溶剂萃取青果粗多酚水溶液,得到乙酸乙酯层(MA)、正丁醇层(MB)、残留水层(MR)3种不同极性萃取层。与粗多酚相比,MA和MB的纯度和抗氧化活性均有了一定的提高,其中,MB的DPPH·清除效果优于人工合成抗氧化剂TBHQ。MA和MB的纯度分别为73.42%和77.05%, MB总抗氧化能力FRAP值为26.97mmol/g,对DPPH·的IC50为0.108mg/mg；MA总抗氧化能力FRAP值为24.25mmol/g,对DPPH·的IC50为0.117mg/mg。(5)AB-8、NKA-9、D4020这3种大孔吸附树脂对青果多酚的静态吸附和洗脱试验结果表明：NKA-9树脂的吸附率和洗脱率分别为为83.79%和84.23%,均高于其它另外的2种树脂,试验选择NKA-9树脂作为多酚纯化用树脂。(6)采用水-乙醇溶液对NKA-9树脂上吸附的青果多酚进行梯度洗脱的结果表明：40%的乙醇洗脱物抗氧化活性最高,其总抗氧化能力FRAP值为29.10mmol/g,对DPPH·的半数清除浓度IC50为0.073mg/mg,纯度可达87.60%。其次,抗氧化活性较强的依次为50%、30%和60%的乙醇洗脱物,30%-60%的乙醇洗脱物DPPH·清除效果均优于抗氧化剂Vc。(7)葡聚糖凝胶Sephadex LH-20对青果多酚分离效果较好,能得到两个组分F-1、F-2；两组分均具有较强的抗氧化活性。其中,组分F-1的FRAP值为32.57mmol/g,DPPH·的IC50为0.070mg/mg；组分F-2的FRAP值为36.83mmol/g,对DPPH·的IC50为0.058mg/mg。(8)采用外标法对青果多酚进行HPLC分析,结果表明：青果多酚组成复杂,除含有没食子酸、原儿茶酸、绿原酸、表儿茶素、香豆酸、阿魏酸、芦丁、根皮苷、鞣花酸等9种单体酚外,还含有三种重要的未知物质。在已确定的单体酚中,含量相对较高的为绿原酸、原儿茶酸、没食子酸、鞣花酸。(9)青果多酚抗氧化活性最强的区间为组分F-2,其获得途径为：青果粗多酚经NKA-9大孔树脂吸附,40%乙醇洗脱,然后葡聚糖凝胶Sephadex LH-20进一步分离。组分F-2的物质组成中能够确定的有：原儿茶酸、绿原酸、香豆酸、阿魏酸、芦丁、根皮苷,其比例分别为：60.21：65.16：3.84：0.45：3.65：1.52；另外尚有几种未确定结构的单体酚,有待进一步研究。更多还原

【Abstract】 Polyphenol is a general name of some polyhydroxy phenols and their derivatives which are widely present in plant. The o-phenolic hydroxyl of polyphenol is easily oxidized and has a strong ability to capture reactive oxygen such as free radicals; so polyphenol has a strong antioxidative activity and free radical scavenging capacity which can be used to prevent cancer, cardiovascular diseases and nerve tissue degradation and so on. Fructus canarii is a kind of special local plant which has been widely distributed in southern China, so it is of great importance to develop and utilize Fructus canarii polyphenols.In this paper, the following items of polyphenols from Fructus canarii were studied:Firstly, polyphenol content and antioxidating activity were compared among Fructus canarii and other 85 Chinese edible herbal materials. And then the antioxidating activity between Fructus canarii polyphenol arid synthetic antioxidant BHT was compared.Secondly, the optimal solvent for extraction of Fructus canarii polyphenol was selected, and Design-expert software was used to establish extraction model of Fructus canarii polyphenol and then checked.Thirdly, the suitable macroporous absorbent resin was chosen to purify polyphenols from Fructus canarii and the antioxidating activity of ethanol eluates with different concentration were studied.Fourthly, Fructus canarii polyphenol was extracted using solvents with different polar. The purity and antioxidating activity of extract were researched, and polarity range of Fructus canarii polyphenol with strong antioxidating activity was determined.Fifthly, Sephadex LH-20 was used to make further separation of Fructus canarii polyphenol, and high-performance liquid chromatography (HPLC) was carried out to analyse the component of Fructus canarii polyphenol.At last, Fructus canarii polyphenol was studied by antioxidating activity tracing method, and antioxidating range of Fructus canarii polyphenol was identified too.The main results were as follows:1. Polyphenol content and antioxidating activity of Fructus canarii polyphenol were both higher than other Chinese edible herbal materials. The antioxidating activity of Fructus canarii polyphenol was higher than that of BHT, and its 50% inhibitory concentration (IC50) of DPPH·was 0.140mg/mg which was 6.336 times higher BHT.2. The extraction capacity of methanol, ethanol and acetone were:methanol> ethanol> acetone. Methanol was selected as the best one regarding its high selective and strong extraction capacity of polyphenol.3. The results based on the central composite design (CCD) of extract of qingguo polyphenol demonstrated that methanol concentration (A), extraction temperature (B), extraction time (C), methanol concentration square of (A2), extraction temperature (B2), extraction time (C2) had strong influences on the extraction effects. The interaction between extraction temperature and extraction time interactive items (BC) was very significant, and so was the interaction between methanol concentration and extraction time of (AC), while methanol concentration and extraction temperature of the interaction of (AB) was not significant, so the impact of 3 factors on the Fructus canarii extraction rate was not a simple linear relationship. Through the Design-Expert software, the extract model of Fructus canarii polyphenol which showed the relationship between the extraction effect and the 3 factors was calculated as follow:Y=0.39+4.883*10-3A+2.514*10-3B+3.064*10-3C-7.919*10-4AB-1.368*10-3AC-1.944*10-3B C-2.419*10-3 A2-1.197*10-3B2-1.298*10-3C2The best parameters of extraction technology were as follows:methanol concentration was 78.2%, extraction temperature 64.4℃, and extraction time 7.4h. In this condition, the model predictive extraction value was 0.3932 g/g DM, while the real average extraction value was0.3926 g/g DM, so the relative error is 1.53% and the model is appropriate and effective. The purity of Fructus canarii polyphenol which was gained from the technology is 62.63%, and the FRAP value 16.15mmol/g, the IC50 of DPPH·0.140mg/mg.4. Fructus canarii crude polyphenol aqueous solution was extracted with ethyl acetate and n-butanol,3 different extraction layers with different polarity were acetoacetate layer (MA), n-butanol layer (MB) and residual water layer (MR).Compared with the crude polyphenol, both the purity and antioxidant activity of MA and MB became higher. The purities of MA and MB were 73.42% and 77.05% respectively, the FRAP value of MB 26.97mmol/g and IC50 of DPPH·0.108mg/mg, while the FRAP value of MA was 24.25mmol/g and IC50 of DPPH·is 0.117mg/mg.5. The results of static adsorption and desorption test of AB-8, NKA-9, D4020 macroporous resin showed that adsorption and elution rates of NKA-9 were 83.79% and 84.23% respectively was better than the other two kinds of resin. In the following experiments, NKA-9 resin was chosen to purify Fructus canarii crude polyphenol. 6. The results of polyphenol gradient elution with water-ethanol solution on the NKA-9 resin showed that the purity eluated with 40% ethanol reached to 87.60% with the highest antioxidant activity, while the FRAP value was 29.10mmol/g and IC50 of DPPH·0.073mg/mg. Besides, eluates with 50%, 30% and 60% ethanol kept strong antioxidant activity. Eluates at different ethanol concentration (30% to 60%)eliminated DPPH·effectively better than Vc.7. Two components with stronger antioxidant activity (F-1, F-2) were obtained, which separated by Sephadex LH-20 from Fructus canarii polyphenol. The FRAP value and IC50 of DPPH·of F-1 were 32:57mmol/g and 0.070mg/mg respectively, while F-2 6.83mmol/g and 0.058mg/mg respectively.8. The Fructus canarii polyphenol are analyzed by HPLC with external standard method, the results indicated that the composition of Fructus canarii polyphenol was complex. There were gallic acid, protocatechuic acid, chlorogenic acid, epicatechin, coumaric acid, ferulic acid, rutin, phlorhizin, ellagic acid and three other important unknown substances in the extractant., chlorogenic acid, protocatechuic acid, gallic acid and ellagic acid were at relatively high concentration among identified phenolic substances.9. The range of F-2 component had the strongest antioxidant activity which was made by following technology. The crude Fructus canarii polyphenol was adsorbed by NKA-9 macroporous resin, and then desorbed with ethanol of 40% and purified by Sephadex LH-20. The phenolic substances identified from F-2 were protocatechuic acid, chlorogenic acid, coumaric acid, ferulic acid, rutin, Phloretin Gambari, and their ratioes 60.21:65.16:3.84:0.45:3.65: 1.52 respectively althought some other substances were not identified, which needed to do further study.更多还原