葡萄组织中白藜芦醇提取工艺优化及含量差异性分析

葡萄组织中白藜芦醇提取工艺优化及含量差异性分析

THE OPTIMIZATION OF RESVERATROL EXTRACT TECHNOLOGY AND CONTENT DIFFERENCES ANALYSIS IN GRAPE TISSUE

作者：孙传艳　　导师：赵新节

山东轻工业学院　　生物化工2011届硕士

摘　要

白藜芦醇是一种多酚类物质，主要存在于虎杖、葡萄、花生等植物体内，具有保护心血管、抗癌、抗氧化、抗菌消炎等多方面的药理作用和保健功能。本实验以酿酒葡萄枝条、叶片、果实为材料，通过正交实验，对有机溶剂回流提取、碱法提取和超声波提取三种白藜芦醇提取工艺的比较研究，探索出白藜芦醇提取得率最高、成本较低的提取方法。在此基础上，通过响应面法优化有机溶剂回流提取，得到最优提取途径；最后采用有机溶剂回流提取法提取赤霞珠葡萄品种的枝条、叶片、果实中的白藜芦醇并检测，分析葡萄不同生长阶段不同组织器官中白藜芦醇含量的差异。结果表明：

1.　采用碱法提取白藜芦醇，考察影响白藜芦醇得率的4个影响因子（浸提温度，浸提时间，pH值，浸提次数），分别进行单因素实验。选取浸提次数为3次，提取剂为氢氧化钠碱液，通过正交实验分析，考察影响葡萄枝条中白藜芦醇提取效果的3个因子（浸提温度、浸提时间，pH），得到最佳提取条件为：浸提时间为2小时，浸提温度为40℃，pH值为11，白藜芦醇提取量最高为50.04 μg/g。

通过正交实验分析，采用超声波法提取白藜芦醇，浸提次数为3次，提取剂为乙酸乙酯，考察影响葡萄枝条中白藜芦醇得率的3个因子（提取温度、提取时间、料液比），最佳工艺参数是浸提时间30 min，浸提温度60 ℃，料液比为1∶20，白藜芦醇提取量最高为71.75 μg/g。采用有机溶剂回流提取白藜芦醇，考察影响白藜芦醇得率的5个影响因子（提取剂、浸提时间、浸提温度、固液比、浸提次数），分别进行单因素实验。选择浸提次数为3次，提取剂为乙酸乙酯，通过正交实验分析，考察影响葡萄枝条中白藜芦醇提取效果的3个因子（浸提温度、浸提时间、固液比），得到最佳提取条件为：浸提时间为7小时，浸提温度为70℃，料液比1∶30，得到白藜芦醇提取量最高为63.68 μg/g。通过三种提取工艺的比较，超声波提取提取率最高而且性质稳定，回流提取次之，碱法提取工艺提取率最低。

2.　在单因素实验的基础上，根据Box-Behnken的中心组合实验设计原理，选取提取温度、提取时间和液固比3个因素，采用3因素3水平的响应面分析方法，对酿酒葡萄枝条中白藜芦醇的回流提取条件进行优化。分析结果表明，提取温度提取时间及液固比与提取含量存在显著的相关性，最优提取工艺条件为提取温度65℃，提取时间6小时，液固比28 mL/g，在该条件下，白藜芦醇的提取量为106.76 μg/g。

3.　采用高效液相色谱（HPLC）分析手段，对不同时间段、不同葡萄组织部位［茎（四年生茎、一年生茎、嫩梢）、叶（嫩叶、老叶，成熟叶）、果梗、果皮、果籽］等白藜芦醇的含量进行检测。结果表明：不同组织部位白藜芦醇含量虽然差异较大，但也呈现一定规律性：果梗>叶片>茎>果皮>果籽；四年生茎>一年生茎>嫩梢；老叶>成熟叶片>嫩叶，果梗中白藜芦醇的平均含量达到139.3μg/g。

关键词　葡萄　白藜芦醇　组织部位　提取工艺　响应面　HPLC

Abstract

Resveratrol is a kind of poly phenolic　substance existing in polygonum,grape,peanut etal,which has many keeping health functions. Resveratrol also have cardiovascular protection,anti-oxidation,anti-cancer,anti-bacterial anti-inflammatory and many other pharmacological effects.

In this experiment,it is aimed chiefly at the branches,the leaves,the fruit. Exploring extraction method of the highest extraction rate of resveratrol and lower costs,we choose three different extraction methods such as reflux extraction,ultrasonic extraction and alkali extraction to research by analysis of orthogonal experiments. On the basis of optimization in reflux extraction by response surface method,we attain the optimal extraction approach;Finally we find the difference by extracting resveratrol content in the branches,leaves and fruits of the same grape variety at different growth stages of different organs. The results showed that:

1.　Resveratrol is extracted in alkali extraction,and inspected the effect of four factors (extraction temperature,extraction time,pH,extraction times) to do the single factor experiments. In analysis of orthogonal experiments,for the sodium hydroxide lye extractant,extraction 3 times,and inspected the effect of three factors (extraction time,extraction temperature,pH) of grape branches to solvent on resveratrol extraction rate in the branches of grape,the optimal technological conditions are as follow: the pH value is 11,extraction temperature is 40 ℃,extraction time is 2 hours,the number of extraction reach the highest rate 50.04μg/g.

In analysis of orthogonal experiments,　resveratrol is extracted in ultrasonic extraction for 3 times using the ethyl acetate extractant,and inspected the effect of three factors (extraction time,extraction temperature,the ratio between solvent and dreg) to solvent on resveratrol extraction rate in the branches of grape,the optimal technological conditions are as follow: the ratio of grape branches to ethyl acetate 1:20,extraction temperature is 60 ,℃ extraction time is 30 minutes,the　highest rate of extraction of resveratrol reach up to 71.75 μg/g.

The effect of five factors　on resveratrol contents (extraction solvent,extraction time,extraction temperature,solid-liquid ratio,extraction times) in resveratrol by reflux extraction were studied to do the single factor experiments,respectively. Resveratrol is extracted in reflux extraction for the ethyl acetate extractant 3 times. In analysis oforthogonal experiments,and inspected the effect of three factors (extraction time,extraction temperature,the ratio between solvent and dreg) of grape branches to solvent on resveratrol extraction rate in the branches of grape,the optimal technological conditions are as follow: the ratio of grape　branches to ethyl acetate 1:30,extraction temperature is 70 ℃,extraction time is 7 hours,the number of resveratrol is 63.68 μg/g in the highest extraction rate. Three extraction methods are compared,ultrasonic extraction is more superior than reflux extraction and alkali extraction for　its highest extraction rate and stable characteristic,followed by reflux extraction,alkali extraction　process the lowest extraction rate.

2.　Based on the single-factor experiments and the principle of Box-Behnken experiment design,extraction temperature,extraction time and the ratio between solvent and dreg were chosen to be the there factors. We used response surface design which was selected three factors and three levels to optimize the extraction condition of trans-resveratrol from grape branches. The results showed that the three factors had noticeable correlation with extraction content and that the optimization extraction condition was that temperature was 65℃,6 hours,the ratio 28 mL/g. On the condition of this,the theory content was 106.76 μg/g.

3.　By analysis of high performance liquid chromatography (HPLC),resveratrol content in different periods,different tissues such as stems (the four-year stem,the one-year stem,young shoots),leaves (young leaves,old leaves,mature leaves),fruit etc were detected. The results showed that: The content of resveratrol changed significantly in different grape tissues,however the general presentation is significantly: Stems>leaves>stems>fruit>fruit seeds;the　four-year stems>the　one-year stems>young shoots,old leaves>mature leaves>young leaves. The average content of leaves reach up to 139.3 μg/g.

Key words　Grape　Resveratrol　Tissue　Extraction　Response surface　HPLC