Resveratrol Grape Leaf Extract And Separation And Purification

Resveratrol is a kind of polyphenols which is widely existed in many natural plants. Because it has many pharmacological effects, such as anti-cancer, anti-oxidation, estrogen regulation, anti-bacterial, anti-inflammatory and prevention cardiovascular diseases, functional foods containing resveratrol have a great favor and demand. At present, extraction and purification resveratrol from plant resources has gradually received more and more attention. resveratrol was mainly extracted from grape skin and its seeds in external and from Chinese traditional medicine Reynoutria japonica Houtt. in internal, both of which were faced with short of raw material used for extraction and high cost. It was reported that there contained resveratrol in several tissues of grape, such as grape skin, grape seed and grape leaf. Although there are abundant grape resources in China, the utilization to grape leaf is insufficiency, and only few part of them are used for animal feed or added to food as adjuvant component and most of them are wasted or burned out, which not only result the waste of agriculture resources, but also induce environmental pollution. So studying and developing resveratrol in grape leaf is significant to enlarge the plant resources containing reaveratrol and improve economic benefit of grape by-products.Grape leaf was used as raw material in our study. The analytical methods, extract technology and purification process of resveratrol from grape leaf were researched, which provide evidence to the development and utilization of grape leaf. The specific contents are as follows:(1) The determination method to resveratrol using high performance liquid chromatography (HPLC) was established, in which acetonitrile-water was selected as mobile phase and gradient elution mode was applied to. The investigation of methodology had showed that the method could use for quantitative determination due to its accuracy and reliability and supply rapidly measurement for subsequent experimental study. There were literatures, which used UV spectrophotometry method to analysis resveratrol content in extract. When HPLC was applied to detection, we founded that there was a large impurity peak before5minutes in grape leaf extract and the peak area of impurity was significantly higher than that of resveratrol. So the amount of resveratrol in extract can not be determined accurately by UV spectrophotometry. Resveratrol content in grape leaf of5varieties nearby xi’an was analyzed by HPLC method that we established in this study. We came to a result that resveratrol amount in grape leaf of’hutai No.8’was relatively higher than that of other4cultivars, which was up to41.08μg/g in October. In different growth months, the content of resveratrol in grape leaf differed and the change tendency of which was as follows:May to June and July, the quantity of resveratrol in grape leaf of cultivars that we detected (except’tengren’) was showed increase; when the grapes were ripe in August, the content in grape leaf was declined; while the amount enhanced greatly before the leaf falling off in October.(2) Resveratrol in grape leaf was extracted by traditional solvent method and the extraction process was optimized in our investigation. Ethanol was used as extraction solvent and the content of resveratrol was used as inspection index, which was determined by HPLC in this paper. On the basis of single factor experiments, the volume fraction of ethanol, the ratio of liquid to solid and the extraction temperature were studied by response surface methodology (RSM). The extraction conditions that we optimized were the following: the volume fractiom of hydrous ethanol was46%, liquid to solid ratio was23mL/g, temperature was62℃and time was60min. The actually extraction content of resveratrol was up to47.96μg/g.(3) Resveratrol was purified by macroporous rein. After considering the two factors of adsorption rate and desorption rate of seven kind of macroporous reins and three kind of polyamide reins, HPD450A macroporous rein was selected for dynamic column chromatography to further purification of resveratrol. The column chromatography conditions were as follows:the feed rate was1.0mL/min, after adsorption thoroughly, firstly3BV30%ethanol was used to remove impurities, then eluant of5BV95%ethanol was collected, elution rate was1.0mL/min. The combination of extraction method and column chromatography was applied to purify resveratrol, in which ligarine was used to get rid of some impurities. After initial measure was taken to remove dopant, column chromatography was utilized to further purification and the yield was5mg/100g, while the content of resveratrol was13.7%.The content of resveratrol in grape leave is not high, but there are rich in related-resources in our country, it is necessary to have a further study on the process conditions of the separation and purification of resveratrol in grape leave, considering comprehensively from raw material drying, storage conditions, experimental design and operation.