摘要
荷叶为睡莲属植物莲(Nelumbo nucifera Gaertn)的叶片,在全世界分布广泛,是药食两用植物。近年来研究表明,荷叶具有降脂减肥、抗自由基、抗氧化、抗衰老、抑菌与抗病毒等多种生理功能,其中黄酮为其主要活性物质之一,而且黄酮类化合物还具有降血糖作用。本文旨在通过对荷叶黄酮的提取、分离纯化,继而对其进行深度研究,对分离得到的荷叶黄酮进行葡萄糖激酶激活研究,为有效利用荷叶资源提供理论基础。为此本论文开展了以下工作:
1通过单因素、Plackett-Burman设计和响应面分析法,研究了超声波辅助提取荷叶黄酮的最佳工艺条件。以荷叶总黄酮提取率为指标,通过单因素实验确定了荷叶黄酮提取工艺的参数范围,通过Plackett-Burman设计和响应面分析法确定了最佳提取条件。结果表明,最佳提取条件为乙醇浓度67%,液固比34∶1,超声提取40 min,提取3次,该条件下荷叶总黄酮提取率为3.17%。
2采用大孔树脂和聚酰胺对荷叶黄酮进行纯化。通过静态吸附—解吸实验,从HPD-100、HPD-100 A、HPD-400 A和聚酰胺中筛选出聚酰胺对荷叶黄酮进行纯化,通过研究径高比、上样液浓度、水洗量、洗脱剂浓度和用量,确定出最佳纯化工艺。结果表明,聚酰胺对荷叶黄酮具有较好的吸附和解吸能力,最佳吸附条件∶径高比1∶6,上样液浓度10.668 mg·mL~(-1);最佳解吸条件:5倍柱体积的70%乙醇作为洗脱剂。
3采用聚酰胺柱层析法分离荷叶黄酮,得到A、B、C和D四个组分,经薄层色谱法检测,组分D中主要含有槲皮素,结合高效液相色谱法测定槲皮素的含量,此方法的相对标准偏差为7.9%,平均回收率为88.6%,测得每克干荷叶粉中槲皮素的含量为0.17 mg。
4采用自制固相萃取小柱结合HPLC法测定荷叶中槲皮素的含量,确定了固相萃取柱的洗脱剂用量为14 BV,该方法的相对标准偏差为9.08%,平均回收率为99.49%。
5采用自制毛细管填充固相微萃取柱结合HPLC法测定荷叶中槲皮素的含量,确定了该固相微萃取柱的上样液体积为25μL,洗脱剂用量为14 BV,该方法的相对标准偏差为4.61%,平均回收率为89.55%。
6对荷叶粗提物及组分A、B、C、D进行GK活性的筛选,得到了具有激活GK活性的组分C,该组分的半激活浓度(EC_(50))为0.0803 mg·ML~(-1)。
The leaf of Nelumbo nucifera Gaertn is widespread in the world.Lotus leaf is a kind of natural plant material used both in food and medicine field,involving many active materials.It has recently been found to display significant antiobesit, anti-hyperlipidemia,antioxidative,free radical scavenging activities,anti-aging, bacteriostasis,antiviral and hypoglycemic action.The flavonoid is one of the main active materials.In this paper,the extraction,separation of flavonoids in lotus leaf and its enzymatic activity of glucokinase have been studied profoundly,which will provide theoretic foundation for exploitation of lotus leaf.The main results were as follow:
1.With the extraction ratio of flavonoids as studying index,based on the signal factor experiment,the optimum parameters and scopes are ensured by the method of Plackett-Burman design and response surface methodology.The results showed that, the ratio of solvent to material,ethanol concentration and extraction times were the main factors to affect the extraction rate of flavonoids.The optimum parameters are as follow:the ethanol concentration was 67%,the ratio of solvent to material was 34:1,the times of extraction was 3,and the ultrasonic extraction time was 40min.The total flavoniods extraction ratio was 3.17%at the optimum conditions.
2.Polyamide resin was chosen by experiment of static adsorption-desorption of four kinds of resin HPD-100、HPD-100 A、HPD-400 A and polyamide,and the factor of affecting the rate of adsorption and desorption of polyamide resin are researched, such as ratio of diameter to height、effluent concentration、volume of eluting water、elution concentration and volume of eluting agents.The optimum absorption conditions were that the ratio of diameter to height was 1:6,the effluent concentration was 10.668 mg·mL~(-1);the optimum desorption conditions were that the eluting solvent was 70%ethanol,and its volume was 5 BV.
3.Fraction A、B、C and D were separated from water and ethanol extracts of lotus leaf by column chromatography.Fraction D was identified of quercetin by the thin layer chromatography.The method reproducibility expressed as relative standard deviation(RSD) was 7.9%for quercetin.The method recovery was 88.6%.Quercetin was qualitative and quantitative determination of 0.17 mg·g~(-1) in lotus leave by HPLC-UV.
4.The quercetin of lotus leaf was purified with polyamide solid-phase extraction (SPE) by HPLC-UV.The volume of eluting solvent was 14 BV.The method reproducibility expressed as relative standard deviation(RSD) was 9.08%,and the average recovery of quercetin was 94.99%for quercetin by SPE.
5.The microextraction in a packed capillary(MEPC) was used to separate and purify flavonoids in lotus leaf as a new method for sample preparation.The volume of eluting solvent was 14 BV.The method reproducibility expressed as relative standard deviation(RSD) was 4.61%,and the average recovery of quercetin was 89.55%for quercetin by MEPC
6.The enzymatic activity of glucokinase was studied in vitro models with the extract and fraction A、B、C、D from lotus leaf.The results showed that only fraction C increased the enzymatical activity of glucokinase,while the extract of lotus leaf and other fractions had decreased the enzymatical activity of glucokinase.EC_(50) of fraction C was 0.0803 mg·mL~(-1).
引文
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