笃斯越桔花青素分离纯化技术及其抗氧化活性研究
摘要
本研究是吉林省科技厅应用基础引导项目(20050567)——长白山道地中药材笃斯越桔有效成分及其功能特性研究课题的一部分。笃斯越桔花青素有许多独特保健功能,如抗氧化作用、抗自由基作用、抗视力退化、抗癌作用及抗动脉硬化和血栓形成等作用。本文首先利用分光光度法对笃斯越桔中花青素进行了含量测定,然后对笃斯越桔花青素提取工艺进行了系统的研究,对比分析了冷浸法与高新技术中的微波法、超声波法和酶法的提取方法对花青素提率的影响,并分别优化出最佳的工艺条件。并采用柱层析法对笃斯越桔花青素进行了纯化研究。在对笃斯越桔花青素的纯度确定的基础上,又对笃斯越桔花青素的种类进行了高效液相色谱的初步鉴定,测得笃斯越桔果实中含有锦葵色素。按照最优的提取及纯化工艺制得笃斯越桔花青素纯化产品,并利用笃斯越桔花青素纯品及粗品展开了体外与体内抗氧化研究。
These years,Vaccinium uliginosum L.is drawing more and more attention in the field of food and health care products industries.There is abundant content of anthocyanins in the fruit skins of Vaccinium uliginosum L.that is of immense benefit to human body,which is due that anthocyanins is a powerful antioxidant agents that can protect the human body against free radical damage.Anthocyanins have been found to exhibit lots of biological functions as antioxidation,anti-free-radical, anti-sight-degrading,antitumouseffect,anti-angiosclerosis and anti-thrombopoiesis and so on.Vaccinium uliginosum L.is widely distributed in Northeast and Inner Mongolia of China,but there are little pure products of anthocyanins from Vaccinium uliginosum L.in the local market of our country,so preparing high purity of anthocyanins and studying the special functions is of great importance to the deep investigation and the industrialization.
The spectrophotometry was employed to determine the contents of anthocyanins from Vaccinium uliginosum L.,which had made up a deficiency for the determination of anthocyanins,where the contents was determined only by chromatographic technique or color scale in the past. On the basis of the qualitative analysis of the anthocyanins,the regression equation was as that:y = 0.0258x -0.0016,with a high linear correlation (coefficient correlation is 0.9998),which indicated that the contents of anthocyanins could be determined accurately and conformed to Beer's law, when the concentration of anthocyanins solution ranged from 4 to 24μg/ml. And then the contents of anthocyanins in different part of Vaccinium uliginosum L. was determined,and the results were that:frozen fruits 2.424%,leaves 0.351%,and the trunk 0.038%as the lowest one.
The extraction technology was studied systematically,comparison between the merceration and advanced technology with respect to microwave,ultrasonic wave and enzymic method was conducted in the article,the optimized technology parameters were studied,respectively.It indicated that the extraction rate of anthocyanins in enzymic method was the highest one that reached 4.55%,increasing by 15%compared with merceration.The moderate methods were ultrasonic wave and merceration and the lowest one was microwave where the extraction rate was 3.783%. As can be seen that,the enzymic method was a potential method,but the conditions of pH values and temperature were hard to control.By the contrary,the merceration was convenient to carry out,especially with a low cost.
The column chromatography was employed to purify anthocyanins after the extraction process.Comparison of the adsorption rate and resolution rate was undertaken for screening five kinds of macroreticular resin,and the AB-8 was selected to purify anthocyanins.The optimized conditions in the process of static adsorption were that,temperature 30℃,pH neutral conditions,alcohol concentration 60%,and the optimized conditions in the process of dynamic adsorption,adsorption flow rate 1.5mL/min,sample volume 5mL(anthocyanins concentration 2.5mg/mL),alcohol concentration of alcohol as the eluant 60%,flow rate of elution 1.5mL/min. The purity of total anthocyanins was 29.8%(UV)after freeze-drying, which was higher than anthocyanins extracts with a purity of 25%in the market.At abroad,25%anthocyanins extracts,usually being used to cure night-blindness,Cerebral vessels block,blood capillary sclerosis,has been recorded in the Pharmacopee Francaisese and gastrelcosis etc.in Italy, France,Spain,Republic of Korea,American and New Zealang and so on.
The categories of anthocyanins from Vaccinium uliginosum L.were identified preliminarily using the High Pressure Liquid Chromatography (HPLC),and the malvidin was found in the fruit of Vaccinium uliginosum L.The standard curve was obtained that,y=766995x-1253.2(R=0.9998). The RSD of precision,reproducibility and recovery rate was 1.377%, 1.339%and 1.039%.It indicated that the methodology authentication was successful for the identification of anthocyanins categories in different part of Vaccinium uliginosum L.and the correlated products.The Malvinidin accounts for 1.53%in purified anthocyanins extracts,and 4.67%in totall anthocyanins.
Comparison of the crude sample and the purified anthocyanins from Vaccinium uliginosum L.that was prepared after extraction and purification according the optimized technology was undertaken with respect to the vitro and vivo antioxidation.In the vitro antioxidation,the inhibition rate scavenging DPPH free radical(0.6mmol/L)was investigated in crude sample,purified sample and Vc(each 100μg/mL)exhibiting the data of 25.51%,63.94%and 44.12%,respectively,the inhibition rate scavenging superoxide free radical(10mmol/L of pyrogallic acid)was examined in crude sample,purified sample,Vc and BHT exhibiting the data of 26.64%, 11.76%,25.15%and 22.49%,respectively,and the reducing power of the samples was investigated,the results were that,the reducing power of purified sample was 7.8 times higher than Vc and 1.8 times than BHT, reducing power of the crude sample was 2.5 times higher than Vc and 0.5 times than BHT.
In the vivo antioxidation,three indexes for antioxidation were investigated in blood serum of mouse,and the purified sample,crude sample and control group were compared.In the aspect of total antioxygen capacity(T-AOC),the antioxidation capacity of purified group was the highest and three times higher than the crude sample.In the aspect of malonaldehyde(MDA),the antioxidation capacity of purified sample was the highest and exceeding 7%than the crude sample.In the aspect of glutathion peroxidase(GSH-PX),the antioxidation capacity of crude sample was the highest and one point five times higher than purified sample.
A systematical research on the extraction and purification of anthocyanins from Vaccinium uliginosum L.was studied in the laboratory; however,the semi-works production remained to be solved in the furore. The antioxidation in this experiment presented herein is only a part of the functional activities of anthocyanins,yet the other activities such as anti-tumor,anti-cancer need to be carried out.
These years,Vaccinium uliginosum L.is drawing more and more attention in the field of food and health care products industries.There is abundant content of anthocyanins in the fruit skins of Vaccinium uliginosum L.that is of immense benefit to human body,which is due that anthocyanins is a powerful antioxidant agents that can protect the human body against free radical damage.Anthocyanins have been found to exhibit lots of biological functions as antioxidation,anti-free-radical, anti-sight-degrading,antitumouseffect,anti-angiosclerosis and anti-thrombopoiesis and so on.Vaccinium uliginosum L.is widely distributed in Northeast and Inner Mongolia of China,but there are little pure products of anthocyanins from Vaccinium uliginosum L.in the local market of our country,so preparing high purity of anthocyanins and studying the special functions is of great importance to the deep investigation and the industrialization.
The spectrophotometry was employed to determine the contents of anthocyanins from Vaccinium uliginosum L.,which had made up a deficiency for the determination of anthocyanins,where the contents was determined only by chromatographic technique or color scale in the past. On the basis of the qualitative analysis of the anthocyanins,the regression equation was as that:y = 0.0258x -0.0016,with a high linear correlation (coefficient correlation is 0.9998),which indicated that the contents of anthocyanins could be determined accurately and conformed to Beer's law, when the concentration of anthocyanins solution ranged from 4 to 24μg/ml. And then the contents of anthocyanins in different part of Vaccinium uliginosum L. was determined,and the results were that:frozen fruits 2.424%,leaves 0.351%,and the trunk 0.038%as the lowest one.
The extraction technology was studied systematically,comparison between the merceration and advanced technology with respect to microwave,ultrasonic wave and enzymic method was conducted in the article,the optimized technology parameters were studied,respectively.It indicated that the extraction rate of anthocyanins in enzymic method was the highest one that reached 4.55%,increasing by 15%compared with merceration.The moderate methods were ultrasonic wave and merceration and the lowest one was microwave where the extraction rate was 3.783%. As can be seen that,the enzymic method was a potential method,but the conditions of pH values and temperature were hard to control.By the contrary,the merceration was convenient to carry out,especially with a low cost.
The column chromatography was employed to purify anthocyanins after the extraction process.Comparison of the adsorption rate and resolution rate was undertaken for screening five kinds of macroreticular resin,and the AB-8 was selected to purify anthocyanins.The optimized conditions in the process of static adsorption were that,temperature 30℃,pH neutral conditions,alcohol concentration 60%,and the optimized conditions in the process of dynamic adsorption,adsorption flow rate 1.5mL/min,sample volume 5mL(anthocyanins concentration 2.5mg/mL),alcohol concentration of alcohol as the eluant 60%,flow rate of elution 1.5mL/min. The purity of total anthocyanins was 29.8%(UV)after freeze-drying, which was higher than anthocyanins extracts with a purity of 25%in the market.At abroad,25%anthocyanins extracts,usually being used to cure night-blindness,Cerebral vessels block,blood capillary sclerosis,has been recorded in the Pharmacopee Francaisese and gastrelcosis etc.in Italy, France,Spain,Republic of Korea,American and New Zealang and so on.
The categories of anthocyanins from Vaccinium uliginosum L.were identified preliminarily using the High Pressure Liquid Chromatography (HPLC),and the malvidin was found in the fruit of Vaccinium uliginosum L.The standard curve was obtained that,y=766995x-1253.2(R=0.9998). The RSD of precision,reproducibility and recovery rate was 1.377%, 1.339%and 1.039%.It indicated that the methodology authentication was successful for the identification of anthocyanins categories in different part of Vaccinium uliginosum L.and the correlated products.The Malvinidin accounts for 1.53%in purified anthocyanins extracts,and 4.67%in totall anthocyanins.
Comparison of the crude sample and the purified anthocyanins from Vaccinium uliginosum L.that was prepared after extraction and purification according the optimized technology was undertaken with respect to the vitro and vivo antioxidation.In the vitro antioxidation,the inhibition rate scavenging DPPH free radical(0.6mmol/L)was investigated in crude sample,purified sample and Vc(each 100μg/mL)exhibiting the data of 25.51%,63.94%and 44.12%,respectively,the inhibition rate scavenging superoxide free radical(10mmol/L of pyrogallic acid)was examined in crude sample,purified sample,Vc and BHT exhibiting the data of 26.64%, 11.76%,25.15%and 22.49%,respectively,and the reducing power of the samples was investigated,the results were that,the reducing power of purified sample was 7.8 times higher than Vc and 1.8 times than BHT, reducing power of the crude sample was 2.5 times higher than Vc and 0.5 times than BHT.
In the vivo antioxidation,three indexes for antioxidation were investigated in blood serum of mouse,and the purified sample,crude sample and control group were compared.In the aspect of total antioxygen capacity(T-AOC),the antioxidation capacity of purified group was the highest and three times higher than the crude sample.In the aspect of malonaldehyde(MDA),the antioxidation capacity of purified sample was the highest and exceeding 7%than the crude sample.In the aspect of glutathion peroxidase(GSH-PX),the antioxidation capacity of crude sample was the highest and one point five times higher than purified sample.
A systematical research on the extraction and purification of anthocyanins from Vaccinium uliginosum L.was studied in the laboratory; however,the semi-works production remained to be solved in the furore. The antioxidation in this experiment presented herein is only a part of the functional activities of anthocyanins,yet the other activities such as anti-tumor,anti-cancer need to be carried out.
引文
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[5]王兆雨,徐美玲,朱蓓薇.蓝莓花青素的提取工艺条件.大连轻工业学院学报,2007,26(3):196
[6]赵玉辉,郭印山,李作轩.果实花青素研究进展.北方园艺,2006(3):46-47
[7]李娟娟.花青素研究进展.中山大学研究生学刊(自然科学、医学版),2007,28(2):1
[8]高爱红,童华荣.天然食用色素-花青素研究进展.保鲜与加工,2001,(3):25-27
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[12]卢钰,董现义,杜景平,等.花色苷研究进展.山东农业大学学报,2004,35(2):315-320
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