扶桑花色素的提取、纯化及性能研究
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摘要
从各种植物的花、果中提取的花色苷类天然色素安全、无毒,具有对人体有益的营养和药理作用,被广泛应用于食品、医药、化妆品等行业,近年来引起人们的广泛关注。本文对扶桑花色素的提取、纯化实验条件、理化性质及抗氧化作用进行了较为系统的研究。
研究结果表明,采用超声波法提取扶桑花色素的最佳提取条件为:提取剂为0.1%HCl-95%乙醇(70:30 V/V),料液的质量与体积比为1:150,提取时间为30min,提取温度为60℃。扶桑花色素的最大吸收波长为525nm,提取率为24.28%。
扶桑花色素粗提物的色价E(525nm)=7.01,为了提高色素的纯度,本文依次采用无水乙醇沉淀法、梯度极性溶剂萃取法和大孔吸附树脂纯化法纯化色素。首先,采用无水乙醇沉淀除去色素中的蛋白质、多糖等杂质;其次,采用梯度极性溶剂萃取法除去色素中的脂质、叶绿素和多酚类杂质;然后,利用大孔吸附树脂进一步纯化色素,通过比较,AB-8大孔吸附树脂适宜于扶桑花色素的纯化,静态吸附动力学及解吸实验的研究结果表明:在8h时几乎达到吸附平衡,90%乙醇(pH=2)溶液的洗脱效果最好,达到了75.18%。对AB-8大孔吸附树脂动态吸附、解吸花色苷色素的条件进行优选,实验结果为:吸附时,宜采用pH值为2.0的色素溶液作为上样溶液,上样流速为1 mL/min;解吸时,宜采用pH值为2.0、乙醇浓度为90%的水溶液为洗脱剂,洗脱流速为1 mL/min,在此条件下解吸率为86.90%。纯化后扶桑花色素的色价E(525nm)=16.74,比纯化前提高了1.39倍。
通过不同pH下色素溶液的颜色比较及紫外-可见吸收光谱谱图和红外光谱谱图分析可初步推断扶桑花色素的主要成分为花色苷;扶桑花色素为水溶性色素;pH值的改变对扶桑花色素稳定性的影响很大;扶桑花色素对直射光比较敏感,对热稳定性较好;色素颜色基本不受常用食品添加剂的影响;Vc对色素具有增色作用,强氧化剂H_2O_2的存在加速其降解;不同金属离子对扶桑花色素稳定性的影响不一样,Ca~(2+)、Al~(3+)等金属离子对其稳定性没有明显的影响,Zn~(2+)使色素的吸光度明显增大,溶液颜色加深,Fe~(3+)、Cu~(2+)等金属离子对色素有降解作用,使溶液颜色变浅。
体外试验结果表明,扶桑花色素具有较强的抗氧化能力。它可以作为氢供体,清除二苯代苦味酰自由基(·DPPH),但清除能力低于Vc和BHT(2,6-二叔丁基对甲酚);具有较强的清除超氧阴离子自由基(O_2)和羟自由基(·OH)的能力;同时对多不饱和脂肪酸(PUFA)过氧化体系有较强的抑制作用;也可作为电子供体,将Fe~(3+)还原为Fe~(2+)。
The safe and nontoxic anthocyanin extracted fro m flowers and fruits of various kinds of plants possess the beneficial nutrition and pharmacologic function to human being.It has been widely used in many industries such as foods,medicine and cosmetics.People pays more and more attention to natural pigment.In this project, extraction,purification and characterization of pigment in flowers of Hibiscus rosa-sinensis L.was systemic studied.
The result showed that the optimum extraction method by ultrasonic as follows: extractant was 0.1%HC1-95 ethanol(70:30 V/V),ratio of solid to liquid was 1:150, extraction time was 30 min,and extraction temperature was 60℃,the maximum absorption wavelength of the pigment was 525 nm,and the extraction rate was 24.28%.
The color scale of crude extract in flowers of Hibiscus rosa-sinensis L.was 7.01.In order to improve the purity of anthocyanin,we used ethanol precipitation,the gradient polar solvent extraction and purification of macroporous resin by turns.First, the protein and polysaccharides impurity in the pigment were removed by ethanol precipitation;second,the lipids,chlorophyll and polyphenols were removed by he gradient polar solvent extraction;then,the pigment was further purified by macroporous resin.AB-8 macroporous resin was suitable for purification of pigment extracted in flowers of Hibiscus rosa-sinensis L.Static adsorption dynamics experiment showed that the absorption equilibrium could be achieved in 8h,and elution result is the best by 90%ehanol,and elution rate is 75.18%.The absorption and desorption experiment of AB—8 macroporous resin showed that the pigment solution(pH2.0) was suitable to use as loading solution and loading flow rate was 1 mL/min when absorbed;the 90%ethanol(pH2.0) was properly used as elution buffer when desorbed.The elution flow rate is 1mL/min and the elution rate is 86.90%.The color scale of purified extract in flowers of Hibiscus rosa-sinensis L.was 16.74, 1.39 times as much as that of crude extract.
We could preliminally deduce that the key component of pigment in flowers of Hibiscus rosa-sinensis L.is anthocyanin by the colors in pigment solution with different pH and the spectrogram of ultra violet-visible light and infrared spectrum. The pigment in flowers of Hibiscus rosa-sinensis L.is water soluble and the stability of the pigment was affected greatly by the alteration of pH,the pigement is sensitive to incident light and possess good heat stability,the color of the pigment was not mainly affected by the frequently used food additives,Vc possess es hyperchromic effect to the pigment,strong oxidant H_2O_2 accelerated the degradation of the pigment, different kinds of metals affected differently on the stability of the pigment,Ca~(2+) and Al~(3+) has no influence on its stability,Zn~(2+) increased the absorbance of the pigment, resulted in the dark color,Fe~(3+) and Cu~(2+) degraded the pigment,resulted in the light color.
In vitro study demonstrated that the pigment in flowers of Hibiscus rosa-sinensis L.possessed good antioxidation.It can be used as hydrogen donator to remove 1,1-diphenyl 2-picrylhydrazyl(·DPPH),but the capability is lower than that of Vc and BHT.It had a strong scavenging activity on superoxide radical(O_2~-) and hydroxyl radical (·OH).Moreover,the restraint result of it on peroxidation from polyunsaturated fatty acid(PUFA) was effective.Also,it can be used as electron donator,reducted Fe~(3+) to Fe~(2+).
研究结果表明,采用超声波法提取扶桑花色素的最佳提取条件为:提取剂为0.1%HCl-95%乙醇(70:30 V/V),料液的质量与体积比为1:150,提取时间为30min,提取温度为60℃。扶桑花色素的最大吸收波长为525nm,提取率为24.28%。
扶桑花色素粗提物的色价E(525nm)=7.01,为了提高色素的纯度,本文依次采用无水乙醇沉淀法、梯度极性溶剂萃取法和大孔吸附树脂纯化法纯化色素。首先,采用无水乙醇沉淀除去色素中的蛋白质、多糖等杂质;其次,采用梯度极性溶剂萃取法除去色素中的脂质、叶绿素和多酚类杂质;然后,利用大孔吸附树脂进一步纯化色素,通过比较,AB-8大孔吸附树脂适宜于扶桑花色素的纯化,静态吸附动力学及解吸实验的研究结果表明:在8h时几乎达到吸附平衡,90%乙醇(pH=2)溶液的洗脱效果最好,达到了75.18%。对AB-8大孔吸附树脂动态吸附、解吸花色苷色素的条件进行优选,实验结果为:吸附时,宜采用pH值为2.0的色素溶液作为上样溶液,上样流速为1 mL/min;解吸时,宜采用pH值为2.0、乙醇浓度为90%的水溶液为洗脱剂,洗脱流速为1 mL/min,在此条件下解吸率为86.90%。纯化后扶桑花色素的色价E(525nm)=16.74,比纯化前提高了1.39倍。
通过不同pH下色素溶液的颜色比较及紫外-可见吸收光谱谱图和红外光谱谱图分析可初步推断扶桑花色素的主要成分为花色苷;扶桑花色素为水溶性色素;pH值的改变对扶桑花色素稳定性的影响很大;扶桑花色素对直射光比较敏感,对热稳定性较好;色素颜色基本不受常用食品添加剂的影响;Vc对色素具有增色作用,强氧化剂H_2O_2的存在加速其降解;不同金属离子对扶桑花色素稳定性的影响不一样,Ca~(2+)、Al~(3+)等金属离子对其稳定性没有明显的影响,Zn~(2+)使色素的吸光度明显增大,溶液颜色加深,Fe~(3+)、Cu~(2+)等金属离子对色素有降解作用,使溶液颜色变浅。
体外试验结果表明,扶桑花色素具有较强的抗氧化能力。它可以作为氢供体,清除二苯代苦味酰自由基(·DPPH),但清除能力低于Vc和BHT(2,6-二叔丁基对甲酚);具有较强的清除超氧阴离子自由基(O_2)和羟自由基(·OH)的能力;同时对多不饱和脂肪酸(PUFA)过氧化体系有较强的抑制作用;也可作为电子供体,将Fe~(3+)还原为Fe~(2+)。
The safe and nontoxic anthocyanin extracted fro m flowers and fruits of various kinds of plants possess the beneficial nutrition and pharmacologic function to human being.It has been widely used in many industries such as foods,medicine and cosmetics.People pays more and more attention to natural pigment.In this project, extraction,purification and characterization of pigment in flowers of Hibiscus rosa-sinensis L.was systemic studied.
The result showed that the optimum extraction method by ultrasonic as follows: extractant was 0.1%HC1-95 ethanol(70:30 V/V),ratio of solid to liquid was 1:150, extraction time was 30 min,and extraction temperature was 60℃,the maximum absorption wavelength of the pigment was 525 nm,and the extraction rate was 24.28%.
The color scale of crude extract in flowers of Hibiscus rosa-sinensis L.was 7.01.In order to improve the purity of anthocyanin,we used ethanol precipitation,the gradient polar solvent extraction and purification of macroporous resin by turns.First, the protein and polysaccharides impurity in the pigment were removed by ethanol precipitation;second,the lipids,chlorophyll and polyphenols were removed by he gradient polar solvent extraction;then,the pigment was further purified by macroporous resin.AB-8 macroporous resin was suitable for purification of pigment extracted in flowers of Hibiscus rosa-sinensis L.Static adsorption dynamics experiment showed that the absorption equilibrium could be achieved in 8h,and elution result is the best by 90%ehanol,and elution rate is 75.18%.The absorption and desorption experiment of AB—8 macroporous resin showed that the pigment solution(pH2.0) was suitable to use as loading solution and loading flow rate was 1 mL/min when absorbed;the 90%ethanol(pH2.0) was properly used as elution buffer when desorbed.The elution flow rate is 1mL/min and the elution rate is 86.90%.The color scale of purified extract in flowers of Hibiscus rosa-sinensis L.was 16.74, 1.39 times as much as that of crude extract.
We could preliminally deduce that the key component of pigment in flowers of Hibiscus rosa-sinensis L.is anthocyanin by the colors in pigment solution with different pH and the spectrogram of ultra violet-visible light and infrared spectrum. The pigment in flowers of Hibiscus rosa-sinensis L.is water soluble and the stability of the pigment was affected greatly by the alteration of pH,the pigement is sensitive to incident light and possess good heat stability,the color of the pigment was not mainly affected by the frequently used food additives,Vc possess es hyperchromic effect to the pigment,strong oxidant H_2O_2 accelerated the degradation of the pigment, different kinds of metals affected differently on the stability of the pigment,Ca~(2+) and Al~(3+) has no influence on its stability,Zn~(2+) increased the absorbance of the pigment, resulted in the dark color,Fe~(3+) and Cu~(2+) degraded the pigment,resulted in the light color.
In vitro study demonstrated that the pigment in flowers of Hibiscus rosa-sinensis L.possessed good antioxidation.It can be used as hydrogen donator to remove 1,1-diphenyl 2-picrylhydrazyl(·DPPH),but the capability is lower than that of Vc and BHT.It had a strong scavenging activity on superoxide radical(O_2~-) and hydroxyl radical (·OH).Moreover,the restraint result of it on peroxidation from polyunsaturated fatty acid(PUFA) was effective.Also,it can be used as electron donator,reducted Fe~(3+) to Fe~(2+).
引文
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