葛藤总黄酮的提取纯化及其抗氧化活性的研究
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摘要
葛根为常用中药,具有多种药理作用,是国家卫生部批准的药食两用植物。葛根含有葛根素、大豆苷、大豆苷元等30多种异黄酮类物质,其中葛根素是本属特有成分和主要有效成分。随着葛根应用的不断扩大,市场需求量大,资源比较紧张。葛藤为野葛的藤茎,其主要成分为异黄酮类化合物,在对野葛地上藤茎部分进行了一系列可行性研究之后发现,野葛藤中含有大量与葛根相同的异黄酮成分,其中葛根素及大豆苷元含量较丰富。而每年秋冬季节葛根采挖后藤茎全都废弃,造成资源极大浪费并破坏自然环境。因此,深入开展野葛藤茎的应用研究对于拓宽葛根药用资源,保持自然界生态平衡、保护环境和资源再利用有重要意义,对于制药和食品工业也具有深远的意义。
本研究以野葛藤为试材,以碱水作溶剂对葛藤总黄酮的提取工艺进行了研究,对提取液的纯化工艺进行了优化,通过HPLC对葛藤总黄酮组分进行了鉴定,并探讨了葛藤总黄酮的体外抗氧化活性。
研究主要内容和结果如下:
(1)对野葛不同部位总黄酮含量进行了对比分析,结果表明野葛的根部总黄酮含量最高,达到4.81%,而秋季以后采集的野葛藤也富含总黄酮成分,含量为2.73%;藤茎的采集时间对于总黄酮含量影响很大,以秋冬季采集的藤中含量最高。
(2)采用颜色反应和薄层色谱法对葛藤总黄酮组分进行了初步定性鉴定,TLC试验发现野葛藤茎中含有多种与葛根相同的异黄酮成分,并初步确定含有葛根素和大豆苷元两种主要异黄酮成分,其R_f值分别为0.45和0.83。
(3)葛藤总黄酮碱水浸提法提取工艺的研究。以总黄酮的提取率为指标,进行了碱水浸提单因素试验和正交试验,结果表明,各因素对总黄酮提取效果影响的主次顺序为:提取时间＞浸提温度＞料液比,提取时间对提取率的影响表现为极显著。最佳浸提工艺条件为:粉碎粒度40目,料液比1:40,浸提温度65℃,浸提时间2.0h。
(4)葛藤总黄酮微波-碱水提取法提取工艺的研究。以总黄酮的提取率为指标,通过单因素试验确定了各因素的试验范围,再由响应面中心旋转组合试验设计,确定了微波-碱水法提取的最优工艺条件:料液比1:22,微波功率728W,微波作用时间47S。各因素对总黄酮提取效果影响的主次顺序是,微波处理时间＞微波功率＞料液比;
(5)以葛藤总黄酮为考察对象,对S-8、AB-8、D-101和NKA-9四种大孔吸附树脂进行了静态吸附解吸性能比较,确定AB-8树脂纯化葛藤总黄酮性能最好。以AB-8树脂为吸附剂,乙醇溶液为洗脱剂,对葛藤总黄酮的纯化工艺进行研究,得到最优纯化工艺条件为:以40g湿树脂装柱量计,总黄酮浓度为1.21mg/mL,流速2.0mL/min,进液量150mL;以180mL70%的乙醇在3.0mL/min的流速下洗脱,洗脱液经真空干燥后,葛藤总黄酮纯度达到80.25%。
(6)采用高效液相色谱法对葛藤总黄酮成分进行了鉴定,试验确定了液相色谱条件为:色谱柱Hypersil BDS C18,柱温30℃,检测波长250nm,进样量20μL,梯度洗脱:0～15min,甲醇:水(25:75);15～30min,甲醇:水(40:60);流速1.0mL/min。在此液相条件下,葛藤提取液中的主要组分得到较好地分离。
(7)通过高效液相色谱分析得出葛藤精黄酮中含有葛根素和大豆苷元两种主要功效成分;通过对纯化前后HPLC图谱的比较发现,葛藤提取液经纯化后,葛根素得到了富集,占总峰面积的比率增加了15.36%,而大豆苷元有些损失,占总峰面积的比率减少了8.25%。
(8)葛藤总黄酮具有较强的·OH清除能力和还原能力,而且经过AB-8大孔吸附树脂纯化后,清除效果和还原能力明显提高,但是,略低于葛根粗提液。
Radix puerariae is traditional Chinese medicine in common use and have many pharmacological effects,Which is proved medical and edible dual purpose by National Ministry of Health.There are more than thirty kinds of isoflavonoids in Radix puerariae,such as puerarin、daidzin and daidzein,of them,puerarin is the major and active ingredient.Along with expanding application,market demand beomes larger,so it is in short supply.The major ingredient in stem of Pueraria lobata(Willd.) Ohw is isoflavonoids.After a series of the feasibility research on stem of Pueraria lobata(Willd.) Ohw,people think out that stem of Pueraria lobata(Willd.) Ohw have many kinds of isoflavonoids which Radix puerariae have.Of which,puerarin and daidzein have high content.However,stem of Pueraria lobata(Willd.) Ohw was all threw away after Radix puerariae was dug,which lead to resources greatly waste,and destroyed the environment.Therefore, deeply research on application of stem of Pueraria lobata(Willd.) Ohw have great meanings on expanding Radix puerariae officinal resource,keeping zoology balance in nature,protecting environment and resource recycle,and also have far-reaching meanings on pharmacy and food industry.
Using the stem ofPueraria lobata(Willd.) Ohw as the main material,and using alkali water as the impregnant to research on its total flavonoids extraction technology,making extraction liquid purifying technology better,identified flavonoids ingredients within the extraction liquid by HPLC, and the antioxidative ability in vitro of total flavonoids was also discussed.
The main content and results of research were as follows:
(1) The contents of total flavonoids in different parts of Pueraria lobata(Willd.) Ohw were contrasted and analyzed,the results indicted Radix puerariae's total flavonoids content was the highest,obtained 4.81%,however,the stem of Pueraria lobata(Willd.) Ohw gathered in the late autumn were also rich in total flavonoids,the content was 2.73%;The season when the stem of Pueraria lobata(Willd.) Ohw were gathered had a great impact on total flavonoids,in autumn and winter,the contents of total flavonoids were the highest.
(2) Using coloration reactions and TLC to qualitative analyze the flavonoid ingredients.The TLC test showed that the stem ofPueraria lobata(Willd.) Ohw had many kinds of isoflavonoids as the same as Radix puerariae,puerarin and daidzein,the two kinds of isoflavonoids were discovered, the Rf value of them was 0.45 and 0.83 respectively.
(3) The alkali water extraction technics of total flavonoids from stem of Pueraria lobata(Willd.) Ohw.Taking the extraction ratio of total flavonoids as the index,single factor test and orthogonal test were made.The results showed that the primary and secondary order of factors influencing extraction efficiency were as follows:the extraction times＞the extraction temperature＞the ratio of material to liquid.The extracting times had very significant difference.The optimum extraction technics parameters was particle size is 40,the ratio of material to liquid is 1:40,the extraction temperature is 65℃,the extraction times is 2.0h.
(4) The microwave-alkali water treatment extraction technics of total flavonoids from stem of Pueraria lobata(Willd.) Ohw.Taking the extraction ratio of total flavonoids as the index,the area of test was ascertained by single factor test.The optimum microwave- alkali water extraction technics were studied by using response surface analysis,the optimum extraction technics parameters was the ratio of material to liquid is 1:22,the microwave power is 728W,the extraction times is 47S.The results showed that the primary and secondary order of factors influencing extraction efficiency were as follows:the extraction times＞the microwave power＞the ratio of material to liquid.
(5) Taking total flavonoids from stem of Pueraria lobata(Willd.) Ohw as viewed object, through the comparison of static absorption and desorption properties of four kinds of macroporous resins,including S-8,AB-8,D-101,NKA-9.AB-8 resin was determined as the best sorbent to purify total flavonoids from stem of Pueraria lobata(Willd.) Ohw.Respectively taking AB-8 resin and ethanol as sorbent and eluant,through the study of purification technics of total flavonoids from stem of Pueraria lobata(Willd.) Ohw,the best purification technics obtained was taking the wet resin 40g as reference,total flavonoids concentration is 1.21 mg/mL,velocity of solution is 2mL/min, the feed volume is 150mL,180mL 70%ethanol,the flow rate on desorption is 3.0mL/min.By vacuum desiccation,The content of total flavonoids obtained 80.25%.
(6) Identified total flavonoids from stem of Pueraria lobata(Willd.) Ohw by HPLC,The conditions of determining liquid phase chromatogram are as follows:chromatogram column Hypersil BDS C18,column temperature at 30℃,the wavelength of detector at 250nm,sample sizes 20μl,adapted gradient desorption:0～15min,methanol:H_2O,(25:75);15～30min,methanol:H_2O (25:75);rate at 1.0mL/min.In this case,major components of extractive from stem of Pueraria lobata(Willd.) Ohw can be separated well.
(7) It is analysised by HPLC that the two major efficent ingredient puerarin and daidzein are in purified flavonoids.Contrasted HPLC graph fore-and-aft purification,it is thought out that with extractive from stem of Pueraria lobata(Willd.) Ohw purified,puerarin and other flavonoids were enriched,the ratio of apex area was increased 15.36%,daidzein was at a 8.25%percent loss.
(8) Total flavonoids from stem ofPueraria lobata(Willd.) Ohw have strong·OH scavenging ability and deoxidize ability,moreover,after purified by AB-8 resin,the ability obviously increased. Whereas,a little weaker than Radix puerariae extractive.
本研究以野葛藤为试材,以碱水作溶剂对葛藤总黄酮的提取工艺进行了研究,对提取液的纯化工艺进行了优化,通过HPLC对葛藤总黄酮组分进行了鉴定,并探讨了葛藤总黄酮的体外抗氧化活性。
研究主要内容和结果如下:
(1)对野葛不同部位总黄酮含量进行了对比分析,结果表明野葛的根部总黄酮含量最高,达到4.81%,而秋季以后采集的野葛藤也富含总黄酮成分,含量为2.73%;藤茎的采集时间对于总黄酮含量影响很大,以秋冬季采集的藤中含量最高。
(2)采用颜色反应和薄层色谱法对葛藤总黄酮组分进行了初步定性鉴定,TLC试验发现野葛藤茎中含有多种与葛根相同的异黄酮成分,并初步确定含有葛根素和大豆苷元两种主要异黄酮成分,其R_f值分别为0.45和0.83。
(3)葛藤总黄酮碱水浸提法提取工艺的研究。以总黄酮的提取率为指标,进行了碱水浸提单因素试验和正交试验,结果表明,各因素对总黄酮提取效果影响的主次顺序为:提取时间＞浸提温度＞料液比,提取时间对提取率的影响表现为极显著。最佳浸提工艺条件为:粉碎粒度40目,料液比1:40,浸提温度65℃,浸提时间2.0h。
(4)葛藤总黄酮微波-碱水提取法提取工艺的研究。以总黄酮的提取率为指标,通过单因素试验确定了各因素的试验范围,再由响应面中心旋转组合试验设计,确定了微波-碱水法提取的最优工艺条件:料液比1:22,微波功率728W,微波作用时间47S。各因素对总黄酮提取效果影响的主次顺序是,微波处理时间＞微波功率＞料液比;
(5)以葛藤总黄酮为考察对象,对S-8、AB-8、D-101和NKA-9四种大孔吸附树脂进行了静态吸附解吸性能比较,确定AB-8树脂纯化葛藤总黄酮性能最好。以AB-8树脂为吸附剂,乙醇溶液为洗脱剂,对葛藤总黄酮的纯化工艺进行研究,得到最优纯化工艺条件为:以40g湿树脂装柱量计,总黄酮浓度为1.21mg/mL,流速2.0mL/min,进液量150mL;以180mL70%的乙醇在3.0mL/min的流速下洗脱,洗脱液经真空干燥后,葛藤总黄酮纯度达到80.25%。
(6)采用高效液相色谱法对葛藤总黄酮成分进行了鉴定,试验确定了液相色谱条件为:色谱柱Hypersil BDS C18,柱温30℃,检测波长250nm,进样量20μL,梯度洗脱:0～15min,甲醇:水(25:75);15～30min,甲醇:水(40:60);流速1.0mL/min。在此液相条件下,葛藤提取液中的主要组分得到较好地分离。
(7)通过高效液相色谱分析得出葛藤精黄酮中含有葛根素和大豆苷元两种主要功效成分;通过对纯化前后HPLC图谱的比较发现,葛藤提取液经纯化后,葛根素得到了富集,占总峰面积的比率增加了15.36%,而大豆苷元有些损失,占总峰面积的比率减少了8.25%。
(8)葛藤总黄酮具有较强的·OH清除能力和还原能力,而且经过AB-8大孔吸附树脂纯化后,清除效果和还原能力明显提高,但是,略低于葛根粗提液。
Radix puerariae is traditional Chinese medicine in common use and have many pharmacological effects,Which is proved medical and edible dual purpose by National Ministry of Health.There are more than thirty kinds of isoflavonoids in Radix puerariae,such as puerarin、daidzin and daidzein,of them,puerarin is the major and active ingredient.Along with expanding application,market demand beomes larger,so it is in short supply.The major ingredient in stem of Pueraria lobata(Willd.) Ohw is isoflavonoids.After a series of the feasibility research on stem of Pueraria lobata(Willd.) Ohw,people think out that stem of Pueraria lobata(Willd.) Ohw have many kinds of isoflavonoids which Radix puerariae have.Of which,puerarin and daidzein have high content.However,stem of Pueraria lobata(Willd.) Ohw was all threw away after Radix puerariae was dug,which lead to resources greatly waste,and destroyed the environment.Therefore, deeply research on application of stem of Pueraria lobata(Willd.) Ohw have great meanings on expanding Radix puerariae officinal resource,keeping zoology balance in nature,protecting environment and resource recycle,and also have far-reaching meanings on pharmacy and food industry.
Using the stem ofPueraria lobata(Willd.) Ohw as the main material,and using alkali water as the impregnant to research on its total flavonoids extraction technology,making extraction liquid purifying technology better,identified flavonoids ingredients within the extraction liquid by HPLC, and the antioxidative ability in vitro of total flavonoids was also discussed.
The main content and results of research were as follows:
(1) The contents of total flavonoids in different parts of Pueraria lobata(Willd.) Ohw were contrasted and analyzed,the results indicted Radix puerariae's total flavonoids content was the highest,obtained 4.81%,however,the stem of Pueraria lobata(Willd.) Ohw gathered in the late autumn were also rich in total flavonoids,the content was 2.73%;The season when the stem of Pueraria lobata(Willd.) Ohw were gathered had a great impact on total flavonoids,in autumn and winter,the contents of total flavonoids were the highest.
(2) Using coloration reactions and TLC to qualitative analyze the flavonoid ingredients.The TLC test showed that the stem ofPueraria lobata(Willd.) Ohw had many kinds of isoflavonoids as the same as Radix puerariae,puerarin and daidzein,the two kinds of isoflavonoids were discovered, the Rf value of them was 0.45 and 0.83 respectively.
(3) The alkali water extraction technics of total flavonoids from stem of Pueraria lobata(Willd.) Ohw.Taking the extraction ratio of total flavonoids as the index,single factor test and orthogonal test were made.The results showed that the primary and secondary order of factors influencing extraction efficiency were as follows:the extraction times＞the extraction temperature＞the ratio of material to liquid.The extracting times had very significant difference.The optimum extraction technics parameters was particle size is 40,the ratio of material to liquid is 1:40,the extraction temperature is 65℃,the extraction times is 2.0h.
(4) The microwave-alkali water treatment extraction technics of total flavonoids from stem of Pueraria lobata(Willd.) Ohw.Taking the extraction ratio of total flavonoids as the index,the area of test was ascertained by single factor test.The optimum microwave- alkali water extraction technics were studied by using response surface analysis,the optimum extraction technics parameters was the ratio of material to liquid is 1:22,the microwave power is 728W,the extraction times is 47S.The results showed that the primary and secondary order of factors influencing extraction efficiency were as follows:the extraction times＞the microwave power＞the ratio of material to liquid.
(5) Taking total flavonoids from stem of Pueraria lobata(Willd.) Ohw as viewed object, through the comparison of static absorption and desorption properties of four kinds of macroporous resins,including S-8,AB-8,D-101,NKA-9.AB-8 resin was determined as the best sorbent to purify total flavonoids from stem of Pueraria lobata(Willd.) Ohw.Respectively taking AB-8 resin and ethanol as sorbent and eluant,through the study of purification technics of total flavonoids from stem of Pueraria lobata(Willd.) Ohw,the best purification technics obtained was taking the wet resin 40g as reference,total flavonoids concentration is 1.21 mg/mL,velocity of solution is 2mL/min, the feed volume is 150mL,180mL 70%ethanol,the flow rate on desorption is 3.0mL/min.By vacuum desiccation,The content of total flavonoids obtained 80.25%.
(6) Identified total flavonoids from stem of Pueraria lobata(Willd.) Ohw by HPLC,The conditions of determining liquid phase chromatogram are as follows:chromatogram column Hypersil BDS C18,column temperature at 30℃,the wavelength of detector at 250nm,sample sizes 20μl,adapted gradient desorption:0～15min,methanol:H_2O,(25:75);15～30min,methanol:H_2O (25:75);rate at 1.0mL/min.In this case,major components of extractive from stem of Pueraria lobata(Willd.) Ohw can be separated well.
(7) It is analysised by HPLC that the two major efficent ingredient puerarin and daidzein are in purified flavonoids.Contrasted HPLC graph fore-and-aft purification,it is thought out that with extractive from stem of Pueraria lobata(Willd.) Ohw purified,puerarin and other flavonoids were enriched,the ratio of apex area was increased 15.36%,daidzein was at a 8.25%percent loss.
(8) Total flavonoids from stem ofPueraria lobata(Willd.) Ohw have strong·OH scavenging ability and deoxidize ability,moreover,after purified by AB-8 resin,the ability obviously increased. Whereas,a little weaker than Radix puerariae extractive.
引文
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