烟草物料中药用成分的分离纯化及鉴定
摘要
烟草中含有绿原酸、芸香苷(芦丁)、烟碱和茄尼醇等多种生物活性成分,这些活性成分经提取纯化后有望用于农业、医药、食品、化工等诸多行业。绿原酸具有抗病毒、抗菌、保肝利胆等多种药理作用；芦丁在医药和食品领域作为心血管保护药物及食品中的抗氧化剂等。
本工作以废弃烟草为研究对象,对绿原酸的提取和纯化工艺进行了研究,同时提取分离芦丁,相关内容如下：
(1)研究了超声波辅助提取烟草中主要药用成分绿原酸的工艺。通过对溶剂种类、浓度、提取时间及温度等因素的试验,确定了适宜的提取条件,在95%乙醇,50℃,60min,pH4和料液比1：10(w:v)的条件下,绿原酸的提取率为92.57%,比较了石油醚萃取与超临界C02萃取去除烟碱的效果,结果表明石油醚萃取对烟碱的去除率为45.5%,对浓缩液还有脱脂的效果。
(2)研究了聚酰胺柱层析分离绿原酸的工艺。采用80-100目的聚酰胺材料,在上样液中绿原酸浓度为10mg/mL时,用4BV30%的乙醇洗脱,所分离的绿原酸纯度为27.7%(收率33.8%)；经聚酰胺柱二次分离,绿原酸产品纯度提高到40.7%(收率为49.5%),同时分离出另一种多酚物质,通过热水重结晶精制,经红外光谱和其他方法鉴定,确定为芦丁(纯度94.8%)。
(3)采用乙酸乙酯萃取对绿原酸粗品进一步富集分离,使用绿原酸浓度12 mg/mL的料液,经1：3(v：v)体积比萃取2次,萃取得到的绿原酸样品纯度为62%(萃取率72%)。
(4)采用常压硅胶柱层析对纯度为62%的绿原酸样品进行精制,在物料比1：30条件下得到纯度为78.2%的绿原酸产品。结合LC-MS技术,1HNMR技术对该绿原酸组成进行分析,结果表明绿原酸(5-CQA)是主要成份(占总绿原酸量的78.2%),同时还存在新绿原酸(3-CQA,1.9%)、隐绿原酸(4-CQA,10.1%)和cis-5-CQA(2.0%)三种绿原酸的同分异构体。通过后续实验的分离,在物料比为1：50的条件下得到更高纯度的绿原酸产品,其中5-CQA含量为89.3%,4-CQA含量为8.4%。
There are many bioactive components in the tobacco, such as chlorogenic acid(CA), rutin, nicotine, solanesol etc. After being isolated and purified, these bioactive compounds can be used in agriculture, medicine, food and chemical industry. Chlorogenic acid is an important bioactive component which possesses many pharmaceutical functions, such as antibacteria, antivirus, protecting liver, cholagogue function and so on. Rutin are also used as medication for blood vessel protection or food antioxidant.
Taking the waste tobacco as the raw material, the extraction and isolation of chlorogenic acid, and the purification of rutin are studied in this paper. The main achievement are as following:
(1) The ultrasound-assisted extraction of principal medicinal composition(chlorogenic acid) in waste tobacco was investigated. Though testting on the major influencing factors, such as extraction solvent type, concentration, extraction time and extraction temperature, the optimum conditions were as following:using 95% ethanol as extration solvent, at 50℃, pH4, extraction for 1 hour, the ratio of solid to liquid 1:10. The extraction ratio of chlorogenic acid was 92.57%. Comparing with the efficiency of supercriticle CO2 extraction, the method of petroleum ether extraction was more suitable for removal of nicotin, the removals was 45.5%; moreover, it had the defatted function.
(2) Polymaid column chromatography was used to purify chlorogenic acid. Through the dynamic experiments, the optimal preparation conditions had been identified:the concentration of sample solution, 10mg/mL; 30% ethanol eluent, eluent volume 4BV. Under this condition the purity of chlorogenic acid was 27.7%(the yield was 38.8%). After secongd purified by polyamide column(80-100mesh), the purity reached 40.7% (yield was 49.5%).Another polyphenolic product (the purity was 94.8%) was obtained by recrystallization of hot water and identified as Rutin by IR and other techniques.
(3) Further experiment was studied by Ethyl acetate extraction to purify chlorogenic acid samples of polymaid resin. The chlorogenic acid concentration of the material liquid was about 12 mg/mL, phase ratio was 1/3 (V/V), extracting 2 times. The purity of chlorogenic acid sample was 62%(the extraction efficiency is 72%).
(4) Finely, the purification of chlorogenic acid by silica gel column chromatography at nomal pressure was investigated. Under the condition of the rate of crude CA to silica 1:30, the purity of chlorogenic acid product reached 78.2%. On the basis of LC-MS and 1HNMR determination, four monocaffeoylquinic acids were detected. While chlorogenic acid (5-CQA,78.2%) was the major component, and other three isomers were neochlorogenic acid (3-CQA,1.9%), cryptochlorogenic acid (4-CQA,10.1%), cis-5 CQA (2.0%) respectively.
Follow-up experiment by the improvement and optimization of the ratio(1:50), the product of chlorogenic acid with higher purity was obtained:the content of 5-CQA is 89.3%, while 4-CQA is 8.4%.
本工作以废弃烟草为研究对象,对绿原酸的提取和纯化工艺进行了研究,同时提取分离芦丁,相关内容如下：
(1)研究了超声波辅助提取烟草中主要药用成分绿原酸的工艺。通过对溶剂种类、浓度、提取时间及温度等因素的试验,确定了适宜的提取条件,在95%乙醇,50℃,60min,pH4和料液比1：10(w:v)的条件下,绿原酸的提取率为92.57%,比较了石油醚萃取与超临界C02萃取去除烟碱的效果,结果表明石油醚萃取对烟碱的去除率为45.5%,对浓缩液还有脱脂的效果。
(2)研究了聚酰胺柱层析分离绿原酸的工艺。采用80-100目的聚酰胺材料,在上样液中绿原酸浓度为10mg/mL时,用4BV30%的乙醇洗脱,所分离的绿原酸纯度为27.7%(收率33.8%)；经聚酰胺柱二次分离,绿原酸产品纯度提高到40.7%(收率为49.5%),同时分离出另一种多酚物质,通过热水重结晶精制,经红外光谱和其他方法鉴定,确定为芦丁(纯度94.8%)。
(3)采用乙酸乙酯萃取对绿原酸粗品进一步富集分离,使用绿原酸浓度12 mg/mL的料液,经1：3(v：v)体积比萃取2次,萃取得到的绿原酸样品纯度为62%(萃取率72%)。
(4)采用常压硅胶柱层析对纯度为62%的绿原酸样品进行精制,在物料比1：30条件下得到纯度为78.2%的绿原酸产品。结合LC-MS技术,1HNMR技术对该绿原酸组成进行分析,结果表明绿原酸(5-CQA)是主要成份(占总绿原酸量的78.2%),同时还存在新绿原酸(3-CQA,1.9%)、隐绿原酸(4-CQA,10.1%)和cis-5-CQA(2.0%)三种绿原酸的同分异构体。通过后续实验的分离,在物料比为1：50的条件下得到更高纯度的绿原酸产品,其中5-CQA含量为89.3%,4-CQA含量为8.4%。
There are many bioactive components in the tobacco, such as chlorogenic acid(CA), rutin, nicotine, solanesol etc. After being isolated and purified, these bioactive compounds can be used in agriculture, medicine, food and chemical industry. Chlorogenic acid is an important bioactive component which possesses many pharmaceutical functions, such as antibacteria, antivirus, protecting liver, cholagogue function and so on. Rutin are also used as medication for blood vessel protection or food antioxidant.
Taking the waste tobacco as the raw material, the extraction and isolation of chlorogenic acid, and the purification of rutin are studied in this paper. The main achievement are as following:
(1) The ultrasound-assisted extraction of principal medicinal composition(chlorogenic acid) in waste tobacco was investigated. Though testting on the major influencing factors, such as extraction solvent type, concentration, extraction time and extraction temperature, the optimum conditions were as following:using 95% ethanol as extration solvent, at 50℃, pH4, extraction for 1 hour, the ratio of solid to liquid 1:10. The extraction ratio of chlorogenic acid was 92.57%. Comparing with the efficiency of supercriticle CO2 extraction, the method of petroleum ether extraction was more suitable for removal of nicotin, the removals was 45.5%; moreover, it had the defatted function.
(2) Polymaid column chromatography was used to purify chlorogenic acid. Through the dynamic experiments, the optimal preparation conditions had been identified:the concentration of sample solution, 10mg/mL; 30% ethanol eluent, eluent volume 4BV. Under this condition the purity of chlorogenic acid was 27.7%(the yield was 38.8%). After secongd purified by polyamide column(80-100mesh), the purity reached 40.7% (yield was 49.5%).Another polyphenolic product (the purity was 94.8%) was obtained by recrystallization of hot water and identified as Rutin by IR and other techniques.
(3) Further experiment was studied by Ethyl acetate extraction to purify chlorogenic acid samples of polymaid resin. The chlorogenic acid concentration of the material liquid was about 12 mg/mL, phase ratio was 1/3 (V/V), extracting 2 times. The purity of chlorogenic acid sample was 62%(the extraction efficiency is 72%).
(4) Finely, the purification of chlorogenic acid by silica gel column chromatography at nomal pressure was investigated. Under the condition of the rate of crude CA to silica 1:30, the purity of chlorogenic acid product reached 78.2%. On the basis of LC-MS and 1HNMR determination, four monocaffeoylquinic acids were detected. While chlorogenic acid (5-CQA,78.2%) was the major component, and other three isomers were neochlorogenic acid (3-CQA,1.9%), cryptochlorogenic acid (4-CQA,10.1%), cis-5 CQA (2.0%) respectively.
Follow-up experiment by the improvement and optimization of the ratio(1:50), the product of chlorogenic acid with higher purity was obtained:the content of 5-CQA is 89.3%, while 4-CQA is 8.4%.
引文
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