中药活性成分提取和分离的研究
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摘要
本论文以中药为研究对象,对中药活性成分的提取分离和测定进行了研究。将超声技术和微波技术应用于中药活性成分的提取,建立了在线超声提取测定方法和微波提取方法。确定了朝鲜淫羊藿提取物的水解实验条件以及水解产物的高效液相色谱分离条件。建立了高速逆流色谱法分离纯化朝鲜淫羊藿水解产物的色谱条件,利用质谱法对水解产物进行了鉴定。
建立了在线超声提取检测方法。在超声提取法的基础上,设计了一套动态超声提取在线测定的实验装置,将超声提取装置与紫外可见光谱测定仪在线联用,可直接测定中药样品中的活性成分并观察提取过程中的动力学过程。应用此装置在线测定了红花中的总红花黄色素和半枝莲中的总黄酮。通过与静态超声提取、振荡提取和索式提取进行比较,证明了该方法提取时间最短、样品用量最小、提取产率相对最高。该方法大大缩短了样品分析时间,简化了分析过程,对实际样品的分析也得到了满意的结果。
研究了应用高压微波提取法、常压微波流动提取法和常压微波回流提取法对提取中药大黄中蒽醌类成分的影响,采用光度法测定了大黄提取液中的蒽醌成分。考查了微波实验参数对提取产率的影响,确定了最佳微波提取条件,同时与索氏提取法和超声提取法的提取结果进行比较,结果说明微波辅助提取法不仅提取时间短,提取产率高,重现性好,而且对环境污染低,是传统的溶剂提取法所不能比拟的。从不同提取方法对大黄总蒽醌提取产率的比较上看,高压微波法>溶剂流动型微波法>微波回流法>超声法>索氏法。从不同提取方法所需要的提取时间上看,高压微波法<溶剂流动型微波法<微波回流法<超声法<索氏法。
建立了朝鲜淫羊藿提取物的最佳水解实验条件以及利用高效液相色谱法梯度洗脱水解产物的色谱分离条件。建立了高速逆流色谱分离纯化淫羊藿两个水解产物的溶剂体系,淫羊藿提取物的两个水解产物经HPLC-MS~n分析,确定这两个化合物分别为淫羊藿次苷和脱水淫羊藿素,这些研究结果为进一步研究淫羊藿生物学功能奠定了基础。
The studies of traditional Chinese medicine involve the extraction, separation and characterization of the active constituents. The extraction plays an important part in traditional Chinese medicine research. At present,of the traditional extraction techniques of major drawbacks of is long extraction time, high solvent consumption.Therefore it is important to develop a fast and effective extraction method. The special features of microwave-assisted extraction and ultrasonica extraction technique over traditional extraction are short time, low temperature, low energy consumption and high extraction yield. Good experimental results obtained by microwave-assisted and ultrasonica extraction show that these extraction methods could be extensively applied to the extraction of active constituents from natural products.
In the thesis, some extraction methods to extract the active constituents from traditional Chinese medicines were developed. There are dynamic ultrasonic extraction method, pressurized microwave-assisted extraction and atmospheric pressure microwave-assisted extraction.
A dynamic ultrasonic extraction (DUE) coupled with on-line detection by spectrophotometry was proposed for the determination of total safflower yellow pigments (caculated against safflor yellow A) in Carthamus tinctorius L.and total flavonoids (calculated against scutellarin) in Scutellaria barbata D. Don.The extraction was performed in a common self-made extraction vessel placed inside an ultrasonic bath and a peristaltic pump was used to deliver the solvent. Several parameters of DUE, including flow rate of extraction solvent, ultrasonic power and sample amount and concentration of extraction solvent were optimized. Compared to the off-line method, the on-line approach has the advantages of minimum sample amount (5mg), on-line monitoring the extraction process and time-saving (10min, 12.5 min).Compared with offline method, the proposed method would be more convenient for obtaining continuous measurements and rapid optimization of the extraction process.
Three microwave-assisted extraction (MAE) methods were studied, including pressurized microwave-assisted extraction, flow microwave-assisted extraction, atmospheric pressure microwave-assisted extraction.MAE process of extracting anthaqinones from Rhubarb were studied.The effects of solvent concentration,radiation time , solid/liquid ratio and microwave power were discussed in orthogonal experiments. The results show that the optimized extraction parameters were 70% ethanol,radiation time 2 min,solid/liquid ratio 1:30 and microwave pressure 300Kpa. the highest extraction yield is 2.86%.The experimental results indicated that the higher extraction yield can be obtained 1.97% by Microwave-assisted dynamic extraction (MADE) when extraction solvent is 70% ethanl,solvent uptake rate is 4 mL/min and irradiation power is 80%. MADE only needs 10 min.
We investigated purification of two flavonoids from hydrolyzed products of Epimedium korenum Nakai extract by high-speed counter-current chomatography (HSCCC).Dried Epimedium korenum Nakai powder was extracted two times with 70% methanol (v/v), crude extract was dried by evaporator to dryness in vacuo. 2mol/L HCll was used to dissolve the sediment.
This solution was heated for 22h.
A two-phase solvent system consisting of hexane–ethylacetate–methanol–water (6:4:5:1, v/v/v/v), showed a baseline separation of icariside from other components in the hydrolysis mixture. Anhydroicaritin was separated from other components in the hydrolysis mixture by using a two-phase solvent system consisting of hexane- ethylacetate–acetonitrile (9:4:6, v/v/v). The structure of icariside and anhydroicaritin were identified by LC-ESI-MSn.
建立了在线超声提取检测方法。在超声提取法的基础上,设计了一套动态超声提取在线测定的实验装置,将超声提取装置与紫外可见光谱测定仪在线联用,可直接测定中药样品中的活性成分并观察提取过程中的动力学过程。应用此装置在线测定了红花中的总红花黄色素和半枝莲中的总黄酮。通过与静态超声提取、振荡提取和索式提取进行比较,证明了该方法提取时间最短、样品用量最小、提取产率相对最高。该方法大大缩短了样品分析时间,简化了分析过程,对实际样品的分析也得到了满意的结果。
研究了应用高压微波提取法、常压微波流动提取法和常压微波回流提取法对提取中药大黄中蒽醌类成分的影响,采用光度法测定了大黄提取液中的蒽醌成分。考查了微波实验参数对提取产率的影响,确定了最佳微波提取条件,同时与索氏提取法和超声提取法的提取结果进行比较,结果说明微波辅助提取法不仅提取时间短,提取产率高,重现性好,而且对环境污染低,是传统的溶剂提取法所不能比拟的。从不同提取方法对大黄总蒽醌提取产率的比较上看,高压微波法>溶剂流动型微波法>微波回流法>超声法>索氏法。从不同提取方法所需要的提取时间上看,高压微波法<溶剂流动型微波法<微波回流法<超声法<索氏法。
建立了朝鲜淫羊藿提取物的最佳水解实验条件以及利用高效液相色谱法梯度洗脱水解产物的色谱分离条件。建立了高速逆流色谱分离纯化淫羊藿两个水解产物的溶剂体系,淫羊藿提取物的两个水解产物经HPLC-MS~n分析,确定这两个化合物分别为淫羊藿次苷和脱水淫羊藿素,这些研究结果为进一步研究淫羊藿生物学功能奠定了基础。
The studies of traditional Chinese medicine involve the extraction, separation and characterization of the active constituents. The extraction plays an important part in traditional Chinese medicine research. At present,of the traditional extraction techniques of major drawbacks of is long extraction time, high solvent consumption.Therefore it is important to develop a fast and effective extraction method. The special features of microwave-assisted extraction and ultrasonica extraction technique over traditional extraction are short time, low temperature, low energy consumption and high extraction yield. Good experimental results obtained by microwave-assisted and ultrasonica extraction show that these extraction methods could be extensively applied to the extraction of active constituents from natural products.
In the thesis, some extraction methods to extract the active constituents from traditional Chinese medicines were developed. There are dynamic ultrasonic extraction method, pressurized microwave-assisted extraction and atmospheric pressure microwave-assisted extraction.
A dynamic ultrasonic extraction (DUE) coupled with on-line detection by spectrophotometry was proposed for the determination of total safflower yellow pigments (caculated against safflor yellow A) in Carthamus tinctorius L.and total flavonoids (calculated against scutellarin) in Scutellaria barbata D. Don.The extraction was performed in a common self-made extraction vessel placed inside an ultrasonic bath and a peristaltic pump was used to deliver the solvent. Several parameters of DUE, including flow rate of extraction solvent, ultrasonic power and sample amount and concentration of extraction solvent were optimized. Compared to the off-line method, the on-line approach has the advantages of minimum sample amount (5mg), on-line monitoring the extraction process and time-saving (10min, 12.5 min).Compared with offline method, the proposed method would be more convenient for obtaining continuous measurements and rapid optimization of the extraction process.
Three microwave-assisted extraction (MAE) methods were studied, including pressurized microwave-assisted extraction, flow microwave-assisted extraction, atmospheric pressure microwave-assisted extraction.MAE process of extracting anthaqinones from Rhubarb were studied.The effects of solvent concentration,radiation time , solid/liquid ratio and microwave power were discussed in orthogonal experiments. The results show that the optimized extraction parameters were 70% ethanol,radiation time 2 min,solid/liquid ratio 1:30 and microwave pressure 300Kpa. the highest extraction yield is 2.86%.The experimental results indicated that the higher extraction yield can be obtained 1.97% by Microwave-assisted dynamic extraction (MADE) when extraction solvent is 70% ethanl,solvent uptake rate is 4 mL/min and irradiation power is 80%. MADE only needs 10 min.
We investigated purification of two flavonoids from hydrolyzed products of Epimedium korenum Nakai extract by high-speed counter-current chomatography (HSCCC).Dried Epimedium korenum Nakai powder was extracted two times with 70% methanol (v/v), crude extract was dried by evaporator to dryness in vacuo. 2mol/L HCll was used to dissolve the sediment.
This solution was heated for 22h.
A two-phase solvent system consisting of hexane–ethylacetate–methanol–water (6:4:5:1, v/v/v/v), showed a baseline separation of icariside from other components in the hydrolysis mixture. Anhydroicaritin was separated from other components in the hydrolysis mixture by using a two-phase solvent system consisting of hexane- ethylacetate–acetonitrile (9:4:6, v/v/v). The structure of icariside and anhydroicaritin were identified by LC-ESI-MSn.
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