黄芪中黄芪皂苷成分的提取分离与纯化工艺研究
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摘要
本文以中药黄芪为原料,主要考察了微波辅助提取法对黄芪中黄芪皂苷成分提取率的影响,以黄芪皂苷Ⅰ-Ⅳ提取率为考察指标,筛选出最佳提取工艺条件,并通过大孔吸附树脂、皂苷衍生物水解转化、硅胶柱层析、低温析晶和重结晶等技术手段对黄芪皂苷成分进行分离与纯化研究,最终建立了一整套黄芪中黄芪皂苷成分提取分离与纯化的最佳工艺,同时确定了黄芪皂苷成分的LC-ESI/MS分析方法,为合理有效的利用黄芪资源提供了科学依据。
1.确定了LC-ESI/MS检测方法同时测定黄芪中黄芪皂苷Ⅰ-Ⅳ的条件：
色谱条件：液相：Agilent 1100高效液相色谱仪；色谱柱：Agilent Eclipse XDB C18柱(150 mm×4.6mm i.d.,5μm)；柱温：30℃;流动相：0.05%甲酸水溶液(A)-乙腈(B)；梯度洗脱：0-6 min 66%(A),6-8 min 66-55%(A),8-13 min 55%(A),13-14min 55-66%(A),14-17 min 66%(A)；流速：0.7 mL/min；进样量：10μL。
质谱条件：质谱仪：API3000三重四级杆质谱仪；电喷雾电离正离子源(ESI+)；多重反应监测模式(MRM);喷雾气(NEG)：12 a.u.；气帘气(CUG)：10 a.u.；碰撞气(COG)：6 a.u.；温度(TEM)：250℃；碰雾电压：5500V。
2.考察了微波辅助提取法对黄芪中黄芪皂苷Ⅰ-Ⅳ提取率的影响,确定了最佳的提取工艺参数。
微波辅助提取黄芪皂苷Ⅰ-Ⅳ工艺参数为：提取溶剂：80%乙醇溶液固液比：1：25提取时间：5min提取温度：70℃微波功率：700W提取次数：3次
在上述优化的条件下,微波辅助提取黄芪皂苷Ⅰ-Ⅳ的提取率分别为0.788mg/g,0.351mg/g,0.206 mg/g和0.278 mg/g,收率分别为91.415%,89.086%,86.193%和94.600%。
3。通过大孔吸附树脂对黄芪中黄芪皂苷Ⅰ-Ⅳ进行了富集和分离,确定了SA-3型大孔吸附树脂富集和分离黄芪皂苷Ⅰ-Ⅳ工艺。
大孔吸附树脂吸附与解吸最佳条件：树脂类型：SA-3型上样浓度：黄芪皂苷Ⅰ为0.483 mg/mL,黄芪皂苷Ⅱ为0.460 mg/mL,黄芪皂苷Ⅲ为0.168 mg/mL,黄芪皂苷Ⅳ为0.320 mg/mL上样体积：30 BV(180mL)上样流速：1 BV/h样品溶液pH值：4解吸溶液：50%乙醇溶液解吸溶液体积：9BV(54mL)解吸流速：1 BV/h
在上述优化的条件下,经过SA-3型大孔吸附树脂处理,黄芪皂苷Ⅰ的含量从0.559%增加到4.908%,提高了8.78倍；黄芪皂苷Ⅱ的含量从0.532%增加到6.173%,提高了11.60倍；黄芪皂苷Ⅲ的含量从0.194%增加到2.043%,提高了10.52倍；黄芪皂苷Ⅳ的含量从0.371%增加到4.181%,提高了11.28倍,收率分别为65.883%,90.920%,84.249%和94.167%。
4.通过皂苷衍生物水解转化技术,将SA-3型大孔吸附树脂处理后的样品溶液中其他类型的黄芪皂苷乙酰化水解为黄芪甲苷(即黄芪皂苷Ⅳ),可以提高黄芪甲苷的含量,该过程的收率为189.861%。再采用硅胶柱层析、低温析晶和重结晶技术对黄芪甲苷进行纯化,最终得到黄芪甲苷晶体,其中,柱层析收率为57.935%,重结晶收率为69.854%。
5.经过微波辅助提取、大孔吸附树脂富集、黄芪皂苷衍生物水解转化、硅胶柱层析纯化、低温析晶和重结晶技术等处理后,黄芪甲苷的纯度达到93%,总收率为68.448%,黄芪甲苷总得率为0.201 mg/g。与传统的方法相比,该方法生产周期短、操作简单、环境污染较小,且所得产品纯度高、得率高,为黄芪甲苷的工业化生产提供了科学参考。
In the present study, the effect of microwave-assisted extraction of astragalosides in Radix Astragali was investigated. Extraction yields of AstragalosidesⅠ-Ⅳ(AGsⅠ-Ⅳ) were used as index, the conditions for extraction of astragalosides were optimized. Preparative separation and purification of astragalosides from Radix Astragali extracts was carried out by macroporous resins, hydrolysis transformation of astragaloside derivatives, silica gel column chromatography, low temperature crystallization and re-crystallization. The optimum process of extraction, separation and purification of astragalosides was established and the LC-ESI/MS method for determination and quantification of astragalosides was developed, which provides the scientific basis for the rational and efficient use of Radix Astragali resources.
1. LC-ESI/MS method for determination and quantification of AGs I-IV was developed as follows:
Chromatographic conditions:Agilent 1100 series HPLC system; column:Agilent Eclipse XDB-C18 column (150 mm×4.6 mm i.d.,5μm); column temperature:30℃; mobile phase: 0.05% formic acid aqueous solution (A)-acetonitrile (B); gradient elution:0-6 min 66% (A),6-8 min 66-55%(A),8-13 min 55% (A),13-14 min 55-66%(A),14-17 min 66%(A); flow rate:0.7 mL/min; injection volume:10μL.
MS conditions:An API3000 triple tandem quadrupole mass spectrometry; electrospray ionization in positive ion mode (ESI+); multiple reaction monitoring mode (MRM); The nebulizing gas (NEB), curtain gas (CUR) and collision gas (COL) were set at 12,10 and 6 a.u., respectively. The ion source was operated at temperature of 250℃. The ion spray voltage was 5500 V.
2. Microwave-assisted extraction method was investigated on the extraction yields of AGs I-IV, the optimum parameters of the extraction process were as follows:
Extraction solvent:ethanol-water (80:20, v/v) solution Ratio of solid/liquid:1:25 Extraction time:5 min Extraction temperatures:70℃Microwave power:700 W Extraction cycles:3 times
Under the above optimum conditions, the extraction yields of AGs I-IV were 0.788 mg/g, 0.351 mg/g,0.206 mg/g and 0.278 mg/g, respectively. The recoveries of AGs I-IV were 91.415%, 89.086%,86.193% and 94.600%, respectively.
3. Enrichment and separation of AGsⅠ-Ⅳby macroporous adsorption resins was studied and the optimum parameters of adsorption and desorption on the optimal SA-3 resin were obtained. Macroporous resin adsorption and desorption of the best conditions as follows: Resin type:SA-3 resin Sample concentration:AGⅠ0.483 mg/mL, AGⅡ0.460 mg/mL, AGⅢ0.168 mg/mL, AG IV 0.320 mg/mL Processing volume:30 BV (180 mL) Adsorption flow rate:1 BV/h pH value of sample solution:4 Desorption solution:ethanol-water (50:50, v/v) solution Desorption solution volume:9 BV (54 mL) Desorption flow rate:1 BV/h
After treated by SA-3 resin under the above optimum conditions, the contents of AGs I-IV in the processing increased from 0.559%,0.532%,0.194% and 0.371% to 4.908%,6.173%,2.043% and 4.181%, which were 8.78-fold,11.60-fold,10.52-fold and 11.28-fold increased, respectively. The recoveries of AGs I-IV were 65.883%,90.920%,84.249% and 94.167%, respectively.
4. By hydrolysis conversion technology, other types of acetylated astragalosides in sample solution after enrichment and separation by SA-3 resin were hydrolyzed into AGⅣ, which improved the content of AG IV, the recovery was 189.861%. Using column chromatography, low temperature crystallization and re-crystallization, the crystal of AG IV was obtained with the recovery of column chromatography 57.935% and the recovery of re-crystallization 69.854%.
5. After microwave-assisted extraction, macroporous adsorption resin, hydrolysis transformation of astragaloside derivatives, column chromatography, low temperature crystallization and re-crystallization, the purity of AGⅣwas 93%, the the total recovery of AGⅣwas 68.448% and the total extraction yield of AG IV was 0.201 mg/g. Compared with traditional methods, this method is short, simple, less environmental pollution, and the product was high purity and high yield. It provides a scientific reference for the industrial production of AG IV.
1.确定了LC-ESI/MS检测方法同时测定黄芪中黄芪皂苷Ⅰ-Ⅳ的条件：
色谱条件：液相：Agilent 1100高效液相色谱仪；色谱柱：Agilent Eclipse XDB C18柱(150 mm×4.6mm i.d.,5μm)；柱温：30℃;流动相：0.05%甲酸水溶液(A)-乙腈(B)；梯度洗脱：0-6 min 66%(A),6-8 min 66-55%(A),8-13 min 55%(A),13-14min 55-66%(A),14-17 min 66%(A)；流速：0.7 mL/min；进样量：10μL。
质谱条件：质谱仪：API3000三重四级杆质谱仪；电喷雾电离正离子源(ESI+)；多重反应监测模式(MRM);喷雾气(NEG)：12 a.u.；气帘气(CUG)：10 a.u.；碰撞气(COG)：6 a.u.；温度(TEM)：250℃；碰雾电压：5500V。
2.考察了微波辅助提取法对黄芪中黄芪皂苷Ⅰ-Ⅳ提取率的影响,确定了最佳的提取工艺参数。
微波辅助提取黄芪皂苷Ⅰ-Ⅳ工艺参数为：提取溶剂：80%乙醇溶液固液比：1：25提取时间：5min提取温度：70℃微波功率：700W提取次数：3次
在上述优化的条件下,微波辅助提取黄芪皂苷Ⅰ-Ⅳ的提取率分别为0.788mg/g,0.351mg/g,0.206 mg/g和0.278 mg/g,收率分别为91.415%,89.086%,86.193%和94.600%。
3。通过大孔吸附树脂对黄芪中黄芪皂苷Ⅰ-Ⅳ进行了富集和分离,确定了SA-3型大孔吸附树脂富集和分离黄芪皂苷Ⅰ-Ⅳ工艺。
大孔吸附树脂吸附与解吸最佳条件：树脂类型：SA-3型上样浓度：黄芪皂苷Ⅰ为0.483 mg/mL,黄芪皂苷Ⅱ为0.460 mg/mL,黄芪皂苷Ⅲ为0.168 mg/mL,黄芪皂苷Ⅳ为0.320 mg/mL上样体积：30 BV(180mL)上样流速：1 BV/h样品溶液pH值：4解吸溶液：50%乙醇溶液解吸溶液体积：9BV(54mL)解吸流速：1 BV/h
在上述优化的条件下,经过SA-3型大孔吸附树脂处理,黄芪皂苷Ⅰ的含量从0.559%增加到4.908%,提高了8.78倍；黄芪皂苷Ⅱ的含量从0.532%增加到6.173%,提高了11.60倍；黄芪皂苷Ⅲ的含量从0.194%增加到2.043%,提高了10.52倍；黄芪皂苷Ⅳ的含量从0.371%增加到4.181%,提高了11.28倍,收率分别为65.883%,90.920%,84.249%和94.167%。
4.通过皂苷衍生物水解转化技术,将SA-3型大孔吸附树脂处理后的样品溶液中其他类型的黄芪皂苷乙酰化水解为黄芪甲苷(即黄芪皂苷Ⅳ),可以提高黄芪甲苷的含量,该过程的收率为189.861%。再采用硅胶柱层析、低温析晶和重结晶技术对黄芪甲苷进行纯化,最终得到黄芪甲苷晶体,其中,柱层析收率为57.935%,重结晶收率为69.854%。
5.经过微波辅助提取、大孔吸附树脂富集、黄芪皂苷衍生物水解转化、硅胶柱层析纯化、低温析晶和重结晶技术等处理后,黄芪甲苷的纯度达到93%,总收率为68.448%,黄芪甲苷总得率为0.201 mg/g。与传统的方法相比,该方法生产周期短、操作简单、环境污染较小,且所得产品纯度高、得率高,为黄芪甲苷的工业化生产提供了科学参考。
In the present study, the effect of microwave-assisted extraction of astragalosides in Radix Astragali was investigated. Extraction yields of AstragalosidesⅠ-Ⅳ(AGsⅠ-Ⅳ) were used as index, the conditions for extraction of astragalosides were optimized. Preparative separation and purification of astragalosides from Radix Astragali extracts was carried out by macroporous resins, hydrolysis transformation of astragaloside derivatives, silica gel column chromatography, low temperature crystallization and re-crystallization. The optimum process of extraction, separation and purification of astragalosides was established and the LC-ESI/MS method for determination and quantification of astragalosides was developed, which provides the scientific basis for the rational and efficient use of Radix Astragali resources.
1. LC-ESI/MS method for determination and quantification of AGs I-IV was developed as follows:
Chromatographic conditions:Agilent 1100 series HPLC system; column:Agilent Eclipse XDB-C18 column (150 mm×4.6 mm i.d.,5μm); column temperature:30℃; mobile phase: 0.05% formic acid aqueous solution (A)-acetonitrile (B); gradient elution:0-6 min 66% (A),6-8 min 66-55%(A),8-13 min 55% (A),13-14 min 55-66%(A),14-17 min 66%(A); flow rate:0.7 mL/min; injection volume:10μL.
MS conditions:An API3000 triple tandem quadrupole mass spectrometry; electrospray ionization in positive ion mode (ESI+); multiple reaction monitoring mode (MRM); The nebulizing gas (NEB), curtain gas (CUR) and collision gas (COL) were set at 12,10 and 6 a.u., respectively. The ion source was operated at temperature of 250℃. The ion spray voltage was 5500 V.
2. Microwave-assisted extraction method was investigated on the extraction yields of AGs I-IV, the optimum parameters of the extraction process were as follows:
Extraction solvent:ethanol-water (80:20, v/v) solution Ratio of solid/liquid:1:25 Extraction time:5 min Extraction temperatures:70℃Microwave power:700 W Extraction cycles:3 times
Under the above optimum conditions, the extraction yields of AGs I-IV were 0.788 mg/g, 0.351 mg/g,0.206 mg/g and 0.278 mg/g, respectively. The recoveries of AGs I-IV were 91.415%, 89.086%,86.193% and 94.600%, respectively.
3. Enrichment and separation of AGsⅠ-Ⅳby macroporous adsorption resins was studied and the optimum parameters of adsorption and desorption on the optimal SA-3 resin were obtained. Macroporous resin adsorption and desorption of the best conditions as follows: Resin type:SA-3 resin Sample concentration:AGⅠ0.483 mg/mL, AGⅡ0.460 mg/mL, AGⅢ0.168 mg/mL, AG IV 0.320 mg/mL Processing volume:30 BV (180 mL) Adsorption flow rate:1 BV/h pH value of sample solution:4 Desorption solution:ethanol-water (50:50, v/v) solution Desorption solution volume:9 BV (54 mL) Desorption flow rate:1 BV/h
After treated by SA-3 resin under the above optimum conditions, the contents of AGs I-IV in the processing increased from 0.559%,0.532%,0.194% and 0.371% to 4.908%,6.173%,2.043% and 4.181%, which were 8.78-fold,11.60-fold,10.52-fold and 11.28-fold increased, respectively. The recoveries of AGs I-IV were 65.883%,90.920%,84.249% and 94.167%, respectively.
4. By hydrolysis conversion technology, other types of acetylated astragalosides in sample solution after enrichment and separation by SA-3 resin were hydrolyzed into AGⅣ, which improved the content of AG IV, the recovery was 189.861%. Using column chromatography, low temperature crystallization and re-crystallization, the crystal of AG IV was obtained with the recovery of column chromatography 57.935% and the recovery of re-crystallization 69.854%.
5. After microwave-assisted extraction, macroporous adsorption resin, hydrolysis transformation of astragaloside derivatives, column chromatography, low temperature crystallization and re-crystallization, the purity of AGⅣwas 93%, the the total recovery of AGⅣwas 68.448% and the total extraction yield of AG IV was 0.201 mg/g. Compared with traditional methods, this method is short, simple, less environmental pollution, and the product was high purity and high yield. It provides a scientific reference for the industrial production of AG IV.
引文
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