“还脑益聪方”的药学工艺研究及川芎中有效成分的分离
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摘要
本文通过单因素及正交实验,对中药复方“还脑益聪方”的提取工艺、含量测定及分离纯化进行了系统的研究。此外,本文采用高速逆流色谱建立了分离纯化中药川芎中有效成分阿魏酸、洋川芎内酯-Ⅰ和洋川芎内酯-H的方法。
实验研究了“还脑益聪方”的提取工艺,通过单因素及正交实验确定最佳提取工艺为：80%乙醇回流3次,料液比为1：15,每次回流90min,并分别采用紫外可见分光光度法和高效液相色谱法对提取液进行的含量测定。论文通过测定5种大孔树脂对“还脑益聪方”中总黄酮的吸附特性,得到AB-8大孔树脂纯化“还脑益聪方”的最佳工艺为：样品液浓度5.13mg/mL, pH为3.3,上样液体积8BV(约125mL),上样流速为3.35mL/min。柱子的径高比为1：5,用5BV水洗脱除杂,再用70%乙醇解吸,解吸剂用量为6BV。可将总黄酮纯度从8.22%提高到34.5%。通过确定的工艺参数按径高比为1：5进行放大实验,结果表明工艺重现性较好。
高速逆流色谱法(HSCCC)是一种无需任何固态支持介质的液液分配色谱,由于不使用固态介质,避免了因不可逆吸附而引起的样品损失、失活、变性等。高速逆流色谱法已广泛应用于天然产物有效成分的分离,本实验成功的一次从川芎乙醇提取物中分离出高纯度的洋川芎内酯-Ⅰ、洋川芎内酯-H和阿魏酸。通过实验可知,溶剂系统的优化对于逆流分离影响很大,同时,增大逆流仪器的管路体积,可以使分离效果得到明显提高。采用EMC-500A型逆流色谱,进样量400.Omg,在正己烷-乙酸乙酯-甲醇-水=(3：7：4：6,v/v)体系下进行分离可得到6.4mg洋川芎内酯-Ⅰ,纯度为98%；1.7mg洋川芎内酯-H,纯度为93%；4.4mg阿魏酸,纯度超过99%,并通过EI-MS,1HNMR光谱数据分析确认其结构。
In this article, the extraction techniques of Huannao Yicong Fang, Chinese medicine compound prescription containing five herbs which are Panax ginseng, Polygonum multiflorum, Grass-leaved sweetflag, Coptis root and Rhizoma Chuanxiong were investigated, including determination, the optimum solution in extraction, separation and purification. Additional, the method to separate three active compounds senkyunolide-Ⅰ, senkyunolide-H and ferulic acid from Rhizoma Chuanxiong by high speed counter-current chromatography (HSCCC) was also studied.
By the results of single and orthogonal experiments, the optimum extraction condition of Huannao Yicong Fang is as follows:80% ethanol, solid to liquid ratio is 1:20, the reflux time is 90min,3 times. To study the optimal technology for the purification of total flavonoids from Huannao Yicong Fang, AB-8 resin was chosen for the separation of total flavonoids because of its higher adsorption capacity and desorption ratio than that of other resins. The highest adsorption capacity of AB-8 was achieved when initial concentration, feed flow rate, feed pH and feed volume were 5.13 mg·mL-1,3.35 mL·min-1, pH 3.3 and 10 bed volumes respectively. The saturated resin was first washed with 5 BV of water to remove impurities, and then a purified product was obtained by eluting the resin with 6 BV of 70%(v/v) aqueous ethanol. The optimal diameter length ratio was 1:5 and AB-8 resin could be reused for 5 times.
High-speed countercurrent chromatography (HSCCC), being a support-free liquid-liquid partition method, eliminates irreversible adsorption of sample onto the solid support, and has been widely used in preparative separation of natural products. Three active compounds senkyunolide-Ⅰ, senkyunolide-H and ferulic acid were successfully isolated and purified from the extracts of Rhizoma Chuanxiong by high-speed counter-current chromatography (HSCCC). Based on the principle of the partition coefficient values (k) for target compounds, an optimized volume ratio of 3:7:4:6 (v/v) was selected for the HSCCC separation. In a single run,400mg of the crude extract yielded pure senkyunolide-I (6.4 mg), senkyunolide-H (1.7 mg), and ferulic acid (4.4 mg) with the purities of 98%,93% and 99%, respectively. The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three active compounds were identified by MS and 1H NMR.
实验研究了“还脑益聪方”的提取工艺,通过单因素及正交实验确定最佳提取工艺为：80%乙醇回流3次,料液比为1：15,每次回流90min,并分别采用紫外可见分光光度法和高效液相色谱法对提取液进行的含量测定。论文通过测定5种大孔树脂对“还脑益聪方”中总黄酮的吸附特性,得到AB-8大孔树脂纯化“还脑益聪方”的最佳工艺为：样品液浓度5.13mg/mL, pH为3.3,上样液体积8BV(约125mL),上样流速为3.35mL/min。柱子的径高比为1：5,用5BV水洗脱除杂,再用70%乙醇解吸,解吸剂用量为6BV。可将总黄酮纯度从8.22%提高到34.5%。通过确定的工艺参数按径高比为1：5进行放大实验,结果表明工艺重现性较好。
高速逆流色谱法(HSCCC)是一种无需任何固态支持介质的液液分配色谱,由于不使用固态介质,避免了因不可逆吸附而引起的样品损失、失活、变性等。高速逆流色谱法已广泛应用于天然产物有效成分的分离,本实验成功的一次从川芎乙醇提取物中分离出高纯度的洋川芎内酯-Ⅰ、洋川芎内酯-H和阿魏酸。通过实验可知,溶剂系统的优化对于逆流分离影响很大,同时,增大逆流仪器的管路体积,可以使分离效果得到明显提高。采用EMC-500A型逆流色谱,进样量400.Omg,在正己烷-乙酸乙酯-甲醇-水=(3：7：4：6,v/v)体系下进行分离可得到6.4mg洋川芎内酯-Ⅰ,纯度为98%；1.7mg洋川芎内酯-H,纯度为93%；4.4mg阿魏酸,纯度超过99%,并通过EI-MS,1HNMR光谱数据分析确认其结构。
In this article, the extraction techniques of Huannao Yicong Fang, Chinese medicine compound prescription containing five herbs which are Panax ginseng, Polygonum multiflorum, Grass-leaved sweetflag, Coptis root and Rhizoma Chuanxiong were investigated, including determination, the optimum solution in extraction, separation and purification. Additional, the method to separate three active compounds senkyunolide-Ⅰ, senkyunolide-H and ferulic acid from Rhizoma Chuanxiong by high speed counter-current chromatography (HSCCC) was also studied.
By the results of single and orthogonal experiments, the optimum extraction condition of Huannao Yicong Fang is as follows:80% ethanol, solid to liquid ratio is 1:20, the reflux time is 90min,3 times. To study the optimal technology for the purification of total flavonoids from Huannao Yicong Fang, AB-8 resin was chosen for the separation of total flavonoids because of its higher adsorption capacity and desorption ratio than that of other resins. The highest adsorption capacity of AB-8 was achieved when initial concentration, feed flow rate, feed pH and feed volume were 5.13 mg·mL-1,3.35 mL·min-1, pH 3.3 and 10 bed volumes respectively. The saturated resin was first washed with 5 BV of water to remove impurities, and then a purified product was obtained by eluting the resin with 6 BV of 70%(v/v) aqueous ethanol. The optimal diameter length ratio was 1:5 and AB-8 resin could be reused for 5 times.
High-speed countercurrent chromatography (HSCCC), being a support-free liquid-liquid partition method, eliminates irreversible adsorption of sample onto the solid support, and has been widely used in preparative separation of natural products. Three active compounds senkyunolide-Ⅰ, senkyunolide-H and ferulic acid were successfully isolated and purified from the extracts of Rhizoma Chuanxiong by high-speed counter-current chromatography (HSCCC). Based on the principle of the partition coefficient values (k) for target compounds, an optimized volume ratio of 3:7:4:6 (v/v) was selected for the HSCCC separation. In a single run,400mg of the crude extract yielded pure senkyunolide-I (6.4 mg), senkyunolide-H (1.7 mg), and ferulic acid (4.4 mg) with the purities of 98%,93% and 99%, respectively. The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three active compounds were identified by MS and 1H NMR.
引文
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[2]邓华明,肖飞,杨丽,等.阿尔兹海默病炎症机制及相关药物的研究概况[J].广东医学,2010,31(16)：2170-2172
[3]Brookmeyer R, Johnson E, Ziegler Graham K, et al. Forecasting the global burden of Alzheimer's disease [J]. A lzheimers Dement,2007,3 (3):186-191
[4]郭静静,廖红.阿尔茨海默病治疗药物的研究进展[J].中国药科大学学报,2010,41(5)：395-400
[5]陈烈,黄君英,薛俐,等.复方何首乌浸膏治疗阿尔茨海默病的疗效研究[J].中南大学学报(医学版),2010,35(6)：612-615
[6]刘港.六味地黄汤和当归芍药散活性组分对MG激活抑制作用的初步研究[D].北京：军事医学科学院,2009
[7]LIM G P, CHU T, YANG F. The curry sp ice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse [J].J Neurosci,2001,21 (21): 8370-8377
[8]P IAO H Z, CHO I I Y, PARK J S, et al. Wogonin inhibit smicroglial cell migration via supp ression of nuclear factor-kappa B activity[J]. Int Immuno pharmacol,2008,8(12): 1658-1662
[9]孙文基,谢世昌.天然药物成分定量分析[M].北京：中国医药科技出版社,2003：233-247
[10]张纯,杨少麟.高效液相色谱法测定何首乌中二苯乙烯苷的含量及其稳定性考察[J].中国中药杂志,1999,24(6)：357-359
[11]刘振丽,宋志前.不同地区制首乌中二苯乙烯苷含量测定及稳定性考察[J].中成药,2002,24(9)：684-687
[12]谭凯丽,廖海民.何首乌的药理作用研究进展[J].山地农业生物学报.2010,29(1)：4-7
[13]杨晓丽,王立为.中药何首乌的药理作用研究进展[J].中医药信息,2004,21(6)：12-14
[14]许爱霞,张振民,葛兵,等.何首乌多糖对氧自由基及抗氧化酶活性的作用研究[J].中国药师,2005,8(11)：900-902
[15]陈计,夏炎兴,杨秋美,等.何首乌吸收成分对大鼠二倍体细胞生长和传代的影响[J].上海中医药杂志,1995(8)：43-44
[16]杨秀伟.何首乌醇提物对易老化小鼠肝脏和脑单胺氧化酶活性的影响[J].中国中药杂 志,1996,21(1)：48-49
[17]谢岚.何首乌及二苯乙烯苷的研究进展[J].天津药学,2010,22(3)：4-6
[18]管淑玉,苏薇薇.何首乌的化学成分和药理作用研究进展[J].中南药学,2008,6(4)：454-456
[19]刘成基,张清华,林军.炮制何首乌对小鼠实验性肝损伤后肝脂代谢影响[J].中国中药杂志,1992,17(10)：595-598
[20]石鹏岩.何首乌对肝脏影响的研究概况[J].江西中医药,2007,38(11)：66-67
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