蟾酥中蟾毒配基类成分的提取纯化工艺研究
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摘要
目的优化蟾酥中脂溶性成分华蟾酥毒基、脂蟾毒配基的提取及纯化工艺。方法以华蟾酥毒基和脂蟾毒配基总量为考察指标,以乙醇浓度、溶剂倍量、提取时间为考察因素,采用正交试验确定最佳提取工艺参数;通过单因素考察并结合Box-Behnken响应面法,以展开剂比例、柱高与直径比、吸附剂与上样量的比值为考察因素,筛选并确定最佳纯化工艺。结果确定最佳提取工艺为加入10倍量85%的乙醇提取90min;最佳纯化工艺为展开剂环己烷-氯仿-丙酮(4∶3∶3),柱高与直径比为7∶1,吸附剂-上样量为5.5∶1,2种蟾毒配基纯化后的总量可达66.51%。通过3批放大工艺验证,表明模型拟合度良好,采用硅胶柱色谱分离纯化蟾毒配基,具有操作简便、分离速度快、分离效果好等优点。结论所选工艺合理、可行,为以后该产品的产业化应用提供技术参考和依据。
Objective To optimize the extraction and purification technology of liposoluble constituents from Bufonis Venenum. Methods The total content of cinobufagin and resibufogenin was taken as the index, and the ethanol potency, solvent multiplication and extraction time were taken as the investigation factors. The optimum extraction process parameters was determined by orthogonal test. Through single factor investigation experiment combined with Box-Behnken response surface method, taking the expansion agent ratio, ratio of diameter to height, adsorbent and sample quantity as the investigation factors, the optimum purification process was selected and determined. Results It was determined that the optimum extraction process for the addition was 10 times 85% ethanol for 90 minutes' extraction, the best purification process for the expansion agent cyclohexane-chloroformacetone was 4∶3∶3, column height and diameter was 7∶1, adsorbent and sample volume was 5.5∶1. The content of two kinds of toad poison base purified was up to 66.51%. Through the verification of three batches of amplification process, it was shown that the model fit well, and the separation and purification of toad poison ligand by silicone column chromatography had the advantages of simple operation, fast separation speed, and good separation effect. Conclusion The selected process is reasonable and feasible, which provides technical reference and basis for the industrialization application of the product in the future.
Objective To optimize the extraction and purification technology of liposoluble constituents from Bufonis Venenum. Methods The total content of cinobufagin and resibufogenin was taken as the index, and the ethanol potency, solvent multiplication and extraction time were taken as the investigation factors. The optimum extraction process parameters was determined by orthogonal test. Through single factor investigation experiment combined with Box-Behnken response surface method, taking the expansion agent ratio, ratio of diameter to height, adsorbent and sample quantity as the investigation factors, the optimum purification process was selected and determined. Results It was determined that the optimum extraction process for the addition was 10 times 85% ethanol for 90 minutes' extraction, the best purification process for the expansion agent cyclohexane-chloroformacetone was 4∶3∶3, column height and diameter was 7∶1, adsorbent and sample volume was 5.5∶1. The content of two kinds of toad poison base purified was up to 66.51%. Through the verification of three batches of amplification process, it was shown that the model fit well, and the separation and purification of toad poison ligand by silicone column chromatography had the advantages of simple operation, fast separation speed, and good separation effect. Conclusion The selected process is reasonable and feasible, which provides technical reference and basis for the industrialization application of the product in the future.
引文
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[18]唐晓霞,贾燕丽,田港,等.干蟾皮中酯蟾毒配基和华蟾酥毒基的纯化及其体外抗结肠癌活性的研究[J].中华中医药学刊,2015,33(9):2243-2245.
[19]Arshadi M,Mousavi S M.Enhancement of simultaneous gold and copper extraction from computer printed circuit boards using Bacillus megaterium[J].Biores Technol,2015,175(1):315-324.
[20]李颖,纪莎,高锦娟,等.星点设计-效应面法优化复方巴戟天生骨颗粒提取工艺[J].中药材,2014,37(10):1863-1867.
[21]姜瑞清,周琳,黎继烈,等.硅胶柱层析纯化花生根中白藜芦醇工艺研究[J].食品科技,2010,35(1):199-202.
[2]Kim J S,Jeong T Y,Cho C K,et al.Antitumor effect of skin of Venenum bufonis in a NCI-H460 tumor regression model[J].J Acupunct Merid Study,2010,3(3):181-187.
[3]寇冠军,秦姿凡,邓雅芳,等.蟾酥的研究进展[J].中草药,2014,45(21):3185-3189.
[4]袁旭江,袁梦泓,沈嘉茵,等.正交法优化蟾酥酒炮制工艺[J].中国实验方剂学杂志,2010,16(6):46-49.
[5]刘冬,杜守颖,何秀峰,等.蟾酥中3种脂溶性有效成分提取工艺及含量测定方法[J].中国实验方剂学杂志,2011,17(3):69-72.
[6]陈瀛澜,郝艳艳,郭夫江,等.蟾酥化学成分及药理活性研究进展[J].中草药,2017,48(12):2579-2588.
[7]张振海,王晋艳,陈彦,等.不同品种及产地蟾皮中抗肿瘤活性成分含量比较[J].中华中医药杂志,2011,26(11):2698-2701.
[8]王佳宝,徐忠伟,王志美,等.蟾酥活性成分下调极光激酶表达并促进肝癌细胞周期阻滞的机制研究[J].中草药,2017,48(18):3796-3801.
[9]陈瀛澜,郭夫江,卞雪莲,等.蟾酥干燥炮制前后化学成分和药效学变化考察[J].中草药,2018,49(8):1816-1822.
[10]左其艳,赵彦敏,李振麟,等.中药蟾酥的研究概况[J].人参研究,2017,13(11):48-51.
[11]马丽,陈家仪,陈稚,等.星点设计-响应面法优选枳实总黄酮的大孔吸附树脂纯化工艺[J].中国实验方剂学杂志,2014,20(8):33-36.
[12]张素娟,张永太,申利娜,等.蟾酥提取工艺优化与提取物体外抗肿瘤活性研究[J].上海中医药杂志,2013,47(5):93-97.
[13]王博,李洪起,连潇嫣,等.星点设计-效应面法优化阿德福韦-β-环糊精包合物制备工艺的研究[J].现代药物与临床,2014,29(11):1227-1230.
[14]裴月梅.高效液相色谱法测定救心滴丸中的华蟾酥毒基和脂蟾毒配基[J].药物评价研究,2014,37(6):526-528.
[15]刘小英,刘昕,陈笑天,等.HPLC法同时测定不同产地蟾酥中的5种活性成分[J].中草药,2013,44(6):760-762.
[16]潘晴,孙京海,李卫国,等.响应面法优化女贞子三萜酸的提取工艺[J].中国药学杂志,2015,50(13):1093-1097.
[17]杜会茹,黄文杰,蒋翠岚,等.厚朴酚中压硅胶柱层析纯化工艺的优化[J].中成药,2017,39(4):835-837.
[18]唐晓霞,贾燕丽,田港,等.干蟾皮中酯蟾毒配基和华蟾酥毒基的纯化及其体外抗结肠癌活性的研究[J].中华中医药学刊,2015,33(9):2243-2245.
[19]Arshadi M,Mousavi S M.Enhancement of simultaneous gold and copper extraction from computer printed circuit boards using Bacillus megaterium[J].Biores Technol,2015,175(1):315-324.
[20]李颖,纪莎,高锦娟,等.星点设计-效应面法优化复方巴戟天生骨颗粒提取工艺[J].中药材,2014,37(10):1863-1867.
[21]姜瑞清,周琳,黎继烈,等.硅胶柱层析纯化花生根中白藜芦醇工艺研究[J].食品科技,2010,35(1):199-202.