血水草总生物碱提取纯化工艺研究
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摘要
血水草是罂粟科白屈菜族血水草属植物，是我国独属、独种的特有物种，系多年生草本植物，广泛分布于我国浙江、江西、四川、湖南等南方各省区，根及根茎入药。《中药志》记载其味苦、性寒、有小毒[1]。具有清热解毒之功效。民间用来治疗急性结肠炎、眼结膜炎、疮痈疗毒以及毒蛇咬伤等症。血水草中含有白屈菜红碱（Chelerythrine）、血根碱（Sanguinarine）、原托品碱（Protopine）、α-别隐品碱（α-allocrypopine）、氧化血根碱（Oxysanguinarine）和白屈菜红默碱（Chelerythridimerine）等生物碱类成分[2~4]，其中白屈菜红碱含量最高。现代药理研究证明血水草中生物碱能阻滞心肌细胞通道，延长有效不应期而抗各种实验性心率失常；抑制体内酶的活性，抗菌、抗病毒，对人的白细胞及多种哺乳动物细胞均有抑制作用，能影响细胞功能发挥作用，抑制细胞有丝分裂等作用[5~11]。本实验为探讨提取工艺的科学性，我们以白屈菜红碱为含量指标，正交实验法优选其提取工艺的最佳条件，从而为实现血水草总生物碱的工业化大生产提供科学依据。
针对血水草的研究基础与现状，结合具体实验条件，本文采用正交设计优选了血水草总生物碱回流提取工艺条件；以血水草总生物碱的静、动态饱和吸附量、洗脱率和精制度为考察指标，系统的研究了大孔吸附树脂富集、纯化总生物碱类成分的吸附性能和洗脱参数以及重复使用周期等。其研究结果主要有：
1．血水草总生物碱回流提取优选出的最佳方案为：以6倍量95EtOH
回流提取3次，每次1.0h。
2．大孔树脂纯化精制工艺研究结果为：从静态吸附和静态解吸实验中优选出AB-8型大孔树脂作为上柱树脂。各项参数优选实验表明：上样液浓度在0.4-0.6mg/ml，PH值调至7.0-8.0之间，过柱速度为3BV/h，依次水、95%EtOH以3BV/h的流速洗脱，收集95%EtOH洗脱液，即可得到总生物碱含量为50.39%的产品，且重现性好，精制度高达596.26%。
结果初步表明，采用大孔吸附树脂这种新技术、新材料可以提高中药材有效成分的分离效率，在中药新药的研制开发中可以推广应用
Studies on Extraction & Purification processes of Total Alkloids of Eomecon chinantha Hance
Mastership candidate:Huang song
Tutors:Prof.Du Fanglu
( Hunan College of TCM , Changsha, China,410007 )
Eomecon chinantha Hance is a many-year living herb and a species of unique category and unique genus in China,extensively distributing in some southern provinces such as Zhejiang,Jiangxi,Sichuang,Hunan etc.Its root and rhizoma are used in medicine.In the traditional Chinese medicines,it can clear away hot and toxic. Eomecon chinantha Hance mainly contains Chelerythrine, Sanguinarine, Protopine,α-allocrypopine, Oxysanguinarine,Chelerythridimerine and other alkaloids.The content of Chelerythrine is the most.The modern pharmacological study indicates that the alkaliods can block cardiac cell passages and prevent various experimemtal virulentcite cardiacarrhythmia.It also inhibits the action of enzymes and WBC in human body and animal cells,fluences cell's fuction,and destroys bacterium and virus. Therefore,the aim of the study on the extraction and purification processes was to provide scientific basis for the manufacture of total Eomecon chinantha Hance alkaloids.
On the bases of previous researches into Eomecon chinantha Hance and
on consideration of specific resources of the laboratory ,this article optimized the parameters of reflux extraction which was screened by the orthogonal design.The purifacation prosess of total alkaloids with macroporous resin was generally studied by taking adsorption ratio ,eluting ratio and purification of total alkaloids as indices.The results were as follow:
1. The optimal parameters of the reflux extraction were that:Eomecon chinantha Hance refluxed in the 95%EtOH(6-folds) for 1.0h ,3 times in all.
2. The study of purification process showed that AB-8 macroresin had the maximum adsorption ratio among three resins:D101A,D101B and AB-8. Its dynamic and static state adsorption ratio were : 3.229±0.012mg.g-1 dried resin, 3.750±0.024mg.g-1 dried resin; the elution ratio was 93.13±0.21%. The experiments showed the optimal parameters of purification were that:original concentration(0.4-0.6mg/ml) ,PH value (7.0-8.0), velocity of flow (3BV/h), then eluted by water and 95%EtOH, gather the part eluted by 95%EtOH, and total alkaloids in the total solid extraction could reach 50.39%, the refine ratio could reach at 596.26%.
The results of the whole experiment has provided the basic data for
preparation process and quality control of modern Chinese pharmaceuticals intend for the treatment of hypertension,which was a referance for process improvement and quality control of similar Chinese herbal pharmaceuticals as well.In the study ,the application of macroporous resin primarily demonstrated that these new techniques and new materials could enhance extrating and purifying ratios of effective components in Chinese herbs,It will be feasible to promote their application to Chinese herbal pharmaceuticals R&D
针对血水草的研究基础与现状，结合具体实验条件，本文采用正交设计优选了血水草总生物碱回流提取工艺条件；以血水草总生物碱的静、动态饱和吸附量、洗脱率和精制度为考察指标，系统的研究了大孔吸附树脂富集、纯化总生物碱类成分的吸附性能和洗脱参数以及重复使用周期等。其研究结果主要有：
1．血水草总生物碱回流提取优选出的最佳方案为：以6倍量95EtOH
回流提取3次，每次1.0h。
2．大孔树脂纯化精制工艺研究结果为：从静态吸附和静态解吸实验中优选出AB-8型大孔树脂作为上柱树脂。各项参数优选实验表明：上样液浓度在0.4-0.6mg/ml，PH值调至7.0-8.0之间，过柱速度为3BV/h，依次水、95%EtOH以3BV/h的流速洗脱，收集95%EtOH洗脱液，即可得到总生物碱含量为50.39%的产品，且重现性好，精制度高达596.26%。
结果初步表明，采用大孔吸附树脂这种新技术、新材料可以提高中药材有效成分的分离效率，在中药新药的研制开发中可以推广应用
Studies on Extraction & Purification processes of Total Alkloids of Eomecon chinantha Hance
Mastership candidate:Huang song
Tutors:Prof.Du Fanglu
( Hunan College of TCM , Changsha, China,410007 )
Eomecon chinantha Hance is a many-year living herb and a species of unique category and unique genus in China,extensively distributing in some southern provinces such as Zhejiang,Jiangxi,Sichuang,Hunan etc.Its root and rhizoma are used in medicine.In the traditional Chinese medicines,it can clear away hot and toxic. Eomecon chinantha Hance mainly contains Chelerythrine, Sanguinarine, Protopine,α-allocrypopine, Oxysanguinarine,Chelerythridimerine and other alkaloids.The content of Chelerythrine is the most.The modern pharmacological study indicates that the alkaliods can block cardiac cell passages and prevent various experimemtal virulentcite cardiacarrhythmia.It also inhibits the action of enzymes and WBC in human body and animal cells,fluences cell's fuction,and destroys bacterium and virus. Therefore,the aim of the study on the extraction and purification processes was to provide scientific basis for the manufacture of total Eomecon chinantha Hance alkaloids.
On the bases of previous researches into Eomecon chinantha Hance and
on consideration of specific resources of the laboratory ,this article optimized the parameters of reflux extraction which was screened by the orthogonal design.The purifacation prosess of total alkaloids with macroporous resin was generally studied by taking adsorption ratio ,eluting ratio and purification of total alkaloids as indices.The results were as follow:
1. The optimal parameters of the reflux extraction were that:Eomecon chinantha Hance refluxed in the 95%EtOH(6-folds) for 1.0h ,3 times in all.
2. The study of purification process showed that AB-8 macroresin had the maximum adsorption ratio among three resins:D101A,D101B and AB-8. Its dynamic and static state adsorption ratio were : 3.229±0.012mg.g-1 dried resin, 3.750±0.024mg.g-1 dried resin; the elution ratio was 93.13±0.21%. The experiments showed the optimal parameters of purification were that:original concentration(0.4-0.6mg/ml) ,PH value (7.0-8.0), velocity of flow (3BV/h), then eluted by water and 95%EtOH, gather the part eluted by 95%EtOH, and total alkaloids in the total solid extraction could reach 50.39%, the refine ratio could reach at 596.26%.
The results of the whole experiment has provided the basic data for
preparation process and quality control of modern Chinese pharmaceuticals intend for the treatment of hypertension,which was a referance for process improvement and quality control of similar Chinese herbal pharmaceuticals as well.In the study ,the application of macroporous resin primarily demonstrated that these new techniques and new materials could enhance extrating and purifying ratios of effective components in Chinese herbs,It will be feasible to promote their application to Chinese herbal pharmaceuticals R&D
引文
[1] 冯瑞芝等. 中药志Ⅱ,1981,9,360
[2] 周天达等. 中草药,1981,12(1):1
[3] 冯瑞芝等. 植物分类学报,1985,23(1):36
[4] 杜方麓等. 中草药,1993,24(4):177
[5] Tolkachcv O N et al. Farmatsiya,1987,27(1):38
[6] Vichhanova S A et al. Khim-Farm.Zh.1978,12(2):101
[7] Lenfeld L et al.Planta Med.198143(2):161
[8] Cerna H etal.Acta.Univ.Palacki.Olomuc,Fac,Med.
1984,107:159
[9] Herbert J M etal.Biochem.Biophys.Res.Commum.
1990,172(3):993
[10] Wolf J et al. Biochemistry,1993,32(48):13334
[11] Simanek V ,Alkaloids(Acadenic Press),1985,26:185
[12] 杜方麓等. 天然产物研究与开发,1999,10(1):37-39
[13] 赵新先，中药注射剂学，第一版，广州：广东科技出版社，2000：96-102
[14] 侯世祥，大孔吸附树脂在中药复方分离纯化工艺中的应用，中药新药与临床药理 2000；11（3）：131-133.
[15] 马振山，大孔吸附树脂在药学领域中的研究应用，中成药
1997；19（12）：40-41.
[16] 李伯庭，大孔吸附树脂在天然产物分离中的应用，中草药 1990；21（8）：4-44.
[17] 中国医学科学院药物研究所植化室，大孔吸附树脂在中草药化学
成分提取分离中的一些应用，中草药 1980；11（3）：138-141.
[18] 冯孝章，天麻化学成分的研究，化学学报 1979；37（3）：175-176.
[19] 金继曙，用大孔吸附树脂分离白芍总苷，中国中药杂志 1994；19（1）：31.
[20] 朱浩， 大孔吸附树脂吸附纯化不同中药有效部位特性研究，中国中药杂志 1998；23（10）：607-609.
[21] 廖工铁，参附注射剂制备工艺的研究，中成药 1989；11（7）：2-3.
[22] 曾宪明，无机盐对大孔吸附树脂人参总皂苷的影响，中国医药工业杂志 1992；23（8）：339-342.
[23] 唐第光，大孔吸附树脂提取三七总皂苷工艺探讨，中成药 1990；（3）：5-6.
[24] 章观德，吸附树脂法测定三七及其制剂冠心宁总皂苷，中草药 1981；12（11）：23-25.
[25] 方亮，吸附树脂法测定梅花参芪精中人参总皂苷的含量，延边大学医学院学报 1998；21（3）：165-166.
[2] 周天达等. 中草药,1981,12(1):1
[3] 冯瑞芝等. 植物分类学报,1985,23(1):36
[4] 杜方麓等. 中草药,1993,24(4):177
[5] Tolkachcv O N et al. Farmatsiya,1987,27(1):38
[6] Vichhanova S A et al. Khim-Farm.Zh.1978,12(2):101
[7] Lenfeld L et al.Planta Med.198143(2):161
[8] Cerna H etal.Acta.Univ.Palacki.Olomuc,Fac,Med.
1984,107:159
[9] Herbert J M etal.Biochem.Biophys.Res.Commum.
1990,172(3):993
[10] Wolf J et al. Biochemistry,1993,32(48):13334
[11] Simanek V ,Alkaloids(Acadenic Press),1985,26:185
[12] 杜方麓等. 天然产物研究与开发,1999,10(1):37-39
[13] 赵新先，中药注射剂学，第一版，广州：广东科技出版社，2000：96-102
[14] 侯世祥，大孔吸附树脂在中药复方分离纯化工艺中的应用，中药新药与临床药理 2000；11（3）：131-133.
[15] 马振山，大孔吸附树脂在药学领域中的研究应用，中成药
1997；19（12）：40-41.
[16] 李伯庭，大孔吸附树脂在天然产物分离中的应用，中草药 1990；21（8）：4-44.
[17] 中国医学科学院药物研究所植化室，大孔吸附树脂在中草药化学
成分提取分离中的一些应用，中草药 1980；11（3）：138-141.
[18] 冯孝章，天麻化学成分的研究，化学学报 1979；37（3）：175-176.
[19] 金继曙，用大孔吸附树脂分离白芍总苷，中国中药杂志 1994；19（1）：31.
[20] 朱浩， 大孔吸附树脂吸附纯化不同中药有效部位特性研究，中国中药杂志 1998；23（10）：607-609.
[21] 廖工铁，参附注射剂制备工艺的研究，中成药 1989；11（7）：2-3.
[22] 曾宪明，无机盐对大孔吸附树脂人参总皂苷的影响，中国医药工业杂志 1992；23（8）：339-342.
[23] 唐第光，大孔吸附树脂提取三七总皂苷工艺探讨，中成药 1990；（3）：5-6.
[24] 章观德，吸附树脂法测定三七及其制剂冠心宁总皂苷，中草药 1981；12（11）：23-25.
[25] 方亮，吸附树脂法测定梅花参芪精中人参总皂苷的含量，延边大学医学院学报 1998；21（3）：165-166.