基于生物毒价检测和化学成分分析的毒性中药附子质量评控研究
摘要
中药质量评价与控制是保证巾药安全性、有效性和稳定可控性的重要手段,也是保障临床合理用药的重要基石。而毒性中药作用峻烈,其质量优劣对临床应用的影响更大。然而,当前以感官评价和化学成分分析为主的质控模式主要体现在毒性中药的真伪鉴别,不能很好地反映其安全性和有效性,未能摆脱“量而不准,难关毒效,难控难评”的困境。
为此,本文以附子为例,从生物毒价检测角度丰富和完善毒性中药质量控制方法体系。建立了基于整体动物最小致死量的毒价测定法、基于相对校正因子的6种生物碱含量同时测定法以及二萜类生物碱的HPLC-MS／MS检测法,并在此方法基础上开展附子炮制减毒科学基础、炮制方法合理性以及探索构建“毒性效应成分指数”等方面的应用研究,并进一步提出增修订附子商品规格和质量检测方法的建议,制定质量标准修订草案,以期为实现毒性中药“量而又准,关联毒效,可控可评”质量评控愿景提供研究模式和方法学实例,为毒性中药的临床合理用药提供参考。
主要研究结果如下：
1.建立和完善了附子生物毒价测定方法
本文尝试建立基于大鼠最小致死量试验的附子生物毒价测定方法,以最小致死量、毒效成分转移率以及给药液的溶血反应、渗透压、有关物质检查等指标,考察了样品的制备工艺、给药途径等关键因素,并取不同炮制品进行验证,结果显示,该法可适用于附子各种炮制品种(可信限率<15%)。
2.建立基于相对校正因子的6种生物碱含量同时测定法
采用“一测多评”思路和方法,尝试以一种对照品同时测定6种成分,结果显示,以毒性较低的苯甲酰次乌头碱为内参物时,苯甲酰新乌头原碱、苯甲酰乌头原碱、新乌头碱、次乌头碱、乌头碱5种成分与其的相对校正因子在不同仪器、色谱柱和检测时间的重现性均良好,分别为1.039(RSD=1.6%),1.026(RSD=2.2%),0.948(RSD=3.7%),0.957(RSD=1.7%),0.876(RSD=1.8%).经15批次样品验证,该法与常规测定法的结果无显著性差异(P>0.05),故可通过苯甲酰次乌头碱为对照同时测定6种成分。
3.结合生物毒价、化学成分含量、HPLC-MS/MS指纹谱研究结果,探讨了附子炮制减毒的科学基础,初步确定了可能的毒性物质基础
从生物毒价测定和化学成分检测两方面入手,比较了生附片和各种炮制附片的毒价和化学成分含量差异,结果表明：附子炮制后毒性显著下降(P<0.01),炮制品的毒价约为生品的1.0%。
与毒价呈显著正相关的7种生物碱为10-羟基乌头碱、去氧乌头碱、乌头碱、10-羟基新乌头碱、次乌头碱、新乌头碱、3-乙酰乌头碱。此7种生物碱在炮制品中均显著下降(P<0.01),其中乌头碱、次乌头碱和新乌头碱三种双酯型生物碱的总量仅为生附片的1.8%-2.6%；而脱水苯甲酰乌头原碱等环氧类生物碱、苯甲酰次乌头原碱等单酯型生物碱、新乌头原碱等胺醇类物质含量显著增加(P<0.01或P<0.05),其中苯甲酰新乌头原碱、苯甲酰次乌头原碱和苯甲酰乌头原碱的总量增加4.5-4.6倍(无胆蒸制、炒制附片)或未见显著性变化(胆制附片),导致三种单酯型生物碱与三种双酯型生物碱的比值增加39.6-184.9倍。
上述结果提示,3-乙酰乌头碱、新乌头碱、乌头碱、次乌头碱、去氧乌头碱、10-羟基新乌头碱、10-羟基乌头碱为附子的主要毒性物质,环氧类生物碱、单酯型生物碱和胺醇类生物碱可能为药效物质,炮制使前者降解为后者而实现“减毒存效”效果。
4.通过比较毒价、毒／效物质以及无机杂质含量,发现无胆蒸制附片的“减毒存效”效果优于传统胆制附片
比较无胆蒸制、炒制附片与传统胆制附片在生物毒价、毒效成分含量以及无机杂质含量三方面的差异,结果表明：
无胆蒸制附片的毒价和毒性成分含量与传统胆制附片无显著性差异;多数药效物质(12/14)含量显著高于传统胆制附片(P<0.01或P<0.05),其中苯甲酰乌头原碱等三种单酯型生物碱含量增加约6倍；无机杂质含量显著低于传统胆制附片。该结果提示,无胆蒸制工艺的“减毒”能力与传统胆制工艺相当,而“存效”效果优于后者,且不会额外引入无机杂质,非常值得推广使用。
无胆炒制附片的毒价和毒效物质含量均显著高于传统胆制附片(P<0.01或P<0.05),其中毒价约为后者的2倍,苯甲酰乌头原碱等三种单酯型生物碱含量约增加6倍。该结果提示,无胆炒制工艺的“减毒存效”效果有待于进一步研究。
5.初步构建了附子的“毒性效应成分指数”
为了提高安全性评控的便捷性和实用性,本文在毒性成分含量与生物毒价相关性研究的基础上,提出探索构建基于主要毒性成分含量综合加权的生物毒价预估值——“毒性效应成分指数”,并初步构建了附子的“毒性效应成分指数”预估模型。经验证,该模型可用于预估附子的生物毒价。
6.整合上述研究结果,提出附子质量标准修订建议,并制定质量标准草案
根据附子质量评控方法的对比研究、毒性物质基础和无胆炮制方法的合理性探讨结果,建议修订商品规格、质量检测方法,即：①规格中增加无胆蒸制附片；②[检查]项修订双酯型生物碱限量检查法；③[含量测定]项修订单酯型生物碱含量测定法；④增加[生物毒价测定]项。
综上所述,本文建立了生物毒价测定方法和“毒性效应成分指数”模型,结合化学成分分析法,提升了评控毒性中药附子安全性的质量标准,丰富和发展毒性中药质量评价与控制研究模式,为客观评价毒性中药的毒性大小提供研究思路和手段,为临床用药安全奠定基础。
Quality control and evaluation of traditional Chinese medicine (TCM) are the important means, which can ensure the safety, effectiveness. stability. controllability and clinical application.Because of the strong toxicity. the quality of toxic TCM will definitely influence its safety and effectiveness in clinical application. However, the current pattern of quality control and evaluation, which is mainly based on the qualitative and quantitative determination of chemical components, is difficult to reflect the safety and efficacy of toxic TCMs.
In this paper, we studied the Aconiti Lateralis Radix (Fuzi) in order to enrich and improve the quality control methodology of toxic TCMs in view of bioassay. The toxic-potency assay of Fuzi was established based on the minimum lethal dose to the whole animal. We also established the determination method for the content of6kinds of alkaloids by the relative correction factor. The21kinds of diterpenoid alkaloids were detected by the HPLC-MS/MS. Based on the toxic-potency assay and chemical composition analysis, we consider the specifications of aconite product and the quality testing method should be modified. The quality standards were revised in draft. Our study also provides a reference for the clinical application of toxic TCMs. The main results are as follows:
1. The toxic-potency assay method was established and verified for the quality control of Fuzi.
Based on the minimum lethal dose, hemolysis reaction, toxic effect ingredient transfer rate, osmotic pressure and relevant physical indicators, this paper studied some critical factors such as drug-delivery way, sample preparation process and the drug concentration in relationship with the minimum lethal dose. And the verification results with different processed products showed that the method can be applied to all kinds of lateral root of aconite processing varieties (FL%<15%).
2. The determination method for the content of6kinds of alkaloids by the relative correction factor was established for the first time.
Benzoyl hypaconitine was used as the reference substance to calculate the contents of other5aconitine. The relative correction factors were1.039(RSD=1.6%),1.026(RSD=2.2%),0.948(RSD=3.7%),0.957(RSD=1.7%),0.876(RSD=1.8%) which indicated the good reproducibility. Validated with the15batches of samples and the results indicated that the multi-components quantities by one marker were no significant difference with the conventional external standard method (P>0.05).
3. The scientific basis of toxic reduction with processed Fuzi was investigated by combination of toxic potency assay, chemical determination and HPLC-MS/MS fingerprinting results.
The results of toxicity studies and HPLC-MS/MS detection showed that the toxicity sequence of7alkaloids:3-acetyl aconitine,mesaconitine,aconitine,hypaconitine, deoxyaconitine,10-hydroxy aconitine mesaconitine. The above7kinds of alkaloids decreased significantly in the processed products (P<0.01), while epoxy alkaloids such as dehydration benzoylaconine alkaloids, MAs such as benzoyl hypacoitine, and new aconine increased significantly (P<0.01or P<0.05).
Content determination results show that the DAs in processed products was only1.8%-2.6%of non-processed products. While the content of MAs increased4.5～4.6times (steaming or stir-frying without Danba, P<0.01) or no insignificant different (steaming or stir-frying with Danba, P>0.05).
These results suggested that3-acetyl aconitine and DAs are toxic components. While the epoxy alkaloids, MAs and amino alcohol alkaloids may be active ingredients. Processing transformed the former into the latter, which is the reason of keeping effectiveness and eliminating toxicity.
4. The "conserving property" effect of different processed method was discussed by comparing the toxic potency, toxic/effective substance and inorganic impurity content.
It was found that the contents of DAs were no insignificant different between "Wu dan fu pian"(steaming without Danba) and "Dan fu pian"(steaming or stir-frying with Danba). Most active substance content (12/14) was significantly higher than "Dan fu pian"(P<0.01or P<0.05), in which3kinds of MAs content increased about6times. The result suggested that the abilities of "eliminating toxicity" of different processing methods were equivalent at all. In contrast, the processing method by steaming or stir-frying without Danba might have better effect for "conserving property" than the method processed with Danba stipulated by China Pharmacopoeia. We believe that the new processing method without Danba can be recommended in further application due to it offers a simple procedure and it will not introduce inorganic impurities in the products.
But, for "Wu dan fu pian"(stir-frying without Danba), the toxicity of and toxic substances content are significantly higher than traditional prepared products (P<0.01or P<0.05). The toxicity of the former is twice as much as that of the latter.
5. Preliminary constructed the toxicity index of Fuzi.
In order to improve the convenient and practical of safety evaluation and control, this paper proposed to explore the biological poison assay estimates,"toxicity index" based on toxic components content comprehensive weighted. And constructed the "aconite toxicity index" prediction model. After verification, the model can be used to predict biological poison of aconite.
6. The revised quality standard draft of Fuzi was proposed.
Based on the results of quality assessment, scientific basis and rationality of processed method, we proposed amendments to product specifications, quality inspection method, i.e.:1) increasing the specifications of "Wu dan fu pian"(steaming without Danba);2) revising the DAs limit test;3) revising the MAs determination method;4) increasing the toxic potency assay.
In summary, the biological poison assay method and "toxic effect index model", which can make up the shortage of chemical analysis methods, enrich and develop the TCM quality control and evaluation model. This paper provided the mentality for the objective toxicity evaluation of TCM. Also, constructed the research model and supplied methodology example for quality control standard of TCM toxicity safety, which will lay the foundation for the clinical rational drug use.
为此,本文以附子为例,从生物毒价检测角度丰富和完善毒性中药质量控制方法体系。建立了基于整体动物最小致死量的毒价测定法、基于相对校正因子的6种生物碱含量同时测定法以及二萜类生物碱的HPLC-MS／MS检测法,并在此方法基础上开展附子炮制减毒科学基础、炮制方法合理性以及探索构建“毒性效应成分指数”等方面的应用研究,并进一步提出增修订附子商品规格和质量检测方法的建议,制定质量标准修订草案,以期为实现毒性中药“量而又准,关联毒效,可控可评”质量评控愿景提供研究模式和方法学实例,为毒性中药的临床合理用药提供参考。
主要研究结果如下：
1.建立和完善了附子生物毒价测定方法
本文尝试建立基于大鼠最小致死量试验的附子生物毒价测定方法,以最小致死量、毒效成分转移率以及给药液的溶血反应、渗透压、有关物质检查等指标,考察了样品的制备工艺、给药途径等关键因素,并取不同炮制品进行验证,结果显示,该法可适用于附子各种炮制品种(可信限率<15%)。
2.建立基于相对校正因子的6种生物碱含量同时测定法
采用“一测多评”思路和方法,尝试以一种对照品同时测定6种成分,结果显示,以毒性较低的苯甲酰次乌头碱为内参物时,苯甲酰新乌头原碱、苯甲酰乌头原碱、新乌头碱、次乌头碱、乌头碱5种成分与其的相对校正因子在不同仪器、色谱柱和检测时间的重现性均良好,分别为1.039(RSD=1.6%),1.026(RSD=2.2%),0.948(RSD=3.7%),0.957(RSD=1.7%),0.876(RSD=1.8%).经15批次样品验证,该法与常规测定法的结果无显著性差异(P>0.05),故可通过苯甲酰次乌头碱为对照同时测定6种成分。
3.结合生物毒价、化学成分含量、HPLC-MS/MS指纹谱研究结果,探讨了附子炮制减毒的科学基础,初步确定了可能的毒性物质基础
从生物毒价测定和化学成分检测两方面入手,比较了生附片和各种炮制附片的毒价和化学成分含量差异,结果表明：附子炮制后毒性显著下降(P<0.01),炮制品的毒价约为生品的1.0%。
与毒价呈显著正相关的7种生物碱为10-羟基乌头碱、去氧乌头碱、乌头碱、10-羟基新乌头碱、次乌头碱、新乌头碱、3-乙酰乌头碱。此7种生物碱在炮制品中均显著下降(P<0.01),其中乌头碱、次乌头碱和新乌头碱三种双酯型生物碱的总量仅为生附片的1.8%-2.6%；而脱水苯甲酰乌头原碱等环氧类生物碱、苯甲酰次乌头原碱等单酯型生物碱、新乌头原碱等胺醇类物质含量显著增加(P<0.01或P<0.05),其中苯甲酰新乌头原碱、苯甲酰次乌头原碱和苯甲酰乌头原碱的总量增加4.5-4.6倍(无胆蒸制、炒制附片)或未见显著性变化(胆制附片),导致三种单酯型生物碱与三种双酯型生物碱的比值增加39.6-184.9倍。
上述结果提示,3-乙酰乌头碱、新乌头碱、乌头碱、次乌头碱、去氧乌头碱、10-羟基新乌头碱、10-羟基乌头碱为附子的主要毒性物质,环氧类生物碱、单酯型生物碱和胺醇类生物碱可能为药效物质,炮制使前者降解为后者而实现“减毒存效”效果。
4.通过比较毒价、毒／效物质以及无机杂质含量,发现无胆蒸制附片的“减毒存效”效果优于传统胆制附片
比较无胆蒸制、炒制附片与传统胆制附片在生物毒价、毒效成分含量以及无机杂质含量三方面的差异,结果表明：
无胆蒸制附片的毒价和毒性成分含量与传统胆制附片无显著性差异;多数药效物质(12/14)含量显著高于传统胆制附片(P<0.01或P<0.05),其中苯甲酰乌头原碱等三种单酯型生物碱含量增加约6倍；无机杂质含量显著低于传统胆制附片。该结果提示,无胆蒸制工艺的“减毒”能力与传统胆制工艺相当,而“存效”效果优于后者,且不会额外引入无机杂质,非常值得推广使用。
无胆炒制附片的毒价和毒效物质含量均显著高于传统胆制附片(P<0.01或P<0.05),其中毒价约为后者的2倍,苯甲酰乌头原碱等三种单酯型生物碱含量约增加6倍。该结果提示,无胆炒制工艺的“减毒存效”效果有待于进一步研究。
5.初步构建了附子的“毒性效应成分指数”
为了提高安全性评控的便捷性和实用性,本文在毒性成分含量与生物毒价相关性研究的基础上,提出探索构建基于主要毒性成分含量综合加权的生物毒价预估值——“毒性效应成分指数”,并初步构建了附子的“毒性效应成分指数”预估模型。经验证,该模型可用于预估附子的生物毒价。
6.整合上述研究结果,提出附子质量标准修订建议,并制定质量标准草案
根据附子质量评控方法的对比研究、毒性物质基础和无胆炮制方法的合理性探讨结果,建议修订商品规格、质量检测方法,即：①规格中增加无胆蒸制附片；②[检查]项修订双酯型生物碱限量检查法；③[含量测定]项修订单酯型生物碱含量测定法；④增加[生物毒价测定]项。
综上所述,本文建立了生物毒价测定方法和“毒性效应成分指数”模型,结合化学成分分析法,提升了评控毒性中药附子安全性的质量标准,丰富和发展毒性中药质量评价与控制研究模式,为客观评价毒性中药的毒性大小提供研究思路和手段,为临床用药安全奠定基础。
Quality control and evaluation of traditional Chinese medicine (TCM) are the important means, which can ensure the safety, effectiveness. stability. controllability and clinical application.Because of the strong toxicity. the quality of toxic TCM will definitely influence its safety and effectiveness in clinical application. However, the current pattern of quality control and evaluation, which is mainly based on the qualitative and quantitative determination of chemical components, is difficult to reflect the safety and efficacy of toxic TCMs.
In this paper, we studied the Aconiti Lateralis Radix (Fuzi) in order to enrich and improve the quality control methodology of toxic TCMs in view of bioassay. The toxic-potency assay of Fuzi was established based on the minimum lethal dose to the whole animal. We also established the determination method for the content of6kinds of alkaloids by the relative correction factor. The21kinds of diterpenoid alkaloids were detected by the HPLC-MS/MS. Based on the toxic-potency assay and chemical composition analysis, we consider the specifications of aconite product and the quality testing method should be modified. The quality standards were revised in draft. Our study also provides a reference for the clinical application of toxic TCMs. The main results are as follows:
1. The toxic-potency assay method was established and verified for the quality control of Fuzi.
Based on the minimum lethal dose, hemolysis reaction, toxic effect ingredient transfer rate, osmotic pressure and relevant physical indicators, this paper studied some critical factors such as drug-delivery way, sample preparation process and the drug concentration in relationship with the minimum lethal dose. And the verification results with different processed products showed that the method can be applied to all kinds of lateral root of aconite processing varieties (FL%<15%).
2. The determination method for the content of6kinds of alkaloids by the relative correction factor was established for the first time.
Benzoyl hypaconitine was used as the reference substance to calculate the contents of other5aconitine. The relative correction factors were1.039(RSD=1.6%),1.026(RSD=2.2%),0.948(RSD=3.7%),0.957(RSD=1.7%),0.876(RSD=1.8%) which indicated the good reproducibility. Validated with the15batches of samples and the results indicated that the multi-components quantities by one marker were no significant difference with the conventional external standard method (P>0.05).
3. The scientific basis of toxic reduction with processed Fuzi was investigated by combination of toxic potency assay, chemical determination and HPLC-MS/MS fingerprinting results.
The results of toxicity studies and HPLC-MS/MS detection showed that the toxicity sequence of7alkaloids:3-acetyl aconitine,mesaconitine,aconitine,hypaconitine, deoxyaconitine,10-hydroxy aconitine mesaconitine. The above7kinds of alkaloids decreased significantly in the processed products (P<0.01), while epoxy alkaloids such as dehydration benzoylaconine alkaloids, MAs such as benzoyl hypacoitine, and new aconine increased significantly (P<0.01or P<0.05).
Content determination results show that the DAs in processed products was only1.8%-2.6%of non-processed products. While the content of MAs increased4.5～4.6times (steaming or stir-frying without Danba, P<0.01) or no insignificant different (steaming or stir-frying with Danba, P>0.05).
These results suggested that3-acetyl aconitine and DAs are toxic components. While the epoxy alkaloids, MAs and amino alcohol alkaloids may be active ingredients. Processing transformed the former into the latter, which is the reason of keeping effectiveness and eliminating toxicity.
4. The "conserving property" effect of different processed method was discussed by comparing the toxic potency, toxic/effective substance and inorganic impurity content.
It was found that the contents of DAs were no insignificant different between "Wu dan fu pian"(steaming without Danba) and "Dan fu pian"(steaming or stir-frying with Danba). Most active substance content (12/14) was significantly higher than "Dan fu pian"(P<0.01or P<0.05), in which3kinds of MAs content increased about6times. The result suggested that the abilities of "eliminating toxicity" of different processing methods were equivalent at all. In contrast, the processing method by steaming or stir-frying without Danba might have better effect for "conserving property" than the method processed with Danba stipulated by China Pharmacopoeia. We believe that the new processing method without Danba can be recommended in further application due to it offers a simple procedure and it will not introduce inorganic impurities in the products.
But, for "Wu dan fu pian"(stir-frying without Danba), the toxicity of and toxic substances content are significantly higher than traditional prepared products (P<0.01or P<0.05). The toxicity of the former is twice as much as that of the latter.
5. Preliminary constructed the toxicity index of Fuzi.
In order to improve the convenient and practical of safety evaluation and control, this paper proposed to explore the biological poison assay estimates,"toxicity index" based on toxic components content comprehensive weighted. And constructed the "aconite toxicity index" prediction model. After verification, the model can be used to predict biological poison of aconite.
6. The revised quality standard draft of Fuzi was proposed.
Based on the results of quality assessment, scientific basis and rationality of processed method, we proposed amendments to product specifications, quality inspection method, i.e.:1) increasing the specifications of "Wu dan fu pian"(steaming without Danba);2) revising the DAs limit test;3) revising the MAs determination method;4) increasing the toxic potency assay.
In summary, the biological poison assay method and "toxic effect index model", which can make up the shortage of chemical analysis methods, enrich and develop the TCM quality control and evaluation model. This paper provided the mentality for the objective toxicity evaluation of TCM. Also, constructed the research model and supplied methodology example for quality control standard of TCM toxicity safety, which will lay the foundation for the clinical rational drug use.
引文
[1]国家药典委员会.中华人民共和国药典[S].2()10年版一部.北京：中国医药科技出版社,2010：3—361.
[2]刘若轩,邓志军,李丽明.某中医院近5年毒性中药使用情况分析[J].今日药学,2012,22(2)：113-115.
[3]白宇明,魏国义,郝近大.常见20种中药饮片的不良反应分析[J].中国药学杂志,2009,44(21)：1673—1675.
[4]张碧华,傅得兴.我院常用的毒性中药及其临床合理应用[J].巾国新药杂志2012,21(13)：1562-1566.
[5]张东风.毒性饮片生产与监管亟待加强[N].中国中医药报.2010-11-05(7).
[6]王兆基,关锡耀,徐树棋,等.毒性中药白降丹质量标准研究[J].中成药.2006,28(1)：31-33.
[7]王兆基,关锡耀,徐树棋,等.毒性中药生川乌质量标准研究[J].中成药.2006,28(5)：651-655.
[8]粟晓黎,林瑞超,王兆基,等.毒性中药鬼臼质量标准研究[J].中成药.2006,28(3)：342-346.
[9]祁文娟,王兆基,吴志成,等.毒性中药天仙子有效成分的液相色谱一质谱法定性定量分析[J].药物分析杂志.2012,32(4)：599-602.
[10]王兆基,粟晓黎,刘秋铭,等.毒性中药蟾酥质量检测方法研究[J].药物分析杂志.2011,31(6)：1027-1030.
[11]粟晓黎,林瑞超,王兆基,等.毒性中药狼毒质量标准研究[J].中成药.2006,28(4)：498-503.
[12]国家药典委员会.中华人民共和国药典[S].2010年版.I部.北京：中国医药科技出版社,2010：177-178.
[13]肖小河,鄢丹,王伽伯,等.关于中药质量生物鉴定的几点商榷[J].世界科学技术一中医药现代化.2009,11(4)：504-508.
[13]肖小河,肖培根,王永炎,等.中药科学研究的几个关键问题[J].中国中药 杂志.2009,34(2)：119-123.
[15]肖小河.巾医药转化医学中的关键科学问题：巾药质量生物评价与控制[J].中1药与临床.2011,1(4)：1-7.
[16]肖小河,金城,鄢丹,等.巾药大质量观及实践[J].中草药.2010,41(4)：505-508.
[17]孙琴,肖小河,金城,等.巾药质量控制和评价模式应多元化[J].中药材.2008,31(1)：1-4.
[18]鄢丹,肖小河.基于道地药材和生物测定的巾药质量控制模式与方法研究——黄连质量生物测定[J].巾成药.2011,46(5)：568-572.
[19]王伽伯,金城,李会芳,等.泻下类中药质量的生物控制方法及基本问题探讨[J].药学学报.2009,44(5)：500-505.
[20]李会芳,王伽伯,曲毅,等.致泻效价检测用于大黄品质评价的方法研究[J].中国中药杂志.2008,33(11)：1309-1312.
[21]李寒冰,鄢丹,王伽伯,等.基于神经氨酸酶活性检测的板蓝根品质的生物评价[J].药学学报.2009,44(2)：162-166.
[22]鄢丹,任永申,骆骄阳,等.中药质量生物测定的思考与实践以板蓝根为例[J].中国中药杂志.2010,35(19)：2637-2640.
[23]张为亮,徐楚江,杨明,等.附子毒效关系的实验研究[J],广西中医药,1997,20(3)：43-44,51.
[24]杨慧,严芳.经方附子炮制及用量探究[J].光明中医.2013,28(2)：397-429.
[25]顾蘅.临床运用附子的毒性问题研究[J].云南中医中药杂志.2012,33(7)：82-83.
[26]石万银.无胆附片加工工艺及其质量控制研究[D].四川：成都中医药大学.2012.
[27]温瑞卿,李东辉,赵昕,等.基于化学分析的毒性中药附子炮制方法的合理性研究[J].药学学报.2013,48(2)：286-290.
[28]张硕峰.附子中三种双酯型生物碱的心脏毒效及甘草苷的干预作用[D].北京：北京中医药大学.2007.
[29]Qin Y, Wang JB, Zhao YL, et al. Establishment of a bioassay for the toxicity evaluation and quality control of Aconitum herbs [J].J Hazard Mater.2012,199-200:350-357.
[：30]王智民,高慧敏,付雪涛,等.“一测多评”法巾药质量评价模式方法学研究[J].中国中药杂志,2006,：31(23)：1925
[31]朱晶晶,王智民,匡艳辉,等.一测多评法同步测定人参和三七药材巾多种人参皂苷的含量[J].药学学报.2008,43(12)：1211-1216.
[32]魏锋,李启艳,马玲云,等.对照品替代法同时测定中药和保健食品中 6种大豆异黄酮类成分的含量[J].药物分析杂志.2009,29(5)：725-730.
[33]李岑,杨红霞,肖远灿,等.一测多评法测定藏药当佐中没食子酸、羟基红花黄色素-A、桂皮醛及胡椒碱的含量[J].药物分析杂志.2011,31(9)：1658-1663.
[34]袁耀佐,顾洁,杭太俊,等.用替代对照品羟苯乙酯高效液相色谱方法测定大蒜辣素[J].分析化学.2008,36(8)：1083-1088.
[35]尹萌,孟月兰,闻琍毓,等.关黄柏中生物碱类成分的“一测多评”[J].中草药.2011,42(6)：1093-1096.
[36]李倩,刘伟,罗祖良,等.一测多评法测定丹参中丹参酮IIA、隐丹参酮、丹参酮I、二氢丹参酮I的含量[J].中国中药杂志.2012,37(6)：824-828.
[37]许佳,金红宇,孙磊,等.替代对照品法测定龙血竭原料中龙血素A和B的含量[J].药物分析杂志.2011,31(11)：2058-2062.
[38]王智民,钱忠直,张启伟,等.一测多评法建立的技术指南[J].中国中药杂志.2011,36(6)：657-658.
[39]王勇,刘志强,宋凤瑞,等.草乌花及其煎煮液中二萜生物碱的电喷雾串联质谱研究[J].药学学报.2003,38(4)：290-293.
[40]图雅,张贵君,王淑敏,等.草乌叶及其煎煮液中生物碱类药效组分的电喷雾串联质谱研究[J].中国中药杂志.2008,33(7)：789-790.
[41]越皓,皮子凤,宋凤瑞,等.生附片化学成分的HPLC/ESI-MSn研究[J].化学学报.2008,66(2)：211-215.
[42]李启艳,朱日然,张学顺,等.附子及其炮制品中生物碱类成分的ESI-MS"研 究[J].中国实验方剂学杂志.2011,17(17)：90-93.
[43]黄勤安,张聿梅,何轶,等.乌头碱水解转化规律的研究[J].巾国中药杂志.2007,32(20)：2143-21,15.
[44]周思思,马增春,梁乾德,等.基于UPLC/Q-TOF-MS分析附了煎煮过程111化学成分的变化[J].巾西医结合学报.2012,10(8)：894-900.
[45]越皓,皮子凤,赵宇峰,等.电喷雾串联质谱分析附子炮制巾的化学成分变化[J].分析化学.200,35(7)：959-963.
[46]谭鹏,刘永刚,关君,等.高效液相色谱-质谱联用结合量子化学计算发现乌头碱一新水解产物[J].中国中药杂志.2011,36(15)：2099-2101.
[47]洪波,仇永清.附子中双酯型乌头碱类成分水解减毒机理的密度泛函理论研究[J].分子科学学报.2008,24(3)：216-219.
[48]包懿,宋凤瑞,刘志强,等.乌头碱类双酯型二萜生物碱水解反应的电喷雾质谱分析[J].质谱学报.2009,30(1)：1-5.
[49]刘文龙,刘志强,宋凤瑞,等.乌头类双酯型生物碱组分转化为单酯水解型及脂型生物碱组分的研究[J].高等学校化学学报.2011,32(3)：717-720.
[50]郑琴,陆浩伟,郝伟伟,等.乌头类双酯型生物碱水解转化规律及含量计算方法研究[J].中国药学杂志.2011,46(9)：652-656.
[51]陈彦林.附子加压蒸制工艺及其饮片的适用性研究[D].北京：北京中医药大学.2007.
[52]李志勇,张硕峰,畅洪异.不同炮制时间附子饮片双酯型生物碱含量变化与饮片安全的相关性研究[J].中国中药杂志.2009,34(9)：1086-1089.
[53]赵纳,侯大斌,刘向鸿.不同炮制方法对附子中乌头总碱和双酯型生物碱含量的影响[J].中药材.2011,34(1)：39-42.
[54]廖勇,张小荣,王盛民,等.超高压水射流对川乌中乌头碱含量的影响[J].时珍国医国药.2011,22(3)：620-621.
[55]郑露露.附子炮制中的成分流失[J].中药通报.1983,8(2)：26-28.
[56]杜厚毅,何家瑜.再论附子黑加工重探古法之真谛.2008北京·第二届扶阳论坛论文集[C],北京,2008：
[57]陈彦琳,杜杰,梁焕,等.道地药材附子炮制加工规范化探讨[J].中国现代中药).2009,11：42-44.
[2]刘若轩,邓志军,李丽明.某中医院近5年毒性中药使用情况分析[J].今日药学,2012,22(2)：113-115.
[3]白宇明,魏国义,郝近大.常见20种中药饮片的不良反应分析[J].中国药学杂志,2009,44(21)：1673—1675.
[4]张碧华,傅得兴.我院常用的毒性中药及其临床合理应用[J].巾国新药杂志2012,21(13)：1562-1566.
[5]张东风.毒性饮片生产与监管亟待加强[N].中国中医药报.2010-11-05(7).
[6]王兆基,关锡耀,徐树棋,等.毒性中药白降丹质量标准研究[J].中成药.2006,28(1)：31-33.
[7]王兆基,关锡耀,徐树棋,等.毒性中药生川乌质量标准研究[J].中成药.2006,28(5)：651-655.
[8]粟晓黎,林瑞超,王兆基,等.毒性中药鬼臼质量标准研究[J].中成药.2006,28(3)：342-346.
[9]祁文娟,王兆基,吴志成,等.毒性中药天仙子有效成分的液相色谱一质谱法定性定量分析[J].药物分析杂志.2012,32(4)：599-602.
[10]王兆基,粟晓黎,刘秋铭,等.毒性中药蟾酥质量检测方法研究[J].药物分析杂志.2011,31(6)：1027-1030.
[11]粟晓黎,林瑞超,王兆基,等.毒性中药狼毒质量标准研究[J].中成药.2006,28(4)：498-503.
[12]国家药典委员会.中华人民共和国药典[S].2010年版.I部.北京：中国医药科技出版社,2010：177-178.
[13]肖小河,鄢丹,王伽伯,等.关于中药质量生物鉴定的几点商榷[J].世界科学技术一中医药现代化.2009,11(4)：504-508.
[13]肖小河,肖培根,王永炎,等.中药科学研究的几个关键问题[J].中国中药 杂志.2009,34(2)：119-123.
[15]肖小河.巾医药转化医学中的关键科学问题：巾药质量生物评价与控制[J].中1药与临床.2011,1(4)：1-7.
[16]肖小河,金城,鄢丹,等.巾药大质量观及实践[J].中草药.2010,41(4)：505-508.
[17]孙琴,肖小河,金城,等.巾药质量控制和评价模式应多元化[J].中药材.2008,31(1)：1-4.
[18]鄢丹,肖小河.基于道地药材和生物测定的巾药质量控制模式与方法研究——黄连质量生物测定[J].巾成药.2011,46(5)：568-572.
[19]王伽伯,金城,李会芳,等.泻下类中药质量的生物控制方法及基本问题探讨[J].药学学报.2009,44(5)：500-505.
[20]李会芳,王伽伯,曲毅,等.致泻效价检测用于大黄品质评价的方法研究[J].中国中药杂志.2008,33(11)：1309-1312.
[21]李寒冰,鄢丹,王伽伯,等.基于神经氨酸酶活性检测的板蓝根品质的生物评价[J].药学学报.2009,44(2)：162-166.
[22]鄢丹,任永申,骆骄阳,等.中药质量生物测定的思考与实践以板蓝根为例[J].中国中药杂志.2010,35(19)：2637-2640.
[23]张为亮,徐楚江,杨明,等.附子毒效关系的实验研究[J],广西中医药,1997,20(3)：43-44,51.
[24]杨慧,严芳.经方附子炮制及用量探究[J].光明中医.2013,28(2)：397-429.
[25]顾蘅.临床运用附子的毒性问题研究[J].云南中医中药杂志.2012,33(7)：82-83.
[26]石万银.无胆附片加工工艺及其质量控制研究[D].四川：成都中医药大学.2012.
[27]温瑞卿,李东辉,赵昕,等.基于化学分析的毒性中药附子炮制方法的合理性研究[J].药学学报.2013,48(2)：286-290.
[28]张硕峰.附子中三种双酯型生物碱的心脏毒效及甘草苷的干预作用[D].北京：北京中医药大学.2007.
[29]Qin Y, Wang JB, Zhao YL, et al. Establishment of a bioassay for the toxicity evaluation and quality control of Aconitum herbs [J].J Hazard Mater.2012,199-200:350-357.
[：30]王智民,高慧敏,付雪涛,等.“一测多评”法巾药质量评价模式方法学研究[J].中国中药杂志,2006,：31(23)：1925
[31]朱晶晶,王智民,匡艳辉,等.一测多评法同步测定人参和三七药材巾多种人参皂苷的含量[J].药学学报.2008,43(12)：1211-1216.
[32]魏锋,李启艳,马玲云,等.对照品替代法同时测定中药和保健食品中 6种大豆异黄酮类成分的含量[J].药物分析杂志.2009,29(5)：725-730.
[33]李岑,杨红霞,肖远灿,等.一测多评法测定藏药当佐中没食子酸、羟基红花黄色素-A、桂皮醛及胡椒碱的含量[J].药物分析杂志.2011,31(9)：1658-1663.
[34]袁耀佐,顾洁,杭太俊,等.用替代对照品羟苯乙酯高效液相色谱方法测定大蒜辣素[J].分析化学.2008,36(8)：1083-1088.
[35]尹萌,孟月兰,闻琍毓,等.关黄柏中生物碱类成分的“一测多评”[J].中草药.2011,42(6)：1093-1096.
[36]李倩,刘伟,罗祖良,等.一测多评法测定丹参中丹参酮IIA、隐丹参酮、丹参酮I、二氢丹参酮I的含量[J].中国中药杂志.2012,37(6)：824-828.
[37]许佳,金红宇,孙磊,等.替代对照品法测定龙血竭原料中龙血素A和B的含量[J].药物分析杂志.2011,31(11)：2058-2062.
[38]王智民,钱忠直,张启伟,等.一测多评法建立的技术指南[J].中国中药杂志.2011,36(6)：657-658.
[39]王勇,刘志强,宋凤瑞,等.草乌花及其煎煮液中二萜生物碱的电喷雾串联质谱研究[J].药学学报.2003,38(4)：290-293.
[40]图雅,张贵君,王淑敏,等.草乌叶及其煎煮液中生物碱类药效组分的电喷雾串联质谱研究[J].中国中药杂志.2008,33(7)：789-790.
[41]越皓,皮子凤,宋凤瑞,等.生附片化学成分的HPLC/ESI-MSn研究[J].化学学报.2008,66(2)：211-215.
[42]李启艳,朱日然,张学顺,等.附子及其炮制品中生物碱类成分的ESI-MS"研 究[J].中国实验方剂学杂志.2011,17(17)：90-93.
[43]黄勤安,张聿梅,何轶,等.乌头碱水解转化规律的研究[J].巾国中药杂志.2007,32(20)：2143-21,15.
[44]周思思,马增春,梁乾德,等.基于UPLC/Q-TOF-MS分析附了煎煮过程111化学成分的变化[J].巾西医结合学报.2012,10(8)：894-900.
[45]越皓,皮子凤,赵宇峰,等.电喷雾串联质谱分析附子炮制巾的化学成分变化[J].分析化学.200,35(7)：959-963.
[46]谭鹏,刘永刚,关君,等.高效液相色谱-质谱联用结合量子化学计算发现乌头碱一新水解产物[J].中国中药杂志.2011,36(15)：2099-2101.
[47]洪波,仇永清.附子中双酯型乌头碱类成分水解减毒机理的密度泛函理论研究[J].分子科学学报.2008,24(3)：216-219.
[48]包懿,宋凤瑞,刘志强,等.乌头碱类双酯型二萜生物碱水解反应的电喷雾质谱分析[J].质谱学报.2009,30(1)：1-5.
[49]刘文龙,刘志强,宋凤瑞,等.乌头类双酯型生物碱组分转化为单酯水解型及脂型生物碱组分的研究[J].高等学校化学学报.2011,32(3)：717-720.
[50]郑琴,陆浩伟,郝伟伟,等.乌头类双酯型生物碱水解转化规律及含量计算方法研究[J].中国药学杂志.2011,46(9)：652-656.
[51]陈彦林.附子加压蒸制工艺及其饮片的适用性研究[D].北京：北京中医药大学.2007.
[52]李志勇,张硕峰,畅洪异.不同炮制时间附子饮片双酯型生物碱含量变化与饮片安全的相关性研究[J].中国中药杂志.2009,34(9)：1086-1089.
[53]赵纳,侯大斌,刘向鸿.不同炮制方法对附子中乌头总碱和双酯型生物碱含量的影响[J].中药材.2011,34(1)：39-42.
[54]廖勇,张小荣,王盛民,等.超高压水射流对川乌中乌头碱含量的影响[J].时珍国医国药.2011,22(3)：620-621.
[55]郑露露.附子炮制中的成分流失[J].中药通报.1983,8(2)：26-28.
[56]杜厚毅,何家瑜.再论附子黑加工重探古法之真谛.2008北京·第二届扶阳论坛论文集[C],北京,2008：
[57]陈彦琳,杜杰,梁焕,等.道地药材附子炮制加工规范化探讨[J].中国现代中药).2009,11：42-44.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |