NIR分析技术在制川乌配方颗粒制备过程中的应用研究
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摘要
中药配方颗粒是以符合炮制规范的优质中药饮片为原料,采用现代制药技术经过提取、浓缩、制粒而成的系列产品。其性味、功效与原中药饮片一致,供中医临床配方使用,具有不需煎煮、直接冲服、服用量少、疗效确切、卫生安全、携带保存方便等优点,符合现代消费与生活需求,是一种安全、可控、高效、方便的现代中药汤剂,是传统中医药“辨证论治、随证加减”核心思想的最好体现。目前,中药配方颗粒年产值达数十亿人民币,产品已在全国2000多家医院广泛使用,并远销国外,创造了良好的社会和经济效益。
随着种植环境的变化,以及中药饮片质量控制标准的不完善,中药饮片的质量在不断下滑；加之2010年版《中国药典》(以下简称为药典)中对制川乌仍然沿用经验法来判断炮制终点的标准主观性较强；中药配方颗粒生产采用离线质量检测,不便于进行实时监控；中药配方颗粒不具原有性状特征,常规检测方法繁琐,难以达到现代工业化生产的快速、便捷、及时的要求。
本课题以制川乌规范化炮制工艺研究为起点,结合高效液相色谱法(HPLC)及近红外光谱法(NIR)考察了各工序下制川乌相关成分的变化规律,建立了制川乌相关成分含量的NIR预测模型,进而对制川乌配方颗粒生产工艺的各个环节进行了监控并制定了相关的终点判断标准,优化了制川乌配方颗粒生产的各个工艺参数,结合药理、毒理研究,为标准化生产提供了有力的实验依据。
主要研究结果如下：
1.应用近红外漫反射光谱法采集119个制川乌样品的NIR数据,采用PLS法分别建立近红外光谱信息与单酯型生物碱、双酯型生物碱、水分之间的定量分析模型。制川乌中单酯型生物碱、双酯型生物碱、水分的校正模型的建模光谱范围为8431.26-4075.93,5086.76～4088.64、7725.44-5924.25,7934.87～4088.64cm-1,校正模型的RMSEC单酯型生物碱=0.098,r单酯型生物碱=0.9559；RMSEC双酯型生物碱=0.034,r双酯型生物碱=0.9694;RMSEC水分=0.002,r水分=0.9767,验证模型的RMSEP单酯型生物碱=0.168,r单酯型生物碱=0.9266；RMSEP双酯型生物碱=0.039,r双酯型牛物碱=0.9826；RMSEP水分=0.004,r水分=0.8363。经统计学检验,三类成分的预测值与参考值的r分别为0.970、0.993、0.918,P<0.001,说明NIR与HPLC测定单酯型生物碱及双酯型生物碱的相关性较好,NIR与烘干法测定水分的相关性较好,可以较准确地预测其覆盖范围的相应物质含量。
2.应用近红外透射光谱法搜集103个制川乌样品的NIR数据,采用PLS法建立NIR与单酯型生物碱之间的定量分析模型。制川乌提取、浓缩液中单酯型生物碱的NIR模型建模的光谱范围为9264.35～7274.11cm-1,校正模型的RMSECV=1.171,r=0.9994；经外部验证,RMSEP:1.321,r=0.9921;经SPSS15.0的相关性统计学分析,单酯型生物碱的NIR预测值与HPLC参考值的r=0.999,P<0.001,说明NIR与HPLC测定单酯型生物碱的相关性较好,可以比较准确地预测出其覆盖范围的单酯型生物碱的含量。
3.以川乌中6种生物碱成分的含量为评价指标,以浸泡条件、蒸制条件及干燥条件为考察因素,采用单因素实验法,对炮制工艺参数进行了优选,其工艺参数为：40℃水浸泡25±1h,111℃(0.05MPa)加压蒸制1h,取出,晾至六成干,切片,60℃烘干,所得样品中的双酯型生物碱及单酯型生物碱含量均达到药典要求。川乌在111℃(0.05MPa)下炮制15-150min内,制川乌中单酯型生物碱时量变化关系曲线拟合回归方程为C(mg/g)=0.273+0.236×1nt(min)(R2=0.753,F=48.818,必0.01),双酯型生物碱为C(mg/g)=0.115×t-0.005(min)(R2=0.850,F=90.660,P<0.01)。
4.根据单因素试验及正交试验设计得到的优选的制川乌配方颗粒提取工艺为加10倍量水,提取3次,每次2h；浓缩工艺为75℃进行减压浓缩；干燥工艺为料液相对密度1.10(60℃)、进风温度165℃、出风温度70℃进行喷雾干燥；成型工艺为取干浸膏粉(物料水分控制在3-4%,制粒过程中适量喷洒95%药用乙醇),置干法制粒机制粒,控制压轮压力0.3-0.35Mpa,,侧封压力0.05-0.06Mpa,装12目筛整粒,取1～4号筛之间的颗粒为合格颗粒。
5.对制川乌炮制终点的研究表明,单酯型生物碱的含量为1.400mg/g左右,且双酯型生物碱的含量为0.100mg/g左右时即可终止炮制,经SPSS15.0的相关性统计学分析,NIR预测值与HPLC参考值的相关系数r单酯型生物碱=0.991,r双酯型生物碱=0.998,NIR预测值与烘干法参考值的相关系数r水分=0.958,P<0.001,说明NIR预测值与HPLC参考值、烘干法参考值的相关性较好,可用于制川乌配方颗粒炮制过程中相关成分的含量预测；对制川乌配方颗粒提取终点的研究表明,第一次提取的提取终点为102～105μg/L,第二次提取的提取终点为37μg/L,第三次提取的提取终点为16μg/L,单酯型生物碱的NIR预测值与HPLC参考值的相关系数r=0.999,P<0.001,说明NIR预测值与HPLC参考值的相关性较好,可用于制川乌配方颗粒提取过程中相关成分的含量预测；对制川乌配方颗粒浓缩终点的研究表明,浓缩的终点为156-159μg/L,单酯型生物碱的NIR预测值与HPLC参考值的相关系数r=0.999,P<0.001,说明NIR预测值与HPLC参考值的相关性较好,可用于制川乌配方颗粒浓缩过程中相关成分的快速检测；在制川乌喷干粉的含量预测中,单酯型生物碱的预测值与参考值的r=0.901,P<0.001,水分的预测值与参考值的r=0.880,P<0.001,说明NIR与药典中的方法测定出来的数据相关性较好,可用于制川乌喷干粉的相关成分快速检测;在制川乌配方颗粒成品的含量预测中,配方颗粒样品中单酯型生物碱的NIR预测值与HPLC参考值的相关系数r=0.957,P<0.001,水分的NIR预测值与烘干法参考值的相关系数r=0.961,P<0.001,说明NIR预测值与HPLC参考值、烘干法参考值的相关性较好,可用于制川乌配方颗粒的相关成分快速检测。
6.在急性毒性试验中,制川乌配方颗粒组及标准煎剂组在小鼠最大给药量为150.00g生药/kg时对小鼠未产生毒性反应,表明制川乌标准煎剂及配方颗粒具有较高安全性,通过科学的炮制方法可确保制川乌用药安全性。在小鼠扭体试验中,制川乌配方颗粒组与制川乌标准煎剂组比较,小鼠潜伏时间延长、扭体次数显著性减少,说明在镇痛效应方面制川乌配方颗粒的作用好于制川乌标准煎剂组。在抗炎药效等效性研究中,在小鼠耳廓肿胀抑制率22.51%-38.65%共同效应范围内,1/2、1、2倍临床等效剂量(195.00、390.00、780.00mg/kg)制川乌标准煎剂等效于0.07、0.20、0.46倍临床等效剂量(27.36、77.67、178.29mg/kg)制川乌配方颗粒所能产生的抗炎效应,制川乌配方颗粒等效剂量DEE制川乌配方颗粒(mg/kg)=-22.950+0.258×DEE制川乌标准煎剂(mg/kg)(R2=0.958,r=0.979,P<0.01),回归系数b0=-22.950,95%可信区间(95%CI)为(4.549,41.351),与总体均值0相比,有显著性差异(P<0.05),常数项b1=0.258,95%CI为(-0.218,0.299),与总体均值1相比,有显著性差异(P<0.05)。可知,制川乌配方颗粒和制川乌标准煎剂的等效应剂量不具有等效性,制川乌配方颗粒等效应剂量较制川乌标准煎剂小。制川乌配方颗粒与制川乌标准煎剂药剂量在1/8-1倍临床等效应剂量范围内(48.75-390.00mg/kg)具有可比性,制川乌配方颗粒的等剂量效应EED制川乌配方颗粒(%)=2.963+1.573×EED制川乌标准煎剂(%)(R2=0.923,r=0.961,P<0.01),回归系数b0=2.963,95%CI为(-5.373,11.300),与总体0比较有显著性差异(P<0.05),常数项b1=1.573,95%CI为(1.230,1.915),与总体均值1相比有显著性差异(P＜0.05)。可知,制川乌配方颗粒和制川乌标准煎剂的等剂量效应不具有等效性,制川乌配方颗粒等剂量效应较制川乌标准煎剂大。
Chinese Medicine formula granules are made using high quality Chinese medicine through modern pharmaceutical technology. These granules provide the same effects as Chinese traditional herbal medicine (without having to boil the medicine). Additionally, the granules are easy to adjust, simple to use and can be effortlessly controlled. With an annual output value of billions of Renminbi, Chinese Medicine formula granules are widely used in more than2,000hospitals and are exported internationally. The advance in technology has created good social and economic benefits.
Owing to the changes in the environment where herbs are planted, imperfect quality control standards in Chinese Medicine, deteriorating quality of Chinese Herbal Medicine, the subjective description of quality control for Chuanwu in the Pharmacopoeia; lack of on-line control of industrial production; formula granules does not have the original appearance. Traditional detection method is over complicated and restricted by modern industrial ized production.
This subject started from dealing with standardized concoct and examines the variation of each process system. Chuanwu ingredients, combined with high performance liquid chromatography (HPLC) and near-infrared spectroscopy (NIR), established NIR models aid to analyse the content to control the process. Then, particles of the Chuanwu recipe are linked in the production process and are monitored and optimized. Particle production process parameters are combined with pharmacological and toxicological experiments. Chuanwu concocted recipe particles are the best technology to date, and to develop relevant standards, provide trustworthy experimental basis for standardized production.
The main conclusions are as follows:
1. Use NIR to collect the data of119Aconiti Radix Cocta samples, use partial least squares regression(PLS) method to establish quantitative analysis model of monoester alkaloids, diester alkaloids and moisture between the information of NIR and HPLC. The spectral ranges of monoester alkaloids, diester alkaloids and moisture models of Aconiti Radix Cocta are;8431.26-4075.93,5086.76-4088.64/7725.44～5924.25and7934.87～4088.64cm-1. The root mean square errors of calibration (RMSEC) and correlation coefficient (r) of the calibration models are; RMSECmonoester alkaloids=0.098, r monoester alkaloids=0.9559, RMSECdiester alkaloidS=0.034, r diester alkaloid=0.9694and RMSEC moisture=0.002, r moisture=0.9767. The root-mean-square error of prediction (RMSEP) and r of the validation models are; RMSEPmonoester alkaloids-0.168, r monoester alkaloids=0.9266, RMSEP diester alkaloids=0.039, r diester alkaloids=0.9826and RMSEP moisture=0.004, r moisture=0.8363. According to the results of statistical tests, r between the predicted value and the reference value of the three types of ingredients are;0.970,0.993and0.918, P<0.001. This means that to analyze these compositions the method of NIR is as good as HPLC and the dehydration method (for testing the value of moisture) of the pharmacopoeia requirements.
2. Using NIR to collect the data of103samples and using PLS method to establish the quantitative analysis model of monoester alkaloid in the information between NIR and monoester alkaloid. The spectral range of monoester alkaloids model is9264.35～7274.11cm-1. The root mean square error of cross validation RMSECV=1.171and r calibration model=0.9994. Through the external validation, RMSEP=1.321and r validation model=0.9921. According to the results of statistical analysis, r of the predicted value and the reference value of monoester alkaloids is0.999, P<0.001. This revealed that the NIR and HPLC methods have a good correlation in determining monoester alkaloids and accurately predict the amount of monoester alkaloids in the covered range.
3. Use content of6Alkaloids compositions of Aconiti Radix as the evaluation index to do single factor experiments to examine factors of soaking, steaming and drying conditions. The result of the best processing technology is; soak Aconiti Radix in40℃water for25±1h, high pressure steam(111℃) for1h, dry at60℃to obtain diester and monoester alkaloid content of the pharmacopoeia requirements. After concocting15～150min in111℃(0.05MPa), the content of diester alkaloid in Aconiti Radix Cocta in the relation curve fitting regression equation was C(mg/g)=0.273+0.236X In t (min)(R2=0.753, F=48.818, P<0.01), monoester alkaloid was C(mg/g)=0.115×t-0.005(min)(R2=0.850, F=90.660, P<0.01).
4. According to single factor tests and the Orthogonal Test Design, Aconiti Radix Cocta formula granule's best process in extraction is; add10times amount of water, extract three times, each time for2hours. The best process in concentration is; concentrate under reduced pressure by75℃. To achieve the best drying process; materials'relative density of1.10(60℃), inlet air temperature of165℃and air-out temperature70℃by spray drying. To achieve a solid form; put extract powder (moisture3-4%, spray little amount of95%medicinal alcohol in granulation process) into dry type granulator, control roller pressure as0.3-0.35Mpa, side sealing pressure as0.05-0.06Mpa, grade with12mesh sieve, take No.1-4sieve as qualified granule.
5. The finishing point of the concocting process is when the content of monoester alkaloids is around1.400mg/g, and diester alkaloid is around0.100mg/g. According to the statistical correlation analysis by SPSS15.0, rbetween predicted value and reference value of monoester alkaloid is r monoesler alkaloid=0.991, diester alkaloid is r diester alkaloid=0.998, moisture is rmoisture=0.958, P <0.001. The finishing point of extracting process is when the content of monoester alkaloids is first time of102～105μg/L, second time by37μg/L, third time around16μg/L. During the extracting process, r monoester alkaloids=0.999, P<.0.001. The finishing point of concentration is when the content of monoester alkaloids is156～159μg/L. During concentration process, rmonoester alkaloids=0.999, P<0.001. During the drying process, r monlester alkaloid=0.901, r moisture=0.880, P <0.001. During the forming process, r monlester alkaloid=0.957,rmoisture=0.961, P <0.001. The data above shows that the correlation between predicted value and reference value is good, and NIR can be used for rapid detection of the preparation of Aconiti Radix Cocta formula granule.
6. The toxicity studies on Aconiti Radix Cocta formula granule shows that Aconiti Radix Cocta formula granule is safe under the highest concentration (150.00g medicine/kg). During the dimethyl acetic acid body turning text, the turning frequency between traditional form group and formula granule group is similar-both are less than model group. While latent time in traditional form group and formula granule group are longer than model group. The formula granule group got closer to Fenbidand better than the traditional form. During the auricular inflammation test, when the mice ear swelling rejection rate was between22.51～38.65%, the effect of1/2,1,2times of the clinical equivalent dose (195,390,780mg/kg) are equal to0.07,0.20,0.46times of Aconiti Radix Cocta formula granule (27.36,77.67,178.29mg/kg), DEEformula granule (mg/kg)=-22.950+0.258×DEEtraditional from(mg/kg)(R2=0.958, r=0.979, P<0.01), b0=-22.950,95%confidence interval is (4.549,41.351)(P<0.05), b1=0.258,95%confidence interval is (-0.218,0.299)(P<0.05). Hence, the dose of equivalent effects of Aconiti Radix Cocta formula granule is less than the traditional form. When the clinical equivalent dose of1/8～1times, EED formula granule(%)=2.963+1.573×EEDtraditional form(%)(R2=0.923, r=0.961, P<0.01),b0=2.963,95%confidence interval is (-5.373,11.300)(P<0.05), b1=1.573,95%confidence interval is (1.230,1.915)(P<0.05). Thus, the effects of the equivalent dose of Aconiti Radix Cocta formula granule is stronger than the traditional form.
随着种植环境的变化,以及中药饮片质量控制标准的不完善,中药饮片的质量在不断下滑；加之2010年版《中国药典》(以下简称为药典)中对制川乌仍然沿用经验法来判断炮制终点的标准主观性较强；中药配方颗粒生产采用离线质量检测,不便于进行实时监控；中药配方颗粒不具原有性状特征,常规检测方法繁琐,难以达到现代工业化生产的快速、便捷、及时的要求。
本课题以制川乌规范化炮制工艺研究为起点,结合高效液相色谱法(HPLC)及近红外光谱法(NIR)考察了各工序下制川乌相关成分的变化规律,建立了制川乌相关成分含量的NIR预测模型,进而对制川乌配方颗粒生产工艺的各个环节进行了监控并制定了相关的终点判断标准,优化了制川乌配方颗粒生产的各个工艺参数,结合药理、毒理研究,为标准化生产提供了有力的实验依据。
主要研究结果如下：
1.应用近红外漫反射光谱法采集119个制川乌样品的NIR数据,采用PLS法分别建立近红外光谱信息与单酯型生物碱、双酯型生物碱、水分之间的定量分析模型。制川乌中单酯型生物碱、双酯型生物碱、水分的校正模型的建模光谱范围为8431.26-4075.93,5086.76～4088.64、7725.44-5924.25,7934.87～4088.64cm-1,校正模型的RMSEC单酯型生物碱=0.098,r单酯型生物碱=0.9559；RMSEC双酯型生物碱=0.034,r双酯型生物碱=0.9694;RMSEC水分=0.002,r水分=0.9767,验证模型的RMSEP单酯型生物碱=0.168,r单酯型生物碱=0.9266；RMSEP双酯型生物碱=0.039,r双酯型牛物碱=0.9826；RMSEP水分=0.004,r水分=0.8363。经统计学检验,三类成分的预测值与参考值的r分别为0.970、0.993、0.918,P<0.001,说明NIR与HPLC测定单酯型生物碱及双酯型生物碱的相关性较好,NIR与烘干法测定水分的相关性较好,可以较准确地预测其覆盖范围的相应物质含量。
2.应用近红外透射光谱法搜集103个制川乌样品的NIR数据,采用PLS法建立NIR与单酯型生物碱之间的定量分析模型。制川乌提取、浓缩液中单酯型生物碱的NIR模型建模的光谱范围为9264.35～7274.11cm-1,校正模型的RMSECV=1.171,r=0.9994；经外部验证,RMSEP:1.321,r=0.9921;经SPSS15.0的相关性统计学分析,单酯型生物碱的NIR预测值与HPLC参考值的r=0.999,P<0.001,说明NIR与HPLC测定单酯型生物碱的相关性较好,可以比较准确地预测出其覆盖范围的单酯型生物碱的含量。
3.以川乌中6种生物碱成分的含量为评价指标,以浸泡条件、蒸制条件及干燥条件为考察因素,采用单因素实验法,对炮制工艺参数进行了优选,其工艺参数为：40℃水浸泡25±1h,111℃(0.05MPa)加压蒸制1h,取出,晾至六成干,切片,60℃烘干,所得样品中的双酯型生物碱及单酯型生物碱含量均达到药典要求。川乌在111℃(0.05MPa)下炮制15-150min内,制川乌中单酯型生物碱时量变化关系曲线拟合回归方程为C(mg/g)=0.273+0.236×1nt(min)(R2=0.753,F=48.818,必0.01),双酯型生物碱为C(mg/g)=0.115×t-0.005(min)(R2=0.850,F=90.660,P<0.01)。
4.根据单因素试验及正交试验设计得到的优选的制川乌配方颗粒提取工艺为加10倍量水,提取3次,每次2h；浓缩工艺为75℃进行减压浓缩；干燥工艺为料液相对密度1.10(60℃)、进风温度165℃、出风温度70℃进行喷雾干燥；成型工艺为取干浸膏粉(物料水分控制在3-4%,制粒过程中适量喷洒95%药用乙醇),置干法制粒机制粒,控制压轮压力0.3-0.35Mpa,,侧封压力0.05-0.06Mpa,装12目筛整粒,取1～4号筛之间的颗粒为合格颗粒。
5.对制川乌炮制终点的研究表明,单酯型生物碱的含量为1.400mg/g左右,且双酯型生物碱的含量为0.100mg/g左右时即可终止炮制,经SPSS15.0的相关性统计学分析,NIR预测值与HPLC参考值的相关系数r单酯型生物碱=0.991,r双酯型生物碱=0.998,NIR预测值与烘干法参考值的相关系数r水分=0.958,P<0.001,说明NIR预测值与HPLC参考值、烘干法参考值的相关性较好,可用于制川乌配方颗粒炮制过程中相关成分的含量预测；对制川乌配方颗粒提取终点的研究表明,第一次提取的提取终点为102～105μg/L,第二次提取的提取终点为37μg/L,第三次提取的提取终点为16μg/L,单酯型生物碱的NIR预测值与HPLC参考值的相关系数r=0.999,P<0.001,说明NIR预测值与HPLC参考值的相关性较好,可用于制川乌配方颗粒提取过程中相关成分的含量预测；对制川乌配方颗粒浓缩终点的研究表明,浓缩的终点为156-159μg/L,单酯型生物碱的NIR预测值与HPLC参考值的相关系数r=0.999,P<0.001,说明NIR预测值与HPLC参考值的相关性较好,可用于制川乌配方颗粒浓缩过程中相关成分的快速检测；在制川乌喷干粉的含量预测中,单酯型生物碱的预测值与参考值的r=0.901,P<0.001,水分的预测值与参考值的r=0.880,P<0.001,说明NIR与药典中的方法测定出来的数据相关性较好,可用于制川乌喷干粉的相关成分快速检测;在制川乌配方颗粒成品的含量预测中,配方颗粒样品中单酯型生物碱的NIR预测值与HPLC参考值的相关系数r=0.957,P<0.001,水分的NIR预测值与烘干法参考值的相关系数r=0.961,P<0.001,说明NIR预测值与HPLC参考值、烘干法参考值的相关性较好,可用于制川乌配方颗粒的相关成分快速检测。
6.在急性毒性试验中,制川乌配方颗粒组及标准煎剂组在小鼠最大给药量为150.00g生药/kg时对小鼠未产生毒性反应,表明制川乌标准煎剂及配方颗粒具有较高安全性,通过科学的炮制方法可确保制川乌用药安全性。在小鼠扭体试验中,制川乌配方颗粒组与制川乌标准煎剂组比较,小鼠潜伏时间延长、扭体次数显著性减少,说明在镇痛效应方面制川乌配方颗粒的作用好于制川乌标准煎剂组。在抗炎药效等效性研究中,在小鼠耳廓肿胀抑制率22.51%-38.65%共同效应范围内,1/2、1、2倍临床等效剂量(195.00、390.00、780.00mg/kg)制川乌标准煎剂等效于0.07、0.20、0.46倍临床等效剂量(27.36、77.67、178.29mg/kg)制川乌配方颗粒所能产生的抗炎效应,制川乌配方颗粒等效剂量DEE制川乌配方颗粒(mg/kg)=-22.950+0.258×DEE制川乌标准煎剂(mg/kg)(R2=0.958,r=0.979,P<0.01),回归系数b0=-22.950,95%可信区间(95%CI)为(4.549,41.351),与总体均值0相比,有显著性差异(P<0.05),常数项b1=0.258,95%CI为(-0.218,0.299),与总体均值1相比,有显著性差异(P<0.05)。可知,制川乌配方颗粒和制川乌标准煎剂的等效应剂量不具有等效性,制川乌配方颗粒等效应剂量较制川乌标准煎剂小。制川乌配方颗粒与制川乌标准煎剂药剂量在1/8-1倍临床等效应剂量范围内(48.75-390.00mg/kg)具有可比性,制川乌配方颗粒的等剂量效应EED制川乌配方颗粒(%)=2.963+1.573×EED制川乌标准煎剂(%)(R2=0.923,r=0.961,P<0.01),回归系数b0=2.963,95%CI为(-5.373,11.300),与总体0比较有显著性差异(P<0.05),常数项b1=1.573,95%CI为(1.230,1.915),与总体均值1相比有显著性差异(P＜0.05)。可知,制川乌配方颗粒和制川乌标准煎剂的等剂量效应不具有等效性,制川乌配方颗粒等剂量效应较制川乌标准煎剂大。
Chinese Medicine formula granules are made using high quality Chinese medicine through modern pharmaceutical technology. These granules provide the same effects as Chinese traditional herbal medicine (without having to boil the medicine). Additionally, the granules are easy to adjust, simple to use and can be effortlessly controlled. With an annual output value of billions of Renminbi, Chinese Medicine formula granules are widely used in more than2,000hospitals and are exported internationally. The advance in technology has created good social and economic benefits.
Owing to the changes in the environment where herbs are planted, imperfect quality control standards in Chinese Medicine, deteriorating quality of Chinese Herbal Medicine, the subjective description of quality control for Chuanwu in the Pharmacopoeia; lack of on-line control of industrial production; formula granules does not have the original appearance. Traditional detection method is over complicated and restricted by modern industrial ized production.
This subject started from dealing with standardized concoct and examines the variation of each process system. Chuanwu ingredients, combined with high performance liquid chromatography (HPLC) and near-infrared spectroscopy (NIR), established NIR models aid to analyse the content to control the process. Then, particles of the Chuanwu recipe are linked in the production process and are monitored and optimized. Particle production process parameters are combined with pharmacological and toxicological experiments. Chuanwu concocted recipe particles are the best technology to date, and to develop relevant standards, provide trustworthy experimental basis for standardized production.
The main conclusions are as follows:
1. Use NIR to collect the data of119Aconiti Radix Cocta samples, use partial least squares regression(PLS) method to establish quantitative analysis model of monoester alkaloids, diester alkaloids and moisture between the information of NIR and HPLC. The spectral ranges of monoester alkaloids, diester alkaloids and moisture models of Aconiti Radix Cocta are;8431.26-4075.93,5086.76-4088.64/7725.44～5924.25and7934.87～4088.64cm-1. The root mean square errors of calibration (RMSEC) and correlation coefficient (r) of the calibration models are; RMSECmonoester alkaloids=0.098, r monoester alkaloids=0.9559, RMSECdiester alkaloidS=0.034, r diester alkaloid=0.9694and RMSEC moisture=0.002, r moisture=0.9767. The root-mean-square error of prediction (RMSEP) and r of the validation models are; RMSEPmonoester alkaloids-0.168, r monoester alkaloids=0.9266, RMSEP diester alkaloids=0.039, r diester alkaloids=0.9826and RMSEP moisture=0.004, r moisture=0.8363. According to the results of statistical tests, r between the predicted value and the reference value of the three types of ingredients are;0.970,0.993and0.918, P<0.001. This means that to analyze these compositions the method of NIR is as good as HPLC and the dehydration method (for testing the value of moisture) of the pharmacopoeia requirements.
2. Using NIR to collect the data of103samples and using PLS method to establish the quantitative analysis model of monoester alkaloid in the information between NIR and monoester alkaloid. The spectral range of monoester alkaloids model is9264.35～7274.11cm-1. The root mean square error of cross validation RMSECV=1.171and r calibration model=0.9994. Through the external validation, RMSEP=1.321and r validation model=0.9921. According to the results of statistical analysis, r of the predicted value and the reference value of monoester alkaloids is0.999, P<0.001. This revealed that the NIR and HPLC methods have a good correlation in determining monoester alkaloids and accurately predict the amount of monoester alkaloids in the covered range.
3. Use content of6Alkaloids compositions of Aconiti Radix as the evaluation index to do single factor experiments to examine factors of soaking, steaming and drying conditions. The result of the best processing technology is; soak Aconiti Radix in40℃water for25±1h, high pressure steam(111℃) for1h, dry at60℃to obtain diester and monoester alkaloid content of the pharmacopoeia requirements. After concocting15～150min in111℃(0.05MPa), the content of diester alkaloid in Aconiti Radix Cocta in the relation curve fitting regression equation was C(mg/g)=0.273+0.236X In t (min)(R2=0.753, F=48.818, P<0.01), monoester alkaloid was C(mg/g)=0.115×t-0.005(min)(R2=0.850, F=90.660, P<0.01).
4. According to single factor tests and the Orthogonal Test Design, Aconiti Radix Cocta formula granule's best process in extraction is; add10times amount of water, extract three times, each time for2hours. The best process in concentration is; concentrate under reduced pressure by75℃. To achieve the best drying process; materials'relative density of1.10(60℃), inlet air temperature of165℃and air-out temperature70℃by spray drying. To achieve a solid form; put extract powder (moisture3-4%, spray little amount of95%medicinal alcohol in granulation process) into dry type granulator, control roller pressure as0.3-0.35Mpa, side sealing pressure as0.05-0.06Mpa, grade with12mesh sieve, take No.1-4sieve as qualified granule.
5. The finishing point of the concocting process is when the content of monoester alkaloids is around1.400mg/g, and diester alkaloid is around0.100mg/g. According to the statistical correlation analysis by SPSS15.0, rbetween predicted value and reference value of monoester alkaloid is r monoesler alkaloid=0.991, diester alkaloid is r diester alkaloid=0.998, moisture is rmoisture=0.958, P <0.001. The finishing point of extracting process is when the content of monoester alkaloids is first time of102～105μg/L, second time by37μg/L, third time around16μg/L. During the extracting process, r monoester alkaloids=0.999, P<.0.001. The finishing point of concentration is when the content of monoester alkaloids is156～159μg/L. During concentration process, rmonoester alkaloids=0.999, P<0.001. During the drying process, r monlester alkaloid=0.901, r moisture=0.880, P <0.001. During the forming process, r monlester alkaloid=0.957,rmoisture=0.961, P <0.001. The data above shows that the correlation between predicted value and reference value is good, and NIR can be used for rapid detection of the preparation of Aconiti Radix Cocta formula granule.
6. The toxicity studies on Aconiti Radix Cocta formula granule shows that Aconiti Radix Cocta formula granule is safe under the highest concentration (150.00g medicine/kg). During the dimethyl acetic acid body turning text, the turning frequency between traditional form group and formula granule group is similar-both are less than model group. While latent time in traditional form group and formula granule group are longer than model group. The formula granule group got closer to Fenbidand better than the traditional form. During the auricular inflammation test, when the mice ear swelling rejection rate was between22.51～38.65%, the effect of1/2,1,2times of the clinical equivalent dose (195,390,780mg/kg) are equal to0.07,0.20,0.46times of Aconiti Radix Cocta formula granule (27.36,77.67,178.29mg/kg), DEEformula granule (mg/kg)=-22.950+0.258×DEEtraditional from(mg/kg)(R2=0.958, r=0.979, P<0.01), b0=-22.950,95%confidence interval is (4.549,41.351)(P<0.05), b1=0.258,95%confidence interval is (-0.218,0.299)(P<0.05). Hence, the dose of equivalent effects of Aconiti Radix Cocta formula granule is less than the traditional form. When the clinical equivalent dose of1/8～1times, EED formula granule(%)=2.963+1.573×EEDtraditional form(%)(R2=0.923, r=0.961, P<0.01),b0=2.963,95%confidence interval is (-5.373,11.300)(P<0.05), b1=1.573,95%confidence interval is (1.230,1.915)(P<0.05). Thus, the effects of the equivalent dose of Aconiti Radix Cocta formula granule is stronger than the traditional form.
引文
[1]国家药典委员会.中华人民共和国药典2010版一部[S].中国医药科技出版社：37-38.
[2]http://www,menet.com.cn/Articles/IEconomy/201011/201011231130513051_2599.html.
[3]曹小玉,彭成.1990年以来川乌研究文献的定量分析[J].时珍国医国药,2005,16(3)：189-i90.
[4]邱启雄.乌头药材的炮制研究进展[J].海峡药学,2009,21(12)：104-106.
[5]侯家玉,方泰惠.中药药理学(M).北京：中国中医药出版社.2007：114-114.
[6]杨世雷.川乌的功能与主治探疑[J].中国现代药物应用,2009,3(5)：]73-174.
[7]杨广民,张志国.川乌草乌附子[M].北京：中国中医药出版社出版,2001.
[8]白向阳.川乌的毒性与抗肿瘤作用概述[J].实用中医药杂志,2005,21(2)：125-126.
[9]张思佳,刘敏卓,刘静涵,等.附子的化学成分研究[J].药学与临床研究,2010,18(3)：262-264.
[10]Lei J,Luo YJ,Bian QQ,etc.Talatisamine,a C(19)-diterpenoid alkaloid from Chinese traditional herbal'Cnuanwu'[J]. Acta Crystallogr Sect E Struct Rep Onl ine.2011 Dec 1:67(Pt 12):3145-3146.
[11]陈红英.川乌头不同组织中挥发油成分分析[J].安徽农业科学,2011,39(6)：3325-3326,3367.
[12]罗霄,彭善贵,文永盛,等.HPLC测定制川乌中的乌头碱、次乌头碱和新乌头碱[J].华西药学杂志,2010,25(4)：472-473.
[13]粟贵,廖林川,颜有仪,等.HPLC同时测定生物样品中新乌头碱、乌头碱、次乌头碱的含量[J].中国药学杂志,2009,44(12)：946-950.
[14]邱葵,吴华,王鹤尧.LC-MS/MS方法同时测定人血浆和尿液中乌头碱与次乌头碱的含量[J].中国药学杂志,2010,45(8)：633-636.
[15]林华,邓广海.川乌HPLC指纹图谱的研究[J].中国实验方剂学杂志,2011,17(3)：73-76.
[16]图雅,张贵君,刘志强,等.蒙药草乌的研究进展[J].时珍国医国药,2008,19(7)：1581-1582.
[17]张世忠,吴博威.乌头碱与钾离子通道激动剂合用对离体大鼠心脏的正性肌力作用研究[J].中国药理学通报,2001,17(5)：570-570.
[18]张惠云,秦林,薛玲,等.川乌配伍白芍的抗炎作用特点研究[J].中国中药杂志,2002,27(6)：449-452.
[19]秦林,张少华,李晓丽.川乌与白芍配伍的免疫调节作用初探[J].中国中药杂志,2002,27(7)：541-544.
[20]黄青,刘启福,李飞,等.川乌不同炮制品提取物的致突变与抗突变作用研究[J].北京中医药大学学报,2002,25(2)：41-43.
[21]曾瑾,罗霞,江南,等.生川乌水煎液抗肿瘤作用的实验研究[J].四川大学学报(自然科学 版),2007,44(6)：1344-1348.
[22]欧明,王宁生.中药及其制剂不良反应大典[M].沈阳：辽宁科学技术出版社,2002：33-35.
[23]楼锦英.乌头类中药的毒性反应原因分析与防治[J].海峡药学,2007,19(12)：122-125.
[24]李梦然,曲玮,梁敬钰.乌头属化学成分和药理作用研究进展[J].海峡药学,2010,22(4)：1-6.
[25]何玉华.乌头、附子的中毒原因探微[J].四川中医,2007,25(12)：51-52.
[26]陈理书,张云鹏,杨悦娅.临床应用附子例探析[J].辽宁中医杂志,2005,32(4)：23-25.
[27]卢豪,王亚其,潘黎.生川乌提取物遗传毒性研究[J].现代预防医学,2008,35(20)：4014-4016.
[28]黄韧.中药生川乌对小鼠卵巢和子宫发育的影响试验研究[J].临床和实验医学杂志,2009,8(10)：3-4.
[29]刘强强,何晓娟,严光焰,等.乌头碱对大鼠卵巢颗粒细胞毒性研究[J].现代预防医学,2010,37(2)：299-301.
[30]赵正航,徐长福.川乌对犬脊髓运动神经元损害的实验研究[J].西安医科大学学报,2000,21(1)：24-25.
[31]Niitsu H, Fujita Y, Fujita S, etc. Distribution of Aconitum alkaloids in autopsy cases of aconite poisoning[J]. Forensic Sci Int.2012 Nov 3. doi:pii:S0379-0738(12)00475-6.
[32]陈宝安,陈嘉勇,梁道明,等.急性中毒2658例流行病学调查[J].中国急救医学,2008,28(5)：410-410.
[33]邓广海,林华.川乌高压蒸制工艺优选[J].中国实验方剂学杂志,2011,17(2)：21-24.
[34]张荣,方庆.川乌加压炮制对乌头类生物碱含量的影响研究[J].中医药学刊,2003,21(1)：156-158.
[35]蒋志斌.川乌、附子、半夏、天南星炮制方法及用量商榷[J].中国药房,2007,18(9)：717-718.
[36]刘文龙,宋凤瑞,刘志强,等.川乌与半夏配伍禁忌的化学研究[J].化学通报,2008,(6)：435-438.
[37]梁泽华,尹丽娜,杨燕,等.不同炮制方法对川乌双酯型生物碱含量的影响[J].中国现代应用药学杂志,2009,26(4)：288-291.
[38]何伟,秦林,司淑媛.川乌与防己配伍前后乌头碱和粉防己碱煎出量的测定[J].中草药,2002,33(7)：600-602.
[39]苏建树,刘白宁,田平芳,等.微生物发酵对川乌、附子中生物碱含量的影响[J].北京化工大学学报(自然科学版),2010,37(3)：97-101.
[40]Zhu H, Wang C, Qi Y, etc. Rapid quality assessment of Radix Aconiti Preparata using direct analysis in real time mass spectrometry[J]. Anal Chim Acta.2012 Nov 8; 752:69-77.
[41]叶定江,原思通.中药炮制学辞典[M].上海：上海科学技术出版社,2005：215-217.
[42]随志刚,陈明玉,刘志强,等.草乌中乌头类生物碱提取方法比较研究[J].时珍国医国药,2009,20(3)：513-514.
[43]刘学湘,陈建伟.川乌中总生物碱提取工艺优化研究[J].中华中医药杂志,2009,24(1)：98-100.
[44]李永生,林强,郑来丽,等.川乌的纤维素酶酶解提取工艺研究[J].中草药,2009,40(11)：1735-1739.
[45]李文兰,王艳萍,范玉奇.中药乌头的实验研究[J].哈尔滨商业大学学报(自然科学版)2006,22(4)：13-17.
[46]余洋,宁玲.炙川乌汤治疗痛痹42例临床体会[J].云南中医中药杂志,2001,22(2)：13-13.
[47]白向阳.川乌的毒性与抗肿瘤作用概述[J].实用中医药杂志,2005,21(2)：125-126.
[48]林颖灵,王德英.生川草乌在疑难疾病中的应用体会[J].江苏中医药,2002,23(5)：35-36.
[49]曹会彦,曹官泽.川乌及草乌塞鼻治疗周围性面瘫326例[J].实用中医药杂志,2009,25(3)：175-175.
[50]涂瑶生,毕晓黎.中药配方颗粒国际化有关问题的思考[J].世界科学技术—中医药现代化,2007,9(2)：77-81.
[51]柳俊,张建军Box-Behnken效应面法优化白芍配方颗粒提取工艺[J].中国实验方剂学杂志,2011,17(1)：9-13.
[52]何健,韩正洲,张跃飞.正交试验法优选苦参配方颗粒的提取工艺[J].海峡药学,2011,23(5)：25-26.
[53]何健,韩正洲,张跃飞.正交实验法优选薄荷配方颗粒的提取工艺[J].时珍国医国药,2007,18(12)：2886-2887.
[54]廖彩震,陈培胜,张文惠,等.正交试验优选菊花配方颗粒的提取工艺[J].世界中西医结合杂志,2010,5(10)：858-860.
[55]李霞,贾晓斌,陈彦,等.五味子配方颗粒工艺研究[J].中成药,2006,28(8)：1122-1126.
[56]凌海慧,刘晓飞.中药配方颗粒的研究进展及应用前景分析[J].云南中医学院学报,2009,32(3)：68-70.
[57]钱丽萍,陈雪萍.阙慧卿,等.不同干燥工艺对制剂影响的研究进展[J].药物评价研究,2012,35(4)：299-303.
[58]田方炳.中药制粒技术进展[J].中国药业,2006,15(5)：62-63.
[59]付静,陈明.中药配方颗粒工艺研究概况[J].北京中医,2007,26(8)：522-524.
[60]涂瑶生.毕晓黎,罗文汇.中药配方颗粒的质量控制研究[J].世界科学技术—中医药现代化,2011,13(1)：41-46.
[61]赵叶东,叶宏军.中药配方颗粒剂与中药饮片药效学等效性研究[J].辽宁中医药大学学报,2010,12(11)：227-229.
[62]赵自明,崔景朝.中药配方颗粒研究进展(III)——药效学研究概况[J].中国实验方剂学杂志,2011,17(5)：270-274.
[63]张惠,胡敏.中药配方颗粒剂的临床应用评价[J].西部医学,2011,23(7)：1355-1358.
[64]魏兰福,田耀洲,夏军权,等.良附丸中药配方颗粒与饮片汤剂治疗胃脘痛临床对比研究[J].时珍国医国药+2009,20(3)：612-612.
[65]孙奇,李占玲,苏迪,等.中药配方颗粒治疗慢性前列腺炎60例临床观察[J].辽宁中医杂志,2009,36(1)：82-83.
[66]张金巍,张延莹,刘岩,等.近红外光谱法在线质量监控白芍工业化提取[J].中草药,2011,42(12)：2459-2461.
[67]丁念亚,黎薇,冯昕韡,等.近红外漫反射光谱在中药分类及真伪鉴别中的应用[J].计算机与应用化学,2008,25(4)：499-502.
[68]陆珺,相秉仁,刘浩,等.二维相关近红外光谱及其应用[J].药学进展,2007,31(7)： 303-308.
[69]曲斌,孟庆华,张婷婷,等.非负矩阵分解在中药鱼腥草近红外光谱定性判别中的应用[J].计算机与应用化学,2009,26(10)：1353-1357.
[70]白雁,刘乐,王东,等.近红外光谱对黄芩药材浸出物的快速测定[J].时珍国医国药,2009,20(5)：1081-1082.
[71]武卫红,王宁,董海平.AOTF—近红外光谱法快速无损分析复方丹参片的均一性[J].华西药学杂志,2011,26(4)：364-366.
[72]倪力军,史晓浩,高秀蛟,等.NIR在线检测、分析技术在丹参水提过程质量监控中的应用[J].中国药学杂志,2004,39(8)：628-630.
[73]瞿海斌,李斌,刘雪松,等.红参醇提液浓缩过程近红外光谱在线分析方法[J].中国药学杂志,2005,40(24)：1897-1899,1903.
[74]郁露,孙素琴,周群,等.白芥子炒制过程的红外及二维相关光谱研究[J].光谱学与光谱分析,2006,26(12)：2181-2182.
[75]刘元芬,李祥,高锦飚,等.石膏炮制前后红外光谱的特征分析与鉴定[J].内蒙古中医药,2007,26(5)：27-29.
[76]白雁,贾永,王东,等.应用近红外漫反射光谱技术测定酒炖熟地黄中的还原糖含量[J].中药材,2006,29(10)：1035-1038.
[77]Li Z, Wang C, Sun S, etc. Analysis of chemical components in Aconitum kusnezoffii leaves and their extracts by infrared spectroscopy[J]. Zhongguo Zhong Yao Za Zhi.2011 Dec;36(23):3281-3285.
[78]李晓明,杨滨.近红外光谱技术的研究进展及其在中药领域的应用[J].中国实验方剂学杂志,2006,12(12)：69-72.
[79]陆婉珍.现代近红外光谱分析技术[M].北京：中国石化出版社,2007,395.
[80]郝勇.近红外光谱微量分析方法研究[D].博士学位论文.南开大学,2009.
[8l]Donald A.Burns, Emil W. Ciurczak. Handbook of Near-Infrared Analysis(Third Edition) [M].CRC Press,2008,795.
[82]Yukihiro Ozaki, W. Fred McClure, Alfred A. Christy. Near-Infrared Spectroscopy in Food Science and Technology [M]. Wiley,2007,408.
[83]Tripathi MM, Hassan E B M, Yueh F Y, et al. Reflection-absorption-based near infrared spectroscopy for predicting water content in bio—oil[M]. Sens Actuator B-Chem,2009, 136:20-25.
[84]Shao Y N, He Y, Gomez A H, et al. Visible/near infrared spectrometric technique for nondestructive assessment of tomato'Heatwave'(Lycopersicum sculentum)quality characteristics[J]. J Food Eng,2007,81:672-678.
[85]Otsuka M. Comparative particle size determination of phenacetin bulk powder by using Kubelka—Munk theory and principal component regression analysis based on near-infrared spectroscopy[J]. Powder Technol,2004,141:24-50.
[86]基药遴选或引入药物经济学药企观望.新华网http://news. xinhuanet. com/health/ 2011-04/20/c_121325466. htm[2011-04-20];2011-04-28.
[87]李端,殷明.药理学[M].北京：人民卫生出版社,2010,第六版：22-24.
[88]陶伟,王昌林,辛海量等.近红外漫反射方法在中药研究领域中的应用[J].实用医学杂志,2008,24(16)：2908-2909.
[89]李文龙,瞿海斌.黄芪提取过程总皂苷质量浓度的在线监测[J].中草药,2012,43(8)：1531-1535.
[90]陈雪英,李页瑞,陈 勇,等.近红外光谱分析技术在赤芍提取过程质量监控中的应用研究[J].中国中药杂志,2009,34(11)：1355-1358.
[91]唐进法,王 星,曹占霞.近红外光谱法测定柴胡药材中柴胡皂苷A含量[J].中国实验方剂学杂志,2012,18(17)：138-141.
[92]董晓强,魏惠珍,饶毅,等.近红外光谱法快速测定六味地黄丸中马钱苷[J].中草药,2011,42(8)：1543-1546.
[93]魏惠珍,方少敏,饶毅,等.近红外光谱技术快速测定白芍药材烘干过程中水分[J].中草药,2011,42(10)：1994-1997.
[94]王钢力,聂黎行,张继,等.应用近红外光谱技术鉴别红参药材[J].中草药,2008,39(3)：438-440.
[95]陈晨,李文龙,瞿海斌,等.近红外透反射光谱法应用于复方苦参注射液渗漉过程在线控制的研究[J].中草药http://www.cnki.net/kcms/detail/12.1108.R.20121117.1459.008.html.
[96]曹小玉,彭成.1990年以来川乌研究文献的定量分析[J].时珍国医国药,2005,16(3)：189-190.
[97]邢跃文.川乌炮制和毒性的关系[J].中国民间疗法,2004(3)：43-44.
[98]邓广海,林华.川乌高压蒸制工艺优选[J].中国实验方剂学杂志,2011,17(2)：21-24.
[99]区炳雄,龚又明,林华,等.川乌微波炮制工艺优选[J].中国实验方剂学杂志,2012,18(1)：39-42.
[100]邱启雄.乌头药材的炮制研究进展[J].海峡药学,2009,21(12)：104-106.
[101]叶定江,原思通.中药炮制学辞典[M].上海：上海科学技术出版社,2005：215-217.
[102]龚千锋.中药炮制学[M].北京：中国中医药出版社.2007：293-293.
[103]张毅,翁代群,王海军,等.乌头碱的水解动力学研究[J].中草药,2009,40：120-120.
[104]黄丹,张强,严芳.紫苏提取物喷雾干燥工艺研究[J].食品与机械,2009,25(5)：160-162.
[105]李学林,刘瑞新.中药剂型改革应加强中药等效性研究[J].中国中药杂志,2008,33(9)：1100-1102.
[106]国家中医药管理局.关于征求《单味中药精制颗粒科研规范(讨论稿)》意见的函.国中医药科研函[1996]78号：1-4.
[107]刘继平.骨愈灵胶囊主要药效及毒理学研究[D].陕西中医学院,2008.
[108]黄良胜.清开灵注射液解热抗炎作用的实验研究[J].临床合理用药,2010,5(1C)：6-7.
[109]沙爱龙,陈红玲,孟庆艳.维药芳香新塔花水提物的抗炎镇痛作用及其急性毒性[J].中国实验方剂学杂志,2012,18(4)：202-204.
[2]http://www,menet.com.cn/Articles/IEconomy/201011/201011231130513051_2599.html.
[3]曹小玉,彭成.1990年以来川乌研究文献的定量分析[J].时珍国医国药,2005,16(3)：189-i90.
[4]邱启雄.乌头药材的炮制研究进展[J].海峡药学,2009,21(12)：104-106.
[5]侯家玉,方泰惠.中药药理学(M).北京：中国中医药出版社.2007：114-114.
[6]杨世雷.川乌的功能与主治探疑[J].中国现代药物应用,2009,3(5)：]73-174.
[7]杨广民,张志国.川乌草乌附子[M].北京：中国中医药出版社出版,2001.
[8]白向阳.川乌的毒性与抗肿瘤作用概述[J].实用中医药杂志,2005,21(2)：125-126.
[9]张思佳,刘敏卓,刘静涵,等.附子的化学成分研究[J].药学与临床研究,2010,18(3)：262-264.
[10]Lei J,Luo YJ,Bian QQ,etc.Talatisamine,a C(19)-diterpenoid alkaloid from Chinese traditional herbal'Cnuanwu'[J]. Acta Crystallogr Sect E Struct Rep Onl ine.2011 Dec 1:67(Pt 12):3145-3146.
[11]陈红英.川乌头不同组织中挥发油成分分析[J].安徽农业科学,2011,39(6)：3325-3326,3367.
[12]罗霄,彭善贵,文永盛,等.HPLC测定制川乌中的乌头碱、次乌头碱和新乌头碱[J].华西药学杂志,2010,25(4)：472-473.
[13]粟贵,廖林川,颜有仪,等.HPLC同时测定生物样品中新乌头碱、乌头碱、次乌头碱的含量[J].中国药学杂志,2009,44(12)：946-950.
[14]邱葵,吴华,王鹤尧.LC-MS/MS方法同时测定人血浆和尿液中乌头碱与次乌头碱的含量[J].中国药学杂志,2010,45(8)：633-636.
[15]林华,邓广海.川乌HPLC指纹图谱的研究[J].中国实验方剂学杂志,2011,17(3)：73-76.
[16]图雅,张贵君,刘志强,等.蒙药草乌的研究进展[J].时珍国医国药,2008,19(7)：1581-1582.
[17]张世忠,吴博威.乌头碱与钾离子通道激动剂合用对离体大鼠心脏的正性肌力作用研究[J].中国药理学通报,2001,17(5)：570-570.
[18]张惠云,秦林,薛玲,等.川乌配伍白芍的抗炎作用特点研究[J].中国中药杂志,2002,27(6)：449-452.
[19]秦林,张少华,李晓丽.川乌与白芍配伍的免疫调节作用初探[J].中国中药杂志,2002,27(7)：541-544.
[20]黄青,刘启福,李飞,等.川乌不同炮制品提取物的致突变与抗突变作用研究[J].北京中医药大学学报,2002,25(2)：41-43.
[21]曾瑾,罗霞,江南,等.生川乌水煎液抗肿瘤作用的实验研究[J].四川大学学报(自然科学 版),2007,44(6)：1344-1348.
[22]欧明,王宁生.中药及其制剂不良反应大典[M].沈阳：辽宁科学技术出版社,2002：33-35.
[23]楼锦英.乌头类中药的毒性反应原因分析与防治[J].海峡药学,2007,19(12)：122-125.
[24]李梦然,曲玮,梁敬钰.乌头属化学成分和药理作用研究进展[J].海峡药学,2010,22(4)：1-6.
[25]何玉华.乌头、附子的中毒原因探微[J].四川中医,2007,25(12)：51-52.
[26]陈理书,张云鹏,杨悦娅.临床应用附子例探析[J].辽宁中医杂志,2005,32(4)：23-25.
[27]卢豪,王亚其,潘黎.生川乌提取物遗传毒性研究[J].现代预防医学,2008,35(20)：4014-4016.
[28]黄韧.中药生川乌对小鼠卵巢和子宫发育的影响试验研究[J].临床和实验医学杂志,2009,8(10)：3-4.
[29]刘强强,何晓娟,严光焰,等.乌头碱对大鼠卵巢颗粒细胞毒性研究[J].现代预防医学,2010,37(2)：299-301.
[30]赵正航,徐长福.川乌对犬脊髓运动神经元损害的实验研究[J].西安医科大学学报,2000,21(1)：24-25.
[31]Niitsu H, Fujita Y, Fujita S, etc. Distribution of Aconitum alkaloids in autopsy cases of aconite poisoning[J]. Forensic Sci Int.2012 Nov 3. doi:pii:S0379-0738(12)00475-6.
[32]陈宝安,陈嘉勇,梁道明,等.急性中毒2658例流行病学调查[J].中国急救医学,2008,28(5)：410-410.
[33]邓广海,林华.川乌高压蒸制工艺优选[J].中国实验方剂学杂志,2011,17(2)：21-24.
[34]张荣,方庆.川乌加压炮制对乌头类生物碱含量的影响研究[J].中医药学刊,2003,21(1)：156-158.
[35]蒋志斌.川乌、附子、半夏、天南星炮制方法及用量商榷[J].中国药房,2007,18(9)：717-718.
[36]刘文龙,宋凤瑞,刘志强,等.川乌与半夏配伍禁忌的化学研究[J].化学通报,2008,(6)：435-438.
[37]梁泽华,尹丽娜,杨燕,等.不同炮制方法对川乌双酯型生物碱含量的影响[J].中国现代应用药学杂志,2009,26(4)：288-291.
[38]何伟,秦林,司淑媛.川乌与防己配伍前后乌头碱和粉防己碱煎出量的测定[J].中草药,2002,33(7)：600-602.
[39]苏建树,刘白宁,田平芳,等.微生物发酵对川乌、附子中生物碱含量的影响[J].北京化工大学学报(自然科学版),2010,37(3)：97-101.
[40]Zhu H, Wang C, Qi Y, etc. Rapid quality assessment of Radix Aconiti Preparata using direct analysis in real time mass spectrometry[J]. Anal Chim Acta.2012 Nov 8; 752:69-77.
[41]叶定江,原思通.中药炮制学辞典[M].上海：上海科学技术出版社,2005：215-217.
[42]随志刚,陈明玉,刘志强,等.草乌中乌头类生物碱提取方法比较研究[J].时珍国医国药,2009,20(3)：513-514.
[43]刘学湘,陈建伟.川乌中总生物碱提取工艺优化研究[J].中华中医药杂志,2009,24(1)：98-100.
[44]李永生,林强,郑来丽,等.川乌的纤维素酶酶解提取工艺研究[J].中草药,2009,40(11)：1735-1739.
[45]李文兰,王艳萍,范玉奇.中药乌头的实验研究[J].哈尔滨商业大学学报(自然科学版)2006,22(4)：13-17.
[46]余洋,宁玲.炙川乌汤治疗痛痹42例临床体会[J].云南中医中药杂志,2001,22(2)：13-13.
[47]白向阳.川乌的毒性与抗肿瘤作用概述[J].实用中医药杂志,2005,21(2)：125-126.
[48]林颖灵,王德英.生川草乌在疑难疾病中的应用体会[J].江苏中医药,2002,23(5)：35-36.
[49]曹会彦,曹官泽.川乌及草乌塞鼻治疗周围性面瘫326例[J].实用中医药杂志,2009,25(3)：175-175.
[50]涂瑶生,毕晓黎.中药配方颗粒国际化有关问题的思考[J].世界科学技术—中医药现代化,2007,9(2)：77-81.
[51]柳俊,张建军Box-Behnken效应面法优化白芍配方颗粒提取工艺[J].中国实验方剂学杂志,2011,17(1)：9-13.
[52]何健,韩正洲,张跃飞.正交试验法优选苦参配方颗粒的提取工艺[J].海峡药学,2011,23(5)：25-26.
[53]何健,韩正洲,张跃飞.正交实验法优选薄荷配方颗粒的提取工艺[J].时珍国医国药,2007,18(12)：2886-2887.
[54]廖彩震,陈培胜,张文惠,等.正交试验优选菊花配方颗粒的提取工艺[J].世界中西医结合杂志,2010,5(10)：858-860.
[55]李霞,贾晓斌,陈彦,等.五味子配方颗粒工艺研究[J].中成药,2006,28(8)：1122-1126.
[56]凌海慧,刘晓飞.中药配方颗粒的研究进展及应用前景分析[J].云南中医学院学报,2009,32(3)：68-70.
[57]钱丽萍,陈雪萍.阙慧卿,等.不同干燥工艺对制剂影响的研究进展[J].药物评价研究,2012,35(4)：299-303.
[58]田方炳.中药制粒技术进展[J].中国药业,2006,15(5)：62-63.
[59]付静,陈明.中药配方颗粒工艺研究概况[J].北京中医,2007,26(8)：522-524.
[60]涂瑶生.毕晓黎,罗文汇.中药配方颗粒的质量控制研究[J].世界科学技术—中医药现代化,2011,13(1)：41-46.
[61]赵叶东,叶宏军.中药配方颗粒剂与中药饮片药效学等效性研究[J].辽宁中医药大学学报,2010,12(11)：227-229.
[62]赵自明,崔景朝.中药配方颗粒研究进展(III)——药效学研究概况[J].中国实验方剂学杂志,2011,17(5)：270-274.
[63]张惠,胡敏.中药配方颗粒剂的临床应用评价[J].西部医学,2011,23(7)：1355-1358.
[64]魏兰福,田耀洲,夏军权,等.良附丸中药配方颗粒与饮片汤剂治疗胃脘痛临床对比研究[J].时珍国医国药+2009,20(3)：612-612.
[65]孙奇,李占玲,苏迪,等.中药配方颗粒治疗慢性前列腺炎60例临床观察[J].辽宁中医杂志,2009,36(1)：82-83.
[66]张金巍,张延莹,刘岩,等.近红外光谱法在线质量监控白芍工业化提取[J].中草药,2011,42(12)：2459-2461.
[67]丁念亚,黎薇,冯昕韡,等.近红外漫反射光谱在中药分类及真伪鉴别中的应用[J].计算机与应用化学,2008,25(4)：499-502.
[68]陆珺,相秉仁,刘浩,等.二维相关近红外光谱及其应用[J].药学进展,2007,31(7)： 303-308.
[69]曲斌,孟庆华,张婷婷,等.非负矩阵分解在中药鱼腥草近红外光谱定性判别中的应用[J].计算机与应用化学,2009,26(10)：1353-1357.
[70]白雁,刘乐,王东,等.近红外光谱对黄芩药材浸出物的快速测定[J].时珍国医国药,2009,20(5)：1081-1082.
[71]武卫红,王宁,董海平.AOTF—近红外光谱法快速无损分析复方丹参片的均一性[J].华西药学杂志,2011,26(4)：364-366.
[72]倪力军,史晓浩,高秀蛟,等.NIR在线检测、分析技术在丹参水提过程质量监控中的应用[J].中国药学杂志,2004,39(8)：628-630.
[73]瞿海斌,李斌,刘雪松,等.红参醇提液浓缩过程近红外光谱在线分析方法[J].中国药学杂志,2005,40(24)：1897-1899,1903.
[74]郁露,孙素琴,周群,等.白芥子炒制过程的红外及二维相关光谱研究[J].光谱学与光谱分析,2006,26(12)：2181-2182.
[75]刘元芬,李祥,高锦飚,等.石膏炮制前后红外光谱的特征分析与鉴定[J].内蒙古中医药,2007,26(5)：27-29.
[76]白雁,贾永,王东,等.应用近红外漫反射光谱技术测定酒炖熟地黄中的还原糖含量[J].中药材,2006,29(10)：1035-1038.
[77]Li Z, Wang C, Sun S, etc. Analysis of chemical components in Aconitum kusnezoffii leaves and their extracts by infrared spectroscopy[J]. Zhongguo Zhong Yao Za Zhi.2011 Dec;36(23):3281-3285.
[78]李晓明,杨滨.近红外光谱技术的研究进展及其在中药领域的应用[J].中国实验方剂学杂志,2006,12(12)：69-72.
[79]陆婉珍.现代近红外光谱分析技术[M].北京：中国石化出版社,2007,395.
[80]郝勇.近红外光谱微量分析方法研究[D].博士学位论文.南开大学,2009.
[8l]Donald A.Burns, Emil W. Ciurczak. Handbook of Near-Infrared Analysis(Third Edition) [M].CRC Press,2008,795.
[82]Yukihiro Ozaki, W. Fred McClure, Alfred A. Christy. Near-Infrared Spectroscopy in Food Science and Technology [M]. Wiley,2007,408.
[83]Tripathi MM, Hassan E B M, Yueh F Y, et al. Reflection-absorption-based near infrared spectroscopy for predicting water content in bio—oil[M]. Sens Actuator B-Chem,2009, 136:20-25.
[84]Shao Y N, He Y, Gomez A H, et al. Visible/near infrared spectrometric technique for nondestructive assessment of tomato'Heatwave'(Lycopersicum sculentum)quality characteristics[J]. J Food Eng,2007,81:672-678.
[85]Otsuka M. Comparative particle size determination of phenacetin bulk powder by using Kubelka—Munk theory and principal component regression analysis based on near-infrared spectroscopy[J]. Powder Technol,2004,141:24-50.
[86]基药遴选或引入药物经济学药企观望.新华网http://news. xinhuanet. com/health/ 2011-04/20/c_121325466. htm[2011-04-20];2011-04-28.
[87]李端,殷明.药理学[M].北京：人民卫生出版社,2010,第六版：22-24.
[88]陶伟,王昌林,辛海量等.近红外漫反射方法在中药研究领域中的应用[J].实用医学杂志,2008,24(16)：2908-2909.
[89]李文龙,瞿海斌.黄芪提取过程总皂苷质量浓度的在线监测[J].中草药,2012,43(8)：1531-1535.
[90]陈雪英,李页瑞,陈 勇,等.近红外光谱分析技术在赤芍提取过程质量监控中的应用研究[J].中国中药杂志,2009,34(11)：1355-1358.
[91]唐进法,王 星,曹占霞.近红外光谱法测定柴胡药材中柴胡皂苷A含量[J].中国实验方剂学杂志,2012,18(17)：138-141.
[92]董晓强,魏惠珍,饶毅,等.近红外光谱法快速测定六味地黄丸中马钱苷[J].中草药,2011,42(8)：1543-1546.
[93]魏惠珍,方少敏,饶毅,等.近红外光谱技术快速测定白芍药材烘干过程中水分[J].中草药,2011,42(10)：1994-1997.
[94]王钢力,聂黎行,张继,等.应用近红外光谱技术鉴别红参药材[J].中草药,2008,39(3)：438-440.
[95]陈晨,李文龙,瞿海斌,等.近红外透反射光谱法应用于复方苦参注射液渗漉过程在线控制的研究[J].中草药http://www.cnki.net/kcms/detail/12.1108.R.20121117.1459.008.html.
[96]曹小玉,彭成.1990年以来川乌研究文献的定量分析[J].时珍国医国药,2005,16(3)：189-190.
[97]邢跃文.川乌炮制和毒性的关系[J].中国民间疗法,2004(3)：43-44.
[98]邓广海,林华.川乌高压蒸制工艺优选[J].中国实验方剂学杂志,2011,17(2)：21-24.
[99]区炳雄,龚又明,林华,等.川乌微波炮制工艺优选[J].中国实验方剂学杂志,2012,18(1)：39-42.
[100]邱启雄.乌头药材的炮制研究进展[J].海峡药学,2009,21(12)：104-106.
[101]叶定江,原思通.中药炮制学辞典[M].上海：上海科学技术出版社,2005：215-217.
[102]龚千锋.中药炮制学[M].北京：中国中医药出版社.2007：293-293.
[103]张毅,翁代群,王海军,等.乌头碱的水解动力学研究[J].中草药,2009,40：120-120.
[104]黄丹,张强,严芳.紫苏提取物喷雾干燥工艺研究[J].食品与机械,2009,25(5)：160-162.
[105]李学林,刘瑞新.中药剂型改革应加强中药等效性研究[J].中国中药杂志,2008,33(9)：1100-1102.
[106]国家中医药管理局.关于征求《单味中药精制颗粒科研规范(讨论稿)》意见的函.国中医药科研函[1996]78号：1-4.
[107]刘继平.骨愈灵胶囊主要药效及毒理学研究[D].陕西中医学院,2008.
[108]黄良胜.清开灵注射液解热抗炎作用的实验研究[J].临床合理用药,2010,5(1C)：6-7.
[109]沙爱龙,陈红玲,孟庆艳.维药芳香新塔花水提物的抗炎镇痛作用及其急性毒性[J].中国实验方剂学杂志,2012,18(4)：202-204.