关于更年颗粒药学部分的探究
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摘要
目的：在更年颗粒工艺及质量控制研究等前期工作基础上，对更年颗粒工艺进一步进行研究和改进，对更年颗粒质量控制进一步进行研究，对有关新的质量控制技术进行探索性研究。采用SFE-CO_2技术、高分子材料包合技术等先进的新工艺、新技术，对中药复方有效成分进行提取、保护研究；对工艺与成分提取的关系进行研究，对药材采用不同的工艺提取进行比较；对高分子材料包合中药复方提取物工艺进行深入研究；对包合物采用多种验证方法进行鉴定，并对其稳定性进行考查，预测其有效期。采用现代指纹图谱质量控制技术的基础上，针对中药质量控制的特点，提出了建立“全指纹图谱系”以控制中药的质量，对更年颗粒进行了“全指纹图谱系”制定的初步探讨。
在中医药“君、臣、佐、使”配伍理论的指导下，利用现代植物化学、计算机技术的研究成果，采用先进的分析手段，对中药制剂的工艺质量进行全面地提高，为研制现代的中药制剂奠定基础。
方法：(1)应用HPLC法测定更年颗粒中当归、白术、香附、砂仁等四味药材(简称“A”)提取物中的阿魏酸、α-香附酮含量，并进行了方法学考查。阿魏酸含量测定方法为：Agilent 1100液相色谱仪，C18柱(Kromasil 250×4.6mm，100-5)，乙腈-0.085％磷酸溶液(17：83)为流动相，流速1.00ml／min，柱温室温25℃，检测波长为316nm，理论塔板数按阿魏酸峰计算应不低于5000，进样量5μl；α-香附酮含量测定方法为：Agilent 1100液相色谱仪，C18柱(Kromasil250×4.6mm，100-5)；甲醇-水(80：20)为流动相，流速1.00ml／min，柱温室温25℃，检测波长为254nm；理论塔板数按α-香附酮峰计算应不低于1500，进样量10μl。
(2)采用单因素试验，以阿魏酸、α-香附酮提取量为考查指标，考查了药材粉碎粒度，夹带剂种类、用量，萃取压力，解析压力，CO_2流量，萃取温度，萃取时间等对SFE-CO_2提取的影响；并进一步采用均匀设计法对A超临界提取的主要影响因素萃取压力、萃取温度、萃取时间、解析压力等工艺条件进行优选。
以提取物提取量为考查指标，选择了A采用水蒸气蒸馏提取是用分提还是用合提；影响水蒸气蒸馏法的因素主要有：加水量、浸泡时间、蒸馏时间，因而以挥发油提取量、α-香附酮提取量为考查指标，采用正交试验对提取时的加水量、浸泡时间及蒸馏时间进行了优选。
以阿魏酸提取量、α-香附酮提取量为考查指标，对A采用SFE-CO_2工艺与蒸馏提取工艺进行了比较研究，考查了两种工艺对提取量的影响。
以阿魏酸提取量为考查指标，探讨了当归提取成分种类与SFE-CO_2提取工艺条件的关系。以阿魏酸提取量为考查指标，比较了当归采用SFE-CO_2提取工艺、水蒸气蒸馏工艺、水蒸气蒸馏后水提工艺、乙醇渗漉工艺、乙醇回流提取工艺等不同工艺对阿魏酸提取量的影响。
(3)以包合物利用率为考查指标，采用单因素试验法，分别考查了包合物种类、包合方法、包合物用量、包合时的温度、包合时间等对A提取物包合物利用率影响。以包合物利用率为考查指标，采用正交试验优选了包合的β-CD用量，包合温度、搅拌时间等三种工艺条件。
对A提取物的β-CD包合物进行了定性试验：对β-CD包合物进行了薄层鉴别，采用硅胶G薄层板，用石油醚-醋酸乙酯(17：3)为展开剂，置紫外灯(365nm)下检视；采用紫外可见分光光度法，以吸收曲线与吸收峰的位置和高度来判断包合物形成情况，采用日本岛津公司UV-2401型可见紫外分光光度计，在200～600nm对β-CD包合物进行扫描。对A提取物的β-CD包合物进行了稳定性试验：对β-CD包合物进行了平衡吸湿率与临界相对湿度的测定；采用化学动力学法——温度系数法(Q_(10)法)预测了β-CD包合物的有效期。
(4)采用HPLC法，对更年颗粒的“全指纹图谱系”制定进行探讨，探讨更年颗粒中苷类成分指纹图谱的制定。以芍药苷为参照物，Agilent 1100高效液相色谱仪，C18色谱柱(Kromasil 250×4.6mm，100-5)，Agilent数据处理软件，乙腈-0.1％磷酸水溶液为流动相进行梯度洗脱，流速1.00ml／min，柱温室温，检测波长225nm，进样10μl，记录时间50分钟，以芍药苷的色谱峰(S峰)的保留时间和峰面积为1计算各峰相对保留时间和相对峰面积比值；并进行了方法学考查；采用国家药典委员会的“中药色谱指纹图谱相似性评价系统”进行相似度评价。
分别制备了更年颗粒的药材供试品溶液、缺味更年颗粒的供试品溶液，按更年颗粒中苷类成分指纹图谱的制定方法绘制各色谱图，与更年颗粒中苷类成分指纹图谱进行对比分析。
结果：(1)阿魏酸方法学考查结果：阿魏酸在0.050～0．800μg范围内具有良好的线性关系，回归方程：Y=4130.8x+4.9042，R~2=0.9999；精密度试验结果表明精密度良好，RSD0.62％；稳定性试验结果表明供试品溶液在12小时内峰面积值基本稳定，RSD1.25％；重现性试验结果表明其重现性良好，RSD3.18％；加样回收率试验结果表明其加样回收率符合规定，平均回收率为96.50％。α-香附酮方法学考查结果；α-香附酮在9.92～79.36μg范围内具有良好的线性关系，标准曲线回归方程的为：Y=273.34X+26.116，R~2=0.9999；精密度试验结果表明精密度良好，RSD1.42％；稳定性试验结果表明供试品溶液在12小时内峰面积值基本稳定，RSD1.09％；重现性试验结果表明其重现性良好，RSD0.76％；加样回收率试验结果表明其加样回收率符合规定，平均回收率为98.47％。
(2)A SFE-C02提取的单因素试验法结果：药材粉碎粒度对阿魏酸、α-香附酮等成分提取量影响不大，为尽量提取出有效成分，采用粗颗粒为宜；夹带剂种类对成分提取量影响差异显著，以加95％乙醇和甲醇成分提取量最高，考虑提取操作的安全性、经济性，采用95％乙醇为宜；夹带剂用量达药材量的20％时，成分提取量保持稳定，考虑提取的经济性，夹带剂用量以20％为宜；萃取压力越高，解析压力越高，CO_2流量越大时，成分提取量增加，但当萃取压力达25～30MPa，解析压力达8～10MPa，CO_2流量达10～12L／h，成分提取量基本达到平衡，考虑提取的经济性和设备安全性，采用萃取压力为25～30MPa，解析压力以8～10MPa，CO_2流量以10～12L／h为宜；萃取温度达40～45℃时，成分提取量保持稳定，考虑提取的经济性、安全性，采用40～45℃为宜；萃取时间达60min时，成分基本提取完全，考虑提取的经济性，采用60min为宜。均匀设计试验结果表明，萃取压力、萃取温度、萃取时间越接近最大值，萃取率会越高，但实际操作中，萃取压力、萃取温度不能过高，萃取压力应在25～30Mpa之间，萃取温度应在40～45℃之间。因而A SFE-CO_2最佳萃取条件为：萃取压力25～30Mpa，萃取温度40～45℃，CO_2流量10～12L／h，萃取时间1小时左右。验证试验结果表明工艺基本合理，重现性良好。
A采用水蒸气蒸馏提取进行分提与合提提取物提取量无显著性差异(P＞0.05)，考虑提取的经济性，选用合提为宜。正交试验优选A采用水蒸气蒸馏提取的最佳工艺条件为加5倍量水，不浸泡，蒸馏提取5小时。阿魏酸等极性较大的成分，蒸馏提取工艺难以提取出，而SFE-C02工艺可以很好的提取；α-香附酮极性较小的成分可以通过两种工艺提取，但两种工艺有极显著性差异(P＜0.01)，SFE-CO_2工艺明显高于水蒸气蒸馏提取工艺。
当归中阿魏酸提取量，随夹带剂乙醇浓度的升高而增加，以甲醇为夹带剂提取量最大，但采用95％乙醇为夹带剂时，已经接近甲醇为夹带剂时的提取量，同时考虑工业生产的安全性和经济性，工业生产以95％乙醇为夹带剂为宜。当归不同提取工艺比较结果：当归中阿魏酸的提取量，以SFE-CO_2提取工艺的提取量最高，其次依次是乙醇渗漉工艺、乙醇回流提取工艺、水提工艺，水蒸气蒸馏工艺几乎提取不出阿魏酸。(3)A提取物包合的单因素试验法结果：包合物种类的包合物利用率以β-CD最高，其次依次为乙基纤维素、硅胶、尿素，β-CD包合物利用率明显高于其它的包合材料；包合方法的包合物利用率以饱和水溶液法最高，其次依次为研磨法、超声波法；包合物用量的包合物利用率随β-CD用量增加而增大，但β-CD用量达到提取物量的8倍量时，包合物利用率基本达到平衡；包合温度在25℃以上，包合物利用率随包合温度升高而减小，因而以室温为宜；包合时间的包合物利用率随包合时间增加而增大，但包合时间达到3小时时，包合物利用率基本达到平衡；同时发现，β-CD用量，包合温度、搅拌时间是三种最重要的影响因素。正交试验结果β-CD包合A提取物的最佳工艺条件为加8倍量β-CD，室温下搅拌3小时。
A提取物的β-CD包合物定性试验结果：薄层试验中提取物石油醚溶液薄层斑点清晰，纯β-CD无斑点，β-CD包合物采用石油醚洗脱只能洗脱表面残留的提取物，首次β-CD包合物石油醚洗脱液只有较模糊的斑点，而再次洗脱液几乎没有斑点，只有热回流供试液才有明显的斑点，从而说明了包合物确已形成；紫外可见扫描结果：提取物样品溶液在260nm左右有最大吸收，纯β-CD溶液有末段吸收，但在260nm左右无最大吸收，首次β-CD包合物石油醚洗脱液只有在260nm左右较小的峰，而再次洗脱液几乎没有吸收，只有热回流供试液在260nm左右才有明显的吸收，从而进一步说明了包合物确已形成。
A提取物的β-CD包合物稳定性试验结果：β-CD包合物临界相对湿度约为68％，因而β-CD包合物宜贮存于相对湿度低于65％的环境中，这与一般的中药制剂相似；β-CD包合物有效期约为1.26年，这接近一般中药制剂的有效期。
(4)制定了更年颗粒中苷类成分的指纹图谱，标注了更年颗粒中苷类成分9个共有指纹峰。方法学考查结果：精密度试验结果，各共有指纹峰的相对保留时间和相对峰面积基本一致，RSD＜2％；稳定性试验结果，在0、2、4、6、8h各共有指纹峰的相对保留时间、相对峰面积基本一致，RSD＜2％，样品液在8h内稳定；重复性试验中，各共有指纹峰的相对保留时间、相对峰面积基本一致，RSD＜2％。相似度计算(全谱)结果10批样品的相似度均在0.80以上。
与更年颗粒中苷类成分指纹图谱进行对比分析结果基本确定了各指纹蜂的来源。
结论：采用SFE-CO_2工艺能明显提高成分的提取量和能使提取成分的种类增加，能明显提高挥发性成分的提取，优于水蒸气蒸馏提取工艺，也优于其它的提取方法；药材中成分提取与SFE-CO_2工艺条件密切相关。挥发性成分采用现代的分子包合工艺稳定可行，其稳定性明显增加，有效期明显延长，达到药剂的要求。通过对更年颗粒中苷类成分指纹图谱的制定，初步探讨了中药“全指纹图谱系”的制定，为提高中药质量控制技术进行了有意义的探索。从而为现代中药的研制采用先进的现代技术手段提供了依据，为中药制剂现代化提供了方法。
Objective: On the formal basis of the technology and quality controlling studying of Gengnian granules, further studying and improving the technology and further study quality controlling of Gengnian granules. Applying new process and new 'technology as SFE-CO2, high molecule wrapping technology, studying extract and protect effective component of compound TCM; studying the relation between technology and components extracting, comparing different technologies to medicinal materials extracting; making a thorough studying on high molecule wrapping extraction of compound TCM; applying several verify methods to identify molecule wrapping, and investigate its stability, calculate its effective time. On the basis of the modem fingerprint atlas quality controlling technology, for the characteristic of TCM quality controlling, put forward setting up "complete fingerprint atlas system" to control the TCM quality, initially probe to formulate the "complete fingerprint atlas system" of Gengnian granules.
In the direct of TCM compatibility theory of "Jun, Chen, Zuo, Shi", utilizing the studying out come modem phytochemistry, computer technology, applying advanced analysis diplomacy, completely improve the technology and quality of Chinese drugs preparation, to establish the basis of modem Chinese drugs preparation.
Method: (1) Applying HPLC determine Ferulic acid and a-cyperone contents of extraction of angelica, atractylodes macrocephala, nutgrass galingale rhizome, amomi of Gengnian granules (call "A"), and review methodology. The content determine method of Ferulic acid: Aginent 1100 HPLC instrument, C18 column (Kromasil 250×4.6mm, 100-5), mobile phase acetonitrile-0.085% phosphoric acid(17: 83), detect wavelength 316nm, theoretical plate number no lower than 5000 refer to Ferulic acid peak, sample size 5μl; The content determine method of a-cyperone: Aginent 1100 HPLC instrument, C18 column (Kromasil 250×4.6mm, 100-5), mobile phase methanol-water(80: 20), detect wavelength 254nm, theoretical plate number no lower than 1500 refer to a-cyperone peak, sample size 10μl.
(2) Deploying mono-factor, taking the extraction as Ferulic acid and a-cyperone as the reviewing index, review the effect of medical materials grinding size, the variety of entrainer, the dosage of entrainer, extracting pressure, resolution pressure, CO_2 flow rate, extracting temperature, extracting time to the SFE-CO_2 extracting; and further, and take uniform design to optimize the technology condition of the main factors as extracting pressure, extracting temperature, extracting time, resolution pressure of SFE of A.
Taking the extracting quantity as the review index, chose the method of steam distillation of A whether extract divide or together. The main factor affecting the method of steam distillation is imbitition quantity, soaking time, steaming time, thus taking the volatile oil extracting quantity and the extracting quantity of a-cyperone as the review index, applying orthogonal experiment optimized the imbitition quantity, soaking time, and steaming time.
Taking the extracting quantity of Ferulic acid and a-cyperone as the review index, comparative studied the two kinds technology of SFE-CO_2 technology and steam distillation technology of A, reviewed the effect of the two kinds technology to extracting quantity.
Taking the extracting quantity of Ferulic acid as the review index, probed the relation between the extracting component variety of angelica and the SFE-CO_2 technology parameter. Taking the extracting quantity of Ferulic acid as the review index, compared the effect of different technology as SFE-CO_2 technology, steam distillation technology, aqua extracting after steam distillation technology, ethanol percolating technology, ethanol backstreaming extracting technology of angelica to the extracting quantity of Ferulic acid.
(3) Taking the clathrate availability as the review index, applying single factor experiment, reviewed the effect of clathrate variety, clathrating method, clathrate dosage, clathrating temperature, clathrating time to the extracting clathrate availability of A. Taking the clathrate availability as the review index, applying orthogonal experiment to optimize the three technology parameter asβ-CD dosage, clathrating temperature, stirring time.
Carried out qualitative test to the extractionβ-CD clathrate of A: carried out lamellar detect toβ-CD clathrate, taking silica G thin layer plate, taking ligarineethyl acetate (17: 3) as developer, putting at uviol lamp(365nm) to postmortem; applying UV method, taking the location and hight of absorbing curve and absorbing peak to judge the forming information of the clathrate, take the UV-2401 UV instrument of Japan DJ, scanned the 13 -CD clathrate in 200～400nm
Carried out stability test to the extractionβ-CD clathrate of A: detected the equilibrium moister absorption rate ofβ-CD clathrate; applying chemical kinetics method——temperature coefficient method (Q10 method)to anticipate the expiration date ofβ-CD clathrate.
(4) Applying HPLC method, probed to formulate "the complete fingerprint system" of Gengnian granules, probed to formulate fingerprint of glycoside of Gengnian granules. Taking peoniflorin as the reference, Aginent 1100 HPLC instrument, C18chromatographic column (Kromasil 250×4.6mm, 100-5), Aginent data handing software, taking acetonitrile-0.1% H_3PO_4 aqueous solution (80: 20) as mobile phase to gradient elution, flow rate 1.00ml/min, column temperature room temperature, detect wavelength 225nm, sample size 10μl, record time 50min, taking the retention time and peak area of peoniflorin chromatographic peak (S peak) as 1 to calculate relative retention time and relative peak area ratios of other peak; also review methodology; and applying " the Chinese medical materials chromatograph fingerprint similarity appreciation system" of nation pharmacopoeia board to appreciate similarity.
Prepared medical materials experiment solution and vacancy medical materials experiment solution of Gengnian granules, referring to the formulating method of formulating fingerprint of glycoside of Gengnian granules, formulated each fingerprint, and carried out to contrast analysis with the fingerprint of glycoside of Gengnian granules.
Results: (1) The results of reviewing methodology of Ferulic acid: there was well linear correlation in the range of 0.050～0.800μg of Ferulic acid, regressionequation: Y=4130.8x+4.9042, R~2=0.9999; precision experiment showed that the precision is well, RSD0.62%; stability experiment showed that the peak area of experiment solution is stable in 12 hour, RSD1.25%; reproducibility experiment showed the reproducibility is well, RSD3.18%; sample spotting recovery experiment showed it correspond with the regulation, the average recovery is 96.50%. The results of reviewing methodology of a-cyperone: there was well linear correlation in the range of 9092～79.36μg of a-cyperone, regression equation: Y=273.34X+26.116, R~2=0.9999; precision experiment showed that the precision is well, RSD1.42%; stability experiment showed that the peak area of experiment solution is stable in 12hour, RSD1.09%; reproducibility experiment showed the reproducibility is well, RSD0.76%; sample spotting recovery experiment showed it correspond with the regulation, the average recovery is 98.47%.
(2) The results of the single factor experiment method of SFE-CO_2 method of A: the grinding size of medical materials can affect little to the extraction quantity of Ferulic acid and a-cyperone, for extracting the active components as far as possible, adopt the coarse grain would be well; the variety of entrainer can affect obviously to the extraction quantity, and the extraction quantity of 95% ethanol and methanol is the max, taking care of the safety and economy of the extracting operation, adopt 95% ethanol would be well; when the dosage of entrainer reach 20% of mecical materials, the extraction quantity keep stability, taking care of the economy, adopt 20% would be well; the more the pressure of extracting, the more the pressure of resolution, the more the CO_2 flow rate, the more the extraction quantity, when thepressure of extracting reach 25～30MPa, the pressure of resolution reach 8～10MPa, the CO_2 flow rate reach 10～12L/h, the extraction quantity can keep stablity, taking care of the safety and economy of the extracting operation, the pressure of extracting adopt 25～30MPa, the pressure of resolution adopt 8～10MPa, the CO_2 flow rate adopt 10～12L/h; when the extracting temperature reach 40～45℃, the extraction quantity keep stability, taking care of the safety and economy of the extracting operation, adopt 40～45℃would be well; when the extracting time reach 60min, the extraction quantity keep stability, taking care of the economy, adopt 60min would be well; the results of uniform experiment, the more the pressure of extracting, the extracting temperature, the extracting time reach to the max, the more the extracting quantity, while in the practice operation, the pressure of extracting and the extractingtemperature can not reach the max, the pressure of extracting ought to be 25～30MPa, the extracting temperature ought to be 40～45℃, therefore, the best technology parameter would be as such: the pressure of extracting adopt 25～30MPa, the extracting temperature adopt 40～45℃, the CO_2 flow rate adopt 10～12L/h, and the extracting time 60min would be well. The results of verification test showed the technology is reasonable, reproducibility is well.
There was no significant difference of extracting quantity of steam distillation adopting extracting separately or together of A (P＞0.05), taking care of the economy, adopt extracting together would be better. The best technology parameter of orthogonal experiment optimizing steam distillation technology of A: adding 5 times aquifer, no soaking, distilling 5 hours. The steam distillation technology was hard to extract the major polarity components as Ferulic acid, while the SFE-CO_2 technology can be well; and the two technologies can extract the minor polarity components as a-cyperone, while there was significant difference (P＜0.01), the SFE-CO_2 technology was more than the steam distillation technology.
The extracting quantity of Ferulic acid of angelica increased with the higher of ethanol density of entrainer, the extracting quantity was the maximum when the entrainer adopting methanol, while the entrainer adopting 95% ethanol, the extracting quantity approached to methanol, taking care of the safety and economy, thus the entrainer adopt 95% ethanol would be better. The results of different technology: the extracting quantity of Ferulic acid of angelica, the SFE-CO_2 technology was the most, next was the ethanol percolating technology, and the ethanol recirculating extracting technology, and the aquifer extracting technology, the steam distillation technology can not extract out Ferulic acid.
(3) The results of the single factor test of extracting clathrate of A: about the clathrate availability of clathrate variety,β-CD was the maximum, next was ethylcellulose, and silica, and carbamide, the availability ofβ-CD clathrate was obviously super to other clathrate materials; about the clathrate availability of clathrate method, the saturate solution method was the maximum, next was the grinding method, and the supersonic method; about the clathrate availability of clathrate dosage, the more the dosage ofβ-CD, the more the clathrate availability, when the clathrate dosage reach 8 times of extraction quantity, the clathrate availability keep stable; when the temperature was higher than 25℃, the clathrate availability can decrease with the temperature stepping up, thus the room temperature would be well; about the clathrate availability of clathrate time, the more the clathrate time, the more the clathrate availability, when the clathrate time reach 3 hours, the clathrate availability keep stable; in the same time, found the dosage ofβ-CD, the temperature of clathrate, the stirring time were the most important factors. The results of orthogonal experiment: the best technology parameter ofβ-CD clathrating the extraction of A, adding 8 timesβ-CD, stirring 3 hours under room temperature.
the qualitative test results of extractingβ-CD. clathrate of A: about the lamellar experiment, the lamellar speckle of the extracting ligarine solution was clear, the pureβ-CD had no speckle, applying ligarine elutingβ-CD clathrate can only elute the rudimentary extracting on the surface ofβ-CD clathrate, the first time ligarine elution solution only had vague speckle, and the next time ligarine elution solution had no speckle, and only the recirculating extracting solution had the conspicuous speckle, therefore, illustrated the clathrate had formed. The scanning results of UV: the extracting solution had the maximum absorption at about 260nm. the pureβ-CD had end piece absorption, while had no absorption at about 260nm. The first time ligarine elution solution only had little peak at about 260nm, and the next timeligarine elution solution had no absorption, and only the recirculating extracting solution had the conspicuous absorption at about 260nm, therefore, further illustrated the clathrate had formed.
The results of stability experiment of the extractionβ-CD clathrate of A: the CRH ofβ-CD clathrate was about 68%, thusβ-CD clathrate would keep under the situation of RH 65%, this approach to the normal Chinese drugs preparation; the expiration date ofβ-CD clathrate was about 1.26 year, this approach to the normal expiration of Chinese drug pharmaceutics.
(4) Formulated the fingerprint of glycosides of Gengnian granules, marked 9 common finger peaks of glycosides of Gengnian granules. The results of reviewing methodology: the precision test results, the relative retention time and relative peak area of each common peak can keep conformity, RSD＜2%; The results of precision test, the relative retention time and relative peak area of can keep conformity in 0、2、4、6、8h, RSD＜2%, the solution can keep stable in 8 hours; the reproducibility test results, the relative retention time and relative peak area of each common peak can keep conformity, RSD＜2%. The results of resemblance calculating (complete spectra) of 10 batches sample products showed the resemblance was all more than 0.80.
The results of contrasting analysis with glycosides of Gengnian granules: probably decided the origin of each finger peak.
Conclusion: Applying the SFE-CO_2 technology can manifestly increase the extracting quantity and can increase variety of extracting components, can manifestly improve the extracting of volatility components, surpass the steaming distillation technology, and also surpass other extracting technology; there is intimate relationship between components extracting of medical materials and the SFE-CO_2 technology technology parameter. It is stable and feasible for the volatility components adopting modem molecule clathrate technology, its stability increases obviously, its expiration date extends obviously, achieves to medicamentous requirement. Through formulating the fingerprint of glycosides of Gengnian granules, initially probed to formulate the "complete fingerprint atlas system" of Chinese material medica, and carried out significant exploring to improve quality controlling technology of Chinese material medica. Accordingly provided foundation for the developing of modem Chinese material medica adopting advanced modern technology and diplomacy, and provided method for the modernization of Chinese drugs pharmaceutics.
在中医药“君、臣、佐、使”配伍理论的指导下，利用现代植物化学、计算机技术的研究成果，采用先进的分析手段，对中药制剂的工艺质量进行全面地提高，为研制现代的中药制剂奠定基础。
方法：(1)应用HPLC法测定更年颗粒中当归、白术、香附、砂仁等四味药材(简称“A”)提取物中的阿魏酸、α-香附酮含量，并进行了方法学考查。阿魏酸含量测定方法为：Agilent 1100液相色谱仪，C18柱(Kromasil 250×4.6mm，100-5)，乙腈-0.085％磷酸溶液(17：83)为流动相，流速1.00ml／min，柱温室温25℃，检测波长为316nm，理论塔板数按阿魏酸峰计算应不低于5000，进样量5μl；α-香附酮含量测定方法为：Agilent 1100液相色谱仪，C18柱(Kromasil250×4.6mm，100-5)；甲醇-水(80：20)为流动相，流速1.00ml／min，柱温室温25℃，检测波长为254nm；理论塔板数按α-香附酮峰计算应不低于1500，进样量10μl。
(2)采用单因素试验，以阿魏酸、α-香附酮提取量为考查指标，考查了药材粉碎粒度，夹带剂种类、用量，萃取压力，解析压力，CO_2流量，萃取温度，萃取时间等对SFE-CO_2提取的影响；并进一步采用均匀设计法对A超临界提取的主要影响因素萃取压力、萃取温度、萃取时间、解析压力等工艺条件进行优选。
以提取物提取量为考查指标，选择了A采用水蒸气蒸馏提取是用分提还是用合提；影响水蒸气蒸馏法的因素主要有：加水量、浸泡时间、蒸馏时间，因而以挥发油提取量、α-香附酮提取量为考查指标，采用正交试验对提取时的加水量、浸泡时间及蒸馏时间进行了优选。
以阿魏酸提取量、α-香附酮提取量为考查指标，对A采用SFE-CO_2工艺与蒸馏提取工艺进行了比较研究，考查了两种工艺对提取量的影响。
以阿魏酸提取量为考查指标，探讨了当归提取成分种类与SFE-CO_2提取工艺条件的关系。以阿魏酸提取量为考查指标，比较了当归采用SFE-CO_2提取工艺、水蒸气蒸馏工艺、水蒸气蒸馏后水提工艺、乙醇渗漉工艺、乙醇回流提取工艺等不同工艺对阿魏酸提取量的影响。
(3)以包合物利用率为考查指标，采用单因素试验法，分别考查了包合物种类、包合方法、包合物用量、包合时的温度、包合时间等对A提取物包合物利用率影响。以包合物利用率为考查指标，采用正交试验优选了包合的β-CD用量，包合温度、搅拌时间等三种工艺条件。
对A提取物的β-CD包合物进行了定性试验：对β-CD包合物进行了薄层鉴别，采用硅胶G薄层板，用石油醚-醋酸乙酯(17：3)为展开剂，置紫外灯(365nm)下检视；采用紫外可见分光光度法，以吸收曲线与吸收峰的位置和高度来判断包合物形成情况，采用日本岛津公司UV-2401型可见紫外分光光度计，在200～600nm对β-CD包合物进行扫描。对A提取物的β-CD包合物进行了稳定性试验：对β-CD包合物进行了平衡吸湿率与临界相对湿度的测定；采用化学动力学法——温度系数法(Q_(10)法)预测了β-CD包合物的有效期。
(4)采用HPLC法，对更年颗粒的“全指纹图谱系”制定进行探讨，探讨更年颗粒中苷类成分指纹图谱的制定。以芍药苷为参照物，Agilent 1100高效液相色谱仪，C18色谱柱(Kromasil 250×4.6mm，100-5)，Agilent数据处理软件，乙腈-0.1％磷酸水溶液为流动相进行梯度洗脱，流速1.00ml／min，柱温室温，检测波长225nm，进样10μl，记录时间50分钟，以芍药苷的色谱峰(S峰)的保留时间和峰面积为1计算各峰相对保留时间和相对峰面积比值；并进行了方法学考查；采用国家药典委员会的“中药色谱指纹图谱相似性评价系统”进行相似度评价。
分别制备了更年颗粒的药材供试品溶液、缺味更年颗粒的供试品溶液，按更年颗粒中苷类成分指纹图谱的制定方法绘制各色谱图，与更年颗粒中苷类成分指纹图谱进行对比分析。
结果：(1)阿魏酸方法学考查结果：阿魏酸在0.050～0．800μg范围内具有良好的线性关系，回归方程：Y=4130.8x+4.9042，R~2=0.9999；精密度试验结果表明精密度良好，RSD0.62％；稳定性试验结果表明供试品溶液在12小时内峰面积值基本稳定，RSD1.25％；重现性试验结果表明其重现性良好，RSD3.18％；加样回收率试验结果表明其加样回收率符合规定，平均回收率为96.50％。α-香附酮方法学考查结果；α-香附酮在9.92～79.36μg范围内具有良好的线性关系，标准曲线回归方程的为：Y=273.34X+26.116，R~2=0.9999；精密度试验结果表明精密度良好，RSD1.42％；稳定性试验结果表明供试品溶液在12小时内峰面积值基本稳定，RSD1.09％；重现性试验结果表明其重现性良好，RSD0.76％；加样回收率试验结果表明其加样回收率符合规定，平均回收率为98.47％。
(2)A SFE-C02提取的单因素试验法结果：药材粉碎粒度对阿魏酸、α-香附酮等成分提取量影响不大，为尽量提取出有效成分，采用粗颗粒为宜；夹带剂种类对成分提取量影响差异显著，以加95％乙醇和甲醇成分提取量最高，考虑提取操作的安全性、经济性，采用95％乙醇为宜；夹带剂用量达药材量的20％时，成分提取量保持稳定，考虑提取的经济性，夹带剂用量以20％为宜；萃取压力越高，解析压力越高，CO_2流量越大时，成分提取量增加，但当萃取压力达25～30MPa，解析压力达8～10MPa，CO_2流量达10～12L／h，成分提取量基本达到平衡，考虑提取的经济性和设备安全性，采用萃取压力为25～30MPa，解析压力以8～10MPa，CO_2流量以10～12L／h为宜；萃取温度达40～45℃时，成分提取量保持稳定，考虑提取的经济性、安全性，采用40～45℃为宜；萃取时间达60min时，成分基本提取完全，考虑提取的经济性，采用60min为宜。均匀设计试验结果表明，萃取压力、萃取温度、萃取时间越接近最大值，萃取率会越高，但实际操作中，萃取压力、萃取温度不能过高，萃取压力应在25～30Mpa之间，萃取温度应在40～45℃之间。因而A SFE-CO_2最佳萃取条件为：萃取压力25～30Mpa，萃取温度40～45℃，CO_2流量10～12L／h，萃取时间1小时左右。验证试验结果表明工艺基本合理，重现性良好。
A采用水蒸气蒸馏提取进行分提与合提提取物提取量无显著性差异(P＞0.05)，考虑提取的经济性，选用合提为宜。正交试验优选A采用水蒸气蒸馏提取的最佳工艺条件为加5倍量水，不浸泡，蒸馏提取5小时。阿魏酸等极性较大的成分，蒸馏提取工艺难以提取出，而SFE-C02工艺可以很好的提取；α-香附酮极性较小的成分可以通过两种工艺提取，但两种工艺有极显著性差异(P＜0.01)，SFE-CO_2工艺明显高于水蒸气蒸馏提取工艺。
当归中阿魏酸提取量，随夹带剂乙醇浓度的升高而增加，以甲醇为夹带剂提取量最大，但采用95％乙醇为夹带剂时，已经接近甲醇为夹带剂时的提取量，同时考虑工业生产的安全性和经济性，工业生产以95％乙醇为夹带剂为宜。当归不同提取工艺比较结果：当归中阿魏酸的提取量，以SFE-CO_2提取工艺的提取量最高，其次依次是乙醇渗漉工艺、乙醇回流提取工艺、水提工艺，水蒸气蒸馏工艺几乎提取不出阿魏酸。(3)A提取物包合的单因素试验法结果：包合物种类的包合物利用率以β-CD最高，其次依次为乙基纤维素、硅胶、尿素，β-CD包合物利用率明显高于其它的包合材料；包合方法的包合物利用率以饱和水溶液法最高，其次依次为研磨法、超声波法；包合物用量的包合物利用率随β-CD用量增加而增大，但β-CD用量达到提取物量的8倍量时，包合物利用率基本达到平衡；包合温度在25℃以上，包合物利用率随包合温度升高而减小，因而以室温为宜；包合时间的包合物利用率随包合时间增加而增大，但包合时间达到3小时时，包合物利用率基本达到平衡；同时发现，β-CD用量，包合温度、搅拌时间是三种最重要的影响因素。正交试验结果β-CD包合A提取物的最佳工艺条件为加8倍量β-CD，室温下搅拌3小时。
A提取物的β-CD包合物定性试验结果：薄层试验中提取物石油醚溶液薄层斑点清晰，纯β-CD无斑点，β-CD包合物采用石油醚洗脱只能洗脱表面残留的提取物，首次β-CD包合物石油醚洗脱液只有较模糊的斑点，而再次洗脱液几乎没有斑点，只有热回流供试液才有明显的斑点，从而说明了包合物确已形成；紫外可见扫描结果：提取物样品溶液在260nm左右有最大吸收，纯β-CD溶液有末段吸收，但在260nm左右无最大吸收，首次β-CD包合物石油醚洗脱液只有在260nm左右较小的峰，而再次洗脱液几乎没有吸收，只有热回流供试液在260nm左右才有明显的吸收，从而进一步说明了包合物确已形成。
A提取物的β-CD包合物稳定性试验结果：β-CD包合物临界相对湿度约为68％，因而β-CD包合物宜贮存于相对湿度低于65％的环境中，这与一般的中药制剂相似；β-CD包合物有效期约为1.26年，这接近一般中药制剂的有效期。
(4)制定了更年颗粒中苷类成分的指纹图谱，标注了更年颗粒中苷类成分9个共有指纹峰。方法学考查结果：精密度试验结果，各共有指纹峰的相对保留时间和相对峰面积基本一致，RSD＜2％；稳定性试验结果，在0、2、4、6、8h各共有指纹峰的相对保留时间、相对峰面积基本一致，RSD＜2％，样品液在8h内稳定；重复性试验中，各共有指纹峰的相对保留时间、相对峰面积基本一致，RSD＜2％。相似度计算(全谱)结果10批样品的相似度均在0.80以上。
与更年颗粒中苷类成分指纹图谱进行对比分析结果基本确定了各指纹蜂的来源。
结论：采用SFE-CO_2工艺能明显提高成分的提取量和能使提取成分的种类增加，能明显提高挥发性成分的提取，优于水蒸气蒸馏提取工艺，也优于其它的提取方法；药材中成分提取与SFE-CO_2工艺条件密切相关。挥发性成分采用现代的分子包合工艺稳定可行，其稳定性明显增加，有效期明显延长，达到药剂的要求。通过对更年颗粒中苷类成分指纹图谱的制定，初步探讨了中药“全指纹图谱系”的制定，为提高中药质量控制技术进行了有意义的探索。从而为现代中药的研制采用先进的现代技术手段提供了依据，为中药制剂现代化提供了方法。
Objective: On the formal basis of the technology and quality controlling studying of Gengnian granules, further studying and improving the technology and further study quality controlling of Gengnian granules. Applying new process and new 'technology as SFE-CO2, high molecule wrapping technology, studying extract and protect effective component of compound TCM; studying the relation between technology and components extracting, comparing different technologies to medicinal materials extracting; making a thorough studying on high molecule wrapping extraction of compound TCM; applying several verify methods to identify molecule wrapping, and investigate its stability, calculate its effective time. On the basis of the modem fingerprint atlas quality controlling technology, for the characteristic of TCM quality controlling, put forward setting up "complete fingerprint atlas system" to control the TCM quality, initially probe to formulate the "complete fingerprint atlas system" of Gengnian granules.
In the direct of TCM compatibility theory of "Jun, Chen, Zuo, Shi", utilizing the studying out come modem phytochemistry, computer technology, applying advanced analysis diplomacy, completely improve the technology and quality of Chinese drugs preparation, to establish the basis of modem Chinese drugs preparation.
Method: (1) Applying HPLC determine Ferulic acid and a-cyperone contents of extraction of angelica, atractylodes macrocephala, nutgrass galingale rhizome, amomi of Gengnian granules (call "A"), and review methodology. The content determine method of Ferulic acid: Aginent 1100 HPLC instrument, C18 column (Kromasil 250×4.6mm, 100-5), mobile phase acetonitrile-0.085% phosphoric acid(17: 83), detect wavelength 316nm, theoretical plate number no lower than 5000 refer to Ferulic acid peak, sample size 5μl; The content determine method of a-cyperone: Aginent 1100 HPLC instrument, C18 column (Kromasil 250×4.6mm, 100-5), mobile phase methanol-water(80: 20), detect wavelength 254nm, theoretical plate number no lower than 1500 refer to a-cyperone peak, sample size 10μl.
(2) Deploying mono-factor, taking the extraction as Ferulic acid and a-cyperone as the reviewing index, review the effect of medical materials grinding size, the variety of entrainer, the dosage of entrainer, extracting pressure, resolution pressure, CO_2 flow rate, extracting temperature, extracting time to the SFE-CO_2 extracting; and further, and take uniform design to optimize the technology condition of the main factors as extracting pressure, extracting temperature, extracting time, resolution pressure of SFE of A.
Taking the extracting quantity as the review index, chose the method of steam distillation of A whether extract divide or together. The main factor affecting the method of steam distillation is imbitition quantity, soaking time, steaming time, thus taking the volatile oil extracting quantity and the extracting quantity of a-cyperone as the review index, applying orthogonal experiment optimized the imbitition quantity, soaking time, and steaming time.
Taking the extracting quantity of Ferulic acid and a-cyperone as the review index, comparative studied the two kinds technology of SFE-CO_2 technology and steam distillation technology of A, reviewed the effect of the two kinds technology to extracting quantity.
Taking the extracting quantity of Ferulic acid as the review index, probed the relation between the extracting component variety of angelica and the SFE-CO_2 technology parameter. Taking the extracting quantity of Ferulic acid as the review index, compared the effect of different technology as SFE-CO_2 technology, steam distillation technology, aqua extracting after steam distillation technology, ethanol percolating technology, ethanol backstreaming extracting technology of angelica to the extracting quantity of Ferulic acid.
(3) Taking the clathrate availability as the review index, applying single factor experiment, reviewed the effect of clathrate variety, clathrating method, clathrate dosage, clathrating temperature, clathrating time to the extracting clathrate availability of A. Taking the clathrate availability as the review index, applying orthogonal experiment to optimize the three technology parameter asβ-CD dosage, clathrating temperature, stirring time.
Carried out qualitative test to the extractionβ-CD clathrate of A: carried out lamellar detect toβ-CD clathrate, taking silica G thin layer plate, taking ligarineethyl acetate (17: 3) as developer, putting at uviol lamp(365nm) to postmortem; applying UV method, taking the location and hight of absorbing curve and absorbing peak to judge the forming information of the clathrate, take the UV-2401 UV instrument of Japan DJ, scanned the 13 -CD clathrate in 200～400nm
Carried out stability test to the extractionβ-CD clathrate of A: detected the equilibrium moister absorption rate ofβ-CD clathrate; applying chemical kinetics method——temperature coefficient method (Q10 method)to anticipate the expiration date ofβ-CD clathrate.
(4) Applying HPLC method, probed to formulate "the complete fingerprint system" of Gengnian granules, probed to formulate fingerprint of glycoside of Gengnian granules. Taking peoniflorin as the reference, Aginent 1100 HPLC instrument, C18chromatographic column (Kromasil 250×4.6mm, 100-5), Aginent data handing software, taking acetonitrile-0.1% H_3PO_4 aqueous solution (80: 20) as mobile phase to gradient elution, flow rate 1.00ml/min, column temperature room temperature, detect wavelength 225nm, sample size 10μl, record time 50min, taking the retention time and peak area of peoniflorin chromatographic peak (S peak) as 1 to calculate relative retention time and relative peak area ratios of other peak; also review methodology; and applying " the Chinese medical materials chromatograph fingerprint similarity appreciation system" of nation pharmacopoeia board to appreciate similarity.
Prepared medical materials experiment solution and vacancy medical materials experiment solution of Gengnian granules, referring to the formulating method of formulating fingerprint of glycoside of Gengnian granules, formulated each fingerprint, and carried out to contrast analysis with the fingerprint of glycoside of Gengnian granules.
Results: (1) The results of reviewing methodology of Ferulic acid: there was well linear correlation in the range of 0.050～0.800μg of Ferulic acid, regressionequation: Y=4130.8x+4.9042, R~2=0.9999; precision experiment showed that the precision is well, RSD0.62%; stability experiment showed that the peak area of experiment solution is stable in 12 hour, RSD1.25%; reproducibility experiment showed the reproducibility is well, RSD3.18%; sample spotting recovery experiment showed it correspond with the regulation, the average recovery is 96.50%. The results of reviewing methodology of a-cyperone: there was well linear correlation in the range of 9092～79.36μg of a-cyperone, regression equation: Y=273.34X+26.116, R~2=0.9999; precision experiment showed that the precision is well, RSD1.42%; stability experiment showed that the peak area of experiment solution is stable in 12hour, RSD1.09%; reproducibility experiment showed the reproducibility is well, RSD0.76%; sample spotting recovery experiment showed it correspond with the regulation, the average recovery is 98.47%.
(2) The results of the single factor experiment method of SFE-CO_2 method of A: the grinding size of medical materials can affect little to the extraction quantity of Ferulic acid and a-cyperone, for extracting the active components as far as possible, adopt the coarse grain would be well; the variety of entrainer can affect obviously to the extraction quantity, and the extraction quantity of 95% ethanol and methanol is the max, taking care of the safety and economy of the extracting operation, adopt 95% ethanol would be well; when the dosage of entrainer reach 20% of mecical materials, the extraction quantity keep stability, taking care of the economy, adopt 20% would be well; the more the pressure of extracting, the more the pressure of resolution, the more the CO_2 flow rate, the more the extraction quantity, when thepressure of extracting reach 25～30MPa, the pressure of resolution reach 8～10MPa, the CO_2 flow rate reach 10～12L/h, the extraction quantity can keep stablity, taking care of the safety and economy of the extracting operation, the pressure of extracting adopt 25～30MPa, the pressure of resolution adopt 8～10MPa, the CO_2 flow rate adopt 10～12L/h; when the extracting temperature reach 40～45℃, the extraction quantity keep stability, taking care of the safety and economy of the extracting operation, adopt 40～45℃would be well; when the extracting time reach 60min, the extraction quantity keep stability, taking care of the economy, adopt 60min would be well; the results of uniform experiment, the more the pressure of extracting, the extracting temperature, the extracting time reach to the max, the more the extracting quantity, while in the practice operation, the pressure of extracting and the extractingtemperature can not reach the max, the pressure of extracting ought to be 25～30MPa, the extracting temperature ought to be 40～45℃, therefore, the best technology parameter would be as such: the pressure of extracting adopt 25～30MPa, the extracting temperature adopt 40～45℃, the CO_2 flow rate adopt 10～12L/h, and the extracting time 60min would be well. The results of verification test showed the technology is reasonable, reproducibility is well.
There was no significant difference of extracting quantity of steam distillation adopting extracting separately or together of A (P＞0.05), taking care of the economy, adopt extracting together would be better. The best technology parameter of orthogonal experiment optimizing steam distillation technology of A: adding 5 times aquifer, no soaking, distilling 5 hours. The steam distillation technology was hard to extract the major polarity components as Ferulic acid, while the SFE-CO_2 technology can be well; and the two technologies can extract the minor polarity components as a-cyperone, while there was significant difference (P＜0.01), the SFE-CO_2 technology was more than the steam distillation technology.
The extracting quantity of Ferulic acid of angelica increased with the higher of ethanol density of entrainer, the extracting quantity was the maximum when the entrainer adopting methanol, while the entrainer adopting 95% ethanol, the extracting quantity approached to methanol, taking care of the safety and economy, thus the entrainer adopt 95% ethanol would be better. The results of different technology: the extracting quantity of Ferulic acid of angelica, the SFE-CO_2 technology was the most, next was the ethanol percolating technology, and the ethanol recirculating extracting technology, and the aquifer extracting technology, the steam distillation technology can not extract out Ferulic acid.
(3) The results of the single factor test of extracting clathrate of A: about the clathrate availability of clathrate variety,β-CD was the maximum, next was ethylcellulose, and silica, and carbamide, the availability ofβ-CD clathrate was obviously super to other clathrate materials; about the clathrate availability of clathrate method, the saturate solution method was the maximum, next was the grinding method, and the supersonic method; about the clathrate availability of clathrate dosage, the more the dosage ofβ-CD, the more the clathrate availability, when the clathrate dosage reach 8 times of extraction quantity, the clathrate availability keep stable; when the temperature was higher than 25℃, the clathrate availability can decrease with the temperature stepping up, thus the room temperature would be well; about the clathrate availability of clathrate time, the more the clathrate time, the more the clathrate availability, when the clathrate time reach 3 hours, the clathrate availability keep stable; in the same time, found the dosage ofβ-CD, the temperature of clathrate, the stirring time were the most important factors. The results of orthogonal experiment: the best technology parameter ofβ-CD clathrating the extraction of A, adding 8 timesβ-CD, stirring 3 hours under room temperature.
the qualitative test results of extractingβ-CD. clathrate of A: about the lamellar experiment, the lamellar speckle of the extracting ligarine solution was clear, the pureβ-CD had no speckle, applying ligarine elutingβ-CD clathrate can only elute the rudimentary extracting on the surface ofβ-CD clathrate, the first time ligarine elution solution only had vague speckle, and the next time ligarine elution solution had no speckle, and only the recirculating extracting solution had the conspicuous speckle, therefore, illustrated the clathrate had formed. The scanning results of UV: the extracting solution had the maximum absorption at about 260nm. the pureβ-CD had end piece absorption, while had no absorption at about 260nm. The first time ligarine elution solution only had little peak at about 260nm, and the next timeligarine elution solution had no absorption, and only the recirculating extracting solution had the conspicuous absorption at about 260nm, therefore, further illustrated the clathrate had formed.
The results of stability experiment of the extractionβ-CD clathrate of A: the CRH ofβ-CD clathrate was about 68%, thusβ-CD clathrate would keep under the situation of RH 65%, this approach to the normal Chinese drugs preparation; the expiration date ofβ-CD clathrate was about 1.26 year, this approach to the normal expiration of Chinese drug pharmaceutics.
(4) Formulated the fingerprint of glycosides of Gengnian granules, marked 9 common finger peaks of glycosides of Gengnian granules. The results of reviewing methodology: the precision test results, the relative retention time and relative peak area of each common peak can keep conformity, RSD＜2%; The results of precision test, the relative retention time and relative peak area of can keep conformity in 0、2、4、6、8h, RSD＜2%, the solution can keep stable in 8 hours; the reproducibility test results, the relative retention time and relative peak area of each common peak can keep conformity, RSD＜2%. The results of resemblance calculating (complete spectra) of 10 batches sample products showed the resemblance was all more than 0.80.
The results of contrasting analysis with glycosides of Gengnian granules: probably decided the origin of each finger peak.
Conclusion: Applying the SFE-CO_2 technology can manifestly increase the extracting quantity and can increase variety of extracting components, can manifestly improve the extracting of volatility components, surpass the steaming distillation technology, and also surpass other extracting technology; there is intimate relationship between components extracting of medical materials and the SFE-CO_2 technology technology parameter. It is stable and feasible for the volatility components adopting modem molecule clathrate technology, its stability increases obviously, its expiration date extends obviously, achieves to medicamentous requirement. Through formulating the fingerprint of glycosides of Gengnian granules, initially probed to formulate the "complete fingerprint atlas system" of Chinese material medica, and carried out significant exploring to improve quality controlling technology of Chinese material medica. Accordingly provided foundation for the developing of modem Chinese material medica adopting advanced modern technology and diplomacy, and provided method for the modernization of Chinese drugs pharmaceutics.
引文
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[3] 范碧亭．中药药剂学．上海：上海科学技术出版社[M]．1997：522-558．
[4] 国家药典委员会．中华人民共和国药典，北京：化学工业出版社[M]．2005，68．
[5] 丁金龙，施少斌，秦春梅，等．克感利咽口服液中挥发油的超临界萃取工艺研究[J]．中草药，2006，37(9)：1325-1328．
[6] 朱日然，李启艳．超临界C02萃取肉桂挥发油的研究[J]．中成药，2006，28(8)：1218-1220．
[7] 张学义．CO_2超临界萃取技术提取中国林蛙卵油的研究[J]．中国林副特产，2006，(5)：17-18．
[8] 董文洪，刘本．超临界流体萃取牛蒡子中牛蒡子苷的实验研究[J]．中国中药杂志，2006，31(15)：1240-1241．
[9] 李慧，王四旺，王剑波．大青叶中靛玉红的超临界萃取工艺及含量测定[J]．第四军医大学学报，2006，27(10)：933-934．
[10] Can Quan, Shufen Li, Songjiang Tian, et al. Supercritical fluid extraction and clean-up of organochlorine pesticides in ginseng [J]. The Journal of Supercritical Fluids. 2004, 31(2): 149.
[11] Rui L. Mendes, Beatriz P. Nobre, Miguel T. Cardoso, et al. Supercritical carbon dioxide extraction of compoundswith pharmaceutical importance from microalgae [J]. Inorganica ChimicaActa. 2003, 356(3): 328.
[12] 国家中医药管理局《中华本草》编委会．中华本草，上海科学技术出版社，1999．
[13] 韩喜江，梁志华，孟祥丽，等．紫草油脂肪酸尿素包合后的组成分析．哈尔滨工业大学学报．2003，35(2)：173～175．
[14] 叶玲，江晓舟，杨华，等．聚酰胺-胺(PAMAM)树状大分子对甲氨蝶呤的包合及缓释研究．高等学校化学学报．2005，26(2)：353～355．
[15] 李树珍，官仕杰．生姜挥发油β-环糊精包结物的稳定性考查[J]．中国中药杂志，1992，17(8)：481-483．
[16] 杨红武，毛衍平，邱应海，等．缬草油β-环糊精包结物的工艺及稳定性研究[J]．中国医院药学杂志，1998，23(11)：676-678．
[17] 陆彬．药物新剂型与新技术．北京：人民卫生出版社[M]．2000，24-51．
[18] Wang Z, Hirayama F, Uekama K. Design and in vitro evaluations of a modified-release oral dosage form of nifidipine by hybridization of hydroxypropyl-β-cyclodextrins and hydroxypropyl cellulose [J]. J Pharm Pharmacol, 1993, (45): 942-946.
[19] Fatouros D G, Hatzidimitriou K, Antimisiaris S G..Liposomes encapsulating prednisolone and prednisolone-cyclodextrin complexes: comparison of mem5rane integrity anddrugrelease[J]. Eur J Pharm Sci, 2001, (13): 287-296.
[20] 肖崇厚．中药化学．上海：上海科学技术出版社[M]．1997：494-506．
[21] Zhang Zhaowang(张兆旺)．The TCM pharmacy(中药药剂学)．Beijing(北京)： China TCM press(中国中医药出版社)[M]．2003：489．
[22] 国家药品监督管理局．关于印发《中药注射剂指纹图谱研究的技术要求(暂行)》的通知[J]．中成药，2000，22(10)：671-675．
[23] 陈强．荆花胃康胶丸的GC指纹图谱研究[J]．海峡药学，2005，17(1)：58-60
[24] 覃洁萍，刘进，廖卓，等．复方扶芳藤合剂HPLC指纹图谱测定方法研究[J]．中成药，2005，27(2)：128-130．
[25] 刘小平，陈笑宇，宋青，等．六味地黄丸的X-衍射指纹图谱鉴定研究[J]．中药材，2005，28(3)：184-186
[26] 王喜军，张宁，孙晖．六味地黄丸指纹图谱的研究[J]．中国中药杂志，2004，29(10)：1004-1005．
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