【作者】 卢彦芳；

【导师】 蒋晔；

【作者基本信息】 河北医科大学 ， 药物分析学， 2011， 硕士

【摘要】 中药的质量控制是保证中药安全性和有效性,促进中药走向国际化的必由之路。要成功构建中药质量评价规范体系,需要解决好三方面的问题:高效快速的样品前处理技术和稳定可靠的分析技术,科学合理的质量控制指标体系以及丰富优质的化学对照品。样品的前处理是中药质量评价关键的第一步,从时间消耗看,约占整个过程的60%⁸0%。目前,常用的浸泡法（渗漉法、冷浸法）和热提取法（煎煮法、回流提取、索氏提取和水蒸气蒸馏等）普遍存在耗时长、溶剂用量大、提取效率低等缺点。超声萃取的提出虽然克服了上述传统提取方法的很多缺点,至今也仍为许多实验室采用,但该技术噪声污染大,并且目前仅局限于实验室的小规模研究,因此已不能满足现代中药发展的需求。而后又提出的超临界流体萃取、加压溶剂萃取等则因存在技术缺陷或设备复杂、运行成本高等问题,而在发展上受到限制。因此,寻找更为简便、快速的新型萃取技术对于中药质量控制的研究具有很重要的意义。微波辅助萃取（Microwave-assisted extraction, MAE）技术,是20世纪80年代兴起的一种新型萃取技术,具有高效快速、选择性好、溶剂用量少、回收率高、重现性好等优点。在中药提取方面,MAE通过离子迁移和偶极子转动对萃取溶剂和物料进行里外同时加热,从根本上改变了传统提取所遵循的加热-渗透进入基体-溶解或夹带-渗透出的模式,解决了传统提取方法萃取时间长、选择性差的问题。通常,传统提取方法需要几小时甚至几十个小时,超声法也需要半个小时到一个小时,而MAE只需要几秒到几分钟,提取速度提高了几十倍至几百倍。本课题旨在将微波萃取技术以及与其它样品前处理技术结合后建立的新型微波辅助萃取技术（如:微波辅助胶束萃取、微波–中空纤维液相微萃取和微波连续抽提）用于中药的样品前处理,解决传统提取方法存在的众多问题,为中药的质量控制提供了高效、简便、快速的样品前处理手段。具体研究如下:（1）补骨脂是种子类药材,外皮坚硬,三黄片中的君药大黄部分以原粉入药,且待测组分脂溶性高,溶解度小,因此二者均存在提取困难,提取时间长的问题,本试验充分利用微波加热速度快、破壁功能强大的特点,在5 min内便可完成提取,大大缩短了样品前处理时间,并且目标组分的提取量与经典方法相当甚至高于经典方法。该方法高效、简便、快速,为那些难提取的药材或中药制剂的分析提供了新手段。（2）马钱子碱、士的宁和柚皮苷均为平消片的指标成分,但其极性、溶解性差别较大,因此使用单一有机溶剂难以满足三指标成分的同时提取,给平消片的质量控制带来一定的困难。本试验建立了微波辅助胶束萃取,以苄泽35 （Brij 35）的胶束水溶液为萃取溶剂,使三种指标成分均得到了良好的萃取效果,方法简便、快速、绿色环保。（3）细辛因含有毒性成分马兜铃酸A而备受关注,但该药材基质复杂、HPLC分离困难,尤其是待测的马兜铃酸A为痕量毒性成分,含量甚低,因此不经富集和纯化处理是无法满足测定需求的。本试验建立的微波–中空纤维液相微萃取,集提取、纯化和富集为一体,具有提取时间短、分析速度快、富集倍数高（152倍）、纯化效果好的优点,为细辛中马兜铃酸A的分离测定提供了新的样品前处理手段。（4）结合索氏提取的原理,建立了新型微波连续抽提法,用于进一步提高微波萃取效率,并将其用于灵芝中三萜类成分的分析。该技术充分结合了微波萃取高效、快速和索氏提取能够连续引入新鲜溶剂的优点,因此比常规的微波萃取更能将药材中的待测成分提取完全,使分析结果更为准确,为中药的成分分析提供了一种新颖的、高效、快速、有效可靠的样品前处理技术。第一部分微波萃取在中药成分分析中的应用一、微波萃取快速分析补骨脂中补骨脂素和异补骨脂素目的:采用自制的开放式微波萃取装置,建立高效、简便、快速的微波萃取法,研究其对于具有坚硬外壳的中药待测成分的提取效果,并用于补骨脂中补骨脂素和异补骨脂素的分析。方法:以补骨脂素和异补骨脂素的提取率为考察指标,对微波萃取的四个参数（萃取溶剂、微波功率、微波萃取时间和液固比）进行单因素试验,以选择最佳的萃取条件,并与传统提取方法进行比较。结果:以甲醇为溶剂,微波功率400 W,液固比400 mL·g^-1的条件下微波萃取3 min,两种待测成分的提取率便可达到最高,而传统的索氏提取、回流提取以及超声提取则需要几十分钟甚至几小时。结论:本文利用自制的开放式微波萃取装置,建立了高效、简便、快速的微波萃取法,提高了萃取效率,缩短了样品前处理时间,装置简便实用,为具有坚硬外壳的难提取中药成分的分析提供了一种新手段。二、微波萃取快速分析三黄片中5种蒽醌类成分目的:研究微波萃取对中药微溶性成分的提取效果,并优化萃取条件,建立高效、简便、快速的微波萃取法用于三黄片中蒽醌类成分的分析。方法:分别以游离蒽醌和结合蒽醌的提取率为考察指标,对微波萃取溶剂、微波功率、萃取时间和液固比进行了优化选择,并将其最优条件下的萃取效果与经典方法进行了比较。结果:微波萃取的最佳条件为:以80%乙醇为溶剂,微波功率300 W,液固比为100:1 (mL·g^-1),游离蒽醌的萃取时间为2 min,结合蒽醌为5 min。本文建立的微波萃取法在5 min中之内便可完成5种游离蒽醌和结合蒽醌的提取,而经典的乙醇回流法则需要60 min才能得到与之相当的提取效果。结论:本文建立的微波萃取法高效、简便、快速,解决了中药微溶性成分提取困难、提取时间长的难题,为含有该类成分的中药材及其制剂的分析提供了有效可靠的前处理手段。第二部分新型微波辅助萃取技术在中药样品前处理中的研究一、微波辅助胶束萃取用于平消片中马钱子碱、士的宁和柚皮苷的分析目的:以表面活性剂的胶束水溶液为萃取溶剂,建立高效、快速、环境友好的微波辅助胶束萃取法,研究其对于中药中性质相差较大的多种指标成分的提取情况,并将其用于平消片中马钱子碱、士的宁和柚皮苷的分析。方法:试验以7% Brij 35水溶液为萃取溶剂,在微波功率300 W,液固比为100: 1 (mL·g^-1)的条件下,萃取2 min,并将该最佳条件下的萃取结果与传统提取方法进行了比较。分析方法采用梯度洗脱,以乙腈-0.4%磷酸水溶液（三乙胺调pH值为3.0）为流动相,检测波长为260 nm。结果:与传统的碱性氯仿回流提取和甲醇超声提取相比,微波辅助胶束萃取缩短了样品前处理时间（2 min:120 min, 2min:30 min）,而各指标成分的提取量与其在最佳条件下的数值相当。马钱子碱、士的宁和柚皮苷分别在0.630⁴0.7μg·mL^-1、0.640⁴1.3μg·mL^-1、9.06⁵80μg·mL^-1浓度范围内线性关系良好（r≥0.999 3）,平均加样回收率为99.7%¹01.1%,RSD为0.7%¹.0%（n=5）。结论:本文建立的微波辅助胶束萃取法高效、简便、快速、环境友好,能够实现多种性质差别较大的指标成分的同时提取,为中药多指标成分的分析提供了一种新的、可靠的样品前处理手段。二、微波–中空纤维液相微萃取用于细辛中痕量毒性成分马兜铃酸A的分析目的:以微波为萃取手段,三相中空纤维液相微萃取为富集和纯化手段,建立快速、灵敏的微波–中空纤维液相微萃取法,研究其对于具有复杂基质的中药痕量成分的前处理效果,并用于细辛中马兜铃酸A的分析。方法:取细辛样品1.0 g,加入70%甲醇25 mL,微波功率400 W条件下萃取6 min,然后采用三相中空纤维液相微萃取技术对提取所得的样品溶液进行富集和纯化处理,条件设为:萃取溶剂为正辛醇,给出相为pH 2.5的HCl水溶液,接收相为pH 12.0的NaOH水溶液,搅拌速度为1200 rpm,萃取时间为50 min,离子强度为5%的NaCl水溶液。结果:在上述条件下,马兜铃酸A的富集倍数可高达152,检测限为0.7 ng·mL^-1。样品的纯化效果显著,色谱分离良好,无基质干扰,回收率良好（97.9%）。结论:本文建立的微波–中空纤维液相微萃取法,既保证了马兜铃酸A从药材中快速提取出来,又实现了该痕量毒性成分的富集和纯化,消除了样品基质的干扰,从而满足了马兜铃酸A高灵敏、高选择性的测定要求。优化后的方法成功用于细辛中痕量毒性成分马兜铃酸A的分析,同时也为其它具有复杂基质的中药痕量成分的分析提供了可靠的样品前处理手段。三、微波连续抽提用于灵芝三萜类成分的分析目的:结合索氏提取的原理,自制了微波连续抽提装置,用于进一步提高微波萃效率。通过与传统方法的比较,研究了微波连续抽提法的特点和萃取效率,并将其用于灵芝三萜类成分的分析方法:微波连续抽提法的萃取条件为:以95%乙醇为萃取溶剂,微波功率200 W,微波时间14.5 min（5个循环）。将上述条件下的萃取结果与传统的回流提取、索氏提取、超声提取和微波萃取法进行比较。结果:微波连续抽提和常规的微波萃取分别需要14.5 min和10 min即可将灵芝三萜类成分的提取量达到最高,而其他非微波提取的方法则需要几十分钟甚至上百分钟。微波萃取10 min与回流提取120 min所得灵芝三萜的量基本一致,明显高于索氏提取和超声提取法,同时又远低于微波连续抽提法。结论:微波连续抽提有效结合了微波萃取和索氏提取的优点,因此比常规的微波萃取更能将药材中的待测成分提取完全,使分析结果更为准确,为中药的成分分析提供了一种新颖的、高效、快速、有效可靠的样品前处理技术。同时由于提高了药材利用率,也可将其用于中药成分的制备和提取工艺的研究。更多还原

【Abstract】 Quality control has played an important role in the aspect of safety and efficacy of traditional Chinese medicines （TCMs）, and it has been considered to be the the only way for TCMs heading into the international market. To successfully build the standard quality evaluation systems of TCMs, problems in the following three parts need to be solved, that is rapid and efficient sample preparation techniques as well as stable and reliable analytical techniques, scientific and rational system of quality control indicators and a rich quality of the chemical reference substances. As we know, sample pretreatment is the critical first step in the quality evaluation of TCMs, and generally, 60%⁸0% of the time is consumed in this process.At present, the commonly used methods can be divided into two main categories: soaking methods （such as percolation method and merceration） and hot extraction methods （such as decocting method, heat reflux extraction, soxhlet extraction and wet distillation）. The traditional methods mentioned above are generally time consuming, low efficiency and require bulk amount of organic solvent. Ultrasonic extraction is a relative fast, low cost and efficient alternative to the conventional extraction methods, and till now it has been extensively applied in the laboratory. However, this technique suffers from a large noise pollution and up to now it is limited in laboratory for small scale researches. Accordingly, it can not meet the requirement for the development of the modern medicine anymore. Due to the technical defect, complicated equipments or high cost, surpercritical fluid extraction and pressurized liquid extraction that raised later has suffered a limitation in their development. Thus, looking for a more simple and rapid extraction technique is of great significance for the quality control of TCMs.Microwave-assisted extraction （MAE） has emerged as an attractive alternative for sample preparation since the 80s in the last twentieth century, because it has the merits: effectiveness, rapidness, minor volume of solvents used, high selectivity and recovery, as well as good reproducibility. In the process of extraction, both of the material and extracting solvent are simultaneously heated by microwave energy, with the mechanisms caused by ionic conduction and dipole rotation. So the principle of MAE differs widely from conventional extraction, whose heating mode can be described as: heating-permeating into the basal body-dissolving or smuggling- permeating out. And then, MAE has successfully overcome these shortcomings such as time-tedious and worse selectivity. In general, several hours or even more are needed in traditional extraction methods, yet half an hour is demanded in ultrasonic extraction. In comparison, MAE only need few seconds or minutes, so the extracting speed has been increased several times to several hundred times.The purpose of our study is to adopt microwave extraction and neotype microwave-assisted extraction, which was established by using microwave extraction combinding with other sample preparation techniques, such as microwave-assisted micelle extraction, microwave extraction coupled with hollow fiber liquid phase microextraction and microwave-assisted continous extraction, for the extraction and analyses of the compotents in TCMs. Many tough problems raised by conventional methods are solved and the experiment procedures are simplified. These proposed techniques provide high efficient, rapid and simple sample preparation methods for the quality control of TCMs. Specific studies are as follows: （1） As is well known, the seeds of Psoralea Corylifolia L. are used for medicine, and then this kind of TCMs has a hard external seed coat. The principal drug of Sanhuang tablets is Radix et Rhizoma Rhei, partly of which is crushed and directly used for preparations. Especially, the analytes are all have a high liposolubility and a low solubility. Therefor, it is quite difficult for extracting the analytes out from the medicial materials, always taking a tediously long time. In this experiment, we fully utilized the character of microwave including fast heating speed and powerful cell wall broken ability, the extraction was therefore finished within 5 minutes and gave a comparable or even higher yield of the analytes than the classical method. The proposed method was high efficient, simple and rapid, and thus can be served as an alternative for the analysis of TCMs that difficult for extracted. （2） Brucine, strychnine and naringin are all the index components of Pingxiao tablets, nevertheless, there is a significant distinction in the polarity and solubility. Consequently a solo organic solvent can not allow the simultaneous extraction of the three index components. That therefore brought some difficulties to the quality control of Pingxiao tablets. In this paper, a microwave-assisted micell extraction was built, aqueous solution of Brij 35 was used as the extracting solvent in order to permit a good extraction result for all of the three analytes. The proposed method was convient, rapid and environmental friendly. （3） Herba Asari has attracted considerable attention for its toxical ingredient aristolochic acid A. However, the matrix of the herbal is complicated and thus brings some difficulties for the separation of HPLC, above all the amount of aristolochic acid A in Herba Asari is trace, and then it is necessary to have a preconcentration and clean-up step before the determination. A pretreatment technique of microwave extraction coupled with hollow fiber liquid microextraction was estabilised in the test, with the merits expressed as short extracting time, rapid analyzing speed, high enrichment factor （152） and effective purification, and thus it provided a new pretreatment means for the analysis of aristolochic acid A in Herba Asari. （4） A neotype microwave-assisted continuous extraction was developed based on the principals of soxhlet extraction, with the purpose to further improve the extraction efficiency of conventional microwave extraction, afterwars it was applied in the analyses of triterpenoids in Ganoderma lucidum. This technique has sufficiently combine the fortes of microwave extraction and soxhlet extraction, which can be summarized as high efficiency and potential to continuously supplying fresh solvent to the sample, respectively. Accordingly, compared with the conventional microwave extraction, the proposed method had a definite ablity to permit a complete extraction of the analytes from TCMs, and a more accurate analytic result was therefore obtained. It has provided a neotype, high efficient, rapid and reliable sample preparation technique for the compotent analysis of TCMs.PART 1 Application of Microwave Extraction in the Analysis of Ingredients of Traditional Chinese Medicines（1） Rapid analysis of psoralen and isopsoralen in Psoralea Corylifolia L. by microwave extractionObjective: To establish a high efficient, convenient and rapid method of microwave extraction （ME） by utilizing a home-made open microwave extraction device, with the purpose of studying the extraction effectiveness of the target compoents of TCMs with hard skin, and finally applied to the analysis of psoralen and isopsoralen from Psoralea corylifolia L..Methods: Several single-factor tests were carried out to investigate the effect of 4 influencial factors （ie. extraction solvent, microwave power, microwave extraction time and the ratio of solvent to material） on the extraction rate of psoralen and isopsoralen, and then the optimal extraction conditions were selected out. Comparison between microwave extraction and other traditional extraction methods were made based on the yields of psoralen and isopsoralen.Results: The optimal parameters of ME can be concluded as follows: 3 min, the ratio of solvent to material was 400 (mL·g^-1) by using methanol as the solvent. Compared with soxhlet extraction, heat reflux extraction, and ultrasound-assisted extraction, MAE only expended 3 min to give the highest yield of psoralen and isopsoralen, while the other extraction methods consumed several hours and gave a lower yield.Conclusion: The proposed method based on a home-made open microwave extraction device has greatly shortened the extraction time and improved the extraction yield, so it provides a high efficient, simple and rapid measure for the analysis of those TCMs with hard skin.（2） Rapid analysis of the 5 anthraquinones in Sanhuang tablets by microwave extractionObjective: To bulid a high efficient, convenient and rapid method of microwave extraction with the purpose of studying the extraction effectiveness of the slightly soluble target compoents of TCMs, and simultaneously optimizing the extracting conditions.Methods: The microwave extraction parameters namely microwave extraction solvent, microwave power, extraction time and the ratio of solvent to material were optimized based on the extraction yield of the free and combined anthraquinones. Under the optimal ME conditions, a comparison with the classical method was made.Results: The optimal microwave extraction conditions obtained were: extraction solvent of 80% ethanol, microwave power of 300 W, liquid/ solid of 100:1 (mL·g^-1), extraction time of 2 min for the free anthraquinones and 5 min for the combined anthraquinones. The extraction of 5 anthraquinones was finished within 5 minutes by using the proposed method, while 60 minutes was needed in the classical heat reflux extraction for giving a comparative extraction yield of the analytes.Conclusion: The proposed method has been proved to be high efficient, simple and rapid. It has successfully solved the difficulty that the extraction of slightly soluble components of TCMs generally need a long time, so it offers an effective and reliable pretreatment means for the analysis of Chinese medical meterials as well as Chinese drugs praeparatums. PART 2 Research on the Neotype Microwave-assisted Extraction methods in Sample Preparation for Traditional Chinese Medicines（1） Simultaneous analysis of brucine, strychnine and naringin in Pingxiao tablets by microwave-assisted miceller extractionObjective: To establish a high efficient, rapid and environmental friendly methodology of microwave-assisted miceller extraction （MAME） using a surfactant as extractant. The performance and application of the proposed method for simultaneously extracting multi-index components with completely different characters of TCMs has been investigated. Meanwhile, a HPLC method is established here for the simultaneous determination of brucine, strychnine and naringin in Pingxiao tablets.Methods: Taking aqueous of 7% Brij 35 as extraction solvent, and under the condition of microwave power as 300 W, ratio of liquid/solid as 100:1(mL·g^-1), the extraction was finished within 2 miniutes. Simultaneously, comparison between the proposed method and the traditional means was carried out in the following experiment. The analytes were eluted with a gradient mode and the mobile phase was composed of acetonitrile and 0.4%phosphoric acid （triethylamine adjusted the pH to 3.0）, meanwhile the detection wavelength was 260 nm.Results: In comparison with the classical heat reflux extraction using alkaline chlorlform and the traditional ultrasonic extraction using methanol, MAME had significantly shortened the extraction time （120 min: 2 min, 30 min: 2 min）, and showed a comparable yields for all of the three index components. Brucine, strychnine and naringin separately showed a good linear relationship at range of 0.630⁴0.7μg·mL^-1, 0.640⁴1.3μg·mL^-1 and 9.06⁵80μg·mL^-1 with the average recovery from 99.7% to 101.1%, and the precision （RSD, n=5） was found to vary from 0.7% to 1.1%.Conclusion: The proposed method of MAME has been proved to be high efficient, convenient, rapid, environmental friendly as well as capable of simultaneously extracting multi-index components of different characters, so it provids a neotype and dependable pretreatment means for the analysis of multi-index components in TCMs.（2） Rapid analysis of toxical and trace amount of aristolochic acid A in Herba Asari using a novel microwave-assisted extraction combined with hollow fiber liquid microextraction techniqueObjective: To develop a rapid and sensitive method （MAE-HF-LPME） using microwave-assisted extraction as the extraction means coupled with hollow fibre liquid microextraction for enrichment and purification. The performance of the proposed method for extracting trace amount of analytes from complex matrices of TCMs has been investigated by utilizing it in the analysis of aristolochic acid A of Herba Asari.Methods: In the experiment, MAE was used for the extraction of aristolochic acid A from 1.0 g Herba Asari powder using 25 mL 70% methanol as the solvent at 400 W for 6 minutes. The extract was subsequently subjectecd to a single step HF-LPME clean-up and enrichment procedure, with the conditions performed as follows: an organic solvent of 1-octanol, a donor phase of aquesous HCl at pH 2.5, an acceptor phase of aquepus NaOH at pH 12.0, a stirring rate of 1200 rpm, extraction time of 50 min and a salt concentration of 5% NaCl.Results: Under these conditions, enrichment factor up to ca. 152-fold was obtained depending on the target analyte. Limit of detection was 0.7 ng·mL^-1. The proposed method provided a notable clean-up, a good separation and a satisfactory recovery as 97.9%.Conclusion: In the proposed method, MAE allowed aristolochic acid A to be rapidly extracted from the herbs, and then the obtained sample was enriched and cleaned via a HF-LPME procedure to directly meet the analysis requirements of aristolochic acid A with sensitivity and selectivity. The optimized technique was successfully applied to the analysis of toxical and trace amount of aristolochic acid A in Herba Asari, and it could potentially be extended to other traditional Chinese medicines.（3） Analysis of triterpenoids in Ganoderma lucidum by microwave-assisted continuous extractionObjective: For further improving the extraction efficiency of microwave extraction, a microwave-assisted continuous extraction （MACE） devic has been designed and utilized. By contrasting with the traditional methods, the characteristics and extraction efficiency of MACE has also been studied. The method is validated by the analysis of the triterpenoids in Ganoderma lucidum.Methods: The extraction conditions of MACE were: using 95% ethanol as solvent, microwave power 200 W and radiation time 14.5min （5 cycles）. The extraction results were subsequently compared with traditional heat reflux extraction （HRE）, soxhlet extraction （SE）, ultrasonic extraction （UE） as well as the conventional microwave extraction （ME）.Results: For triterpenoids, the two methods based on the microwaves （ME and MACE） were in general capable of finishing the extraction in 10 min and 14.5 min, respectively, while other methods should consume 60 min and even more than 100 min. Additionally, ME can produce comparable extraction results as the classical HRE and higher extraction yield than both SE and UE, however, notably lower extraction yield than MASE.Conclusion: MACE can effectively combine the advantages of microwave extraction and soxhlet extraction, thus enabling a more complet extraction of the analytes of TCMs in comparison with ME. And therefore makes the analytic result more accurate. It provides a novle, high efficient, rapid and reliable pretreatment technique for the analysis of TCMs, and it could potentially be extended to ingredient prepareation or extracting techniques of TCMs.更多还原