| 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.
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