| Traditional Chinese Medicine (TCM) is a treasure in China. With five thousands years of history, TCM has played an important role in the aspects of treatment and prevention of disease, tonification, healthcare and so on. However, some difficult problems including the separation of active ingredients and quality control of TCM have been existed in the course of its development. Therefore, it is necessary to establish a new TCM extraction and quality control technique method with simple, rapid, high accuracy and sensitivity characterizes, thus contributing to its pharmacological research, clinical applications and international development.Dysosma versipellis, Sinopodophyllum hexandrum and Diphylleia sinensis are TCMs in podophyllum, they exhibit of a lot of activities, such as detoxification, anti-inflammatory analgesic, dissipating phlegm and resolving masses, removing blood stasis and detumescence, et al. As a mutual bioactive ingredient in the three herbs, podophyllotoxin played an important role in the medical field. It is not only a good drug to treat genital warts, but also a precursor to synthesize a variety of anti-cancer drugs. In this paper, podophyllotoxin was selected as the object of research, it was extracted from the podophyllum plants via modern new sample preparation techniques. Selective separation and extraction technique of podophyllotoxin has also been investigated. With the purpose of providing reference to the quality control of TCMs, a two-dimensional fingerprint of D. versipellis medicine has been established. Details are as follows:1. Application of ionic liquids in the microwave-assisted extraction of podophyllotoxin from Chinese herbal medicineA micelle-mediated extraction technique, i.e. ionic liquid-based microwave-assisted extraction (ILs-MAE) technique has been developed for the extraction of podophyllotoxin from D. versipellis, S. hexandrum and D. sinensis. Several operating parameters were successively optimized by single-factor and L9 (34) orthogonal array experiments. The highest extraction yields of podophyllotoxin in these three TCMs were 96.8%, 89.1% and 82.4%, respectively. Compared with other extraction techniques, such as ionic liquids-based maceration extraction (ILs-ME), heat extraction (ILs-HE) and ultrasound-assisted extraction (ILs-UAE), the ILs-MAE technique not only took a shorter time but also afforded a higher extraction rate of podophyllotoxin. Reversed phase high performance liquid chromatography was employed for the analysis of podophyllotoxin. The results showed that the linearity for analyzing podophyllotoxin in the three herbs was in the concentration range of 0.005-0.4 mg/mL with the correlation coefficients between 0.9993 and 0.9996. LODs were 2.05-2.58 mg/mL. The recoveries were in the range of 97.1-102% and all RSDs were lower than 3.0%. Repeatability and intermediate precision were both satisfactory. Furthermore, the mechanism of ILs-MAE was preliminarily studied by means of kinetic mechanism, surface structures and chemical compositions of samples before and after different extraction techniques. Employing this method to analyze real samples, it could be found that the contents of podophyllotoxin in ten samples were quite distinct due to their different origins.2. Molecularly imprinted polymers-solid phase extraction (MISPE) method for the selective enrichment of podophyllotoxin from traditional Chinese medicinesMicrowave heating initiated precipitation polymerization was developed to prepare podophyllotoxin molecularly imprinted polymers (MIPs), and the synthesized parameters were respectively optimized. Using podophyllotoxin as the template, acrylamide as the functional monomer; ethylene glycol dimethacrylate and divinylbenzene as the bi-crosslinker (with mole ratio of 1:6:40); acetonitrile as the porogen; 0.01 mol/L azobisisobutyronitrile as the initiator, the polymer with optimal adsorption property was obtained by synthesizing for 150 min at 60℃. The recognition mechanism of MIPs was studied by spectroscopy analysis. FTIR, SEM and thermal analysis were used to characterize the MIPs. Equilibrium rebinding experiments, competitive adsorption experiments and Scatchard analysis combined with HPLC detection technology were employed to evaluate their adsorption properties. Using the prepared MIPs as the SPE sorbent, podophyllum samples were analyzed. The selectivity coefficients of the MIPs were respectively 16.93 and 5.60 for arctigenin and 4'-demethylepipodophyllotoxin. LODs of podophyllotoxin in D. versipellis, S. hexandrum and D. sinensis were 0.15, 0.12 and 0.18μg/mL. The recoveries were 91.1%, 89.5% and 90.6% (n=3). All these results showed that this study could provide a new approach for efficient separation and enrichment of podophyllotoxin from TCMs.3. Establishment of two-dimensional fingerprint profile of D. versipellisIn the study, two-dimensional fingerprint of D. versipellis including chromatography and spectrography data was established. In comparison with single-dimensional fingerprint at a fixed wavelength, it compiled more information data and could be more comprehensive and objective to control the quality of TCMs. The respective chromatography and mutual mode of 10 baches of herbs were evaluated by the software"SOP of Similarity evaluation system for chromatographic fingerprint of TCM". Results showed that these medicinal samples originated from different places were divided into three clusters by their correlation coefficients, which were 0.9674-0.9814, 0.8725-0.8878 and 0.5506, respectively. Thus, it could be preliminarily judged that D. versipellis from the south was superior to that from the North. Cluster analysis and principal component analysis of samples were processed by Matlab 7.0, and the obtained results were in accordance with similarity evaluation. Instrumental precision, methodological repeatability and sample stability were both satisfactory. These methods provided scientific proof for evaluating origins and qualities of D. versipellis. |
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