| The active constituents screening and quality control of traditional Chinese medicine (TCM) has been the focus of the research of TCM, and it is also a key problem in the development of the modernization to TCM. In order to find out the pharmacodynamic material accurately and quickly, and to better control the quality of TCM, a new strategy based on component knock-out/knock-in was introduced in this paper to study the pharmacodynamic material screening and the quality control of TCM. A quality evaluation model of Rhizoma coptidis (R. Coptidis) for antibacterial (Shigella dysenteriae, S. dysenteriae) active constituents recognition and quality control was built.The main research contents and results are as follows:(1) There’s ahuge difference in the content of alkaloids from different source and different varieties of R. coptidis.The contents of the main alkaloids from different source and different varieties of R. coptidis were analysed by high performance liquid chromatography (HPLC). There was a significant difference among the contents of each alkaloid from the samples of R. coptidis, and there was also be obvious different in the contents of total alkaloids of different varieties of R. coptidis.(2) It will contribute to identifying the active constituents using chromatographic fingerprintings (CFs) combined with biological fingerprintings (BFs).The chemical information of different concentrations of the multi-source R. coptidis was determined by HPLC. The inhibitory effect of those samples on S. dysenteria growth has been investigated by microcalorimetry. The results of BFs showed that the antibacterial activity sequence of R. coptidis of different sources was Coptis teeta Wall.(CT)> Coptis chinensis Franch.(CC)> Coptis deltoidea C.Y. Cheng et Hsiao (CD). The "spectrum-activity" relationships between CFs and BFs were investigated using the canonical correlation analysis (CCA). It revealed that there are a close correlation between the spectrum-activity relationships and several of the major anti-bacterial components in the CFs, including Jatrorrhizine (Jat), Coptisine (Cop), Palmatine (Pal), and Berberine (Ber). The results of the current study are consistent with the index components stipulated in China PharmaCopoeia.(3) Identifying the key antibacterial active constituents of R. coptidis through the composition knock out strategyThe samples of R. coptidis was analysed and separated by using thin layer chromatography (TLC). Target compositions which were determined to knock out were Columbamine (Col), Jat, Epiberberine (Epi), Ber, Pal, Cop, respectively. In order to evaluate the thoroughness of "knock out" experiment and identify the target compositions before and after the "knock out" experiment is changed or not, we adopted ultra high performance liquid chromatography (UPLC)-mass spectrometry (MS) to identify "knock out" compositions and their negative samples. The result showed that, each target component was well separated, and there was no residue in the negative samples, it showed "knock out" of target component was proceed thoroughly and the method of "knock out" was high efficiency. And because of the ion fragments of each target composition are same before and after that the TLC separation, it means that TLC separation did not destroy the chemical structure of the target component.The inhibitory effect of each target component which was obtained in the knock out experiment on S. dysenteria growth has been investigated by microcalorimetry. The antibacterial activity sequence of target components was Cop> Ber> Epi> Jat+Col> Pal> Non-alkaloids. Comparing the inhibitory effect of the positive components, the negative components and the extract of R. coptidis on S. dysenteria growth, we can find that, there are some the inhibitory activities on S. dysenteria growth in six kinds of alkaloids from in natural ratio of extract of R. coptidis; The inhibitory effects of Ber and Cop were stronger than the other four alkaloids, and Ber> Cop. So we can make Ber and Cop as the key investigation object in the next study, and then study the interaction relationship between Ber, Cop and other alkaloids. The results showed that, in0.8mg/mL extract of R. coptidis (1) some constituents with antagonistic effect may exist in R. coptidis,(2) there was synergy effect between Ber and Cop,(3) there was some additive effect between (Ber+Cop) and other alkaloids.(4) Setting the quality control standard of R. coptidis through the composition knock in strategy.Different concentrations of Ber or Cop were gradually added into the negative solutions of R. coptidis with a lack of corresponding active constituent as the knock-in samples. The chemical information and biological activity of these samples were evaluated by HPLC and microcalorimetry. Then we set up the corresponding "quantity-effect" relation curve to establish preliminarily the percentage content of key active constituent Ber and Cop:8.08%31.92%and8.08%-14.45%.(5) Determining the best dose proportion of Ber and Cop to provide a new way of thinking and method for the development of multiple-component Chinese medicine of R. coptidis.The protocol was designed with checkerboard method. The inhibitory effect of different ratio of Ber and Cop on S. dysenteria growth has been investigated by the fluorescence absorption method. We found out the best dose ratio of Cop and Ber is2:1, and better dose ratios of Cop and Ber are4:1,1:2,1:4.In summary, a quality evaluation strategy for active constituents recognition and quality control of R. Coptidis by constituent knock-out/knock-in strategy is set up preliminary. And we find out the active constituents of R. Coptidis and set their content standards and the best dose proportion in this paper. These results will offer some useful references for the further development of quality control of R. Coptidis and also provide the ideas and methods for the research of active constituents recognition and quality control of TCM. |
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