| The quality of Traditional Chinese medicine (TCM) has been a major problem in our country pharmaceutical industry, and the quality will directly affect its clinical efficacy. So how to control the quality of TCM more objectively and accurately, how to identify active components of TCM, establishing a scientific and feasible quality assessment method is the key issue in TCM development process. In this paper, under the guidance of traditional Chinese medicine theory and modern analytical methods, Angelica for the study, adopting multi-level IR macro-fingerprints analysis and multi-component pharmaceutical analysis, exploring the overall infrared spectral characterization and the trends in partial metabolism, screening therapeutic basis for Angelica quality evaluation gradually, so as to provide new ideas and methods for the studies of TCM quality evaluation. The major experimental contents and achievements are as follows:1. IR characterization of Angelica therapeutic basis. Fourier transform-infrared spectroscopy (FT-IR) and the second derivative infrared spectroscopy (SD-IR) were applied to analyze and identify Angelica raw materials, the volatile oil and water-soluble components, observing the macro molecular structure information of Chinese Angelica. The result showed that Angelica contained large amounts of sucrose. Ether extraction of the distillate mainly contained ester components, while petroleum ether extract mainly contained lactones ingredients such as ligusticum. And the main components of water extraction of the distillation residue were polysaccharides. Compared to direct water extraction, the process optimization of water extraction of the distillation residue could get more liposoluble constituents, but also get more Angelica polysaccharides and other water-soluble constituents. This method could widen the scope of medication and avoid the waste of herbs. The absorption peak was less obvious in water extraction and ethanol extraction of Angelica, so their main components were polysaccharides. Residue after ethanol extraction mainly contained matrix components such as starch, cellulose and protein. The major components in Angelica can be identified first by infrared spectroscopy. By extraction of raw materials in different ways, the chemical compositions become relatively concentrated, contributing to determine the specific components contained in Angelica, to establish a quality evaluation system of Angelica based on IR.2. IR characterization of Angelica aqueous extract. In this study, the method of "multi-level IR macro-fingerprint" was adopted to solve infrared spectral analysis problem of the complex systems of TCM. The macroscopic IR fingerprint method including Fourier transform infrared spectroscopy (FT-IR), the second derivative infrared spectroscopy (SD-IR) and two-dimensional correlation infrared spectroscopy (2D-IR) can enhance the resolution to achieve the purpose of parsing complex systems. Through studying the decoction and different concentrations of ethanol eluted production of AB-8 macroporous resin. FT-IR spectrum indicated that Angelica raw material was rich in sucrose and the correlation coefficient was 0.8465. The decoction of Angelica contained varieties of polysaccharides components and the content was gradually decreased with increasing concentration of ethanol. In addition, the decoction of Angelica contained a certain amount of protein components and 50% ethanol eluate had more protein than other eluates. Their second derivative spectra amplified the differences and revealed the potentially characteristic IR absorption bands, and we concluded that the decoction of Angelica might contain a certain amount of ferulic acid and ligustilide. The characteristic absorption peak of ferulic acid can be obviously observed in the high concentration ethanol eluted production, and 30% ethanol eluate,50% ethanol eluate and 70% ethanol eluate were similar to ligustilide. In 2D-IR spectra, we could clearly observe that all ethanol eluates have two obvious auto-peaks and they form 2*2 peak cluster. They were respectively assigned to amide II and amide I bands of protein.3. Selection of the quality control ingredients of Angelica preliminarily. To investigate metabolism of the chemical constituents of Angelica aqueous extract, everted rat gut sacs was carried out as well as the blood was taken from abdominal aorta, and all samples were analyzed by HPLC. There were 23 components absorbed by intestinal wall of rat, including 19 components which were absorbed in prototype forms directly, in which three components were identified as ferulic acid, senkyunolide I, senkyunolide H by comparing with the standards. And four components were new ones from intestinal wall metabolism. On the other hand, six components could be absorbed into blood through analysis serum samples obtaining from abdominal aorta of rats. Four of them could be absorbed directly by intestine, while two components in new form were absorbed into blood. The content of one of prototype components was increased in the blood, which showed that it might be the metabolites of intestine and liver. Based on the intestinal wall absorption experiment and analyzing the components in blood, we were able to preliminarily select the three prototype components as the quality control ingredients of Angelica.4. Sequential metabolism in rat of multicomponent in Angelica. In vitro incubation, in situ closed-loop methods and in vivo blood sampling were used to build the whole process. The artificial digestive juice was prepared and the stability of components was determined at 37 ℃. In situ closed-loop methods and by analyzing HPLC fingerprints of intestinal metabolism and liver metabolism, we could infer multicomponent changes of Angelica aqueous extract in the Sequential metabolic process. The incubation experiment showed that totally 15 components were basically stable in the digestive juice, four of them which might be metabolized by intestine, seven of them were metabolized by liver. And we found that one new component were the metabolites of liver, seven new components were the metabolites of intestine, but five of which were metabolized by liver and could not be absorbed into blood. Finally, we identified that of four prototype components of Angelica water extract and three metabolites could be absorbed into blood. Based on of the multi-component metabolic profiling of Angelica, we initially selected four prototype components including senkyunolide I as the quality control components, and we finally established a quality evaluation method based on components metabolism.To sum up, IR macroscopic characterization showed that Angelica contained polysaccharides and protein. The decoction of Angelica contained large amounts of polysaccharides, but also contains a certain amount of ester components and a small amount of ferulic acid. By studying the trends of partial multi-component, we were able to preliminarily select the four quality control components in vitro experiments and then select four quality control components in vivo experiments. Finally, we determined three kinds of the same components can be used as the standard component for quality control of Angelica, one of which was identified as senkyunolide I. In this paper, from macroscopic characterization to the trace components, from the overall structure to specific component, combined infrared spectroscopy and high performance liquid chromatography, based on the multi-component metabolic profiling in vivo and in vitro, we can screen therapeutic basis for Angelica. This method provided a new way of thinking for evaluation and quality control of TCM. |
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