| With the rapid development of natural medicine research over the world, it is an important goal of TCM modernization to develop traditional Chinese medicine compounds into a highly efficient, rapidly available, low toxicity of modern pharmaceutical preparations. It can be realized by clarifying the pharmacodynamic effects, improving the internal quality, stable and reliable in quality, and improving the preparation process. Extraction and separation of high purity active ingredients or monomer is a premise and key technology of modern TCM compound preparations. Macroporous resin, as an important extraction technology, has been widely used in modern TCM industrial production, and has played an important role in enrichment of active ingredients. Compared with other traditional extraction technology, it has obvious advantages, such as easy operation, high extraction efficiency, lower operation expense, and environment friendliness. It applied in the natural plant extract more and more widely, and even replaced the traditional extraction technology. For example, for Ginkgo biloba flavonoids extract production, macroporous resins have completely displaced solvent extraction developed abroad, and to achieve better results.However, there also exist some serious problems in macroporous resin technology. The matrix of resins applied in industrial production was usually of hydrophobic polystyrene, and their absorption mechanism primarily relied on the hydrophobic force in the aqueous solution, which may lead to the low adsorption selectivity. In the TCM compounds, the active ingredients and impurities of each herb are extraordinarily different. Thus, high selective resin is necessary to obtain high purity extract of each active ingredient. Higher requirements was put forward in designing and adjusting adsorbent structure, that is, how to develop synergistic effect of various weak interactions by the structure and functional group of resins, to prepare the resins with high selectivity to specific active ingredients, according to molecular structures of active ingredients and the expected separation effects. In addition, it is necessary to consider many factors, such as the environment where active ingredients are being located, the interference of impurities on the separation process, even whether synthetic resin can be used in large-scale industrial production.In this paper, the anti-cancer TCM compound of Bai Ban Tang was selected as the research object. According to the structure and properties of anti-tumor active components in the TCM compound, that is flavonoids in Scutellaria barbata and triterpenoid acids in Hedyotis diffusa, the macroporous resins with different structure and function were designed and synthesized to greatly enhance their selectivity. On the other hand, polymeric adsorbents based on the synergistic effect of the hydrophobic and dipole interactions were synthesized to separate the individual flavonoid components in Scutellaria barbata. And then, six extracts of two herbs were screened for anti-cancer test in vitro. Furthemore, the growth inhibitory effects were evaluated for the compatibility of the best efficacy extracts in two herbs.First, in order to avoid some hydrophobic contribution of polystyrene in the resin matrix, a commonly used crosslinking agent divinylbenzene (DVB) was changed to ethylene glycol dimethacrylate (EGDMA). A series of resins with weak hydrophobic of polyacrylate matrix were synthesized, based on the copolymerization of methyl acrylate (MA) and EDGMA. And specific functional group was introduced onto adsorbents to produce hydrogen bonding to phenolic hydroxyl groups in flavonoids molecules. The relationship among hydrophobicity, functional groups and adsorption selectivity to flavonoids of the resins was investigated. The results showed that adsorbent ME-3B with 15% EGDMA content and amide group performed the most appropriate power to purify flavonoids. FTIR spectroscopy and adsorption thermodynamics analysis confirmed the existence of hydrogen bonding, which played an important role in adsorption process of flavonoids. The universality of the method could find its full expression via its good effects in the purification of total flavonoids from Ginkgo biloba L, Radix puerariae, and Hypericum perforation L. The optimal process of extracting total flavonoids in Scutellaria barbata by adsorbent ME-3B, was determined through ptimizing the conditions of adsorption and desorption. And its repeatability and stability was tested. In the final extract, the content of total flavonoids is about 56%, much better than purification effect by commercial adsorbent.After extraction of total flavonoids, a series of methyl acrylate-co-divinylbenzene (MA-co-DVB) macroporous resins were synthesized, based on the synergistic effect of the hydrophobic and dipole interactions. The resins were applied to separate the individual flavonoid components with similar structure by adjusting adsorbents’polarity with MA content. The influence of the ratio of DVB and MA on the adsorption selectivity was investigated. The results showed that only under the appropriate hydrophobic condition, hydrophobic and dipole interaction could play the synergistic effect. Thus, the affinity differences between adsorbents and adsorbates appeared. Because the contribution of dipole interaction on the adsorption would greatly overshadowed by the high hydrophobicity, but it couldn’t be expressed due to extremely weak hydrophobicity. It was related to strong interference of water molecules on dipole interaction when adsorption occurred in aqueous solution. The separation process of individual flavone compound was designed by investigating the influence of MA content of adsorbents on the adsorption and desorption of the four flavonoids. Firstly, adsorbent MD-4 coupled with the method of gradient elution separated the four flavonoids into three fractions, namely, scutellarin, scutellarein and the mixture of luteolin and apigenin. Afterwards, the mixture solution of luteolin and apigenin was separated by adsorbent MD-2 completely. The optimal process of four individual flavonoids in Scutellaria barbata separated by adsorbent MD-2 and MD-4, was determined by optimizing the conditions of adsorption and desorption. In the final four extracts, four flavonoids achieved complete separation, and had good recoveries.According to chemical structure of triterpenoid acids in Hedyotis diffusa, a series of macroporous resins with polystyrene matrix and amino groups were synthesized based on adjusting type and content of amino group. The relationship among crosslink degree, type and content of amino groups and adsorption selectivity to triterpenoid acids of the resins was investigated. The results showed that adsorbent R-3 with 10% crosslink degree and dimethylamine group performed the most appropriate power to purify triterpenoid acids. FTIR spectroscopy confirmed the existence of static electricity. Selective desorption test showed high concentrations of ethanol could elute a large number of impurities, and whether high concentrations of ethanol or 10% acetic acid aqueous solution could not elute triterpene acid, only 80% ethanol solution contained 10% acetic acid desorbed the triterpene acids completely. This results not only proved the adsorption mechanism of the triterpenoid acid and impurities on the adsorbents is completely different, that is, the adsorption of impurities on resin is mainly hydrophobic adsorption, while the adsorption of the triterpene acids are based on a common result of hydrophobic and acid-base interaction, also provided the condition to obtain high purity extract by step elution. For this very reason, we further studied the elution effect by different concentration ethanol aqueous in order to enhance the purity of triterpene acids in final production. The optimal process of triterpene acids in Hedyotis diffusa extracted by adsorbent R-3 was determined by optimizing the conditions of adsorption and desorption. And its repeatability and stability was tested. In the final extract, the content of triterpene acids is above 90%, much better than purification effect by commercial adsorbent.Finally, the anti-cancer effect of above six extracts in vitro was studied by MTT and SRB assay. All extracts expressed cytotoxic effect on human hepatocellular carcinoma A549 and human colon carcinoma LOVO in vitro, and showed a certain dose-effect relationship. By comparing IC50 value, we found that the cytotoxic effect apigenin extract from Scutellaria barbata and triterpene acids extract from Hedyotis diffusa against cancer cell line was best. We further studied the compatibility extracts’ cytotoxic effect against cancer cell line. The compatibility extracts were composed of total flavonoids, apignin from Scutellaria barbata and triterpene acids from Hedyotis diffusa, respectively. The result showed that cytotoxic effect of apigenin and triterpene acids compatibility extract was superior to individual extracts, and proved the synergistic effect between the anti-cancer active ingredients in two herbs. The IC50 value of apigenin and triterpene acids compatibility extract on A549 and LOVO was 49.54μg/mL and 34.48μg/mL, the cytotoxic effect was the best. It had a potential value to be developed into novel anti-cancer agent. |
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