高选择性吸附树脂结构设计及在中药复方活性成分提取中的应用
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摘要
随着天然药物研究的迅速发展,将传统的中药复方以现代科技阐明药效作用,完善内在质量,强化可控、稳定,提高制剂工艺,研制成高效、速效、低毒的现代中药复方制剂成为中国中药现代化的重要目标。高纯度中药有效成分的提取或有效成分中单体组分的分离是现代中药复方制剂研究的前提与关键技术。大孔吸附树脂分离技术广泛应用于现代中药的工业生产中,起到了去粗取精、富集有效成分的作用,相较于其他传统的提取技术,它具有操作工艺简单、环境友好、提取效率高、生产成本低等明显的技术优势,在天然植物提取中应用越来越广泛,甚至替代了传统提取技术,例如在银杏黄酮提取物的生产中,吸附树脂分离工艺已完全替代了国外开发的溶剂萃取工艺,并得到更好的提取效果。
但是,吸附分离技术也存在着亟待解决的问题,主要体现在目前工业生产中使用的树脂多为聚苯乙烯型骨架,因此吸附机理比较单一,主要以疏水性作用力与吸附质分子发生相互作用,在水溶液中的吸附过程,疏水性作用力是非特异性的吸附结合力,这就势必造成对树脂有效成分的吸附选择性较差。在复方中药中,每一味药材的有效成分、甚至杂质组成都不相同,为了得到每一种有效成分的高纯度提取物,就需要高选择性的吸附树脂,这就给我们设计和调控树脂结构提出了更高的要求,即如何根据有效成分的分子结构和要达到的分离结果,综合考虑有效成分所处的环境、杂质成分对分离的干扰、甚至所合成的树脂能否真正用于大规模的工业化生产等等因素,调控树脂的骨架结构和功能基,将多种弱相互作用的协同效应引入树脂结构的设计和调控过程中,合成一类针对特定有效成分具有高选择性的吸附树脂。
本论文选择了抗肿瘤中药复方白半汤为研究对象,针对方剂中两味药材中抗肿瘤的有效成分,即半枝莲中的黄酮成分和白花蛇舌草中的三萜酸成分的结构和性质,分别设计合成不同结构、功能的大孔吸附树脂,大大提高对两味药材中有效成分的选择性,从而较大程度地提高产品纯度。另一方面,通过对大孔吸附树脂极性的精细调节实现了半枝莲中四种单体黄酮的完全分离。最后,在各提取物的体外抗肿瘤活性筛选的基础上,将两位药材中药效最佳的成分进行配伍,进一步研究其在体外对肿瘤细胞的抑制作用。
首先,为了避免树脂骨架中聚苯乙烯部分对疏水性的贡献,我们改变了合成吸附树脂常用的交联剂二乙烯苯(DVB),合成了一系列以双甲基丙烯酸乙二醇酯(EGDMA)为交联剂、丙烯酸酯(MA)为共聚单体的弱疏水性聚丙烯酸酯骨架的吸附树脂,并在树脂骨架上引入能与黄酮分子的酚羟基发生氢键作用的特殊功能基团,研究了不同疏水性、不同功能基对树脂吸附性能和吸附选择性的影响,发现EGDMA含量为15%的酰胺树脂ME-3B可充分发挥疏水作用力和氢键之间的协同效应,对黄酮的选择性最佳。红外光谱和吸附热力学的研究证实了氢键作用的存在,并且在弱疏水性的酰胺树脂中氢键作用在吸附黄酮过程中起了很重要的作用。为了验证ME-3B树脂对黄酮分子的选择性,我们将其用于多种植物体系中,如葛根、银杏和贯叶连翘,纯化效果均显著优于商品化的大孔吸附树脂,证明了这种弱疏水性骨架的酰胺树脂在天然植物中黄酮类有效成分的提取具有普适性,可开发一类黄酮提取的专用树脂。通过吸附、解吸条件的优化,确定了ME-3B树脂纯化半枝莲总黄酮的最佳工艺流程,且检验了工艺的可重复性和稳定性。最终产品中,总黄酮含量可达56%左右,大大优于商品化树脂的纯化效果。
在分离得到总黄酮的基础上,基于疏水和偶极的协同作用,设计合成了一系列MA和DVB共聚的树脂,通过MA含量变化调节树脂的极性,将其用于半枝莲总黄酮中结构极为相近的四种单体黄酮,即野黄芩苷、野黄芩素、木犀草素和芹菜素。考察了树脂骨架中DVB和MA的比例对吸附选择性的影响,发现只有在适宜的疏水作用力范围内,疏水和偶极作用才能发挥协同效应,使得树脂对不同极性的吸附质吸附结合力的差别得以体现,过高的疏水性将大大掩盖了偶极作用对吸附的贡献,而过低的疏水性又无法帮助偶极作用发挥出来,这与水溶液中吸附时强极性的水分子对吸附剂和吸附质之间的偶极作用产生了强烈的干扰有关。通过疏水部分和弱极性部分比例的调控,考察了不同MA含量变化的吸附树脂对四种黄酮吸附解吸性能的影响,设计了半枝莲黄酮的分离过程。首先MA含量为45%的MD-4树脂可将半枝莲四种黄酮分为三个部分,即野黄芩苷、野黄芩素、木犀草素和芹菜素的混合物。然后再将木犀草素和芹菜素的混合物通过MA含量为25%的MD-2树脂,只需简单的吸附-解吸即可实现木犀草素和芹菜素的分离。根据这样的设计,通过优化影响两种树脂吸附解吸性能的条件,建立了半枝莲单体黄酮的制备分离工艺,实现了四种黄酮的完全分离。
针对白花蛇舌草中三萜酸类有效成分的结构特点,合成了一系列聚苯乙烯骨架、带有胺基的大孔吸附树脂,研究了树脂的交联度、胺基含量和胺基结构变化对三萜酸的吸附性能的影响。结果表明交联度为10%、引入二甲胺的R-3树脂对三萜酸的吸附选择性最佳。红外光谱的研究证明了静电力作用的存在。通过选择性解吸实验,我们发现高浓度乙醇水溶液能够解吸大量杂质,而无论是高浓度乙醇水溶液还是含有10%醋酸的水溶液都无法将三萜酸解吸,只有含10%醋酸的80%乙醇水溶液才能将三萜酸完全解吸,这不仅间接证明了树脂对三萜酸和杂质成分的吸附机理完全不同,即树脂对大部分杂质的吸附是疏水性吸附,而对三萜酸的吸附是疏水和酸碱作用的共同结果,单纯破坏疏水作用或酸碱作用都不能将三萜酸从树脂柱上有效解吸,同时也给我们利用分步解吸制备高纯度三萜酸提取物创造了条件。为此,我们进一步研究了不同浓度乙醇洗脱液对杂质的洗脱性能,进而提高产品中三萜酸的纯度。通过对吸附流速、洗脱流速等条件的考察,确定了R-3树脂纯化白花蛇舌草总三萜酸的最佳工艺流程,且检验了工艺的重复性和稳定性。最终产品中,总三萜酸含量可达90%以上,大大优于商品化树脂的纯化效果。
最后,我们通过MTT法和SRB法研究了半枝莲总黄酮、野黄芩苷提取物、野黄芩素提取物、木犀草素提取物、芹菜素提取物和白花蛇舌草总三萜酸提取物的体外抗肿瘤活性,发现在一定范围内它们对人肝癌A549和人结肠癌LOVO细胞株均有一定的抑制作用,且存在一定的量效关系。通过6种提取物对肿瘤细胞株的IC50值考察,发现半枝莲的芹菜素提取物和白花蛇舌草的总三萜酸提取物对肿瘤细胞株的抑制效果最佳。我们又进一步考察了半枝莲的总黄酮提取物和芹菜提取物分别与白花蛇舌草的总三萜酸提取物配伍,对肿瘤细胞株的体外抑制作用。研究发现,芹菜素提取物与总三萜酸提取物配伍后的药物对肿瘤细胞株的抑制作用明显优于这两种有效成分单独使用时的效果,其对A549和LOVO细胞株的IC5o值49.54μg/mL和34.48μg/mL,抑制作用最佳,证明了二者之间具有良好的协同作用,并有进一步开发成新型抗肿瘤制剂的潜力与价值。
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.
但是,吸附分离技术也存在着亟待解决的问题,主要体现在目前工业生产中使用的树脂多为聚苯乙烯型骨架,因此吸附机理比较单一,主要以疏水性作用力与吸附质分子发生相互作用,在水溶液中的吸附过程,疏水性作用力是非特异性的吸附结合力,这就势必造成对树脂有效成分的吸附选择性较差。在复方中药中,每一味药材的有效成分、甚至杂质组成都不相同,为了得到每一种有效成分的高纯度提取物,就需要高选择性的吸附树脂,这就给我们设计和调控树脂结构提出了更高的要求,即如何根据有效成分的分子结构和要达到的分离结果,综合考虑有效成分所处的环境、杂质成分对分离的干扰、甚至所合成的树脂能否真正用于大规模的工业化生产等等因素,调控树脂的骨架结构和功能基,将多种弱相互作用的协同效应引入树脂结构的设计和调控过程中,合成一类针对特定有效成分具有高选择性的吸附树脂。
本论文选择了抗肿瘤中药复方白半汤为研究对象,针对方剂中两味药材中抗肿瘤的有效成分,即半枝莲中的黄酮成分和白花蛇舌草中的三萜酸成分的结构和性质,分别设计合成不同结构、功能的大孔吸附树脂,大大提高对两味药材中有效成分的选择性,从而较大程度地提高产品纯度。另一方面,通过对大孔吸附树脂极性的精细调节实现了半枝莲中四种单体黄酮的完全分离。最后,在各提取物的体外抗肿瘤活性筛选的基础上,将两位药材中药效最佳的成分进行配伍,进一步研究其在体外对肿瘤细胞的抑制作用。
首先,为了避免树脂骨架中聚苯乙烯部分对疏水性的贡献,我们改变了合成吸附树脂常用的交联剂二乙烯苯(DVB),合成了一系列以双甲基丙烯酸乙二醇酯(EGDMA)为交联剂、丙烯酸酯(MA)为共聚单体的弱疏水性聚丙烯酸酯骨架的吸附树脂,并在树脂骨架上引入能与黄酮分子的酚羟基发生氢键作用的特殊功能基团,研究了不同疏水性、不同功能基对树脂吸附性能和吸附选择性的影响,发现EGDMA含量为15%的酰胺树脂ME-3B可充分发挥疏水作用力和氢键之间的协同效应,对黄酮的选择性最佳。红外光谱和吸附热力学的研究证实了氢键作用的存在,并且在弱疏水性的酰胺树脂中氢键作用在吸附黄酮过程中起了很重要的作用。为了验证ME-3B树脂对黄酮分子的选择性,我们将其用于多种植物体系中,如葛根、银杏和贯叶连翘,纯化效果均显著优于商品化的大孔吸附树脂,证明了这种弱疏水性骨架的酰胺树脂在天然植物中黄酮类有效成分的提取具有普适性,可开发一类黄酮提取的专用树脂。通过吸附、解吸条件的优化,确定了ME-3B树脂纯化半枝莲总黄酮的最佳工艺流程,且检验了工艺的可重复性和稳定性。最终产品中,总黄酮含量可达56%左右,大大优于商品化树脂的纯化效果。
在分离得到总黄酮的基础上,基于疏水和偶极的协同作用,设计合成了一系列MA和DVB共聚的树脂,通过MA含量变化调节树脂的极性,将其用于半枝莲总黄酮中结构极为相近的四种单体黄酮,即野黄芩苷、野黄芩素、木犀草素和芹菜素。考察了树脂骨架中DVB和MA的比例对吸附选择性的影响,发现只有在适宜的疏水作用力范围内,疏水和偶极作用才能发挥协同效应,使得树脂对不同极性的吸附质吸附结合力的差别得以体现,过高的疏水性将大大掩盖了偶极作用对吸附的贡献,而过低的疏水性又无法帮助偶极作用发挥出来,这与水溶液中吸附时强极性的水分子对吸附剂和吸附质之间的偶极作用产生了强烈的干扰有关。通过疏水部分和弱极性部分比例的调控,考察了不同MA含量变化的吸附树脂对四种黄酮吸附解吸性能的影响,设计了半枝莲黄酮的分离过程。首先MA含量为45%的MD-4树脂可将半枝莲四种黄酮分为三个部分,即野黄芩苷、野黄芩素、木犀草素和芹菜素的混合物。然后再将木犀草素和芹菜素的混合物通过MA含量为25%的MD-2树脂,只需简单的吸附-解吸即可实现木犀草素和芹菜素的分离。根据这样的设计,通过优化影响两种树脂吸附解吸性能的条件,建立了半枝莲单体黄酮的制备分离工艺,实现了四种黄酮的完全分离。
针对白花蛇舌草中三萜酸类有效成分的结构特点,合成了一系列聚苯乙烯骨架、带有胺基的大孔吸附树脂,研究了树脂的交联度、胺基含量和胺基结构变化对三萜酸的吸附性能的影响。结果表明交联度为10%、引入二甲胺的R-3树脂对三萜酸的吸附选择性最佳。红外光谱的研究证明了静电力作用的存在。通过选择性解吸实验,我们发现高浓度乙醇水溶液能够解吸大量杂质,而无论是高浓度乙醇水溶液还是含有10%醋酸的水溶液都无法将三萜酸解吸,只有含10%醋酸的80%乙醇水溶液才能将三萜酸完全解吸,这不仅间接证明了树脂对三萜酸和杂质成分的吸附机理完全不同,即树脂对大部分杂质的吸附是疏水性吸附,而对三萜酸的吸附是疏水和酸碱作用的共同结果,单纯破坏疏水作用或酸碱作用都不能将三萜酸从树脂柱上有效解吸,同时也给我们利用分步解吸制备高纯度三萜酸提取物创造了条件。为此,我们进一步研究了不同浓度乙醇洗脱液对杂质的洗脱性能,进而提高产品中三萜酸的纯度。通过对吸附流速、洗脱流速等条件的考察,确定了R-3树脂纯化白花蛇舌草总三萜酸的最佳工艺流程,且检验了工艺的重复性和稳定性。最终产品中,总三萜酸含量可达90%以上,大大优于商品化树脂的纯化效果。
最后,我们通过MTT法和SRB法研究了半枝莲总黄酮、野黄芩苷提取物、野黄芩素提取物、木犀草素提取物、芹菜素提取物和白花蛇舌草总三萜酸提取物的体外抗肿瘤活性,发现在一定范围内它们对人肝癌A549和人结肠癌LOVO细胞株均有一定的抑制作用,且存在一定的量效关系。通过6种提取物对肿瘤细胞株的IC50值考察,发现半枝莲的芹菜素提取物和白花蛇舌草的总三萜酸提取物对肿瘤细胞株的抑制效果最佳。我们又进一步考察了半枝莲的总黄酮提取物和芹菜提取物分别与白花蛇舌草的总三萜酸提取物配伍,对肿瘤细胞株的体外抑制作用。研究发现,芹菜素提取物与总三萜酸提取物配伍后的药物对肿瘤细胞株的抑制作用明显优于这两种有效成分单独使用时的效果,其对A549和LOVO细胞株的IC5o值49.54μg/mL和34.48μg/mL,抑制作用最佳,证明了二者之间具有良好的协同作用,并有进一步开发成新型抗肿瘤制剂的潜力与价值。
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.
引文
[1]赵守训,中药现代化,中国中西医结合杂志,2001,5(21)：323～325
[2]甘师俊,李振吉,邹健强,中药现代化发展战略,北京,科学技术文献出版社,1998.110-132
[3]辉国钧,葛发欢,王海波等,超临界CO2萃取工艺在紫苏子脂肪油提取中的应用研究,中国医药工业杂志,1996,27(2)：51～53
[4]I.Nykanen, L.Nykanen, M. Alkio, J. Essential Oil Rev.,1991,3:229
[5]姚煜东,金波,向志敏,超临界CO2萃取穿心莲中穿心莲内酯和脱水穿心莲内酯成分的研究,中草药,2000,(31)：69～72
[6]全山从,钱俊,王金政,超滤法和水醇法制备补骨脂注射液的实验比较研究,中成药,1990,12(1)：3-6
[7]宋洪涛,韩家荣,超滤法用于复方当归注射液制备工艺的改革,药学情报通讯,1993,11(4)：41～43
[8]刘洪谦,屈凌波,贾金付,生脉饮口服液超滤技术研究,中草药,1996,27(4):209～211
[9]王世岭,郑殿宝,中草药,超滤法提取黄芩苷的初步考察,中成药研究,1988,(3):5-7
[10]夏超,韩英,张文等,不同大孔吸附树脂对复方脑脉康提取物吸附容量初步比较,时珍国医国药,2000,11(12)：1082～1083
[11]钟玲,尹蓉莉,张仲林,超声提取技术在中药提取中的研究进展,西南军医,2007,9(6)：118～121
[12]J.Pare, Microwave-assisted extraction from materials containing organic matter [P]. US: 5458897,1995-10-7
[13]缪宏良,微波辐照诱导萃取天然香料,上海轻工业,1994(1):29～32
[14]J. Lutisan, M.Micov, J. Cvengros, Separation Science and Technology,1998,33 (1):83-89
[15]王君武,许松林,徐世民,分子蒸馏技术的应用现状,化工进展,2002,21(7):499～501
[16]许松林,王君武,徐世民,Fractionating pharmaceutical material by short path distillation,化工学报,2003,54(9)：1343～1344
[17]高昆玉,色谱法在精细化工中的应用,北京,中国石化出版社,1997
[18]M.Bruno, F. Piozzi, B. Rodriguez et al. Phytochemistry,1996,42:1059
[19]Q.Yue, Q.Fang, X. Liang et al. Planta. Med.,1995,61:375
[20]J. N. Grech, Q. Li, C. C. Duke, et al. J. Nat. Prod.1994,57:1682
[21]L. R. Snyder, In:E. Heftmann, ed. Chromatography,5th ed. Amsterdam:Elsevier,1992,1
[22]韩金玉,李海静,王华等,中草药,2000,31(2)：652～653
[23]D.Wu, K. Lohse, H. C. Greenblatt, J. Chromatogr. A,1995,702,233
[24]杨庆隆,许荣顺,浅谈酶在中药制剂中的应用,中成药,1995,17(6):4-5
[25]张学兰,张兆旺,徐霞,黄柏SBE法与WE法的成分比较,中国中药杂志,1999,24(10)：600～602
[26]冯颖,大孔吸附树脂新工艺在中药制剂中的应用,中医药学刊,2003,21(2):315～316
[27]张虹,柳正良,王洪泉,大孔吸附树脂在药学领域的应用,中国医药工业杂志,2001,32(1)：41～47
[28]严永清主编,中药现代研究的思路与方法,北京,化学工业出版社,2006
[29]Farrall M J, Frechet M J, Functionalization of Polystyrene Resins by Chemical Modification Characterization of Halogenated Polystyrenes by Carbon-13 Nuclear Magnetic Resonance Spectroscopy, Macromolecules,1979,12 (3):426-428
[30]许名成,刘菊湘,史作清等,聚乙烯醇/明胶复合型吸附树脂的合成极其对鞣酸的吸附性能.离子交换与吸附,1999,15(6)：524-530
[31]李彦锋,张树江,李耀增等,PS/PVC互贯大孔吸附树脂对脲酸的吸附研究,高分子学报,2002(2)：153-156
[32]Dolfner K, Ion Exchanger, Berlin, New York:Walter de Gruyter,1991
[33]北川浩,铃木谦一郎,吸附的基础与设计,鹿政理译,北京,化学工业出版社,1983
[34]Nirmalya M., Gregory F. P., Adsorption from aqueous solutions based on a combination of hydrogen bonding and hydrophobic interactions, Langmuir,1991,7,1241-1254
[35]陆志科,谢碧霞,大孔树脂对竹叶黄酮的吸附分离特性研究,经济林研究,2003,21(3)：1-4
[36]李增标,董善年,液固萃取及其在样品处理中的应用Ⅱ：大孔吸附树脂用于液固萃取及其与十八烷基键合硅胶的比较,药物分析杂志,1991,11(6):339～341
[37]吴梅林,周春山,钟世安等,大孔吸附树脂纯化银杏活性化合物的工艺研究,中南药学,2005,3(2)：75～77
[38]曹群华,瞿伟菁,李家贵等,大孔树脂吸附纯化沙棘籽渣总黄酮的研究,中国中药杂志,2004,29(3)：225～228
[39]毕岳琦,侯世祥,毛声俊等,大孔吸附树脂对中药提取液中3种苷类成分的吸附规律考察,中国药学杂志,2007,42(16):1211-1213,1217
[40]欧来良,史作清,施荣富等,吸附树脂对葛根中葛根素的分离研究,中草药,2003,34(2)：134～136
[41]何琦,及元乔,丁立生等,D140大孔吸附树脂银杏黄酮提取纯化性能研究,天然产物研究与开发,2001,13(1)：56～59
[42]李国华,李晨曦,刘晓航等,大孔吸附树脂对盐酸阿霉素吸附性能的研究,离子交换与吸附,2000,16(9)：9～15
[43]任海,查丽杭,秦川等,大孔树脂分离提取麻黄碱的研究,离子交换与吸附,2002,18(2)：97～99
[44]孟琴,张兵,大孔吸附树脂对紫草宁的吸附性的研究及从细胞培养液中提出紫草宁的探索,离子交换与吸附,1999,15(14)：36～42
[45]黄建明,郭济贤,陈万生等,大孔树脂对草乌生物碱的吸附性能及提纯工艺,复旦学报(医学版),2003,30(3)：267～269
[46]刘映,李志忠,大孔吸附树脂预分离后测定黄连及左金丸含量,中国药师,2001,4(2):113～114
[47]侯世祥,田恒康,大孔吸附树脂在中药复方分离纯化工艺中的应用,中药新药与临床药理,2000,11(3)：131～133
[48]卢艳花,中药有效成分提取分离技术,北京：化学工业出版社,2005
[49]Schwabe W., Kloss P., Vasoactive drugs from Ginkgo biloba leaves, DE1767098,1971-10-12
[50]史作清,施荣富,范云鸽等,树脂吸附法在中草药有效成分提取中的应用,中草药,2001,32(7)：660-662
[51]史作清,许名成,施荣富等,树脂吸附法制备银杏叶提取物,中国食品添加剂,1997,3：38-40
[52]Zhang Ying, Li Shufen, Wu Xiwen, et al. Macroporous Resin Adsorption for Purification of Flavonoids in Houttuynia cordata Thunb., Chin. J. Chem. Eng.2007,15 (6):872-876
[53]Yujie Fu, Yuangang Zu, Wei Liu et al., Optimization of luteolin separation from pigeonpea [Cajanus cajan (L.) Millsp.] leaves by macroporous resins, Journal of Chromatography A, 2006,1137:145-152
[54]Yujie Fu, Yuangang Zu, Wei Liu et al., Preparative separation of vitexin and isovitexin from pigeonpea extracts with macroporous resins, Journal of Chromatography A,2007,1139: 206-213
[55]Min Gao, Wei Huang, Chunzhao Liu, Separation of scutellarin from crude extracts of Erigeron breviscapus (vant.) Hand. Mazz. by macroporous resins, Journal of Chromatography B,2007,858:22-26
[56]Jiang Xinyu, Zhou Jinhua, Zhou Chunshan, Study on adsorption and separation of Naringin with macroporous Resin, Front. Chem. China,2006,1:77-81
[57]G F Payne, M L. Shuler. Selective adsorption of plant Products. Biotechnology and Bioengineering,1988,31(9):922-928.
[58]王瑞芳,李家政,史作清等.树脂吸附层析法分离喜树果中的喜果甙.高等学校化学学报,2003,24(8)：1534-1536
[59]王瑞芳,色层分离树脂的合成及其分离性能的研究[博士学位论文],天津：南开大学,2004
[60]Guopei Pi, Ping Ren, Jiamei Yu et al. Separation of sanguinarine and chelerythrine in Macleaya cordata (Willd) R. Br. based on methyl acrylate-co-divinylbenzene macroporous adsorbents, Journal of Chromatography A,2008,1192:17-24
[61]Xuegong Qin, Yuanjin Yuan, Jinchuan Wu et al. Separation of alkaloids from Sophora alopecuroides L. by adsorption using macroporous resins, Journal of Chemical engineering of Japan,2007,40 (2):93-97
[62]张扬,杨磊,牛卉颖等,大孔树脂吸附分离长春花中的文多灵、长春质碱和长春碱,化工学报,2008,59(3)：607～614
[63]何炳林,刘永宁.史作清等.吸附树脂AB-8对甜菊苷的吸附性能及在其提取纯化中的应用.应用化学,1994,11(1)：16～19
[64]马建标,王利民,李建敏等.新型吸附树脂S-038对绞股蓝皂甙的吸附性能及其在绞股蓝皂甙和三七皂甙提取、纯化中的应用.离子交换与吸附,1993,9(2):97-101
[65]施荣富,史作清,范云鸽等.季铵基吸附树脂的合成及其在甜菊甙提取分离中的应用.离子交换与吸附,2001,17(2)：117～122
[66]姜换荣,裴旭红,胡海伟.大孔吸附树脂用于赤芍总甙分离方法的探索.中成药,1999,21(12)：644-645
[67]Bin Zhang, Ruiyuan Yang, Yan Zhao et al. Separation of chlorogenic acid from honeysuckle crude extracts by macroporous resins, Journal of Chromatography B,2008,867:253-258
[68]艾心灵,王新洪,朱松,大孔树脂吸附分离烟草绿原酸的研究,天然产物研究与开发,2008,20：161～164
[69]Xueling Du, Qipeng Yuan, Ye Li et al. Preparative Purification of Solanesol from Tobacco Leaf Extracts by Macroporous Resins, Chem. Eng. Technol.,2008,31 (1):87-94
[70]郭克琳,史作清,何炳林.栀子黄色素的分离与提纯.中国食品添加剂.1996 No.2.4-7.
[71]赵维亮,孙明珍,石秀伟等,大孔树脂纯化红花黄色素的影响因素考察,中成药,2008,30(10)：1557～1559
[72]侯世祥,田恒康.大孔吸附树脂在中药复方分离纯化中的应用.中药新药与临床药理,2000,11(3)：131～133
[73]臧琛,聂其霞,赵小妹等,不同精制方法对抗感颗粒剂中芍药苷的影响,中国实验方剂学杂志,2000,6(2)：1-5
[74]刘中秋,赖小平,吴燕红等.大孔树脂吸附法富集保和丸中橙皮苷的工艺研究.中成药,2001,23(8)：550～554
[75]夏超,韩英,张文等.不同大孔吸附树脂对复方脑脉康提取物吸附容量的初步比较.时珍国药研究.2000,11(12)：1082～1085
[76]朱浩,侯世祥,孙毅毅等.大孔吸附树脂吸附纯化不同中药有效部位特性研究.中国中药杂志.1998,23(10)：607～611
[77]饶品昌,罗文华.右归煎剂用大孔树脂D1300精制的工艺研究.中成药,1999,21(8):388～392
[78]郭立玮,彭国平,王天山,等.大孔树脂吸附与超滤联用对六味地黄丸中丹皮酚和马钱素含量的影响.南京中医药大学学报,1999,15(2):86.
[79]周国平主编,《癌症秘方验方偏方大全》,中国医药科技出版社,1992年1月出版
[80]芦柏震,林能明,何晓波,白花蛇舌草在我院抗肿瘤治疗中的应用分析,中国中药杂志,2006,31(18)：1550～1552
[81]谭萍,芦柏震,包文龙,半枝莲在我院抗肿瘤治疗中的应用分析,江西中医药,2006,37(3)：57～58
[82]游雪秋,半枝莲与白花蛇舌草对癌细胞株以及自发性肝肿瘤的抗癌作用.国外医学中医中药分册,2001,23(2):92
[83]国家中医药管理局中华本草编委会.中华本草精选本.上海：上海科学技术出版社：1998.6.433.
[84]Sung Hoon Kim, Byung-Zun Ahn, Shi Yong Ryu. Antitumour effects of ursolic acid isolated from Oldenlandia diffusa. Phytother. Res.1998,12,553-556
[85]Shaun Willimott, James Barker, Lucy A. Jones et.al. Apoptotic effect of Oldenlandia diffusa on the leukaemic cell line HL60 and human lymphocytes. Journal of Ethnopharmacology, 2007,114:290-299
[86]Jenq-Jer Yang, Hsue-Yin Hsu, Yau-Hui Ho et.al. Comparative study on the immune competent activity of three different kinds of Peh-Hue-Juwa-Chi-Cao, Hedyotis diffusa, H. corymbosa and Mollugo pentaphylla after sublethal whole body X-irradiation. Phytother. Res. 1997,11:428-432
[87]Y. Yoshida, M. Q. Wang, J. N. Liu et.al. Immunomodulating activity of Chinese medicinal herbs and Oldenlandia diffusa in particular, Int. J.lmmunopharmac.,1997,19 (7):359-370
[88]雅达斌,李少钦,白花蛇舌草激活肿瘤细胞产生超氧化物和激活半胱氨酸天冬氨酸查白酶诱导肿瘤细胞凋亡,中西医结合学报,2006,4(5):485～489
[89]张民庆,龚惠,抗肿瘤中药的临床作用,北京：人民卫生出版社,1997:42～43
[90]M. O. Hengartner, The biochemistry of apoptosis, Nature,2000,407:770-776
[91]高冬,高永琳,白平等,白花蛇舌草对宫颈癌细胞钙信号的影响,福建中医学院学报,2002,12(4)：37
[92]D. Sadava, J. Ahn, M. Zhan, et al. Effects of four Chinese herbal extracts on drug-sensitive and multidrug-resistant small-cell lung carcinoma cells. Cancer Chemother Pharmacol,2002, 49(4):261-268
[93]朱大诚,尹小明,王建红,蟾酥诱导白血病细胞凋亡的研究进展,实用癌症杂志,2004,19(4)：447～451
[94]蔡楚伧,钱秀丽,姜达衢,白花蛇舌草的化学成分研究Ⅰ.药学学报,1964,11(12)：809～814
[95]黄建荣,刘咏海,喻志标等,白花蛇舌草化学成分和药理活性研究进展,中成药,2005,27(11)：1329～1331
[96]Yoshida Y, Wang M Q, Liu J N et al. International Journal of Immunopharmacology,1997, 19(7):359-370
[97]Kim Sun Hoon, Ann Byung Zun, Ryu Shi Yong. Antitumour effects of ursolic acid isolated from Oldenlandia diffusa, Phytotherapy Research,1998,12(8):553-556
[98]黄镜,孙燕,熊果酸的抗肿瘤活性,中国新药杂志,1997,6(2)：101～104
[99]C. Chandramu, Rao D. Manohar, David G. L. Krupadanam. Isolation, characterization and biological activity of betulinic acid and ursolic acid from Vitex negundo L, Phytother. Res. 2003,17,129-134
[100]夏文娟等,白花蛇舌草联合化疗治疗晚期恶性肿瘤的疗效分析,四川省卫生管理干部学院学报,2001,6:93
[101]王刚,董梅,刘秀书等,半枝莲醇提物抗肿瘤活性的研究,现代中西医结合杂志,2004,13(9)：1041～1042
[102]张春玲,胡峻峰,Ames试验检测几种中草药及绿茶的抗诱变作用,卫生毒理学杂志,2002,16(1)：66
[103]谢珞琨,邓涛,半枝莲提取物诱导白血病K562细胞凋亡,武汉大学学报(医学版),2004,25(2)：115～118
[104]林敬明,刘煜,罗荣城,半枝莲提取物抗人肝癌Hep-G2细胞增殖及其机制研究,南方医科大学学报,2006,26(7)：975～977
[105]吴东风,林明宝,舒虹等,复方半枝莲胶囊抗肿瘤作用的实验研究,实用中西医结合临床,2006,6(6)：79～80
[106]张妮娜,卜平,朱海杭等,半枝莲抑制肿瘤血管生成的作用及其机制研究,癌症,2005,24(12)：1459～1463
[107]李洁,石俊英,半枝莲对人肺巨细胞癌细胞株PG细胞端粒酶活性的影响,中药药理与临床,2006,22(3)：113～114
[108]李洁,半枝莲对人肺豆细胞癌细胞株PG细胞bcl-2基因表达的影响,中药药理与临床,2004,23(1)：46～47
[109]张春玲,胡俊峰,曲江斌等,几种中草药及绿茶对B(α)P和NNK的抗诱变作用,癌变·畸变·突变,2003,15(2)：101～103
[110]高冬,高永琳,白平,半枝莲对宫颈癌细胞钙信号系统的影响,中药材,2003,26(10):730～732
[111]杨容,黄志,习洋等,中药半枝莲中黄酮类化合物的抗氧化活性,河北大学学报(自然科学版),2007,27(5)：489～491
[112]王洪琦,崔娜娟,胡玲等,清热解毒和补益中药对小鼠腹水肝癌H-22细胞的作用及免疫学机制比较,广州中医药大学学报,2006,23(2):156～159
[113]蒋小岗,顾振纶,半枝莲化学成分和药理作用,中国野生植物资源,2004,23(1):3-5
[114]林敬明,刘煜,罗荣城.半枝莲的化学成分及其抗肿瘤作用的研究现状,2006,29(4):407～410
[115]Sonoda M, Nishiyama T, Matsukwa Y. Cytotoxic activities of flavonoids from two Scutellaria plants in Chinese medicine. J Ethnopharmcol,2004,91(1):65-68.
[116]糜松元,栾连军,半枝莲抗肿瘤活性成分及其药代动力学研究,浙江大学硕士论文,2006年5月
[117]Cardenas M., Marder M., et al. Antitumor activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines. Bioorganic & Medicinal Chemistry,14:2966-2971
[118]余建清,邹国林,半枝莲生物活性及相关化学成分研究,武汉大学博士论文,2005年4月
[119]张才华,郭兴杰,李芳等,HPLC法同时测定白花蛇舌草中齐墩果酸和熊果酸的含量,中草药,2004,21(5)：358～360
[120]罗新根,蒋琳兰,赵树进,高效液相色谱法同时测定白花蛇舌草中两组分的含量,药品检验,2004,15(3)：175～176
[121]谢艳,林圣云,任其龙,白花蛇舌草中齐墩果酸和熊果酸的HPLC测定,中国医药工业杂志,2006,37(8)：559～561
[122]薛梅,王自军,周静,半枝莲中总黄酮和多糖的微波提取与含量测定,时珍国医国药, 2005,16(10)：965～966
[123]李省,杨保华,陈悦,中药半枝莲总黄酮含量分析,中国公共卫生,2005,21(7):847-848
[124]李守拙,潘海峰,HPLC测定半枝莲中野黄芩苷的含量,中草药,2003,34(2)：183～184
[125]乔春峰,韩全斌,宋景政,RP-HPLC测定半枝莲药材中4种主要黄酮类成分的含量,中国药学杂志,2006,41(17)：1342～1344
[126]N. Maity, G. F. Payne. Adsorption from aqueous solutions based on a combination of hydrogen bonding and hydrophobic interactions. Langmuir.1991,7:1247-1254
[127]C. Renner, J. Piehler, T. Schrader. Arginine- and Lysine-Specific Polymers for Protein Recognition and Immobilization. J. Am. Chem. Soc.2006,128 (2):620-628
[128]严永清,中药现代研究的思路与方法,北京：化学工业出版社,2006,44～52
[1]M. Andersen, K. R. Markham. Flavonoids-Chemistry, Biochemistry and Applications, Taylor & Francis Group,2006
[2]朱英,何远,李丽,大孔树脂对地锦草、金银花叶与杭白菊总黄酮吸附分离性能研究,医学研究杂志,2007,37(1)：56～59
[3]于志,王敏,大孔树脂精制苦荞总黄酮工艺,中国中药杂志,2007,32(7)：585～589
[4]M. C. Xu, Z. Q. Shi, R. F. Shi, et al. Synthesis of the adsorbent based on macroporous copolymer MA-DVB beads and its application in purification for the extracts from Ginkgo biloba leaves. React. Funct. Polym.2000,43:297-304
[5]N. Maity, G. F. Payne. Adsorption from aqueous solutions based on a combination of hydrogen bonding and hydrophobic interactions. Langmuir.1991,7:1247-1254
[6]C. Renner, J. Piehler, T. Schrader. Arginine- and Lysine-Specific Polymers for Protein Recognition and Immobilization. J. Am. Chem. Soc.2006,128 (2):620-628
[7]乔春峰,韩全斌,宋景政等,RP-HPLC测定半枝莲药材中4种主要黄酮类成分的含量,中国中药杂志,2006,41(17)：1342～1344
[8]L. P. Li, H. D. Jiang. Determination and assay validation of luteolin and apigenin in human urine after oral administration of tablet of Chrysanthemum morifolium extract by HPLC. Journal of Pharmaceutical and Biomedical Analysis,2006,41:261-265
[9]何炳林,黄文强,离子交换与吸附树脂,上海科技出版社,1995
[10]国家药典委员会.中国药典2005年版,北京：化学工业出版社,234
[11]国家药典委员会.中国药典2005年版,北京：化学工业出版社,234
[12]A. Hasler, O. Sticher, B. Meier. Identification and determination of the flavonoids from Ginkgo biloba by high-performance liquid chromatography. Journal of chromatography 1992, 605:41-48
[13]R. A. Garcla-Delgado, L. M. Cotouelo-Minguez, J. J. Rodfiguez. Equilibrium study of single-solute adsorption of anionic surfactants with polymeric XAD resins. Sep Sci Techn, 1992,27 (7):975-987
[14]G. F. Payne, N. Maity. Solute Adsorption from water onto a "modified" sorbent in which the hydrogen binding site Is protected from water, Thermodynamics and Separations. Ind Eng Chem Res,1992,31:2024-2033
[15]王庆文,杨玉桓,高鸿宾.有机化学中的氢键问题.天津：天津大学出版社,1993
[16]冯年平,郁威.中药提取分离技术原理与应用.中国医药科技出版社,北京,2005.124～128
[1]Q.Li, J. H. Wu, D. J. Guo, et.al. Suppression of diet-induced hypercholesterolemia by scutellarin in rats. Planta Med,2009,75 (11):1203-1208
[2]L. L. Lin, A. J. Liu, J. H. Liu. Protective effects of scutellarin and breviscapine on brain and heart ischemia in rats. Journal of Cardiovascular Pharmacology,2007,50 (3):372-332
[3]H. Hong, G. Q. Liu. Protection against hydrogen peroxide-induced cytotoxicity in PC 12 cells by scutellarin. Life Sciences,2004,74 (24):2959-2973
[4]赵铁华,邓淑华,高巍等.野黄芩苷镇痛作用的实验研究.中国中医药信息杂志,2001,8(6)：28～29
[5]G. H. Zhang, Q. Wang, J. J. Chen, et al. The anti-HIV-1 effect of scutellarin. Biochemical and Biophysical Research Communications,2005,334 (3):812-816
[6]J. Y. Chan, B. K. Tan, S. C. Lee. Scutellarin Sensitizes Drug-evoked Colon Cancer Cell Apoptosis through Enhanced Caspase-6 Activation. Anticancer Research,2009,29 (8): 3043-3047
[7]崔建梅,吴淞.野黄芩苷的研究进展.天然产物研究与开发,2003,15(3)：255～263
[8]W. Xu, Z. P. Zha, W. Y. Wang, et al. Effects of scutellarin on PKCy in PC12 cell injury induced by oxygen and glucose deprivation. Acta. Pharmacol. Sin.2007,28:1573-1579
[9]B. H.Zhu. Scutellarein, a potent preventer of diabetic complications. Journal of Pharmacological Sciences,109,1:231-235
[10]K. Plochmann, G. Korte, E. Koutsilieri, et al. Structure-activity relationships of flavonoid-induced cytotoxicity on human leukemia cells. Arch. Biochem. Biophys.,2007, 460:1-3
[11]J. H. Kim, E. O. Lee, H. J. Lee, et al. Annals of the New York Academy of Sciences,1095, Issue Signal Transduction Pathways, Part C,598
[12]H. J. Lee, C. J. Wang, H. C. Kuo, et al. Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of BaxBak and activation of JNK. Toxicol. Appl. Pharmacol.2005,203,124-131
[13]W. Q. Wang, L. Heideman, C. S. Chung, et al. Cell-Cycle Arrest at G2M and Growth Inhibition by Apigenin in Human Colon Carcinoma Cell Lines, Mol. Carcinog.,2000,28: 102-110
[14]I. K. Wang, S. Y. Lin-Shiau, J. K. Lin, Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells, Eur. J. Cancer,1999,35 (10):1517-1525
[15]S. J. Dai, L. Shen, Y. Ren, J. Integ. Plant Bio.2008,50:699-704
[16]S. A.M. Abdelgaleil, M. A. Abbassy, A. S. H. Belal, M. A.A. Abdel Rasoul, Bioresour. Technol.2008,99:5947-5955
[17]Y. W. Ha, Y. S. Kim, Phytochem. Anal.2009,20:207-212
[18]D. J. Comeskey, M. Montefiori, P. J. B. Edwards, T. Mcghie, J. Agric. Food Chem.2009,57: 2035-2038
[19]G. T. Jia, X. Y. Lu, J. Chromatogr. A.2008,1193:136-142
[20]C. Y. Ma, J. Tang, H. X. Wang, G. J. Tao, X. H. Gu, L. M. Hu, J. Sep. Sci.2009,32:185-192
[21]Y. J. Fu, Y. G. Zu, W. Liu, T. Efferth, N. J. Zhang, X. N. Liu, Y. Kong, J. Chromatogr. A.2006, 1137:145-152
[22]Y. Zhao, B. Chen, S. Z. Yao, Sep. Purif. Technol.2007,52:533-541
[23]R. Sun, K. Fu, Y. J. Fu, Y. G. Zu, Y. Wang, M. Luo, S. M. Li, H. Luo, Z. N. Li, J. Sep. Sci., 2009,32:1284-1288
[24]J.B. Wan, Q.W. Zhang, W.C. Ye, Y. T. Wang, Sep. Purif. Technol.2008,60:198-205
[25]F. E. Bernardin. Experimental design and testing of adsorption and adsorbates. In:F L Slejko (ed). Adsorption Technology, New York:Marcel Dekker,1985,1-5
[26]方宾,阚显文.近代分离方法导论.合肥：安徽人民出版社,2006.95-96
[27]W. J. Weber. Adsorption theory, concepts, and models. In:F L Slejko (ed). Adsorption Technology. New York:Marcel Dekker,1985.31-35
[1]黄炜,黄济群,张东等,18p-甘草次酸和甘草酸对人肝癌细胞增殖的抑制和诱导分化作用,中国中医药科技,2002,9(2)：92～93
[2]Baek J H, Lee YS, Kang CM, et al. Intracellular Ca2+ release mediates ursolic acid-induced apoptosis in human leukemic HL-60 cells. Int. J. Cancer,1997,73 (5):725-728
[3]黄炜,黄济群,张东等,五环三萜类化合物抗人肺癌细胞侵袭和诱导细胞凋亡的研究,中国肺癌杂志,2003,6(4)：254～257
[4]张瑞玲,黄炜,黄济群,中药五环三萜类有效成分抗人肺癌细胞增殖和侵袭作用的研究,中医药学刊,2004,22(11)：2065～2067
[5]孟艳秋,赵临襄,王趱等,五元三萜类化合物的构效关系,中国新药杂志,2004,13(12):1098～1102
[6]张才华,郭兴杰,李芳等,HPLC法同时测定白花蛇舌草中齐墩果酸和熊果酸的含量,沈阳药科大学学报,2004,24(9)：358～360,370
[7]Liu Jie. Oleanolic acid and ursolic acid:Research perspectives. Journal of Ethnopharmacology, 2005, (100):92-94
[8]Young HS, Chung HY, Lee CK, et al. Ursolic acid inhibits aflatoxin Bl-induced mutagenicity in a Salmonella assay system. Biol. Pharm. Bull.,1994,17 (7):990-992
[9]Lee HY, Chung HY, Kim KH, et al. Induction of differentiation in the cultured F9 terato-carcinoma stem cells by triterpene acids. J. Cancer Res. Clin. Oncol.,1994,120 (9):513-518
[10]Baek J H, Lee YS, Kang CM, et al. Intracellular Ca2+ release mediates ursolic acid-induced apoptosis in human leukemic HL-60 cells. Int. J. Cancer,1997,73 (5):725-728
[11]A. A. Ramos, C. F. Lima, M. L. Pereira et al. Antigenotoxic effects of quercetin, rutin and ursolic acid on HepG2 cells Evaluation by the comet assay. Toxicology Letters,2008,177: 66-73
[12]E. K. Yim, M. J. Lee, K.H. Lee et al. Antiproliferative and antiviral mechanisms of ursolic acid and dexamethasone in cervical carcinoma cell lines. Int. J. Gynecol. Cancer,2006,16: 2023-2031
[13]Irene Bonaccorsi, Fabio Altieri, Ilaria Sciamanna, et al. Endogenous reverse transcriptase as a mediator of ursolic acid's anti-proliferative and differentiating effects in human cancer cell lines. Cancer Letters,2008,263:130-139
[14]Hsue Yin Hsu, Jenq Jer Yang, Chun Ching Lin. Effects of oleanolic acid and ursolic acid on inhibiting tumor growth and enhancing the recovery of hematopoietic system postirradiati on in mice. Cancer letters,1997,2(1):7-13
[15]任秀莲,周春山,魏琦峰等,苦丁茶中熊果酸的分离纯化工艺研究,中国药学杂志,2004,39(8)：571～574
[16]谷芳芳,任秀莲,吴梅林,大孔吸附树脂对熊果酸吸附性能的研究,广州化学,2006,31(2)：24～27
[17]何思煌,祝红,大孔树脂吸附提取五味子的有效成分乌索酸,临床和实验医学杂志,2007,6(1)：143～144
[18]李丽,陈剑锋,林国荣,枇杷叶中熊果酸的分离纯化,现代化工,2007,2:415～147
[19]谢艳,林圣云,任其龙,白花蛇舌草中齐墩果酸和熊果酸的HPLC测定,中国医药工业杂志,2006,37(8)：559～561
[20]何炳林,黄文强,离子交换与吸附树脂,上海：上海科技出版社,1995
[21]刘永宁,史作清,施荣富等,极性修饰的聚苯乙烯吸附树脂的结构和性能,高等学校化学学报,1994,15(3)：450～453
[1]张勇,胡笑克,陈维真等,激素难治性前列腺癌细胞对化疗药物敏感性的比较,标记免疫分析与临床,2003,10(2)：159～261
[2]李经伦,刘长征,3H-TdR应用研究现状,放射免疫学杂志,2004,17(1)：64～67
[3]H. Tominaga, Ishiyama M, et al. A water-soluble tetrazolium salt useful for colorimetric cell viabiLity assay, Anal. Commum.,1999,36:47-50
[4]S. Indah, F. K. Tantular. An improved simple screening for detection of glucose-6-phosphate dehydrogenase deficiency, Tropical Medicine,2003,8(6):569-675
[5]T. Kuzuko, O. Motoyasu, U. Hideo, A simple bioassay for evaluating immunotoxic properties of chemicals by use of in vitro antibody production system. Journal of Health Science,2002,48(2):161-167
[6]陈学清,杨晓鸥,潘国宗等,酸性磷酸酶法在体外化疗药物筛选中的应用,癌症,1998,17(6)：477～478
[7]D. T. Connolly, M. B. Knight, N. Harakas, et al. Determination of number of endothelial cells in culture using an acid phosphatase assay, Anal. Biochem.,1986,152:136
[8]J. D. Moyer, J. F. Henderson, Ultrasensitive assay for ribonucleo side triphosphates in 50-1000 cells. Biochem. Pharmacol.,1983,32:3831
[9]陈历排,周雯,李志阳,用微量板ATP生物发光法检测肿瘤细胞对化疗药物的敏感性.肿瘤,2000,20(2)：103～105
[10]司徒镇强,吴军正,细胞培养,西安：世界图书出版社,1996,136.
[11]T. Mosmann, Rapid colorimetric assay for celluar growth and surival. J. Immumol. Methods, 1983,65:551
[12]吴慧,抗肿瘤药物筛选方法,医学理论和实践,2005,18(2)：146～147
[13]P. Skehan, R. Storeng, D. Scudiero, et al. New colorimetric cytotoxicity assay for anticancer drug screening, J. Nat. Cancer Ins.,1990,82(13):1107-1112
[14]谭卫东,金红,罗第祥等,抗肿瘤药物筛选中MTT法与SRB法的比较,天然产物研究与开发,1999,11(3)：217～221
[15]于永洲,白花蛇舌草DNA指纹图谱与体外抗肿瘤活性的相关性研究,沈阳药科大学硕士论文,2004.5
[16]于春艳,李薇,刘玉和等,白花蛇舌草提取物体外抗肿瘤作用及机制研究,北华大学学报(自然科学版),2004,5(5)：412～416
[17]周志愉,王兆雷,不同乙醇浓度半枝莲提取物抗肿瘤作用的相关性研究,实用中西医结合临床,2007,7(6)：89～90
[2]甘师俊,李振吉,邹健强,中药现代化发展战略,北京,科学技术文献出版社,1998.110-132
[3]辉国钧,葛发欢,王海波等,超临界CO2萃取工艺在紫苏子脂肪油提取中的应用研究,中国医药工业杂志,1996,27(2)：51～53
[4]I.Nykanen, L.Nykanen, M. Alkio, J. Essential Oil Rev.,1991,3:229
[5]姚煜东,金波,向志敏,超临界CO2萃取穿心莲中穿心莲内酯和脱水穿心莲内酯成分的研究,中草药,2000,(31)：69～72
[6]全山从,钱俊,王金政,超滤法和水醇法制备补骨脂注射液的实验比较研究,中成药,1990,12(1)：3-6
[7]宋洪涛,韩家荣,超滤法用于复方当归注射液制备工艺的改革,药学情报通讯,1993,11(4)：41～43
[8]刘洪谦,屈凌波,贾金付,生脉饮口服液超滤技术研究,中草药,1996,27(4):209～211
[9]王世岭,郑殿宝,中草药,超滤法提取黄芩苷的初步考察,中成药研究,1988,(3):5-7
[10]夏超,韩英,张文等,不同大孔吸附树脂对复方脑脉康提取物吸附容量初步比较,时珍国医国药,2000,11(12)：1082～1083
[11]钟玲,尹蓉莉,张仲林,超声提取技术在中药提取中的研究进展,西南军医,2007,9(6)：118～121
[12]J.Pare, Microwave-assisted extraction from materials containing organic matter [P]. US: 5458897,1995-10-7
[13]缪宏良,微波辐照诱导萃取天然香料,上海轻工业,1994(1):29～32
[14]J. Lutisan, M.Micov, J. Cvengros, Separation Science and Technology,1998,33 (1):83-89
[15]王君武,许松林,徐世民,分子蒸馏技术的应用现状,化工进展,2002,21(7):499～501
[16]许松林,王君武,徐世民,Fractionating pharmaceutical material by short path distillation,化工学报,2003,54(9)：1343～1344
[17]高昆玉,色谱法在精细化工中的应用,北京,中国石化出版社,1997
[18]M.Bruno, F. Piozzi, B. Rodriguez et al. Phytochemistry,1996,42:1059
[19]Q.Yue, Q.Fang, X. Liang et al. Planta. Med.,1995,61:375
[20]J. N. Grech, Q. Li, C. C. Duke, et al. J. Nat. Prod.1994,57:1682
[21]L. R. Snyder, In:E. Heftmann, ed. Chromatography,5th ed. Amsterdam:Elsevier,1992,1
[22]韩金玉,李海静,王华等,中草药,2000,31(2)：652～653
[23]D.Wu, K. Lohse, H. C. Greenblatt, J. Chromatogr. A,1995,702,233
[24]杨庆隆,许荣顺,浅谈酶在中药制剂中的应用,中成药,1995,17(6):4-5
[25]张学兰,张兆旺,徐霞,黄柏SBE法与WE法的成分比较,中国中药杂志,1999,24(10)：600～602
[26]冯颖,大孔吸附树脂新工艺在中药制剂中的应用,中医药学刊,2003,21(2):315～316
[27]张虹,柳正良,王洪泉,大孔吸附树脂在药学领域的应用,中国医药工业杂志,2001,32(1)：41～47
[28]严永清主编,中药现代研究的思路与方法,北京,化学工业出版社,2006
[29]Farrall M J, Frechet M J, Functionalization of Polystyrene Resins by Chemical Modification Characterization of Halogenated Polystyrenes by Carbon-13 Nuclear Magnetic Resonance Spectroscopy, Macromolecules,1979,12 (3):426-428
[30]许名成,刘菊湘,史作清等,聚乙烯醇/明胶复合型吸附树脂的合成极其对鞣酸的吸附性能.离子交换与吸附,1999,15(6)：524-530
[31]李彦锋,张树江,李耀增等,PS/PVC互贯大孔吸附树脂对脲酸的吸附研究,高分子学报,2002(2)：153-156
[32]Dolfner K, Ion Exchanger, Berlin, New York:Walter de Gruyter,1991
[33]北川浩,铃木谦一郎,吸附的基础与设计,鹿政理译,北京,化学工业出版社,1983
[34]Nirmalya M., Gregory F. P., Adsorption from aqueous solutions based on a combination of hydrogen bonding and hydrophobic interactions, Langmuir,1991,7,1241-1254
[35]陆志科,谢碧霞,大孔树脂对竹叶黄酮的吸附分离特性研究,经济林研究,2003,21(3)：1-4
[36]李增标,董善年,液固萃取及其在样品处理中的应用Ⅱ：大孔吸附树脂用于液固萃取及其与十八烷基键合硅胶的比较,药物分析杂志,1991,11(6):339～341
[37]吴梅林,周春山,钟世安等,大孔吸附树脂纯化银杏活性化合物的工艺研究,中南药学,2005,3(2)：75～77
[38]曹群华,瞿伟菁,李家贵等,大孔树脂吸附纯化沙棘籽渣总黄酮的研究,中国中药杂志,2004,29(3)：225～228
[39]毕岳琦,侯世祥,毛声俊等,大孔吸附树脂对中药提取液中3种苷类成分的吸附规律考察,中国药学杂志,2007,42(16):1211-1213,1217
[40]欧来良,史作清,施荣富等,吸附树脂对葛根中葛根素的分离研究,中草药,2003,34(2)：134～136
[41]何琦,及元乔,丁立生等,D140大孔吸附树脂银杏黄酮提取纯化性能研究,天然产物研究与开发,2001,13(1)：56～59
[42]李国华,李晨曦,刘晓航等,大孔吸附树脂对盐酸阿霉素吸附性能的研究,离子交换与吸附,2000,16(9)：9～15
[43]任海,查丽杭,秦川等,大孔树脂分离提取麻黄碱的研究,离子交换与吸附,2002,18(2)：97～99
[44]孟琴,张兵,大孔吸附树脂对紫草宁的吸附性的研究及从细胞培养液中提出紫草宁的探索,离子交换与吸附,1999,15(14)：36～42
[45]黄建明,郭济贤,陈万生等,大孔树脂对草乌生物碱的吸附性能及提纯工艺,复旦学报(医学版),2003,30(3)：267～269
[46]刘映,李志忠,大孔吸附树脂预分离后测定黄连及左金丸含量,中国药师,2001,4(2):113～114
[47]侯世祥,田恒康,大孔吸附树脂在中药复方分离纯化工艺中的应用,中药新药与临床药理,2000,11(3)：131～133
[48]卢艳花,中药有效成分提取分离技术,北京：化学工业出版社,2005
[49]Schwabe W., Kloss P., Vasoactive drugs from Ginkgo biloba leaves, DE1767098,1971-10-12
[50]史作清,施荣富,范云鸽等,树脂吸附法在中草药有效成分提取中的应用,中草药,2001,32(7)：660-662
[51]史作清,许名成,施荣富等,树脂吸附法制备银杏叶提取物,中国食品添加剂,1997,3：38-40
[52]Zhang Ying, Li Shufen, Wu Xiwen, et al. Macroporous Resin Adsorption for Purification of Flavonoids in Houttuynia cordata Thunb., Chin. J. Chem. Eng.2007,15 (6):872-876
[53]Yujie Fu, Yuangang Zu, Wei Liu et al., Optimization of luteolin separation from pigeonpea [Cajanus cajan (L.) Millsp.] leaves by macroporous resins, Journal of Chromatography A, 2006,1137:145-152
[54]Yujie Fu, Yuangang Zu, Wei Liu et al., Preparative separation of vitexin and isovitexin from pigeonpea extracts with macroporous resins, Journal of Chromatography A,2007,1139: 206-213
[55]Min Gao, Wei Huang, Chunzhao Liu, Separation of scutellarin from crude extracts of Erigeron breviscapus (vant.) Hand. Mazz. by macroporous resins, Journal of Chromatography B,2007,858:22-26
[56]Jiang Xinyu, Zhou Jinhua, Zhou Chunshan, Study on adsorption and separation of Naringin with macroporous Resin, Front. Chem. China,2006,1:77-81
[57]G F Payne, M L. Shuler. Selective adsorption of plant Products. Biotechnology and Bioengineering,1988,31(9):922-928.
[58]王瑞芳,李家政,史作清等.树脂吸附层析法分离喜树果中的喜果甙.高等学校化学学报,2003,24(8)：1534-1536
[59]王瑞芳,色层分离树脂的合成及其分离性能的研究[博士学位论文],天津：南开大学,2004
[60]Guopei Pi, Ping Ren, Jiamei Yu et al. Separation of sanguinarine and chelerythrine in Macleaya cordata (Willd) R. Br. based on methyl acrylate-co-divinylbenzene macroporous adsorbents, Journal of Chromatography A,2008,1192:17-24
[61]Xuegong Qin, Yuanjin Yuan, Jinchuan Wu et al. Separation of alkaloids from Sophora alopecuroides L. by adsorption using macroporous resins, Journal of Chemical engineering of Japan,2007,40 (2):93-97
[62]张扬,杨磊,牛卉颖等,大孔树脂吸附分离长春花中的文多灵、长春质碱和长春碱,化工学报,2008,59(3)：607～614
[63]何炳林,刘永宁.史作清等.吸附树脂AB-8对甜菊苷的吸附性能及在其提取纯化中的应用.应用化学,1994,11(1)：16～19
[64]马建标,王利民,李建敏等.新型吸附树脂S-038对绞股蓝皂甙的吸附性能及其在绞股蓝皂甙和三七皂甙提取、纯化中的应用.离子交换与吸附,1993,9(2):97-101
[65]施荣富,史作清,范云鸽等.季铵基吸附树脂的合成及其在甜菊甙提取分离中的应用.离子交换与吸附,2001,17(2)：117～122
[66]姜换荣,裴旭红,胡海伟.大孔吸附树脂用于赤芍总甙分离方法的探索.中成药,1999,21(12)：644-645
[67]Bin Zhang, Ruiyuan Yang, Yan Zhao et al. Separation of chlorogenic acid from honeysuckle crude extracts by macroporous resins, Journal of Chromatography B,2008,867:253-258
[68]艾心灵,王新洪,朱松,大孔树脂吸附分离烟草绿原酸的研究,天然产物研究与开发,2008,20：161～164
[69]Xueling Du, Qipeng Yuan, Ye Li et al. Preparative Purification of Solanesol from Tobacco Leaf Extracts by Macroporous Resins, Chem. Eng. Technol.,2008,31 (1):87-94
[70]郭克琳,史作清,何炳林.栀子黄色素的分离与提纯.中国食品添加剂.1996 No.2.4-7.
[71]赵维亮,孙明珍,石秀伟等,大孔树脂纯化红花黄色素的影响因素考察,中成药,2008,30(10)：1557～1559
[72]侯世祥,田恒康.大孔吸附树脂在中药复方分离纯化中的应用.中药新药与临床药理,2000,11(3)：131～133
[73]臧琛,聂其霞,赵小妹等,不同精制方法对抗感颗粒剂中芍药苷的影响,中国实验方剂学杂志,2000,6(2)：1-5
[74]刘中秋,赖小平,吴燕红等.大孔树脂吸附法富集保和丸中橙皮苷的工艺研究.中成药,2001,23(8)：550～554
[75]夏超,韩英,张文等.不同大孔吸附树脂对复方脑脉康提取物吸附容量的初步比较.时珍国药研究.2000,11(12)：1082～1085
[76]朱浩,侯世祥,孙毅毅等.大孔吸附树脂吸附纯化不同中药有效部位特性研究.中国中药杂志.1998,23(10)：607～611
[77]饶品昌,罗文华.右归煎剂用大孔树脂D1300精制的工艺研究.中成药,1999,21(8):388～392
[78]郭立玮,彭国平,王天山,等.大孔树脂吸附与超滤联用对六味地黄丸中丹皮酚和马钱素含量的影响.南京中医药大学学报,1999,15(2):86.
[79]周国平主编,《癌症秘方验方偏方大全》,中国医药科技出版社,1992年1月出版
[80]芦柏震,林能明,何晓波,白花蛇舌草在我院抗肿瘤治疗中的应用分析,中国中药杂志,2006,31(18)：1550～1552
[81]谭萍,芦柏震,包文龙,半枝莲在我院抗肿瘤治疗中的应用分析,江西中医药,2006,37(3)：57～58
[82]游雪秋,半枝莲与白花蛇舌草对癌细胞株以及自发性肝肿瘤的抗癌作用.国外医学中医中药分册,2001,23(2):92
[83]国家中医药管理局中华本草编委会.中华本草精选本.上海：上海科学技术出版社：1998.6.433.
[84]Sung Hoon Kim, Byung-Zun Ahn, Shi Yong Ryu. Antitumour effects of ursolic acid isolated from Oldenlandia diffusa. Phytother. Res.1998,12,553-556
[85]Shaun Willimott, James Barker, Lucy A. Jones et.al. Apoptotic effect of Oldenlandia diffusa on the leukaemic cell line HL60 and human lymphocytes. Journal of Ethnopharmacology, 2007,114:290-299
[86]Jenq-Jer Yang, Hsue-Yin Hsu, Yau-Hui Ho et.al. Comparative study on the immune competent activity of three different kinds of Peh-Hue-Juwa-Chi-Cao, Hedyotis diffusa, H. corymbosa and Mollugo pentaphylla after sublethal whole body X-irradiation. Phytother. Res. 1997,11:428-432
[87]Y. Yoshida, M. Q. Wang, J. N. Liu et.al. Immunomodulating activity of Chinese medicinal herbs and Oldenlandia diffusa in particular, Int. J.lmmunopharmac.,1997,19 (7):359-370
[88]雅达斌,李少钦,白花蛇舌草激活肿瘤细胞产生超氧化物和激活半胱氨酸天冬氨酸查白酶诱导肿瘤细胞凋亡,中西医结合学报,2006,4(5):485～489
[89]张民庆,龚惠,抗肿瘤中药的临床作用,北京：人民卫生出版社,1997:42～43
[90]M. O. Hengartner, The biochemistry of apoptosis, Nature,2000,407:770-776
[91]高冬,高永琳,白平等,白花蛇舌草对宫颈癌细胞钙信号的影响,福建中医学院学报,2002,12(4)：37
[92]D. Sadava, J. Ahn, M. Zhan, et al. Effects of four Chinese herbal extracts on drug-sensitive and multidrug-resistant small-cell lung carcinoma cells. Cancer Chemother Pharmacol,2002, 49(4):261-268
[93]朱大诚,尹小明,王建红,蟾酥诱导白血病细胞凋亡的研究进展,实用癌症杂志,2004,19(4)：447～451
[94]蔡楚伧,钱秀丽,姜达衢,白花蛇舌草的化学成分研究Ⅰ.药学学报,1964,11(12)：809～814
[95]黄建荣,刘咏海,喻志标等,白花蛇舌草化学成分和药理活性研究进展,中成药,2005,27(11)：1329～1331
[96]Yoshida Y, Wang M Q, Liu J N et al. International Journal of Immunopharmacology,1997, 19(7):359-370
[97]Kim Sun Hoon, Ann Byung Zun, Ryu Shi Yong. Antitumour effects of ursolic acid isolated from Oldenlandia diffusa, Phytotherapy Research,1998,12(8):553-556
[98]黄镜,孙燕,熊果酸的抗肿瘤活性,中国新药杂志,1997,6(2)：101～104
[99]C. Chandramu, Rao D. Manohar, David G. L. Krupadanam. Isolation, characterization and biological activity of betulinic acid and ursolic acid from Vitex negundo L, Phytother. Res. 2003,17,129-134
[100]夏文娟等,白花蛇舌草联合化疗治疗晚期恶性肿瘤的疗效分析,四川省卫生管理干部学院学报,2001,6:93
[101]王刚,董梅,刘秀书等,半枝莲醇提物抗肿瘤活性的研究,现代中西医结合杂志,2004,13(9)：1041～1042
[102]张春玲,胡峻峰,Ames试验检测几种中草药及绿茶的抗诱变作用,卫生毒理学杂志,2002,16(1)：66
[103]谢珞琨,邓涛,半枝莲提取物诱导白血病K562细胞凋亡,武汉大学学报(医学版),2004,25(2)：115～118
[104]林敬明,刘煜,罗荣城,半枝莲提取物抗人肝癌Hep-G2细胞增殖及其机制研究,南方医科大学学报,2006,26(7)：975～977
[105]吴东风,林明宝,舒虹等,复方半枝莲胶囊抗肿瘤作用的实验研究,实用中西医结合临床,2006,6(6)：79～80
[106]张妮娜,卜平,朱海杭等,半枝莲抑制肿瘤血管生成的作用及其机制研究,癌症,2005,24(12)：1459～1463
[107]李洁,石俊英,半枝莲对人肺巨细胞癌细胞株PG细胞端粒酶活性的影响,中药药理与临床,2006,22(3)：113～114
[108]李洁,半枝莲对人肺豆细胞癌细胞株PG细胞bcl-2基因表达的影响,中药药理与临床,2004,23(1)：46～47
[109]张春玲,胡俊峰,曲江斌等,几种中草药及绿茶对B(α)P和NNK的抗诱变作用,癌变·畸变·突变,2003,15(2)：101～103
[110]高冬,高永琳,白平,半枝莲对宫颈癌细胞钙信号系统的影响,中药材,2003,26(10):730～732
[111]杨容,黄志,习洋等,中药半枝莲中黄酮类化合物的抗氧化活性,河北大学学报(自然科学版),2007,27(5)：489～491
[112]王洪琦,崔娜娟,胡玲等,清热解毒和补益中药对小鼠腹水肝癌H-22细胞的作用及免疫学机制比较,广州中医药大学学报,2006,23(2):156～159
[113]蒋小岗,顾振纶,半枝莲化学成分和药理作用,中国野生植物资源,2004,23(1):3-5
[114]林敬明,刘煜,罗荣城.半枝莲的化学成分及其抗肿瘤作用的研究现状,2006,29(4):407～410
[115]Sonoda M, Nishiyama T, Matsukwa Y. Cytotoxic activities of flavonoids from two Scutellaria plants in Chinese medicine. J Ethnopharmcol,2004,91(1):65-68.
[116]糜松元,栾连军,半枝莲抗肿瘤活性成分及其药代动力学研究,浙江大学硕士论文,2006年5月
[117]Cardenas M., Marder M., et al. Antitumor activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines. Bioorganic & Medicinal Chemistry,14:2966-2971
[118]余建清,邹国林,半枝莲生物活性及相关化学成分研究,武汉大学博士论文,2005年4月
[119]张才华,郭兴杰,李芳等,HPLC法同时测定白花蛇舌草中齐墩果酸和熊果酸的含量,中草药,2004,21(5)：358～360
[120]罗新根,蒋琳兰,赵树进,高效液相色谱法同时测定白花蛇舌草中两组分的含量,药品检验,2004,15(3)：175～176
[121]谢艳,林圣云,任其龙,白花蛇舌草中齐墩果酸和熊果酸的HPLC测定,中国医药工业杂志,2006,37(8)：559～561
[122]薛梅,王自军,周静,半枝莲中总黄酮和多糖的微波提取与含量测定,时珍国医国药, 2005,16(10)：965～966
[123]李省,杨保华,陈悦,中药半枝莲总黄酮含量分析,中国公共卫生,2005,21(7):847-848
[124]李守拙,潘海峰,HPLC测定半枝莲中野黄芩苷的含量,中草药,2003,34(2)：183～184
[125]乔春峰,韩全斌,宋景政,RP-HPLC测定半枝莲药材中4种主要黄酮类成分的含量,中国药学杂志,2006,41(17)：1342～1344
[126]N. Maity, G. F. Payne. Adsorption from aqueous solutions based on a combination of hydrogen bonding and hydrophobic interactions. Langmuir.1991,7:1247-1254
[127]C. Renner, J. Piehler, T. Schrader. Arginine- and Lysine-Specific Polymers for Protein Recognition and Immobilization. J. Am. Chem. Soc.2006,128 (2):620-628
[128]严永清,中药现代研究的思路与方法,北京：化学工业出版社,2006,44～52
[1]M. Andersen, K. R. Markham. Flavonoids-Chemistry, Biochemistry and Applications, Taylor & Francis Group,2006
[2]朱英,何远,李丽,大孔树脂对地锦草、金银花叶与杭白菊总黄酮吸附分离性能研究,医学研究杂志,2007,37(1)：56～59
[3]于志,王敏,大孔树脂精制苦荞总黄酮工艺,中国中药杂志,2007,32(7)：585～589
[4]M. C. Xu, Z. Q. Shi, R. F. Shi, et al. Synthesis of the adsorbent based on macroporous copolymer MA-DVB beads and its application in purification for the extracts from Ginkgo biloba leaves. React. Funct. Polym.2000,43:297-304
[5]N. Maity, G. F. Payne. Adsorption from aqueous solutions based on a combination of hydrogen bonding and hydrophobic interactions. Langmuir.1991,7:1247-1254
[6]C. Renner, J. Piehler, T. Schrader. Arginine- and Lysine-Specific Polymers for Protein Recognition and Immobilization. J. Am. Chem. Soc.2006,128 (2):620-628
[7]乔春峰,韩全斌,宋景政等,RP-HPLC测定半枝莲药材中4种主要黄酮类成分的含量,中国中药杂志,2006,41(17)：1342～1344
[8]L. P. Li, H. D. Jiang. Determination and assay validation of luteolin and apigenin in human urine after oral administration of tablet of Chrysanthemum morifolium extract by HPLC. Journal of Pharmaceutical and Biomedical Analysis,2006,41:261-265
[9]何炳林,黄文强,离子交换与吸附树脂,上海科技出版社,1995
[10]国家药典委员会.中国药典2005年版,北京：化学工业出版社,234
[11]国家药典委员会.中国药典2005年版,北京：化学工业出版社,234
[12]A. Hasler, O. Sticher, B. Meier. Identification and determination of the flavonoids from Ginkgo biloba by high-performance liquid chromatography. Journal of chromatography 1992, 605:41-48
[13]R. A. Garcla-Delgado, L. M. Cotouelo-Minguez, J. J. Rodfiguez. Equilibrium study of single-solute adsorption of anionic surfactants with polymeric XAD resins. Sep Sci Techn, 1992,27 (7):975-987
[14]G. F. Payne, N. Maity. Solute Adsorption from water onto a "modified" sorbent in which the hydrogen binding site Is protected from water, Thermodynamics and Separations. Ind Eng Chem Res,1992,31:2024-2033
[15]王庆文,杨玉桓,高鸿宾.有机化学中的氢键问题.天津：天津大学出版社,1993
[16]冯年平,郁威.中药提取分离技术原理与应用.中国医药科技出版社,北京,2005.124～128
[1]Q.Li, J. H. Wu, D. J. Guo, et.al. Suppression of diet-induced hypercholesterolemia by scutellarin in rats. Planta Med,2009,75 (11):1203-1208
[2]L. L. Lin, A. J. Liu, J. H. Liu. Protective effects of scutellarin and breviscapine on brain and heart ischemia in rats. Journal of Cardiovascular Pharmacology,2007,50 (3):372-332
[3]H. Hong, G. Q. Liu. Protection against hydrogen peroxide-induced cytotoxicity in PC 12 cells by scutellarin. Life Sciences,2004,74 (24):2959-2973
[4]赵铁华,邓淑华,高巍等.野黄芩苷镇痛作用的实验研究.中国中医药信息杂志,2001,8(6)：28～29
[5]G. H. Zhang, Q. Wang, J. J. Chen, et al. The anti-HIV-1 effect of scutellarin. Biochemical and Biophysical Research Communications,2005,334 (3):812-816
[6]J. Y. Chan, B. K. Tan, S. C. Lee. Scutellarin Sensitizes Drug-evoked Colon Cancer Cell Apoptosis through Enhanced Caspase-6 Activation. Anticancer Research,2009,29 (8): 3043-3047
[7]崔建梅,吴淞.野黄芩苷的研究进展.天然产物研究与开发,2003,15(3)：255～263
[8]W. Xu, Z. P. Zha, W. Y. Wang, et al. Effects of scutellarin on PKCy in PC12 cell injury induced by oxygen and glucose deprivation. Acta. Pharmacol. Sin.2007,28:1573-1579
[9]B. H.Zhu. Scutellarein, a potent preventer of diabetic complications. Journal of Pharmacological Sciences,109,1:231-235
[10]K. Plochmann, G. Korte, E. Koutsilieri, et al. Structure-activity relationships of flavonoid-induced cytotoxicity on human leukemia cells. Arch. Biochem. Biophys.,2007, 460:1-3
[11]J. H. Kim, E. O. Lee, H. J. Lee, et al. Annals of the New York Academy of Sciences,1095, Issue Signal Transduction Pathways, Part C,598
[12]H. J. Lee, C. J. Wang, H. C. Kuo, et al. Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of BaxBak and activation of JNK. Toxicol. Appl. Pharmacol.2005,203,124-131
[13]W. Q. Wang, L. Heideman, C. S. Chung, et al. Cell-Cycle Arrest at G2M and Growth Inhibition by Apigenin in Human Colon Carcinoma Cell Lines, Mol. Carcinog.,2000,28: 102-110
[14]I. K. Wang, S. Y. Lin-Shiau, J. K. Lin, Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells, Eur. J. Cancer,1999,35 (10):1517-1525
[15]S. J. Dai, L. Shen, Y. Ren, J. Integ. Plant Bio.2008,50:699-704
[16]S. A.M. Abdelgaleil, M. A. Abbassy, A. S. H. Belal, M. A.A. Abdel Rasoul, Bioresour. Technol.2008,99:5947-5955
[17]Y. W. Ha, Y. S. Kim, Phytochem. Anal.2009,20:207-212
[18]D. J. Comeskey, M. Montefiori, P. J. B. Edwards, T. Mcghie, J. Agric. Food Chem.2009,57: 2035-2038
[19]G. T. Jia, X. Y. Lu, J. Chromatogr. A.2008,1193:136-142
[20]C. Y. Ma, J. Tang, H. X. Wang, G. J. Tao, X. H. Gu, L. M. Hu, J. Sep. Sci.2009,32:185-192
[21]Y. J. Fu, Y. G. Zu, W. Liu, T. Efferth, N. J. Zhang, X. N. Liu, Y. Kong, J. Chromatogr. A.2006, 1137:145-152
[22]Y. Zhao, B. Chen, S. Z. Yao, Sep. Purif. Technol.2007,52:533-541
[23]R. Sun, K. Fu, Y. J. Fu, Y. G. Zu, Y. Wang, M. Luo, S. M. Li, H. Luo, Z. N. Li, J. Sep. Sci., 2009,32:1284-1288
[24]J.B. Wan, Q.W. Zhang, W.C. Ye, Y. T. Wang, Sep. Purif. Technol.2008,60:198-205
[25]F. E. Bernardin. Experimental design and testing of adsorption and adsorbates. In:F L Slejko (ed). Adsorption Technology, New York:Marcel Dekker,1985,1-5
[26]方宾,阚显文.近代分离方法导论.合肥：安徽人民出版社,2006.95-96
[27]W. J. Weber. Adsorption theory, concepts, and models. In:F L Slejko (ed). Adsorption Technology. New York:Marcel Dekker,1985.31-35
[1]黄炜,黄济群,张东等,18p-甘草次酸和甘草酸对人肝癌细胞增殖的抑制和诱导分化作用,中国中医药科技,2002,9(2)：92～93
[2]Baek J H, Lee YS, Kang CM, et al. Intracellular Ca2+ release mediates ursolic acid-induced apoptosis in human leukemic HL-60 cells. Int. J. Cancer,1997,73 (5):725-728
[3]黄炜,黄济群,张东等,五环三萜类化合物抗人肺癌细胞侵袭和诱导细胞凋亡的研究,中国肺癌杂志,2003,6(4)：254～257
[4]张瑞玲,黄炜,黄济群,中药五环三萜类有效成分抗人肺癌细胞增殖和侵袭作用的研究,中医药学刊,2004,22(11)：2065～2067
[5]孟艳秋,赵临襄,王趱等,五元三萜类化合物的构效关系,中国新药杂志,2004,13(12):1098～1102
[6]张才华,郭兴杰,李芳等,HPLC法同时测定白花蛇舌草中齐墩果酸和熊果酸的含量,沈阳药科大学学报,2004,24(9)：358～360,370
[7]Liu Jie. Oleanolic acid and ursolic acid:Research perspectives. Journal of Ethnopharmacology, 2005, (100):92-94
[8]Young HS, Chung HY, Lee CK, et al. Ursolic acid inhibits aflatoxin Bl-induced mutagenicity in a Salmonella assay system. Biol. Pharm. Bull.,1994,17 (7):990-992
[9]Lee HY, Chung HY, Kim KH, et al. Induction of differentiation in the cultured F9 terato-carcinoma stem cells by triterpene acids. J. Cancer Res. Clin. Oncol.,1994,120 (9):513-518
[10]Baek J H, Lee YS, Kang CM, et al. Intracellular Ca2+ release mediates ursolic acid-induced apoptosis in human leukemic HL-60 cells. Int. J. Cancer,1997,73 (5):725-728
[11]A. A. Ramos, C. F. Lima, M. L. Pereira et al. Antigenotoxic effects of quercetin, rutin and ursolic acid on HepG2 cells Evaluation by the comet assay. Toxicology Letters,2008,177: 66-73
[12]E. K. Yim, M. J. Lee, K.H. Lee et al. Antiproliferative and antiviral mechanisms of ursolic acid and dexamethasone in cervical carcinoma cell lines. Int. J. Gynecol. Cancer,2006,16: 2023-2031
[13]Irene Bonaccorsi, Fabio Altieri, Ilaria Sciamanna, et al. Endogenous reverse transcriptase as a mediator of ursolic acid's anti-proliferative and differentiating effects in human cancer cell lines. Cancer Letters,2008,263:130-139
[14]Hsue Yin Hsu, Jenq Jer Yang, Chun Ching Lin. Effects of oleanolic acid and ursolic acid on inhibiting tumor growth and enhancing the recovery of hematopoietic system postirradiati on in mice. Cancer letters,1997,2(1):7-13
[15]任秀莲,周春山,魏琦峰等,苦丁茶中熊果酸的分离纯化工艺研究,中国药学杂志,2004,39(8)：571～574
[16]谷芳芳,任秀莲,吴梅林,大孔吸附树脂对熊果酸吸附性能的研究,广州化学,2006,31(2)：24～27
[17]何思煌,祝红,大孔树脂吸附提取五味子的有效成分乌索酸,临床和实验医学杂志,2007,6(1)：143～144
[18]李丽,陈剑锋,林国荣,枇杷叶中熊果酸的分离纯化,现代化工,2007,2:415～147
[19]谢艳,林圣云,任其龙,白花蛇舌草中齐墩果酸和熊果酸的HPLC测定,中国医药工业杂志,2006,37(8)：559～561
[20]何炳林,黄文强,离子交换与吸附树脂,上海：上海科技出版社,1995
[21]刘永宁,史作清,施荣富等,极性修饰的聚苯乙烯吸附树脂的结构和性能,高等学校化学学报,1994,15(3)：450～453
[1]张勇,胡笑克,陈维真等,激素难治性前列腺癌细胞对化疗药物敏感性的比较,标记免疫分析与临床,2003,10(2)：159～261
[2]李经伦,刘长征,3H-TdR应用研究现状,放射免疫学杂志,2004,17(1)：64～67
[3]H. Tominaga, Ishiyama M, et al. A water-soluble tetrazolium salt useful for colorimetric cell viabiLity assay, Anal. Commum.,1999,36:47-50
[4]S. Indah, F. K. Tantular. An improved simple screening for detection of glucose-6-phosphate dehydrogenase deficiency, Tropical Medicine,2003,8(6):569-675
[5]T. Kuzuko, O. Motoyasu, U. Hideo, A simple bioassay for evaluating immunotoxic properties of chemicals by use of in vitro antibody production system. Journal of Health Science,2002,48(2):161-167
[6]陈学清,杨晓鸥,潘国宗等,酸性磷酸酶法在体外化疗药物筛选中的应用,癌症,1998,17(6)：477～478
[7]D. T. Connolly, M. B. Knight, N. Harakas, et al. Determination of number of endothelial cells in culture using an acid phosphatase assay, Anal. Biochem.,1986,152:136
[8]J. D. Moyer, J. F. Henderson, Ultrasensitive assay for ribonucleo side triphosphates in 50-1000 cells. Biochem. Pharmacol.,1983,32:3831
[9]陈历排,周雯,李志阳,用微量板ATP生物发光法检测肿瘤细胞对化疗药物的敏感性.肿瘤,2000,20(2)：103～105
[10]司徒镇强,吴军正,细胞培养,西安：世界图书出版社,1996,136.
[11]T. Mosmann, Rapid colorimetric assay for celluar growth and surival. J. Immumol. Methods, 1983,65:551
[12]吴慧,抗肿瘤药物筛选方法,医学理论和实践,2005,18(2)：146～147
[13]P. Skehan, R. Storeng, D. Scudiero, et al. New colorimetric cytotoxicity assay for anticancer drug screening, J. Nat. Cancer Ins.,1990,82(13):1107-1112
[14]谭卫东,金红,罗第祥等,抗肿瘤药物筛选中MTT法与SRB法的比较,天然产物研究与开发,1999,11(3)：217～221
[15]于永洲,白花蛇舌草DNA指纹图谱与体外抗肿瘤活性的相关性研究,沈阳药科大学硕士论文,2004.5
[16]于春艳,李薇,刘玉和等,白花蛇舌草提取物体外抗肿瘤作用及机制研究,北华大学学报(自然科学版),2004,5(5)：412～416
[17]周志愉,王兆雷,不同乙醇浓度半枝莲提取物抗肿瘤作用的相关性研究,实用中西医结合临床,2007,7(6)：89～90