摘要
来源于大自然的中药是凝聚了人类千百年智慧的结晶,已成为我国临床广泛使用的特色药品。在2009年发布的《国家基本药物目录》里,中成药占全部品种的三分之一。由于历史上受限于当时的科技水平,许多中成药的药效物质及作用机制均不甚明了,导致难以确保这些药品的安全、有效和质量可控。近十几年来,我国政府大力推进中药现代化研究,倡导采用现代科技手段对中成药进行二次开发,从而提高中药质量标准和临床疗效;另一方面,实施国家科技重大专项,开展现代中药新药创制研究,努力实现“药效物质基本明确、作用机制基本清楚”的目标。这就对中药及天然药物化学研究提出了极大挑战,同时也为生物化学工程学科开辟新方向提供了历史机遇。
寻找发现中药或药用植物活性成分,揭示其药效物质基础及其作用机制是中药研究领域的核心科学问题之一,其关键点是建立先进适宜的分离分析方法。换句话说,中药或药用植物化学物质的有效分离和成分鉴定是中药现代化研究重点。高效液相制备色谱具有高分离效率和多种分离模式等特点,已成为分离复杂物质体系的有力工具,决定其分离效能的关键因素是色谱操作条件。目前,制备液相色谱的分离条件主要凭经验选定,存在较大的盲目性。为此,极有必要深入研究液相制备色谱分离理论模型及洗脱规律,进而创新发展液相制备色谱分离条件优化方法。
本文首先选取荚莲和鸡血藤为具体研究对象,采用传统的分离方法制备得到化学组分。通过对荚蒾的甲醇提取物中化学成分的分析,鉴定,发现了一个新的化合物2'-(β-glucopyranosyloxy)-benzyl 3-(β-glucopyranosyloxy)-benzoate。通过体外细胞试验,筛选出鸡血藤中抗癌活性组分,并采用RP-HPLC-MSn对活性组分进行结构分析,推断出相应的化学组成主要为9种黄酮类物质。其中,7-羟基-3',4'-二甲氧基-异黄酮、7-羟基-6,2',4'-三甲氧基-异黄酮和3'-羟基-7,4'-二甲氧基-异黄酮在该植物中均为首次报导。
在分析型RP-HPLC对鸡血藤黄酮类化合物分离分析的基础上,根据吸附分离原理,结合现有的液相色谱保留模型,采用matlab软件建立了计算模块,实现了对同种物质在制备型RP-HPLC上保留时间的预测。在设定分离指标后,可优化选择制备型RP-HPLC的分离操作条件。与凭借色谱操作者经验确定制备液相色谱分离条件的传统方法相比,本文所建方法可在药用植物种类变化和化学组成波动的情况下,便捷、可靠地优化选择制备色谱分离条件,提高植物成分的分离效率,节约大量的样品和溶剂。
在上述制备液相色谱分离条件预测与优化方法研究的基础上,作者进一步研究开发了新的SID集成方法,用以分离和鉴定植物中的酚酸类成分,并能高通量、便捷地测定复杂物质体系中传统方法较难测定的重要物性参数pKa值。该流程由酚酸物质判定、pKa预测定、制备色谱分离条件优化、纯品制备、物质鉴定和pKa确定等六个步骤组成。本文选取丹参酚酸体系开展实例研究,采用SID集成方法测定了丹参素、咖啡酸、紫草酸、迷迭香酸、丹酚酸B和丹酚酸A的pKa值。在优化的制备色谱分离条件下,制得了未知样的纯品,并且采用HPLC-MS和'HNMR鉴定了未知酚酸类物质为丹酚酸A。该方法可以明显提高酚酸类成分的分离效率,且在无需获得酚酸纯品的前提下便可以得到其解离常数pKa,因此具有良好的应用前景。
Traditional Chinese medicine coming from the nature is the intelligence gathering of human beings and has been used widely in clinic as a special drug. Chinese patent medicines account for one thirds of all drugs listed in the catalogue of basic medicines of the state promulgate in 2009. Limited by the level of technology in history, many bioactive components and the corresponding mechanism of action of the Chinese patent medicines are not clearly understood. Thus it is hard to make sure the security, the effectiveness and the quality control of these drugs. Over the past decade, our government promoted the researches of the traditional Chinese medicine with modern technologies to improve the quality standard and the clinic curative effect of them. On the other hand, the state carries out major science and technology projects and promotes the development of new traditional and herbal drugs for the aim of bioactive components basically known and the corresponding mechanism of action basically clear. So it poses a rigorous challenge for the study of traditional Chinese medicine and natural pharmaceutical chemistry. At the same time, it also affords a chance to open up a new field for biochemical engineering discipline.
It is the heart of the matter to find out the active components from traditional Chinese medicines or medicinal plants and reveal the bioactive components and the corresponding mechanism of action in the field of the research of Chinese medicine. The key point is to build up the suitable method for separation and analysis. In other words, it is the emphasis of the modern investigation of traditional Chinese medicines or medicinal plants to separating the bioactive components and identifying the corresponding compounds. Preparative high performance liquid chromatography (HPLC), with high separation effect and a lot of separation modes, has been powerful tool for separating complex systems including many compounds. The separation effect of preparative HPLC is decided by operation conditions of chromatography. At present, the operation conditions for the preparative HPLC are mainly selected by operators according their experiences, which is blindfold. So it is necessary to study the theoretical model of separation and the elution rules of preparative HPLC intensively and to develop the new method for optimizing the operation conditions of chromatography.
In this paper, we selected Viburnum dilatatum Thunb. and Caulis Spatholobi as subjects investigated at first and separated compounds with traditional methods. To analyze compounds extracted from Viburnum dilatatum Thunb. with methanol, a new compound,2-(β-glucopyranosyloxy)-benzyl 3-((3-glucopyranosyloxy)-benzoate, was found out. According to the anti-tumor experiment in vitro, we screened out bioactive components and analyzed the structures of the corresponding compounds with HPLC-MSn. The results showed that the bioactive components mainly contained nine flavones and three isoflavones:7-hydroxy-3',4'-dimethoxy-isoflavone, 7-hydroxy-6,2',4'-trimethoxy-isoflavone and 3'-hydroxy-7,4'-dimethoxy-isoflavone were reported at first time in this plant.
Basing on the analysis of flavones from Caulis Spatholobi with analytical reverse-phase HPLC (RP-HPLC) and according to the retention models of chromatography, we predicted the retention time and the peak width of the same substance in preparative RP-HPLC with the computer programs wrote with matlab soft. The optimized operation conditions would be predicted after setting the separation index. Compared with traditional method for deciding the operation conditions of chromatography, the method developed in this paper was with advantages of flexible, convenient, high effective and economical.
On the base of the study of the new method for prediction of separation conditions of preparative RP-HPLC, SID, a new integration method, was developed again, which could be used to separate and identify phenolic acids from plants and determine parameter of pKa of compounds in complex system at the same time. The SID method included processes of phenolic acids judgement, pKa prediction, conditions of preparative RP-HPLC optimization, sample purification, substance identification and pKa determination. In this paper, we selected phenolic acids from Danshen as subject investigated and determined the pKa values of danshensu, caffeic acid, lithospermic acid, rosmarinci acid, salvianic acid B and salvianic acid A. The unknown compound prepared with preparative RP-HPLC was identified with HPLC-MS and 'H NMR. The result showed it was salvianic acid A. The method could improved the separation effect of phenolic acids and test the pKa values of them without getting pure samples of phenolic acids. So the SID method was a promising method.
引文
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