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青岛能源所等开发出中成药生物成分鉴定新技术

摘要 : 近日,由中国科学院青岛生物能源与过程研究所单细胞研究中心生物信息学团队与山东省医学科学院药物研究所组成的联合研究团队提出了基于高通量测序数据分析的中成药生物成分分析新技术,并在国际上首次评估了该技术对中成药“六味地黄丸”进行全面生物成分(处方物种和杂质物种)分析的可行性。相关成果于6月3日在线发表于Nature出版集团旗下期刊Scientific Reports 。

中成药(Traditional Chinese Medicine Preparation)是以中药材为原料,经加工制成的各种不同剂型的药品,是我国历代医药学家经过千百年医疗实践创造、总结的有效方剂的精华。中成药的成分分析一般包括生物成分(即物种组成)分析和化学成分分析。然而,目前我国中成药的成分分析主要聚焦在后者,通过光谱和色谱手段对中成药的化学成分进行测定,但不能鉴别出其中的杂质成分。

近日,由中国科学院青岛生物能源与过程研究所单细胞研究中心生物信息学团队与山东省医学科学院药物研究所组成的联合研究团队提出了基于高通量测序数据分析的中成药生物成分分析新技术,并在国际上首次评估了该技术对中成药“六味地黄丸”进行全面生物成分(处方物种和杂质物种)分析的可行性。相关成果于6月3日在线发表于Nature出版集团旗下期刊Scientific Reports 。

基于高通量测序及元基因组分析对不同厂商生产的“六味地黄丸”中处方物种和杂质物种数量的分析。

基于高通量测序及元基因组分析对不同厂商生产的“六味地黄丸”中处方物种和杂质物种数量的分析。蓝色代表处方物种,红色代表杂质物种。(a) 采用ITS2序列作为生物标记序列的结果;(b) 采用trnL序列作为生物标记序列的结果。

助理研究员苏晓泉和硕士研究生程新玮等利用高通量测序和元基因组数据分析手段,对中成药的测序数据中处方物种和杂质物种的ITS2以及trnL生物标记序列同时进行无差别识别,从而实现了对中成药生物成分的全面分析和品质初步评估。通过该方法对不同厂商、不同批次的“六味地黄丸”进行了一致性比较,从而对其品质进行了评价。

由于此种方法通用性较强,可在无需组织形态鉴定的专业知识背景下,基于分子标记和信息学分析对物种进行鉴定,解决了中成药中处方物种难以准确检测和杂质物种难以全面评估等瓶颈问题,故在中药及其相关制剂的生物成分分析方面有着巨大的市场潜力。研究人员将继续针对中成药多种复杂成分的现实,开发整合生物和化学方法的新一代中成药成分分析技术,从而更全面地评估中成药品质。

目前,相关研究成果已经分别发表在Scientific Reports、Genomics,Proteomics & Bioinformatics等期刊,相关技术也已申请专利保护。该项目由宁康研究员和山东省医学科学院药物研究所白虹副研究员共同主持完成。

原文摘要:

Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughput sequencing: the story for Liuwei Dihuang Wan

Xinwei Cheng, Xiaoquan Su, Xiaohua Chen, Huanxin Zhao, Cunpei Bo, Jian Xu, Hong Bai &Kang Ning

Although Traditional Chinese Medicine (TCM) preparations have long history with successful applications, the scientific and systematic quality assessment of TCM preparations mainly focuses on chemical constituents and is far from comprehensive. There are currently only few primitive studies on assessment of biological ingredients in TCM preparations. Here, we have proposed a method, M-TCM, for biological assessment of the quality of TCM preparations based on high-throughput sequencing and metagenomic analysis. We have tested this method on Liuwei Dihuang Wan (LDW), a TCM whose ingredients have been well-defined. Our results have shown that firstly, this method could determine the biological ingredients of LDW preparations. Secondly, the quality and stability of LDW varies significantly among different manufacturers. Thirdly, the overall quality of LDW samples is significantly affected by their biological contaminations. This novel strategy has the potential to achieve comprehensive ingredient profiling of TCM preparations.

DNA Extraction Protocol for Biological Ingredient Analysis of LiuWei DiHuang Wan

Xinwei Cheng, Xiaohua Chen, Xiaoquan Su, Huanxin Zhao, Maozhen Han, Cunpei Bo, Jian Xu,Hong Bai, Kang Ning

Traditional Chinese medicine (TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients, which often include some mis-identified herbal materials, adulterants or even some biological contaminants. For biological ingredient analysis, the efficiency of DNA extraction is an important factor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and the DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation, Liuwei Dihuang Wan (LDW), as an example to develop a TCM-specific DNA extraction method. An optimized CTAB method (TCM-CTAB) and three commonly-used extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer (ITS2) and the chloroplast genome trnL intron was carried out. The results have shown that PCR amplification was successful only with templates of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3∼4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting TCM-CTAB method could facilitate follow-up DNA-based examination of TCM preparations.

来源: 中科院 浏览次数:93

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