Molecular Authentication of Chinese Medicinal Materials in Decoctions and Concentrated Chinese Medicine Granules

• Other Authors: Lo, Yat Tung (author.)
• Format: Others
• Language: English; Chinese
• Published: 2016
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292169

中醫藥用作醫療用途已有數千年的歷史，其中服用傳統湯劑及現代中藥顆粒是目前最主要的治療方法。不過，由於他們的藥材表徵已被破壞，傳統的性狀及顯微鑑定方法並不適用。儘管化學分析技術已被廣泛採納作藥材鑑定用途，湯劑或中藥顆粒所產生的化學圖譜是非常複雜，而且其結果會受到不同的成長環境所影響。此外，親緣關係相關的物種，其化學成分十分相似，故此化學方法並不能作準確區分。相比之下，採用分子鑑定方法分析物種獨特的DNA序列是較爲準確。2010年版的《中國藥典》亦首次加入分子鑑定技術，爲藥材分子鑑定提供指引。 === 雖然分子鑑定技術提供了諸多優勢，傳統認爲DNA在藥材煎煮或生產過程中會被降解，而且藥材中的PCR抑制物會導致假陰性結果，故此目前分子鑑定仍局限於原藥材層面。因此，本研究旨在開發分子鑑定技術，爲湯劑及中藥顆粒提供有效的鑑別及質控指引。 === 本研究模擬一般中藥湯劑的製作過程，把藥材以水煮製不同時間。我們發現DNA的完整性會隨煮製時間而下降，故此鑑定湯劑或市購湯包需要透過擴增短DNA片段來完成。此外，我們亦利用多重PCR（multiplex PCR）作爲有效的鑑定方法。 === 爲制定最佳的DNA提取方法，我們比較了傳統CTAB法及試劑盒方法來提取中藥顆粒中的DNA。結果表明合併使用上述兩種方法能大大改善DNA的質量和PCR的成功率，而提取高純度的DNA比提取高濃度的DNA更爲重要。另一方面，透過設計特異引物，中藥顆粒中的藥材基原能與目標偽品區分出來。 === 本研究亦通過定量PCR（qPCR）以測定湯劑藥材及中藥顆粒的份量。重複性試驗顯示使用10-100毫克的中藥顆粒能產生較爲準確的結果。而在測定混合中藥顆粒樣本時，誤差值少於3毫克。不過，在湯劑中qPCR只能粗略地顯示原藥材的重量。 === DNA條形碼（DNAbarcode）常用於鑑定未知物種，不過完整的DNA條形碼片段不容易於湯劑及中藥顆粒中找到。此外，微型DNA條形碼（DNA mini-barcode）通常難以提供足夠的物種分辨能力。透過改良外源接頭介導PCR （adaptor ligation-mediated PCR），樣本中位於目標位置附近不同長度的DNA片段能夠擴增，並且提供足夠的鑑定能力。 === 本研究把分子鑑定技術延伸到中藥產品。本研究的成果更可以進一步開發成診斷試劑盒及爲檢測及認證實驗室提供技術指引。 === Traditional Chinese medicine (TCM) have been used for therapeutic purposes over thousands of years and the two major ways of TCM consumption are decoctions and concentrated Chinese medicine granules (CCMG). In terms of authentication, traditional methods such as organoleptic and microscopic identification are less applicable for them since their visual characteristics are destroyed. Although chemical identification may be applied, chemical profiles are complicated and usually indistinguishable between related adulterants. Additionally, chemical constitutes change upon different developmental stages. Molecular authentication is comparatively more accurate since it is based on unique DNA sequences, and it is first stated officially in the 2010 edition of the Chinese Pharmacopoeia. === Although molecular authentication gives several advantages, its applicability on processed herbal products is not yet clear and it is now restricted for crude herbal materials. Extensive DNA fragmentation and co-extraction of PCR inhibitory substances may lead to false-negative results. This study is thus undertaken to find the availability of DNA in decoctions and CCMG, and find the applicability of various molecular techniques for identification and quality control. === In this study, decoctions were made by boiling multi-herbs formulations in water. It was observed that intactness of DNA decreased with prolonged boiling. By designing species specific primers targeting on short DNA sequences, constituent herbs could be identified in the decoctions as well as a commercial soup product. In addition, multiplex PCR was developed for effective authentication. === To formulate a DNA extraction protocol for removing various inhibitory substances in CCMG, CTAB extraction and column kit extraction were tested. It was found that combination of CTAB and column kit extraction methods greatly improved the quality of DNA extracted and PCR successful rate. In addition, it was observed that extracting high quality DNA was more important than extracting a large amount of DNA. Besides, diagnostic primers could differentiate between genuine and adulterant species. === This project also aimed at quantifying herbal amount by quantitative PCR (qPCR). It was found that herbal quantities determined between different batches of 10 to 100 mg of CCMG were consistent. These findings thus enable determination of constitute herbal amount in mixed CCMG with low discrepancy less than 3 mg. However, only rough quantification results were given for herbal materials in decoction. === Determination of unknown species identity in herbal products is difficult since full-length DNA barcodes cannot be obtained. Moreover, mini-barcodes do not give sufficient differentiation power. To solve this problem, a modified adapter ligation-medic === Lo, Yat Tung. === Thesis Ph.D. Chinese University of Hong Kong 2016. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.