Yang Huan, Zheng Jie, Wang Hai-Yan, Li Nan, Yang Ya-Ya, Shen Yu-Ping
Department of Chinese Medicine and Pharmacy, School of Pharmacy, Jiangsu University, Zhenjiang 212013, China.
Research Centre for Herbalomics and Drug Discovery, School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
Pharmacogn Mag. 2017 Oct-Dec;13(52):663-667. doi: 10.4103/pm.pm_54_17. Epub 2017 Nov 13.
Gelatinous Chinese medicines (GCMs) including Asini Corii Colla, Testudinis Carapacis ET Plastri Colla, and Cervi Cornus Colla, were made from reptile shell or mammalian skin or deer horn, and consumed as a popular tonic, as well as hemopoietic and hemostatic agents. Misuse of them would not exert their functions, and fake or adulterate products have caused drug market disorder and affected food and drug safety. GCMs are rich in denatured proteins, but insufficient in available DNA fragments, hence commonly used cytochrome c oxidase I barcoding was not successful for their authentication.
In this study, we performed comparative proteomic analysis of them and their animal origins to identify the composition of intrinsic proteins for the first time.
A reliable and convenient approach was proposed for their authentication, by the incorporation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional electrophoresis, and matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS).
A total of 26 proteins were identified from medicinal parts of original animals, and GCMs proteins presented in a dispersive manner in electrophoresis analyses due to complicated changes in the structure of original proteins caused by long-term decoction and the addition of ingredients during their manufacturing. In addition, by comparison of MALDI-TOF/TOF-MS profiling, 19 signature peptide fragments originated from the protein of GCM products were selected according to criteria.
These could assist in the discrimination and identification of adulterates of GCMs and other ACMs for their form of raw medicinal material, the pulverized, and even the complex.
Comparative proteomic analysis of three gelatinous Chinese medicines was conducted, and their authentications were based on tryptic-digested peptides profiling using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. GCMs: Gelatinous Chinese medicines, COI: Cytochrome c oxidase I, SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2-DE: Two-dimensional electrophoresis, MALDI-TOF/TOF-MS: Matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry, LC: Liquid chromatography, ChP: Chinese Pharmacopoeia, HPLC: High performance liquid chromatography, LC-ESI-MS: Liquid chromatography-electro spray ionization-mass spectrometry, IEF: isoelectric focusing, HCCA: α-Cyano-4-hydroxycinnamic acid.
胶类中药包括阿胶、龟甲胶和鹿角胶，由爬行动物的壳、哺乳动物的皮或鹿角制成，作为一种受欢迎的滋补品以及造血和止血剂使用。滥用它们不会发挥其功效，而假冒或掺假产品已导致药品市场混乱并影响食品药品安全。胶类中药富含变性蛋白质，但可用的DNA片段不足，因此常用的细胞色素c氧化酶I条形码技术无法成功对其进行鉴定。
在本研究中，我们首次对它们及其动物来源进行了比较蛋白质组学分析，以鉴定其内在蛋白质的组成。
通过结合十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳、二维电泳和基质辅助激光解吸/电离飞行时间/飞行时间质谱（MALDI - TOF/TOF - MS），提出了一种可靠且便捷的鉴定方法。
从原始动物的药用部位共鉴定出26种蛋白质，由于长期煎煮以及生产过程中添加成分导致原始蛋白质结构发生复杂变化，胶类中药蛋白质在电泳分析中呈分散分布。此外，通过比较MALDI - TOF/TOF - MS图谱，根据标准选择了19个源自胶类中药产品蛋白质的特征肽片段。
这些有助于鉴别和识别胶类中药及其他类似中药原料形式、粉碎形式甚至复杂形式的掺假品。
对三种胶类中药进行了比较蛋白质组学分析，并基于使用基质辅助激光解吸/电离飞行时间/飞行时间质谱的胰蛋白酶消化肽图谱对其进行鉴定。GCMs：胶类中药，COI：细胞色素c氧化酶I，SDS - PAGE：十二烷基硫酸钠聚丙烯酰胺凝胶电泳，2 - DE：二维电泳，MALDI - TOF/TOF - MS：基质辅助激光解吸/电离飞行时间/飞行时间质谱，LC：液相色谱，ChP：《中国药典》，HPLC：高效液相色谱，LC - ESI - MS：液相色谱 - 电喷雾电离质谱，IEF：等电聚焦，HCCA：α - 氰基 - 4 - 羟基肉桂酸
