Objective: To establish an effective method for detecting the components of Lychas mucronatus and investigating instances of adulteration with whole scorpions in Chinese patent medicines. Methods: Thirty Chinese patent medicines containing whole scorpion components, as documented in the Pharmacopoeia of the People's Republic of China, were collected. DNA was extracted, and differences in the mitochondrial cytochrome C oxidase subunit Ⅰ(COⅠ) gene sequences were compared. Specific primers for the identification of Mesobuthus martensii and Lychas mucronatus DNA were designed. Annealing temperature, cycle number, Taq polymerase, and DNA concentration were optimized to establish the most suitable PCR identification system and conditions. The optimal PCR conditions for distinguishing the sharp-tailed wolf scorpion were finally determined to be an annealing temperature of 52 ℃, 36 cycles, 2×PCR Mix Taq enzyme, and 1 μL of DNA template. The developed method was applied to identify the presence of Centruroides gracilis components in 30 commercially available Chinese patent medicines. Results: Gel electrophoresis revealed the presence of authentic DNA bands specific to the Mesobuthus martensii in all 30 whole scorpion-containing Chinese patent medicines. Eighteen medicines (60%) showed a single specific DNA band between 100-250 bp, indicative of Lychas mucronatus, while the blank control exhibited no bands. Sequencing of the PCR products containing the identified Lychas mucronatus -specific bands, after TA cloning, revealed sequences with high similarity (92%-98%) to the reference sequences of Lychas mucronatus. Phylogenetic analysis indicated that the obtained sequences belonged to the same lineage as Lychas mucronatus and were distinct from the Mesobuthus martensii. Therefore, the presence of a single band around 120 bp indicates the identification of Lychas mucronatus. Conclusion: The presence of Lychas mucronatus components mixed with authentic scorpion materials in Chinese patent medicines indicates a need for strengthened supervision. The method established in this study enables the rapid and accurate detection of Lychas mucronatus components in Chinese patent medicines. This is beneficial for improving the quality control of whole scorpion-containing Chinese patent medicines, providing assurance for their clinical applications.
[1] 中华人民共和国药典2020年版. 一部[S]. 2020: 149
ChP 2020. Vol Ⅰ[S]. 2020: 149
[2] NNUES-NESI A, ARAUJO WL, OBATA T, et al. Regulation of the mitochondrial tricarboxylic acid cycle[J]. Curr Opin Plant Biol, 2013, 16(3): 335
[3] 卫秋阳, 邓小书, 竭航, 等. 药用全蝎毒液的蛋白差异分析[J]. 亚太传统医药, 2023, 19(5): 42
WEI QY, DENG XS, JIE H, et al. Analysis of protein differences in the venom of medicinal scorpions[J]. Asia Pac Tradit Med, 2023, 19(5): 42
[4] KOCH LE. The taxonomy, geographic distribution and evolutionary radiation of Australo-papuan scorpions[J]. Rec West Aust Mus, 1977, 5(2): 83
[5] 陈爱娟. 新发现的一种全蝎伪品细尖狼蝎[J]. 河南中医, 2015, 35(12): 3197
CHEN AJ. Identification and preliminary study of a newly discovered counterfeit scorpion Lychas mucronatus[J]. Henan Tradit Chin Med, 2015, 35(12): 3197
[6] 李文鑫. 蝎生物学与毒素[M]. 北京: 科学出版社, 2016: 216
LI WX. Scorpion Biology and Toxins[M]. Beijing: Science Press, 2016: 216
[7] 陈广玉, 田慧, 赵成坚, 等. 蛤蚧分子鉴定技术研究进展[J]. 沈阳药科大学学报, 2021, 38(3): 328
CHEN GY, TIAN H, ZHAO CJ, et al. Advances in molecular identification techniques of Gekko gecko[J]. J Shenyang Pharm Univ, 2021, 38(3): 328
[8] 刘晶晶, 杨晶凡, 姚令文, 等. 基于COⅠ基因对中成药中土鳖虫的分子鉴别研究[J]. 药物分析杂志, 2023, 43(5): 880
LIU JJ, YANG JF, YAO LW, et al. Molecular identification of Eupolyphaga steleophaga in Chinese patent medicine based on COⅠ gene[J]. Chin J Pharm Anal, 2023, 43(5): 880
[9] 王丽, 金艳, 蒋超, 等. 猪胆粉及其中成药的特异性PCR鉴别方法[J]. 中国实验方剂学杂志, 2019, 25(17): 136
WANG L, JIN Y, JIANG C, et al. Specific PCR method for identification of Suis Fellis Pulvis and its Chinese patent medicines[J]. Chin J Exp Tradit Med Form, 2019, 25(17): 136
[10] 苟惠, 王译伟, 郑茜, 等. 含当归中成药的DNA提取及其分子鉴定[J]. 中国实验方剂学杂志, 2018, 24(1): 44
GOU H, WANG YW, ZHENG Q, et al. DNA extraction and molecular identification in Angelicae Sinensis Radix preparations[J]. Chin J Exp Tradit Med Form, 2018, 24(1):44
[11] 胡力, 袁媛, 张辉, 等. 烫水蛭(蚂蟥)配方颗粒的位点特异性PCR鉴别[J]. 中国现代中药, 2023, 25(8): 1676
HU L, YUAN Y, ZHANG H, et al. Identification of Whitmania pigra Whitman formula granules by allele-specific PCR[J]. Mod Chin Med, 2023, 25(8): 1676
[12] 石旸, 胡力, 赵玉洋, 等. 地骨皮的多重位点特异性PCR鉴别[J]. 中国实验方剂学杂志, 2024, 30(4): 35
SHI Y, HU L, ZHAO YY, et al. Identification of original plants of Lycii Cortex by multiplex allele-specific PCR[J]. Chin J Exp Tradit Med Form, 2024, 30(4): 35
[13] 胡力, 陈梓媛, 赵玉洋, 等. 五倍子配方颗粒的位点特异性PCR鉴别[J]. 中国现代中药, 2023, 25(8): 1668
HU L, CHEN ZY, ZHAO YY, et al. Dentification of Wubeizi formula granules by allele-specific PCR[J]. Mod Chin Med, 2023, 25(8): 1668
[14] 莫静, 王文斌, 程华春, 等. 利用分子方法鉴别白花蛇舌草及其伪品伞房花耳草[J]. 药物分析杂志, 2023, 43(4): 676
MO J, WANG WB, CHENG HC, et al. Identification of Hedyotis diffusa and its adulterant Hedyotis corymbosa by molecular method[J]. Chin J Pharm Anal, 2023, 43(4): 676
[15] 段庆梓, 王巍, 尚柯, 等. 一种用于鉴别湖北麦冬的PCR-RFLP方法研究[J]. 药物分析杂志, 2023, 43(11): 1974
DUAN QZ, WANG W, SHANG K, et al. A new method based on PCR-RFLP for identification of root of Liriope spicata[J]. Chin J Pharm Anal, 2023, 43(11): 1974
[16] KUMAR S, STECHER G, LI M, et al. MEGA X: molecular evolutionary genetics analysis across computing platforms[J]. Mol Biol Evol, 2018, 35(6): 1547
