中药药理与临床

目的:明确日本医蛭(Hirudo nipponica Whitman)的新型形态鉴别特征，构建基于形态学、生物效价与分子生物学的多维度鉴别体系，为其品种鉴定、质量控制提供科学依据。方法:收集我国11个省份19批市售日本医蛭样品，采用传统性状鉴别法观察样品背部纵纹形态差异；提取样品基因组DNA,扩增细胞色素C氧化酶亚基Ⅰ(COI)基因序列，进行变异位点检测与Blast序列比对，结合K2P模型计算种内与种间遗传距离，构建邻接(NJ)系统发育树。通过凝血酶滴定法测定各样品抗凝血活性，量化其生物效价。结果:性状鉴别显示，市售样品可依据背部纵纹形态明确分为两类，即虚线型与实线型。分子生物学分析表明，虚线型日本医蛭与日本医蛭标准COI序列匹配度高，种内遗传距离小，符合正品特征；实线型日本医蛭与正品COI序列存在显著变异，种间遗传距离超出种内变异阈值，在NJ系统发育树中单独聚类。生物效价测定结果显示，虚线型日本医蛭抗凝血酶活性为115～402 U/g,实线型日本医蛭抗凝血酶活性为233～3 444 U/g。整体而言，实线型日本医蛭的抗凝血活性显著高于虚线型日本医蛭，且实线型日本医蛭活性离散度更大，稳定性相对较差。结论:结合形态学、分子生物学及活性评价多维度分析，虚线型日本医蛭与实线型日本医蛭在性状鉴定和含量测定方面均符合2025年版《中华人民共和国药典》正品日本医蛭的相关标准，二者均可作为药用水蛭使用；但实线型日本医蛭与虚线型日本医蛭相比存在显著的遗传分化和一定的形态差异，且二者野生种群存在明确的地理分布界限，据此推测实线型日本医蛭为日本医蛭的地理亚种。鉴于实线型日本医蛭的药理活性范围跨度较大，从保障临床用药效果稳定的角度考量，更推荐使用抗凝活性范围较为稳定的虚线型日本医蛭。

Objective:To clarify the novel morphological characteristics of Hirudo nipponica Whitman for identification and establish a multi-dimensional identification system encompassing morphology, biological potency, and molecular biology, thereby providing a scientific basis for its variety identification and quality control. Methods:Nineteen batches of commercially available H. nipponica samples were collected from 11 provinces in China. The conventional morphological identification method was employed to observe the morphological differences of dorsal longitudinal stripes. Genomic DNA was extracted from the samples for amplification of the cytochrome C oxidase subunit Ⅰ(COI) gene sequence. The mutation site detection and Blast sequence alignment were performed, and intraspecific and interspecific genetic distances were calculated based on the K2P model. A neighbor-joining(NJ) phylogenetic tree was then constructed. The thrombin titration method was adopted to determine the anticoagulant activity of each sample and quantify its biological potency. Results:Morphological identification showed that the commercially available samples could be clearly classified into two types—the dotted-line type and the solid-line type—according to the morphology of dorsal longitudinal stripes. Molecular biological analysis indicated that dotted-line type H. nipponica had high homology with the standard COI sequence of H. nipponia and a low intraspecific genetic distance, which conformed to the characteristics of genuine medicinal materials. Solid-line type H. nipponica presented significant variations from the COI sequence of H. nipponia,with the interspecific genetic distance exceeding the intraspecific variation threshold and forming an independent clade in the NJphylogenetic tree. The results of biological potency assay revealed that the antithrombin activity of dotted-line type H. nipponica was 115～402 U/g, while that of solid-line type H. nipponica was 233～3 444 U/g. Overall, the anticoagulant activity of solid-line type H. nipponica was significantly higher than that of dotted-line type H. nipponica,and solid-line type H. nipponica exhibited larger activity dispersion with poor stability. Conclusion:Comprehensive analysis based on morphology, molecular biology, and activity evaluation demonstrates that both dotted-line type H. nipponica and solid-line type H. nipponica meet the relevant quality standards for genuine H. nipponia specified in the Pharmacopoeia of the People's Republic of China(2025 Edition) in terms of morphological identification and content determination, and both could be used as medicinal leeches. However, solid-line type H. nipponica has significant genetic differentiation and certain morphological differences compared with dotted-line type H. nipponica,and there is a clear geographical distribution boundary between their wild populations. According to these findings, solid-line type H. nipponica is inferred to be a geographical subspecies of H. nipponia. In view of the wide variation range of pharmacological activity of solid-line type H. nipponica,dotted-line type H. nipponica with a stable range of anticoagulant activity is more recommended for clinical application to ensure the stability of therapeutic effects.