Leeches are a well-known animal-derived health supplement commonly used as an anticoagulation and antithrombosis agent; however, adulteration and counterfeiting are often made for illegal profits. To identify leech species, this study developed a rapid, simple, and visualized method based on loop-mediated isothermal amplification (LAMP), which relies on a specific primer set designed according to the mitochondrial DNA control region of the target species. Quantitative polymerase chain reaction (qPCR) was also employed in parallel to compare the sensitivity and confirm the primer specificity. Primer sets with high specificity were successfully screened for LAMP reactions against four common leech species on the market. All of them have produced typical amplification profiles of the target sequences in qPCR reactions with significantly lower amplification sensitivity than LAMP assay. The newly established LAMP method in this study can be accomplished within 1 h, and it could be successfully applied for on-site visual identification of mislabeling and adulteration in the leech market.

目的 鉴定小儿复方鸡内金散中鸡内金的真伪.方法 根据家鸡、麻鸭、家鹅的线粒体基因序列设计和筛选引物,优化反应条件和体系,建立基于特异性引物的多重PCR法进行鉴别.结果 鸡内金、鸭内金、鹅内金DNA在优化条件下,分别在94、124、155 bp处产生了清晰、明亮的条带,并且未观察到非特异性扩增,呈现出较好的特异性,检测灵敏度均达到 0.1 ng/μL.来源于 8 个厂家的 11 批样品中,有 2 批检出了鸭内金掺伪,检出率为18.2%.结论 该方法灵敏度高,特异性强,可为更好地监控含鸡内金中药制剂的质量提供新颖的途径和有力的手段.

Ethnopharmacological relevance: The prevalence of cardiovascular disease (CVD) is increasing worldwide. Despite significant improvements in novel targeted treatment agents, natural products purified from medicinal animals with minimal side effects have attracted much attention. Several native proteins explored from suck-blood leeches, such as non-thermostable hirudin and its variants, revealed potent anticoagulant activity. Traditional Chinese medicine clinics have proved that non-suck-blood leech Whitmania pigra Whitman (W. pigra) also played notable roles in CVD treatments even after decoction. However, only a few natural proteins and peptides have been identified from the fresh material of this medicinal species. Aim of the study: We aimed to purify and characterize thermostable anticoagulant proteins from W. pigra for further development of a therapeutic agent for thrombosis. Materials and methods: W. pigra crude extract was prepared by decoction in water. Anticoagulant proteins were purified by DEAE cellulose DE-52, Sephadex G-75, and reversed-phase liquid chromatography sequentially and analyzed by SDS-PAGE and LC-MS/MS for structural information. In addition, we conducted in vitro anticoag-ulant experiments, including plasma recalcification time (PRT) assay, fibrinolytic assay, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fib) assay, and cell viability assays. Furthermore, a carrageenan-induced chronic thromboembolism model was employed in ICR mice, and four coagulation factors (APTT, PT, TT, and Fib) activities were determined after intragastric administration. Results: The anticoagulant protein WP-77 has a relative molecular weight of ca. 20.8 kDa. It was effective over a broad temperature range from 20 degrees C to 100 degrees C and a pH 2-8 condition. The anticoagulant activity of WP-77 was retained after incubation with pepsin but was greatly inhibited by trypsin (P < 0.01). It significantly prolonged APTT and TT (P < 0.05) but had little effect on PT and Fib in vitro. Furthermore, WP-77 of a low concentration resulted in the recovery of injured EA.hy926 by thrombin. The protein also significantly prolonged APTT and TT (P < 0.01) and inhibited thrombus formation in carrageenan-induced thrombosis mice, demonstrating its antithrombotic effect in vivo. Conclusion: Our results suggest that WP-77 from W. pigra plays a distinct role in treating thrombotic diseases, and it is an essential substance of anticoagulant activity of non-suck-blood medicinal leeches. This thermostable anticoagulant protein could be a promising candidate for the development of clinical antithrombosis medicines.

目的 鉴定鹿角与鹿茸蛋白质.方法 通过SDS-PAGE、2-DE分析鹿角与鹿茸蛋白质组成,比较两者电泳图谱,找出各自稳定的蛋白质条带或斑点及其差异.将两者SDS-PAGE蛋白质条带以胰蛋白酶进行胶内消化后,采用MALDI-TOF/TOF质谱分析,通过MASCOT检索匹配以鉴定蛋白质.结果 在SDS-PAGE图谱中,鹿角在约75、66.2 kD处呈现出2个清晰的蛋白质条带,而鹿茸则在约75、61、45、20 kD处呈出现4个蛋白质条带;2-DE分析显示,鹿角蛋白质斑点集中于Ⅰ区(pH5～7,Mw 45 kD)以及Ⅱ区(pH10,Mw 66.2 kD);鹿茸蛋白质斑点则主要分布在Ⅲ区(pH5～7,Mw 45 kD),另外,在pH5～6和Mw 20 kD的区域内存在3个稳定出现的蛋白质斑点.通过生物质谱分析,分别从鹿角与鹿茸中鉴定出2、4个蛋白质,评分高于100.结论 鹿角与鹿茸蛋白质电泳图谱存在明显差异,可为两者鉴别提供科学依据.