应用磁性固定化酶“垂钓”水蛭素技术进行中药水蛭鉴定研究

Leech is an important animal Chinese traditional medicine for blood-activating and stasis-dissolving, its main active ingredients are hirudin analogues, which are extremely strong thrombin inhibitors. Natural hirudin analogues are peptides with low molecular weight, accurately determination of the activity and content of hirudin analogues is very important for both the quality control of traditional Chinese medicine leech and clinical application of hirudin analogues. Chinese Pharmacopoeia uses the method of the thrombin titration based on the principle of specific binding of hirudin and thrombin, which manifests distinctive advantages of bioactivity detection, while it is not satisfactory on the sensitivity and stability of this method. Given this, the present study will keep the advantages of the above method, firstly prepare the magnetic nanoparticles (Fe3O4 core-shell structure) and immobilize the thrombin on the magnetic carrier to specially fishing hirudin, accomplishing the rapid extraction and purification, then employ the technology of mass spectrometry (MS) for accurate identifying of hirudin. Comparative research of the obtained huridin analogues is proposed on different varieties and different processing methods. Chinese medicine leech peptide mapping will be developed originally to point to a new direction for quality control of leech and the identification of animal medicine.

中药水蛭是重要的活血化瘀动物类药材，其有效成分是水蛭素及类似物，是极强的凝血酶抑制剂。天然水蛭素类为低分子量多肽，无论是中药水蛭的质量控制还是水蛭素类的临床应用，准确检测水蛭素类的活性与含量都至关重要。《中国药典》采用凝血酶滴定法，依据水蛭素与凝血酶特异性结合的原理而建立，突出了生物活性检定技术的优势，但灵敏度和稳定性都不够理想。鉴于此，本课题兼顾水蛭素的活性评测，拟制备磁性纳米载体（Fe3O4的核壳结构），将凝血酶固定于磁珠上，定点“垂钓”水蛭素，实现快速提取与纯化，并运用质谱（MS）技术进行准确鉴定，进一步拟运用该方法对不同品种、不同炮制方法所得的水蛭素类进行比较研究，首次建立中药水蛭的“肽图谱”，为水蛭的质量评价提供新方法，为动物药的鉴定提供了新的思路。

中药水蛭是重要的活血化瘀动物类药材，其活性成分水蛭素是天然最有效的抗凝剂。天然水蛭素为一类含65个氨基酸的多肽，有三种变异体（HV 1，HV 2，HV 3）。无论是中药水蛭的质量控制还是水蛭素类的临床应用，准确检测水蛭素类的活性与含量都至关重要。《中华人民共和国药典》采用凝血酶滴定法，依据水蛭素与凝血酶特异性结合的原理而建立，突出了生物活性检定技术的优势，但灵敏度和稳定性都不够理想。鉴于此，本课题兼顾水蛭素的活性评测，制备磁性琼脂糖NHS微球，将凝血酶间接固定于磁珠上，定点“垂钓”水蛭素及其衍生物。本课题结合磁性固定化酶提取与液相色谱-质谱（HPLC-MS/MS）检测技术，建立一套快速、特异和灵敏的水蛭素及其衍生物的定量方案。该方法定量限低（10 ng/mL）, 线性范围宽（10 ng/mL~10 ug/mL），精密度高，重复性好，日内和日间的相对标准偏差均不大于10%。该技术为水蛭的质量评价提供新方法，为动物药的鉴定提供了新的思路。