Identification of characteristic peptides from different primitive leeches
The 2020 edition of the Pharmacopoeia of the People’s Republic of China (hereinafter referred to as the “Pharmacopoeia”) stipulates that the traditional Chinese medicine leech is a dried whole of the leech family animal, the leech Whitman pigra Whitman, the leech Hirudo nipponica Whitman, or the willow leaf leech Whitman acranulata Whitman. Its taste is salty, bitter, and mild; There is a small toxin, which belongs to the liver meridian. It has the function of breaking blood and promoting circulation, removing blood stasis and eliminating stagnation. It is commonly used for blood stasis and meridian obstruction, treating masses, stroke hemiplegia, and falls and injuries. In recent years, the number of patients with cardiovascular and cerebrovascular diseases has significantly increased, resulting in an increasing demand for leech medicinal materials. However, there are many complex and varying quality leech medicinal materials on the market. In addition to leeches, there is a counterfeit product called Poecilobdra manillensis Lesson in the market. Currently, leeches and leeches are both dried goods with significant differences in appearance and characteristics, making them easy to distinguish. Therefore, this study only focuses on studying different origins of leeches. Through literature review, it has been found that current research on leeches focuses on anticoagulant mechanisms and amino acid determination, while there is relatively little comprehensive study on the quality of different primitive leeches and their mixtures. Identification studies between different primitive leeches are mostly based on trait identification under the 2020 edition of the Chinese Pharmacopoeia. Some studies have found through DNA molecular identification and protein analysis techniques that leech DNA molecular identification technology can be established through PCR, and proteins of different primitive leeches can be distinguished using SDS-PAGE and 2-DE. In addition, there are studies that can also distinguish them based on HPLC characteristic spectrum methods or high-throughput sequencing methods, but such methods have problems such as long detection time and low popularity of detection instruments. This study used LC-MS method to detect leech peptide I and leech peptide II. The established method has strong specificity, high sensitivity, short analysis time, and can be used to distinguish leeches with different origins, improve their quality control level, and provide reference for the safety and effectiveness of clinical use of related drugs.

The identification of animal medicinal materials has always been a key and difficult issue in traditional Chinese medicine research. However, current identification methods mainly rely on traditional identification methods, which have strong subjectivity and require strong professional knowledge and experience from the discriminator. There is a lack of scientific, effective, and accurate identification methods based on modern detection methods. Leech is a precious traditional Chinese medicine with strong medicinal and research value. The medicinal value of leeches has also been recognized worldwide, and leeches have been used to treat diseases in Europe, Egypt, and India. The 2020 edition of the Chinese Pharmacopoeia records that leeches contain three basic elements, but currently the Chinese Pharmacopoeia only controls them through tests such as moisture, total ash content, acidity and alkalinity, and antithrombin activity. There is currently no record of differential characterization between different basic elements, which lacks scientific basis and cannot identify the decoction or formulation of medicinal materials after boiling.
This study tested 22 batches of leeches with different origins, including 15 batches of leeches, 2 batches of willow leaf leeches, and 5 batches of leeches. The results showed that all 15 batches of leeches could detect leech peptide I and leech peptide II chromatographic peaks, 2 batches of willow leaf leeches could only detect leech peptide II, and 5 batches of leeches could only detect leech peptide I. From the inspection results, it can be seen that leeches with different origins can be distinguished by measuring leech peptide I and leech peptide II. Through PLS-DA analysis, it can be seen that leeches with different origins can be clearly divided into three categories, and the analysis results are consistent with the identification results of the origins. According to other documents, the activity of hirudo polypeptide is mainly anticoagulant activity, and also involves protease inhibition, anti-inflammatory, hydrolase, inhibition of macrophage migration in atherosclerosis and other pharmacological activities. This indicates that in addition to amino acids and nucleosides, hirudo polypeptide is also one of the main components of its pharmacological effect. However, the above documents are focused on the pharmacological study of polypeptide, and no detection method for polypeptide has been established. This paper uses LC-MS method to determine hirudo polypeptide with reference to the extraction and inspection methods of other varieties of polypeptide, providing a scientific and technical means for quality control, which can reflect the quality of its pharmacological effect by measuring the peak area of hirudo polypeptide; In addition, under the content determination item of leeches in the Chinese Pharmacopoeia, it is stipulated that “the anti thrombin activity of leeches should not be less than 16.0U per 1g; leeches and willow leaf leeches should not be less than 3.0U”. This shows that leeches have significant differences in anti thrombin activity compared to leeches and willow leaf leeches, that is, leeches with different origins have significant quality differences. Therefore, using this method to quickly and accurately distinguish leech medicinal materials with different origins and their water decoctions, formula preparations, etc. that have lost their basic characteristics is of great significance for improving the quality of leeches and related products.
The method established by this research institute has the characteristics of speed, effectiveness, and reproducibility, which can improve its quality control level and provide reference for the safety and effectiveness of clinical use of leech related drugs. It effectively solves the problem of leech that can only distinguish different origins through traditional identification methods for a long time.