# A Comprehensive Quality Evaluation System for Medicinal Leeches by Integrating Macromolecular Protein Analysis and Small-Molecule Marker Detection as Well as Quantitative Bioassays

## TL;DR

This study develops a new system to evaluate the quality of medicinal leeches using protein analysis, small molecule detection, and bioassays to distinguish genuine from counterfeit samples.

## Contribution

The study introduces an integrated quality evaluation strategy combining macromolecular and small-molecule analysis with bioassays for medicinal leeches.

## Key findings

- Eight small molecules, including a novel compound SZ-1, and seven differential proteins were identified as quality markers.
- SDS-PAGE, HPLC, and TLC methods were established for rapid and accurate authentication of medicinal leeches.
- Hirudo nipponica showed strong antithrombin activity, while Whitmania pigra exhibited weak activity and promoted platelet aggregation.

## Abstract

Background/Objectives: Medical leech (Hirudo in the Chinese Pharmacopoeia) is renowned in traditional medicine for its significant antithrombin activity. As an animal-derived medicine with complex and incompletely understood composition, its insufficient quality control measures are met with widespread counterfeiting caused by limited animal resources and rising demand. Methods: In this study, an integrated quality evaluation strategy guided by “Totality of the Evidence” (TOE) method is proposed. This strategy combines chemical characterization of small and macromolecular components with bioassays relevant to its clinical functions. A total of 28 batches of samples were analyzed, comprising 23 genuine and 5 counterfeit batches. Species origins were identified by morphology and DNA barcoding. Chemical characterization included TLC, HPLC and UPLC-QTOF-MS/MS for small molecules, and SDS-PAGE with HPLC-Orbitrap Fusion Lumos Tribrid-MS for macromolecules. Antithrombotic activity was assessed by thrombin titration and platelet aggregation assays. Results: Several characteristic components were discovered and identified as key quality control markers, including eight small molecules such as an unreported compound SZ-1, plus seven major differential proteins across species. Based on these markers, accurate and rapid authentication methods were established using SDS-PAGE for macromolecules, and both HPLC and TLC for small molecules. Furthermore, using bioassay methods we established for quality evaluation, Hirudo nipponica exhibits potent anti-thrombin activity and inhibits platelet aggregation, while Whitmania pigra shows weak anti-thrombin activity and promotes platelet aggregation. Conclusions: This quality evaluation strategy is not only applicable for the quality assessment of genuine Hirudo products of different origins, but also for distinguishing medical leeches from their counterfeits.