Hirudin Extraction of Iranian Leech Saliva by Freezing and Lyophilization Methods

Leech saliva contains a diverse array of bioactive compounds, most notably hirudin, a potent natural thrombin inhibitor. Despite the clinical utility of hirudotherapy, inherent risks such as localized infection, uncontrolled hemorrhage, and leech migration limit its widespread application. To overcome these limitations, this study presents a refined extraction, partial purification, and stabilization protocol for hirudin-containing salivary extract derived from the Iranian leech, Hirudo orientalis. Saliva was harvested using 0.001 M arginine in 0.15 M saline under controlled temperatures (23 °C vs. 37-40 °C) and subsequently stored at 4 °C. The crude extract underwent fractionation/purification via centrifugation, followed by stabilization with sucrose, fructose, and mannitol, and final lyophilization (freeze-drying). Optimal extraction efficiency was observed at 37-40 °C. The short-term stability (44 days at 4 °C) and antibacterial efficacy of the resulting lyophilizate were evaluated using High-Performance Liquid Chromatography (HPLC) against authentic reference parameters and Brain Heart Infusion (BHI) broth assays. The findings demonstrate that this stabilization matrix preserves the short-term chromatographic profile and in-vitro biological activity of the salivary protein extract, providing a preliminary foundation for future in-vivo evaluation and development of standardized leech-derived biotherapeutics.