Biological activities of saliva extract from the medicinal Malaysian leech hirudinaria manillensis

Abstract

The work presented in this thesis describes selected biological activities of the salivary gland secretion of the medicinal Malaysian leech Hirudinaria manillensis (Lesson, 1842). A new device for leech feeding was developed using common laboratory tools consisting of a glass funnel wrapped with a parafilm membrane and filled with the phagostimulatory solution. Many phagostimulatory solutions were examined and only those containing sodium chloride and arginine were accepted by leeches. It was found that 71% of the tested leeches continued sucking for 15-45 min and 86% of them experienced 1-5 folds increments in body weight. For collection of a high quality and quantity LSE, we starved leeches for three weeks. A new method for LSE collection was described. The fed leeches were forced to regurgitate whatever they sucked by immersing them in ice containers. By using this method, leeches stayed alive and regain their activity after 15-30 min. The maximum concentration (A280 = 0.342) was provided within the first five minutes of sucking action. Gel electrophoresis (SDSPAGE and Tricine-SDS-PAGE) methods revealed the existence of various peptides and proteins with molecular weights ranging from 2.3-250 kDa. Amidolytic activity assay showed that LSE inhibited thrombin-induced release of p-nitroanilide from the synthetic substrate S-2238 with IC50 of 49.391±2.219 ?g/ml. LSE was shown to prolong thrombin time in vitro. The maximum antithrombin activity was obtained during the dry season (IC100 =16.081±0.079 ?g/ml). A longer starvation period yielded a lower antithrombin activity. An optimization of lyophilization conditions revealed that pre-freezing at -20