The salivary gland secretion of the haematophagous animals, leeches, has attracted the attention of therapists since the extreme old ages due to its wide range of medical properties. Thus, many researches have been done to develop and optimize new methods to collect leech saliva with high quality and quantity. In the present study, we aimed to evaluate the effects of starvation period and repeated collection on the quality and quantity of leech saliva extract LSE and its contents of proteins during the rainy season. Protein recovery in the LSE was also studied after first collection. It was found that leeches are able to produce protein-containing saliva whenever fed during the whole study period of 18 weeks with varied protein concentrations. The results showed that the highest protein concentrations (105-91 μg/mL) were produced after 12-15 weeks of starvation. The results of successive collection showed that leeches are able to produce proteins and peptides whenever they suck the solution after first collection with some varies in the concentrations. The concentrations varied between 0 and 72% of the initial concentration. Gel electrophoresis results showed absence for some bands when the concentrations are too low. Also the results showed that leeches are able to recover about 42% of their initial proteins concentration within four weeks of starvation after first feeding. The gel electrophoresis results showed the closeness between the first and second collections. To conclude, all test factors (starvation period, successive collection and recovery test) were shown to have an important impact on protein concentration of leech saliva and therefore its medicinal affectivity. The mentioned results are reported for the first time and they open the gate for further studies.
The medicinal Malaysian leeches have been used in traditional medicine to treat many different ailments. In this study, leech saliva extract (LSE) was collected from the medicinal Malaysian leech Hirudinaria manillensis. Gel electrophoresis of LSE was carried out to estimate the peptide and protein molecular weights of its content. Results showed that LSE contains more than 60 peptides and proteins with molecular masses ranging from 1.9-250kDa. Thrombin time assay in vitro was employed to assess the collected LSE antithrombin activity. First, to study its stability, LSE was lyophilized under the following different conditions: pre-freezing temperature, type of container and lyophilization cycle. Pre-freezed LSE sample at -20°C and lyophilized for 24 hours retained about 100-95% of its original biological activities. Second, the LSE antithrombin activity was monitored for a period of six months. Storage temperature, type of the container and photosensitivity effects on antithrombin activity of the lyophilized (solid state) and non-lyophilized (liquid state) were investigated. Results showed that storage temperature drastically affected the biological activity of LSE with -20 °C as the optimum temperature. Samples stored at ambient temperature and +4 °C were light photosensitive and adversely affected when stored in polypropylene tubes. Lyophilized samples were more stable than non-lyophilized ones over the period of study. To sum up, in order to have a biologically active stock of LSE, it has to be lyophilized for no more than 24 hours following freezing at -20°C and has to be stored at -20°C in glass tubes protected from light.