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 anticoagulant effect of leech saliva was traditionally employed in the treatment of diabetes mellitus complications such as peripheral vascular complications. This study was carried out to examine the effect of leech saliva extract (LSE) on blood glucose levels in alloxan-induced diabetic rats. First, LSE was collected from leeches which were fed on a phagostimulatory solution. Second, total protein concentration was estimated using the Bradford assay. Third, diabetic rats were injected subcutaneously (sc) with LSE at doses of 500 and 1 000 μg·kg(-1) body weight (bw). Other diabetic rats were injected sc with insulin at doses of 10 and 20 U·kg(-1) bw. Another group was injected simultaneously with LSE (250 μg·kg(-1) bw) and insulin (10 U·kg(-1) bw). Fasting blood glucose (FBG) concentrations were monitored during a study period of eight hours at regular intervals. Findings showed that both doses of LSE resulted in a significant and gradual decrease in FBG starting from 10%-18% downfall after two hours of injection reaching the maximal reduction activity of 58% after eight hours. Remarkably, LSE was sufficient to bring the rats to a near norm-glycemic state. The high dose of insulin induced a severe hypoglycemic condition after 2-4 h of injection. The lower dose was able to decline FBG for 2-6 h in rats which became diabetic again after 8 h. On the other hand, the concurrent injection of low doses of LSE and insulin produced a hypoglycemic effect with all rats showing normal FBG levels. Taken together, these findings indicated that the subcutaneous injection of LSE of the medicinal Malaysian leech was able to provide better glycemic control compared with insulin. Moreover, the synergism between LSE and insulin suggests that LSE could be utilized as an adjuvant medication in order to reduce insulin dosage or to achieve better control of blood glucose.