Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis / 中国天然药物

A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (K

Comparison of the Effects on Angiogenesis Activity between Whitmania Pigra Whitman and Hirudinaria Manillensis Lesson Based on a Zebrafish Model / 中国药师

Objective:To investigate and compare the effects of water extracts of Whitmania pigra Whitman and Hirudinaria ma-nillensis Lesson on the angiogenesis of Tg (kdrl:mCherry) zebrafish. Methods:The zebrafish embryos 6-8 hours after fertilization (6-8hpf) were transferred to the culture medium containing Whitmania pigra Whitman or Hirudinaria manillensis Lesson extract at different concentrations, and the culture medium containing the drugs was replaced every 24 h. And then, at 72 hpf, the larvalmorphology and intersegmental vessels were observed under a microscope. The hatchability of 48-and 72-hpf embryos, and the number of intersegmen-tal vessels and the heart rate of 72-hpf juveniles were measured. Results:Compared with the control group, when the concentration of Whitmania pigra Whitmanis was higher than 30μg· ml-1 , and the concentration of Hirudinaria manillensis Lesson was higher than 20μg· ml-1, the number of intersegmental vessels was significantly reduced (P<0. 01). Compared with the control group, at 48 hpf, when the concentration of the drug groups was higher than 40 μg· ml-1 , the hatchability of the two groups significantly decreased ( P<0. 01);at 72 hpf, the hatchability of Whitmania pigra Whitman decreased significantly at the concentration of 100 μg·ml-1 (P<0.01), while the hatchability of Hirudinaria manillensis Lesson decreased significantly at the concentration of 80 μg· ml-1(P <0. 01). There was no obvious yolk sac edema, pericardial edema and spine curvature in the two groups. The heart rate decreased sig-nificantly (P<0. 01), while was still within the normal range. Conclusion:Both Whitmania pigra Whitman and Hirudinaria manillen-sis Lesson have notable anti-angiogenic activity, and the anti-angiogenesis activity of Hirudinaria manillensis Lesson is stronger. They both have effects on the development of zebrafish embryos, while the toxicity is not obvious.

In vivo anti-hyperglycemic activity of saliva extract from the tropical leech Hirudinaria manillensis / 中国天然药物

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.

Acute subcutaneous toxicity study of Hirudinaria manillensis (Lesson) LEECH homogenate in mice

Background: The vosoactive effects of proteins isolated from leech saliva and leech homogenate have been the focus of many recent studies because of its therapeutic potential. Possible nonlethal and lethal toxic effects have to be determined before any therapeutic effects could be tested. The general objective is to take the first step in drug design by performing an acute toxicity studyMethods: In this randomized, double-blind trial, Swiss mice were administered subcutaneously with varying doses of leech Hirudinaria manillensis (Lesson) homogenate and observed for possible acute toxicity. Toxidromes appearing within the two-week period after the administration of the substance were noted. Necropsy was performed on all the mice subjects. The LD(50) was computed using the log dose-response probit analysis. The doses (in g/kg body weight) were 3.54, 5.0 and 7.06Results: The LD(50) at Day 2 of Hirudinaria manillensis extract was 4.6124 g/kg body weight. The LD(50) at Day 14 was not obtained, because the test animals incurred mortalities beyond Day 2, which could be explained by a delayed toxicity of the test substance. Mice injected with 7.06 g/kg showed the most number of observable toxidromes, involving several organ systems, which generally had an early onset and persisted until the time of death of the mice. Mice treated with 3.54 g/kg showed toxidromes with early onset time, but they were not as consistent as the highest dose and the effects were generally not long-lasting. Motor activity was the most affected among the toxidromes. Gross pathologic findings revealed that the intestine, liver, heart, stomach, lungs and kidney are the commonly affected organs, which had marked changes in all dose groupsConclusions: The crude H. manillensis leech extract caused toxicity in mice, with toxidromes involving mainly the nervous system, specifically the somatosensory and neuromuscular systems. The LD(50) could not be determined in this study. However, using the data at Day 2, LD(50) was 4.6124 g/kg body weight. The LOAEL could be less than or equal to 3.54 g/kg body weight. No observable adverse effect level could not be determined. (Author)

Studies on growth and reproduction of Hirudinaria manillensis in Guangdong Province / 中草药

Object To study the dynamic growth and reproduction of Hirudinaria manillensis Lesson in laboratory with providing the data of artificially breeding leech in the field. Methods The newly hatched juvenile from same cocoon were bred in 80 cm?60 cm?50 cm aquaria with natural water from rice paddy and weighted every ten days. Water temperature was recorded daily and water was exchanged monthly. Ten chemical elements of water from rice paddy were analyzed in the laboratory. Leeches were allowed to engorge the blood on living rabbits and the amount of engorged blood was measured every time to observe the growth and development, record the copulation and cocoon-deposit, and make the curve of dynamic growth of the leech. Results The newly hatched juvenile grew and developed for 14 months to sexual maturation period by engorging rabbit's blood of five or six times. The leeches were still juvenile in the first three times of engorging, became subadult in the fourth. Then some developed to the sexual maturation period individually in the fifth, and some individually in the sixth. The curve of dynamic growth displayed rising and falling with the record of copulation and cocoon deposit. Conclusion The above study elucidate the dynamic growth, reproduction and the successfully breeding of leeches in the door.