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
Amino Acid Sequence ; Animals ; Leeches/chemistry* ; Pancreatic Elastase/antagonists & inhibitors* ; Protease Inhibitors/pharmacology* ; Proteins

The present study was designed to identify immunomodulatory components from the leech salivary gland of Haemadipsa sylvestris. The Sephadex G-50, Resource(TM) S column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC) were used to isolate and purify the salivary gland extracts (SGE). Structural analysis of isolated compounds was based on Edman degradation and matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The cDNA encoding the precursor of the compound was cloned from the cDNA library of the salivary gland of H. sylvestris. The levels of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1) were assayed using an enzyme-linked immunosorbent assay (ELISA). The effects on cell proliferation and cell viability were observed using MTT assay. A novel neuropeptide Y (Neuropeptide Y-HS) from the leech salivary gland of H. sylvestris was purified and characterized. It was composed of 36 amino acid residues and the amino acid sequence was determined to be FLEPPERPAVFTSVEQMKSYIKALNDYYLLLGRPRF-NH2, containing an amidated C-terminus. It showed significant inhibitory effects on the production of inflammatory cytokines including TNF-α, IFN-γ, IL-6, and MCP-1. Neuropeptide Y was identified from leeches for the first time. The presence of neuropeptide Y-HS in leech salivary gland may help get blood meal from hosts and inhibit inflammation.
Amino Acid Sequence ; Animals ; Immunologic Factors ; administration & dosage ; chemistry ; genetics ; Inflammation ; drug therapy ; immunology ; Interferon-gamma ; immunology ; Interleukin-6 ; immunology ; Leeches ; chemistry ; Mass Spectrometry ; Mice ; Molecular Sequence Data ; Neuropeptide Y ; administration & dosage ; chemistry ; genetics ; Peptide Mapping ; Salivary Glands ; chemistry ; Tumor Necrosis Factor-alpha ; immunology

Animal medicines mainly contain protein which was organic molecule with quaternary structure and had the property of thermal denaturation. When suffering from heat for a consistent time, the native conformation of protein would be destroyed. After denaturation the biological activity of protein will lose and some physicochemical and biochemical properties will be changed. Leech was a classical animal medicine in the views of traditional Chinese medicine (TCM) which had the functions of breaking stagnant and eliminating blood stasis. In the usage history, it was processed for a long time. No matter stir-frying leech with talc powder embodied in Chinese Pharmacopoeia or stir-baking with wine as a distinctive method in Beijing district, the process procedure was basically performed under high temperature. The purposes and intentions of process are mostly limited to technology conditions at specific historical period. In this article, based on existing processing procedure and its character of Leech, the changes of active components and pharmacological activities before and after processing under high temperature were summarized. The results demonstrate that the protein of leech would be denaturated; some active peptide such as hirudin were partly or totally destroyed; some toxic mineral elements, such as Pb, Hg, Cd, were decreased; at the same time, heating can promote some chemical components transforming into hypoxanthine which had the function of antihypertensive, antiasthmatic and antalgic. Consequently, after processed under high temperature, the purpose of decreasing toxicity and alleviating the strong property was achieved. Pharmacological changes of leech processed under high temperature were mainly manifested in the anticoagulant and antithrombotic activity, etc. Based on current processing research status about animal medicine leech, future research methods and directions on scientific connotation of leech processed under high temperature were put forward in this article.
Animals ; Biological Factors ; chemistry ; isolation & purification ; pharmacology ; Chemistry, Pharmaceutical ; methods ; Hot Temperature ; Humans ; Leeches ; chemistry

Mouthpart developmental histology of Whitmania pigra at different month of age were studied by paraffin section, HE staining combined alcian blue and periodic acid schifts reaction procedure (AB-PAS). The following results was obtained: Change ranges: oral width 0.6 mm (1-3 month), 1.2 mm (34 month); oral diameter 0.3 mm (1-3 month); 1.2 mm (34 month), the oral size reached maximum during 4-6 months and unchanged thereafter. Oral lip had a thin protective film located in the front of the mouthpart. The W. pigra possessed three jaws in oral cavity, the big one was in dorsum, the other two separated on both side of abdomen respectively. Jaws and muscular pharynx were interrelated closely. The jaws were composed by cuticle, epithelial layer, muscularis and jaw cavity from outside to inside. In the front of jaws had mastoid abdomen with function of secreting acidophilic granule from 2 month age. Oral cavity was composed by mucosa, submucosa and muscularis inside and outside. Oral cavity was rich of peristomial nerves. And pharynx was composed of mucosa, muscularis, adventitia from inside to outside. The folds height and width become heighten and thicken. Mucosa epithelium from complex flat epithelium changed into columnar epithelium, muscularis gradually developed into thickened along with growing. Muscular thickness reached maximum at 4 months. Mucous cells of W. pigra were classified into I-IV types based on different staining and two mainly morphological shapes (Tubular, Pear-shaped). Jaws, oral cavity, pharynx by AB-PAS staining showed little changes at different month of age. Mucous cells were few at 1 month age, and type II cells were increased rapidly in 2-3 month age in oral lip. Oral cavity contains more mucous gland cells type I. Under the muscularis there were connective tissues which distributed a few of mucous cells type II.
Animals ; Female ; Histology ; Leeches ; anatomy & histology ; chemistry ; classification ; growth & development ; Male ; Mouth ; anatomy & histology ; chemistry ; Mucous Membrane ; chemistry

The dried Whitmania pigra is used for the treatment of cardiovascular and cerebrovascular diseases in traditional Chinese medicine. Bellamya purificata is widely distributed in the Chang Jiang River basin, it is natural diets of W. pigra. Current study was conducted to compare and analyze the nutritional ingredient in W. pigra, body fluid and flesh of B. purificata. Results showed that the contents of protein, crude fat and total sugar in W. pigra, body fluid and flesh of B. purificata were significantly different (P < 0.05). Protein content in W. pigra accounts up to 65.01%. The contents of inorganic elements and amino acid were abundant in W. pigra, body fluid and flesh of B. purificata. The content of essential amino acids in them were 32.6, 221.59, 40.78 mg x g(-1), respectively. The content of flavor amino acid in them were 27.51, 14.5, 32.03 mg x g(-1), while the coresponding content of antioxidant amino acid were 8.81, 5.91, 9.73 mg x g(-1), respectively. The individual amino acids of high content in them were Glu, Asp and Leu. Macro elements Ca, P, Mg and trace elements Zn, Si, Fe were abundant. It could be speculated that W. pigra may be a promising novel food, and the present results provide a foundation to develop artificial feed for W. Pigra.
Amino Acids ; analysis ; Animals ; Gastropoda ; chemistry ; Leeches ; chemistry ; Medicine, Chinese Traditional

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.
Animals ; Biological Factors ; administration & dosage ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Humans ; Hypoglycemic Agents ; administration & dosage ; Leeches ; chemistry ; Male ; Rats ; Rats, Sprague-Dawley ; Saliva ; chemistry

OBJECTIVETo study the extraction system of hirudin emulsion liquid membrane with the Poecilobdella manillensis as raw material, di-(2-ethylhexyl) phosphate (D2EHPA) as carrier, Span 80 as emulsifier, octane and D2EHPA mixed to constitute membrane solution, diluted HCl solutions as internal aqueous phase.

METHODUsing the orthogonal experiment to optimize the extraction conditions of hirudin reference substance such as membrane phase, internal aqueous phase volume ratio (MIPVR), external aqueous phase pH, internal aqueous phase pH, mobile carrier concentration and so on, and then using hirudin crude extracts to do purifying experiment, and gaining experimental samples.

RESULTThe optimal conditions of hirudin extraction were as follows: MIPVR 10: 3, internal aqueous phase pH 2.6, external aqueous phase pH 3.4, the mass fraction of carrier D2EHPA 2%. In the optimal extraction conditions, when the initial concentration of hirudin was one anti-thrombin activity units (ATU) x mL(-1), ATU recovery rate of the reference substance was 83.06%. In the purifying experiment of crude extracts, ATU recovery rate was 82.99%, and the specific activity of sample was 3 289.48 the ATU x mg(-1). Discontinuous polyacrylamide gel electrophoresis and spectral scanning, the results showed that the purity and reference substance were considerable.

CONCLUSIONThe method of preparation hirudin was relatively simple, the purity of the experimental samples and ATU recovery were both high.

Animals ; Emulsions ; chemistry ; Hirudins ; analysis ; isolation & purification ; Leeches ; chemistry ; Membranes, Artificial ; Solid Phase Extraction ; instrumentation ; methods

Shuxuetong (SXT) injection is the first animal original injection in traditional Chinese medicine, the main component are the leech and the earthworm. SXT has got five national invention patents, and is recognized as the most potent medicine expelling blood stasis agent, which indication is the only one be clearly approved by SFDA as the acute phase of cerebral infarction. Modern biological extraction technology is adopted to prepare SXT, the entire production process using only saline as a solvent. Patented product process is induced to maximize the retention of medicinal components and activity, as well as to remove invalid substances as variant protein,high molecular weight substances which causes allergic reactions. SXT have been isolated and identified class 7 of56 compounds, the molecular weight is from 100 to 1 700 Da, mainly including peptides, glycopeptides, endogenous small molecules.
Animals ; Drug Therapy ; standards ; Drugs, Chinese Herbal ; administration & dosage ; adverse effects ; chemistry ; standards ; Humans ; Leeches ; chemistry ; Medicine, Chinese Traditional ; standards ; Molecular Weight ; Oligochaeta ; chemistry ; Quality Control

OBJECTIVETo establish fingerprints for Poecilobdella manillensis from Guangxi province using the high performance capillary electrophoresis (HPCE) method.

METHODElectrophoresis was performed on a fused silica capillary column (75 microm x 56 cm), with 25 mmol x L(-1) Na2HPO4-120 mmol x L(-1) Tris-16 mmol x L(-1) SDS (adjusted to pH 12.0 with 1 mol x L(-1) NaOH ) as the running buffer. The applied voltage was 17 kV, the temperature was 25 degrees C and the detection wavelength was 254 nm. The sample's hydrodynamic injection was 3.4 kPa x 6s and the duration was 27 min.

RESULTHPCE fingerprint was established with 13 common peaks. The similarity between fingerprints of P. manillensis in 10 batches and control fingerprints was more than 0.98.

CONCLUSIONThe method is so precise, reproducible and stable that it could be used as a new means for the quality control of P. manillensis.

Animals ; China ; Electrophoresis, Capillary ; methods ; Leeches ; chemistry ; Medicine, Chinese Traditional ; standards ; Quality Control ; Reproducibility of Results

OBJECTIVECurrent study was conducted to investigate and compare the impact of temperature and pH on the activities of amylase, protease and lipase in alimentary tract of Whitmania pigra.

METHODThe responses of amylase, protease, and lipase activities were determined over a wide range of temperatures (7-52 degrees C) and pH gradient (2.2-11.2).

RESULTThe highest lipase activity was found under 37 degrees C, pH 8.2, and the highest amylase activity was detected under 37 degrees C, pH 5.2, while protease activity peaked at 42 degrees C, pH 3.2 or pH 9.2.

CONCLUSIONThe optimal temperature in alimentary tract of Wh. pigra for lipase and amylase was 37 degrees C, and the responding temperature for protease was 42 degrees C. The optimal pH value in alimentary tract of Wh. pigra for lipase and amylase was pH 8.2 and pH 5.2, respectively. While pH 3.2 or 9.2 seems to be both favorable for high protease activity.

Amylases ; chemistry ; metabolism ; Animals ; Digestive System ; chemistry ; enzymology ; Enzyme Stability ; Hydrogen-Ion Concentration ; Leeches ; chemistry ; enzymology ; Lipase ; chemistry ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Temperature