OBJECTIVETo study the anticoagulant constituents in dried leech (Whitmania pigra Whitman).

METHODThe plasma recalcification time (PRT) as index, the constituents with anticoagulant activity were isolated and purified by anion-exchange chromatography on Sephadex DEAE A-50, gel permeation chromatography on Sephadex G-25 and Sephadex LH-20 columns, and then reversed phase high-performance liquid chromatography successively.

RESULTThree anticoagulant polypeptides were isolated and purified. Compounds 1 and 2 can be translated each other in natural conditions, and their molecular weights are 7100 and 5531, respectively. Compound 3 was identified as a pure polypeptide by HPLC and SDS-PAGE, and its molecular weight was determined as 8 608 by MALDI-TOF-MS. The amino acid composition of compound 3 was also determined.

CONCLUSIONCompound 3 was inferred to be a novel anticoagulant, and named whitmanin.

Animals ; Anticoagulants ; analysis ; chemistry ; isolation & purification ; Leeches ; chemistry ; Molecular Weight

OBJECTIVEUsing HPLC To determine hypoxanthine in co-hirudo injection for establishing its HPLC fingerprint, and evaluating its internal quality.

METHODThe chromatographic separation was performed on a Kromasil C18 column (4.6 mm x 250 mm,5 microm). A linear gradient elution with A (0.01 mol x L(-1) x KH2PO4) and B (50% methanol) was used, the flow rate was 0.8 mL x min(-1), the detection wavelength was set at 254 nm, and the column temperature was at normal.

RESULTHypoxanthine was used as the reference substance in the fingerprint of co-hirudo injection, it showed 15 common peaks and theirs similarity threshod was 0.97.

CONCLUSIONThis method was accurate, repeatable and useful for the quality control of co-hirudo injection.

Animals ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Hypoxanthine ; chemistry ; Leeches ; chemistry ; Reproducibility of Results

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

OBJECTIVETo determine the contents of hirudin's hydrolysates in processed leeches, set up a new evaluating method of dot blotting to evaluate the qualities of those processed Chinese medicines as leeches.

METHODContents of hirudin's hydrolysates in processed leeches were determined by dot blotting with rat antibody of anti-hirudin as the first antibody. Blotting signal was analyzed by software of Quantity One.

RESULTContents of hirudin's hydrolysates in four batches of processed leeches were 296.51, 165.47, 95.58, and 298.05 microg g(-1), respectively.

CONCLUSIONDifference among four batches of processed leeches was significant in the content of hirudin's hydrolysates. Dot blotting, as a convenient and accurate method can be broadly used for evaluating processed products of Chinese crude drugs similar to leeches.

Animals ; Drugs, Chinese Herbal ; chemistry ; Hirudins ; immunology ; metabolism ; Immunoblotting ; methods ; Leeches ; chemistry ; Reproducibility of Results

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

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

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

OBJECTIVETo evaluate the genetic differentiation and phylogenetic relationship between Whitmania pigra Whitman and Hirudo nipponia Whitman.

METHODBy the sequences of the rDNA internal transcribed spacer (ITS) the molecular phylogenetic tree was constructed by MP method using software MEGA 4.0.

RESULTThe average length of ITS was 857.2-861.2 bp. The A, T, G and C contents in this fragment were 25.12%, 28.28%, 17.34%, 29.29%, respectively. The GC content was higher than the AT content. Little sequence variation was observed in ITS gene fragments with in species, and transition in only 45 loci was revealed in 14 populations. 14 W. pigra and H. nipponia populations were clustered into 2 groups by MP phylogenetic tree.

CONCLUSIONThe results also showed that the intraspecific variation was dominated in variation types of W. pigra and H. nipponia. The classification result of W. pigra and H. nipponia and its adulterants based on DNA sequences are not totally consistent with those based on classification. It showed that a little of mutation of base in ITS sequences had occurred in the process of evolution, such as transition cites, transvertion cites among base or base gap.

Animals ; Base Sequence ; DNA, Ribosomal Spacer ; chemistry ; Leeches ; classification ; genetics ; Molecular Sequence Data ; Phylogeny

OBJECTIVEDynamic changes of quality and growth of Whitmania pigra were investigated to provide theoretical basis for quality control and determination of optimal harvest time.

METHODThe contents of moisture, ethanol-soluble extractive, total ash, and acid-insoluble ash, as well as antithrombin activity were determined according to Chinese pharmacopoeia (2005 edition).

RESULTQualities of W. pigra collected from different growth stages met the standards of Chinese pharmacopoeia (2005 edition). The highest amount of ethanol-soluble extract was found in 9-month-old, W. pigra, followed by 6-month-aged ones. And there was significant difference between 9-month-old and 4-month-old, 7 month-old and 11 month-old (P < 0.05). The highest antithrombin activity was detected in 6 month-old W. pigra, followed by 10 month-old. Significant differences of antithrombin activity were found between 6-month-old and 4 month-old ones, and 5 month-old (P < 0.05), however, there were no significant difference between 6 month-old ones with other samples. Eleven-month-old W. pigra got the most dry weight, and there were significant difference between 11 month-old ones with other samples (P < 0.05).

CONCLUSIONNovember, namely for 6 month old artificial cultivated W. pigra was the best harvest time in Jiangsu and Zhejiang provinces.

Animals ; Antithrombin III ; Antithrombins ; analysis ; China ; Leeches ; chemistry ; growth & development ; Medicine, Chinese Traditional ; Peptide Fragments

OBJECTIVETo examine the quality of Whitmania pigra from the different populations to provide the basis for new species selection.

METHODThe contents of the moisture, alcohol extractive, total ash, acid-insoluble ash, and the activity of antiplatelet aggregation enzyme of wild and the breeding W. pigra were determined by the methods were determined in Chinese Pharmacopoeia (2005 edition). The contents xanthine and hypoxanthine by HPLC.

RESULTIt showed that the contents of the alcohol extractive, total ash, acid-insoluble ash, antiplatelet aggregation enzyme, and xanthine and hypoxanthine content of the breeding population in Nanjing are lations. Many quality indexes of the breeding in Zhejiang Tongxiang base were beffer than the wild.

CONCLUSIONThe breeding W. pigra are good in quality, It suggested large-scale promotion of aquaculture.

Animals ; Breeding ; Female ; Hypoxanthine ; analysis ; Leeches ; chemistry ; physiology ; Male ; Xanthine ; analysis