Dysbiosis of intestinal homeostasis contribute to Whitmania pigra edema disease.

Whitmania pigra is widely used in traditional Chinese medicine. However, W. pigra is being threatened by an edema disease with unknown causes (WPE). In this study, a comprehensive exploration of virome, microbiome, and metabolome aberrations in the intestine of W. pigra was performed to address the aetiology of WPE. Virome analysis indicated that eukaryotic viruses did not contribute to WPE, whereas an expansion of Caudovirales was observed in WPE. Compared to the control, the microbial richness and diversity in diseased W. pigra decreased remarkably. Nine genera, including Aeromonas, Anaerotruncus, Vibrio, Proteocatella, Acinetobacter, and Brachyspira were overrepresented in WPE, whereas eleven genera, including Bifidobacterium, Phascolarctobacterium, Lactobacillus, Bacillus and AF12, were enriched in healthy individuals. Furthermore, certain metabolites, especially amino acids, short-chain fatty acids, and bile acids, were found to be linked to intestinal microbiota alterations in WPE. An integration of the microbiome and metabolome in WPE found that dysbiosis of the gut microbiota or metabolites caused WPE. Notably, W. pigra accepted intestinal microbiota transplantation from WPE donors developed WPE clinical signs eventually, and the dysbiotic intestinal microbiota can be recharacterized in this recipient W. pigra. Strikingly, pathological features of metanephridium and uraemic toxin enrichment in the gut indicated a putative interconnection between the gut and metanephridium in WPE, which represents the prototype of the gut-kidney axis in mammals. These finding exemplify the conservation of "microecological Koch's postulates" from annelids to insects and other vertebrates, which provides a direction of prevention and treatment for WPE and opens a new insight into the pathogenesis of aquatic animal diseases from an ecological perspective.

Characterization of the natural peptidome of four leeches by integrated proteogenomics and pseudotargeted peptidomics.

Animal-derived drugs are an indispensable part of folk medicine worldwide. However, their chemical constituents are poorly approached, which leads to the low level of the quality standard system of animal-derived drugs and further causes a chaotic market. Natural peptides are ubiquitous throughout the organism, especially in animal-derived drugs. Thus, in this study, we used multi-source leeches, including Hirudo nipponica (HN), Whitmania pigra (WP), Whitmania acranulata (WA), and Poecilobdella manillensis (PM), as a model. A strategy integrating proteogenomics and novel pseudotargeted peptidomics was developed to characterize the natural peptide phenotype and screen for signature peptides of four leech species. First, natural peptides were sequenced against an in-house annotated protein database of closely related species constructed from RNA-seq data from the Sequence Read Archive (SRA) website, which is an open-sourced public archive resource. Second, a novel pseudotargeted peptidomics integrating peptide ion pair extraction and retention time transfer was established to achieve high coverage and quantitative accuracy of the natural peptides and to screen for signature peptides for species authentication. In all, 2323 natural peptides were identified from four leech species whose databases were poorly annotated. The strategy was shown to significantly improve peptide identification. In addition, 36 of 167 differential peptides screened by pseudotargeted proteomics were identified, and about one-third of them came from the leucine-rich repeat domain (LRR) proteins, which are widely distributed in organisms. Furthermore, six signature peptides were screened with good specificity and stability, and four of them were validated by synthetic standards. Finally, a dynamic multiple reaction monitoring (dMRM) method based on these signature peptides was established and revealed that one-half of the commercial samples and all of the Tongxinluo capsules were derived from WP. All in all, the strategy developed in this study was effective for natural peptide characterization and signature peptide screening, which could also be applied to other animal-derived drugs, especially for modelless species that are less studied in protein database annotation.

Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis.

Haematophagous leeches express a broad variety of secretory proteins in their salivary glands, among them are hirudins and hirudin-like factors. Here, we describe the identification, molecular and initial functional characterization of Tandem-Hirudin (TH), a novel salivary gland derived factor identified in the Asian medicinal leech, Hirudinaria manillensis. In contrast to the typical structure of hirudins, TH comprises two globular domains arranged in a tandem-like orientation and lacks the elongated C-terminal tail. Similar structures of thrombin inhibitors have so far been identified only in kissing bugs and ticks. Expression of TH was performed in both cell-based and cell-free bacterial systems. A subsequent functional characterization revealed no evidence for a thrombin-inhibitory potency of TH.

Life without blood: Molecular and functional analysis of hirudins and hirudin-like factors of the Asian non-hematophagous leech Whitmania pigra.

BACKGROUND: Several leech species of the genera Hirudo, Hirudinaria, and Whitmania are widely used in traditional Chinese medicine (TCM) for the oral treatment of disorders associated with blood stasis. Among them, the non-hematophagous leech Whitmania pigra expresses a variety of components that have the potential to act on the vertebrate blood coagulation system. OBJECTIVE: Whether the thrombin inhibitor hirudin, probably the most prominent leech-derived anticoagulant, is actually present in Whitmania pigra, is still a matter of debate. To answer that open question was the aim of the study. METHODS: We identified several putative hirudin-encoding sequences in transcriptome data of Whitmania pigra. Upon gene synthesis and molecular cloning the respective recombinant proteins were expressed in Escherichia coli, purified, processed, and eventually functionally characterized for thrombin-inhibitory potencies in coagulation assays. RESULTS: We were successful in the identification and functional characterization of several putative hirudins in Whitmania pigra. Some, but not all, of these factors are indeed thrombin inhibitors. Whitmania pigra hence expresses both hirudins (factors that inhibit thrombin) and hirudin-like factors (that do not or only very weakly inhibit thrombin). Furthermore, we revealed the exon/intron structures of the corresponding genes. Coding sequences of some putative hirudins of Whitmania pigra were present also in transcriptome datasets of Hirudo nipponia, a hematophagous leech that is likewise used in TCM. CONCLUSIONS: Based on both structural and functional data we provide very strong evidence for the expression of hirudins in Whitmania pigra. This is the first description of hirudins in a non-hematophagous leech.

A study of the influence of lead pollution on the anticoagulant activity of Whitmania pigra based on pharmacodynamics and metabolomics research.

Whitmania pigra Whitman (leech, also called Shuizhi in China, abbreviated as SZ), which has been used as a traditional Chinese medicine in the treatment of blood stasis syndrome (BSS) for a long time, is vulnerable to lead pollution in aquaculture environments. SZ has good anticoagulant activity. However, there are few studies on the influence of lead pollution on it. Therefore, we carried out the following researches to explore the influence of lead pollution on the anticoagulant activity of SZ and its mechanism. Firstly, the acute blood stasis model of rats was established by subcutaneous injection of adrenaline hydrochloride and ice water bath. Then unpolluted SZ (UPS) and lead-polluted SZ (LPS) were extracted. Next, the blood stasis model rats were administrated by gavage and the rats in normal control (NC) group and blood stasis model (BM) group were given the same amount of normal saline. Finally, the blood of the rats was collected to detect the coagulation function and hemorheology indexes. The metabolomics of rat plasma was studied by ultra-high-performance liquid chromatography coupled with orbitrap mass spectrometry (UPLC-Orbitrap-MS) technology. Principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and Hierarchical clustering analysis (HCA) were used to perform metabolomics analysis. MetPA analysis was used to search for related metabolic pathways. The results of coagulation function and hemorheology showed that lead pollution could decrease the anticoagulant activity of SZ. The OPLS-DA score plots indicated that the plasma metabolites of rats in LPS group were close to BM group, while UPS group tended to be close to NC group both in the positive and negative ion mode. Hierarchical cluster analysis (HCA) suggested that UPS group and NC group were clustered into a branch, while LPS group and BM group were clustered into a branch. To sum up, lead pollution will reduce the anticoagulant activity of SZ. And lead pollution reduces the anticoagulant activity of SZ probably by influencing the metabolic pathways such as sphingolipid metabolism, amino acid metabolism and energy metabolism in rats.

Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis.

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 (Ki) of 1.69 × 10-8 mol·L-1. Further study showed that HMEI-A inhibited the formation of neutrophil extracellular trap (NET). These results suggested that HMEI-A from the leech of H. manillensis is a novel elastase inhibitor which can suppress NET formation. It may play a significant role in blood-sucking of leeches and is a potential candidate as an anti-inflammatory agent.

Affinity ultrafiltration and UPLC-HR-Orbitrap-MS based screening of thrombin-targeted small molecules with anticoagulation activity from Poecilobdella manillensis.

This study aims to screen potential anticoagulant components from leeches, a representative animal-sourced traditional Chinese medicine using thrombin (THR)-targeted ultrafiltration combined with ultrahigh performance liquid chromatography and high-resolution Orbitrap mass spectrometry (UPLC-HR-Orbitrap-MS). As a result, five small molecules in leech extract were discovered to interact with THR for the first time. Among them, two new compounds were isolated and their structures were identified by IR, HR-MS and NMR data. Furthermore, their THR inhibitory activity was confirmed with IC50 values of 4.74 and 8.31 µM, respectively. In addition, molecular docking analysis showed that the active (catalytic) site of THR might be the possible binding site of the two hits. Finally, reverse screening analysis indicated that LTA4-H, ACE and ALOX5AP were potential anticoagulant targets of the two new compounds. This study will broaden our understanding of the medicinal substance basis in leeches and further contribute to the discovery and development of clinical anticoagulant drugs from leeches.

Uncovering the mechanisms of leech and centipede granules in the treatment of diabetes mellitus-induced erectile dysfunction utilising network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetes mellitus-induced erectile dysfunction (DMED) is one of the most common complications of diabetes mellitus. Leech and centipede granules (LCG) have traditionally been used as blood-activating agents in various ethnomedicinal systems of East Asia, especially in China. It is often used to regulate bodily functions and considered as adjuvant therapy for promoting blood circulation, alleviating blood coagulation, activating meridians, and relieving stasis. AIM OF THE STUDY: This study aimed to identify potential genes and mechanisms of LCG on DMED from the network pharmacological perspective. MATERIALS AND METHODS: The active components of LCG were identified by UHPLC-Q-TOF-MS, TCMID, and the BATMAN-TCM databases, and the disease targets of DMED were obtained from the DisGeNET, CooLGeN, GeneCards databases. After identifying DMED targets of LCG, a protein-protein interaction (PPI) network was constructed. Hub genes and significant modules were identified via the MCODE plug-in of Cytoscape software. Then, significant signaling pathways of the modules were identified using the Metascape database. The probable interaction mode of compounds-hub genes is examined using Molecular Operating Environment (MOE) docking software. Besides, we investigated the effects and mechanisms of LCG on improving erectile function in the streptozotocin (STZ)-induced diabetic rats model. RESULTS: Combined UHPLC-Q-TOF-MS analysis with network pharmacology study, 18 active compounds were selected for target prediction. There are 97 common target genes between LCG and DMED. Enrichment of the KEGG pathway mainly involves in the calcium signaling pathway, NF-kappa B signaling pathway, cGMP-PKG signaling pathway, HIF-1 signaling pathway, PI3K-Akt signaling pathway, and mTOR signaling pathway. Nine hub genes were regulated by LCG in DMED, including CXCL8, NOS3, CRH, TH, BDNF, DRD4, ACE, CNR1, and HTR1A. The results of molecular docking analysis showed that the tyrosin, ursolic acid, and L-Histidine has a relatively stable interaction with corresponding hub genes via generating hydrogen bonds, H-π, and π-π interactions. Significantly, the results in docking predicted a higher affinity of vardenafil to the hub genes compared to the tyrosin, ursolic acid, and L-Histidine. Furthermore, LCG increased the testosterone, erection frequency, the ratio of ICP and MAP, SOD, cGMP, cAMP as well as decreased the MDA, and AGEs expression levels. And, LCG ameliorated the histological change of penile tissues in DMED rats. Hence, LCG attenuates oxidative stress, increases NO production; For the mechanism exploration, LCG could significantly upregulate the mRNA and protein expression of CNR1, NOS3, CRH, TH, BDNF, and DRD4, whereas CXCL8, ACE, and HTR1A levels were significantly higher than those in the DMED group. Moreover, LCG activates the NO/cGMP/PKG pathway, PI3K/Akt/nNOS pathway, cAMP/PKA pathway, and inhibits the HIF-1α/mTOR pathway to improve erectile function. CONCLUSIONS: Our results suggest that LCG maybe offer a new therapeutic basis for the treatment of DMED via altering the gene expression of involved metabolic pathways.

Co-administration of Erythromycin and Leech Salivary Extract Alleviates Osteomyelitis in Rats Induced by Methicillin-Resistant Staphylococcus aureus.

OBJECTIVE: Erythromycin (Ery) and leech saliva (LS) can inhibit Staphylococcus aureus growth in in vitro conditions. This study aimed to evaluate the activities and synergy between Ery and LS on chronic osteomyelitis in male Wistar rat's tibia induced by methicillin-resistant S. aureus (MRSA). MATERIALS AND METHODS: Four weeks after osteomyelitis induction, rats were divided into four groups including no treatment (control), Ery monotherapy (orally), LS monotherapy, or Ery + LS twice daily for 2 weeks. Staphylococcus aureus growth, pathological signs and inflammatory cytokine tumour necrosis factor-alpha (TNF-α) levels were assessed. RESULTS: Rats tolerated all therapeutic strategies well during the experiment. The Ery treatment alone significantly decreased bacterial growth, pathological signs and TNF-α levels. Leech saliva alone reduced TNF-α level significantly, but did not produce a significant reduction in bacterial growth and pathological signs. Ery + LS treatment significantly decreased bacterial growth, considerably alleviated bone pathological signs and decreased TNF-α levels compared with other groups. Statistical analysis suggested that there was a stronger efficiency and synergistic action of Ery and LS when combined against MRSA-induced osteomyelitis in rats. CLINICAL SIGNIFICANCE: The present study suggests that LS may have clinical utility to treat MRSA-induced osteomyelitis when combined with Ery or other therapeutics.

The hirudin-like factors HLF3 and HLF4-hidden hirudins of European medicinal leeches.

The hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis, Hirudo verbana, and Hirudo orientalis, respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of hirudin and HLF molecules for thrombin inhibition.

Characterization of a monoacylglycerol lipase in the medicinal leech, Hirudo verbana.

Endocannabinoids are a class of lipid neuromodulators found throughout the animal kingdom. Among the endocannabinoids, 2-arachydonoyl glycerol (2-AG) is the most prevalent endocannabinoid and monoacylglycerol lipase (MAGL) is a serine hydrolase primarily responsible for metabolizing 2-AG in mammals. In the medicinal leech, Hirudo verbana, 2-AG has been found to be an important and multi-functional modulator of synaptic transmission and behavior. However, very little is known about the molecular components of its synthesis and degradation. In this study we have identified cDNA in Hirudo that encodes a putative MAGL (HirMAGL). The encoded protein exhibits considerable sequence and structural conservation with mammalian forms of MAGL, especially in the catalytic triad that mediates 2-AG metabolism. Additionally, HirMAGL transcripts are detected in the Hirudo central nervous system. When expressed in HEK 293 cells HirMAGL segregates to the plasma membrane as expected. It also exhibits serine hydrolase activity that is blocked when a critical active site residue is mutated. HirMAGL also demonstrates the capacity to metabolize 2-AG and this capacity is also prevented when the active site is mutated. Finally, HirMAGL activity is inhibited by JZL184 and MJN110, specific inhibitors of mammalian MAGL. To our knowledge these findings represent the first characterization of an invertebrate form of MAGL and show that HirMAGL exhibits many of the same properties as mammalian MAGL's that are responsible for 2-AG metabolism.

A novel protease-activated receptor 1 inhibitor from the leech Whitmania pigra.

Whitmania pigra has been used as a traditional Chinese medicine (TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the therapeutic components of this species, especially proteins and peptides were poorly exploited. Until now only a few of them were obtained by using chromatographic isolation and purification. In recent decade, transcriptome techniques were rapidly developed, and have been used to fully reveal the functional components of many animal venoms. In the present study, the cDNA of the salivary gland of Whitmania pigra was sequenced by illumina and the transcriptome was assembled by using Trinity. The proteome were analysed by LC-MS/MS. Based on the data of the transcriptome and the proteome, a potential antiplatelet protein named pigrin was found. Pigrin was cloned and expressed using P. pastoris GS115. The antiplatelet andantithrombotic bioactivities of pigrin were tested by using aggregometer and the rat arterio-venous shunt thrombosis model, respectively. Thebleeding time of pigrin was measured by a mice tail cutting method. The docking of pigrin and protease-activated receptor 1 (PAR1) or collagen were conducted using the ZDOCK Server. Pigrin was able to selectively inhibit platelet aggregation stimulated by PAR1 agonist and collagen. Pigrin attenuated thrombotic formation in vivo in rat, while did not prolong bleeding time at its effective dosage. There are significant differences in the key residues participating in binding of Pigrin-Collagen complex from Pigrin-PAR1 complex. In conclusion,a novel PAR1 inhibitor pigrin was found from the leech Whitmania pigra. This study helped to elucidate the mechanism of the leech for the treatment of cardiovascular disorder.

Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches.

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

[Preliminary study on changes of sex steroid hormones in periodic development of Whitmania pigra gonad].

The present study was conducted to investigate the rule of sex steroid hormones dynamic in the periodic development of Whitmania pigra gonad. The dynamic of sex steroid hormones in different age of Wh. pigra were detected by enzymolysis, extraction and UPLC-MS/MS. The results showed that the concentrations of estrone, estriol, testosterone and progesterone in the Wh. pigra showed M-type curve and peaked in 6-month-old Wh. pigra. The concentrations of above-mentioned four steroid hormones were the lowest in 9-month-old and then increased slightly. The another peaks of testosterone and progesterone were found in 2-month-old and the another peaks of estrone and estriol were obtained in 8-month-old. The concentrations of testosterone and progesterone are slightly higher than estriol during 1-month-old to 4-month-old, and thereafter strone and estriol showed higher concentration than testosterone and progesterone. In summary, the concentrations of four sex steroid hormones in Wh. pigra increased gradually with the maturation of gonads and decreased rapidly after the discharge period, which indicated that the ratio between estrogen, androgen and progesterone may be greatly related to the specificity of gonad development.

[Effect of ingestion on antithrombin activity in different tissues of Whitmania pigra].

To study the effects of ingestion on antithrombin activities in different tissues of Whitmania pigra, the salivary glands, ingluvies, intestine and muscle of adult leeches were weighted on the 1st d, 3rd d, 5th d, 7th d and 11th d after feeding, respectively, and meanwhile antithrombin activity was measured by antithrombin titration method. The results showed that the antithrombin activity of salivary glands in different stages was significantly higher than that in other tissues (P<0.05). The activity of antithrombin in muscle tissue increased initially and then decreased with the prolongation of the time after feeding, and the peak was observed on the 5th day after feeding (P<0.05). The activity of antithrombin in the salivary glands, gluvies and intestine were found the highest on the 1st day after feeding(P<0.05), and then gradually decreased with the prolongation of the time of stopping the diet. The total amount of antithrombin activity on the 5th day was increased by 49.5%, 73.5%, 69.1% and 126.0% comparing with the 1st, 3rd, 7th and 11th day after feeding, respectively (P<0.05). In summary, both the feeding behavior and the food can induce the secretion of anticoagulant substances in the salivary glands and the digestive tract. The total amount of antithrombin activity was the highest on the 5th day after feeding and the 5th day after feeding was suggested as harvesting time.

Identification and characterization of a novel neuropeptide (neuropeptide Y-HS) from leech salivary gland of Haemadipsa sylvestris.

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.

A novel selective inhibitor to thrombin-induced platelet aggregation purified from the leech Whitmania pigra.

BACKGROUND/AIM: The dried whole body of the leech Whitmania pigra, a well-known traditional Chinese medicine, has been widely used to treat thrombus diseases for thousands of years. However, its bioactive constituents were reported rarely. The aim of our study was to investigate antithrombotic components of it. METHODS: The antithrombotic peptide was purified using a combination of anion-exchange chromatography, ultrafiltration and reverse-phase HPLC. the sequence of the peptide was determined using MALDI-TOF-MS-MS. Anti-platelet aggregation activity in vitro was evaluated using a turbidimetric method, and antithrombotic effect in vivo was assessed in an arterio-venous shunt thrombosis model in rats. RESULTS: A novel antithrombotic peptide named WP-30, with the sequence VISRTQSNVQAAWGQVGGHAADYSAVAIER, was isolated from the dried whole body of the leech W. pigra. WP-30 selectively inhibited thrombin-induced anti-platelet aggregation in vitro, and potently attenuated thrombus formation in rats in vivo. CONCLUSIONS: Taken together, we found a novel peptide from leech bodies, and this peptide showed antiplatelet aggregation and antithrombotic effects.

[In vitro anticoagulant activity of different processed products of Whitmania pigra by water extraction and bionic extraction].

In order to determine the scientificalness of traditionally processed Whitmania pigra, water extraction method and bionic extraction method were used respectively to extract the anticoagulating active components in W. pigra hanging dry products, talcum powder fried products and wine immersing-baked products. Activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and antithrombin activity were selected as the activity indexes to evaluate the anticoagulant activities of different processed W. pigra. Then the contents of protein in different processed W. pigra were measured by Coomassie brilliant blue method to preliminarily explain the reason of anticoagulant activity changes. When water extraction method was used, the results of APTT, PT, TT and antithrombin activity showed that the anticoagulant activities of W. pigra were decreased both in talcum powder fried products and wine immersing-baked products, and the activity order was as follows: hanging dried products> wine immersing-baked products>talcum powder fried products. This order was same as the protein content order. While when bionic extraction was used, APTT was shortened in talcum powder fried products, but all the other results indicated the anticoagulant activities of W. pigra processed products were increased, and the activity order was as follows: wine immersing-baked products>talcum powder fried products>hanging dry products. As compared with water extraction, the bionic extraction was more similar to the absorption process of W. pigra in human digestive system after oral administration and was more scientific. Therefore, the traditional processing method can not only modify the taste and smell, but also enhance the anticoagulant activity of W. pigra.

[Review on scientific connotation of leech processed under high temperature].

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.