OBJECTIVEApplying for the activity of enzyme in vitro,the research optimized the best preparation procedure for the anticoagulated blood region from Lumbricus.

METHODAll through our experiment, the content of protein and theactivity of enzyme were examined. The extraction process, the refining technology, concentration processes of Lumbricus were optimized with single factor checking and orthogonal design method.

RESULTAt 37 degrees C, the coarse powder of Lumbricus soaking with 15 fold of 0.9% sodium chloride and ultrasonic extracting 40 minites for three times was the best ultrasonic extraction. Utrafiltration membrane with molecular weights of 30 x 10(3) for refining and 10 x 10(3) for concentrating were selected.

CONCLUSIONUltrasonic extraction and membrane separation technology, to well improve the effect of purification for the anticoagulant site of Lumbricus, is conducive to further study.

Animals ; Anticoagulants ; chemistry ; isolation & purification ; Drug Compounding ; methods ; Oligochaeta ; chemistry ; enzymology ; Temperature ; Ultracentrifugation ; Ultrasonics

OBJECTIVETo study the factors influencing the activity of fibrinolytic enzymes from earthworm and to obtain the better way to extract fibrinolytic enzymes as well as keep its optimum activity.

METHOD75% alcohol, 0.9% NaCl and 10% saccharose was used to extract the crude fibrinolytic enzymes from earthworm, the method of urokinase gelose-fibrin plate was used to measure the activity of fibrinolytic enzymes from earthworm. and the method of 3,3'-diaminobezidine tetrahydrochloride colorimetry to was used measure the content of selenium. The method use ts of measuring the content of arsenic was silver diethyldithiocarbamate colorimetry.

RESULTThe fibrinolytin of earthworms reared with cattle soils had higher activity than that reared with garbage. The arsenic in the earthworm's body could improve the activity of earthworm's fibrinolytin. However, the selenium had litter influence on it. Among the three methods of extraction, the 75% alcohol one was the most efficient, the 0.9% NaCl was next, and the 10% saccharose was the lowest. The influence of dialysis on the activity of fibrinolytin was less than that of ultrafiltration, when the earthworm's fibrinolytin enzyme was further sublimated.

CONCLUSIONThe activity of the earthworm's fibrinolytin will be increased earthworm is reared with the fitting baits and when appropriate methods, of extraction and purification are used.

Animal Feed ; Animals ; Endopeptidases ; isolation & purification ; metabolism ; Oligochaeta ; chemistry ; Selenium ; pharmacology

In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.
Animals ; Chromatography, Liquid ; Electrophoresis, Polyacrylamide Gel ; Gene Ontology ; Mass Spectrometry ; Oligochaeta ; chemistry ; Proteome ; Proteomics

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 explore the antivirus function in vitro of earthworm coelomic fluid (ECF) by researching its effect on inhibiting respiratory syncytial virus (RSV).

METHODSBy the method of Hep-2 cell culture and using ribavirin as a positive control, the anti-RSV effect of ECF was investigated by observing cytopathic effect (CPE) with MTT colorimetric assay.

RESULTSIn Hep-2 cells, the CC50 of ECF and ribavirin were 3.11 mg/ml and 1.35 mg/ml separately. In the experiment of ECF directly killing RSV, the IC50 of ECF was 184.1 microg/ml, SI was 16.87; In the experiment of ECF preventing RSV invasion, no antiviral function of ECF within the experimental concentration range was observed; In the experiment of ECF inhibiting RSV replication, the IC50 of ECF was 1555. 8 microg/ml, SI was 1.99.

CONCLUSIONECF couldn't prevent virus from invading into host cell, but showed direct killing-virus function and inhibition of the virus replication.

Animals ; Antiviral Agents ; chemistry ; pharmacology ; Cell Line ; Humans ; Inhibitory Concentration 50 ; Oligochaeta ; chemistry ; Respiratory Syncytial Viruses ; drug effects ; Virus Replication ; drug effects

In this paper, actinomycetes were isolated from vermicompost by tablet coating method. Antimicrobial activities of actinomycetes were measured by the agar block method. Strains with high activity were identified based on morphology and biochemical characteristics, as well as 16S rDNA gene sequence analysis. The results showed that 26 strains of actinomycetes were isolated, 16 of them had antimicrobial activities to the test strains which accounts for 61.54% of all strains. Among the 16 strains, the strain QYF12 and QYF22 had higher antimicrobial activity to Micrococcus luteus, with a formed inhibition zone of 27 mm and 31 mm, respectively. While the strain QYF26 had higher antimicrobial activity to Bacillus subtilis, and the inhibition zone diameter was 21 mm. Based on the identification of strains with high activity, the strain QYF12 was identified as Streptomyces chartreusis, the strain QYF22 was S. ossamyceticus and the strain QYF26 was S. gancidicus. This study provided a theoretical basis for further separate antibacterial product used for biological control.
Actinobacteria ; chemistry ; classification ; genetics ; isolation & purification ; Animals ; Anti-Bacterial Agents ; isolation & purification ; metabolism ; Bacteria ; drug effects ; Feces ; microbiology ; Molecular Sequence Data ; Oligochaeta ; Phylogeny ; Quality Control

OBJECTIVETo explore the effect of compound decoction on notoginsenosides in Panax notoginseng.

METHODNotoginsenoside R1, Rg1, Re, Rb1 and pH were used as the parameters to investigate the changes on the content of notoginsenosides in different compound extractions by heating for two hours and their correlation with pH.

RESULTWhen the pH values of solution of P. notoginseng with Fructus ligustri, P. notoginseng with Eupolyphaga seu steleophaga, P. notoginseng with Pheretima asiatica, and Zhitangjiang Fang (free of Hirudo) were rept higher than 5.7, the reserved rate (RR) of notoginsenside were higher than 90%; When the pH values of decoetion of P. notoginseng with Salvia miltiorrhiza, P. notoginseng with Paeonia lactiflora, P. notoginseng with Platycodon grandiflorum, P. notoginseng with Arctium lappa were kept 4.5-5.5, their RR of notoginsenside were 60% - 85%; When the pH values of the decotction of P. notoginseng with Hirudo nipponica was decreased to 3.4, its RR of of notoginsenside was 38.4%; When the pH values of Zhitangjiang Fang extraction was regulated by 0.1% NaOH solution to pH 6. 3, and the RR of notoginsenside increased to 97%.

CONCLUSIONThe pH of other Chinese herbal medicines extraction with P. notoginseng compound is a critical effect on the stability and yields of notoginsensides.

Animals ; Arctium ; chemistry ; Cockroaches ; chemistry ; Drug Combinations ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Ginsenosides ; analysis ; Hirudo medicinalis ; chemistry ; Hot Temperature ; Hydrogen-Ion Concentration ; Ligustrum ; chemistry ; Materia Medica ; chemistry ; isolation & purification ; Oligochaeta ; chemistry ; Paeonia ; chemistry ; Panax ; chemistry ; Platycodon ; chemistry ; Salvia miltiorrhiza ; chemistry

OBJECTIVETo explore the antineoplastic effect in vitro of earthworm coelomic fluid (ECF)on growth inhibition and its mechanism for the tumor cell lines Siha, SW480, Colo205 and PC12.

METHODSMTT colorimetric assay, flow cytometry and morphological analysis were used to test its antitumor activity on tumor cell lines and normal cell line Cos7 in vitro.

RESULTSECF can inhibit the cell growth of Siha, SW480, Colo205, PC12 and Cos7. But different tumor cell lines showed different sensitivity.

CONCLUSIONEFC can significantly inhibit the proliferation of tumor cells in vitro by inducing tumor cells apoptosis.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Body Fluids ; chemistry ; COS Cells ; Cell Growth Processes ; drug effects ; Cell Line ; Cell Line, Tumor ; Cercopithecus aethiops ; Oligochaeta ; chemistry ; PC12 Cells ; Rats

There are four different types of N-terminal amino acid sequences (F-I-0, F-I, F-II, F-III) in the multicomponents of earthworm fibrinolytic enzymes (EFE). In GenBank 21 nucleic acid sequences of EFE have been reported. Among them, most of the N-terminal amino acid sequences belong to the F-III type,few belong to the F-II type. Only one is similar to the F-I type, but none to F-I-0. In this research we hoped to obtain the gene encoding component F-I-0 of EFE by the bioinformatics tools. Based on the N-terminal amino acid sequence VVGGSDTTIGQYPHQL of the F-I-0 type from Lumbricus rubellus, a nucleic acid sequence was obtained by in silico cloning from dbEST of Lumbricidae using the software DNAMAN. A new gene of EFE from Eisenia foetida was successfully obtained by RT-PCR using specific primers designed according to this sequence. The new gene named EfP-0 was cloned in pMAL-c2x and expressed as the fusion protein MBP-EfP-0 in the supernatant of lysate. The fusion protein MBP-EfP-0 purified by affinity chromatography had hydrolytic activity on casein plate. Sequencing result shows, EfP-0 has 678bp and encodes a protein of 225 amino acids. The protein is a serine protease belonging to trypsin family. It has similar amino acid composition to F-I-0. BLAST in GenBank shows that the similarity is lower than 40% between EJP-0 gene and other EFE genes. By this we conclude that EfP-0 gene of EFE is a novel gene and it is the first time to be reported, its accession number for Genbank is DQ836917.
Amino Acid Sequence ; Animals ; Cloning, Molecular ; Computational Biology ; Databases, Genetic ; Endopeptidases ; biosynthesis ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; Expressed Sequence Tags ; metabolism ; Molecular Sequence Data ; Oligochaeta ; enzymology ; genetics ; Open Reading Frames ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVE: To develop a convenient method for rapid purification of fresh Pheretima proteins and assess the inhibitory effect of these proteins against pulmonary fibrosis. METHODS: The crude extract of fresh Pheretima was obtained by freeze-drying method and then purified by size exclusion chromatography. The composition of the purified proteins was analyzed by mass spectrometry. MRC-5 cells were treated with 5 ng/mL TGF-β1 alone (model group) or in combination with SB431542 (2 μmol/L) or the purified proteins (13.125 μg/mL), and the cytotoxicity of purified proteins and their inhibitory effects on cell proliferation were detected with CCK8 assay. Flow cytometry was used to detect the changes in cell apoptosis, and the cellular expressions of α-SMA, Vimentin, E-cadherin, collagen I, Smad2/3 and P-Smad2/3 were detected using RT-PCR and Western blotting. In the animal experiment, adult male C57BL/6 mice were subjected to intratracheal instillation of bleomycin followed by treatment with the purified proteins (5 mg/mL) for 21 days, after which HE and Masson staining was used to observe the pathological changes in the lung tissue of the mice. RESULTS: We successfully obtained purified proteins from fresh Pheretima protein by size exclusion chromatography. Treatment with the purified proteins significantly inhibited TGF-β1-induced proliferation of MRC-5 cells (P < 0.01), reduced the cellular expressions of α-SMA, Vimentin and collagen I (P < 0.001 or P < 0.01), increased the expression of E-cadherin (P < 0.01), and inhibited the expressions of Smad2/3 and P-Smad2/3 (P < 0.001 or P < 0.01). In male C57BL/6 mice models of bleomycin-induced pulmonary fibrosis, treatment with the purified proteins obviously reduced the number of inflammatory cells and fibrotic area in the lungs. CONCLUSION The purified proteins from fresh Pheretima obtained by size exclusion chromatography can inhibit pulmonary fibrosis in mice by regulating the TGF-β/ Smad pathway.
Animals ; Biological Products/pharmacology* ; Bleomycin/adverse effects* ; Cadherins/metabolism* ; Collagen Type I ; Lung/pathology* ; Male ; Mice ; Mice, Inbred C57BL ; Oligochaeta/chemistry* ; Pulmonary Fibrosis/drug therapy* ; Transforming Growth Factor beta1/metabolism* ; Vimentin/metabolism*