Intestinal Absorption Mechanism And Its Anticoagulant Substance Basis

As a traditional Chinese medicine, leeches were widely used to treat various thrombosis diseases.The dried bodies of three species of animals usually, including Hirudo nipponica Whitman, Whitmania pigra Whitman and Whitmania acranulata Whitman were used.The Whitmania pigra Whitman is widely used in Traditional Chinese Medicine and widely cultivated in our country.At present the studys on the Whitmania pigra Whitman often used parasite overall as experiment subject, Whitmania pigra enzymatic extract was few reported.So for further research Whitmania pigra Whitman were selected as the experiment subject, the absorption characteristics and the anticoagulant components were studied. The major findings of this study are as follows.1, Enzymatic hydrolysis techniqueIn order to select an appropriate index for study the enzymolysis process, the APTT, PT, and TT of enzymatic products were determined before and after intestinal absorption, respectively, using an isolated everted intestine model. TT was an appropriate index.The emzymolysis of W. pigra was carried out by pepsin, trypsin, and the complex enzyme of pepsin and trypsin. According to the selected indexes, the enzymolysis process was optimized under different conditions, including the kind of enzyme, the ratio of enzymes to substrates, the time of enzymolysis, and the temperature of enzymolysis. Enzymatic products with high activity could be obtained only using trypsin. After optimization, the best enzymolysis condition was as follows:10% trypsin was added in the solution of W. pigra. The reaction was proceeded under 73℃ for 8 h.2-Absorption characteristics of Whitmania pigra enzymatic extractIsolated rat everted gut sac model was used to study the intestinal absorption of Whitmania pigra enzymatic extract in different segments, different drug concentration, different absorption enhancers and p-glycoprotein (P-gp) inhibitor. Thrombin times of samples were determined, and the drug concentration was calculated based on the elongation rate of thrombin time.he enzymatic extract has an absorption ratio less than 15%, that the intestine absorption rate constant (Ka) increased with Whitmania pigra enzymatic extract’s concentration. The absorption rate had no significant difference between different regions of intestines. The cumulative amount (Q) and Ka. increased with the increase of drug concentrations,The absorption was not saturated at 50.0 mg/ml.The absorption promoters, tween-80, SDS, chitosan, had almost no absorption-enhancing effect on Ka and Pappat the commonly used concentration. While, p-glycoprotein (P-gp) inhibitorverapamil,a well-known P-glycoprotein inhibitor was able significantly to improve the absorption of Whitmania pigra enzymatic extract across ileum.we concluded the absorption ofWhitmania pigra enzymatic extract is higer than most of the protein and peptide drugs.The absorption of Whitmania pigra enzymatic extract may be an passive transportation in dependent on of P-glycoprotein-Mediated efflux systems.According to the results of MALDI-TOF-MS and the absorption of large molecular weight substances,we presumed that the low molecular weight peptides may play anticoagulant efficacy.3、Anticoagulant component separationTaken TT as the index, the anti-coagulate active components were separated from the Whitmania pigra enzymatic extract by organic solvent precipitation,membrane separation,DEAE-Sepharose Fast Flow and SP-Sepharose Fast Flow ion exchange chromatography,SephadexG-50 and SephadexG-25 gel filtration chromatography.A component F3-2 can separated by Sephadex G-50 and Sephadex G-25 gel filtration chromatography,showed strong anti-coagulant activity.Conclusion:An appropriate method was constructed to extracted the anticoagulative effective parts of Whitmania pigra,which provides references for high extraction efficiency of Whitmania pigra enzymatic extract;The intestinal absorption characteristics of Whitmania pigra enzymatic extract were determined,which provides a theoretical basis for the formulation design of Whitmania pigra enzymatic extract;The anti-coagulate active components were obtained,which lays the foundation for scorpion’s anti-coagulation pharmacodynamieal research.