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Objective:To investigate and compare the effects of water extracts of Whitmania pigra Whitman and Hirudinaria ma-nillensis Lesson on the angiogenesis of Tg (kdrl:mCherry) zebrafish. Methods:The zebrafish embryos 6-8 hours after fertilization (6-8hpf) were transferred to the culture medium containing Whitmania pigra Whitman or Hirudinaria manillensis Lesson extract at different concentrations, and the culture medium containing the drugs was replaced every 24 h. And then, at 72 hpf, the larvalmorphology and intersegmental vessels were observed under a microscope. The hatchability of 48-and 72-hpf embryos, and the number of intersegmen-tal vessels and the heart rate of 72-hpf juveniles were measured. Results:Compared with the control group, when the concentration of Whitmania pigra Whitmanis was higher than 30μg· ml-1 , and the concentration of Hirudinaria manillensis Lesson was higher than 20μg· ml-1, the number of intersegmental vessels was significantly reduced (P<0. 01). Compared with the control group, at 48 hpf, when the concentration of the drug groups was higher than 40 μg· ml-1 , the hatchability of the two groups significantly decreased ( P<0. 01);at 72 hpf, the hatchability of Whitmania pigra Whitman decreased significantly at the concentration of 100 μg·ml-1 (P<0.01), while the hatchability of Hirudinaria manillensis Lesson decreased significantly at the concentration of 80 μg· ml-1(P <0. 01). There was no obvious yolk sac edema, pericardial edema and spine curvature in the two groups. The heart rate decreased sig-nificantly (P<0. 01), while was still within the normal range. Conclusion:Both Whitmania pigra Whitman and Hirudinaria manillen-sis Lesson have notable anti-angiogenic activity, and the anti-angiogenesis activity of Hirudinaria manillensis Lesson is stronger. They both have effects on the development of zebrafish embryos, while the toxicity is not obvious.
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Objective:To compare the anti-thrombin activity and the effects on the coagulation pathway between Whitmania pigra Whitman and Hirudinaria manillensis to provide scientific reference for the anticoagulation mechanism revelation for Hirudo. Methods:Anti-thrombin titration and chromogenic substrate assay-extraction-HPLC were applied to study the anti-thrombin activity of Whitmania pigra Whitman and Hirudinaria manillensis. APTT, PT and TT were determined by a clotting assay to compare the effects on the path-way of blood clotting. Results:The anticoagulation activity order measured by anti-thrombin titration was living Hirudinaria manillensis> dried Hirudinaria manillensis > > dried Whitmania pigra Whitman. The results of chromogenic substrate assay-extraction-HPLC in-dicated that the low dose of aqueous extract promoted the thrombin activity, while the high dose inhibited the thrombin activity. Hirudi-naria manillensis significantly inhibited the activity of thrombin, while Whitmania pigra Whitman showed weak anti-thrombin activity only at the higher dose. All leeches could prolong APTT, PT and TT. However, living Hirudinaria manillensis mainly affected TT, and dried Hirudinaria manillensis mainly affected APTT. Dried Whitmania pigra Whitman dramatically influenced APTT and TT. All the results indicated that the anticoagulant activity of Whitmania pigra Whitmanis was significantly higher than that of Hirudinaria manillen-sis. Conclusion:There are notable differences in the anti-thrombin activity and the effect on the pathway of blood clotting between Whitmania pigra Whitman and Hirudinaria manillensis.
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OBJECTIVE: To assay the anticoagulant activity of leeches by using biological standardization method with the aim to explore the quality control method which can reflect the biological activity. METHODS: Heparin was used as the reference substance, and APTT value was used as the index of anticoagulant activity evaluation. Four species of leeches were determined, including Hirudo nipponia Whitman, Poecilobdella manillensis Lesson, Poecilobdella javanica Wahlberg, and Whitmania pigra Whitman. The determination results were calculated with standard curve and bioassay statistics. RESULTS: The concentration-response curves of APTT of the four species of leeches were similar to that of heparin, and their variation ranges were parallel. CONCLUSION: APTT values can reflect the comprehensive anticoagulant activity of different species of leeches, which may have more clinical significance. Biological standardization is a good supplement to the current quality control methods, also a proper technology for the quality control of TCM.
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Objective: To analyze the influencing factors in thrombin titration for the determination of anticoagulant activity of Whitmania Pigra Whitman. Methods: The white porcelain plates were used as the titration carriers instead of tubes in the titration ( called white porcelain method for short) . The effect of different carriers, interval time of titration and thrombin concentration on the results of anticoagulant activity test was studied. Results:Under the same conditions, the anticoagulant activity was more accurate and stable using white porcelain method. Using white porcelain method with 20 u·ml-1 or 10 u·ml-1 as the thrombin concentration and titrating 5μl each time, once every minute, the thrombin consumption volume was linear with the sample concentration within the range of 0. 125-0. 333 g·ml-1(r20 =0. 961 and r10 =0. 992), and the anticoagulant activity respectively was (33. 08 ± 2. 64) and (31. 24 ±1.32) u·g-1(RSD20 =8.0% and RSD10 =4.2%). As for a certain sample concentration (0.333 g·ml-1), the theoretical error of determination was not more than 10% and 5%. Conclusion:The improved white porcelain method is more suitable for determining anticoagulant activity of Whitmania Pigra Whitman with more stable results and accurate end point states than tube method. Under the conditions of 10 u·ml-1 thrombin concentration, titrating 5μl each time, once every minute, the linearity, accuracy and precision are all promising.
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Objective: To optimize the enzymolysis process for Whitmania pigra. Methods: In order to select an appropriate index for study on the enzymolysis process, the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of enzymatic products were determined before and after intestinal absorption, respectively, using an isolated everted intestine model. 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, such as the kind of enzyme, ratio of enzymes to substrates, time of enzymolysis, and temperature of enzymolysis. Results: TT was an appropriate index. Enzymatic products with high activity could be obtained only using trypsin. After optimization, the best enzymolysis conditions were as follows: 10% trypsin was added in the solution of W. pigra, and the reaction was proceeded under 73℃ for 8 h. Conclusion: TT is a reliable and sensitive anticoagulation index for enzymolysis. The enzymolysis technology is simple and stable, which could supply the supports for reasonable extraction of W. pigra.