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Objective: To investigate the anti-gout effect of hirudin and its mechanism. Methods: Hypoxanthine was used to replicate mouse model of hyperuricemia. Sodium uric induced acute gouty inflammation in rats to observe the effect of hirudin on the level of uric acid and inflammation induced by acute hyperuricemia. The serum uric acid, serum urea nitrogen, serum xanthine oxidase activity, and liver xanthine oxidase activity were observed in chronic hyperuricemia mouse model induced by potassium oxonate. The changes of renal pathology, the level of C-reactive protein (CRP) and the expression of glucose transporter 9 (GLUT9) were also be tested. Results: Hirudin could significantly reduce the serum uric acid level in hyperuricemia mice induced by hypoxanthine and significantly inhibit acute toe swelling induced by sodium uric acid in rats. It could significantly reduce the level of serum hyperuricemia, decrease the level of blood urea nitrogen (BUN), significantly inhibit the expression of GLUT9 and alleviate the pathological changes of kidney in chronic hyperuricemia mice induced by potassium oxazinate. Conclusion: Hirudin has significant anti-hyperuricemia and anti-gout effects, and its mechanism may be related to the protection of kidney and the regulation of GLUT9 expression.
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Objective: To study the anti-gout effect of active ingredients in Poecilobdella manillensis. Methods: Hypoxanthine was used to replicate mouse model of hyperuricemia, and xylene was used to induce mouse auricle swelling model. The hot plate method and writhing method were used to screen the active site of Poecilobdella manillensis, and then the active ingredients were screened. The material basis of anti-gout effect of Poecilobdella manillensis was observed. Results: The water-soluble fraction of Poecilobdella manillensis was the active site against gout, which could reduce the level of serum uric acid in hypoxanthine-induced hyperuricemic mice and inhibit xylene-induced auricular swelling in mice, deduce acetic acid-induced writhing reaction in mice and increase the hot plate pain threshold in mice; Hirudin was the main active ingredient in water-soluble parts. Poecilobdella manillensis active ingredient 0.8 g/kg and 0.4 g/kg and Poecilobdella manillensis residue 2.0 g/kg could significantly reduce serum uric acid levels. The serum uric acid levels decreased from232.73 ± 50.93 umol/L in model group to 140.70 ± 25.97 umol/L, 149.07 ± 39.28 umol/L, 176.45 ± 44.33 umol/L, respectively (P ＜ 0.01) . Poecilobdella manillensis active ingredients 0.8 g/kg, 0.4 g/kg and 0.2 g/kg and Poecilobdella manillensis residue 2.0 g/kg could significantly inhibit xylene-induced ear auricle swelling in mice. The swelling degree was inhibited from 22.80 ± 2.86 mg to 20.10 ± 2.18 mg, 19.80 ± 2.57 mg, 20.10 ± 1.66 mg and 20.85 ± 1.60 mg respectively (P ＜ 0.05) . Poecilobdella manillensis 0.8 g/kg active ingredient could significantly reduce the number of writhing mice caused by acetic acid. The number of times was reduced from 22.80 ± 2.86 times to 20.10 ± 2.18 times (P ＜0.05) . Conclusion: Poecilobdella manillensis anti-gout activity is in water-soluble parts, and Hirudin is the main active ingredient.
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The effects of harvest and different processing methods on the anti-thrombin activity of Poecilobdella manillensis were respectively studied. The indicators included processing methods (vacuum freeze drying, fresh homogenate, drying under sunlight, freezing, scalding, baking under different temperatures), different parts (entire body, cephalon, pygidium, exudate) and body weights (≤10, 10-20, 20-30, 30-40, ≥40 g). The anti-thrombin activities of P. manillensis with different processing methods were evaluated by direct anti-thrombin titration. The results indicated that the processing methods significantly affected the anti-thrombin activities of P. manillensis. Among the 11 groups, the anti-thrombin activity of P. manillensis processed with vacuum freeze drying (1 303.56 U•g⁻¹) was significantly highest than the other groups (P<0.05), and that processed with baking under 90 ℃ (15.44 U•g⁻¹) was the lowest. The anti-thrombin activity of the cephalon of P. manillensis (226.42 U•g⁻¹) was the highest, and that of the pygidium (102.12 U•g⁻¹) was lowest; the anti-thrombin activities for different body weights were significantly different (P<0.05); and among the five groups, the body weight of ≤10 g (328.86 U•g⁻¹) was the highest (P<0.05), and the body weight of ≥40 g (87.71 U•g⁻¹) was the lowest. In conclusion, harvest and different processing methods had a significant impact on the anti-thrombin activities of P. manillensis. In the study, for the optimal processing method for P. manillensis, the body weight between 20-30 g is recommended, and the vacuum freeze drying is preferred, which is followed by the drying under sunlight.
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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 optimize the formula of artificial diets for Poecilobdella manillensis. Methods: Survival rate and individual body weight growth rate of P. manillensis were chosen as indexes. The three factors [NP-90 spray-dried hemoglobin powder, NP-2002 spray-dried plasma powder, and vitamin C (VC)] of the formula of artificial diets were investigated by L9(34) orthogonal test. AHP was applied to statistical analysis on non-interacting orthogonal test, the effect weight of each factor for test result was obtained, and principal-secondary sequence of factors and optimal project was given. Results: The effects of the three components in artificial diets on the survival rate were in the following decreasing order: VC>NP-90>NP-2002. And the optimization of components of artificial diets was 0 NP-90 spray-dried hemoglobin powder, 1% NP-2002 spray-dried plasma powder, and 0.05% VC. When the individual body weight growth rate was chosen as the index, the effects of the three components in artificial diets on the individual body weight growth rate were in the following decreasing order: NP-2002>NP-90>VC. The optimization of components of artificial diets was 0.5% NP-90 spray-dried hemoglobin powder, 3% NP-2002 spray-dried plasma powder, and 0.05% VC. The weight of principal-secondary sequence of factors and levels influences on the tests' results was NP-90>NP-2002>VC. The weight of 0.5% NP-90 spray-dried hemoglobin powder, 3% NP-2002 spray-dried plasma powder and 0.05% VC was maximum. And the optimization of components in artificial diets was 0.5% NP-90 spray-dried hemoglobin powder, 3% NP-2002 spray-dried plasma powder, and 0.05% VC. According to the results, P. manillensis bred with the artificial diets (0.5% NP-90 spray-dried hemoglobin powder, 3% NP-2002 spray-dried plasma powder, 3% NP-2002 spray-dried plasma powder, 0.05% VC, and other components) had the survival rate of 100% and the individual body weight growth rate was 19.91%. Conclusion: The optimal artificial diets could be used to feed P. manillensis.
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Objective: To observe the therapeutic effects on gout and the acute toxicity of Poecilobdella manillensis. Methods: The anti-gout effects of P. manillensis were studied by observing its effects on the hyperuricacidemia model and the level of blood uric acid. And we observed the effect of P. manillensis on gouty inflammation via injection of MSU to the foot of rats. The analgesic effect and toxicity of P. manillensis will be reported in this work too. Results: Comparing with the model group, P. manillensis could significantly reduce the level of the blood uric acid in the hyperuricemic and normal mice at 3.00 and 1.50 g/kg doses (P < 0.01), and acute gouty arthritis at 0.75 g/kg dose (P < 0.05, 0.01). It reduced the number of writhing in mice at 3.00 and 0.75 g/kg doses (P < 0.05, 0.01). Obvious toxic reaction was not observed and the maximum tolerant dose was 23.09 g/kg for mice in acute toxicity experiment. Conclusion: P. manillensis is proved to have the therapeutic effect on gout. Further research and development will be continued.
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OBJECTIVE: To establish an extraction method of hirudin by double aqueous phase combined with gel chromatography. METHODS: Anti-thrombin activity units (ATU) was adopted as the index. On the basis of optimizing the double aqueous phase extraction conditions, a new extraction technology of hirudin was established by employing double aqueous phase system combined with gel chromatography. The double aqueous phase was composed of polyethylene glycol and ammonium sulfate and took the digestive juice of Poecilobdella manillensis from Guangxi Province as the raw material. RESULTS: In the studied experimental range, the optimum technological conditions of double aqueous phase extraction of hirudin reference substantce were as follows: the contents of PEG 2000, (NH4)2SO4 and NaCl were 22%, 20% and 0.04%, respectively. The extraction temperature was 35°C, and pH was 6.0. The obtained extracts were further purified by gel chromatography. The recoveries of ATU reached 81.92%, 80.34% and 80.36% for hirudin reference substance, hirudin crude extracts and scale-up experiment, respectively. The specific activity of obtained products reached 3210.27 ATU · mg-1. The results of discontinuous polyacrylate gel electrophoresis and spectral scan for the extract were the same with for the reference substance. CONCLUSION: The double aqueous phase extraction combined with gel chromatography is good for hirudin. Copyright 2012 by the Chinese Pharmaceutical Association.