RESUMO
This study attempts to establish a method for the anti-thrombin activty bioassay of musk, explore the impact of species and producing areas on the anti-thrombin activty of musk, and provide scientific basis for its biological quality evaluation. Anti-thrombin activty of musk was analyzed by thrombin titration, and the influence factors such as musk solution concentration, fibrinogen concentration, thrombin concentration and titration interval were optimized to evaluate the effect of different species and producing areas on anti-thrombin activty of musk. As a result, there was a good linear relationship between musk solution concentration and thrombin consumption volume within the range of 0.01-0.02 g·mL⁻¹ (=0.991 4) under the experimental condition as follows: fibrinogen concentration was 0.5%, the thrombin concentration was 10 U·mL⁻¹； titration time interval was once every minute, and each titration volume was 2 μL. The average anti-thrombin activty potency of Moschus berezovskii from different producing areas was (105.0±10.4) U·g⁻¹, (102.4±5.5) U·g⁻¹ for M. sifanicus from different producing areas, (97.7±6.6) U·g⁻¹ for M. moschiferus from Anhui province, and (58.6±6.4) U·g⁻¹ for artificial musk. The results indicated that this anti-thrombin activty bioassay method could be applied to evaluate the anti-thrombin activty of musk quickly, conveniently, sensitively and exactly. It was also suggested that different species and producing areas had effects on the anti-thrombin activty of musk, so it is necessary to pay attention to species and producing areas in the process of musk domestication; meanwhile, the artificial musk and natural musk also showed great differences in anti-thrombin activty, suggesting that ratio in artificial musk prescription needs to be further optimized.
RESUMO
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.