三种含水蛭中药制剂溶出曲线、体外抗凝活性与胃肠稳定性试验

Dissolution curves,in vitro anticoagulant activities and gastrointestinal stabilities of three traditional Chi⁃nese medicines containing medicinal leech

目的:评估3种含水蛭中药制剂脑血康滴丸、活血通脉胶囊、脉血康胶囊体外溶出曲线、抗凝活性与胃肠稳定性。方法:采用4种p H值溶出介质和浆板法,及Lowry法测定3种含水蛭中药制剂不同时间点的水蛭多肽含量,计算溶出曲线,相似因子f_(2)评估溶出相似性;通过HPLC方法测定产物对硝基苯胺(p-nitroaniline,p NA)生成量,评估3种含水蛭中药制剂对凝血酶的抑制活性;测定3种含水蛭中药制剂与胃蛋白酶温孵之后凝血酶抑制活性的变化,以及枯草菌素、酪蛋白和脱氧胆酸钠加入后对制剂抗凝活性下降的改善。结果:在水介质中,脑血康滴丸和活血通脉胶囊的溶出行为相似(f_(2)=56.1);在其他溶出介质中,3种中药制剂溶出曲线均不相似,脑血康滴丸的溶出速度最快,且在测定时间内溶出量更多。水蛭多肽质量浓度均为10 mg·m L^(–1)时,脑血康滴丸、活血通脉胶囊、脉血康胶囊对凝血酶平均抑制率分别为97.9%、85.5%、59.4%。与胃蛋白酶温孵30 min后,脑血康滴丸、脉血康胶囊、活血通脉胶囊对凝血酶抑制活性平均保留率分别为64.2%、33.0%、10.7%。枯草菌素、酪蛋白和脱氧胆酸钠3种成分均具有阻止胃蛋白酶对活血通脉胶囊和脉血康胶囊抗凝活性影响的作用。脱氧胆酸钠、胃蛋白酶和脉血康胶囊共同温孵后,抗凝活性相较于无脱氧胆酸钠提高了4.0倍;枯草菌素、胃蛋白酶和活血通脉胶囊共同温孵后,抗凝活性提高了2.4倍。结论:本研究体外评估了3种含水蛭中药制剂的溶出曲线、抗凝活性与胃肠道稳定性,为3种中药制剂体内药效活性研究提供了数据参考。

OBJECTIVE To evaluate the in vitro dissolution profiles,anticoagulant activities and gastrointestinal stabilities of three traditional Chinese medicines containing medicinal leech,i.e..Naoxuekang Dropping Pill(NDP),Huoxuetongmai Cap⁃sules(HC)and Maixuekang Capsules(MC).METHODS Four physiological pH dissolution media and paddle method were employed for dissolution measurement.At different timepoints,the amount of leech peptides was determined by Lowry method for calculating the dissolution curves.Similarity factor f_(2) was utilized for assessing dissolution similarity.High performance liquid chromatography(HPLC)was employed for determining the generation amount of p-nitroaniline(pNA)and evaluating the inhibi⁃tory activities of NDP/HC/MC on thrombin.The changes in thrombin inhibitory activities of NDP/HC/MC were recorded after incubating with gastric protease.And the improvements of lower anticoagulant activity were observed after adding subtilin,casein and sodium deoxycholate.RESULTS In water medium,dissolution behaviors of NDP and HC were similar(f_(2)=56.1)while dis⁃solution curves of NDP/HC/MC in other dissolution media differed.NDP exhibited the fastest dissolution rate and the highest cumulative amount within the specified testing time.At a leech peptide concentration of 10 mg·mL^(–1),average inhibition rates of thrombin by NDP/HC/MC were 97.9%,85.5%and 59.4%respectively.After 30-min incubation with gastric protease,aver⁃age retention rates of thrombin inhibition were 64.2%,33.0%and 10.7%for NDP/MC/HC respectively.Subtilin,casein and sodium deoxycholate could arrest the inhibition of gastric protease on HC/MC.After co-incubation with sodium deoxycholate,gastric protease and MC,anticoagulant activity spiked 4.0 folds as compared with no sodium deoxycholate.Similarly,coincubations with subtilin,gastric protease and HC resulted in a 2.4-fold rise in anticoagulant activity.CONCLUSION Dissolu⁃tion profiles,anticoagulant activity and gastrointestinal stability in vitro of NDP/HC/MC have been profiled.It provided refer⁃ences for in-depth studies of their in vivo pharmacological activities.