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首页> 外文期刊>International Journal of Pharmaceutical Sciences and Research >SENSITIVE AND SELECTIVE METHODS FOR DETERMINATION OF PROTEOLYTIC ACTIVITY OF FORMULATION CONTAINING BROMELAIN AND TRYPSIN AS PROTEOLYTIC ENZYMES
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SENSITIVE AND SELECTIVE METHODS FOR DETERMINATION OF PROTEOLYTIC ACTIVITY OF FORMULATION CONTAINING BROMELAIN AND TRYPSIN AS PROTEOLYTIC ENZYMES

机译：测定含溴和胰蛋白酶作为蛋白水解酶制剂蛋白水解活性的灵敏和选择性方法

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Accurate and sensitive assay methods for quantitative determination of Rutoside trihydrate (rutin), Trypsin and Bromelain in tablet formulation based on UV-spectrophometric analysis were developed and validated. This three drug combination has anti-inflammatory properties and hence is used for treatment of edema and inflammations. Rutin was analyzed directly by UV-Vis spectrophotometer at 257nm because Trypsin and Bromelain are not UV-active therefore they don’t interfere in the analysis of rutin. Trypsin and Bromelain being proteolytic enzymes, Casein was used as substrates for their analysis. Casein was standardized according to Bradford assay using Bovine serum albumin as a standard. Casein was hydrolyzed to amino acid tyrosine by both the proteolytic enzymes. Tyrosine formed was further reacted with Folin-Ciolcalteu (F-C) reagent and absorbance was recorded at 785nm. Trypsin was inhibited by specific trypsin inhibitor Nα-p-tosyl-L-lysine chloromethyl Ketone (TLCK) and specific proteolytic activity of Bromelain was analysed using casein. Bromelain was inactive towards N-a-benzoyl-DL-arginine p-nitroaniline (BApNA) therefore; BApNA was used as a specific substrate for analysis of Trypsin. BApNA was hydrolyzed to pNA by Trypsin which was further diazotized with N (1 Naphthyl) Ethylene Diamine Dihydochloride (NEDD) and absorbance was recorded at 545nm. Intra- and inter-day precision of all the methods developed were within acceptable limits. All methods were validated as per ICH guidelines and can be adopted for the routine analysis of Trypsin, Rutoside trihydrate and Bromelain in tablet formulations.
机译：开发并验证了基于紫外分光光度法定量测定片剂中三水合芸香苷，芦丁和胰蛋白酶的准确，灵敏的测定方法。这三种药物的组合具有抗炎特性，因此可用于治疗水肿和炎症。芦丁是在257nm处用紫外可见分光光度计直接分析的，因为胰蛋白酶和菠萝蛋白酶不具有紫外线活性，因此它们不会干扰芦丁的分析。胰蛋白酶和菠萝蛋白酶是蛋白水解酶，酪蛋白用作其分析的底物。根据酪蛋白测定法，以牛血清白蛋白为标准对酪蛋白进行标准化。酪蛋白被两种蛋白水解酶水解为氨基酸酪氨酸。使形成的酪氨酸进一步与Folin-Ciolcalteu（F-C）试剂反应，并在785nm处记录吸光度。胰蛋白酶被特异性胰蛋白酶抑制剂Nα-对甲苯磺酰基-L-赖氨酸氯甲基酮（TLCK）抑制，并使用酪蛋白分析菠萝蛋白酶的特定蛋白水解活性。菠萝蛋白酶对N-α-苯甲酰基-DL-精氨酸对硝基苯胺（BApNA）无活性。 BApNA用作分析胰蛋白酶的特异性底物。通过胰蛋白酶将BApNA水解为pNA，然后将其进一步用N（1萘基）乙二胺二氯化物（NEDD）重氮化，并在545nm处记录吸光度。所有开发方法的日内和日间精度均在可接受的范围内。所有方法均已按照ICH指南进行了验证，可用于片剂中胰蛋白酶，三糖Rutoside三水合物和菠萝蛋白酶的常规分析。

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