SENSITIVE AND SELECTIVE METHODS FOR DETERMINATION OF PROTEOLYTIC ACTIVITY OF FORMULATION CONTAINING BROMELAIN AND TRYPSIN AS PROTEOLYTIC ENZYMES

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.