Published online in http://ijam.co.in ISSN: 0976-5921 International Journal of Ayurvedic Medicine, 2013, 4(1), 17-26 17 Urokinase: Isolation, Purification, Characterization, New Spectrophotometric Bioassay Method and In-vitro Blood Clot Dissolving Activity from Cow Urine Research article Badhe Ravindra V 1* , Badhe Sonali R 2 , Bhujbal Mayur N 1 , Nanda Rabindra K 1 , Shirolkar Satish V 1. 1. Pharm. Chem. Dept., Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune – 411018. 2. Pharmacology Dept. Modern College of Pharmacy, Yamunanagar, Nigdi, Pune 411044. Abstract Urokinase, a potent plasminogen activator is an enzyme present in cowurine attributing to the thromboembolic properties of cow urine but its presence in cow urine has never been convincingly documented. In our work, we did isolation, purification, partial characterization and quantitation of urokinase in cowurine using newly developed spectrophotometric bioassay method and studied its invitro blood clot dissolving activity. The cow urine was collected from cows of Indian origin (Bos indicus). Isolation of Urokinase was done by ammonium sulfate precipitation and dialysis. Purification was done by using Sephadex G-200 Gel Filtration Chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Gelatin Zymography, HPLC and TLC of tryptic digest of the purified urokinase was carried out for partial characterization studies which conformed the molecular weight of 59 kD of the purified Urokinase. Quantification with the help of new spectrophotometric bioassay method showed 956 IU/ml and with the help of HPLC it was 910 IU/ml of urokinase in cow urine. The invitro blood clot dissolving activity on a cotton cloth confirmed the activity and efficacy of the isolated and purified urokinase. Keywords: Cow urine, Plasminogen activator, New Spectrophotometric Thrombolytic Bio Assay, Urokinase, In-vitro clot dissolving activity, Tryptic digest fingerprint. 1. Introduction: Cow urine from Indian cows (Bos indicus) has a unique place in Ayurveda and has been described in many ancient manuscripts like Atharva Veda, Charaka Samhita, Rajni Ghuntu, Vridhabhagabhatt, Amritasagar, Bhavaprakash, Sachitra Ayurveda, and Sushruta Samhita to be the most effective substance / secretion of animal origin with therapeutic values. It has been recognized as ‘Water of Life’ or ‘Amrita’. In India, drinking of cow urine has been practiced for thousands of years (1-3). Cow Urine Treatment And Research Center, Indore, and Go Vigyan Anusandhan Kendra, Nagpur, India has conducted a lot of research over the past few years and has reached the conclusion that it is capable of curing thromboembolic diseases, diabetes(4), blood pressure, asthma, psoriasis, eczema, heart attack, blockage in arteries, fits, cancer (5), Immunomodulatory (6) piles, prostrate, arthritis, migraine, thyroid, ulcer, acidity, *Corresponding Author: Badhe Ravindra V Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune-18. Tel: +91-9422432038, Fax no.: 020-27420261 Email: badheravi@rediffmail.com