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https://doi.org/10.5713/ab.22.0249    [Accepted] Published online January 11, 2023.
Identification of bioactive components behind the antimicrobial activity of cow urine by Peptide and metabolite profiling
Rohit Kumar1  , Jai Kumar Kaushik1  , Ashok Kumar Mohanty1,2  , Sudarshan Kumar1,* 
1ICAR-National Dairy Research Institute, Cell Biology and Proteomics Lab, Animal Biotechnology Center (ABTC), Karnal, Haryana, 132001, India
2Current affiliation: ICAR-Indian Veterinary Research Institute, Mukteshwar 263138, Nainital, Uttarakhand, India
Correspondence:  Sudarshan Kumar, Tel: +91-9254912456, Fax: +91-184-2250042, Email: kumarsudershan@gmail.com
Received: 22 June 2022   • Revised: 19 August 2022   • Accepted: 6 January 2023
Abstract
Objective
Cow urine possesses several bioactive properties but the responsible components behind these bioactivities are still far from identified. In our study, we tried to identify the possible components behind the antimicrobial activity of cow urine by exploring the peptidome and metabolome.
Methods
We extracted peptides from the urine of Sahiwal cows belonging to three different physiological states viz heifer, lactation, and pregnant, each group consisting of 10 different animals. The peptides were extracted using the Solid Phase Extraction technique followed by further extraction using ethyl acetate. The antimicrobial activity of the aqueous extract was evaluated against different pathogenic strains like S. aureus, E. coli, and S. agalactiae. The safety of urinary aqueous extract was evaluated by haemolysis and cytotoxicity assay on BuMEC cell line. The urinary peptides were further fractionated using HPLC to identify the fraction(s) containing the antimicrobial activity. The HPLC fractions and ethyl acetate extract were analysed using nLC-MS/MS for the identification of the peptides and metabolites.
Results
A total of three fractions were identified with antimicrobial activity, nLC-MS/MS analysis of fractions resulted in the identification of 511 sequences. While 46 compounds were identified in the metabolite profiling of organic extract. The urinary aqueous extract showed significant activity against E.coli as compared to S.aureus and S.agalactiae and was relatively safe against mammalian cells.
Conclusions
The antimicrobial activity of cow urine is a consequence of the feeding habit. The metabolites of plant origin with several bioactivities are eliminated through urine and are responsible for its antimicrobial nature. Secondly, and the plethora of peptides generated from the activity of endogenous proteases on protein shed from different parts of tissues also find their way to urine. Some of these sequences possess antimicrobial activity due to their amino acid composition.
Keywords: Antimicrobial Peptides; Cow Urine; Mass Spectrometry; Metabolites; Solid-phase Extraction
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