DOI QR코드

DOI QR Code

Identification of bioactive components behind the antimicrobial activity of cow urine by peptide and metabolite profiling

  • Rohit Kumar (ICAR-National Dairy Research Institute, Cell Biology and Proteomics Lab, Animal Biotechnology Center (ABTC)) ;
  • Jai Kumar Kaushik (ICAR-National Dairy Research Institute, Cell Biology and Proteomics Lab, Animal Biotechnology Center (ABTC)) ;
  • Ashok Kumar Mohanty (ICAR-National Dairy Research Institute, Cell Biology and Proteomics Lab, Animal Biotechnology Center (ABTC)) ;
  • Sudarshan Kumar (ICAR-National Dairy Research Institute, Cell Biology and Proteomics Lab, Animal Biotechnology Center (ABTC))
  • 투고 : 2022.06.22
  • 심사 : 2023.01.06
  • 발행 : 2023.07.01

초록

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 Staphylococcus aureus, Escherichia coli, and Streptococcus agalactiae. The safety of urinary aqueous extract was evaluated by hemolysis and cytotoxicity assay on the BuMEC cell line. The urinary peptides were further fractionated using high-performance liquid chromatography (HPLC) to identify the fraction(s) containing the antimicrobial activity. The HPLC fractions and ethyl acetate extract were analyzed using nLC-MS/MS for the identification of the peptides and metabolites. Results: A total of three fractions were identified with antimicrobial activity, and 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. Conclusion: 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 their antimicrobial nature. Secondly, 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.

키워드

과제정보

The authors acknowledge the director of the Institute for funding the work. The author also acknowledges the Department of Biotechnology for the fellowship grant.

참고문헌

  1. Pisitkun T, Johnstone R, Knepper MA. Discovery of urinary biomarkers. Mol Cell Proteomics 2006;5:1760-71. https://doi.org/10.1074/mcp.R600004-MCP200 
  2. Kumar R, Ali SA, Singh SK, et al. Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties. Sci Rep 2021;11:12427. https://doi.org/10.1038/s41598-021-91684-4 
  3. Kumar R, Ali SA, Singh SK, et al. Antimicrobial peptides in farm animals: an updated review on its diversity, function, modes of action and therapeutic prospects. Vet Sci 2020;7:206. https://doi.org/10.3390/vetsci7040206 
  4. Sharma A, Nigam R, Kumar A, Singh S. Mass spectrometry-based identification of urinary antimicrobial peptides in dairy cows. Protein Pept Lett 2020;27:225-35. https://doi.org/10.2174/0929866526666191025105038 
  5. Minocheherhomji FP, Vyas BM. Study of the antimicrobial activity of cow urine and medicinal plant extracts on pathogenic human microbial strains. Int J Adv Pharm Biol Chem 2014;3:836-40. 
  6. Rana R, De, S. In vitro antimicrobial screening of cow urine-a potential natural antimicrobial agent. Int J Bioassays 2013;2:436-39.
  7. Ahuja A, Kumar P, Verma A, Tanwar R. Antimicrobial activities of cow urine against various bacterial strains. Int J Recent Adv Pharm Res 2012;2:84-7. 
  8. Singh UP, Maurya S, Singh A, Nath G, Singh M. Antimicrobial efficacy, disease inhibition and phenolic acid-inducing potential of chloroform fraction of cow urine. Arch Phytopathol Plant Prot 2012;45. https://doi.org/10.1080/03235408.2012.681247 
  9. Gotora T, Masaka L, Sungirai M. Effect of cow urine on the growth characteristics of fusarium lateritium, an important coffee fungus in Zimbabwe. Int J Agron 2014;2014:Article ID 986068. https://doi.org/10.1155/2014/986068 
  10. Hoh JM, Dhanashree B. Antifungal effect of cow's urine distillate on Candida species. J Ayurveda Integr Med 2017;8:233-7. https://doi.org/10.1016/j.jaim.2017.04.009 
  11. Jain NK, Gupta VB, Garg R, Silawat N. Efficacy of cow urine therapy on various cancer patients in Mandsaur District, India-A survey. Int J Green Pharm 2010;4:29-35. https://doi.org/10.4103/0973-8258.62163 
  12. Dutta P, Das S. Mammalian antimicrobial peptides: promising therapeutic targets against infection and chronic inflammation. Curr Top Med Chem 2016;16:99-129. https://doi.org/10.2174/1568026615666150703121819 
  13. Kekuda PTR, Nishanth BC, Kumar P SV. Kamal D, Sandeep M, Megharaj HK. Cow urine concentrate : a potent agent with antimicrobial and anthelmintic activity. J Pharm Res 2010;3:1025-7. 
  14. Lata S, Sharma BK, Raghava GPS. Analysis and prediction of antibacterial peptides. BMC Bioinformatics 2007;8:263. https://doi.org/10.1186/1471-2105-8-263 
  15. Lohner K, Latal A, Lehrer RI, Ganz T. Differential scanning microcalorimetry indicates that human defensin, HNP-2, interacts specifically with biomembrane mimetic systems. Biochemistry 1997;36:1525-31. https://doi.org/10.1021/bi961300p 
  16. Dathe M, Wieprecht T. Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells. Biochim Biophys Acta Biomembr 1999;1462:71-87. https://doi.org/10.1016/S0005-2736(99)00201-1 
  17. Michael Henderson J, Lee KYC. Promising antimicrobial agents designed from natural peptide templates. Curr Opin Solid State Mater Sci 2013;17:175-92. https://doi.org/10.1016/j.cossms.2013.08.003 
  18. Ozma MA, Khodadadi E, Pakdel F, et al. Baicalin, a natural antimicrobial and anti-biofilm agent. J Herb Med 2021;27:100432. https://doi.org/10.1016/J.HERMED.2021.100432 
  19. Wang ZZ, Jia Y, Wang G, et al. Dynamic covalent hydrogel of natural product baicalin with antibacterial activities. RSC Adv 2022;12:8737-42. https://doi.org/10.1039/D1RA07553E 
  20. Barreca D, Bellocco E, Lagana G, Ginestra G, Bisignano C. Biochemical and antimicrobial activity of phloretin and its glycosilated derivatives present in apple and kumquat. Food Chem 2014;160:292-7. https://doi.org/10.1016/J.FOODCHEM.2014.03.118 
  21. Goc A, Niedzwiecki A, Ivanov V, Ivanova S, Rath M. Inhibitory effects of specific combination of natural compounds against SARS-CoV-2 and its Alpha, Beta, Gamma, Delta, Kappa, and Mu variants, Eur J Microbiol Immunol 2022;11:87-94. https://doi.org/10.1556/1886.2021.00022 
  22. You J, Li H, Fan P, et al. Inspiration for COVID-19 treatment: network analysis and experimental validation of baicalin for cytokine storm. Front Pharmacol 2022;13:853496. https://doi.org/10.3389/FPHAR.2022.853496 
  23. Wang CZ, Zhang CF, Chen L, Anderson S, Lu F, Yuan CS. Colon cancer chemopreventive effects of baicalein, an active enteric microbiome metabolite from baicalin. Int J Oncol 2015;47:1749-58. https://doi.org/10.3892/IJO.2015.3173 
  24. Zheng WX, Wang F, Cao XL, et al. Baicalin protects PC-12 cells from oxidative stress induced by hydrogen peroxide via anti-apoptotic effects. Brain Inj 2014;28:227-34. https://doi.org/10.3109/02699052.2013.860469 
  25. He XW, Yu D, Li WL, et al. Anti-atherosclerotic potential of baicalin mediated by promoting cholesterol efflux from macrophages via the PPARγ-LXRα-ABCA1/ABCG1 pathway. Biomed Pharmacother 2016;83:257-64. https://doi.org/10.1016/J.BIOPHA.2016.06.046 
  26. Kong F, Luan Y, Zhang ZH, Cheng GH, Qi TG, Sun C. Baicalin protects the myocardium from reperfusion-induced damage in isolated rat hearts via the antioxidant and paracrine effect. Exp Ther Med 2014;7:254-9. https://doi.org/10.3892/ETM.2013.1369 
  27. Xu M, Chen X, Gu Y, et al. Baicalin can scavenge peroxynitrite and ameliorate endogenous peroxynitrite-mediated neurotoxicity in cerebral ischemia-reperfusion injury. J Ethnopharmacol 2013;150:116-24. https://doi.org/10.1016/J.JEP.2013.08.020 
  28. Zandi K, Musall K, Oo A, et al. Baicalein and baicalin inhibit SARS-CoV-2 RNA-Dependent-RNA polymerase. Microorg 2021;9:893. https://doi.org/10.3390/MICROORGANISMS9050893 
  29. Gosch C, Halbwirth H, Stich K. Phloridzin: biosynthesis, distribution and physiological relevance in plants. Phytochemistry 2010;71:838-43. https://doi.org/10.1016/J.PHYTOCHEM.2010.03.003 
  30. Barrera-Hemandez G, Wanke IE, Wong NCW. Phlorizin or vanadate treatment reverses impaired expression of albumin and hepatocyte nuclear factor 1 in diabetic rats. Diabetes 1996;45:1217-22. https://doi.org/10.2337/DIAB.45.9.1217 
  31. Takii H, Matsumoto K, Kometani T, Okada S, Fushiki T. Lowering effect of phenolic glycosides on the rise in postprandial glucose in mice. Biosci Biotechnol Biochem 1997;61:1531-5. https://doi.org/10.1271/BBB.61.1531 
  32. Rezk BM, Haenen GRMM, Van der Vijgh WJF, Bast A. The antioxidant activity of phloretin: the disclosure of a new antioxidant pharmacophore in flavonoids. Biochem Biophys Res Commun 2002;295:9-13. https://doi.org/10.1016/S0006-291X(02)00618-6 
  33. Sukhorukov VL, Kurschner M, Dilsky S, et al. Phloretin induced changes of lipophilic ion transport across the plasma membrane of mammalian cells. Biophys J 2001;81:1006-13. https://doi.org/10.1016/S0006-3495(01)75758-X 
  34. Jiang WL, Zhang SP, Hou J, Zhu HB. Effect of loganin on experimental diabetic nephropathy. Phytomedicine 2012;19:217-22. https://doi.org/10.1016/J.PHYMED.2011.08.064 
  35. Park CH, Tanaka T, Kim JH, et al. Hepato-protective effects of loganin, iridoid glycoside from Corni Fructus, against hyperglycemia-activated signaling pathway in liver of type 2 diabetic db/db mice. Toxicology 2011;290:14-21. https://doi.org/10.1016/J.TOX.2011.08.004 
  36. Kim A, Ma JY. Isoliquiritin apioside suppresses in vitro invasiveness and angiogenesis of cancer cells and endothelial cells. Front Pharmacol 2018;9:1455. https://doi.org/10.3389/FPHAR.2018.01455 
  37. Luo J, Li Z, Wang J, Weng Q, Chen S, Hu M. Antifungal activity of isoliquiritin and its inhibitory effect against Peronophythora litchi Chen through a membrane damage mechanism. Molecules 2016;21:237. https://doi.org/10.3390/molecules21020237