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Journal of Complementary and Integrative Medicine

Editor-in-Chief: Lui, Edmund

Ed. by Ko, Robert / Leung, Kelvin Sze-Yin / Saunders, Paul / Suntres, PH. D., Zacharias

CiteScore 2017: 1.41

SCImago Journal Rank (SJR) 2017: 0.472
Source Normalized Impact per Paper (SNIP) 2017: 0.564

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Evaluation of the antioxidant properties of tryptophan and its metabolites in in vitro assay

Bichitra N. Nayak
  • Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, Manitoba, Canada
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Harpal S. Buttar
Published Online: 2015-12-08 | DOI: https://doi.org/10.1515/jcim-2015-0051


Background: Human milk contains a number of nutrients and bioactive ingredients which play an important role in the growth and development of infants. One important nutrient and bioactive ingredient of human milk is L-tryptophan. L-Tryptophan is an essential aromatic α-amino acid and is required in the diet of children and adult humans. As an essential amino acid, it is needed for protein synthesis and as a precursor of key biomolecules such as serotonin, melatonin, tryptamine, niacin, quinolinic acid and kynurenic acid, nicotinamide adenine dinucleotide. The aim of the study was to evaluate the antioxidant, anti-inflammatory and antiproliferative properties of tryptophan isolated from enzymatic hydrolysates from human milk and its metabolites on human glioma U251 cells and to evaluate the effects of human recombinant (hrIFNγ) on molecular ions of tryptophan and its metabolites in human glial U251 cells.

Methods: The cytotoxicity was determined by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. The antioxidant property was assessed by the oxygen radical scavenging capacity (ORAC) method. The anti-inflammatory effect was determined by the enzyme-linked immunosorbent assay (ELISA) against cytokines IL-6 and TNF-α. The effects of recombinant human (rhIFNγ) on molecular ions of tryptophan and its catabolites were evaluated by mass spectrometry. The tryptophan was isolated from milk peptides following enzymatic digestion, followed by separation by chromatographic and mass spectrometric methods.

Results: Tryptophan from human milk exhibited profoundly higher oxygen radical absorption capacity (7,986±468 µm Trolox equivalent (TE)/g) than that of whole human milk (80.4±13.3 µm TE/g). Tryptophan showed a moderate degree of anti-inflammatory activity against TNF-α and IL-6. rhIFNγ inhibited tryptophan metabolism. A low concentration of L-tryptophan (10–25 μg/mL) inhibited nearly 25% of cell growth. When U251 cells were treated with 25 μg/mL L-tryptophan and subsequently challenged with 30 ng/mL of human recombinant IFNγ, a significant inhibitory effect on cell growth was observed. Low concentrations of Xanthurenic acid, L-kynurenine, and 3-OH DL kynurenine were found to inhibit cell growth except melatonin and 3-OH anthranilic acid. Melatonin was a strong inducer of TNF-α in RAW cells, whereas 3-OH kynurenine at 25, 50 and 100 µg/mL inhibited IL-6 in RAW cells. No significant change was observed in the IL-8 profile in tryptophan-treated U251 cells except that L-kynurenine at 10 µg/mL produced significantly high level of an inflammatory cytokine IL-8. Melatonin, 3-OH, DL kynurenine at high concentrations (100 µg/mL) induced proliferation of U251 cells. Melatonin seemed to show synergistic effects with recombinant human IFNγ (rhINFγ) in promoting growth of human glioma cells. While treatment of U251 cells with tryptophan alone and subsequent treatment with rhIFNγ inhibited the growth of human cancer glioma cells, and conversely melatonin combined with rhIFNγ promoted growth of the U251 cells.

Conclusions: The findings from this study suggest that human milk-derived tryptophan and its metabolites possess strong antioxidant properties. Such effects might play a significant role in regulating the cell proliferation and growth of human cancer cells in a concentration-dependent manner.

Keywords: antioxidant; human milk; L-tryptophan; ORAC; TNF-α; U251 cells; recombinant human IFNγ


  • 1. Kuhn J, Somerville RL, Mutant strains of Escherichia coli k12 that use D-Amino Acids. Proc Nat Acad Sci (USA) 1971;68:2484–7.Google Scholar

  • 2. Ruddick JP, Evans AK, Nutt DJ, Lightman SL, Rook GA, Lowry CA.,Tryptophan metabolism in the central nervous system: medical implications. Expert Rev Mol Med 2006;8:1–27.Google Scholar

  • 3. Boutin JA, Audinot V, Ferry G. Delagrange, Molecular tools to study. Melatonin pathways and actions. Trends Pharmacol Sci 2005;26: 412–9.Google Scholar

  • 4. Tsopmo A, Diehl−Jones BW, Aluko RE, Kitts DD, Elisia I, Friel JK. Tryptophan released from mother’s milk has antioxidant properties. Pediatr Res 2009;66:614–8.Google Scholar

  • 5. Krause D, Suh HS, Tarassishin L, Cui QL, Durafourt BA, Choi N, et al. The tryptophan metabolite 3-hydroxyanthranilic acid plays anti-inflammatory and neuroprotective roles during inflammation: role of hemeoxygenase-1. Am J Pathol 2011;179:1360–72.Web of ScienceGoogle Scholar

  • 6. Huang D, Ou B, Hampsch-Woodill M, Flanagan J, and Prior R. High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. J Agric Food Chem 2002;50:4437–44.Google Scholar

  • 7. Elisia I, Tsopmo A, Friel JK, Diehl-Jones W, Kitts DD. Tryptophan from human milk Induces oxidative stress and upregulates the Nrf-2-mediated stress response in human intestinal cell lines. J Nutr 2011;141:1417–23.Web of ScienceGoogle Scholar

  • 8. Christen S, Peterhans E, Stocker R. Antioxidant activities of some tryptophan metabolites: Possible implication for inflammatory diseases. Proc Natl Acad Sci USA 1990;87:2506–10.Google Scholar

  • 9. Yamada K, Miyazaki T, Shibata T, Hara N, Tsuchiya M. Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2008;867:57–61.Google Scholar

  • 10. Shii Y, Ogara A, Katsumata T, Umemura T, Nishikawa A, Iwasaki Y, et al. Quantification of nitrated tryptophan in proteins and tissues by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. J Pharm Biomed Anal 2007;44:150–9.Google Scholar

  • 11. Stone TW, Forrest CM, Stoy N, Darlington LG. Involvement of kynurenines in Huntington’s disease and stroke-induced brain damage. J Neural Transm 2012;119:261–74.Web of ScienceGoogle Scholar

  • 12. Takikawa O, Habara-Ohkubo A, Yoshida R. IFN- gamma is the inducer of indoleamine 2, 3-dioxygenase in allografted tumor cells undergoing rejection. J Immunol 1990;145:1246–50.Google Scholar

  • 13. O’Mahony SM, Clarke G, Borre YE, Dinan TG, Cryan JF. Serotonin, tryptophan metabolism and the brain-gut microbiome axis. Behav Brain Res 277:32–48.Web of Science

  • 14. Friel J.K., Diehl-Jones B, Nayak BN., Yang H, Tsopmo A. Anti-inflammatory and anti-oxidant properties of human milk derived peptides. Presented at the Society for Free Radical Biology and Medicine (USA), Nov 18, 2008.

  • 15. Weber WP, Feder-Mengus C, Chiarugi A, Rosenthal R, Reschner A, Schumacher R, et al. Differential effects of the tryptophan metabolite 3-hydroxy anthranilic acid on the proliferation of human CD 8+T cells induced by TCR triggering or homeostatic cytokines. Eur J Immunol 2006;36:296–304.Google Scholar

About the article

Received: 2015-07-06

Accepted: 2015-11-10

Published Online: 2015-12-08

Published in Print: 2016-06-01

Citation Information: Journal of Complementary and Integrative Medicine, Volume 13, Issue 2, Pages 129–136, ISSN (Online) 1553-3840, ISSN (Print) 2194-6329, DOI: https://doi.org/10.1515/jcim-2015-0051.

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