Indian Journal of Clinical Biochemistry

, Volume 34, Issue 1, pp 68–75 | Cite as

A Phenolic Acid and Flavonoid Fraction Isolated from Lolium multiflorum Lam. Prevents d-Galactosamine-Induced Liver Damages through the Augmentation of Nrf2 Expression

  • Young-Ok Son
  • Jung-Min Hwang
  • Ki-Choon ChoiEmail author
  • Jeong-Chae LeeEmail author
Original Research Article


The aims of this study were to explore whether a phenolic acid and flavonoid fraction (named PAFF) isolated from Lolium multiflorum Lam. protects against d-galactosamine (GalN)-induced liver damages in mice and to investigate the associated mechanisms. ICR mice received oral administration with various concentrations (50, 100, and 200 mg/kg body weight) of PAFF once per 2 days for seven times before intraperitoneal injection with 800 mg/kg GalN. After a day of GalN challenge, blood and tissue samples were analyzed by biochemical, histopathological, real time RT-PCR, and Western blot methods. GalN challenge induced severe damage to hepatocytes with hepatocellular vacuolization and necrosis. GalN treatment increased serum ALT, ALP, AST, and LDH levels and hepatic MDA levels and stimulated mRNA and protein expressions of Nrf2 and HO-1 in the liver. GalN treatment also diminished the levels of GSH and the activities of CAT, SOD, and GPx in the liver. However, combined treatment with PAFF inhibited GalN-mediated increases in the histological damages and the levels of serum enzymes and hepatic MDA, restored the activities of hepatic antioxidant enzymes up to those in the control values, and augmented the GalN-stimulated expression of Nrf2 and HO-1 in the liver. Furthermore, PAFF treatment alone increased the cellular SOD activity and the expression of Nrf2 and HO-1 in the liver. Our results suggest that PAFF may protect against GalN-induced liver damage by decreasing oxidative stress and increasing cellular antioxidant activities through an activation of Nrf2/HO-1-dependent pathway.


Italian ryegrass d-Galactosamine Hepatic damage Nrf2 Bioactive compounds 



This work was supported by a grant from the Rural Development Administration, Ministry of Agriculture and Forestry, South Korea (Grant No.: PJ010903032015).

Compliance with Ethical Standards

Conflict of interest

Young-Ok Son, Jung-Min Hwang, Ki-Choon Choi and Jeong-Chae Lee declares that they have no conflict of interest.

Human and Animal Rights

This study was carried out in strict accordance with the recommendations in the Guide for the Animal Care and Use of the Chonbuk National University. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The protocol in this study was approved by the University Committee on Ethics in the Care and Use of Laboratory Animals (Permit No. CBU 2012–0039).


  1. 1.
    Wang Y, Wan Y, Ye G, Wang P, Xue X, Wu G, et al. Hepatoprotective effects of AdipoRon against d-galactosamine-induced liver injury in mice. Eur J Pharm Sci. 2016;93:123–31.CrossRefGoogle Scholar
  2. 2.
    Lu J, Ren DF, Wang JZ, Sanada H, Egashira Y. Protection by dietary Spirulina platensis against d-galactosamine- and acetaminophen-induced liver injuries. Br J Nutr. 2010;103(11):1573–6.CrossRefGoogle Scholar
  3. 3.
    Taslidere E, Vardi N, Esrefoglu M, Ates B, Taskapan C, Yologlu S. The effects of pentoxifylline and caffeic acid phenethyl ester in the treatment of d-galactosamine-induced acute hepatitis in rats. Hum Exp Toxicol. 2016;35:353–65.CrossRefGoogle Scholar
  4. 4.
    Vasanth Raj P, Nitesh K, Sagar Gang S, Hitesh Jagani V, Raghu Chandrashekhar H, Venkata Rao J, et al. Protective role of catechin on d-galactosamine induced hepatotoxicity through a p53 dependent pathway. Indian J Clin Biochem. 2010;25(4):349–56.CrossRefGoogle Scholar
  5. 5.
    Liu X, Wang T, Liu X, Cai L, Qi J, Zhang P, et al. Biochanin A protects lipopolysaccharide/d-galactosamine-induced acute liver injury in mice by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation. Int Immunopharmacol. 2016;38:324–31.CrossRefGoogle Scholar
  6. 6.
    Matsuda H, Ninomiya K, Shimoda H, Yoshikawa M. Hepatoprotective principles from the flowers of Tilia argentea (linden): structure requirements of tiliroside and mechanisms of action. Bioorg Med Chem. 2002;10(3):707–12.CrossRefGoogle Scholar
  7. 7.
    Lekić N, Canová NK, Hořínek A, Farghali H. The involvement of heme oxygenase 1 but not nitric oxide synthase 2 in a hepatoprotective action of quercetin in lipopolysaccharide-induced hepatotoxicity of d-galactosamine sensitized rats. Fitoterapia. 2013;87:20–6.CrossRefGoogle Scholar
  8. 8.
    Ilavenil S, Arasu MV, Lee JC, Kim DH, Vijayakumar M, Lee KD, et al. Positive regulations of adipogenesis by Italian ryegrass [Lolium multiflorum] in 3T3-L1 cells. BMC Biotechnol. 2014;14:54.CrossRefGoogle Scholar
  9. 9.
    Valan Arasu M, Ilavenil S, Kim DH, Gun Roh S, Lee JC, Choi KC. In vitro and in vivo enhancement of adipogenesis by Italian ryegrass (Lolium multiflorum) in 3T3-L1 cells and mice. PLoS ONE. 2014;9(1):e85297.CrossRefGoogle Scholar
  10. 10.
    Hwang JM, Choi KC, Bang SJ, Son YO, Kim BT, Kim DH, et al. Anti-oxidant and anti-inflammatory properties of methanol extracts from various crops. Food Sci Biotechnol. 2013;22(1):265–72.CrossRefGoogle Scholar
  11. 11.
    Choi KC, Hwang JM, Bang SJ, Kim BT, Kim DH, Chae M, et al. Chloroform extract of alfalfa (Medicago sativa) inhibits lipopolysaccharide-induced inflammation by downregulating ERK/NF-κB signaling and cytokine production. J Med Food. 2013;16(5):410–20.CrossRefGoogle Scholar
  12. 12.
    Choi KC, Hwang JM, Bang SJ, Son YO, Kim BT, Kim DH, et al. Methanol extract of the aerial parts of barley (Hordeum vulgare) suppresses lipopolysaccharide-induced inflammatory responses in vitro and in vivo. Pharm Biol. 2013;51(8):1066–76.CrossRefGoogle Scholar
  13. 13.
    Choi KC, Son YO, Hwang JM, Kim BT, Chae M, Lee JC. Antioxiant, anti-inflammatory and anti-septic potential of phenolic acids and flavonoid fractions isolated from Lolium multiflorum. Pharm Biol. 2017;55(1):611–9.CrossRefGoogle Scholar
  14. 14.
    Zhang Q, Pi J, Woods CG, Andersen ME. A systems biology perspective on Nrf2-mediated antioxidant response. Toxicol Appl Pharmacol. 2010;244(1):84–97.CrossRefGoogle Scholar
  15. 15.
    Kleszczyński K, Zillikens D, Fischer TW. Melatonin enhances mitochondrial ATP synthesis, reduces reactive oxygen species formation, and mediates translocation of the nuclear erythroid 2-related factor 2 resulting in activation of phase-2 antioxidant enzymes (γ-GCS, HO-1, NQO1) in ultraviolet radiation-treated normal human epidermal keratinocytes (NHEK). J Pineal Res. 2016;61(2):187–97.CrossRefGoogle Scholar
  16. 16.
    Nath KA. Heme oxygenase-1 and acute kidney injury. Curr Opin Nephrol Hypertens. 2014;23(1):17–24.CrossRefGoogle Scholar
  17. 17.
    Firuzi O, Miri R, Tavakkoli M, Saso L. Antioxidant therapy: current status and future prospects. Curr Med Chem. 2011;18(25):3871–88.CrossRefGoogle Scholar
  18. 18.
    Christofidou-Solomidou M, Muzykantov VR. Antioxidant strategies in respiratory medicine. Treat Respir Med. 2006;5(1):47–78.CrossRefGoogle Scholar
  19. 19.
    Tan KK, Bang SL, Vijayan A, Chiu MT. Hepatic enzymes have a role in the diagnosis of hepatic injury after blunt abdominal trauma. Injury. 2009;40(9):978–83.CrossRefGoogle Scholar
  20. 20.
    Preetha SP, Kanniappan M, Selvakumar E, Nagaraj M, Varalakshmi P. Lupeol ameliorates aflatoxin B1-induced peroxidative hepatic damage in rats. Comp Biochem Physiol C: Toxicol Pharmacol. 2006;143(3):333–9.Google Scholar
  21. 21.
    Yener Z, Celik I, Ilhan F, Bal R. Effects of Urtica dioica L. seed on lipid peroxidation, antioxidants and liver pathology in aflatoxin-induced tissue injury in rats. Food Chem Toxicol. 2009;47(2):418–24.CrossRefGoogle Scholar
  22. 22.
    Larsson P, Busk L, Tjälve H. Hepatic and extrahepatic bioactivation and GSH conjugation of aflatoxin B1 in sheep. Carcinogenesis. 1994;15(5):947–55.CrossRefGoogle Scholar
  23. 23.
    Singha I, Das SK. Free radical scavenging properties of skin and pulp extracts of different grape cultivars in vitro and attenuation of H2O2-induced oxidative stress in liver tissue ex vivo. Indian J Clin Biochem. 2015;30(3):305–12.CrossRefGoogle Scholar
  24. 24.
    Johnson F, Giulivi C. Superoxide dismutases and their impact upon human health. Mol Aspects Med. 2005;26(4–5):340–52.CrossRefGoogle Scholar
  25. 25.
    Xiao BH, Shi M, Chen H, Cui S, Wu Y, Gao XH, et al. Glutathione peroxidase level in patients with Vitiligo: a meta-analysis. Biomed Res Int. 2016;2016:3029810.PubMedPubMedCentralGoogle Scholar
  26. 26.
    Maithili Karpaga Selvi. N, Sridhar MG, Swaminathan RP, Sripradha R. Efficacy of turmeric as adjuvant therapy in type 2 diabetic patients. Indian. J Clin Biochem. 2015;30(2):180–6.CrossRefGoogle Scholar
  27. 27.
    Wei L, Ren F, Zhang X, Wen T, Shi H, Zheng S, et al. Oxidative stress promotes d-GalN/LPS-induced acute hepatotoxicity by increasing glycogen synthase kinase 3β activity. Inflamm Res. 2014;63(6):485–94.CrossRefGoogle Scholar
  28. 28.
    Wen T, Wu ZM, Liu Y, Tan YF, Ren F, Wu H. Upregulation of heme oxygenase-1 with hemin prevents d-galactosamine and lipopolysaccharide-induced acute hepatic injury in rats. Toxicology. 2007;237(1–3):184–93.CrossRefGoogle Scholar
  29. 29.
    Park G, Oh DS, Lee MG, Lee CE, Kim YU. 6-Shogaol, an active compound of ginger, alleviates allergic dermatitis-like skin lesions via cytokine inhibition by activating the Nrf2 pathway. Toxicol Appl Pharmacol. 2016;310:51–9.CrossRefGoogle Scholar
  30. 30.
    Wu S, Yue Y, Peng A, Zhang L, Xiang J, Cao X, et al. Myricetin ameliorates brain injury and neurological deficits via Nrf2 activation after experimental stroke in middle-aged rats. Food Funct. 2016;7(6):2624–34.CrossRefGoogle Scholar

Copyright information

© Association of Clinical Biochemists of India 2017

Authors and Affiliations

  1. 1.Cell Dynamics Research Center and School of Life SciencesGwangju Institute of Science and TechnologyGwangjuSouth Korea
  2. 2.Research Center of Bioactive MaterialsInstitute of Oral Biosciences and School of Dentistry, Chonbuk National UniversityJeonjuSouth Korea
  3. 3.Grassland and Forages Research CenterNational Institute of Animal ScienceCheonanSouth Korea

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