Pomegranate peel polyphenols inhibits inflammation in LPS-induced RAW264.7 macrophages via the suppression of TLR4/NF-κB pathway activation

  • Lin Du
  • Jianke Li
  • Xitong Zhang
  • Lifang Wang
  • Weimin Zhang
  • Mi Yang
  • Chen Hou
Keywords: pomegranate peel polyphenols; punicalagin; ellagic acid; anti-inflammation; TLR4-NF-κB


Backgrounds: Inflammatory response mediated by activated immune cells is a vital process in host defense system while responding to various stresses. Our previous studies have indicated that pomegranate peel polyphenols (PPPs) and their main components punicalagin (PC) and ellagic acid (EA) decreased pro-inflammatory cytokines and inflammatory mediators by regulating the mitogen-activated protein kinases (MAPKs) pathway, but whether these tested polyphenols play an important role in NF-κB signaling pathway, another crucial pathway of inflammation, remains unclear.

Objective: In this study, we analyzed the anti-inflammatory effect of these polyphenols via TLR4-NF-κB pathway in lipopolysaccharide (LPS)-induced RAW264.7 macrophages.

Methods: Different concentrations of PPPs, PC, and EA were pre-incubated with RAW264.7 macrophages and then stimulated with LPS (1 μg/mL), and the effects of reactive oxygen species and TLR4 were investigated. Moreover, NF-κB p65 nuclear translocation and phosphorylation, and degradation of IκB were measured by Western blot. Furthermore, the influence of pro-inflammatory cytokines was detected by enzyme-linked immunosorbent assay (ELISA).

Results: Our data showed that PPPs, PC, and EA inhibited LPS-induced intracellular ROS production and suppressed the mRNA and protein expression levels of TLR4 in a dose-dependent manner. Moreover, the anti-inflammatory mechanism was involved in blocking LPS-induced phosphorylation, degradation of IκB, and nuclear translocation of p65. Additionally, PPPs and PC exhibited a stronger anti-inflammatory effect than that of EA.

Conclusion: The results indicated that PPPs possess potent anti-inflammatory effect, and PC was the main effective component in PPPs, which provided new insights into the utilization of PPPs to prevent inflammation- associated disorders.


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  1. Shao J, Li YQ, Wang ZY, Xiao MM, Yin PH, Lu YH, et al. 7b, a novel naphthalimide derivative, exhibited anti-inflammatory effects via targeted-inhibiting TAK1 following down-regulation of ERK1/2- and p38 MAPK-mediated activation of NF-κB in LPS-stimulated RAW264.7 macrophages. Int Immunopharmacol 2013; 17(2): 216–28. doi:10.1016/j.intimp.2013.06.008.

  2. Himaya SWA, Ryu BM, Qian ZJ, Kim SK. Paeonol from Hippocampus kuda Bleeler suppressed the neuro-inflammatory responses in vitro via NF-κB and MAPK signaling pathways. Toxicol in Vitro 2012; 26(6): 878–87. doi:10.1016/j.tiv.2012.04.022.

  3. Park HH, Kim MJ, Li Y, Park YN, Lee J, Lee JL, et al. Britanin suppresses LPS-induced nitric oxide, PGE2 and cytokine production via NF-κB and MAPK inactivation in RAW 264.7 cells. Int Immunopharmacol 2013; 15(2): 296–302. doi:10.1016/j.intimp.2012.12.005.

  4. Bai SK, Lee SJ, Na HJ, Ha KS, Han JA, Lee HS, et al. β-Carotene inhibits inflammatory gene expression in lipopolysaccharide-stimulated macrophages by suppressing redox-based NF-κB activation. Exp Mol Med 2005; 37(4): 323–34. doi:10.1038/emm.2005.42.

  5. Suh SJ, Chung TW, Son MJ, Kim SH, Moon TC, Son KH, et al. The naturally occurring biflavonoid, ochnaflavone, inhibits LPS-induced iNOS expression, which is mediated by ERK1/2 via NF-κB regulation, in RAW 264.7 cells. Arch Biochem Biophys 2006; 447(2): 136–46. doi:10.1016/j.abb.2006.01.016.

  6. Kim HG, Yoon DH, Lee WH, Han SK, Shrestha B, Kim CH, et al. Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-κB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophage. J Ethnopharmacol 2007; 114(3): 307–15. doi:10.1016/j.jep.2007.08.011.

  7. Olivera A, Moore TW, Hu F, Brown AP, Sun A, Liotta DC, et al. Inhibition of the NF-κB signaling pathway by the curcumin analog, 3, 5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties. Int Immunopharmacol 2012; 12(2): 368–77. doi:10.1016/j.intimp.2011.12.009.

  8. Blonska M, Lin X. NF-κB signaling pathways regulated by CARMA family of scaffold proteins. Cell Res 2011; 21(1): 55–70. doi:10.1038/cr.2010.182.

  9. Wu SJ, Chen YW, Wang CY, Shyu YT. Anti-inflammatory properties of high pressure-assisted extracts of Grifola frondosa in lipopolysaccharide-activated RAW 264.7 macrophages. Int J Food Sci Tech 2017; 52(3): 671–8. doi:10.1111/ijfs.13320.

  10. Zhang JX, Xing JG, Wang LL, Jiang HL, Guo SL, Liu R. Luteolin inhibits fibrillary β-Amyloid1-40-induced inflammation in a human blood-brain barrier model by suppressing the p38 MAPK-mediated NF-κB signaling pathways. Molecules 2017; 22(3): 334–53. doi:10.3390/molecules22030334.

  11. Shukla MSM, Gupta PDK, Rasheed PDZ, Khan PDKA, Haqqi PDTM. Consumption of hydrolyzable tannins-rich pomegranate extract suppresses inflammation and joint damage in rheumatoid arthritis. Nutrition 2008; 24(7): 733–43. doi:10.1016/j.nut.2008.03.013.

  12. Lee CJ, Chen LG, Liang WL, Wang CC. Anti-inflammatory effects of Punica granatum Linne in vitro and in vivo. Food Chem 2010; 118(2): 315–22. doi:10.1016/j.foodchem.2009.04.123.

  13. Ismail T, Sestili P, Akhtar S. Pomegranate peel and fruit extracts: a review of potential anti-inflammatory and anti-infective effects. J Ethnopharmacol 2012; 143(2): 397–405. doi:10.1016/j.jep.2012.07.004.

  14. Shaban NZ, El-Kersh MAL, El-Rashidy FH, Habashy NH. Protective role of Punica granatum (pomegranate) peel and seed oil extracts on diethylnitrosamine and phenobarbital-induced hepatic injury in male rats. Food Chem 2013; 141(3): 1587–96. doi:10.1016/j.foodchem.2013.04.134.

  15. Wu D, Ma X, Tian W. Pomegranate husk extract, punicalagin and ellagic acid inhibit fatty acid synthase and adipogenesis of 3T3-L1 adipocyte. J Funct Foods 2013; 5(2): 633–41. doi:10.1016/j.jff.2013.01.005.

  16. Al-Muammar MN, Khan F. Obesity: the preventive role of the pomegranate (Punica granatum). Nutrition 2012; 28(6): 595–604. doi:10.1016/j.nut.2011.11.013.

  17. Gonzalez-Trujano ME, Pellicer F, Mena P, Moreno DA, Garcra-Viguera C. Antinociceptive and anti-inflammatory activities of a pomegranate (Punica granatum L.) extract rich in ellagitannins. Int J Food Sci Nutr 2015; 66(4): 395–9. doi:10.3109/09637486.2015.1024208.

  18. Li JK, He XY, Li MY, Zhao W, Liu L, Kong XH. Chemical fingerprint and quantitative analysis for quality control of polyphenols extracted from pomegranate peel by HPLC. Food Chem 2015; 176: 7–11. doi:10.1016/j.foodchem.2014.12.040.

  19. Lv O, Wang LF, Li JK, Ma QQ, Zhao W. Effects of pomegranate peel polyphenols on lipid accumulation and cholesterol metabolic transformation in L-02 human hepatic cells via the PPARγ-ABCA1/CYP7A1 pathway. Food Funct 2016; 7(12): 4976–83. doi:10.1039/c6fo01261b.

  20. Zhao YH, Li JK, Li GR. Purification with macroporous adsorbent resins and in vitro antioxidant evaluation of pomegranate peel polyphenols. Food Sci 2010; 31(11): 31–7. doi:10.7506/spkx1002-6630-201011007.

  21. Li JK, Li GX, Zhao YH, Yu CZ. Composition of pomegranate peel polyphenols and its antioxidant activities. Sci Agric Sin 2009; 42(11): 4035–41. doi:10.3864/j.issn.0578-1752.2009.11.034.

  22. Song BB, Li J, Li JK. Pomegranate peel extract polyphenols induced apoptosis in human hepatoma cells by mitochondrial pathway. Food Chem Toxicol 2016; 93: 158–66. doi:10.1016/j.fct.2016.04.020.

  23. Liu R, Li J, Cheng Y, Huo T, Xue J, Liu Y, et al. Effects of ellagic acid-rich extract of pomegranates peel on regulation of cholesterol metabolism and its molecular mechanism in hamsters. Food Funct 2015; 6(3): 780–7. doi:10.1039/c4fo00759j.

  24. Zhao W, Li J, He X, Lv O, Cheng Y, Liu R. In vitro steatosis hepatic cell model to compare the lipid-lowering effects of pomegranate peel polyphenols with several other plant polyphenols as well as its related cholesterol efflux mechanisms. Toxicol Rep 2014; 1: 945–54. doi:10.1016/j.toxrep.2014.10.013.

  25. Zhao SJ, Li JK, Wang LF, Wu XX. Pomegranate peel polyphenols inhibit lipid accumulation and enhance cholesterol efflux in raw264.7 macrophages. Food Funct 2016; 7(7): 3201–10. doi:10.1039/c6fo00347h.

  26. Chun J, Choi RJ, Khan S, Lee DS, Kim YC, Nam YJ, et al. Alantolactone suppresses inducible nitric oxide synthase and cyclooxygenase-2 expression by down-regulating NF-κB, MAPK and AP-1 via the MyD88 signaling pathway in LPS-activated RAW 264.7 cells. Int Immumopharmacol 2012; 14(4): 375–83. doi:10.1016/j.intimp.2012.08.011.

  27. Lee HS, Ryu DS, Lee GS, Lee DS. Anti-inflammatory effects of dichloromethane fraction from Orostachys japonicas in RAW 264.7 cells: suppression of NF-κB activation and MAPK signaling. J Ethnopharmacol 2012; 140(2): 271–6. doi:10.1016/j.jep.2012.01.016.

  28. Du L, Li J, Zhang X, Wang L, Zhang W. Pomegranate peel polyphenols inhibits inflammation in LPS-induced RAW264.7 macrophages via the suppression of MAPKs activation. J Funct Foods 2018; 43: 62–9. doi:10.1016/j.jff.2018.01.028.

  29. Gasparrini M, Forbes-Hernandez TY, Giampieri F, Afrin S, Alvarez-Suarez JM, Mazzoni L, et al. Anti-inflammatory effect of strawberry extract against LPS-induced stress in RAW 264.7 macrophages. Food Chem Toxicol 2017; 102: 1–10. doi:10.1016/j.fct.2017.01.018.

  30. Kumar A, Wu H, Collier-Hyams LS, Hansen JM, Li T, Yamoah K, et al. Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species. EMBO J 2007; 26(21): 4457–66. doi:10.1038/sj.emboj.7601867.

  31. Baker RG, Hayden MS, Ghosh S. NF-κB, inflammation and metabolic disease. Cell Metab 2011; 13: 11–22. doi:10.1016/j.cmet.2010.12.008.

  32. Fu Y, Liu B, Zhang N, Liu Z, Liang D, Li F, et al. Magnolol inhibits lipopolysaccharide-induced inflammatory response by interfering with TLR4 mediated NF-κB and MAPKs signaling pathways. J Ethnopharmacol 2013; 145(1): 193–9. doi:10.1016/j.jep.2012.10.051.

  33. Kim HS, Kim YJ, Lee HK, Ryu HS, Kim JS, Yoon MJ, et al. 
Activation of macrophages by polysaccharide isolated from Paecilomyces cicadae through toll-like receptor 4. Food Chem Toxicol 2012; 50(9): 3190–7. doi:10.1016/j.fct.2012.05.051.

  34. Wang X, Hu D, Zhang L, Lian G, Zhao S, Wang C, et al. Gomisin A inhibits lipopolysaccharide-induced inflammatory responses in N9 microglia via blocking the NF-κB/MAPKs pathway. Food Chem Toxicol 2014; 63: 119–27. doi:10.1016/j.fct.2013.10.048.

  35. Zhang H, Chen MK, Lia K, Hu C, Lu MH, Situ J. Eupafolin nanoparticle improves acute renal injury induced by LPS through inhibiting ROS and inflammation. Biomed Pharmacother 2017; 85: 704–11. doi:10.1016/j.biopha.2016.11.083.

  36. Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling. Cell 2008; 132: 344–62. doi:10.1016/j.cell.2008.01.020.

  37. Lawrence T. The nuclear factor NF-κB pathway in inflammation. CSH Perspect Biol 2009; 1(6): a001651. doi:10.1101/cshperspect.a001651.

  38. Xue Y, Wang Y, Feng DC, Xiao BG, Xu LY. Tetrandrine suppresses lipopolysaccharide-induced microglial activation by inhibiting NF-κB pathway. Acta Pharmacol Sin 2008; 29(2): 245–51. doi:10.1111/j.1745-7254.2008.00734.x.

  39. Lo JY, Kamarudin MNA, Hamdi OAA, Awang K, Kadir HA. Curcumenol isolated from Curcuma zedoaria suppresses Akt-mediated NF-κB activation and p38 MAPK signaling pathway in LPS-stimulated BV-2 microglial cells. Food Funct 2015; 6(11): 3550–9. doi:10.1039/c5fo00607d.

  40. Mandal A, Bhatia D, Bishayee A. Anti-inflammatory mechanism involved in pomegranate- mediated prevention of breast cancer: the role of NF-κB and Nrf2 signaling pathways. Nutrients 2017; 9(5): 436. doi:10.3390/nu9050436.

  41. Fischer UA, Carle R, Kammerer DR. Identification and quantification of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently produced juices by HPLC-DAD–ESI/MS. Food Chem 2011; 127(2): 807–21. doi:10.1016/j.foodchem.2010.12.156.

  42. Gil MI, Tomás-Barberán FA, Hess-Pierce B, Holcroft DM, Kader AA. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J Agr Food Chem 2000; 48(10): 4581–9. doi:10.1021/jf000404a.

  43. Faria A, Calhau C. The Bioactivity of pomegranate: impact on health and disease. Crit Rev Food Sci 2011; 51(7): 626–34. doi:10.1080/10408391003748100.

  44. Karwasra R, Kalra P, Gupta YK, Saini D, Kumar A, Singh S. Antioxidant and anti-inflammatory potential of pomegranate rind extract to ameliorate cisplatin-induced acute kidney injury. Food Funct 2016; 7(7): 3091–101. doi:10.1039/c6fo00188b.

  45. Shah TA, Parikh M, Patel KV, Patel KG, Joshi CG, Gandhi GJ. Evaluation of the effect of Punica granatum juice and punicalagin on NFκB modulation in inflammatory bowel disease. Mol Cell Biochem 2016; 419(1–2): 65–74. doi:10.1007/s11010-016-2750-x.

  46. Ramlagan P, Rondeau P, Planesse C, Neergheen-Bhujun VS, Fawdar S, Bourdon E. Punica granatum L. mesocarp suppresses advanced glycation end products (AGEs) -and H2O2 -induced oxidative stress and pro-inflammatory biomarkers. J Funct Foods 2017; 29: 115–26. doi:10.1016/j.jff.2016.12.007.

  47. Romier-Crouzet B, Walle JVD, During A, Joly A, Rousseau C, Henry O, et al. Inhibition of inflammatory mediators by polyphenolic plant extracts in human intestinal Caco-2 cells. Food Chem Toxicol 2009; 47(6): 1221–30. doi:10.1016/j.fct.2009.02.015.

  48. Dell’Agli M, Galli GV, Bulgari M, Basilico N, Romeo S, Bhattacharya D. Ellagitannins of the fruit rind of pomegranate (Punica granatum) antagonize in vitro the host inflammatory response mechanisms involved in the onset of malaria. Malaria J 2010; 9(1): 208. doi:10.1186/1475-2875-9-208.

  49. Xiang J, Apea-Bah FB, Ndolo VU, Katundu MC, Beta T. Profile of phenolic compounds and antioxidant activity of finger millet varieties. Food Chem 2019; 275: 361–8. doi:10.1016/j.foodchem.2018.09.120.

  50. Landete JM. Ellagitannins, ellagic acid and their derived metabolites: a review about source, metabolism, functions and health. Food Res Int 2011; 44(5): 1150–60. doi:10.1016/j.foodres.2011.04.027.

  51. Seeram NP, Adams LS, Henning SM, Niu Y, Zhang Y, Nair MG, et al. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. J Nutr Biochem 2005; 16(6): 360–7. doi:10.1016/j.jnutbio.2005.01.006.

  52. Winand J, Schneider YJ. The anti-inflammatory effect of a pomegranate husk extract on inflamed adipocytes and macrophages cultivated independently, but not on the inflammatory vicious cycle between adipocytes and macrophages. Food Funct 2014; 5(2): 310–8. doi:10.1039/c3fo60443h.

  53. Mo J, Panichayupakaranant P, Kaewnopparat N, Nitiruangjaras A, Reanmongkol W. Topical anti-inflammatory and analgesic activities of standardized pomegranate rind extract in comparison with its marker compound ellagic acid in vivo. J Ethnopharmacol 2013; 148(3): 901–8. doi:10.1016/j.jep.2013.05.040.

  54. Park S, Seok JK, Kwak JY, Suh HJ, Kim YM, Boo YC. Anti-inflammatory effects of pomegranate peel extract in THP-1 cells exposed to particulate matter PM10. Evid Based Compl Alternat Med 2016; 51(3): 469–78. doi:10.1155/2016/6836080.

  55. Lee CJ, Chen LG, Liang WL, Wang CC. Multiple activities of Punica granatum Linne against acne vulgaris. Int J Mol Sci 2017; 18(1): 141–52. doi:10.3390/ijms18010141.

How to Cite
Du L., Li J., Zhang X., Wang L., Zhang W., Yang M., & Hou C. (2019). Pomegranate peel polyphenols inhibits inflammation in LPS-induced RAW264.7 macrophages via the suppression of TLR4/NF-κB pathway activation. Food & Nutrition Research, 63. https://doi.org/10.29219/fnr.v63.3392
Original Articles