Ferric-Induced Pancreatic Injury Involves Exacerbation of Cholinergic and Proteolytic Activities, and Dysregulation of Metabolic Pathways: Protective Effect of Caffeic Acid


The protective effect of caffeic acid on ferric-induced pancreatic injury was investigated using ex vivo and in silico models. Incubation of pancreatic tissues with Fe2+ led to significant depleted levels of glutathione (GSH) and SOD and catalase activities, with concomitant elevated levels of malondialdehyde (MDA) and nitric oxide (NO) and acetylcholinesterase and α-chymotrypsin activities. Treatment with caffeic acid led to significant reversion of these levels and activities. Molecular docking revealed a higher binding affinity of caffeic acid with acetylcholinesterase via hydrogen bonding, Pi-Pi stacking, and Van der Waals interactions. FTIR spectroscopy of pancreatic metabolite revealed little or no effect by caffeic acid on functional groups in ferric-induced injured pancreas. The LC-MS analysis of the metabolites revealed Fe2+ caused a 20% depletion of the normal metabolites, with concomitant generation of glyceraldehyde and 3,4-dihydroxymandelaldehyde. Treatment with caffeic acid led to the restoration of TG(22:4(7Z,10Z,13Z,16Z)/24:0/22:5(7Z,10Z,13Z,16Z,19Z)) and dTDP-d-glucose, while depleting glyceraldehyde as well as activating gluconeogenesis. These results indicate the ability of caffeic acid to protect against ferric toxicity by exacerbating antioxidative activities, with concomitant inhibition of MDA and NO levels while deactivating metabolic pathways linked to oxidative stress.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7



Reduced glutathione


Superoxide dismutase




Nitric oxide


Fourier transform infrared


Liquid chromatography-mass spectroscopy

FeSO4 :

Iron(II) sulfate


Diabetes mellitus


Type 1 diabetes


Type 2 diabetes


Sodium chloride


Kyoto Encyclopedia of Genes and Genomes

O2•− :

Superoxide anion


Perhydroxyl radical



H2O2 :

Hydrogen peroxide


Hydroxyl radicals


  1. 1.

    Bommer C, Heesemann E, Sagalova V, Manne-Goehler J, Atun R, Bärnighausen T, Vollmer S (2017) The global economic burden of diabetes in adults aged 20–79 years: a cost-of-illness study. Lancet Diabetes Endocrinol 5(6):423–430

    PubMed  Google Scholar 

  2. 2.

    IDF (2018) IDF Diabetes Atlas, 8th edn. The International Diabetes Federation, Brussels

    Google Scholar 

  3. 3.

    Erukainure OL, Hafizur R, Kabir N, Choudhary I, Atolani O, Banerjee P, Preissner R, Chukwuma CI, Muhammad A, Amonsou E (2018) Suppressive effects of Clerodendrum volubile P Beauv. [Labiatae] methanolic extract and its fractions on type 2 diabetes and its complications. Front Pharmacol 9:8. https://doi.org/10.3389/fphar.2018.00008

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Araki E, Nishikawa T (2010) Oxidative stress: a cause and therapeutic target of diabetic complications. J Diabetes Investig 1(3):90–96

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Erukainure OL, Sanni O, Islam MS (2018) Clerodendrum volubile: phenolics and applications to health. In: Polyphenols: mechanisms of action in human health and disease. Elsevier, pp 53–68

  6. 6.

    Magnani C, Isaac VLB, Correa MA, Salgado HRN (2014) Caffeic acid: a review of its potential use in medications and cosmetics. Anal Methods 6(10):3203–3210

    CAS  Google Scholar 

  7. 7.

    Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L (2004) Polyphenols: food sources and bioavailability. Am J Clin Nutr 79(5):727–747

    CAS  PubMed  Google Scholar 

  8. 8.

    Gülçin İ (2006) Antioxidant activity of caffeic acid (3, 4-dihydroxycinnamic acid). Toxicol 217(2–3):213–220

    Google Scholar 

  9. 9.

    Genaro-Mattos TC, Maurício ÂQ, Rettori D, Alonso A, Hermes-Lima M (2015) Antioxidant activity of caffeic acid against iron-induced free radical generation—a chemical approach. PLoS One 10(6):e0129963

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Huang D-W, Shen S-C (2012) Caffeic acid and cinnamic acid ameliorate glucose metabolism via modulating glycogenesis and gluconeogenesis in insulin-resistant mouse hepatocytes. J Funct Foods 4(1):358–366

    CAS  Google Scholar 

  11. 11.

    Huang D-W, Shen S-C, Wu JS-B (2009) Effects of caffeic acid and cinnamic acid on glucose uptake in insulin-resistant mouse hepatocytes. J Agric Food Chem 57(17):7687–7692

    CAS  PubMed  Google Scholar 

  12. 12.

    Jung UJ, Lee M-K, Park YB, Jeon S-M, Choi M-S (2006) Antihyperglycemic and antioxidant properties of caffeic acid in db/db mice. J Pharmacol Exp Ther 318(2):476–483

    CAS  PubMed  Google Scholar 

  13. 13.

    Salau VF, Erukainure OL, Ibeji CU, Olasehinde TA, Koorbanally NA, Islam MS (2019) Ferulic acid modulates dysfunctional metabolic pathways and purinergic activities, while stalling redox imbalance and cholinergic activities in oxidative brain injury. Neurotox Res. https://doi.org/10.1007/s12640-019-00099-7

  14. 14.

    Erukainure OL, Mopuri R, Oyebode OA, Koorbanally NA, Islam MS (2017) Dacryodes edulis enhances antioxidant activities, suppresses DNA fragmentation in oxidative pancreatic and hepatic injuries; and inhibits carbohydrate digestive enzymes linked to type 2 diabetes. Biomed Pharmacother 96:37–47

    CAS  PubMed  Google Scholar 

  15. 15.

    Erukainure OL, Oyebode OA, Sokhela MK, Koorbanally NA, Islam MS (2017) Caffeine–rich infusion from Cola nitida (kola nut) inhibits major carbohydrate catabolic enzymes; abates redox imbalance; and modulates oxidative dysregulated metabolic pathways and metabolites in Fe 2+-induced hepatic toxicity. Biomed Pharmacother 96:1065–1074

    CAS  PubMed  Google Scholar 

  16. 16.

    Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82(1):70–77

    CAS  PubMed  Google Scholar 

  17. 17.

    Chance B, Maehly A (1955) Assay of catalases and peroxidases. Methods Enzymol 2:764–775

    Google Scholar 

  18. 18.

    Kakkar P, Das B, Viswanathan P (1984) A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 21:130–132

    CAS  PubMed  Google Scholar 

  19. 19.

    Chowdhury P, Soulsby M (2002) Lipid peroxidation in rat brain is increased by simulated weightlessness and decreased by a soy-protein diet. Ann Clin Lab Sci 32(2):188–192

    CAS  PubMed  Google Scholar 

  20. 20.

    Tsikas D (2005) Review Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids. Free Radic Res 39(8):797–815

    CAS  PubMed  Google Scholar 

  21. 21.

    Ellman GL, Courtney KD, Andres V Jr, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7(2):88–95

    CAS  PubMed  Google Scholar 

  22. 22.

    Saleem H, Ahmad I, Ashraf M, Gill MSA, Nadeem MF, Shahid MN, Barkat K (2016) In vitro studies on anti-diabetic and anti-ulcer potentials of Jatropha gossypifolia (Euphorbiaceae). Trop J Pharm Res 15(1):121–125

    CAS  Google Scholar 

  23. 23.

    Strange RW, Antonyuk SV, Hough MA, Doucette PA, Valentine JS, Hasnain SS (2006) Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu–Zn, Zn–Zn and as-isolated wild-type enzymes. J Mol Biol 356(5):1152–1162

    CAS  PubMed  Google Scholar 

  24. 24.

    Laursen M, Yatime L, Nissen P, Fedosova NU (2013) Crystal structure of the high-affinity Na+, K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site. Proc Natl Acad Sci 110(27):10958–10963

    CAS  PubMed  Google Scholar 

  25. 25.

    Yang B, Hao F, Li J, Chen D, Liu R (2013) Binding of chrysoidine to catalase: spectroscopy, isothermal titration calorimetry and molecular docking studies. J Photochem Photobiol B 128:35–42

    CAS  PubMed  Google Scholar 

  26. 26.

    Kryger G, Silman I, Sussman JL (1999) Structure of acetylcholinesterase complexed with E2020 (Aricept®): implications for the design of new anti-Alzheimer drugs. Structure 7(3):297–307

    CAS  PubMed  Google Scholar 

  27. 27.

    Fujinaga M, Sielecki AR, Read RJ, Ardelt W, Laskowski M Jr, James MN (1987) Crystal and molecular structures of the complex of α-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 Å resolution. J Mol Biol 195(2):397–418

    CAS  PubMed  Google Scholar 

  28. 28.

    Sanner MF (1999) Python: a programming language for software integration and development. J Mol Graph Model 17(1):57–61

    CAS  PubMed  Google Scholar 

  29. 29.

    Frisch M, Trucks G, Schlegel HB, Scuseria G, Robb M, Cheeseman J, Scalmani G, Barone V, Mennucci B, Petersson G (2009) Gaussian 09, revision D. 01. Gaussian, Inc., Wallingford CT,

  30. 30.

    Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30(16):2785–2791

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Chan CX, Khan AA, Choi JH, Ng CD, Cadeiras M, Deng M, Ping P (2013) Technology platform development for targeted plasma metabolites in human heart failure. Clin Proteomics 10(1):7

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Wishart DS, Jewison T, Guo AC, Wilson M, Knox C, Liu Y, Djoumbou Y, Mandal R, Aziat F, Dong E (2012) HMDB 3.0—the human metabolome database in 2013. Nucleic Acids Res 41(D1):D801–D807

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Chong J, Soufan O, Li C, Caraus I, Li S, Bourque G, Wishart DS, Xia J (2018) MetaboAnalyst 4.0: towards more transparent and integrative metabolomics analysis. Nucleic Acids Res 46(W1):W486–W494

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Maritim A, Sanders R, Watkins J III (2003) Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxicol 17(1):24–38

    CAS  PubMed  Google Scholar 

  35. 35.

    Gallego FQ, Sinzato YK, Miranda CA, Iessi IL, Dallaqua B, Volpato GT, Scarano WR, SanMartín S, Damasceno DC (2018) Pancreatic islet response to diabetes during pregnancy in rats. Life Sci 214:1–10

    CAS  PubMed  Google Scholar 

  36. 36.

    Gerber PA, Rutter GA (2017) The role of oxidative stress and hypoxia in pancreatic beta-cell dysfunction in diabetes mellitus. Antioxid Redox Signal 26(10):501–518

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Latunde-Dada GO (2017) Ferroptosis: role of lipid peroxidation, iron and ferritinophagy. Biochim Biophys Acta Gen Subj 1861(8):1893–1900

    CAS  PubMed  Google Scholar 

  38. 38.

    Skrypnik K, Bogdański P, Schmidt M, Suliburska J (2019) The effect of multispecies probiotic supplementation on iron status in rats. Biol Trace Elem Res 1–10. https://doi.org/10.1007/s12011-019-1658-1

  39. 39.

    Erukainure OL, Sanni O, Islam MS (2018) Clerodendrum volubile: phenolics and applications to health. In: Watson R, Preedy V, Zibadi S (eds) Polyphenols: mechanisms of action in human health and disease, 2nd edn. Elsevier, Amsterdam. https://doi.org/10.1016/B978-0-12-813006-3.00006-4

    Chapter  Google Scholar 

  40. 40.

    Singh B, Singh JP, Kaur A, Singh N (2018) Phenolic compounds as beneficial phytochemicals in pomegranate (Punica granatum L.) peel: a review. Food Chem 261:75–86

    CAS  PubMed  Google Scholar 

  41. 41.

    Rajpathak SN, Crandall JP, Wylie-Rosett J, Kabat GC, Rohan TE, Hu FB (2009) The role of iron in type 2 diabetes in humans. Biochim Biophys Acta Gen Subj 1790(7):671–681

    CAS  Google Scholar 

  42. 42.

    Andrews NC (1999) Disorders of iron metabolism. N Engl J Med 341(26):1986–1995

    CAS  PubMed  Google Scholar 

  43. 43.

    Kanias T, Acker JP (2010) Biopreservation of red blood cells–the struggle with hemoglobin oxidation. FEBS J 277(2):343–356

    CAS  PubMed  Google Scholar 

  44. 44.

    Aslan M, Thornley-Brown D, Freeman BA (2000) Reactive species in sickle cell disease. Ann N Y Acad Sci 899(1):375–391

    CAS  PubMed  Google Scholar 

  45. 45.

    Paraoanu LE, Layer PG (2008) Acetylcholinesterase in cell adhesion, neurite growth and network formation. FEBS J 275(4):618–624

    CAS  PubMed  Google Scholar 

  46. 46.

    Zhang B, Yang L, Yu L, Lin B, Hou Y, Wu J, Huang Q, Han Y, Guo L, Ouyang Q (2012) Acetylcholinesterase is associated with apoptosis in β cells and contributes to insulin-dependent diabetes mellitus pathogenesis. Acta Biochim Biophys Sin 44(3):207–216

    CAS  PubMed  Google Scholar 

  47. 47.

    Melo JB, Agostinho P, Oliveira CR (2003) Involvement of oxidative stress in the enhancement of acetylcholinesterase activity induced by amyloid beta-peptide. Neurosci Res 45(1):117–127

    CAS  PubMed  Google Scholar 

  48. 48.

    Rodríguez-Fuentes G, Rubio-Escalante FJ, Noreña-Barroso E, Escalante-Herrera KS, Schlenk D (2015) Impacts of oxidative stress on acetylcholinesterase transcription, and activity in embryos of zebrafish (Danio rerio) following Chlorpyrifos exposure. Comp Biochem Physiol C Toxicol Pharmacol 172:19–25

    PubMed  Google Scholar 

  49. 49.

    Anwar J, Spanevello RM, Thomé G, Stefanello N, Schmatz R, Gutierres J, Vieira J, Baldissarelli J, Carvalho FB, da Rosa MM (2012) Effects of caffeic acid on behavioral parameters and on the activity of acetylcholinesterase in different tissues from adult rats. Pharmacol Biochem Behav 103(2):386–394

    CAS  PubMed  Google Scholar 

  50. 50.

    Rezg R, Mornagui B, El-Fazaa S, Gharbi N (2008) Caffeic acid attenuates malathion induced metabolic disruption in rat liver, involvement of acetylcholinesterase activity. Toxicol 250(1):27–31

    CAS  Google Scholar 

  51. 51.

    Oboh G, Agunloye OM, Akinyemi AJ, Ademiluyi AO, Adefegha SA (2013) Comparative study on the inhibitory effect of caffeic and chlorogenic acids on key enzymes linked to Alzheimer’s disease and some pro-oxidant induced oxidative stress in rats’ brain-in vitro. Neurochem Res 38(2):413–419

    CAS  PubMed  Google Scholar 

  52. 52.

    Tonon J, Cecchini AL, Brunnquell CR, Bernardes SS, Cecchini R, Guarnier FA (2013) Lung injury-dependent oxidative status and chymotrypsin-like activity of skeletal muscles in hamsters with experimental emphysema. BMC Musculoskelet Disord 14(1):39

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Hagopian K, Ramsey JJ, Weindruch R (2008) Enzymes of glycerol and glyceraldehyde metabolism in mouse liver: effects of caloric restriction and age on activities. Biosci Rep 28(2):107–115

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M (2001) Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Investig 108(9):1341–1348

    CAS  PubMed  Google Scholar 

  55. 55.

    Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414(6865):813

    CAS  PubMed  Google Scholar 

  56. 56.

    Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M (2000) High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C--dependent activation of NAD (P) H oxidase in cultured vascular cells. Diabetes 49(11):1939–1945

    CAS  PubMed  Google Scholar 

  57. 57.

    Ipson BR, Fisher AL (2016) Roles of the tyrosine isomers meta-tyrosine and ortho-tyrosine in oxidative stress. Ageing Res Rev 27:93–107

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Sgaravatti ÂM, Vargas BA, Zandoná BR, Deckmann KB, Rockenbach FJ, Moraes TB, Monserrat JM, Sgarbi MB, Pederzolli CD, Wyse AT (2008) Tyrosine promotes oxidative stress in cerebral cortex of young rats. Int J Dev Neurosci 26(6):551–559

    CAS  PubMed  Google Scholar 

  59. 59.

    Grimsrud PA, Xie H, Griffin TJ, Bernlohr DA (2008) Oxidative stress and covalent modification of protein with bioactive aldehydes. J Biol Chem 283(32):21837–21841

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Berg JM, Tymoczko JL, Stryer L (2002) Biochemistry, 5th edn. W H Freeman, New York

    Google Scholar 

Download references


This work was supported by funding from the Research Office, University of KwaZulu-Natal, Durban, and the National Research Foundation-the World Academy of Science (NRF-TWAS), Pretoria, South Africa.

Author information



Corresponding author

Correspondence to Md. Shahidul Islam.

Ethics declarations

The study was carried out in accordance with the approved guidelines of the Animal Ethics Committee of the University of KwaZulu-Natal, Durban, South Africa (protocol approval number: AREC/020/017D).

Conflict of Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Salau, V.F., Erukainure, O.L., Ibeji, C.U. et al. Ferric-Induced Pancreatic Injury Involves Exacerbation of Cholinergic and Proteolytic Activities, and Dysregulation of Metabolic Pathways: Protective Effect of Caffeic Acid. Biol Trace Elem Res 196, 517–527 (2020). https://doi.org/10.1007/s12011-019-01937-7

Download citation


  • Antioxidant
  • Caffeic acid
  • Functional chemistry
  • Metabolomics