Chlorogenic Acid Mitigates Phenylhydrazine-Induced Haemolytic Anaemia and Associated Oxidative Stress in Swiss Mice
Archives of Advances in Biosciences,
Vol. 17 No. 1 (2026),
28 Ordibehesht 2026
,
Page 1-12
https://doi.org/10.22037/aab.v17i1.51567
Abstract
Background and Aim: Haemolytic anaemia has been the bane of several blood borne diseases like malaria, and it appears intractable especially in endemic areas like the tropics and developing economies. It is characterized by decreased red blood cells, severe haemoglobinuria, jaundice, and high mortality rates in young subjects without prompt medical intervention. The potential ability of several nutraceuticals and plant extracts is being investigated as affordable haematinic in the management of the condition in different models of anaemia. Chlorogenic acid (CGA) is a phytonutrient with therapeutic capacities. This study is designed to investigate the haematinic property of chlorogenic acid, a plant-based polyphenol widely distributed in the diet, using Phenylhydrazine-induced haemolytic anaemia model in mice.
Methods: Fifty-four adult male Swiss mice were used for the study. Forty-eight mice were administered 40 mg/kg dose of phenylhydrazine (PHZ) intraperitoneally twice at 12-hour interval to induce anaemia. Those animals that were confirmed to be anaemic were weight-matched into seven groups (n = 6/group) as follows: Group I- untreated non-anaemic animals received distilled water only, Group II- PHZ only, Group III- PHZ + ferrous sulphate (2 mg/kg), Group IV- PHZ + CGA (3.75 mg/kg), Group V- PHZ + CGA (7.5 mg/kg), Group VI- PHZ + CGA (15 mg/kg), Group VII- PHZ + CGA (30 mg/kg). The treatment with chlorogenic acid was by oral gavage for 5 consecutive days. On day 6, blood specimens were obtained for haematological parameters. The liver and spleen were also excised, weighed and processed for antioxidant assay, oxidative stress markers and liver function test.
Results: Chlorogenic acid reversed the haemolytic anaemia, reduced oxidative stress and liver damage and elevated bilirubin associated with exposure to phenylhydrazine at doses of 15 and 30 mg/kg.
Conclusion: Chlorogenic acid possesses significant haematinic, antioxidant and hepatoprotective potential at moderate doses.
- Chlorogenic acid
- Haematology
- Spleen
- Oxidative stress
- Liver function test
How to Cite
References
1. World Health Organization. Anaemia. 2025. (LINK)
2. Baldwin C, Pandey J, Olarewaju O. Hemolytic Anemia. 2023. (LINK)
3. WebMD Editorial Contributors. Anemia. 2023. (LINK)
4. Al-Halani AA, Edrees WH, Alrahabi LM, Thabit JM, Al-Bahloul SM, Alwashali FA, et al. Prevalence of intestinal parasites, malnutrition, anemia, and their risk factors among orphaned children in Sana’a City, Yemen. Univers J Pharm Res. 2023;8(2). (DOI: 10.22270/ujpr.v8i2.923)
5. Zekar L, Sharman T. Plasmodium falciparum malaria. 2023. StatPearls. (LINK) (Accessed January 30, 2026).
6. Goorani S, Shariatifar N, Seydi N, Zangeneh A, Moradi R, Tari B, et al. The aqueous extract of Allium saralicum RM Fritsch effectively treat induced anemia: experimental study on Wistar rats. Orient Pharm Exp Med. 2019;19(4):403-13. (DOI: 10.1007/s13596-019-00361-5)
7. Coulibaly A, Gnangoran NB, Oussou J-B N, Bleyere MN. Evaluation of Moringa oleifera Lam leaves (Moringaceae) diets against induced anemia in Wistar rats. EAS J Nutr Food Sci. 2020;2(3):101-6. (DOI: 10.36349/easjnfs.2020.v02i03.004)
8. Azeez OI, Fatodu OT, Oridupa OA. Parquetina nigrescens reverses haemorrhagic and haemolytic anaemia with reduction of erythrocyte osmotic fragility in adult male Wistar rats. Trop Vet. 2023;41(2):15-22. (LINK)
9. Santana-Galvez J, Cisneros-Zevallos L, Jacobo-Velazquez DA. Chlorogenic acid: Recent advances on its dual role as a food additive and a nutraceutical against metabolic syndrome. Molecules. 2017;22(3):358. (DOI: 10.3390/molecules22030358)
10. Tajik N, Tajik M, Mack I, Enck P. The potential effects of chlorogenic acid, the main phenolic components of coffee, on health: a comprehensive review of the literature. Eur J Nutr. 2017;36(7):2215-44. (DOI: 10.1007/s00394-017-1379-1)
11. Naveed M, Hejazi V, Abbas M, Kamboh AA, Khan GJ, Shumzaid M, et al. Chlorogenic acid (CGA): A pharmacological review and call for further research. Biomed Pharmacother. 2018;97:67-74. (DOI: 10.1016/j.biopha.2017.10.064)
12. Adeyemo-Salami OA, Afonja OJ, Adeleke OF, Adedara AO, Abolaji AO. Ameliorative potential of chlorogenic acid on rotenone-induced neurotoxicity in Drosophila melanogaster model. JPHI. 2021;4(3):55-66. (DOI: 10.14302/issn.2641-4538.jphi.21-3993)
13. Nguyen V, Taine EG, Meng D, Cui T, Tan W. Chlorogenic acid: A systematic review on the biological functions, mechanistic actions and therapeutic potentials. Nutrients. 2024;16(7):924. (DOI: 10.3390/nu16070924)
14. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972;247:3170-75. (PMID: 4623845)
15. Claiborne A. Catalase activity. In: Greenwald RA, editor. Handbook of Methods for Oxygen Radical Research. Boca Raton, Florida: CRC Press; 1985: 243–47.
16. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: biochemical role as a component of glutathione peroxidase. Science. 1973;179:588-90. (DOI: 10.1126/science.179.4073.588)
17. Habig WH, Pabst MJ, Jakoby WB. Glutathione – S – transferase: The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249:7130-9. (PMID: 4436300)
18. Beutler E, Duron DJ, Kelly MB. Improved method for the determination of blood glutathione. J Lab Clin Med. 1963;61:882-8. (PMID)
19. Jagota SK, Dani HM. A colorimetric technique for the estimation of vitamin C using folin phenol reagent. Anal Biochem. 1982;127:178-82. (DOI: 10.1016/0003-2697(82)90162-2)
20. Varshney R, Kale RK. Effect of calmodulin antagonists on radiation – induced lipid peroxidation in microsomes. Int J Radiat Biol. 1990;58:733-43. (DOI: 10.1080/09553009014552121)
21. Wolff SP. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol. 1994;233:182–9. (DOI: 10.1016/S0076-6879(94)33021-2)
22. Cleveland Clinic. Hematopoiesis. 2022. (LINK)
23. Kapila V, Wehrle CJ, Tuma F. Physiology, Spleen. 2023. StatPearls. Link (Accessed September 12, 2024).
24. Shena TY, Chy UK, Sultana A, Faruq MO. Acute hemolytic anaemia following naphthalene poisoning in a G6PD deficient patient. BCCJ. 2022;10(1):71–4. (DOI: 10.3329/bccj.v10i1.59209)
25. Malik S, Bora J, Dhasmana A, Kishore S, Nag S, Preetam S, et al. An update on current understanding of the epidemiology and management of the re-emerging endemic Lassa fever outbreaks. Int J Surg. 2023;109(3):584–86. (DOI: 10.1097/JS9.0000000000000178)
26. Mezieobi KC, Alum EU, Ugwu OP, Uti DE, Alum BN, Egba SI, et al. Economic burden of malaria on developing countries: a mini review. Parasite Epidemiol Control. 2025;30:e00435. (DOI: 10.1016/j.parepi.2025.e00435)
27. Ekweogu CN, Ude VC, Nwankpa P, Emmanuel O, Ugbogu EA. Ameliorative effect of aqueous leaf extract of Solanum aethiopicum on Phenylhydrazine-induced anaemia and toxicity in rats. Toxicol Res. 2020;36:227–38. (DOI: 10.1007/s43188-019-00021-5)
28. Ajibade TO, Oyagbemi AA, Omobowale TO, Asenuga ER, Saba AB. Telferia Occidentalis and vitamin C attenuate phenylhydrazine-induced haemolytic anaemia and associated cardio-renal dysfunctions via inhibition of oxidative stress and proapoptotic-protein (Bax) expressions. Drug Res. 2018;68(2):104–12. (DOI: 10.1055/s-0043-119070)
29. Oladele JO, Oyeleke OM, Awosanya OO, Olowookere BD, Oladele OT. Fluted Pumpkin (Telfairia occidentalis) protects against Phenylhydrazine-induced anaemia and associated toxicities in rats. Adv Tradit Med. 2021;21(4):739–45. (DOI: 10.1007/s13596-020-00499-7)
30. Banerjee A, Dey T, Ghosh AK, Mishra S, Bandyopadhyay D, Chattopadhyay A. Insights into the ameliorative effect of oleic acid in rejuvenating Phenylhydrazine induced oxidative stress mediated morphofunctionally dismantled erythrocytes. Toxicol Rep. 2020;7:1551–63. (DOI: 10.1016/j.toxrep.2020.10.022)
31. Aloke C, Emelike CU, Obasi NA, Ogbu PN, Edeogu CO, Uzomba CG, et al. HPLC profiling and studies on Copaifera salikounda methanol leaf extract on Phenylhydrazine-induced hematotoxicity and oxidative stress in rats. Arab J Chem. 2021;14(12):103428. (DOI: 10.1016/j.arabjc.2021.103428)
32. Nandi A, Yan LJ, Jana CK, Das N. Role of catalase in oxidative stress and age associated degenerative diseases. Oxid Med Cell Longev. 2019;2019:9613090. (DOI: 10.1155/2019/9613090)
33. Zheng M, Liu Y, Zhang G, Yang Z, Xu W, Chen Q. The applications and mechanisms of superoxide dismutase in medicine, food, and cosmetics. Antioxidants. 2023;12(9):1675. (DOI: 10.3390/antiox12091675)
34. Sheehan D, Meade G, Foley VM, Dowd CA. Structure, function and evolution of glutathione transferases: Implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem J. 2001;360(Pt1):1–16. (DOI: 10.1042/0264-6021:3600001)
35. Sarikaya E, Dogan S. Glutathione peroxidase in health and diseases. In: Bagatini MD, editor. Glutathione system and oxidative stress in health and disease. 2020. (LINK)
36. Gegotek A, Skrzydlewska E. Antioxidative and anti-inflammatory activity of ascorbic acid. Antioxidants (Basel). 2022;11(10):1993. (DOI: 10.3390/antiox11101993)
37. Matuz-Mares D, Riveros-Rosas H, Vilchis-Landeros MM, Vazquez-Meza H. Glutathione participation in the prevention of cardiovascular diseases. Antioxidants. 2021;10(8):1220. (DOI: 10.3390/antiox10081220)
38. Ndrepepa G. Aspartate aminotransferase and cardiovascular disease – a narrative review. J Lab Precis Med. 2021;6:6. (DOI: 10.21037/jlpm-20-93)
39. McGill MR. The past and present of serum aminotransferases and the future of liver injury biomarkers. EXCLI J. 2016;15:817–28. (DOI: 10.17179/excli2016-800)
40. Lowe D, Sanvictores T, Zubair M, John S. Alkaline Phosphatase. 2023. StatPearls. (LINK)
41. Chaphekar S, Jadhav ND, Rajurkar SR, Chigure GM, Rathod PR, Jadhav MD. Evaluation of anti-anemic activity of ferrous sulphate and copper sulphate in Phenylhydrazine induced anemia in Wistar rats. J Ayu Herb Med. 2024;10(3):81–8. (DOI: 10.31254/jahm.2024.10303)
42. Allahmoradi M, Alimohammadi S, Cheraghi H. Protective effect of Cynara scolymus L. on blood biochemical parameters and liver histopathological changes in Phenylhydrazine-induced hemolytic anemia in rats. Pharm Biomed Res. 2019;5(4):53–62. (DOI: 10.18502/pbr.v5i4.2397)
43. Ezeigwe OC, Nzekwe FA, Nworji OF, Ezennaya CF, Iloanya EL, Asogwa KK. Effect of aqueous extract of F. capensis leaves and its combination with C. aconitifolius leaves on essential biochemical parameters of Phenylhydrazine-induced anemic rats. J Exp Pharmacol. 2020;12:191–201. (DOI: 10.2147/JEP.S254003)
44. Winterbourn CC. The biological chemistry of hydrogen peroxide. Methods Enzymol. 2013;528:3–25. (DOI: 10.1016/B978-0-12-405881-1.00001-X)
45. Yaman OA, Ayhanci A. Lipid peroxidation. In: Atukeren P, editor. Accenting lipid peroxidation. 2021. (LINK)
46. Madhikarmi NL, Murthy KR. Biochemical studies on Phenylhydrazine induced experimental anemic albino rats. JUCMS. 2015;3:41–7. (DOI: 10.3126/jucms.v3i1.13258)
47. Ezeigwe OC, Nzekwe FA, Nworji OF, Ezennaya CF, Iloanya EL, Asogwa KK. Effect of aqueous extract of F. capensis leaves ant its combination with C. aconitifolius leaves on essential biochemical parameters of phenyl hydrazine-induced anemic rats. J Exp Pharamcol. 2020; 12: 191-201. )DOI: 10.2147/JEP.S254003.eCollection2020(.
48. Winterbourn C.C. The biological chemistry of hydrogen peroxide. Methods Enzymol. 2013; 528: 3-25. )DOI: 10.1016/B978-0-12-405881-1.00001-X(.
49. Yaman OA, Ayhanci A. Lipid peroxidation. In: Accenting lipid peroxidation Atukeren P, editor. 2021. (LINK)
50. Madhikarmi NL, Murthy KR. Biochemical studies on phenyl hydrazine induced experimental anemic albino rats. JUCMS. 2015; 3: 41-7. (Doi:10.3126/jucms.v.3i1.13258).
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