Hematological and Biochemical Evaluation of Acute and Sub-acute Oral Toxicity of Azanza garckeana Hydroethanolic Fruit Extract in Male Wistar Rats

• Akinyele Olubiyi Akinsola

  Redeemer's University

  Author

• Matthew Obaineh Ojezele

  Author

• Anthony Taghogho Eduviere

  Author

• Celestine Ogheneruro Akpovwre

  Author

Background: Azanza garckeana is a widely used African medicinal plant, particularly for reproductive health and various ailments. Despite its ethnomedicinal applications and pharmacological evidence, comprehensive data on the safety of its hydroethanolic fruit extract remain limited. This study evaluated the acute and sub-acute (15 and 30 days) oral toxicity of hydroethanolic fruit extract of Azanza garckeana (AZG) in male Wistar rats, focusing on hematological, biochemical, and oxidative stress markers.

Methods: Acute toxicity was assessed using Lorke's method. For sub-acute studies, rats received daily oral doses of hydroethanolic fruit extract of Azanza garckeana (200, 400, or 800 mg/kg) or distilled water for 15 or 30 days (n = 5/group). Body weight, food/fluid intake, hematology, serum biochemistry, and tissue oxidative stress markers were evaluated. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test (p<0.05).

Results: No mortality or overt toxicity was observed. The oral LD50 was estimated to be >5000 mg/kg, indicating low acute toxicity. At 15 days, minor changes included decreased platelet distribution width (all doses) and mean platelet volume (800 mg/kg), increased HDL-cholesterol (800 mg/kg), reduced blood urea nitrogen (all doses), and enhanced antioxidant markers (e.g., glutathione and glutathione peroxidase) in liver, kidney, and heart, with no evidence of organ damage. After 30 days, changes included improved lipid profile (decreased total/LDL-cholesterol, increased HDL), mild hematological modulations (e.g., decreased red cell distribution width), reduced food intake (400–800 mg/kg) without weight loss, and sustained catalase elevation in heart (800 mg/kg). No hepatotoxicity or nephrotoxicity was observed.

Conclusion: AZG exhibited no significant toxicity at doses up to 800 mg/kg/day for 30 days. The no-observed-adverse-effect level (NOAEL) under the present experimental conditions is 800mg/kg/day. These findings support its relative safety, with observed changes suggesting no adverse effects but potential hypolipidemic, antioxidant, and hematological modulations.

1. Mojeremane W, Tshwenyane SO. Azanza garckeana: a valuable edible indigenous fruit tree of Botswana. Pak J Nutr. 2004; 3(5):264–267. DOI: https://doi.org/10.3923/pjn.2004.264.267

2. Yusuf AA, Lawal B, Alozieuwa UB, Onikanni AS, Lukman HY, Fadaka AO, Olawale F, Osuji O, Sani S, Owolabi MS, Adewuyi AH, Yusuf DH, Batiha GE, Ataya FS, Fouad D.Attenuating effects of Azanza garckeana fractions on glycemo-impaired-associated dyslipidemia, hepatopathy, and nephropathy. Am J Transl Res. 2023; 15(10):5997–6014.

3. Sulieman A. Azanza garckeana L.: distribution, composition, nutritive value, and utilization. In: Mariod AA, editor. Wild fruits: composition, nutritional value and products. Cham: Springer; 2019. p. Chapter 30. doi:10.1007/ 978-3-030-31885-7_30.

4. Jacob C, Shehu Z, Danbature W, Karu E. Proximate analysis of the fruit Azanza garckeana (“gorontula”). Bayero J Pure Appl Sci. 2016; 9(2):221–224. DOI: https://doi.org/10.4314/bajopas.v9i2.38

5. Ahmed MU, Umaru IJ, Bazza JA, Adamu AA. Effect of aqueous fruit extract of Azanza garckeana on some biochemical and hematological parameters in Wistar rats. Asian Sci Bull. 2024; 2(4):298–303. DOI: https://doi.org/10.3923/asb.2024.298.303

6. Elshiekh Y, Ali M. Preliminary phytochemical screening, antibacterial and antioxidant activities of Azanza garckeana fruits. GSC Biol Pharm Sci. 2020; 11(3):125–129. DOI: https://doi.org/10.30574/gscbps.2020.11.3.0179

7. Titus S, Christian N, Samuel KB, Ishaya SG, Francis A, Leyoa AA. Chemical constituents and bioactivities of Azanza garckeana: a review. J Multidiscip Sci. 2025; 3(1):146–160. DOI: https://doi.org/10.58578/mikailalsys.v3i1.4911

8. Dakwa R, Mozirandi W, Mukanganyama S. Antibacterial activity of Azanza garckeana extracts (Malvaceae) in vitro and their potential use in respiratory infections. Microb Pathog. 2024; 198:107170. doi:10.1016/j.micpath.2024.107170. DOI: https://doi.org/10.1016/j.micpath.2024.107170

9. Magaji P, Muhammad A, Shadrach P. Antioxidant activities and FT-IR of Azanza garckeana leaves extracts. Afr J Biochem Mol Biol Res. 2024; 1(1):459–470. doi:10.58578/ajbmbr.v1i1.3486. DOI: https://doi.org/10.58578/ajbmbr.v1i1.3486

10. Salih A, Al-Qurainy F, Tarroum M, Shaikhaldein H, Hashimi A. Screening and estimation of bioactive compounds of Azanza garckeana (Jakjak) fruit using GC-MS, UV–visible spectroscopy, and HPLC analysis. Separations. 2022; 9(7):172. doi:10.3390/separations9070172. DOI: https://doi.org/10.3390/separations9070172

11. Okogbule F, Abadi P, Opiah J, Ihunnaya C, Martins N, Ogunrombi O. Anti-nutrients and mineral contents of Azanza garckeana (Goron Tula). J Biol Genet Res. 2024; 10(3):10–17.

12. Chawafambira A. Extraction and characterization of pectin from snot apple (Azanza garckeana) fruits with potential use in Zimbabwe. Int J Fruit Sci. 2021; 21(1):791–803. doi:10.1080/15538362.2021.1932693. DOI: https://doi.org/10.1080/15538362.2021.1932693

13. Sun W, Shahrajabian M. Therapeutic potential of phenolic compounds in medicinal plants: natural health products for human health. Molecules. 2023; 28(4):1845. doi:10.3390/molecules28041845. DOI: https://doi.org/10.3390/molecules28041845

14. Ojezele MO, Agunbiade S. Phytochemical constituents and medicinal properties of different extracts of Anacardium occidentale and Psidium guajava. Asian J Biomed Pharm Sci. 2013; 3(16):20–23.

15. Yusuf AA, Lawal B, Sani S, Garba R, Mohammed BA, Oshevire DB, Adesina DA. Pharmacological activities of Azanza garckeana (Goron Tula) grown in Nigeria. Clin Phytosci. 2020; 6:27. doi:10.1186/s40816-020-00173-0. DOI: https://doi.org/10.1186/s40816-020-00173-0

16. Adam R, El-Kamali H. Physicochemical properties, antioxidant activity, and GC-MS profiling of ethanol extract from Azanza garckeana seed oil. Trends Biol Sci. 2025; 1(1):92–100. DOI: https://doi.org/10.21124/tbs.2025.92.100

17. Pawlowska E, Szczepanska J, Blasiak J. Pro- and antioxidant effects of vitamin C in cancer in correspondence to its dietary and pharmacological concentrations. Oxid Med Cell Longev. 2019. doi:10.1155/2019/7286737. DOI: https://doi.org/10.1155/2019/7286737

18. Sallau M, Maiha B, Ejiofor I. Sub-acute toxicity studies of methanol root bark extract of Azanza garckeana in rats. Niger J Pharm Res. 2024; 20:51–63. DOI: https://doi.org/10.4314/njpr.v20iS.6s

19. Singarayar M, Chandrasekaran A, Neethirajan V, Balasundaram D, Veerasamy V, Thilagar S. Solvent polarity-driven phytochemical profiling and antioxidant evaluation of Diospyros kaki fruit extracts. J Phytol. 2025; 17:91–104. DOI: https://doi.org/10.25081/jp.2025.v17.9612

20. Erhirhie E, Ihekwereme C, Ilodigwe E. Advances in acute toxicity testing: strengths, weaknesses, and regulatory acceptance. Interdiscip Toxicol. 2018; 11(1):5–12. DOI: https://doi.org/10.2478/intox-2018-0001

21. Lee E, Richards N, Harrison J, Barnes J. Prevalence of use of traditional, complementary and alternative medicine by the general population: a systematic review. Drug Saf. 2022; 45(7):713–735. DOI: https://doi.org/10.1007/s40264-022-01189-w

22. Akinsola AO, Adeneye AA, Olorundare OE, Salahdeen HM, Murtala BA, Mukhtar H, Albrecht RM. Vasorelaxant mechanisms of Clerodendrum volubile ethanol leaf extract. Asian J Pharm Clin Res. 2022; 15(7):135–143. DOI: https://doi.org/10.22159/ajpcr.2022.v15i7.44887

23. Ekor M. The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol. 2014;4:177. DOI: https://doi.org/10.3389/fphar.2013.00177

24. Ugwah-Oguejiofor C, Okoli C, Ugwah M, Umaru M, Ogbulie C, Mshelia H, Umar M, Njan AA. Acute and sub-acute toxicity of Caralluma dalzielii extract. Heliyon. 2019; 5(1):e01179. DOI: https://doi.org/10.1016/j.heliyon.2019.e01179

25. Murtala A, Akindele A, Oreagba I. Ninety-day toxicological assessment of Newbouldia laevis preparation. Nat Prod Commun. 2024;19(5):1–11. DOI: https://doi.org/10.1177/1934578X241257080

26. Lippi G, Cervellin G, Sanchis-Gomar F. Red blood cell distribution width: a marker potentially associated with atrial fibrillation. World J Cardiol. 2019;11(12):292–304. DOI: https://doi.org/10.4330/wjc.v11.i12.292

27. Ahmed R, El-Hassan M, El-Hadi H. Potential capability of Azanza garckeana aqueous extract on iron absorption. Int J Adv Res Biol Sci. 2016;3(3):245–250.

28. Abuaraki H, Elbadawi S, Elsheikh H, Baiuomy A. Hematinic effects of medicinal plants used in anemia. Saudi J Health Sci. 2024;13(3):201–207. DOI: https://doi.org/10.4103/sjhs.sjhs_161_24

29. Felix JO, Nkwocha CC, Oruchukwu ML. Assessment of nutrients and antioxidant studies of Azanza garckeana. Research Square [Preprint]. 2024. doi:10.21203/rs.3.rs-3996423/v1. DOI: https://doi.org/10.21203/rs.3.rs-3996423/v1

30. Jin J, Wu G, Ruan C, Ling H, Zheng X, Ying C, et al. Platelet distribution width-to-platelet ratio as predictor in papillary thyroid carcinoma. J Clin Lab Anal. 2022;36(6):e24443. DOI: https://doi.org/10.1002/jcla.24443

31. Pogorzelska K, Krętowska A, Krawczuk-Rybak M, Sawicka-Żukowska M. Characteristics of platelet indices and prognostic significance. Adv Med Sci. 2020;65(2):310–315. DOI: https://doi.org/10.1016/j.advms.2020.05.002

32. Obia O, Ezekiel-Hart H, Laz-Okenwa JO, Reuben E, Dan-Jumbo D, Wami-Amadi CF. Effects of Azanza garckeana seed extract on lipid profile and oxidative stress markers. Int J Med Eval Phys Rep. 2024;8(5).

33. Amaduruonye W, Ikwunze K, Charles PC, Egbo AB. Direct and residual effect of Azanza garckeana fruit meal on lipid profiles of rabbits. In: Proc Nig Soc Anim Prod. 2024. DOI: https://doi.org/10.51791/njap.vi.5033

34. Salem MA, Hamdan DI, Mostafa I, Adel R, Elissawy A, El Shazly AM. Natural products in metabolic disorders. In: Natural products in clinical trials. Bentham Science; 2020.

35. Xiao MY, Li S, Pei WJ, Gu YL, Piao XL. Natural saponins on cholesterol-related diseases. Phytother Res. 2025;39(3):1292–1318. DOI: https://doi.org/10.1002/ptr.8432

36. Dash L, Singh A, Roychoudhury A. Non-enzymatic antioxidant defense systems in plants. In: Role of antioxidants in mitigating plant stress. Academic Press; 2025. DOI: https://doi.org/10.1016/B978-0-443-26799-4.00013-X

37. Surniyantoro HNE, Kisnanto T, Tetriana D, Yusuf D, Basri IKH, Lusiyanti Y. Immune response and malondialdehyde levels in irradiated rats supplemented with Curcuma xanthorriza Roxb extract. Asian Pac J Cancer Prev. 2023;24(5):1717–1723. DOI: https://doi.org/10.31557/APJCP.2023.24.5.1717

38. Rroji M, Kasa M, Spahia N, Kuci S, Ibrahimi A, Sula H. Acute kidney injury biomarkers. Diagnostics (Basel). 2025;15(19):2438. DOI: https://doi.org/10.3390/diagnostics15192438

39. Dennis JM, Witting PK. Protective role for antioxidants in acute kidney disease. Nutrients. 2017;9(7):718. DOI: https://doi.org/10.3390/nu9070718

40. Itodo JI, Jolayemi KO, Sani D, Ocheja JO, Ibrahim S, Ojeamiren MT. Effects of Azanza garckeana and melatonin on biochemical changes. Emerg Anim Species. 2023;9:100031. DOI: https://doi.org/10.1016/j.eas.2023.100031

41. Abolfazli S, Karav S, Johnston TP, Sahebkar A. Regulatory effects of resveratrol on nitric oxide signaling. Pharmacol Rep. 2025;77(2):355–374. DOI: https://doi.org/10.1007/s43440-025-00694-w

42. Saad B, Ghareeb B, Kmail A. Metabolic and epigenetic mechanisms of anti-obesity medicinal plants. Evid Based Complement Alternat Med. 2021;2021:9995903. DOI: https://doi.org/10.1155/2021/9995903

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