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Greener Journal of Biomedical and Health Sciences
Vol. 9(1), pp. 132-144, 2026
ISSN: 2672-4529
Copyright ©2026, Creative Commons Attribution 4.0 International.
https://gjournals.org/GJBHS
DOI: https://doi.org/10.15580/gjbhs.2026.1.072826127
1 Department of Human Physiology, Faculty of Basic Medical Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli Campus, Nigeria
Type: Research
Full Text: PDF, PHP, HTML, EPUB, MP3
DOI: 10.15580/gjbhs.2026.1.072826127
Accepted: 30/07/2026
Published: 22/08/2026
*Corresponding Author
Gospel Onyinyeamara Okereke
Address:
Department of Human Physiology, Faculty of Basic Medical Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli Campus, Nigeria
E-mail: okerekeg097@gmail.com
Keywords: Immunoglobulin, Interleukin, Immunity, Ethanol, Hibiscus Sabdariffa (zobo) Wistar rats, antioxidants.
Background; Hibiscus sabdariffa, broadly referred to by the common name Roselle, is a species indigenous to West Africa and selected parts of Asia. Its historical record of human utilization extends to Java, where a French botanist documented its edibility as early as 1576. At present, the plant is under large-scale cultivation across multiple countries for fiber, food, and medicinal purposes. Within Nigeria, it is colloquially designated “Zobo,” a term of Hausa linguistic derivation.
Methods; This study constituted of three experimental groups which are the following:
Control Group: No active pharmacological treatment; vehicle alone (distilled water or physiological saline) was administered. This group provided the comparative reference baseline.
Low Dose Group: Received 100 mg/kg body weight of Hibiscus sabdariffa extract daily, to assess minimum effective biological activity.
High Dose Group: Received 300 mg/kg body weight of extract daily, to evaluate maximal pharmacological response and any potential adverse effects.
All treatments were administered once daily via oral gavage for 28 consecutive days.
Results; The results showed that the immunoglobin level increased at low dose of extract for decreased at high dose of extract, the interleukin 2 and 6 level increased compared to the control groups when extract was administered. The SOD level decreased compared to the control group while the GSH level decreased with low dose of extract and decreased with high dose extract and the WBC decreased with low dose and increased with high dose of extract.
Conclusion; This study provides evidence that the ethanolic leaf extract of Hibiscus sabdariffa significantly alters immune and antioxidant indices in male Wistar rats in a dose-dependent manner. Low-dose administration was associated with enhanced immunoglobulin synthesis, improved glutathione status, and potentiated immune signaling, all indicative of immunostimulatory and antioxidant benefit. High-dose administration, in contrast, resulted in reduced
List of Abbreviations:
Superoxide Dismutase (SOD),
Immunoglobulin G (IgG)
White Blood Cell (WBC)
Reduced Glutathione (GSH)
Interleukin (IL)
Hibiscus sabdariffa, broadly referred to by the common name Roselle, is a species indigenous to West Africa and selected parts of Asia. Its historical record of human utilization extends to Java, where a French botanist documented its edibility as early as 1576. At present, the plant is under large-scale cultivation across multiple countries for fiber, food, and medicinal purposes. Within Nigeria, it is colloquially designated “Zobo,” a term of Hausa linguistic derivation.
The introduction of Hibiscus sabdariffa into Nigeria is generally attributed to merchants who traversed ancient trans-Saharan trade corridors linking North Africa to western sub-Saharan regions, transporting the plant’s seeds as part of broader commercial exchanges. The plant proved highly adaptive to the Nigerian climate, establishing itself particularly in semi-arid northern zones. Key cultivating states like Kano, Kaduna, Katsina, and Sokoto remain important production centers owing to their climatically suitable conditions.(Rao, 1996)
Several factors account for Zobo’s widespread acceptance in Nigeria. From a nutritional standpoint, the plant offers substantial quantities of vitamin C, anthocyanins, and organic acids, qualifying it as both a dietary supplement and a traditional remedy. Culturally, it has been assimilated into the national cuisine as a popular thirst-quenching drink and a constituent of indigenous medicinal formulations. Economically, it sustains the livelihoods of smallholder farmers and micro-scale enterprises whose operations depend on calyx-based products such as beverages, syrups, and jams. Low input requirements and adaptability to local climatic conditions render it an accessible crop across commercial and subsistence farming scales.
In spite of the plant’s established global and historical relevance, the particular ways in which Zobo contributes to Nigerian culinary heritage, therapeutic practice, and rural economies remain incompletely understood and deserve more focused inquiry. The present investigation was therefore undertaken to examine the prospective health-related properties of Hibiscus sabdariffa, with emphasis on its effects upon hematological indicators and its suitability as an adjunct or alternative therapeutic resource.(Rao, 1996)
Aim: The primary aim of this study is to ascertain the impact of the ethanolic leaf extract of Hibiscus sabdariffa (Zobo) on Immunoglobulin G (IgG), White Blood Cell (WBC) count, Interleukins (IL-2 and IL-6), Superoxide Dismutase (SOD) and Reduced Glutathione (GSH) in male albino Wistar rats.
Specific Objectives:
Research Questions.
1. How does administration of the ethanolic leaf extract of Hibiscus sabdariffa influence IgG levels in male Wistar rats?
2. What changes in IL-2 and IL-6 levels are observed in male Wistar rats following treatment with the extract?
3. In what way does exposure to the extract alter total WBC counts in male Wistar rats?
4. What are the measurable effects of this extract on SOD activity and GSH levels in male Wistar rats?
Research Hypotheses
Null Hypothesis (H₀):
Administration of the ethanolic leaf extract of Hibiscus sabdariffa will produce no statistically significant changes in IgG, IL-2, IL-6, white blood cell count, SOD, or GSH levels in male Wistar rats.
Alternative Hypothesis (H₁):
Administration of the ethanolic leaf extract of Hibiscus sabdariffa will yield statistically significant changes in one or more of the following parameters in male Wistar rats: IgG, IL-2, IL-6, white blood cell count, SOD and GSH levels.
Study Area;
A quantity of dried Hibiscus sabdariffa (Zobo) leaves sufficient for experimental requirements was sourced from the main market in Onitsha, Anambra State, Nigeria.
Experimental Animals
Male albino Wistar rats were selected as the animal model for this study.
Other Materials
The following were used in the study:
(A) Extraction Method
Dried Zobo leaves from Onitsha Main Market, Anambra State, were washed under running tap water to remove particulate contamination. After washing, the leaves were laid flat for ambient air-drying over a two-week period before being processed into fine powder in an electric blender. Maceration in 1000 mL of 95% absolute ethanol (JHD Chemicals, Guangdong, China) was carried out over 48 hours. Coarse filtration was accomplished using clean porcelain cloth, followed by fine filtration via Whatman Qualitative Filter Paper (Grade 1, Sigma Aldrich, WHA1001042, USA). The filtrate was concentrated under reduced pressure using a rotary evaporator (Digital TT-52, Techmel & Techmel, USA) and the resulting residue was air-dried to a powder. The extract powder was preserved in sealed containers in a domestic refrigerator (Nexus model) until use. The procedure was adapted from Attar and Abu-Zeid (2013) with minor protocol adjustments.
(B) Acute Toxicity Study
The lethal dose (LD50) of the ethanolic Hibiscus sabdariffa leaf extract was established at the Department of Physiology, Faculty of Basic Medical Sciences, Nnamdi Azikiwe University, Nnewi Campus, following the method of Lorke (1983). Thirteen rats were enrolled in two successive phases; all doses were administered by the oral route.
Phase I
Nine animals were divided equally into three groups. Group 1 received 10 mg/kg; Group 2, 100 mg/kg; Group 3, 1000 mg/kg. All animals were kept under observation for 24 hours post-dosing. No clinical abnormalities or fatalities were detected in any group during Phase I.
Phase II
Four animals (one per group) received 1200, 1600, 2900, and 5000 mg/kg respectively. Observation was maintained for 24 hours. No mortality was observed in any Phase II animal.
Table 1: Acute Toxicity Study Analysis
100 mg/kg
1000 mg/kg 0/3
0/3
0/3 All rats clinically normal
All rats clinically normal
All rats clinically normal 2 1200 mg/kg
1600 mg/kg
2900 mg/kg
5000 mg/kg 0/1
0/1
0/1 Animal remained healthy
Animal remained healthy
No mortality recorded
LD50 is computed as the geometric mean of the maximum dose with 0% mortality and the minimum dose with 100% mortality: LD50 = √(A × B). Since no fatalities occurred at any dose level, the LD50 of the ethanolic extract of Hibiscus sabdariffa is above 5000 mg/kg, indicating a wide margin of safety for animal and human use.
(C) Experimental Design
Three experimental groups were constituted as follows:
(D) Animal Source and Care
Wistar rats were obtained from a certified breeding establishment. On arrival at the facility, animals were housed in clean, ventilated cages in the animal house of the Department of Physiology, Nnamdi Azikiwe University. Housing conditions were maintained at a 12-hour photoperiod cycle, 22–25°C ambient temperature, and 40–60% relative humidity. A two-week acclimatization period was observed before experimentation commenced; throughout this period and the study itself, animals received standard rat chow and unrestricted water. All animal procedures complied with approved ethical protocols, and formal institutional ethical clearance was secured before the study began.
(E) Statistical Analysis
Experimental data were analyzed by inferential statistical methods to determine the significance of inter-group differences. Statistical computations were carried out using SPSS (Statistical Package for the Social Sciences) and Microsoft Excel. Continuous data are presented as group mean ± standard deviation (SD). Between-group comparisons were performed using one-way Analysis of Variance (ANOVA); where significant main effects were found, pairwise distinctions were identified using Tukey’s or Bonferroni’s post hoc tests. The threshold for statistical significance was set at p < 0.05, corresponding to a less than 5% probability of observed differences due to chance. Findings are presented in both tabular and graphical form to facilitate interpretation.
This research was carried out at the animal facility of the Human Physiology Department, Nnamdi Azikiwe University, College of Health Sciences, Nnewi, Anambra State. The total study span was five weeks comprising two weeks of acclimatization followed by a three-week active experimental phase. During acclimatization, all animals received standard feed and free access to water. Male albino Wistar rats with an initial body mass of 90–120 g were housed in adequately spaced, ventilated cages maintained at appropriate temperatures throughout the study.
Ethical Consideration
Ethical approval was obtained from the Research Ethics Committee of the Faculty of Basic Medical Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli Campus.
Evaluation of Body Weights of the Experimental Animals
Table 2: Descriptive Table for the initial weights of Animals
Table 3: Descriptive Table for the Final Weights of Animals
166.5833
EVALUATION OF HEAMATOLOGICAL PARAMETERS
ANOVA COMPARISON FOR IMMUNOGLOBULIN
Table 4.1: Descriptive Table for Immunoglobulin
Table 4.2: ANOVA
Post Hoc Tests
Table 4.3: Multiple Comparisons
Variable: Reading
Bonferroni Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval (I) Group (J) Group Lower Bound Upper Bound A B -38.87500 17.27223 .153 -89.5400 11.7900 C 19.55000 17.27223 .861 -31.1150 70.2150 B A 38.87500 17.27223 .153 -11.7900 89.5400 C 58.42500* 17.27223 .024 7.7600 109.0900 C A -19.55000 17.27223 .861 -70.2150 31.1150 B -58.42500* 17.27223 .024 -109.0900 -7.7600 *. The mean difference is significant at the 0.05 level.
ANOVA COMPARISON FOR INTERLEUKINS 2
Table 5.1: Descriptive Table for Interleukins 2
Table 5.2: ANOVA for Interleukins 2
Table 5.3: Multiple Comparisons for Interleukins 2
Bonferroni Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval (I) Group (J) Group Lower Bound Upper Bound A B -2.10000* .54032 .011 -3.6849 -.5151 C -1.17500 .54032 .173 -2.7599 .4099 B A 2.10000* .54032 .011 .5151 3.6849 C .92500 .54032 .363 -.6599 2.5099 C A 1.17500 .54032 .173 -.4099 2.7599 B -.92500 .54032 .363 -2.5099 .6599 *. The mean difference is significant at the 0.05 level.
ANOVA COMPARISON FOR INTERLEUKINS 6
Table 6.1: Descriptive Table for Interleukins 6
Table 6.2: ANOVA for Interleukins 6
Table 6.3: Multiple Comparisons for Interleukins 6
Bonferroni Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval (I) Group (J) Group Lower Bound Upper Bound A B -9.05000 3.80338 .124 -20.2065 2.1065 C -6.05000 3.80338 .438 -17.2065 5.1065 B A 9.05000 3.80338 .124 -2.1065 20.2065 C 3.00000 3.80338 1.000 -8.1565 14.1565 C A 6.05000 3.80338 .438 -5.1065 17.2065 B -3.00000 3.80338 1.000 -14.1565 8.1565
ANOVA COMPARISON FOR SUPEROXIDE DISMUTASE (SOD)
Table 7.1: Descriptive Table for SOD
Table 7.2: ANOVA for SOD
Table 7.3: Multiple Comparisons for SOD
Bonferroni Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval (I) Group (J) Group Lower Bound Upper Bound A B .57500 1.11971 1.000 -2.7095 3.8595 C 2.40000 1.11971 .182 -.8845 5.6845 B A -.57500 1.11971 1.000 -3.8595 2.7095 C 1.82500 1.11971 .413 -1.4595 5.1095 C A -2.40000 1.11971 .182 -5.6845 .8845 B -1.82500 1.11971 .413 -5.1095 1.4595
ANOVA COMPARISON FOR GLUTATHIONE (GSH)
Table 8.1: Descriptive Table for GSH
37.0000
Table 8.2: ANOVA for GSH
Table 8.3: Multiple Comparisons for GSH
Bonferroni Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval (I) Group (J) Group Lower Bound Upper Bound A B -1.52500 1.46036 .971 -5.8087 2.7587 C 3.40000 1.46036 .135 -.8837 7.6837 B A 1.52500 1.46036 .971 -2.7587 5.8087 C 4.92500* 1.46036 .025 .6413 9.2087 C A -3.40000 1.46036 .135 -7.6837 .8837 B -4.92500* 1.46036 .025 -9.2087 -.6413 *. The mean difference is significant at the 0.05 level.
ANOVA COMPARISON FOR WHITE BLOOD CELLS (WBC)
Table 9.1: Descriptive Table for WBC
Table 9.1: ANOVA for WBC
Table 9.3: Multiple Comparisons for WBC
Variable: Reading Bonferroni Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval (I) Group (J) Group Lower Bound Upper Bound A B -1.52500 1.46036 .971 -5.8087 2.7587 C 3.40000 1.46036 .135 -.8837 7.6837 B A 1.52500 1.46036 .971 -2.7587 5.8087 C 4.92500* 1.46036 .025 .6413 9.2087 C A -3.40000 1.46036 .135 -7.6837 .8837 B -4.92500* 1.46036 .025 -9.2087 -.6413 *. The mean difference is significant at the 0.05 level.
EVALUATION OF BODY WEIGHTS OF EXPERIMENTAL ANIMALS
Table 10. Initial and Final Body Weights of Experimental Rats
156.50 ± 15.12*
Values presented as mean ± SD
* Significantly different from control values (p < 0.05)
Pre- and post-treatment body weight measurements were obtained for all Wistar rats. Analysis of initial versus final weight data revealed a significant net decrease in body mass across both treatment groups. The control group showed mixed weight trajectories (both gains and losses) during the experimental period, whereas both the low-dose and high-dose groups exhibited consistent significant weight reductions by study end.
Figure 1: Bar chart of initial and final body weights across Control, Low Dose, and High Dose groups (n=4 per group), with color-coded pre- and post-treatment values
EVALUATION OF HEMATOLOGICAL PARAMETERS ANOVA COMPARISON FOR IMMUNOGLOBULIN (mg/dL)
Table 11. Serum Immunoglobulin G (IgG) Levels across Study Groups
Values presented as mean ± SD; * significantly different from control (p < 0.05)
Serum IgG concentrations differed significantly between the control and treatment groups (p = 0.023). A dose-dependent biphasic pattern was observed: the low-dose group demonstrated elevated IgG, while the high-dose group exhibited a reduction relative to control values, consistent with immunostimulatory effects at low extract concentrations and immunosuppression at high concentrations.
ANOVA COMPARISON FOR INTERLEUKINS 2 (pg/mL)
Table 12. Interleukin-2 (IL-2) Levels Across Study Groups
Treatment Group
IL-2 concentrations reached statistical significance in the low-dose group (p = 0.012); the high-dose group change did not achieve significance. These results indicate a concentration-dependent variation in the extract’s capacity to modulate IL-2-mediated immune signaling.
ANOVA COMPARISON FOR INTERLEUKINS 6 (pg/mL)
Table 13. Interleukin-6 (IL-6) Levels Across Study Groups
IL-6 concentrations did not show a statistically significant difference between control and treatment groups at either dose level (p = 0.104), suggesting that the extract did not substantially alter IL-6 secretion under the conditions of this experiment.
ANOVA COMPARISON FOR SUPEROXIDE DISMUTASE (SOD) (U/g Hb)
Table 14. Superoxide Dismutase (SOD) Activity Across Study Groups
No statistically significant differences in SOD enzymatic activity were observed between control and extract-treated groups (p = 0.137), indicating that the extract did not produce a measurable alteration in SOD levels at the doses and duration employed.
ANOVA COMPARISON FOR GLUTATHIONE (GSH) (mg/dL)
Table 15. Reduced Glutathione (GSH) Concentrations Across Study Groups
37.00 ± 2.55
GSH levels showed a statistically significant difference across groups (p = 0.022). The low-dose group exhibited a marginal gain in GSH, whereas the high-dose group demonstrated a significant depletion, supporting a dose-dependent biphasic antioxidant response similar to that observed for IgG.
ANOVA COMPARISON FOR WHITE BLOOD CELLS (WBC) (cells/L)
Table 16. White Blood Cell (WBC) Counts Across Study Groups
Values presented as mean ± SD; * p < 0.05; ** p < 0.01
Total WBC counts differed highly significantly across experimental groups (p = 0.000). The low-dose group showed a slight decrease while the high-dose group exhibited a marked leukocytosis, suggestive of either immune activation or a pro-inflammatory systemic response at the higher extract concentration.
The present investigation was carried out to characterize the effects of the ethanolic leaf extract of Hibiscus sabdariffa on immune and antioxidant parameters specifically IgG, WBC, IL-2, IL-6, SOD, and GSH in male Wistar rats. Results indicated that the extract elicited concentration-dependent effects across measured hematological and immunological endpoints.
IgG levels increased at the low dose but fell at the high dose, suggesting that dilute concentrations of the extract upregulate antibody production while supraphysiological phytochemical loads suppress it. This pattern is consistent with observations made by Okereke (2015), who noted a dose-threshold relationship in the immune-modulating properties of medicinal herbs. The polyphenol-mediated immunomodulatory activity of H. sabdariffa in a dose-dependent fashion has also been established by Da-Costa-Rocha et al. (2014). The suppressive effect at high dose may represent immune exhaustion or oxidative overload and contrasts with the sustained immunostimulatory response reported by Gurrola-Díaz et al. (2010) in metabolic syndrome subjects, pointing to possible population- or condition-specific variability in response.
Both treatment groups exhibited elevations in IL-2 and IL-6 compared to the control, implying that H. sabdariffa extract activates interleukin-mediated immune cascades. This aligns with findings reported by Augustine et al. (1998) relating to hibiscus polyphenol-driven cytokine production. The persistence of elevated IL-6 is, however, a feature of chronic inflammation and may indicate that high-dose administration tips the physiological balance toward a pro-inflammatory rather than a protective immune state.
SOD activity was reduced in both low- and high-dose groups, a result partially at odds with Crawford et al.’s (1998) report of enhanced antioxidant enzyme function. A plausible explanation is that the extract augments cellular oxidative burden, accelerating enzymatic consumption beyond the rate of replenishment; the greater SOD reduction at the high dose supports dose-proportionate oxidative stress accumulation.
GSH showed a marginal increase at low dose but a significant decline at high dose, reinforcing the hypothesis that moderate H. sabdariffa intake bolsters antioxidant defenses while excessive intake depletes them a pattern consistent with Da-Costa-Rocha et al. (2014).
WBC counts were slightly reduced at the low dose but rose markedly at the high dose. The observed leukocytosis corroborates Haji and Haji (1999), who documented stimulation of immune cell proliferation by hibiscus extract; however, excessive leukocytosis may reflect systemic inflammatory stress, reinforcing the concept that high-dose use poses a potential health risk.
In aggregate, these observations confirm the immunomodulatory and antioxidant nature of Hibiscus sabdariffa, beneficial at conservative doses but potentially detrimental in excess, providing scientific grounding for dosage-conscious applications in traditional and integrative medicine.
This study provides evidence that the ethanolic leaf extract of Hibiscus sabdariffa significantly alters immune and antioxidant indices in male Wistar rats in a dose-dependent manner. Low-dose administration was associated with enhanced immunoglobulin synthesis, improved glutathione status, and potentiated immune signaling, all indicative of immunostimulatory and antioxidant benefit. High-dose administration, in contrast, resulted in reduced IgG, depleted glutathione, decreased SOD activity, and elevated WBC counts and interleukin levels, collectively suggesting immune suppression and oxidative imbalance. These findings are consistent with a dose-dependent biphasic pharmacological profile, underscoring the importance of dosage precision in therapeutic applications of Hibiscus sabdariffa.
Competing Interests
There is no conflict of interest.
Authors’ Contributions
GO; wrote the study design, CM; analysed the data generated from the study, MO; wrote the introduction of the study, OP and CC; wrote the literature review, NC and MI discussed the results.
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