1,4,6 Department of Human Anatomy, School of Basic Medical Sciences, Federal University of Technology, Akure, Ondo State. P.M.B 704.
2 Department of Biology, Federal University of Technology Akure, Ondo State
3 Department of Medical Biochemistry, Federal University of Technology Akure, Ondo State
5 Department of Human Anatomy, Faculty of Allied Health Sciences, Elizade University, Ilara-Mokin, Ondo State, Nigeria.
Aluminum chloride (AlCl?) is a known environmental and industrial toxin, has been implicated in stress and immunotoxicity, particularly affecting the spleen and hematopoietic system. Composite tea, rich in polyphenols and antioxidants, may offer protective benefits against heavy metal-induced toxicity. This study investigated the ameliorative effects of herbal composite teas on aluminum chloride-induced splenic and hematopoietic cells toxicity in adult male Wistar rats. Seventy adult male Wistar rats (200-250g) were randomly divided into seven groups (n=10): Control group (distilled water), AlCl? group (200 mg/kg body weight), AlCl? (200 mg/kg body weight) + date tea, AlCl? (200 mg/kg body weight) (without sacrifice), AlCl? (200 mg/kg body weight) + clove tea, AlCl? (200 mg/kg body weight) + garlic tea, ), AlCl? (200 mg/kg body weight) + tumeric tea, AlCl? (200 mg/kg body weight) + ginger tea. Treatment will last for 28 days and the weight of the rats were taken every 3-day interval, after which the rats were allowed to fast overnight sacrifice by decapitation. Hematological parameters, splenic histoarchitecture, oxidative stress markers, and immunological parameters were evaluated. AlCl? administration significantly reduced hemoglobin concentration, red blood cells count, and white blood cells count. Equally, histological examination revealed splenic lymphoid depletion and structural disorganization. Co-administration
Aluminum is a common metal in the crust of the earth and is widely used in industries as such causes a great exposure to humans in many ways such as drinking water, food additives, cosmetics, pharmaceutical products among others1. Aluminum chloride (AlCl3), the most frequently used compound of aluminum has been widely researched regarding its possible toxic impact on the functioning of different organ systems2. Scientific research conducted recently has revealed that chronic exposure to AlCl3 causes substantial histopathological changes and oxidative stress in various organs, but these are dose- and time-specific2.
Susceptibility to aluminum toxicity is especially high in the hematopoietic system and spleen since they have high metabolic rates and contribute to immune defense. Past studies have indicated that aluminum exposure produces immunotoxicity to spleens and elevates serum norepinephrine levels in rats, but its mechanisms remain unknown3. Immunological studies on cultured T and B cells of the spleen have shown that aluminum exposure has been shown to readily influence a dose- dependent manner immune functions3. A recent study by Akinola et al. revealed strong protective effects of natural extracts in combating reproductive toxicity of aluminum chloride, indicating that exposure to aluminum leads to oxidative damage and tissue degeneration, which may be managed when antioxidant-rich plant extracts are administered4.
Extramedullary hematopoiesis primarily occurs in the spleen, which also has its own in situ hematopoietic stem cells. Like bone marrow, splenic hematopoietic stem cells have the capacity to self-renew and proliferate5, 22.Under normal circumstances, the primary source of hematopoiesis is bone marrow hematopoietic stem cells.6, 22 The spleen, however, is essential for emergency hematopoiesis.5, 7, 22 About half of the myeloid cells in atherosclerosis are actually produced by the spleen, an important organ for red blood cell filtration and immune surveillance. Aluminum-induced splenic toxicity can thus have far-reaching effects on both immunological and hematology processes. The processes of aluminum toxicity involve the induction of oxidative stress, impairment of cellular membrane integrity, disturbances of enzymatic activity, and change of gene expression patterns8.
There has been an emphasis on the possible health advantages of tea specifically composite tea blends, owing to the high concentration of bioactive compounds in tea such as polyphenols, catechins, theaflavins and thearubigins9,10. Tea polyphenols may exert some of their functions as antioxidants by removing the reactive oxygen/nitrogen species, and also by binding transition metal ions, redox active ions11, 12. Recent studies have proved that tea extracts have the potential of alleviating hematopoietic damage by means of antioxidant activity and that their administration helps considerably in increasing survival rates13.
The health benefits of tea have been reported by numerous studies. These benefits include cardiovascular protection, hepatoprotection, anti-inflammatory, immuno-regulatory, anticancer, anti-diabetic, anti-obesity, and antioxidant properties.11, 13, 14. Tea polyphenols protective actions are via direct antioxidant action, control of cellular signaling, strengthening of the endogenous antioxidant responses, and control of inflammatory pathways12,14. Due to the alarming concerns on the effects of aluminum exposure and its toxicity and the growing interest on protective natural agents, this study set out to explore the prospect of composite tea extract alleviating the effect of aluminum chloride induced splenic and hematopoietic stress in mature male Wistar rats.
MATERIALS AND METHODS
Eighty male Wistar rats 200-250g of adult age were purchased in the Animal House of the Department of Human Anatomy, Federal University of Akure, Ondo state, Nigeria. These animals were kept in standard laboratory conditions lasting 12 hours light/12 hours dark, and on maintaining temperature of 25±2°C, and relative humidity of 50-60%. The rats were given usual and normal pelleted food and clean drinking water at the discretion of the rats. The study was performed in line with the Centre of Research and Development (CERAD) of the Federal University of Akure, Ondo state, Nigeria, and all experimental practices were done under the conditions of the institution compliance with animal care and use.
To prepare the composite tea, blends were made between Date tea, Clove tea, Garlic tea, Tumeric tea, and Ginger tea in different ratios. Herbal teas (2 g) were prepared and bagged, a summary of the preparation procedure is shown in Table 1.
Table 1: Showing different formulations of different tea bags
|
Tea Name |
Tea Name Formulations (%) |
||||
|
|
Date seed |
Clove seed |
Garlic root |
Tumeric root |
Ginger root |
|
date tea |
30 |
25 |
20 |
15 |
10 |
|
ginger |
25 |
20 |
15 |
10 |
30 |
|
tumeric tea |
20 |
15 |
10 |
30 |
25 |
|
garlic tea |
15 |
10 |
30 |
25 |
20 |
|
clove tea |
10 |
30 |
25 |
20 |
15 |
4g teabags2 → 400ml (24 hours)
Dosage was given individually according to each rat’s weight:
mass per rat(g) ×5ml concentrated extract1000 (g)
∴ an average of 1.0ml of solution was given to each rat
Two tea bags each (date tea, ginger tea, turmeric tea, garlic tea, clove tea formulations) were soaked in separate air tight containers and kept in an enclosed space for a period of 24 hours with warm water to get a concentrated solution (containers were correctly labelled with their respective extract). After 24 hours, they were then kept in a moderately cold environment and dosage commenced.
After two weeks of acclimatization, the rats were randomly divided into seven groups of ten animals each:
All treatments were administered daily through oral gavage for 42 consecutive days. Body weights were recorded weekly, and general health status was monitored throughout the experimental period.
Twenty-four hours later the rats were deprived of food overnight and sacrificed under cervical dislocation. The blood samples were collected through the retro-orbital sinus, and drawn into EDTA tubes and plain tubes to do a hematological and serum biochemical analysis respectively. The spleens were removed carefully, weighed and subjected to similar portions as histological observations and biochemical evaluations.
Automated hematology analyzer (Sysmex KX-21N, Japan) was used to measure complete blood count of hemoglobin concentration (Hb), red blood cell count (RBC), and white blood cell count (WBC), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).
Histologic sections of the splenic tissues were fixed in 10% neutral buffer formalin, specimens were processed routinely, and embedded in paraffin wax. Thicknesses of 5μm cut were prepared and stained using hematoxylin and eosin (H&E) and examined using light microscope. The assessment and grading of histological changes were performed in terms of alterations in splenic architecture, lymphoid removal, and cellular accretion15.
Data were expressed as mean ± standard error of mean (SEM). Statistical analysis was performed using SPSS version 25.0. Multiple groups comparisons were done using one-way analysis of variance (ANOVA), followed by Tukey post hoc tests. The significance of P-values was less than 0.05.
RESULTS
Throughout the experimental period, no mortality was observed in any of the groups. Rats in the AlCl?-treated group showed reduced food intake and appeared less active compared to control animals. The composite tea pre-treatment group maintained normal behavior and appetite patterns similar to the control group. Body weight gain was significantly reduced in the AlCl? group compared to the control group (p<0.05). Co-administration of composite tea extract significantly improved body weight gain compared to the AlCl? alone group (p<0.05), though it remained slightly lower than the control group.
Fig 1 Shows the final and initial body weight of the groups: - A decrease in body weight can be observed in all groups that received AlCl3 either alone or with treatment Tea.
Fig 2 Shows the final body weight of the groups as compared using ANOVA: - AlCl3 treatment however shows a statistically significant difference (p<.01) in body weight when compared to the control group. This is indicative that AlCl3 significantly reduced body weight. Also, D. Tea treated group also shows a significant difference (p<.05) in the final body weight when compared to the control group.
Fig 3 shows the ANOVA comparison of spleen weights across all groups: - There is a noticeable increase of spleen weight in groups that received the tea treatments especially the Tu. Tea and also a noticeable decrease in the group that received only AlCl3. The differences in spleen weight across groups is not statistically significantly different (p>.05).
The phytochemical analysis revealed the presence of various bioactive compounds in the composite tea blend, including flavonoids, phenolic compounds, and other antioxidant molecules.
Fig 4 shows a comparison of flavonoids content (mgRE/g) between all the contents of the composite tea: - Tumeric stands out having the most amount of flavonoids content (mgRE/g) and also showing a statistically significant difference when compared to the other components. Clove is next in line having second the greatest number of Flavonoids after Tumeric with Garlic having the least.
Fig 5 Shows the diagrammatical representation of phenol and tannins content of each composition of the composite tea (Ginger. Garlic, Date, Tumeric, Clove): - Clove stands out as having the highest amounts of both contents (Phenol and Tannins). Garlic follows in line having the second largest amount of both contents (Phenol and Tannins). Tumeric on the other hand has more phenol content when compared to Date and Ginger. However, date has a slightly higher amount of Tannins when compared to Tumeric and Ginger. Ginger has the least Tannins while Date has the least Phenol content.
Fig 6 shows the percentage content of Alkanoids and Saponin present in each composition of the composite Tea: - Ginger stands out having an outstanding high amount of Alkanoids followed by Turmeric and then cloves with garlic having the least percentage. Tumeric however, has the highest percentage of saponin content and Garlic the least. Date has a higher percentage of Saponin when compared to Tumeric and Clove.
AlCl? administration caused significant alterations in hematological parameters. Hemoglobin concentration was significantly decreased in the AlCl?-treated group compared to controls, indicating the development of anemia. WBC count was also significantly decreased in the AlCl? group, these changes were indicative of both anemia and immunosuppression3. Composite tea pre-treatment significantly ameliorated these hematological alterations. In the composite tea + AlCl? group, hematological parameters were significantly higher than the AlCl? alone group and approached normal values.
Fig 7 shows the levels of WBC across all groups. The chart shows seemingly equal WBC levels across the groups: - It can be observed that the group exposed to AlCl3 alone shows lower WBC level when compared to the other groups. However, this difference is not statistically significant (p>.05).
Fig 8 shows RBC levels as compared among all groups: A statistically significant increase (p<.05) in RBC can be observed in rats treated with Cl. Tea when compared to the control group. Also there exist a significant difference (p<.05) between Cl tea + AlCl3 group that the following groups-AlCl3, D. tea + AlCl3, Gi. Tea + AlCl3 and Tu + AlCl3. There is also no significant difference between the control group and other groups except Cl. Tea + AlCl3.
Fig 9 shows the diagrammatic representation of HGB levels across all groups: The AlCl3 group shows the least amount of HGB level and Gi. Tea + AlCl3 group shows the highest amount. However, ANOVA reveals the observable differences are not statistically significant (p>.05).
Fig 10 shows the diagrammatic representation of HCT (%) across all groups: - Cl tea + AlCl3 groups shows the highest level of HCT (%), with AlCl3 group having the lease level. There is a significant difference (p<.05) between Cl tea + AlCl3 and the control and D tea + AlCl3 groups.
Fig 11 shows a comparison of MCV (fl) levels among the groups : - The bar chart shows relatively equal levels. This is confirmed by the ANOVA test results (p>.05).
Fig 12 Shows a Bar Chart representation of MCH levels: - Differences can be observed abount all groups except the control and D.tea group which show a relatively equal level. There is a significant different (p<.05) between the control group and the Tu. Tea, Ga tea and Cl tea groups. There was also a significant difference between the Alcl3 group and Cl tea group, with AlCl3 group showing closer to normal MCH level.
Fig 13 show a bar chart comparison of MCHC (g/L): - Fig. 13 show a significant difference (p<.00) in MCHC between the Control and Ga tea and Cl tea groups. The AlCl3 group was significant difference higher than Ga tea and Cl tea groups.
Fig 14 represents the PLT in a bar chart: There is significant difference (p<.05) between AlCL3 group and Cl tea and Ga tea groups. The Cl tea + AlCl3 group also shows a significant difference when compared with the control group.
Plate 1: Representative micrograph of the spleen in the control group showing normal histological features, including clearly defined white pulp (WP) surrounding the central artery (CA), distinct germinal centers (GC), and well-demarcated mantle (MnZ) and marginal zones (MgZ). The red pulp (RP) exhibits normal sinusoidal architecture interspersed with blood vessels (BV), while the trabeculae (T) and capsule (C) remain intact. No signs of vacuolation (V) or necrotic areas (E) are visible. H& E, Mgx. 40 & x100.
Plate 2: Representative micrograph from rats treated with AlCl3 shows significant histological disruption. The white pulp (WP) appears atrophic with poorly defined germinal centers (GC), marginal (MgZ) and mantle zones (MnZ). Notable vacuolation (V) and necrotic foci (E) are observed, with disrupted central artery (CA) and irregular red pulp (RP) structure. Trabeculae (T) and capsule (C) appear thinned or distorted. H& E, Mgx, x40 & x100.
Plate 3: The spleen from the clove tea and AlCl3-treated group representative demonstrates substantial recovery of splenic integrity. The white pulp (WP) shows well-organized germinal centers (GC) and distinct marginal (MgZ) and mantle zones (MnZ). The central artery (CA) and trabeculae (T) are intact, while the red pulp (RP) displays normal sinusoidal arrangement. Minimal vacuolation (V) and rare necrotic areas (E) are observed. H& E, Mgx, x40 & x100.
Plate 4: Representative micrograph of Wistar rat spleen in date tea + AlCl3 group shows partial preservation of architecture. The WP retains recognizable germinal centers (GC) and modestly defined mantle (MnZ) and marginal zones (MgZ). However, scattered vacuolation (V) and necrotic zones (E) remain visible. The CA and trabeculae (T) appear moderately preserved. H& E, Mgx, x40 & x100.
Plate 5: Representative of the micrograph spleen in the garlic tea and AlCl? group (Ga tea + AlCl3) reveals improved tissue preservation. The white pulp (WP) shows fairly organized germinal centers (GC) with discernible MnZ and MgZ, though some sections display minor vacuolation (V). The red pulp (RP), central artery (CA), trabeculae (T), and capsule (C) are largely maintained. Foci of necrosis (E) are infrequent. H& E, Mgx, x40 & x100.
Plate 6: Representative of the micrograph of Wistar rat spleen in (Gi tea + AlCl3) group reveals notable structural preservation. The white pulp (WP) retains prominent germinal centers (GC) and clearly delineated mantle (MnZ) and marginal zones (MgZ). The red pulp (RP) exhibits normal architecture, with intact central artery (CA), trabeculae (T), and capsule (C). Minimal vacuolation (V) and necrotic regions (E) are observed. H&E; Mgx. X40 & X100.
Plate 7: Representative of the micrograph of Wistar rat spleen in (Tu tea + AlCl3) group showing significant histological improvement. The white pulp (WP) displays well-preserved germinal centers (GC), along with intact mantle (MnZ) and marginal zones (MgZ). The central artery (CA), trabeculae (T), and capsule (C) appear structurally sound. The red pulp (RP) remains undistorted, with very few vacuolated (V) or necrotic (E) regions. H&E; Mag. x40 & x100.
DISCUSSION
The current study examined the protective effects of composite tea on splenic and hematopoietic toxicity effects AlCl3 in Wistar rats in vitro, and the study revealed significant protective effects of tea extracts. This is in tandem to past studies that have shown that the exposure to AlCl3 causes oxidative stress and extensive tissue damage, especially in the spleen and the hematopoietic system3, 16, 17. The group treated with AlCl3 presented significant changes regarding hematological measures and splenic histology, similar to Kadhim et al., who also found the depletion of lymphoid elements and splenic tissue alterations due to aluminum exposure1. The findings confirm the hypothesis that aluminum causes splenotoxicity due to oxidative stress and immune-suppressing mechanisms.
Lower body weight in the AlCl3 group shows toxicity, which is, in accordance with existing literature on the subject matter 4, 18. Part of the body weight was regained in the composite tea-treated group, especially in the turmeric and clove tea groups, implying that antioxidant-rich teas could be used to alleviate the effects of aluminum-induced stress. The increased weight of the spleen in tea-treated groups is a characteristic of compensatory splenomegaly, a reaction to tissue injury or stress12. This observation can be interpreted as the bioactive constituents of turmeric preserve the physiology of the spleen by regulating the immune system and attenuating inflammation. These findings agree with that of Khadim et al. who opined that splenic hypertrophy due to antioxidant therapy was possible, depending on the protective mechanisms in response to oxidative stress1.
In the group exposed to AlCl3, histopathological examination showed extreme evidence of lymphoid depletion, necrosis and excessive connective tissue deposition in the spleen, which is a characteristic of oxidative injury19. This is consistent with previous findings, which recommend that aluminum-induced oxidative stress leads to cell apoptosis and loss of cell order 2, 18. Nevertheless, in both the composite tea groups, specifically in the turmeric and clove tea-treated groups, the splenic architecture was significantly conserved and lymphoid deficiency as well as inflammatory cell infiltration was minimal. These findings are supported by the experiments of Ferdousi et al. that proved polyphenol-containing teas could be characterized as antioxidative and prevent the occurrence of oxidative damage in tissues and enhance immune activity10.
Regarding the hematological parameters, hemoglobin (Hb), the red blood cell (RBC) count and the white blood cell (WBC) count were highly affected by AlCl3 exposure, similar to the observations of Igbokwe et al. who also described that aluminum caused anemia and immunosuppression in rodents due to exposure16. Conversely, the administration of composite tea particularly clove tea showed the RBC count, hemoglobin levels, and WBC count improvement significantly thus indicating a possible restoration of hematological functioning by these teas. This is in line with previous studies which found that plant extracts rich in antioxidants has a protective effect on aluminum-induced anaemia in the form of increased red blood cell production and minimized oxidative stress20, 21.
CONCLUSION
In summary, the results presented in this study reveal that the administration of composite tea extract has important protective benefits on the aluminum chloride-induced splenic and hematopoietic toxicity in rats of the Wistar strain. The intervention was able to improve oxidative stress, maintain the splenic architecture, and normalise hematological parameters, specifically red blood cells and hemoglobin numbers. These findings illuminate the powerful antioxidant and immunomodulatory effects of the active ingredients in the mixed tea, mainly of turmeric and clove teas. The research shows great promise in the potential of natural plant extracts as therapeutic agents in treating heavy metal intoxication and it is important to note that diet rich in antioxidants should be used to reduce the toxic effects of environmental pollutants such as Aluminum.
AUTHOR'S CONTRIBUTION
Conceptualization and design: OTS and AJA; Experimentation, data collection, data analysis, and data interpretation: OIS, IBO, FRA; Draft manuscript: FRA; Vetting and approval of the manuscript for submission: OTS
ACKNOWLEDGMENTS
Authors are thankful to the authorities of Federal University of Technology Akure for the animal house and laboratory facilities for this study. The authors equally appreciate the service of Mr Ige, Department of Cell Biology, College of Medicine, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria for the production of the histological slides in their Laboratory. We also thank Tertiary Education Trust Fund (TETFund), Institution Research based (IBR) grant for providing the necessary support for this study.
FINANCIAL SUPPORT AND SPONSORSHIP
This research was supported by grants from the Federal University of Technology Akure Tertiary Education Trust Fund (TET Fund), Institution Research based (IBR) grant.
CONFLICT OF INTEREST
The authors declare no conflict of interest regarding the publication of this manuscript.
REFERENCES
Toluwase Solomon Olawuyi, Joseph A. Adeyemi, Idowu Sunday Oyeleye, Babatunde Oluwaseun Ibitoye, Rukayat Adesewa Farinde, Hadiza Enyo Amidu, Ameliorative Effects of Herbal Composite Teas on Aluminium Chloride-Induced Toxicity in The Spleen and Hemopoietic Cells of Adult Male Wistar Rats, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 11, 1362-1377. https://doi.org/10.5281/zenodo.17571501
10.5281/zenodo.17571501
