Biochemical Effect of Aqueous Leaf Extract of Detarium microcarpum in Wistar Rats

Medicinal plants serve as a reservoir of plant-based drugs and natural products with diverse pharmacological activities (Musa et al., 2024; Abaka et al., 2024). Moreover, their abundance, affordability, and accessibility provide an alternative for managing various ailments (Dahiru et al., 2024). Herbal remedies represent the main treatments used over millennia for preventing and treating a wide variety of ailments (Petran et al., 2024). Since ancient times, people have prepared their herbal medicines or acquired them from local traditional healers (Che et al., 2024). It is highly suggestive that about 80% of the population in developing countries still use herbal medicine to meet their primary healthcare needs (Zhang et al., 2019). Furthermore, in the past few decades, people have been rediscovering more traditional and predominantly herbal medicine (Murgia et al., 2021), either as an alternative to or in conjunction with modern drugs (Santucci et al., 2021). Many herbal remedies obtained from plants are processed and administered without scientific evaluation of their safety (Ahmed et al., 2022).
D. microcarpum, a perennial tree, also called small detar or sweet detar, can grow up to 10 m tall and occurs naturally in the arid regions of West and Central Africa (Kouyate and Van Damme, 2006; Rouamba et al., 2016). It is found in shrub savannas, wooded savannas, open forests, and dry forests, as well as in fallows (Arbonnier, 2009). It generally grows on marginal soils such as sandy and lateritic soils (Kouyate, 2005), thereby involving little to no land use competition with crops. It reproduces through rejection, suckers, and spontaneously sprouted seeds (Kouyate, 2005). This capacity suggests the possibility of vegetative propagation of D. microcarpum, which is necessary for its domestication. Regarding the species' growth, it can reach a height of 50 cm after one year and 120 cm after seven years (Bastide and Ouedraogo, 2008). It is also a species that withstands stress, such as cutting (Bationo et al., 2001; Sawadogo et al., 2002). In folklore medicine, D. microcarpum is considered a potent medicinal herb and is traditionally used to cure and prevent many diseases, including oxidative stress-related ailments such as cancer. Scientific studies have shown that D. microcarpum possesses antimicrobial, hepatoprotective, cytotoxic, and antidiabetic effects (Shofian et al., 2011; Hamza et al., 2014; Rouamba et al., 2016). This study, however, aimed to evaluate the effects of D. microcarpum aqueous leaf extract on liver and kidney function parameters.
MATERIALS AND METHODS
Plant material
D. microcarpum was collected from a Vimtim community in the Mubi North Local Government Area, Adamawa State, Nigeria. The plant material was taxonomically identified by a botanist from the Department of Botany, Adamawa State University, Mubi, where the herbarium voucher was deposited.


Laboratory animals
Twenty-five male Wistar rats weighing between 120 and 150 g used for the experiment were obtained from the Animal House of Adamawa State University, Mubi. The animals were acclimatized for one week, and all animals were treated in a manner that complied with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH publication, 1985). The animals were allowed free access to grower mash and water where a 23 -250C temperature and a 12 h light/dark light cycle was maintained throughout the experiment period. An ethical clearance for experimenting on research animals was secured from the University Ethical Committee before the initiation of the experiment with an approval number of ………………………………….
Plant extraction
The leaves of D. microcarpum were washed and air dried for 14 days, after which it was pulverized into fine powder. The method used for the extraction was as described by Adebayo et al. (2006). The powdered leaves of D. microcarpum (800 g) were soaked in 8 L of distilled water for four days, after which the extract was filtered using a Whatman no. 1 filter paper. It was further concentrated at 50°C using a rotary evaporator and further concentrated using a water bath at 48°C. The weight of the extract obtained was 67 g, giving a percentage yield of 8.9%.
Experimental design
Twenty-five (25) male albino Wistar rats were used for the study. The rats were divided into four groups of seven rats per group. The crude extract was dissolved in normal saline (0.9%) before the treatment. Groups 1, 2, 3, 4, and 5 were used for the sub-chronic experiment. Groups 2, 3, 4, and 5 were, respectively, administered with 200, 400, 600, and 800 mg/kg body weight of aqueous leaf extract of D. microcarpum for 21 days orally. Animals in group 1 (control group) did not receive the extract, but were treated with normal saline. After this period, the rats were subjected to an overnight fast and were subsequently anaesthetized with diethyl-ether, and the blood sample was collected by cardiac puncture into EDTA and were analyzed for liver and kidney parameters based on the method described by Rietman and Frankel (1957), Wright et al. (1972), Penhaker et al. (2013), and Rifai (2018).
RESULTS
The phytochemical composition of aqueous D. microcarpum leaf extract are presented in Table 1. The phytochemicals assayed for include alkaloids, flavonoids, tannins, saponins, and sterols. The result disclosed the presence of all the phytochemicals assayed for

Table 1. Qualitative phytochemical constituents of the aqueous leaf extract of D. micropapum
Phytochemical Inference
Alkaloids +
Flavonoids +
Saponins +
Tannins +
Sterols +
Key: + = present
Table 2 showed the serum liver indices of rats treated with the aqueous D. microcarpum leaf extract. The total protein levels increased significantly (P<0.05) across all the treatment groups in a dose dependent manner and when compared to the control group. Albumin levels showed a significant increase (p<0.05) in a dose dependent manner and when compared to the control groups, however the groups treated with 200 and 300 mg/kg body weight of the extract were not significantly different (p<0.05). There was a dose dependent significant increase (p<.0.5) in ALT levels when the treatment groups were compared and when compared to the control groups but groups that received 200 and 400 mg/kg body weight of the extract were not significantly different (p<0.05). AST levels showed significant decrease (p<0.05) in all the groups in a dose dependent manner and when compared to the control group. ALP levels showed significant increase (p<0.05) dose dependently and when compared to the control group, howbeit, groups that were treated with 600 and 800 mg/kg body weight of the extract were not significantly different (p<0.05).



Table 2. Serum liver indices of rats administered with aqueous leaf extract of D. microcarpum
Group T. protein (g/dL) Albumin (g/dL) ALT (UL) AST (UL) ALP (UL)
Control 63.00 ± 0.61a 38.33 ± 0,85a 33.67 ± 1.53a 53.00 ± 1.00a 64.67 ± 1.53a
200 mg/kg b.wt. 82.33 ± 0.91b 56.87 ± 0.15b 52.33 ± 1.53b 154.67 ± 0.58b 151.32 ± 1.15b
400 mg/kg b.wt. 84.97 ± 0.31c 58.63 ± 0.51b 55.00 ± 1.00b 157.33 ± 0.58c 156.67 ± 1.53c
600 mg/kg b.wt. 83.73 ± 0.45d 62.13 ± 1.17c 59.00 ± 2.00c 163.67 ± 2.08d 160.67 ± 1.53d
800 mg/kg b.wt. 88.47 ± 0.53e 66.53 ± 3.27d 65.33 ± 2.08d 166.67 ± 0.58e 166.33 ± 1.51d
All data are presented as mean ± SEM. Different superscripts down the column indicate that they are significantly different at (p<0.05), n=5
Table 3 showed the serum kidney indices of rats treated with D. microcarpum aqueous leaf extract. The serum urea levels of the groups that received extract showed a significant decrease (p<0.05) as the dose of the extract decreases and when compared with the control group. However, the group the received the lowest extract (200 mg/kg b.wt) was not significantly different (p<005) when compared with the control group. Creatinine levels also showed a significant decrease (p<0.05) when compared with the control group. H+CO3 significantly increased (p<005) with increase in dose of the extract and when compared with the control group. Significant increase (p<0.05) was observed in the serum levels of Cl- as dose of the extract increases and when compared with the extract. The serum levels of Na+ increased significantly (p<0.05) with increase in dose of the extract and when compared with the control group. Significant increase (p<0.05) was observed in the serum K+ levels as dose of the extract increases and when compared with the control group.



Group Urea (mmol/L) Creatinine (mmol/L) H+CO3 (mmol/L) Cl- (mmol/L) Na+ (mmol/L) K+ (mmol/L)
Control 19.20 ± 0.75b 1.13 ± 0.01d 24.67 ± 0.88a 95.60 ± 0.70a 139.67 ±0.58a 3.80 ± 0.10a
200 mg/kg b.wt. 18.83 ± 0.95b 0.57 ± 0.06a 25.33 ±0.58a 98.87 ± 0.32b 142.00 ± 1.00b 4.40 ± 2.00b
400 mg/kg b.wt. 17.80 ± 0.40ab 0.67 ± 0.06ab 25.33 ± 0.58a 99.83 ± 0.31b 142.67 ± 0.58b 4.57 ± 0.15b
600 mg/kg b.wt. 17.20 ± 1.25a 0.77 ± 0.06bc 26.67 ± 0.58b 102.46 ± 2.05c 144.33 ± 0.58c 4.90 ± 0.17c
800 mg/kg b.wt. 17.07 ± 1.25a 0.83 ± 0.06c 27.00 ± 1.00c 106.83 ± 0.31d 145.38 ± 0.58c 5.00 ± 0.10c
Table 3. Serum kidney indices of rats administered aqueous leaf extract of D. microcarpum

All data are presented as mean ± SEM. Different superscripts down the column indicate that they are significantly different at (p<0.05), n=5
DISCUSSION
The liver maintains homeostasis in living systems. It is involved in biochemical pathways necessary for growth and for fighting against diseases (Ward and Daly, 1999). Aspartate aminotransferase (AST) and alkaline phosphatase (ALT) indicate cellular leakage and loss of the functional integrity of the hepatocyte membrane architecture (Fki et al., 2020), and these two enzymes are considered suitable markers for liver inflammation and necrosis (Fki et al., 2020). The current results disclose that D. microcarpum aqueous leaf extract altered the membrane architecture by damaging the hepatocytes, as the levels of ALT and AST escalated significantly in a dose-dependent manner. Alkaline phosphatase (ALP) is a hydrolytic enzyme responsible for the removal of phosphate groups from many types of molecules, including nucleotides and proteins, and is particularly concentrated in the liver, bile duct, kidney, bone, and placenta (Chukwudoruo et al., 2021). An increase in ALP indicates an obstruction of the bile duct, consequently affecting the liver. An increase in ALP may also result from celiac disease (Tamas et al., 2002). The concentrations of total protein and albumin may indicate the state of the liver and the type of damage (Yakubu et al., 2005). The study also suggests that toxic metabolites in D. microcarpum aqueous leaf extract may be responsible for the significantly high values of total protein and albumin in rats administered with the extract. The albumin and total protein concentrations significantly increased (p<0.05), indicating conditions that cause overproduction of proteins.
The functional capacity of the kidney can be assessed through dye excretion tests, clearance tests, concentration and dilution tests, and methods for examining blood concentrations of excretory and electrolyte constituents (Yakubu et al., 2003). Furthermore, renal function tests are necessary either to demonstrate the presence or absence of active lesions in the kidney or to evaluate the normal functional capacity of various parts of the functioning unit (Talwar et al., 2002). The functional capacity was compromised in the nephron, with a significant increase (p<0.05) in Na+ concentration, likely resulting from excessive loss of the Na+ pool from body fluids due to the toxic effects of the aqueous leaf extract of D. microcarpum. This is corroborated by the significant decrease in Cl-. Potassium ions play a crucial role in the propagation of nerve impulses along nerve cells and their transmission to receptor cells. The sodium pump maintains the intracellular K+ concentration against the extracellular K+ concentration (Burtis and Ashwood, 1999). The hyperkalemia observed suggests a possible adverse effect on the pump that regulates its extracellular concentration due to the renal impairment caused by the extract. Urea is the primary nitrogen-containing metabolic product of protein catabolism. The significant reduction in serum urea concentration throughout the experimental period may be attributed to hindered urea cycle function, which leads to reduced production of this metabolic product. This is particularly evident in the significant reduction observed in creatinine, another product of protein metabolism. Thus, these findings indicate an abnormality in the physiological excretion of urea caused by a non-renal factor. Additionally, since urea synthesis converts toxic ammonia into nontoxic urea, defects in urea synthesis, as observed in this study, may result in ammonia intoxication. In this study, there was a significant increase (p<0.05) in the rats treated with higher doses of the plant extract. An increase in serum bicarbonate level within the normal range was associated with a reduced risk of dialysis initiation among the late-stage CKD population (Shah et al., 2009; Raphael et al., 2011). This may suggest reduced waste product removal and excess fluid from the blood due to impaired kidney function resulting from the extract.
CONCLUSION
In conclusion, this study confirms that aqueous leaf extract of D. microcarpum exhibits some levels of toxicity, posing significant risks to human health and the environment. Further research is necessary to understand the full scope of its toxicological profile and develop strategies for safe handling and mitigation. The findings of this study underscore the importance of cautious handling and responsible management of D. microcarpum to prevent potential harm.
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