Tamarindus indica Ameliorates Neural Aluminum Chloride Toxicity in Neonatal Wistar rats

Objective: This was to determine levels of heavy metal expression following Tamarindus indica activity in neonatal rats. Pregnant timed rats were divided into 5 groups (n =4) and neonatal rats were used. Group 1 (negative control), Groups II-V were experimental groups treated with 100 mg/kg of AlCl 3 s/c. Group II (positive control), Groups III and IV receiving 400mg/kg and 800mg/kg of Tamarindus indica respectively, and Group 5 receiving 30mg/kg of Vitamin E (comparative control) for 3 weeks. Brain metal; copper, zinc, iron, and calcium (Cu, Zn, Fe, and Ca respectively) quantication was done using atomic absorption spectrophotometer. Results: Oleic Acid, n-Hexadecanoic acid 11.03%, Phenol, 3,5-bis (1,1-dimethyl ethyl)- 8.48 %, and cis-9-Hexadecenal 7.79% as the main components in Tamarindus indica. The differential expression of brain metals in the treatment groups on post gestation day 7 and 21 revealed signicantly high mean Zn, Fe, and Cu and lower Ca expression in the positive control compared to the negative control; but signicantly lower mean Zn, Fe, and Cu and higher Ca expression was observed in the group treated with 400 and 800mg/kg bw of EATI and comparative control when compared to values observed in positive control.


Introduction
Pharmacological investigations on Tamarindus indica extracts revealed antibacterial and antifungal properties (1), however, there is limited information on the effect of the Tamarindus indica on the expression of trace elements in the brain following prenatal aluminum chloride exposure in Wistar rats, despite its rich phytochemical composition. Trace elements are often required in well-regulated and moderate quantity (2,3). Iron (Fe 2+ ) acts as a coenzyme essential in phosphorylation in the body (4)(5)(6).
Zinc (Zn 2+ ) is associated with presynaptic vesicles of glutaminergic neurons and is often released into the synaptic cleft where they modulate the activities of various post-synaptic receptors (7,8), and regulation of oxidant generation in the neuronal cell (9). Copper (Cu 2+ ) is an essential micronutrient essential for the maintenance of cellular integrity, production of energy, cell signaling, cellular proliferation, defense against oxidation, and effect of radiation (10) while Calcium (Ca 2+ ) plays a role in gene transcription, neurotransmission, memory processing, synaptic plasticity, and apoptotic cell death (11,12). During pregnancy, the demand for energy and nutrients is usually increased especially for micronutrients such as Fe 2+ , Zn 2+ , Cu 2+ , and Ca 2+ (13). One of the potential mechanisms of aluminum (Al 3+ ) induced toxicity involves its ability to interfere with neural ionic homeostasis (14). The ability of aluminum chloride (AlCl 3 ) to cross the blood placental barrier, with subsequent ability to cross the fragile developing blood-brain barrier, gives it more access to the brain (15). The objective of the study was to establish effects of Tamarindus indica on expression of heavy metals following prenatal exposure to AlCl 3 in neonatal Wistar rats.

Chemical and Drug Preparation
Aluminum chloride (CAS Number: 7446-70-0) was obtained from Sigma-Aldrich. Aluminum chloride stock solution was prepared by dissolving 1g of Aluminum chloride in 10mls of distilled water. Capsules of vitamin E (Gujarat liquid pharmacaps Pvt) were obtained from a reputable drug store in Zaria, Kaduna, Nigeria. The stock solution was prepared using Tween 80, containing 60 mg of vitamin E in 0.2 ml of the suspension. The stock solution containing vitamin E was then shielded from direct light to avoid photodegradation.

Plant Material Acquisition and Extraction
Tamarindus indica leaves were collected from the Botanical garden of Ahmadu Bello University, Zaria. The leaves were authenticated in the Herbarium unit of the Department of Botany and assigned a veri cation number of 2417. The extraction of the leaves was carried out by maceration, followed by subsequent fractionation (16).

Phytochemical Screening
Phytochemical screening of the crude extract and the fractions was carried out using standard methods (17), to reveal the presence of chemical constituents. Gas chromatography-mass spectrometry (GC-MS) analysis on the chemical composition of ethyl acetate leaf fraction of Tamarindusindica was done using GCMS-QP2010 PLUS SHIMADZU, Japan) (see additional le 1).

Experimental Animals
Apparently healthy (20) adult non-pregnant females and 10 adult male Wistar rats were acclimatized for two weeks in the Animal house of Human Anatomy Department, Ahmadu Bello University, after which, the vaginal smear was taken from all the female rats and examined under a light microscope for the staging of their estrous cycle. The female rats in the proestrus phase were caged overnight with the mature male in the ratio of 2:1 (female: male); the presence of vaginal plugs the following morning indicated mating and assumed to be day zero of pregnancy (18)(19)(20). The dams and their pups were allowed free access to feed and water before and during the experiment, under similar ventilated and spacious housing condition. A total of 40 male pups were used in the study.

Dosage Determination
A dosage of 200 mg/kg bw was adopted for AlCl 3 based on previous studies in Wistar rats (21,22). The adopted dosage for Ethyl acetate leaf fraction of Tamarindus indica (EATI) were 400 and 800 mg/kg bw (low and high dose respectively). A dosage of 300 mg/kg bw was adopted for vitamin E based on previous studies using the Wistar rat model (23).

Experimental Design
Gestational rats on day 7 day (day 0 of the experiment) were administered the extracts for two weeks i.e. day 21 of gestation. To reduce biases, animals were assigned random numbers and independently assigned to groups (n=4) in line with the ARRIVE guidelines on experimental animals. Neonatal rats on post gestation days 7 and 21 were euthanized using thiopental sodium since was ethically acceptable in experimental animals (24,25), by intraperitoneal injection of 5mg/kg thiopental sodium. The skull was dissected and the brain tissues were harvested from neonates for quanti cation of brain total Cu, Zn, Fe, and Ca.

Sample Preparation
Two grams of the harvested brain tissue were weighed and transferred to a beaker, then 10 ml of solvent (NHO 3 +HCl) was added. The mixture was then heated for 45 minutes to 1 hour at 100 0 C, to allow complete digestion. The digested mixture was then allowed to cool for 15-20 minutes. The mixture was then ltered and readied for brain metal quanti cation using Atomic Absorption Spectrophotometer (AAS) (Perkin-Elmer Corp., Norwalk, Conn. 06856) and the concentrations were recorded in ppm.

Statistical analysis
Data obtained were analyzed using the GRAPH PAD prism. The results were expressed as mean ± SEM; differences in means were compared using one-way analysis of variance (ANOVA) and considered signi cantly different at p ≤ 0.05.

Phytochemical Screening of ethyl acetate of Tamarindus indica
This showed that the ethyl acetate fraction of Tamarindus indica had secondary metabolites (Additional le 1). There was a positive reaction for carbohydrates and avonoids. The gas chromatography revealed 10 major peaks (Additional le 1), representing 10 chemical compounds in the ethyl acetate fraction of Tamarindus indica leaves. The mass spectrometry identi ed the following chemical components were Oleic Acid 61.63 %, n-Hexadecanoic acid 11.03%, Phenol, 3,5-bis (1,1-dimethyl ethyl)-8.48 %, and cis-9-Hexadecenal 7.79% as the main components of our plant fraction (Table 1).

Brain metal expression in brain tissue of neonatal rats
On days 7 and 21, there were signi cant differences in ionic concentrations between the negative and positive controls (Table 2) in which the positive control was associated with high ionic levels compared to other experimental groups. EATI lowered Zn (Fig 1A) concentrations comparable to the comparative control (P > 0.05). EATI lowered Cu levels and there were signi cant differences in the dosages at which 800 mg/kg bw was most effective on day 7, although this wasn't reproduced on day 21 ( Fig 1B). Furthermore, Fe levels in the positive control were the highest, however, EATI at 400 mg/kg bw and 800 mg/kg bw reduced Fe concentrations in the brain tissue on both days 7 and 21 ( Fig 1C). Signi cant decreases in Fe levels were signi cant on day 7 at 400 mg/kg. Furthermore, the positive control Ca levels were lowest, however, total Ca levels were increased by EATI at 800 mg/kg to the comparative control levels (P > 0.05) (Fig 1D).

Discussion
The current study showed that Tamarindusindica contains avonoids known for their unprecedented antioxidant property. Flavonoids also possess anti-in ammatory and anti-mutagenic properties (26).
Flavonoids-rich plant materials are shown to signi cantly improve cognition (27), These effects range from memory and learning enhancement to the said general cognition improvements (28). Ethyl acetate leave fraction of Tamarindusindica was reported to contain oleic acid. Oleic acid is involved in some very important developmental processes in the nervous system (29). Although, the nervous system can produce enough quantities it needs for these processes; however, the importance of dietary sourced oleic acid to other tissues of the body cannot be overruled. The reported n-Hexadecanoic acid present in ethyl acetate leave fraction of Tamarindusindica is said to possess hypo-cholesterolemic, anti-in ammatory, antioxidant, and antitumor activities (30).
During pregnancy, demand for energy and nutrients is usually increased especially for micronutrients such as Cu, Zn, Fe, and Ca (13). In the present study prenatal exposure to AlCI 3 adversely antagonized neural homeostasis of essential elements and this was in agreement with previous ndings (31). The study showed that administration of ethyl acetate leaves fraction of Tamarindusindica (EATI) at both 400 and 800mg/kg bw improved neural ionic homeostasis. Al exerts its toxicity by displacing Zn from the tissues demonstrating the importance of EATI in protecting neural tissue. Since Zn plays a crucial role in DNA and RNA enzyme activity, neural transmission, and antioxidant balance (7)(8)(9), ndings in the study would help in alternative and complementary medicine. Also, the study showed that EAT lowered Cu levels especially at 800 mg/kg bw in the rst week demonstrating its effects to be associated with chronic effects. Returning Cu levels to baseline levels is essential in mammals for the maintenance of cellular integrity, cell signaling, and antioxidation, (10,32). Furthermore, EATI lowered Fe levels to comparable levels to the negative and comparative controls. Since Fe acts as a coenzyme essential in phosphorylation in the body (4-6) and the body lacks mechanisms to metabolize excessive levels, administration of EATI offers protective effects. The study also showed that Al toxicity was associated with a suppression of Ca 2+ levels. Ca 2+ is essential for neural transmission, cellular signaling, and apoptosis (11,12), showing that EATI administration helps to restore tissue physiological function. Since trace elements are needed in moderate quantities (2,3), administration of EATI showed that this helped to modulate and promote physiological function following AlCl 3 toxicity. The current study, though basic offers a preview of the roles of EATI in preventing heavy metal toxicity. Furthermore, A1 toxicity was associated with elevation of trace elements in the tissues except Ca 2+ demonstrating a need to investigate cellular mechanisms responsible for ionic e ux/in ux.

Limitations
The study fails to investigate in-vitro trace element concentrations following challenges in infrastructure.
Also, limited funding meant a thorough phytochemical analysis on the plant was impossible thus creating room for further research on EATI. Cellular mechanisms interrupted and gene expression studies were not investigated, and these would help offer a pluralistic interpretation of our study ndings.  Figure 1 Mean concentration of brain metals following EATI administration following aluminum chloride exposure.

Supplementary Files
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