Unveiling phytochemical diversity and safety profile of hot water extract from Tetrapleura tetraptera fruit (2024)

Background

Tetrapleura tetraptera, a widely used medicinal plant in West Africa, has been traditionally employed for various ailments. Despite its folkloric significance, scientific validation of its safety and potential neuroactive properties remains limited.

Objectives

This study aimed to investigate the acute and subchronic toxicity of Tetrapleura tetraptera hot water extract (HWETTF) in rats and to elucidate its phytochemical composition.

Methods

Acute oral toxicity was assessed in mice using the OECD guideline 423, while a 14-day repeat-dose toxicity study was conducted in rats. The phytochemical analysis included HPLC, FT-IR, and GC–MS.

Results

HWETTF exhibited no significant toxicity in acute or subchronic studies, even at high doses. Phytochemical analysis revealed a diverse array of compounds, including those with potential GABAergic and CNS depressant activities.

Conclusion

Tetrapleura tetraptera demonstrated a favourable safety profile in rodents and possesses a rich phytochemical composition. Further research is warranted to explore its potential neuroactive properties and develop therapeutic applications.

For millennia, plants have served as humanity’s natural remedies, their therapeutic secrets whisper across generations. Tetrapleura tetraptera, a Nigerian spice woven into the fabric of culinary and medicinal traditions, is now captivating scientific interest due to its enigmatic bioactive potential. This study opens up a new frontier in its story, venturing beyond folkloric lore to rigorously assess its safety profile and unveil its promising neuroactive landscape.

Renowned as Aridan in Nigeria, T. tetraptera transcends its culinary prowess. Deeply embedded in local medicine, it has been employed to combat diverse ailments like diabetes, hypertension, and epilepsy [1, 2]. Recent scientific explorations have illuminated its phytochemical secrets, revealing a treasure trove of antioxidants, antiproliferative agents, and neuroprotective compounds [3,4,5,6]. Irondi et al. [3] reported that fully ripe T. tetraptera pods were more potent in scavenging free radicals and inhibiting pancreatic alpha-amylase than green pods. Moukette et al. [4] found that extracts from T. tetraptera could protect against oxidative-mediated ion toxicity. Saliu et al. [5] identified various bioactive phytochemicals in T. tetraptera using HPLC–DAD analysis. Aikins et al. [6] reported that fractions of T. tetraptera had antiproliferative activity. The spotlight, however, falls on aridanin, a constituent showcasing potent GABAergic and CNS depressant activity, hinting at its potential in neurological and mental health realms [7].

Transforming promising botanical resources into safe and efficacious therapeutics demands rigorous safety assessments. This study embarks on a critical mission: deciphering the acute and 14-day toxicity trial with repeated oral dose profiles of HWETTF in rodent models. Acute and 14-day toxicity trials are crucial for understanding HWETTF’s therapeutic potential. Acute trials determine short-term effects and lethal doses, while 14-day trials assess sub-acute toxicity. These trials help establish safety profiles, identify dose–response relationships, target organ toxicity, and risk–benefit ratio, ultimately guiding decisions about further development and clinical trials. By meticulously mapping its safety boundaries, we pave the way for responsible exploration of HWETTF’s neuroactive potential, unlocking the gateway to its therapeutic applications.

Ethical approval and guidelines

All animal procedures were approved by the Imo State University Owerri Animal Experimentation Committee (IMSU-AEC) of the Department of Biochemistry with ethical number: IMSU/EC/01/2022. The study adhered to the National Institute of Health Care Guide for the Care and Use of Laboratory Animals [8]. The study was carried out per ARRIVE guidelines for reporting animal experiments.

Animals

Fifty-nine healthy male Wistar rats and 12 mice were obtained from the Veterinary Research Institute, Vom, Jos, Nigeria, for their established suitability in biomedical research. To minimize stress, transportation used commercial carriers with individual housing and secondary containment. Upon arrival at Imo State University, rats were acclimated for 2weeks in standard colony conditions (12h light/dark cycle, 25–27°C, 40–60% humidity) with individual housing and free access to food and water. Wistar strains exhibit lower body weight but comparable reproductive performance compared to Sprague–Dawley rats and Swiss mice. These characteristics, coupled with their well-characterized physiology, established genetic background, and extensive use in preclinical studies, facilitate data comparison and translational potential. Wistar rats and mice have emerged as the de facto animal models in toxicological research, as evidenced by their widespread utilization and consistent representation in the peer-reviewed literature.

Plant material and extraction

Fresh T. tetraptera fruits were procured from local Imo State farmers and authenticated by a plant taxonomist; Dr. C.I.N Unamba at the Department of Plant Science and Biotechnology, Imo State University Owerri by comparing them with herbarium specimens. Voucher specimen was subsequently deposited at the Department of Plant Science and Biotechnology, Imo State University Owerri Herbarium. Dried for 72h (4% moisture) at 50°C, pulverized fruits underwent decoction (10% w/v) for 30min at 100°C. After cooling, filtration, and lyophilization, a dark brown, crude hot water extract (HWETTF) was obtained and stored at 4°C. Aliquots were dissolved in distilled water for subsequent assays.

HPLC analysis of extract

HWETTF was analyzed using Agilent Technologies’ 1200 series HPLC with a PDA detector, employing a modified method by Kim et al. (2010). Separation occurred on a Zorbax SB-C18 column (80Å, 5μm, 4.6 × 150mm) with a linear gradient elution of water, acetic acid, and acetonitrile. Parameters were: flow rate 1.0mL/min, column temperature 30°C, detection wavelength 210nm. LC solution software recorded results.

Identification of compounds with potential GABAergic and CNS depressant activity in extract using GC–MS analysis

HWETTF underwent GC–MS analysis using a Perkin-Elmer Clarus 680 GC equipped with an Elite-5ms capillary column. Helium gas (99.99%) served as the carrier at 1mL/min. Electron ionization (70eV) with scan time 0.2s and fragments 40–600m/z enabled spectral detection. An injection volume of 1 μL (split ratio 10:1) was used with a maintained injector temperature of 250°C. The column temperature was held at 50°C for 30min, then increased at 10°C/min to 280°C, followed by a final increase to 300°C for 10min. Bioactive compound identification relied on comparing retention times, peak values, peak heights, and mass spectral patterns with the National Institute of Standards and Technology Mass Spectral Database (NIST) and the WILEY library of GC–MS [9].

FT-IR analysis

A Cary 630 spectrometer (Agilent) investigated the HWETTF (centrifuged 3000rpm, 10min; filtered, vacuum-assisted) diluted 1:10. Spectra were acquired in the 4000–650nm range, with peak identification and reporting following established protocols.

One-Time oral dose toxicity investigation

An acute oral toxicity study of the hot water extract of T. tetraptera (Aidan Fruit) was performed using mice according to the Organisation for Economic Cooperation and Development (OECD) guideline 423 [10]. Nine male mice were randomly divided into 3 groups of 3 mice each. The Hot water extract and distilled water were orally administered to the mice after overnight fasting at a volume of 10ml/kg body weight [11]. The mice in group I were administered 300mg/kg body weight of the HWETTF dissolved in distilled water. The mice were observed for general behaviour changes; symptoms of toxicity and mortality after treatment for the first 4h, then over 48h. Group 2 was administered sequentially at a 48-h interval with the next higher dose of 2000mg/kg body weight of the HWETTF in distilled water when there were no signs of toxicity or mortality showed in group 1 after 48h of treatment. In parallel, group 3 mice were treated with vehicle (distilled water) to establish a comparative negative control group. All animals were observed at least once during the first 30min in the first 4h following vehicle or HWETTF administration and then once a day for 14days. This observation was done to check the onset of clinical or toxicological symptoms according to the OECD guidelines [10]. All observations include changes in skin and fur, eyes and mucous membranes and behavioural patterns were systematically recorded and maintained with an individual record. In addition, consideration was given to observations of convulsions, tremors, diarrhoea, salivation, lethargy, sleep, coma and mortality. At the end of the study, mice in each group were sacrificed by decapitation with a rodent guillotine (Harvard Apparatus, USA) and their heart, liver and kidney were quickly dissected out and rinsed in ice-cold saline (0.9% NaCl). Samples from the vital organs (liver, kidney and heart) of acute oral toxicity tests were then subjected to histopathological evaluation.

14-Day toxicity trial with repeated oral doses

Rats were used in a 14-day repeat-dose toxicity investigation of the HWETTF. Although 28-day research is advised by OECD guideline 407 [12], the purpose of this low-resource exploratory experiment was to collect preliminary data on the effects of the extract and help determine whether to proceed with a full 28-day investigation. Shorter study duration, such as the exploratory experiment described, can have limitations in assessing long-term toxicity. These limitations include an incomplete assessment of toxic effects, underestimation of risk, limited data for risk assessment, difficulty in extrapolating to humans, and the need for further evaluation. To ensure the safety of substances, it is generally recommended to conduct comprehensive studies that evaluate potential toxic effects over a longer duration. For a 14-day repeat-dose toxicity study, healthy male Wistar rats were randomly assigned to four groups (5/sex/group). Vehicle (distilled water) or graded doses of the hot water extract (500, 1000 and 2000mg/kg of body weight) were administered to rats by oral gavage once daily for 14days at a dose of 10ml/kg of body weight. The food composition and water intake are recorded daily. The body weights of animals were recorded shortly before the administration of the tested substance and at the end of each week. The percentage of body weight change is calculated according to the equation [11]:

On the 15th day, about 5ml of blood was collected from the retro-orbital plexus without the use of topical anaesthesia after overnight fasting and sera were prepared from 4ml of the collected blood by centrifugation at 640g for 10min and then stored at 4°C for different biochemical parameters (Urea, Creatinine, Total protein, Albumin, Globulin, Total bilirubin, Conjugated bilirubin, Alkaline phosphatase, Alanine transaminase and Aspartate aminotransferase). The remaining uncoagulated blood from the 5ml collected was used for analysing haematological parameters such as haemoglobin, white blood cells, Neutrophils, lymphocytes, monocytes, Eosinophils and basophils. After the collection of blood samples from rats, rats in each group were killed by asphyxiation with CO₂ followed by decapitation with a rodent guillotine (Harvard Apparatus, USA) and their Heart, Liver and Kidneys were quickly dissected out and rinsed in ice-cold saline (0.9% NaCl). The Heart, Liver and Kidney were harvested, cleaned of fat blotted with clean tissue paper, and weighed immediately. The relative organ ‘s weight (ROW) was calculated and recorded in proportion to the body weight according to the equation [11]:

Samples from the vital organs (liver, kidney and heart) of acute oral toxicity tests were subjected to histopathological evaluation. They were fixed in 10% buffered formalin, routinely processed and embedded in paraffin wax. Paraffin Sects.(5µm) were cut on glass slides and stained with haematoxylin and eosin. An experienced pathologist who was unaware of the experimental groups to which each section belonged conducted the analysis. The slides were examined under a light microscope as earlier stated by Ekeanyanwu and Njoku [11].

Statistical analysis

Biochemical data were presented as mean ± SEM and analyzed by one-way ANOVA (SPSS v.20) followed by Bonferroni’s post hoc test. Differences with p < 0.05 were considered statistically significant.

The HPLC analysis of HWETTF

The HPLC analysis of hot water extracts of HWETTF is presented in Fig.1. It displayed the major significant peaks at a wavelength of 210nm (Highest component concentration) at retention periods (min) of 0.2376, 1.341, 1.488, 1.575, 1.757, 1.912, 2.650, 2.879, 2.975, 3.168, 3.690, 4.067, 4.672, 5.508, 6.018 and 6.534 respectively.

FT-IR chromatogram of HWETTF

Based on peak values in the infrared region, FT-IR spectroscopy (Aye et al., 2019) is a dependable and practical method for detecting various bonds/stretches and phytochemical functional groups. The HWETTF was analysed using FT-IR in the study. The peak ratio was used to distinguish between the functional classes of biomolecular components. The FT-IR spectrum chromatogram profile of HWETTF is shown in Fig.2. The FT-IR peak values, intensity, and functional groups are shown in Table1. There are eight major peaks between 900 and 3500cm⁻¹. Each band addresses an overall overlap of specific functional group absorption peaks in the test sample. Every absorption spectrum of different components exhibited significant overlap in the spectra.

Alcohols (-OH stretching) in HWETTF showed a peak at 3268.9cm⁻¹, while alkane (C-H stretch) showed a peak at 2929.7cm⁻¹. The peak for alkynes (C–C stretching) was 2102.2cm⁻¹, while the peak for aromatic (C–C stretching) was 1595.2cm⁻¹. At the peak of 1036.2cm⁻¹, alkoxy (C-O stretch) was also observed.

GC–MS Chromatogram of HWETTF

GC–MS chromatogram of hot water extract of HWETTF showed 23 peaks which indicated the presence of 23 different bioactive/phytochemical compounds (Fig.3). The results revealed that the percentage of major bioactive compounds viz., 1, 2-propanedithiol—(1.08%), Dodecanoic acid methyl ester—(5.39%), 2-Hexyn-1-ol—(1.21%), 3-(prop-2-enoyloxy) dodecane-(2.68%), 2-propanol, 1- (2-butoxyethoxy)—(1.69%), n-Hexadecanoic acid – (0.60%), 1-hexadecanesulphonamide, N-(2-aminoethyl) – (0.42%), pentafluoropropionic acid, 10-undecenyl ester – (0.92%), methyl 2,6 – anhydro – alpha – d – altroside – (0.81%), d-glycerol – glucoheptose – (1.95%), methyl – beta – D – thiogalactoside – (14.86%), methyl – 4-O-methyl – d – arabinopyranoside (2.20%), d – glucoheptose (20.01%) – hexadecanoic acid, methyl ester (13.91%), chloro-methyl-methoxy-amine (0.19%), 12-bromododecanoic acid (0.63%), cyclononene (25.19%), cyclopentaneundecanoic acid (1.10%), oxiraneundecanoic acid, 3-pentyl-methyl ester trans (0.99%), 3-octenoic acid, methyl ester (2.26%), 8-nonynoic acid, methyl ester (2.26%) and 9-octadecynoic acid (0.52%) were found as the major compounds in the HWETTF (Table2).

Acute toxicity study of HWETTF

The acute toxicity test revealed that oral administration of a single 2000mg/kg dose of HWETTF to rats did not bring about any signs of toxicity or mortality in treated animals during the 14-day observation period. During the entire observation period, they did not present any significant clinical alteration. Furthermore, no significant difference was observed between the weight gains in these groups with the control (Table3).

14-day toxicity trial with repeated oral dose study on HWETTF

Body Weight, Dietary, and Water Intake: The 14-day toxicity trial with repeated oral dose study on HWETTF at all doses used did not produce any obvious symptoms of toxicity or, mortality in all the treated rats. Besides, no significant changes occurred in food and water consumption in rats treated acutely with repeated oral doses of the aqueous extract (500, 1000, or 2000mg/kg). Both the control and treated rats appeared healthy at the end and throughout the 14days of the study. According to data presented in Table4 after the whole experiment period, significant changes occurred in the weight of treated groups with a dose up to the maximum of 2000mg/kg compared to the control group (P < 0.05). However, the body weight gains of rats treated with 1000, and 2000mg/kg were all higher than those of the control group, being 14.83% and 14.44% respectively, as compared with the control group (8.95%).

Organ weight and relative organ weight

Organ weight and relative organ weights of 14-day treated rats are shown in Table5. No significant differences in organ weight and relative organ weight of liver, kidney and heart were observed between the vehicle control and treatment group administered 500mg/kg b.wt HWETTF. A similar result was recorded in the treatment group administered 2000mg/kg b.wt however; this was not the case for the treatment group administered 1000mg/kg b.wt as significant differences were observed between the vehicle control and treatment group administered 1000mg/kg b.wt of HWETTF.

Haematological parameters

Table 6 shows the effect of HWETTF on haematological parameters in rats. After 14days of treatment, there was no significant (P > 0.05) effect on total white blood cells in the treated group compared to the control group. There was no significant (P > 0.05) effect on the lymphocyte count in the treated group administered 2000mg/kg b.wt HWETTF compared to the control group. However, significant differences (P < 0.05) were observed in the Monocytes and Granulocytes count in the treated group administered 2000mg/kg b.wt the HWETTF compared to the control group administered distilled water only. Red blood cells, haemoglobin, erythrocyte sedimentation rats, and packed cell volume were not significantly different (P > 0.05) in the test groups compared to the control group. There was also no significant (P > 0.05) effect on mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration in the treated groups compared with the control group.

Biochemical parameters

Table 7 shows the results of serum biochemical parameters of wistar rats orally administered with HWETTF in a 14-day repeat dose toxicity study. After 14days of oral administration, a significant decrease (P < 0.05) in the values of serum urea, serum creatinine, and serum globulin in the 1000mg/kg b.wt treated group compared to the control group administered distilled water only. Similarly, a significant decrease in the serum protein level was observed in the group administered 2000mg/kg b.wt compared to the control group administered distilled water. However, a significant increase (P < 0.05) in the serum alkaline phosphatase and alanine transaminase activities compared to the control group was observed.

Histopathological changes

Histopathological examinations were performed on the liver, kidney and heart to assess possible organ damage (Fig.4). The normal rat liver has a microscopic architecture structured in hexagonal lobules and acini. The hexagonal lobules are centred on the central vein (CV), with surrounding hepatocyte cords and have a portal triad containing branches of the hepatic artery (HA), the bile duct (BD), and the portal vein (PV). The photomicrograph of the liver section of the normal rats shows a normal central vein, lamella of hepatocytes and sinusoids. Other stromal elements appear normal. Adverse effects were not found in the liver of rats administered 500mg/kg b.wt. of HWETTF orally. However, the histological section of the liver of rats administered 1000mg/kg b.wt. of HWETTF shows hepatocytes with hyperchromatic nuclei and increased nucleocytoplasmic ratio. Also, there is stromal proliferation with focal areas of haemorrhage. The central vein is slightly enlarged. Similarly, a histological section of the liver of rats administered 2000mg/kg b.wt. HWETTF show stromal proliferation with haemorrhage. There are few cystic degenerative changes and oedema. The hepatocytes are equally undergoing balloting degeneration.

The kidneys of normal rats showed normal glomeruli, Bowman’s capsule, tuft, tubules and stromal cells. Adverse effects were not found in the kidneys of rats administered 500mg/kg and 1000mg/kg b.wt. HWETTF, however, histological section of the kidney of rats show areas of haemorrhage, congested tubules and normal glomeruli with a slight increase in Bowman’s capsule space.

The histological section of the heart tissues of normal rats shows unremarkable cardiac muscle cells within an intact tissue stroma. Adverse effect was not found in rats administered 500mg/kg b.wt, 1000mg/kg b.wt and 2000mg/kg b.wt HWETTF.

Herbal medicines are subject to significant quality variations influenced by factors such as harvest season, plant origins, drying processes, and potential contamination. Rigorous phytochemical analysis is imperative to ensure the reliability, reproducibility, and safety of these products [13]. The phytochemical composition of herbal products is a critical determinant of their efficacy, safety, consistency, and therapeutic properties. Variations in phytochemical content can be attributed to plant species, growth conditions, harvesting practices, and processing methods. To mitigate these challenges, manufacturers often employ techniques like standardization, authentication, purity testing, and potency assays. The quality of the HWETTF (hot water extract of Tetrapleura tetraptera fruit), as evidenced by its phytochemical composition and associated properties, is crucial for its potential therapeutic applications. By employing a multi-faceted analytical approach, this study aimed to elucidate the phytochemical profile of HWETTF and its implications for therapeutic development.

Phytochemicals derived from diverse plant sources have demonstrated promising anxiolytic properties through their interactions with the central nervous system (CNS). Phootha et al. [14] comprehensively reviewed these compounds, highlighting their potential to modulate various neurotransmitter systems, including GABAergic, dopaminergic, serotonergic, and noradrenergic pathways. This multifaceted engagement suggests a complex interplay between phytochemicals and the neural mechanisms underlying anxiety. Moreover, the study identified a broad range of chemical classes, such as flavonoids, alkaloids, saponins, and polyphenols, contributing to the diverse pharmacological profiles of these natural compounds. HPLC analysis enabled the identification and quantification of individual components within our extract, paving the way for standardization and consistent biological activity in future pharmaceutical preparations [15]. FT-IR spectroscopy revealed a diverse array of functional groups, including aliphatic, aromatic, hydroxyl, and potentially carbonyl-containing moieties. This spectrum of functionalities suggests a molecule with potential for versatile chemical reactivity and a broad range of applications. Further structural elucidation using NMR spectroscopy would be necessary to confirm the precise arrangement of these functional groups and fully elucidate the structure of the active metabolites in HWETTF. GC–MS analysis identified key phytochemical constituents, including 1-hexadecane sulfonamide, n-(2-aminoethyl), d-glycerol-glucoheptose, methyl-β-d-thiogalactoside, d-glucoheptose, and n-hexadecanoic acid. Notably, these compounds have been reported to possess GABAergic and/or CNS depressant effects in Dr Duke‘s phytochemical and ethnobotanical information database [16], aligning with the study’s focus on potential neuroactive properties.

Herbal medicines have acquired greater importance as a substitute for conventional therapy due to their potential efficacy and minimal side effects [17]. As the use of medicinal plants increases, screening plant products to assess their toxic characteristics is considered an essential initial step [17]. During the evaluation of the toxic characteristics of medicinal plants, an initial assessment of toxic manifestations is one of the screening experiments performed with all compounds. In addition, data from the acute toxicity study may serve as the basis for the classification and labelling of the test material [18]. Thus, part of the current study was to evaluate the acute and 14-day toxicity trial with repeated oral dose studies on HWETTF in an animal model. The oral route administration is the most prevalent and appropriate one while conducting toxicity studies due to its similarity to human consumption [19]. As the crude extracts are administered orally, the animals need to fast before administration to minimize the influence of food and other chemicals within the digestive system on the tested materials’ reactions [19]. All procedures were performed based on the appropriate OECD guidelines [19].

While acute toxicity information provides valuable insights, it may not fully capture the potential cumulative effects at lower doses. Consequently, 14-day toxicity trials with repeated oral dose studies involving repeated exposure are crucial for evaluating the safety profile of phytomedicines like HWETTF [20]. This study employed a 14-day toxicity trial with repeated oral dose tests, acknowledging that body weight changes serve as key indicators of adverse side effects [20]. A loss exceeding 20% of body weight is considered critical and defines one of the humane endpoints in international guidelines [21, 22].

The initial dose of 300mg/kg body weight is commonly used in situations where limited information suggests potential toxicity [10]. In this study, both control and HWETTF-treated rats did not exhibit any mortality at this dose. Therefore, the next higher dose, 2000mg/kg, was chosen as stipulated by the OECD Guidelines 423 [10]. Throughout the 14-day observation period, rats receiving HWETTF at both doses displayed no noticeable signs of distress, toxicity, or death. Additionally, no significant changes in wellness parameters were observed. Physical appearance features like skin, fur, eyes, mucous membranes, salivation, and behavioural patterns remained normal in both control and treated groups (300mg/kg and 2000mg/kg). Notably, the absence of lethargy, tremors, diarrhoea, and coma further reinforced the lack of adverse effects. Although all rats displayed weight gain, there were no statistically significant differences in body weight gain between the groups during the weekly single-dose oral test.

A 14-day toxicity trial with repeated oral dose study was conducted to evaluate the adverse effects of testing medicinal plant HWETTF and was carried out to provide information about the possible health threats that are probable to arise from the 14-day toxicity trial with repeated oral dose exposure over some time, the possibilities of cumulative effects, and an estimate of the dose at which there is no observed adverse effect. Evaluation of the safety margin between different dose levels that produce the therapeutic effect and that which produce the adverse effects is necessary. Evaluation of safety is exactly to provide benefit to risk assessment. The animal experiment model is the only method that can assess this matter [23].

Comprehensive safety evaluations are crucial for herbal medicine development, particularly through detailed analysis of haematological and biochemical parameters [24]. Blood components serve as primary transporters for drugs and xenobiotics, exposing them to potentially significant concentrations of compounds that could induce toxicity [25]. Even subtle changes in haematological parameters, even within normal limits, can be significant indicators of safety concerns. By carefully monitoring these parameters, healthcare providers can proactively identify and mitigate potential risks associated with drug or chemical exposure, especially for herbal medicines, whose safety profiles may not be as well-established as those of synthetic drugs. This study investigated the 14-day toxicity trial with repeated oral dose study on HWETTF, a promising phytomedicine, in Wistar rats, focusing on haematological and biochemical parameters alongside gross and histopathological analyses.

The 14-day toxicity trial with repeated oral dose study revealed no mortality or observed clinical signs of distress in any treatment groups receiving HWETTF at doses up to 2000mg/kg body weight. Importantly, haematological parameters remained largely unchanged between control and treated groups, except monocytes and granulocytes exhibiting statistically significant differences (P < 0.05). However, these changes remained within the established normal range for Wistar rats [24, 26], potentially attributable to inter-individual variations rather than indicating HWETTF-induced toxicity.

Evaluation of kidney and liver function is equally vital for assessing phytomedicine safety due to their critical roles in organismal survival [27]. Serum levels of urea and creatinine, key indicators of renal function, showed no significant differences between the control and HWETTF groups (P > 0.05). Similarly, analysis of liver function markers, including total protein, albumin, globulin, bilirubin, ALP, AST, and ALT, revealed no statistically significant variations between groups (P > 0.05). These findings suggest that HWETTF did not adversely affect either kidney or liver function at the tested doses.

Histopathological evaluations of major organs further corroborated the lack of toxicity observed in haematological and biochemical analyses. To assess the effects of HWETTF on various organs, we conducted a comprehensive histopathological evaluation. Tissues were fixed, embedded, sectioned, and stained with H&E. The stained sections were examined under a light microscope to identify and characterize any lesions or abnormalities. While H&E staining provided valuable information, immunohistochemistry and morphometric analysis could have been incorporated to enhance the study’s depth and provide more quantitative data. Overall, the histopathological evaluation in this study provided valuable insights into the substance ‘s impact on the target organs. No abnormalities were detected in the liver, kidney, or heart of animals treated with any HWETTF dose, further supporting the safety profile of this phytomedicine.

Previous studies have investigated the safety and potential cytotoxic effects of T. tetraptera. Bonsou et al. [28] found that the fruit and its most active compounds exhibited cytotoxic properties in vitro, while an acute oral dose of 5000mg/kg was well-tolerated in vivo. Dongmo et al. [29] reported that the aqueous extract from the stem bark was safe at therapeutic doses (200mg/kg) but induced liver toxicity at higher doses (400mg/kg) administered for extended periods. Imade et al. [30] further examined the safety and antileiomyoma effects of the ethanol extract from the fruit and concluded that while acute administration was well-tolerated, long-term, high-dose consumption may pose risks to organs such as the heart and uterus.

This study provides valuable insights into the safety profile and phytochemical composition of T. tetraptera. The absence of significant toxicity in both acute and subchronic trials underscores its potential for further exploration as a medicinal resource. The identified phytochemical constituents, including those with potential neuroactive properties, warrant further investigation to elucidate their specific mechanisms of action and therapeutic applications. To fully realize the potential of T. tetraptera, the following recommendations are suggested:

1. 1.

   In-depth pharmacological studies: Conduct more comprehensive studies to evaluate the neuroactive properties of T. tetraptera, focusing on its potential effects on neurotransmitters, such as GABA, and their implications for neurological disorders.

2. 2.

   Isolation and characterization of bioactive compounds: Isolate and characterize the specific compounds responsible for the observed biological activities to facilitate the development of standardized herbal preparations.

3. 3.

   Preclinical and clinical trials: Conduct preclinical studies in animal models to assess the efficacy of T. tetraptera extracts or isolated compounds in treating neurological disorders. Subsequently, well-designed clinical trials can be initiated to evaluate its safety and efficacy in human subjects.

4. 4.

   Traditional knowledge preservation: Document and preserve traditional knowledge associated with T. tetraptera to ensure the sustainable use of this valuable medicinal plant.

5. 5.

   Drug development: Explore the potential for developing novel drug candidates based on the bioactive compounds identified in Tetrapleura tetraptera, considering their therapeutic potential and safety profiles.

ANOVA:

Analysis of Variance

BAS:

Basophils

BD:

Bipolar Disorder

CNS:

Central Nervous System

EOS:

Eosinophils

FT-IR:

Fourier Transform Infrared Spectroscopy

GC–MS:

Gas Chromatography-Mass Spectrometry

HB:

Haemoglobin

HCT:

Haematocrit

HPLC:

High-Performance Liquid Chromatography

HWETTF:

Hot water extracts of T. tetraptera fruit

LYM:

Lymphocytes

MCH:

Mean Corpuscular Haemoglobin

MCHC:

Mean Corpuscular Haemoglobin Concentration

MCV:

Mean Corpuscular Volume

MONO:

Monocytes

MPV:

Mean Platelet Volume

NEUT:

Neutrophils

OECD:

Organisation for Economic Cooperation and Development

PCT:

Procalcitonin

PDW:

Platelet Distribution Width

P-LCR:

Platelet Large Cell ratio

PLT:

Platelets

RBC:

Red Blood Cell

RDW-CV:

Red Cell Distribution Width–Coefficient of Variation

RDW-SD:

Red Cell Distribution Width–Standard Deviation

ROW:

Relative Organ Weight

SPSS:

Statistical Package for Social Sciences

TWBC:

Total White Blood Cells

WHO:

World Health Organisation

Authors and Affiliations

1. Department of Biochemistry, Imo State University Owerri, Owerri, Imo State, Nigeria

   Chukwuma Raphael Ekeanyanwu&Chidinma Lynda Ekeanyanwu

2. Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria

   Chinelo Chinenye Nkwocha

Contributions

R.C. Ekeanyanwu: Writing-original draft, Conceptualization, Resources. C.C. Nkwocha: Supervision, Methodology, Visualization, Project Administration, C.L. Ekeanyanwu: Formal Analysis, and Validation.

Corresponding author

Correspondence to Chukwuma Raphael Ekeanyanwu.

Ethics approval and consent to participate

All animal procedures received ethical approval from the Department of Biochemistry’s Animal Experimentation Committee (IMSU/EC/01/2022). The study was carried out per ARRIVE guidelines for reporting animal experiments.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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