Kolaviron offers significant neuroprotection on potassium dichromate-induced neurobehavioral, biochemical, histopathological, and immunohistochemical changes in rats

Abstract

Potassium dichromate (K₂Cr₂O₇) exposure has been linked to neurotoxicity in various animal models, raising concerns about its implications for human health, particularly neurodegenerative diseases. Kolaviron, a biflavonoid complex from Garcinia kola seeds, has demonstrated antioxidant and anti-inflammatory properties, but its neuroprotective effects against potassium dichromate-induced neurotoxicity remain underexplored. This study investigated the neuroprotective potential of Kolaviron in Wistar rats exposed to potassium dichromate toxicity. Thirty-two adults male Wistar rats were divided into four groups: control, potassium dichromate (2 mg/kg), Kolaviron (100 mg/kg) plus potassium dichromate (2 mg/kg), and Kolaviron (100 mg/kg) alone. Neurobehavioral assessments, oxidative stress biomarkers, antioxidant parameters, histopathological, and immunohistochemical staining of Glial Fibrillary Acidic Protein (GFAP) analyses were conducted. Results indicated that potassium dichromate induced significant neurobehavioral deficits, heightened oxidative stress, neuroinflammation, astrocytosis, and histopathological alterations. However, Kolaviron treatment attenuated oxidative stress, improved cognitive function, and abrogated neuroinflammation, demonstrating its potential as a neuroprotective agent. These findings suggest that Kolaviron could be a promising therapeutic agent for mitigating neurotoxicity and cognitive impairments associated with neurodegenerative diseases.

1. Introduction

Neuroprotection encompasses various approaches to safeguard neurons from damage, potentially leading to rescue, recovery, or nervous system regeneration (Hasler and Inta, 2024). The significance of neuroprotection is underscored by the increasing prevalence of neurodegenerative diseases and acute neurological injuries, which contribute significantly to global morbidity and mortality (Cantarero and Del Río, 2025). Neurodegenerative diseases (NDs), such as Alzheimer's and Parkinson's, are characterized by progressive neuronal loss and are associated with oxidative stress and neuroinflammation (Magalingam et al., 2018). Environmental contaminants, including potassium dichromate induce oxidative stress and neuronal damage (Dashti et al., 2016).

Potassium dichromate (K₂Cr₂O₇), a potassium salt of dichromic acid, is a compound known for its various roles, including being an oxidizing agent, allergen, and sensitizer (Pastorino et al., 2021, Kądziołka et al., 2022). Potassium dichromate contains chromium in the hexavalent state - Cr (VI) and as with all hexavalent compounds, it is acutely and chronically harmful to health (DesMarias and Costa, 2019). It is widely used in numerous industrial processes and, as a result, is a contaminant of many environmental systems (Kasumagi-Halilovic and Ovaina-Kurtovic, 2018). The sources of exposure to potassium dichromate are diverse, ranging from industrial processes to everyday products (Azeez and Braimah, 2020). Widely used in industries such as leather, chrome-plating, and dye production, potassium dichromate, along with other Cr (VI) salts, is recognized for its versatile applications but also for its associated health and environmental risks (Gregoviou et al., 2020).

Potassium dichromate poses significant neurotoxic risks to both humans and animals, with its neurotoxic effects primarily attributed to the generation of reactive oxygen species (ROS), which can lead to oxidative stress and subsequent neuronal damage (Dashti et al., 2016). Also, it induces neurotoxicity characterized by alterations in neurotransmitter levels and neuronal apoptosis, which can disrupt normal neuronal function and lead to cognitive deficits (Saleh et al., 2022). Potassium dichromate is widely used in industrial processes and is known to cause severe neurotoxic effects, including cognitive deficits and motor dysfunction (Aboul-Fotouh et al., 2018). Additionally, potassium dichromate is classified as a potential human carcinogen, with studies suggesting that it may contribute to the development of neurodegenerative diseases due to its ability to induce oxidative stress and inflammation (Casalegno et al., 2015). The neurotoxic effects observed in animal models and human studies underscore the need for neuroprotective agents to treat potassium dichromate-induced neural defects (Duarte et al., 2023).

Oxidative stress induced by potassium dichromate results in lipid peroxidation and DNA damage, which are critical events in the pathogenesis of neurodegeneration (Dashti et al., 2016). Exposure to potassium dichromate has been shown to result in elevated levels of oxidative stress markers, such as malondialdehyde (MDA), which is a byproduct of lipid peroxidation (Luo et al., 2016, Dashti et al., 2016). In addition to oxidative stress, potassium dichromate has been shown to activate inflammatory pathways that further exacerbate neuronal injury. Exposure to potassium dichromate can trigger the activation of nuclear factor kappa B (NF-kB) and mitogen-activated protein kinases (MAPKs), which are key mediators of inflammatory responses (Xiao et al., 2013). Potassium dichromate exposure has been associated with increased levels of tumor necrosis factor-alpha (TNF-α) and interleukin−6 (IL−6), which are known to mediate neuroinflammation (García-Niño et al., 2015, Dashti et al., 2016).

However, several natural compounds have shown promising neuroprotective effects against oxidative stress-induced neurotoxicity, including that caused by potassium dichromate intoxication. Compounds, such as flavonoids, alkaloids, and polyphenols, can modulate ROS production and mitochondrial function (Chen and Zhang, 2022). Antioxidants from natural extracts (e.g., Acai berry, Ginseng) and synthetic compounds (e.g., N-acetyl-L-cysteine, tocopherol) have demonstrated effective antioxidant properties in lipid-rich environments, suggesting their potential as neuroprotective agents (Vrbovská and Babincová, 2016). Antioxidants can neutralize free radicals’ generation and reduce induction of oxidative stress, which is a key factor in neurodegeneration (Kanwugu et al., 2022). Various antioxidants, including polyphenols, carotenes, vitamins, and hormones like melatonin, have been investigated as adjunctive therapies for neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases (Moren et al., 2022). Kolaviron, a biflavonoid complex extracted from Garcinia kola seeds, has numerous biological and pharmacologic activities including antioxidant and anti-inflammatory properties in experimental models (Farombi et al., 2013). Kolaviron supplementation has been documented to exert neuroprotective effects by protecting against cognitive deficits and neuronal perturbations, particularly in prefrontal cortex and hippocampal regions (Omotoso et al., 2019). Studies indicate that Kolaviron can inhibit neuroinflammation in microglial cells through the Nrf2/ARE (Nuclear factor erythroid 2-related factor 2/Antioxidant Response Element) antioxidant protective mechanism, which is crucial for mitigating oxidative stress in the brain (Onasanwo et al., 2016). Additional research finding showed that Kolaviron possesses anti-inflammatory properties and can mitigate oxidative stress and lipid peroxidation, which are critical factors in various pathological conditions (Erukainure et al., 2021). Despite its potential, the efficacy of Kolaviron in mitigating potassium dichromate-induced neurotoxicity has not been thoroughly investigated. This study aims to explore the neuroprotective effects of Kolaviron against potassium dichromate-induced neurotoxicity in rats, focusing on neurobehavioral, biochemical, and histopathological outcomes in the brain of exposed male Wistar rats. In a clinical setting, supplementation and adequate intake of Kolaviron, a major bioflavonoid extract from Garcinia kola seeds, holds promise in supporting cognition, memory, and learning due to its neuroprotective and neuro-enhancing properties. The structures of both Kolaviron and potassium dichromate are shown in Figs. 1a and 1b, respectively. While preclinical evidence is promising, more clinical trials are needed to confirm its efficacy and optimal usage in humans.

Fig. 1.

a: Structure of Kolaviron. 1b: Structure of Potassium dichromate.

2. Materials and methods

2.1. Experimental animals

This research was conducted at the experimental laboratory of the Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, University of Ibadan, Oyo State, Nigeria. Thirty-two male Wistar rats (150–180 g) of 6 weeks of age were used for the study. The rats were sourced from a purebred rat colony in the Experimental Animal House of the Faculty of Veterinary Medicine, University of Ibadan. They were housed in spacious cages (8 rats/per cage) to minimize stress and enhance normal species-specific behavior. The cages were kept in a well-ventilated room under natural lighting conditions of 12 h light and 12 h dark daily throughout the experimental period. They were fed commercially formulated broiler finisher feed produced by Top Feeds®. Feed and clean water were provided ad libitum. Dry wood shavings were used as litter material, and this was regularly changed as required upon visual inspection. The rats were acclimatized for 14 days and maintained under standard laboratory conditions with free access to food and water. Ethical approval for the study and consent for the use of animals was obtained from the University of Ibadan, Animal Care and Use Research Committee (UI-ACUREC) with approval number NHREC/UIACUREC/05/12/2022 A.

2.2. Extraction of Garcinia kola and isolation of Kolaviron (Kv)

Kolaviron was extracted from the seeds of Garcinia kola according to the method of Iwu et al. [1987] with slight modification. The seeds were sliced, air-dried and powdered. The powdered seeds were defatted by extraction using n-hexane in a Soxhlet extractor apparatus for 24 h. The defatted dried marc was repacked and extracted with methanol. Kolaviron was fractionated from concentrated methanol extract using chloroform to give a golden tallow solid, which consists of Garcinia biflavanones-GB1, GB2 and kolaflavanone (Marouani et al., 2017).

2.3. Experimental design

A completely randomized design was used for the study. After fourteen (14) days of acclimatization. They were then randomly assigned to four (4) treatment groups of eight (8) rats each. All treatments were administered for 14 consecutive days. The various treatment groups were as follows

The rats were randomly divided into four groups (n = 8):

• •Group A: Control (0.2 mL distilled water, orally by gavage)
• •Group B: Potassium dichromate (2 mg/kg, intraperitoneal)
• •Group C: Potassium dichromate (2 mg/kg) + kolaviron (100 mg/kg, orally by gavage)
• •Group D: Kolaviron (100 mg/kg, orally by gavage)

The dosage of potassium dichromate was based on the work of Marouani et al., (2017) while that of kolaviron was selected from the findings of Adedara et al. (2020). Neurobehavioural evaluations (novel object recognition and hanging wire tests) were performed on day 12 – 14 of the experiment.

2.4. Neurobehavioral assessments

The following tests were conducted to assess neurobehavioural function: the novel object recognition test and the hanging wire test.

2.4.1. Novel object recognition (NOR) test

This test is used to evaluate cognition in rats as it relates to different aspects of learning and memory (Lueptow, 2017). Trial tests (training) were done on days 12 and 13, and the final test on day 14.

A general description for the test is as follows: in an open field box, the rats are first familiarized with two identical objects (habituation phase on days 12 &13). Twenty-four hours later (test phase – day 14), one of the objects is replaced with a new object, and the rats are allowed to explore both objects for 5 min and the time spent exploring each object was recorded. The recognition index (%) was calculated by dividing the time spent exploring the novel object by the total exploration time (novel + familiar) as previously described by Oyagbemi et al., (2025).

$$\mathbf{R}\mathbf{I}=\frac{{\mathbf{T}}_{\mathbf{n}\mathbf{o}\mathbf{v}\mathbf{e}\mathbf{l}\mathbf{X}}\left(\mathbf{100}\%\right)}{{\mathbf{T}}_{\mathbf{n}\mathbf{o}\mathbf{v}\mathbf{e}\mathbf{l}+}{\mathbf{T}}_{\mathbf{f}\mathbf{a}\mathbf{m}\mathbf{i}\mathbf{l}\mathbf{i}\mathbf{a}\mathbf{r}}}$$

Tnovel = time spent exploring the novel object

Tfamiliar = time spent exploring the familiar object

2.4.2. Hanging wire test

The hanging wire test, as described (Onukak et al., 2025) is used to evaluate muscle strength and motor coordination in rats. Each rat was hung with its fore paw on a wire support, at a height of about 60 cm, and the time until it drops to the floor is recorded (Hoffman and Winder, 2016). Each rat was allowed three trials, and a rest period of 5 min between consecutive attempts. The average duration for each rat was then recorded. The hanging wire test was performed on experimental day 14 immediately after the NOR test.

2.5. Animal sacrifice

All rats were sacrificed on day 15 of the experiment. Rats were sacrificed by quick cervical dislocation, and brains were harvested for biochemical, histopathology, and immunohistochemistry. Brain tissues were harvested and fixed in 10% neutral buffered formalin, and thereafter subjected to histological procedures for histopathological and immunohistochemical analysis (Igado et al., 2020).

2.6. Biochemical assays

2.6.1. Tissue homogenization

The brain samples were homogenized in buffer (0.1 M Phosphate Buffer, pH 7.4) at a dilution ratio of 1:8 using a Teflon® homogenizer. The resulting homogenate was then centrifuged using a cold centrifuge at 10,000 rpm for 10 min at 4 °C. The resulting supernatant was collected into plain sample bottles and stored at −20 °C until use.

2.6.2. Determination of biomarkers of oxidative stress and antioxidant status

Lipid peroxidation was evaluated by measuring the formation of Thiobarbituric Acid Reactive Substances (TBARS) according to previously described methods (Varshney and Kale, 1990). Briefly, 0.2 mL of sample was added to a solution containing 0.8 mL Tris-potassium chloride and 0.25 mL trichloroacetic acid, before the addition of 0.25 mL of thiobarbituric acid. Then, the reaction mixture was placed in a boiling water bath for 30 min. Thereafter, the mixture was allowed to cool on ice, centrifuged for 15 mins at 4000 r/min, and read spectrophotometrically at 532 nm wavelength.

Hydrogen Peroxide concentration was determined as previously described (Wolff, 1994). The assay for hydrogen peroxide (H₂O₂) was carried out by adding 10 µL of sample to a reaction mixture of 0.1 M potassium phosphate buffer (pH 7.4), 50 μL ammonium ferrous sulfate, 20 µL sorbitol, 20 μL of xylenol orange and 10 μL sulfuric acid, and subsequently vortexed. Thereafter, the reaction mixture was incubated at room temperature for 30 mins and read the absorbance at 560 nm.

Reduced glutathione content was measured (Beutler, 1969). The reduced glutathione in the sample was assayed based on the principle that the thiol moiety in glutathione exhibits a reduction reaction with 5,5′-dithiobis−2-nitrobenzoic acid (DTNB) to form 5-thio−2-nitrobenzoic acid which is yellow in color and can be detected spectrophotometrically at 412 nm wavelength. Add 0.1 mL of sulfosalicylic acid into all the test tubes followed by the addition of 0.1 mL of sample and centrifuge the mixture at 4000 rpm for 5 mins. 20 µL of the supernatant was pipetted into micro-titer plates and 180 µL of Ellman’s reagent was added and subsequently incubated for 30 min at room temperature. The absorbance was read at 412 nm. Final results were extrapolated from GSH standard curve.

The glutathione peroxidase activity was determined as previously described (Rotruck et al., 1973). The reaction mixture containing 0.5 mL of phosphate buffer, 0.1 mL sodium nitrate (NaNO₃), 0.2 mL glutathione (GSH), 0.1 mL hydrogen peroxide (H₂O₂ min. min. The absorbance was read at 412 nm wavelength against blank. One unit of GPx activity is defined as the amount of enzyme required to utilize 1 nmole of NADPH/minute at 25^OC.

The activity of superoxide dismutase (SOD) was determined as previously described (Misra and Fridovich, 1977) with slight modification (Oyagbemi et al., 2015). Briefly, 800 µL of Tris-KCl buffer was added into a test tube, then 250 µL of trichloroacetic acid was added followed by an addition of 250 µL of thiobarbituric acid, then 20 µL of sample was added. The mixture formed was incubated in a water bath at 80˚C for 45 min, cooled on ice and centrifuged at 4000 rpm for 5 min. Subsequently, the supernatant was decanted and placed in micropipette plate reader and the absorbance was measured against a reference blank of distilled water at 490 nm wavelength. One unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of adrenaline to adrenochrome during 1 min.

Glutathione S-transferase was determined as previously described (Habig et al., 1974). the activity of glutathione S-transferase was quantified as described by the ability of the enzyme to catalyze the conjugation of GSH with 1-chloro−2, 4-dinitrobenzene (CDNB) over a time period of about 3 min with absorbance read at 340 nm in 30 s intervals.

The concentration of nitric oxide (NO) was determined using Griess reagent (Vodovotz, 1996). The level of serum nitric oxide (NO) was evaluated as in a reaction involving the addition of Griess reagent (100 mL), sample (300 mL), and deionized water 2.6 mL in a cuvette and allowing the reaction to proceed for 30 min at room temperature. Then, the absorbance was read at 540 nm wavelength.

2.6.3. Determination of acetylcholinesterase (AChE) activity

The activity of AChE in brain samples was determined as previously described (Dingova et al., 2014). The reaction mixture contained 0.10 mL of buffered Ellman’s reagent, 5,5’-dithiobis−2-nitrobenzoic acid [DTNB (10 mmol/L), NaHCO₃ mL s interval for 3 min and read at 410 nm. The activities of AChE were expressed as mmole of substrate/min/mg protein.

2.7. Histopathology

Brain tissues were fixed in 10% neutral buffered formalin (NBF), processed, and stained with cresyl stain (Igado et al., 2020). Histopathological changes in the cerebellum, hippocampus, and cerebrum were examined under a light microscope.

2.8. Immunohistochemistry

Immunohistochemical staining was performed on neutral buffered formalin (NBF) fixed brain tissues and probed with anti-glial fibrillary acidic protein (GFAP; Cat Number: E-AB−65069) antibody as recently described [40]. Slight modification was adopted using a 2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System with DAB solution (Catalog number: E-IR-R217 from Elabscience Biotechnology®, China) was employed.

Section (5–6 µm) were prepared on charged slides from formalin-fixed, paraffin-embedded blocks, deparaffinized in xylene, rehydrated through a graded ethanol series (100%, 90%, 70%) for 1 min each, and rinsed in distilled water for 2 min. For antigen retrieval, sections were briefly boiled in 10 mM sodium citrate buffer (pH 6.0) and cooled for 20 min at room temperature. The sections were treated with endogenous peroxide (H₂O₂) for 10 min, rinsed in phosphate-buffered saline (PBS), and incubated with goat serum for 30 min to block non-specific binding. The primary antibody (GFAP) was diluted at 1:200 in PBS and incubated in a humidified chamber at room temperature for 2 h. After PBS washes, the secondary antibody detection system (2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection) was applied for 20 min. Slides were then incubated with DAB chromogen for 3 min; the reaction was terminated with deionized water, and sections were counterstained with HIGHDEF® IHC hematoxylin (Enzo Life Sciences, NY, USA) for 3 s. Sections were dehydrated in ascending ethanol concentrations (70%, 90%, 100%) for 30 s, cleared in xylene, and mounted with DPX mountant. Positive immunoreactive expression of GFAP was examined on each slide under × 100 magnification using a version 3 digital microscope (Leica microscope). The immunoreactivity was quantified with ImageJ software.

2.9. Statistical analysis

All values obtained were expressed as mean ± standard error. One-way analysis of variance (ANOVA) was used as a test of significance at p < 0.05, while Tukey posthoc test was used for pair-wise comparison. Analysis was performed using “GraphPad Prism version 9.0.” statistical software.

3. Results

3.1. Toxicity of on body weights and relative organ weights

The body weights were assessed following potassium dichromate (K₂Cr₂O₇)-induced neurotoxicity (Fig. 2a). From the results obtained, there was no significant (P > 0.05) difference in body weights across all treatment groups when compared to the control. The brain mass was assessed following potassium dichromate-induced neurotoxicity (Fig. 2b). From the results obtained, potassium dichromate-induced neurotoxicity caused a significant (P < 0.05) increase in the brain mass of the group administered with K₂Cr₂O₇ compared to the control group. The relative organ mass was assessed following potassium dichromate-induced neurotoxicity (Fig. 2c). From the results obtained, there was a significant (P < 0.05) reduction in relative organ mass in K₂Cr₂O₇-intoxicated rats compared to the control. However, a significant (P < 0.05) increase in relative brain mass was obtained in rats co-administered K₂Cr₂O₇ and KV and rats that received only KV (Fig. 2c).

Fig. 2.

a: The Effects of Kolaviron on Body Weights in Potassium dichromate-induced Neurotoxicity in Rats. No statistically significant differences across groups. Mean ± SD (n = 5). 2b: The Effects of Kolaviron on Brain mass in Potassium dichromate-induced Neurotoxicity in Rats. No statistically significant differences across groups. Mean ± SD (n = 5). 2c: The Effects of Kolaviron on Relative Brain Weights in Potassium dichromate-induced Neurotoxicity in Rats. No statistically significant differences across groups. Mean ± SD (n = 5).

3.2. Effects of Kolaviron on novel object recognition test following potassium dichromate toxicity

Results obtained from the neurobehavioural assessment showed that K₂Cr₂O₇ toxicity caused a significant (P < 0.05) decrease in the recognition index compared to the control (Fig. 3a). There was also a significant (P < 0.05) increase in the recognition index of groups administered K₂Cr₂O₇ + Kolaviron (KV) and KV only when compared to the control group. Our findings showed an increase in the recognition index of rats treated with Kolaviron was able to mitigate K₂Cr₂O₇ toxicity-induced neuroinflammation, which indicates that Kolaviron was effective in ameliorating K₂Cr₂O₇ neurotoxicity (Fig. 3a).

Fig. 3.

a: The Effects of Kolaviron on Recognition Index in Potassium dichromate-induced Neurotoxicity in Rats. P value 0.0001 ****. Mean ± SD (n = 5). 3b: The Effects of Kolaviron on Wire Hang Test in Potassium dichromate-induced Neurotoxicity in Rats. P value - *0.0211, *** 0.0068, ****0.0001. Superscript (a) indicates a significant difference when compared with control, while superscript (b) indicates a significant difference when compared with K₂Cr₂O₇. Mean ± SD (n = 5).

3.3. Potassium dichromate toxicity on hanging wire assessment and neuroprotective effects of Kolaviron

The results showed that K₂Cr₂O₇ toxicity caused a significant (P<0.05) decrease in time spent on hanging wire when compared to the control group (Fig. 3b). There was a significant (P<0.05) increase in time spent on the hanging wire by animals co-administered K₂Cr₂O₇ + Kolaviron (KV) and KV only when compared to the control group (Fig. 3b).

3.4. Effects of Kolaviron on nitric oxide (NO) content and AChE activity in potassium dichromate-induced neurotoxicity

From this study, our results showed that toxicity associated K₂Cr₂O₇ caused a significant (P < 0.05) increase in AChE activity when compared to the control (Fig. 4a). We also observed a significant (P < 0.05) reduction in AChE activity in rats co-treated with Kolaviron and those that received Kolaviron, respectively. Brain nitric oxide content is another biomarker of neuroinflammation and oxidative stress. The data from the study revealed that K₂Cr₂O₇ toxicity caused a significant (P < 0.05) increase in NO levels when compared to the control. We also observed a significant (P<0.05) reduction in NO levels in rats treated with Kolaviron and those that received Kolaviron only (Fig. 4b).

Fig. 4.

a: The Effects of Kolaviron on brain acetylcholinesterase activity in Potassium dichromate-induced Neurotoxicity in Rats. P value - *0.0011. Mean ± SD (n = 5). 4b: The Effects of Kolaviron on brain nitric oxide (NO) content in Potassium dichromate-induced Neurotoxicity in Rats. P value - *** 0.0001. Mean ± SD (n = 5).

3.5. Antioxidative action of Kolaviron on potassium dichromate-induced oxidative stress

From the results, K₂Cr₂O₇-induced neurotoxicity caused a significant (P < 0.05) increase in the contents of malondialdehyde (MDA) when compared to the control (Fig. 5a). Furthermore, a significant (P < 0.05) decrease in MDA levels were observed in the co-administration of K₂Cr₂O₇ + Kolaviron (KV) and KV-only groups when compared to the control group and the K₂Cr₂O₇-intoxicated untreated rats. Results from Fig. 4b show that K₂Cr₂O₇-induced neurotoxicity caused a significant (P < 0.05) increase in H₂O₂ generation when compared to the control. Furthermore, a significant (P < 0.05) decrease in H₂O₂ generation was observed in the co-administration of K₂Cr₂O₇ + Kolaviron (KV) and KV-only groups when compared to the control group and K₂Cr₂O₇-intoxicated untreated rats. Again, we assessed the effects of K₂Cr₂O₇-induced neurotoxicity on brain reduced glutathione (GSH) (Fig. 5c). The results showed that there was a significant (P<0.05) reduction in the GSH content of rats intoxicated with K₂Cr₂O₇ in comparison to the control. However, a significant improvement in the GSH content was recorded in rats administered K₂Cr₂O₇ + Kolaviron (KV) relative to the K₂Cr₂O₇ untreated rats. The in vivo antioxidant activity of glutathione peroxidase (GPx) was assessed as indicated in Fig. 6a. From the results obtained, there was a significant (P < 0.05) increase in GPx activity of the K₂Cr₂O₇-untreated group in comparison to the control group. It is important to note that the observed increase in GPx activity may reflect an adaptive response to K₂Cr₂O₇-induced toxicity. Also, there was a significant (P < 0.05) decrease in GPx activity of the K₂Cr₂O₇ + KV-treated group in comparison to the K₂Cr₂O₇ group. From this study, results from Fig. 6b show a significant (P<0.05) decrease in GST activity of rats exposed to K₂Cr₂O₇ only when compared to the K₂Cr₂O₇ + KV and KV-treated groups. The activity of Superoxide dismutase (SOD) was assessed as shown in Fig. 6c. From the results obtained, there was a significant (P < 0.05) decrease in SOD activity was recorded in K₂Cr₂O₇ only group in comparison to the control group. However, there was an improvement in the activity of SOD in animals co-treated with Kolaviron and Kolaviron only, respectively.

Fig. 5.

a: The Effects of Kolaviron on brain malondialdehyde (MDA) content in Potassium dichromate-induced Neurotoxicity in Rats. P value - **0.0478, *** 0.0002. Mean ± SD (n = 5). 5b: The Effects of Kolaviron on brain hydrogen peroxide (H₂O₂) generation in Potassium dichromate-induced Neurotoxicity in Rats. P value - ****0.0001. Mean ± SD (n = 5). 5c: The Effects of Kolaviron on brain reduced glutathione (GSH) content in Potassium dichromate-induced Neurotoxicity in Rats. P value - **0.0024, ****0.0001. Mean ± SD (n = 5).

Fig. 6.

a: The Effects of Kolaviron on brain glutathione peroxidase (H₂O₂) activity in Potassium dichromate-induced Neurotoxicity in Rats. P value - *0.001, **0.002. Mean ± SD (n = 5). 6b: The Effects of Kolaviron on brain glutathione S-transferase (GST) activity in Potassium dichromate-induced Neurotoxicity in Rats. P value - **0.0029, ****0.0001. Mean ± SD (n = 5). 6c: The Effects of Kolaviron on brain superoxide dismutase (SOD) generation in Potassium dichromate-induced Neurotoxicity in Rats. P value - **0.0044. Mean ± SD (n = 5).

3.6. Potassium dichromate-induced histopathological changes and the protective role of Kolaviron

Histopathology revealed loss of the Purkinje cell layer of the cerebellum, necrosis of the hippocampus, and severely shrunken neurons in the cerebrum in the K₂Cr₂O₇-intoxicated rats (Fig. 7, Fig. 8, Fig. 9). Several research findings have attributed the loss of the Purkinje cell layer to neurodegeneration, neurobehavioural deficit, motor dysfunction, cognitive impairment, and memory loss. The loss of the Purkinje cell layer in the K₂Cr₂O₇ toxicity group was rescued with the concurrent administration of Kolaviron.

Fig. 7.

Histopathology revealed mild neuronal degeneration in the white matter with granular layer degeneration (hypochromic neurons), pyknosis and necrosis of Purkinje cells with segmental disruption of the Purkinje cell layer and moderate astrocytosis (black arrows) in the cerebellum of the K2Cr2O7 intoxicated rats while rats treated with kolaviron showed intact neurons, intact uniformly stained Purkinje cell layer, and mild astrocytosis. (Cresyl stain, scale bar – 50 µm).

Fig. 8.

Photomicrograph of hippocampus showing necrosis and shrinkage of single and clusters of neurons and characterized by cytoplasmic eosinophilia and marked astrocytosis (black arrows), in the hippocampus of the K₂Cr₂O7 intoxicated rats while rats treated with kolaviron showed mild shrinkage of most neurons though neurons are normal (note the slightly angular nature of the large pyramidal neurons) (Cresyl stain, scale bar – 50 µm).

Fig. 9.

Photomicrograph of cerebrum showing severe shrunken neurons with marked cytoplasmic eosinophilia especially the large pyramidal neurons in the deeper layers with chromatolysis; marked astrocytosis on all layers of the cortex in the K2Cr2O7 intoxicated rats while rats treated with kolaviron showed intact large pyramidal neurons but with mild astrocytosis. (Cresyl stain, scale bar – 50 µm).

3.7. Effects of Kolaviron on immunolocalization of glia fibrillary acidic protein (GFAP) in potassium dichromate toxicity

Immunolocalization of glia fibrillary acidic protein (GFAP) severe astrocytosis in the cerebellum and hippocampus (CA2) region, mild astrocytosis in the cerebral cortex K₂Cr₂O₇ intoxicated rats (Fig. 9, Fig. 10, Fig. 11). Again, K₂Cr₂O₇ toxicity was associated with significant reduction in neuronal cell count, while rats co-treated with Kolaviron exhibited significant improvement in neuronal cell counts with concomitant significant reduction in astrocytosis as indicated with lower GFAP immunoreactivity (Fig. 10, Fig. 11, Fig. 12).

Fig. 10.

Immunostaining of Glia fibrillary acidic protein (GFAP) in the cerebral cortex and neuronal cell counts. Scale bar – 50 µm. Groups were compared with Group B (K₂Cr₂O₇) group, which was statistically significantly different from other groups. Arrows indicate astrocytes. **** 0.0001, *** 0.001, ** 0.005, * 0.01.

Fig. 11.

Immunostaining of Glia fibrillary acidic protein (GFAP) in the Hippocampus (CA2) and neuronal cell counts. Scale bar – 50 µm. Groups were compared with Group B (K₂Cr₂O₇). ** 0.0034.

Fig. 12.

Immunostaining of Glia fibrillary acidic protein (GFAP) in the cerebellum and neuronal cell counts. Scale bar – 50 µm. Groups were compared with Group B (K₂Cr₂O₇) group, which was statistically significantly different from other groups. **** 0.0001, * 0.023.

4. Discussion

Alzheimer's disease (AD) and Parkinson's disease (PD) are complex neurodegenerative disorders influenced by both genetic and environmental factors, which may interact and impact the epigenome (Boyd et al., 2022, Schaffner and Kobor, 2022). Recent studies have highlighted the complex relationship between environmental toxins, oxidative stress, and neurodegenerative diseases (Oluwafunmilayo et al., 2023). Potassium dichromate (K₂Cr₂O₇) is a hexavalent chromium compound widely used in industry but poses significant health and environmental risks (Al-Qarni et al., 2024). Hexavalent chromium (Cr(VI)) is a highly toxic environmental contaminant capable of inducing severe adverse health effects in mammals (Pokhrel and Pokhre, 2022). Hexavalent chromium readily crosses cell membranes, damages DNA, increases reactive oxygen species (ROS) generation, and alters signaling pathways (Younus et al., 2024). The study of Ding et al. (2024) reported that Cr(VI) crossed the blood-brain barrier, particularly in the hypothalamus, causing neurological damage through oxidative stress and inflammation.

Evidence indicates that Cr(VI) exposure is associated with deficits in learning, attention, and social memory across both human populations and experimental animal models (Wise et al., 2022). Age-related differences in Cr(VI) neurotoxicity were observed in rats, with adult rats being most affected, especially in memory-related tasks (Vielee et al., 2024). Occupational exposure to chromium and its compounds has been linked to genetic alterations, respiratory issues, cancer, and dermatitis, with hexavalent chromium being the most frequently implicated agent (Domingo-Pueyo et al., 2014). Other studies have highlighted the neurotoxic effects of hexavalent chromium [Cr(VI)] exposure. Vielee et al. (2024) documented that Cr(VI) in drinking water significantly impacted rat behaviors, particularly memory, even at levels considered safe by regulatory agencies. Exposure to Cr(VI) has been linked to various diseases, including neurological disorders, through epigenetic alterations and oxidative stress (Iyer et al., 2023). Saleh et al. (2022) demonstrated that Cr(VI) exposure in rats led to decreased neurotransmitter levels, DNA damage, and increased apoptosis in brain tissue. Another study found that Cr accumulated in the hypothalamus of mice exposed to Cr(VI) via intraperitoneal injection, caused neuropathologies and disrupted the blood-brain barrier (Ding et al., 2024). In another study, Cr(VI) induced neurotoxicity by generating ROS, leading to cellular injury, DNA damage, and apoptosis (Saleh et al., 2022).

Kolaviron, a flavonoid-rich extract from Garcinia kola seeds, demonstrates significant therapeutic potential across various health conditions exhibiting antioxidant, anti-inflammatory, and immunomodulatory properties (Farombi et al., 2022). Studies have indicated that flavonoids, including those found in Kolaviron, possess structural properties that allow them to traverse the BBB effectively (Nazari-Serenjeh et al., 2024). Kolaviron (KV), a biflavonoid extract from Garcinia kola seeds, demonstrates significant neuroprotective effects against various forms of neurotoxicity.

Previous research findings showed that KV modulated complex I activity, enhanced dopamine metabolism, and facilitated glutamate clearance in rotenone-induced neurotoxicity (Akinmoladun et al., 2018). Kolaviron also attenuates cognitive decline in okadaic acid-induced Alzheimer's-like conditions by mitigating tau hyperphosphorylation, apoptosis, neuroinflammation, and oxidative stress (Nazari-Serenjeh et al., 2024). Furthermore, KV ameliorates busulfan-induced chemo-brain and testicular damage by inhibiting oxidative stress and down-regulating inflammatory and apoptotic mediators (Tesi et al., 2022). These neuroprotective effects are consistent with the general mechanisms of flavonoids, which include reducing oxidative stress, inhibiting monoamine oxidases, stimulating neuroprotective pathways, and suppressing neuroinflammation (Bellavite, 2023). In light of these, our study investigated the neuroprotective effects of Kolaviron on K₂Cr₂O₇-induced neurotoxicity in rats. As reported, K₂Cr₂O₇has been reported to induce neurotoxicity (Ayuwardani et al., 2024).

The novel object recognition (NOR) test is a widely used behavioral assay for assessing non-spatial memory in rodents and has been applied to octopuses (Vergara-Ovalle et al., 2023). It evaluates an animal's ability to distinguish between familiar and novel objects, relying on their natural exploratory behavior (Tamijani et al., 2023). This study showed that KV effectively mitigated cognitive impairment as indicated by an increase in the recognition index. Findings from the study show that administration of KV together with K₂Cr₂O₇ caused a significant improvement in the recognition index, thereby mitigating neurobehavioural deficit and cognitive impairment. Several studies have reported the induction of neurobehavioural deficit and cognitive impairment by K₂Cr₂O₇ toxicity (Jansone et al., 2016, Ayuwardani et al., 2024). Hence, the use of KV as a dietary supplement for the management of cognitive impairment could open a novel therapeutic regimen in neuroprotection. The wire hang test is performed to assess muscle strength and motor function as an indicator of neuromuscular abnormalities in rats (Jasone et al., 2023). The findings of this study indicate that KV supplementation significantly increased the time spent on the hanging wire, suggesting its potential to alleviate Parkinsonian-like symptoms associated with K₂Cr₂O₇-induced neurotoxicity.

Data from the study revealed an exaggerated increase in nitric oxide content of rats exposed to K₂Cr₂O₇ toxicity. This is indicative of neuroinflammation and neurotoxicity. However, treatment with KV significantly reduced the content of NO. This finding attests to the aforementioned anti-inflammatory and neuroprotective action of KV (Tesi et al., 2022, Tauchen et al., 2023, Nazari-Serenjeh et al., 2024). Toxicity associated with K₂Cr₂O₇ was further assessed using an AChE activity assay as a biomarker of neurotoxicity. From the present study, AChE activity was significantly higher following K₂Cr₂O₇ toxicity. Previous studies have reported that inhibition of AChE activity is an effective strategy for managing cognitive impairment and associated behavioral deficits (Jiang et al., 2018, Chvojkova et al., 2024). From our study, the treatment of rats with KV caused significant inhibition of AChE activity. This also confirms the neuroprotective action of KV as previously reported (Ijomone and Obi, 2013). Nitrosative stress and neuroinflammation could also trigger increased AChE activity as observed in the research finding of Liu et al. (2024). Together, KV mitigated neurotoxicity, neuroinflammation, and nitrosative stress via the reduction of the contents of NO and AChE activity.

Biomarkers of oxidative stress were assessed following induction of neurotoxicity by K₂Cr₂O₇. We observed that K₂Cr₂O₇ induced- neurotoxicity precipitated oxidative stress with exaggerated production of H₂O₂. It is noteworthy that the treatment of rats exposed to K₂Cr₂O₇ toxicity together with KV ameliorated oxidative stress as indicated by a significant reduction in H₂O₂ generation. Our results also conform with the documentation on the anti-oxidative and anti-inflammatory properties of KV on locomotor impairment and coordination in Drosophila melanogaster (Farombi et al., 2018, Okoko, 2018).

The glutathione (GSH) system plays a crucial role in cellular defense against oxidative stress and detoxification of ROS across various organisms (Cassier-Chauvat et al., 2023). Lower levels of GSH were recorded in rats administered only K₂Cr₂O₇. The GSH is an intracellular antioxidant defense system (Oyagbemi et al., 2023). However, there was a significant increase in the content of GSH in rats supplemented with KV. The observable improvement in the content of GSH is an indication of the antioxidant property of KV. The depletion of GSH content has been reported as a biomarker of oxidative stress in many disease conditions, including neurodegenerative diseases (Lana et al., 2024, Mikashinovich et al., 2024). Hence, the depletion of the content of GSH caused by K₂Cr₂O₇ is indicative of oxidative stress.

Glutathione S-transferase GST plays a major role in cellular defense by decreasing the toxicity of many hydrophobic and electrophilic intermediates (Kalinina, 2024). They accomplish this by catalyzing the conjugation of GSH, which occurs with electrophilic intermediates (Kalinina, 2024). We recorded an increase in the activity of GST in rats intoxicated with K₂Cr₂O₇. The observed increase in GST activity could be attributed to oxidative stress-induced adaptive response, which has been documented elsewhere (Bae et al., 2024). However, we also observed improvement in GST activity in rats treated with KV. Furthermore, the activities of SOD were found to decrease significantly in rats administered K₂Cr₂O₇. Superoxide dismutase (SOD) is in the first line of defense against oxidative stress by dismutating superoxide anion radicals to hydrogen peroxide and oxygen. Therefore, the inhibition of SOD activity by K₂Cr₂O₇ toxicity could have aggravated oxidative stress. Final detoxification of H₂O₂ and hydroperoxides is performed by the GPx. However, there was a significant reduction in the activity of GPx in the rats administered with K₂Cr₂O₇ + KV.

Histopathology revealed the loss of the Purkinje cell layer in the cerebellum, loss of large pyramidal neurons in the hippocampus and cerebrum of K₂Cr₂O₇ intoxicated rats, accompanied by reactive gliosis (astrocytosis) in multiple brain regions. Several research findings have attributed the loss of the Purkinje cell layer to neurodegeneration, neurobehavioral deficit, motor dysfunction, cognitive impairment, and memory loss (Rademakers et al., 2021; Stroobanti et al., 2021; Todd et al., 2022). We discovered that concurrent administration of KV with K₂Cr₂O₇ rescued the cerebellum, hippocampus, and cerebrum from neuronal cell loss and reduction in gliosis. Our findings support reports that K₂Cr₂O₇ induced-neurotoxicity in tandem with oxidative stress and apoptosis (Sedik and Elgohary, 2023, Zhang et al., 2024). The observed neuronal cell depopulation could be responsible for the neurobehavioural changes, such as the reduction in muscular strength and cognitive impairment.

Qualitative and quantitative analysis of glial fibrillary acidic protein (GFAP) has been reported as a sensitive and specific biomarker of neurotoxic conditions, such as neuroinflammatory and neurodegenerative processes (Rauk et al., 2025). The immunoreactivity of GFAP has been linked with oxidative stress, neuroinflammation, and neurodegenerative conditions elsewhere (Dai et al., 2025; Wnag et al., 2025). From this study, we observed that K₂Cr₂O₇ toxicity precipitated significant astrocytosis in the hippocampal CA3 region and the cerebellum. The finding could be linked to the observed neurobehavioural changes associated with K₂Cr₂O₇ neurotoxicity. More importantly, co-administration of KV significantly attenuates astrocytosis associated with K₂Cr₂O₇ neurotoxicity, probably via its antioxidant, anti-inflammatory, and free radical scavenging activities.

5. Conclusion

The findings from this study revealed that potassium dichromate-induced neurotoxicity, neurobehavioural changes including cognitive decline and neurobehavioural deficit, oxidative stress, and neuroinflammation. Toxicity of potassium dichromate also caused exaggerated increase in nitric oxide levels and acetylcholinesterase activity. However, supplementation with Kolaviron attenuated oxidative stress, improved neuroinflammation, and enhanced cognitive function. Taken together, Kolaviron could be a potential therapeutic agent for managing neurodegenerative diseases associated with neuroinflammation and cognitive impairment.

CRediT authorship contribution statement

Temitayo Olabisi Ajibade: Writing – review & editing, Visualization, Project administration, Methodology, Investigation, Formal analysis, Data curation. Oluwaseun Olanrewaju Esan: Writing – review & editing, Investigation, Data curation. Shammah Oluwaseyi Adeyemi: Methodology, Data curation. Taiwo Olaide Oyagbemi: Writing – review & editing, Methodology. Olumayowa Olawumi Igado: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation. Femi-Olabisi Fehintola Joy: Writing – review & editing, Investigation. Ebenezer Oyedele Ajiboye: Writing – original draft, Methodology. Adewumi Victoria Adeogun: Writing – review & editing, Project administration, Methodology, Investigation, Data curation. Olanrewaju Samuel Olaifa: Writing – review & editing, Investigation, Formal analysis, Data curation. Glory Oluomachi Uruakpa: Methodology, Formal analysis, Data curation. Sunday Samuel Adewumi: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Ademola Adetokunbo Oyagbemi: Writing – review & editing, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Data curation, Conceptualization. Ishmael Festus Jaja: Writing – review & editing, Methodology. Oguntibeju Olufemi Omoniyi: Writing – review & editing, Visualization, Validation, Methodology. Temidayo Olutayo Omobowale: Writing – review & editing, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Momoh Audu Yakubu: Writing – review & editing, Visualization, Methodology. Evaristus Nwulia: Writing – review & editing, Methodology.

Ethics Statement

Ethical approval for the study and consent for the use of animals was obtained from University of Ibadan, Animal Care and Use Research Committee (UI-ACUREC) with approval number NHREC/UIACUREC/05/12/2022 A.

Compliance with ethical standards

The ethical approval for this study was obtained from the Animal Care and Use Research Ethics Committee (ACUREC) of the University of Ibadan, Nigeria. All animals were humanely handled so as to eliminate unnecessary pain or discomfort. Guidelines for handling experimental animals according to the NIH were strictly followed.

Compliance with ethical standards

The ethical approval for this study was obtained from the Animal Care and Use Research Ethics Committee (ACUREC) of the University of Ibadan, Nigeria, with approval number NHREC/UIACUREC/05/12/2022 A.

All animals were humanely handled so as to eliminate unnecessary pain or discomfort. Guidelines for handling experimental animals according to the NIH were strictly followed.

Consent to Participate

Not Applicable

Consent to Publish

Not Applicable

Funding

The authors affirm that they did not receive any money from grants or other sources to conduct their studies in the laboratory or to prepare this manuscript.

Declaration of Competing Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Data availability

The data used to support the findings of this study is available from the corresponding author upon request.