Neuroprotective Effects of Rauwolfia vomitoria Against 3-nitropropionic Acid-induced Motor Impairment and Oxidative Stress in Mice

1.Introduction
The cerebellum and the basal ganglia have a major impact on movement control and coordination. Cerebellar damage causes obvious deficits in motor control and coordination, but no observable deficits in perception or emotion [1]. Motor dysfunction, which is marked by deficiencies in the initiation, coordination, and execution of movement as observed in Parkinson’s disease, is also caused by dysfunctions of the basal ganglia [2]. Although the exact origin of these disorders is still unknown, oxidative stress and mitochondrial dysfunction have been found to be important contributors to neuronal degeneration and the resulting motor deficits [3].
Animal models have been crucial in helping to clarify the fundamental causes of motor dysfunction and investigate possible treatment options. Because it can mimic important aspects of neurodegenerative diseases marked by motor deficits, the 3-nitropropionic acid (3-NP)-induced neurotoxicity model has become well-known among these models [4]. 3-NP, a mitochondrial toxin, inhibits succinate dehydrogenase, resulting in energy depletion and neuronal cell death, culminating in motor impairment [5].
An imbalance between a biological system’s capacity to detoxify these reactive products and the generation and accumulation of reactive oxygen species (ROS) in cells and tissues causes oxidative stress. The onset and progression of motor impairment in a number of neurodegenerative diseases are significantly influenced by oxidative stress. Neuronal death and functional impairment result from ROS’ damage to mitochondria, lipids, proteins, and DNA. Neuropathological lesions and brain damage can arise from the reaction of free radicals, such as ROS, with substrates such as lipids, proteins, and nucleic acids [6, 7]. The brain is extremely vulnerable to peroxidation due to its high polyunsaturated fatty acid content, increased oxygen demand, and insufficient defenses [8, 9]. Therefore, in order to create therapeutic strategies targeted at lowering oxidative damage and maintaining the integrity of motor function in affected individuals, it is essential to comprehend the processes by which oxidative stress contributes to motor dysfunction. The effects of oxidative stress on motor performance may be mitigated by antioxidant treatments, mitochondrial protectors, and anti-inflammatory drugs.
Natural compounds have gained significant attention in the quest for innovative medicinal agents. Among these is Rauwolfia vomitoria (RV), a plant with a long ethnomedical history that has shown neuroprotective qualities in early research. The Nigerian rainforest shrub RV has clusters of small flowers and oval leaves with straight venations [10, 11]. Common names include swizzle stick, African snake root, African serpent wood, and deadly devil’s pepper. In Yoruba, it is known as Asofeyeje, Ira in Igbo, and Wadda in Hausa; in Efik and Ibibio, it is either mmoneba ebot or utoenyin, respectively [11]. It is also possible to powder the root of RV and consume it with pap or as a decoction [12]. According to other reports, RV may have antipsychotic and anticonvulsant effects [13]. Due in large part to its extensive phytochemical makeup, RV is well known for its wide range of pharmacological characteristics. Numerous bioactive substances, such as alkaloids, flavonoids, terpenoids, and saponins, are present in the plant. These substances add to the overall pharmacological profile of the plant, with alkaloids being the most common class that confers their medicinal properties [14]. These alkaloids have been shown to have anti-inflammatory, hypotensive, and neuroprotective effects [15]. This study aims to explore the potential neuroprotective effects of RV against 3-NP-induced motor dysfunction and oxidative stress in mice, but its precise effects on motor coordination deficits caused by neurotoxins are still poorly understood. This study offers insights into the antioxidant mechanisms of action of the plant and its potential as a therapeutic agent for neurodegenerative disorders characterized by motor impairments.

2. Materials and Methods 
2.1. Drugs and chemicals
ELISA Abcam reagent kits were acquired from Randoux Laboratory in the United Kingdom, while 3-NP was acquired from Sigma Aldrich LTD. in Canada. Every chemical and reagent employed in this investigation was of analytical grade.

2.2. Extraction preparation
The roots of RV were acquired from the University of Calabar’s botanical garden in Calabar. The plant sample was verified using the voucher number Bot/Herb/UCC/0576. The roots were cleaned of sand and grime using flowing tap water. The bark was scraped off and the roots were dried in the fresh air. An electric kitchen blender was used to powder the scraped root bark, yielding 700 g of powdered plant material. Five L of distilled water were used to fully submerge 700 g of RV powdered root bark, and the mixture was then agitated briskly. For a whole day, it was left to stand at room temperature, with periodic stirring. A rotary evaporator was used to allow the filtrate to concentrate after a 24-hour period, and an Astell Hearson oven was used to further dry the concentrate at 40–45 degrees Celsius. The extract was filtered multiple times, particularly using No. 1 Whatman filter paper with pore sizes of 0.45 micrometres and a funnel. This was done to guarantee that the extract completely evaporated and turned into the crude extract, a pasty brown residue. A spatula was then used to gather the RV root bark extract into a container, and an electric weighing scale was used to measure the amount. After processing, 17.5 g of RV paste was prepared and stored in a refrigerator until it was needed for research.

2.3. Animals 
The study employed 30 Swiss male mice, weighing between 19 and 35 g, which were 10 weeks of age. They were purchased from the University of Calabar’s Zoology Department. The animals were housed in normal cages at the University of Calabar’s Physiology Department. Their cages were regularly cleaned, and they were given commercial feed and unlimited access to tap water. The animals were given a week to become used to their new surroundings before the experimental treatments started. The University of Calabar Committee on Animal Use and Care granted ethical clearance for the experiments, and they were conducted in compliance with the regulations governing the use of laboratory animals.

2.4. Experimental design
Three groups of male Swiss mice, each consisting of ten animals, were randomly allocated and given the following treatments: Group II received 3-NP 15 mg/kg intraperitoneally, group III received 3-NP 15 mg/kg intraperitoneally, and group I received RV 20 mg/kg orally. Group I was also given 2 mL of distilled water as a control. Motor coordination was assessed using the beam walk test and the open field test on the final day of the 21-day course of therapy. The animals were put to sleep in a closed chamber using a chloroform inhalation approach following the neurobehavioral examinations. When the animals were completely anesthetized, they were placed in a supine position. The heart was then located by making an incision along the linea alba on their abdomen. Blood was collected via cardiac puncture with the use of a syringe. To prevent clotting, the obtained specimen was kept in EDTA (ethylenediaminetetraacetic acid) sample bottles before being used to test for indicators of oxidative stress.

2.5. Neurobehavioral studies (beam walk test)
Fine motor coordination and balance were tested using the beam walking test. This exercise is very helpful in identifying subtle balance and motor skill deficiencies that other motor tests might miss. The mouse must remain upright during this test in order to traverse an elevated beam and reach a secure platform [16, 17]. The beam walk equipment is raised roughly 50 cm above the ground and measures 1 to 2 cm in width and 50 to 100 cm in length. There is a goal box with food at the end of the beam, and the test environment is peaceful and distraction-free. First, the mice spent sixty seconds getting used to the goal box and beam. During testing, each mouse was placed at the starting location and permitted to walk across the beam. Foot slips (occurrences of paws slipping off the beam), reversals (changes in walking direction), and line crossings (number of preset segments traversed) are important parameters that were noted. An observer recorded these measurements, and they were also captured on video for further examination. 

2.6. Tissue antioxidant assessment 
After adding 1.15% potassium chloride to 100 mL of tissue homogenate in 50 mM Tris-HCl buffer (pH 7.4), the mixture was centrifuged for 15 minutes at 10,000 rpm and 4 °C. Using hydrogen peroxide as the substrate, the supernatant was collected for the catalase (CAT) test. Reduced glutathione (GSH) was measured at 340 nm using the technique of Luchese et al. (2009) [18]. Hydrogen peroxide was used as the substrate for the glutathione peroxidase (GPx) test [18]. The technique outlined by Misra and Fridovich (1972) [19] was used to measure superoxide dismutase (SOD). Choudhary et al. (2007) [20] and Okhawa et al. (1979) [21] provided an explanation of how malondialdehyde (MDA) was measured in thiobarbituric acid-reactive substances (TBARS). Following that, 0.2 mL of 8.1% sodium dodecyl sulphate was added in the reaction mixture. To 0.2 mL of 10% (w/v) homogenate, 1.5 mL of a 20% acetic acid solution that was adjusted to PH 3.5 with sodium hydroxide and 1.5 mL of a 0.8% aqueous solution of thiobarbituric acid were added. Using distilled water, the mixture was increased to 4.0 mL and heated for 60 minutes at 95 °C. After adding around 1.0 mL of distilled water and 0.5 mL of the n-butanol and pyridine mixture (15.1 v/v), the mixture was centrifuged at 4000 rpm with ice chilling. After discarding the supernatant layer, absorbance at 532 nm was measured against MDA standards. Absorbance measurements were used to compute the concentrations.

2.7. Statistical analysis
The results were analyzed using a one-way analysis of variance (ANOVA) and a post-hoc multiple comparison test to compare the significance levels between the control and other groups. The data were expressed as Mean±SEM. The analysis was conducted using Microsoft Excel and SPSS software, version 20, with a significance level of P<0.05.

3. Results
3.1. Effect of 3-NP and RV on motor coordination
Prior research has confirmed that performance on the balance beam is a valuable indicator of balance and fine coordination. The beam test can reveal motor deficiencies due to age, central nervous system injuries, genetic, or pharmacological alterations in young and older animals [17]. The effects of administering 3NP and RV alone and in combination with 3NP on motor coordination are depicted in Figures 1A, 1B, and 1C, respectively.

In comparison to the control group, mice administered 3NP experienced a significant (P<0.01) increase in foot slips and a decrease in line crossing and reversals during the beam-walking test. However, as demonstrated in Figures 1A, 1B, and 1C, when compared to the control and 3NP groups, the co-administration of RV with 3NP (3NP+RV group) led to a significant (P<0.05) decrease in the number of foot slips and an increase in the number of line crossings and reversals in the mice’s beam-walking test.

3.2. Effect of 3-NP and RV on biomakers of oxidative stress
The effect of 3NP and RV on oxidative stress and antioxidant enzyme levels was assessed by measuring the concentrations of CAT, SOD, GPX, and MDA in the different groups. Treatment with 3NP resulted in a significant (P<0.001) decrease in the levels of CAT, SOD, and GPX, and a significant increase in MDA levels when compared to the control group. Specifically, the 3NP group showed a marked reduction in CAT levels compared to the control (P<0.001). The co-administration of RV with 3NP (3NP+RV group) significantly (P<0.05) increased CAT levels compared to the 3NP group (Figure 2A).

Similar to CAT, the SOD levels were significantly reduced in the 3NP group compared to the control (P<0.001). The 3NP+RV group exhibited a significant (P<0.05) increase in SOD levels compared to the 3NP group (Figure 2B). The GPx levels were also significantly lower in the 3NP group compared to the control (P<0.001). The administration of RV significantly (P<0.05) improved GPx levels compared to the 3NP group (Figure 2C). In contrast, the MDA levels, were significantly elevated in the 3NP group compared to the control (P<0.001). The 3NP+RV group showed a significant (P<0.05) reduction in MDA levels compared to the 3NP group, suggesting an antioxidant effect (Figure 2D).

​​​​​​​4. Discussion
An increase in foot slips and a decrease in line crossings and reversals in the beam-walking test compared to the control group demonstrated the study’s conclusions that 3-NP significantly impairs motor performance. These findings are consistent with earlier research showing that 3-NP’s neurotoxic effects on the striatum led to motor impairment [22].
Additionally, co-treatment with RV reversed the decrease in line crossings and reversals and significantly reduced the increase in foot slips caused by 3-NP. This implies that RV has neuroprotective qualities that can help alleviate 3-NP-induced motor impairments. 
An improvement in balance and coordination is indicated by the notable decrease in foot slips seen in the 3NP+RV group. This suggests that RV has a positive impact on cerebellar function, which is essential for motor control. Furthermore, a decrease in anxiety-like behavior and an improvement in exploratory behaviour are suggested by the rise in line crossings and reversals in the same group. These results lend more credence to RV’s neuroprotective properties.
When compared to the control group, 3-NP significantly increased levels of MDA, a marker of lipid peroxidation, while significantly lowering levels of antioxidant enzymes such as GPX, CAT, and SOD. These results are in line with earlier observations that 3-NP produces ROS, leading to oxidative stress [23].
Crucially, co-treatment with RV reduced MDA levels and greatly mitigated the 3-NP-induced decline in CAT, SOD, and GPx levels. It is possible that the extract’s bioactive components’ capacity to inhibit nitric oxidase and restrict the production of ROS in response to oxidative stress is what allows it to restore the level of GPx and the activity of the enzymes (CAT and SOD). Terpenoids, steroids, alkaloids, saponins, tannins, flavonoids, and vitamin C have all been identified through phytochemical examination of the RV [14]. Vitamin C is an antioxidant that either directly or indirectly reduces lipid peroxidation by regenerating vitamin E, as demonstrated by the study of Padayatty et al. (2003) [24]. Inhibiting lipid peroxidation [24] allows vitamin C to scavenge free radicals from ROS through a quick electron transfer, acting as an indirect antioxidant. The inclusion of phytochemicals such as terpenoids and alkaloids, which have been shown to have antioxidant activity, may also be responsible for the observed antioxidant effect of RV [15]. This implies that RV has antioxidant qualities that lessen the oxidative damage caused by 3-NP.
The 3NP+RV group’s markedly elevated levels of CAT, SOD, and GPx suggest a strengthened antioxidant defense mechanism, which is essential for shielding cellular constituents from oxidative damage and slowing the progression of neurodegeneration. The antioxidant capability of RV and its capacity to lessen lipid peroxidation are further supported by the decrease in MDA levels in the same group.
To sum up, the current study shows that RV has antioxidant qualities, which help explain its neuroprotective effects against 3-NP-induced motor impairment in mice. According to the research, RV may be a viable therapeutic option for the prevention and management of oxidative stress-related neurodegenerative diseases such as Parkinson’s disease [25]. To clarify the precise mechanisms behind RV’s antioxidant benefits and assess its effectiveness in other animal models of neurodegenerative diseases, more research is necessary.

Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Committee on Animal Use and Care of the University of Calabar, Calabar, Nigeria (Code: FARE C/PA/[UC/049]/181PHY122).

Data availability
Data supporting this study’s findings are available upon a reasonable request from the corresponding author.

Funding
The authors confirm that they received no funding for the research, authorship, and publication of this article. 

Authors' contributions
Conceptualisation and study design: Esu Ukpai Enene; Writing the original draft: Saminu Samaila; Review and editing: Lawrence Dayo Adedayo; Analysis and data interpretation: Ekementeabasi Aniebo Umoh.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgements
The authors would like to express their gratitude to the laboratory technical staff of the Department of Physiology, University of Calabar, Calabar, Nigeria, for their invaluable support. 

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