Protective Effects of Tricin and Alpha-tocopherol on Behavioral Alterations and Dopaminergic Neuron Loss in Rotenone-Induced Neurotoxicity in Rats

Sushil Giri*, Phool Chandra

Teerthanker Mahaveer College of Pharmacy, Teerthanker Mahaveer University, Moradabad - 244001,

Uttar Pradesh, India.

*Corresponding Author E-mail: susilgiri4@gmail.com

ABSTRACT:

Background: Parkinson’s disease (PD) is a gradually worsening neurological disorder that primarily emerges in older adults. It is marked by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc), resulting in reduced striatal dopamine (DA) content. Flavonoids are becoming increasingly recognized as novel therapeutics in treating neurological illnesses due to their antioxidant and neuroprotective properties. Tricin, a bioactive flavonoid combined with alpha-tocopherol, is proposed to offer a promising therapeutic approach for PD. Objectives: This study focused on assessing the therapeutic potential of tricin and alpha-tocopherol in a rotenone (ROT) induced animal model of PD. Material and methods: ROT mediated PD model was used to test the neuroprotective activity of tricin and alpha-tocopherol. Behavioral activity, such as open field test, narrow beam walk, rota-rod, actophotometer, catalepsy bar test, grip strength test, footprint analysis, antioxidant activity, mitochondrial function and mounts of DA and its metabolic byproducts in rat SNpc, was used to ascertain the neuroprotective impact of these compounds. Results: Tricin, in combination with alpha-tocopherol, enhances efficacy compared to their single dosage forms and alleviates behavioral anomalies induced by ROT in experimental rats. Furthermore, it mitigated the dopaminergic toxicity caused by ROT in the animal's striatum and SNpc. Subcellularly protected the functionality and stability of mitochondria in the rats' SNpc from the deterioration caused by ROT. Further, investigations on the individual ingredients concerning their antioxidant activity, alpha-tocopherol showed the most antioxidant activity. The results suggest that tricin may exhibit neuroprotective effects against ROT-induced damage in rats, maybe through the Nrf2-mediated pathway. Consequently, tricin and alpha-tocopherol may be regarded as a feasible option for the treatment of PD. Conclusion: The in-vivo findings suggest that the combination of tricin and alpha-tocopherol may exert synergistic neuroprotective effects, presenting a potential new therapeutic approach for managing PD.

Graphical Abstract:

KEYWORDS: Parkinson's disease, Tricin, Rotenone, Alpha-tocopherol, Substantia nigra, Mitochondria.

INTRODUCTION:

Parkinson’s disease is a progressive neurological condition mainly impairs motor function and tends to occur more frequently in older adults. It manifests as bradykinesia, gait difficulties, resting tremors, muscle rigidity and balance problems.^1,2These symptoms stem from the degeneration of the nigrostriatal dopaminergic pathway, leading to decreased DA levels in the striatum. PD affects 10 million individuals worldwide, including 1-2% of those over 65. Although several medications are available, their long-term use is limited by adverse effects that hinder treatment adherence.³ As a result, natural compounds are being explored for their neuroprotective potential.

Tricin, a flavonoid found in rice bran and sugarcane, possesses antioxidant and anti-inflammatory properties.^4,5 It is readily obtainable from various natural sources, making it more accessible than other natural compounds like isorhamnetin⁶, Baicalein⁷, resveratrol⁸, β-caryophyllene, gastrodin⁹, Wogonin and cannabidiol.¹⁰ Tricin has the benefit of reduced molecular weight (330.29), potentially facilitating its passage across the blood-brain barrier and augmenting its therapeutic efficacy.¹¹ Alpha-tocopherol, a lipid-soluble antioxidant, is widely available in dietary. sources such as vegetable oils, nuts, and green leafy vegetables, and is recognized for its neuroprotective properties The combination of tricin and alpha-tocopherol was chosen due to their complementary antioxidant and anti-inflammatory actions. Tricin scavenges free radicals and modulates inflammation, while alpha-tocopherol protects lipid membranes from oxidative damage. Together, they may offer enhanced neuroprotection compared to single antioxidants used in rotenone, MPTP, or 6-OHDA PD models.¹²

Together, tricin and alpha-tocopherol show promise for PD management. However, tricin’s role in counteracting ROT-induced neurotoxicity has not been studied. Oxidative stress (OS) activates the Nrf2 pathway, which plays a key role in cellular defence.¹³ Nrf2 induces the expression of antioxidant enzymes like glutathione peroxidase (GPx) and heme oxygenase-1 (HO-1), helping neutralize ROS and maintain redox balance.¹⁴

Impaired Nrf2 signaling increases the vulnerability of dopaminergic cells, whereas stable Nrf2 activity promotes neuroprotection. Given this, Nrf2 is a potential target in treating PD. This study examined the neuroprotective effects of tricin and alpha-tocopherol in rats with PD induced by ROT.

MATERIALS AND METHODS:

Experimental animals and ethical considerations:

42 adult male Wistar albino rats (6–8 weeks old, 250–300g) were housed in polyacrylic cages under regulated conditions. The environment was maintained at 22±2°C, 55±5% relative humidity, and a 12hour light-dark cycle. The Moradabad Educational Trust Group of Institutions, Faculty of Pharmacy, animal facility provided the rats. They were randomly assigned to seven six-rat groups. The animal got free food and water during the investigation. The Institutional Animal Ethics Committee approved the experimental protocol (Letter No. 1867/PO/Re/S/16/CPCSEA/METFOP/IAEC/10/23) and followed CPCSEA guidelines.

Chemicals and reagents:

Rotenone, tricin, selegiline, and alpha-tocopherol were obtained from Sigma-Aldrich (St. Louis, USA). Additional reagents and chemicals were procured from TCI Pvt. Ltd. (India).

ROT injection in SN:

Using a stereotaxic apparatus, rats were anesthetized intraperitoneally with ketamine and xylazine. A 2μL volume of ROT, prepared in a 1:1 mixture of DMSO and PEG, was infused into the right SNpc at a controlled rate of 0.2μL/min. The stereotaxic coordinates relative to the bregma were: lateral 0.20mm, anteroposterior 0.53mm, and dorsoventral 0.75mm. Sham-operated rats received 2μL of the vehicle solution (DMSO:PEG, 1:1) in the same region. All animals were provided with appropriate post-operative care until complete recovery (Figure 1).¹⁵

Research protocol:

The study lasted 14 days, with the first 7 days for animal acclimatization. Rats were then randomly assigned to seven groups: control, sham, and five treatment groups receiving different combinations of ROT, tricin (T), alpha-tocopherol (A), and selegiline (S). From Day 1, tricin (60mg/kg, i.p.)¹⁶, was given to the ROT+T, and ROT+T+A groups. Similarly, alpha-tocopherol (50 mg/kg, i.p.)¹⁷,was administered to the ROT+A and ROT+T+A groups, while selegiline (10mg/kg, i.p.) was given to the ROT+S group. The control and sham groups received only the respective vehicle. On Day 14, behavioral tests were conducted, followed by euthanasia via cervical dislocation. Brains were dissected to isolate the striatum and SNpc, which were preserved at −80°C for biochemical analysis.¹⁸

Figure 1: Graphical depiction of the detailed experimental timeline.

EVALUATION PARAMETERS:

Behavioral assessments:

Narrow beam walking assessment:

The narrow beam test, used to assess balance and fine motor coordination, evaluated hind-limb impairment in rats. It measured foot slip frequency and time to traverse a narrow-elevated beam. Rats were trained twice on day -1, about two hours before ROT injection, to acclimate to the setup. On day 14, the test was conducted using a 120cm long, 3cm diameter beam elevated 60cm above the ground. One end led to an open field, and the other to a shadowy goal box (25 × 20 × 18cm). Each rat was placed at one site, and the number of rear paw slips and crossing time were recorded, with a 120-second time limit.¹⁹

Actophotometer:

Locomotor activity was assessed using an actophotometer equipped with photoelectric cells linked to a counter. Each interruption of the light beam by the animal was recorded as a count. On Day 14, each rat was placed individually in the device, and total activity was measured over five minutes as photobeam counts.²⁰

Rotarod test:

Motor coordination was evaluated using a rotarod apparatus. Rats were acclimatized in the test room 20–30minutes before evaluation. Each rat was placed on a rotating rod and had to maintain balance to avoid falling. Pre-training was conducted over five days, with speed increasing from 4 RPM to higher levels over 10minutes until a stable baseline was reached. During testing, which lasted up to three minutes, the RPM at the time of fall and the latency to fall were recorded.²¹

Grip strength assessment:

Musculoskeletal grip was assessed on Day 14 using a standard protocol.²² A 90cm long, 1mm diameter metal wire was fixed horizontally 50cm above a flat surface. Each rat was placed at the center, gripping with its forepaws, and scored as follows: 0-falls immediately; 1-grips with forepaws; 2-grips and attempts to climb; 3-uses forepaws and one/ both hind paws; 4-grips with all paws and wraps tail; 5-falls after evading the setup.

Bar catalepsy test:

Bar catalepsy is used to assess muscle rigidity and the inability to correct imposed postures.²³ Rats are placed upright with their front paws on a bar 10cm above the surface, and the time taken to remove one or both paws is recorded. This is repeated over three trials, each with a 60-second time limit.

Open Field Test (OFT):

OFT is widely used to assess spontaneous locomotion, movement initiation, and emotional responses in rats.²⁴ A custom hardwood field (50 × 50 × 40cm) was used to evaluate total distance traveled, average speed, immobility frequency, and asymmetric rotations. Each rat was placed in the center, and movement was recorded over 5 minutes. Distance, speed, line crossings, and rotations were quantified from video analysis.²⁵

Assessment of footprints:

Footprint patterns were analyzed to assess gait. Rats were trained to walk through a narrow 100 × 10cm pathway with 20cm high walls, lined with white paper and ending in a dimly lit enclosure. Forepaws were painted red and hind paws green to distinguish movements. As rats walked the path, footprints were recorded and evaluated for forepaw and hind paw base width, stride length, and paw overlap.²⁶

Evaluating the SNpc's antioxidant enzyme activity: Following the completion of the treatment period, rats were euthanized under moderate anesthesia, and brain tissues were harvested. The SNpc was carefully dissected to evaluate the activities of glutathione reductase (GR) and glutathione peroxidase (GPx). Tissues were homogenized in ice-cold 0.9% saline, followed by centrifugation at 800 × g for 10 minutes to remove debris, and then at 12,000 × g for 15 minutes to obtain the mitochondrial-rich supernatant, which was used for enzymatic assays.²⁷

Assessment of GR:

GR activity was measured spectrophotometrically following standard protocols. The reaction mixture included 80 mM GSSG, 6 mM NADPH, and 50 mM phosphate buffer with 6.3 mM EDTA (pH 7.4). Fifty microliters of cell lysate were added, and absorbance at 340 nm was monitored for four minutes after a 60-second incubation. Enzyme activity was calculated by NADPH consumption per mg protein per minute.²⁸

Assessment of GPx:

GPx levels were measured using a standard method based on its ability to oxidize reduced glutathione (GSH) to GSSG. This reaction is coupled with NADPH-dependent GR, which regenerates GSH. The reaction was monitored at 340 nm using UV-visible spectrophotometry at 37 °C, and activity was expressed as micromolar NADPH consumption per minute per mg protein.²⁹

Assessment of DA and its metabolites:

The high-performance liquid chromatography (HPLC) with an electrochemical detector was used to measure striatal DA and its metabolic byproducts in rats. The protein content was quantified via a colorimetric assay.³⁰

Evaluation of Rat SNpc Mitochondrial Dysfunction:

Mitochondria extraction in Rat SNpc:

Mitochondria were extracted from the SNpc using a conventional protocol. The tissues were first homogenized in an isolation buffer composed of 215 mM mannitol, 75mM sucrose, 0.1% bovine serum albumin (BSA), 20mM HEPES, and 1mM EGTA (pH 7.2), followed by centrifugation at 1300×g for 5 minutes at 4°C. The supernatant obtained was combined with freshly prepared EGTA-containing buffer and subjected to a second centrifugation. The final mitochondrial pellet was washed by resuspending in buffer without EGTA and centrifuged again for 10 minutes to remove any remaining EGTA.³¹

Evaluation of the activity of complex I (NADH: ubiquinone oxidoreductase) in the mitochondrial respiratory chain:

The function of Complex I was evaluated by monitoring the rate at which NADH is converted to NAD⁺. Fluorescence measurements were taken using an excitation wavelength of 350nm and an emission wavelength of 470nm. The assay mixture consisted of 200µL of 10mM potassium ferricyanide, 60µL of 1mM NADH prepared in 2mM phosphate buffer, and 2.64mL of 0.12 M phosphate buffer. The enzymatic activity of NADH dehydrogenase was calculated based on the rate of NADH oxidation, expressed as micromoles per minute per milligram of protein.³²

Statistical analysis for behavioural test:

The data are presented as the mean ± standard error of the mean (SEM; N = 6). Statistical analysis was performed using GraphPad Prism 5.01 software (GraphPad Inc., San Diego, CA). A one-way ANOVA followed by the Newman–Keuls post hoc test was used for all biochemical and behavioral evaluations. Statistical significance was considered at *p< 0.05, **p < 0.01, and ***p<0.001.

RESULTS:

Protective effect of tricin and alpha-tocopherol on ROT-induced motor impairments under narrowing beam walking test (NBWT):

Figure 2 shows the effects of tricin and alpha-tocopherol on ROT-induced hind-limb impairments during the narrow beam test, including time to reach the goal (A) and rear paw slips (B, C). One-way ANOVA revealed significant group differences in transfer latency [F (6,36) =92.33, ***p<0.001], left paw slips [F (6,36)=26.64, ***p<0.001], and right paw slips F (6,36)=16.36]. ROT exposure increased crossing time and slip frequency versus control. Tricin alone reduced these deficits more effectively than alpha-tocopherol. Their combination showed superior efficacy, comparable to the standard drug.

Table 1: Effect of tricin and alpha-tocopherol on ROT-induced behavioral changes in the footprint test

Experimental Groups	Fore-paw base width (cm)	Hind-paw base width (cm)	Left fore-paw stride length (cm)	Left hind-paw stride length (cm)	Left overlap (cm)
Control	3.05± 0.33	3.45± 0.18	9.98± 0.23	9.73± 0.10	1.09± 0.11
Sham	3.10± 0.43	3.44± 0.24	9.90± 0.23	9.70± 0.10	1.13± 0.10
ROT	2.68 ± 0.14	4.40 ± 0.16	6.90 ± 0.17***	5.86 ± 0.10***	2.16 ± 0.16***
ROT +T	2.77 ± 0.26	4.27 ± 0.10	9.41 ± 0.47***	8.16 ± 0.27***	1.28 ± 0.10***
ROT+A	2.74 ± 0.22	4.32 ± 0.12	9.01 ± 0.31***	7.40 ± 0.37***	1.46 ± 0.15***
ROT+T+A	2.83 ± 0.28	4.23 ± 0.11	9.53 ± 0.48***	8.45 ± 0.38***	1.27 ± 0.10***
ROT+S	2.92 ± 0.31	4.18 ± 0.12	9.61 ± 0.47***	9.28 ± 0.82***	1.23 ± 0.10***

Tricin and alpha-tocopherol ameliorate rot-induced disruptions in mitochondrial complex I activity in the SNpc: Figure 6 shows the impact of tricin and alpha-tocopherol on ROT-induced changes in mitochondrial complex I activity in the SNpc. One-way ANOVA revealed significant differences in NADH dehydrogenase activity [F (6,36) =38.79, ***p<0.001, **p<0.01]. Tricin significantly countered the ROT-induced decline, while its combination with alpha-tocopherol further improved enzyme activity, restoring it to levels comparable with the standard drug.

Figure 6: Depicts the effect of tricin on ROT-induced alterations in mitochondrial complex I activity in the rat SNpc.

DISCUSSION:

PD is a chronic neurodegenerative disorder characterized by the gradual loss of dopamine-producing neurons, with OS accelerating its progression through ROS accumulation, mitochondrial dysfunction, and antioxidant system impairment. Prompt diagnosis and timely action are essential for improving treatment outcomes, enhancing patients' quality of life, and optimizing prognosis. Flavonoids are increasingly recognized for their neuroprotective properties, offering a viable alternative to synthetic drugs, including anticholinergic agents such as trihexyphenidyl (Artane).Selegiline and other monoamine oxidase-B (MAO-B) inhibitors,³³ and catechol-O-methyltransferase (COMT) inhibitors like entacapone, which are utilized to alleviate PD symptoms, yet are associated with notable side effects such as dyskinesia and wearing-off effects.

PD is studied in neurotoxin-induced animal models like 6-hydroxydopamine (6-OHDA)³⁴, ROT and 1-methyl-4-phenyl-1, 2, 3,6-tetrahydropyridine (MPTP)³⁵ induced models. In the current study, ROT is used in PD models because it mimics environmental toxin exposure, selectively damages DA neurons by inhibiting mitochondrial complex I. It also causes progressive neurodegeneration, resembling the chronic nature of PD and can be administered systemically without direct brain injection.

Our results indicate that the combination of tricin and alpha-tocopherol considerably mitigated ROT-induced motor and behavioral deficits. For example, narrow beam walk, rotarod test, grip strength test, and footprint analysis showed improved motor coordination and symptoms in PD rats after treatment with tricin and alpha-tocopherol. In addition, open field test, actophotometer and catalepsy bar test^36,can provide information on both motor activity and cognitive aspects, confirming that the combination of tricin and alpha-tocopherol enhanced the PD rats' capacity for learning and memory. This combined treatment effectively preserved DA neurons in the SNpc and restored the activities of antioxidant enzymes GR and GPx, indicating potent antioxidant properties. These outcomes suggest that the beneficial impact of tricin and alpha-tocopherol on neuronal survival may be associated with the activation of the Nrf2 pathway, which is essential for mitochondrial function and integrity in PD models.

Moreover, tricin outperformed alpha-tocopherol in countering ROT-induced neurochemical alterations in the dopaminergic system, significantly restoring striatal DA concentrations and their metabolites. This improvement in dopaminergic activity could be attributed to tricin's ability to modulate the activity of dopaminergic metabolism enzymes, thereby enhancing motor functions in ROT-challenged animals. Collectively, these findings suggest that tricin, especially when combined with alpha-tocopherol, offers neuroprotective effects against ROT-induced damage in rats, potentially via the Nrf2-mediated pathway. This combination therapy addresses multiple pathogenic mechanisms in PD, including OS, mitochondrial dysfunction, and dopaminergic neuronal loss. Therefore, tricin and alpha-tocopherol represent promising candidates for further research and development as therapeutic agents for PD.

CONCLUSION:

The combination of tricin and alpha-tocopherol effectively countered ROT-induced behaviour and biochemical impairments in rats. The observed neuroprotective effects appear to involve antioxidant activity and Nrf2 pathway modulation, restoring dopaminergic function and mitochondrial activity. These findings position tricin and alpha-tocopherol as promising natural therapeutic agents for managing PD.

CONFLICT OF INTEREST:

The authors declare that they have no conflicts of interest

ACKNOWLEDGMENTS:

The writers express gratitude to Teerthanker Mahaveer University, Moradabad, for supplying the requisite facilities. We would also like to express our gratitude to Jay Prakash, research scholar at CSIR-IIIM, Jammu, India, for helping with biochemical estimation.

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Received on 24.04.2025 Revised on 09.08.2025

Accepted on 15.12.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2039-2046.

DOI: 10.52711/0974-360X.2026.00292

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Experimental Groups	DA (ng/mg protein)	DOPAC (ng/mg protein)	HVA (ng/mg protein)
Control	185.8 ± 16.53	11.90 ± 1.322	29.17 ± 1.643
Sham	188.0 ± 45.50	11.75 ± 1.438	28.65 ± 1.598
ROT	91.00 ± 7.416***	4.833 ± 0.6088***	17.17 ± 1.920***
ROT+T	154.4 ± 4.775***	5.967 ± 0.4457	21.44 ± 3.850*
ROT+A	145.4± 4.561***	4.982 ± 0.5741	18.40 ± 2.780
ROT+T+A	160.2 ± 3.493***	6.417 ± 0.3920*	23.57 ± 2.269***
ROT+S	164.4 ± 3.647***	7.300 ± 0.4243**	26.75 ± 2.809***