Investigating the neuroprotective effects of strawberry extract against diesel soot-induced motor dysfunction in Drosophila: an in-vivo and in-silico study

Abstract

Environmental pollutants including diesel soot, have been known to contribute to neurological disorders. Previous studies highlight the neuroprotective effects of strawberry-derived compounds. This work explores the impacts of diesel soot and strawberry extract in movement-related disorders. In-silico analysis assessed compounds from HPLC/GCMS in the literature of soot and strawberry extract for ADME properties and blood–brain barrier permeability, selecting six compounds and four motor function-related proteins (SOD1, TARDBP, FUS, MAPT) with D. melanogaster orthologs. Homology modeling generated protein structures, molecular docking assessed binding affinities. MLSD examined combined interactions, with RMSD validating accuracy. Docking scores matched neuroprotective controls (quercetin, resveratrol), while differed for negative control (formaldehyde). Phenanthrene and anthocyanin strongly bound to FUS (− 7.60 ± 0.26 kcal/mol, − 7.1 ± 0.26 kcal/mol) and cocoon (− 6.5 ± 0.39 kcal/mol, − 7.23 ± 0.45 kcal/mol). MLSD yielded − 3.00 ± 0.24 kcal/mol and − 3.12 ± 0.11 kcal/mol respectively. In-vivo assays in D. melanogaster exhibited soot impaired movement (p = 0.0006), while strawberry improved it (p = 0.0003) with partial recovery in combined exposure (p = 0.0003). Strawberry enhanced cold stress recovery (p = 0.0048), climbing (p < 0.0001), and vortex recovery (p = 0.0003). One-way ANOVA confirmed significant effects on crawling in males (F (9,20) = 37.67, p < 0.0001, η² = 0.53) and female flies (F (9,20) = 70.10, p < 0.0001), with normality confirmed by Shapiro–Wilk test (p > 0.05). Toxicant exposure accelerated mortality, while strawberry improved thermotolerance. Combined exposure provided partial protection with minor sex differences. Findings highlight strawberries’ neuroprotective role in counteracting diesel soot toxicity, even under combined exposure.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40203-025-00344-2.

Introduction

Effects of environmental toxicants have been implicated in the pathogenesis of various neurological disorders. Human and animal studies suggest that air pollution may cause developmental neurotoxicity, and may contribute to the etiology of neurodevelopmental disorders (Costa et al. 2020). Among these neurological conditions, movement disorders represent a diverse spectrum of clinical, pathological, and genetic manifestations, all leading to an alteration of normal motor function (Singh et al. 2021). These disorders pose unique challenges to individuals hindering their daily activities. An estimated 3.40 billion individuals had a condition affecting the nervous system in 2021, corresponding to 43.1% of the world population (Steinmetz et al. 2024). In the past three decades, most studies in India have shown a high disease burden for specific neurological diseases with a % increase including stroke (37.9%), epilepsy (11.3%), Parkinson's disease (1.8%), Alzheimer’s disease and other dementia (4.6%), motor neuron diseases (0.1%) and other neurological disorders (1.3%) (Mehndiratta et al. 2021; Singh et al. 2021). A comprehensive strategy is necessary for the effective management of these disorders in order to support patients in maintaining their functional independence and leading a healthy lifestyle.

Long-term air pollution (AP) exposure, including diesel exhaust (DE) exposure, is increasingly being recognized as a major contributor to the development of neurodegenerative diseases (Ha et al. 2022; Feng et al. 2024). Abnormalities in white matter and the activation of microglia have been observed in individuals exposed to air pollution, suggesting these factors might contribute to cognitive and motor impairments (Aderinto et al. 2025). Air pollution derived from diesel exhaust has been linked with cognitive decline and cerebrovascular diseases (Tang-Tan et al. 2025). DE is composed of gases, notably including soot particles (Zhang et al. 2022), nitrogen dioxide (NO₂), and carbon monoxide (CO) (Long et al. 2022), and is recognised as an increasingly abundant air pollutant in urbanized communities. Qualitative analysis of extracts of different soot samples (Viteri et al. 2019) has been explored in the past to identify different compounds from soot. Intriguing data has come out of regions that experience heavy air pollution, suggesting that exposure to DE may contribute to protein aggregation and increase markers of neurodegeneration (Barnhill et al. 2020). Previous studies have revealed that DE soot exposure is neurotoxic and induces neuroinflammation (Ha et al. 2022). Particulate matter from diesel exhaust is especially concerning as it can penetrate into the brain and absorb chemicals including PAHs, which exacerbate inflammatory responses (Jäntti et al. 2024).

Fruits are regarded as a valuable source of bioactive compounds with a therapeutic potential against neuronal disorders (Borowiec et al. 2021). The potential of natural polyphenols against oxidative stress and BBB disruptive pathology has been explored (Kim et al. 2022a, b). Numerous studies investigate the impact of plant-derived polyphenols on brain cell metabolism, neutralization of reactive oxygen species, and cognitive functions related to signal transduction and neuronal plasticity (Grabska-Kobyłecka et al. 2023). Epidemiological and clinical studies highlight the potential of polyphenol-rich diets to decrease the risk and alleviate symptoms of neurodegenerative disorders and neuroinflammation (Jalouli et al. 2025). Fruits such as berries are a rich source of natural antioxidants and polyphenols, such as anthocyanins, vitamins, and other phytochemicals, which are actively involved in slowing down the aging process (Nuzzo et al. 2021). Among different forms of berries, strawberry is considered an important fruit owing to its nutritional value. It is a rich source of vitamins C, folates, and many phenolic compounds. Recent studies have revealed that the ratio of unsaturated to saturated fatty acids (UFA/SFA) was higher in strawberries as compared to other analysed fruits (cranberries, goji berries, etc.) (Bajramova et al. 2022), which can influence inflammation, oxidation, and immunity (Chaaba et al. 2023). Currently, strawberry consumption has gained attention due to the presence of significant dietary compounds involved in neuroprotective function, and prospective epidemiological evidence indicates that habitual consumption of strawberries is associated with a diminished rate of age-related cognitive decline (Singh et al. 2024; Krikorian et al. 2023). Despite these promising findings, the molecular mechanisms underlying these protective effects remain underexplored.

Research focusing on investigating the role of dietary compounds and their interactions with environmental pollutants to better understand their potential in mitigating motor neuronal damage has not been explored in detail. In this context, employing cost-effective, rapid, reliable, and efficient in-vitro and in-vivo assays will facilitate analyzing these compounds' behavioral response, dosage response, and pharmacological and toxicological profiles (Lopez-Ortiz et al. 2023). Simpler organisms like Drosophila melanogaster (D. melanogaster) could also be favored for toxicity studies, as 75% of the genes related with human diseases are known to have homologs in D. melanogaster, which facilitates research into different anomalies (Huang et al. 2023). D. melanogaster offers a great opportunity to test complex motor functions (Mariano et al. 2020; Welch et al. 2022).

This work aims to explore the neuroprotective effects of strawberry extract in D. melanogaster exposed to soot compounds, with a focus on how dietary compounds can counteract pollutant-induced motor dysfunction. A detailed protocol has been followed to assess the effects of these compounds and toxicants, which involves multiple ligand interactions and docking studies along with various experimental procedures involving D. melanogaster flies. Experimental concentrations were chosen based on previous in vivo studies using D. melanogaster to model air pollution toxicity, ensuring doses were within physiologically relevant ranges. The selected concentrations (2.5, 5,10 mg/ml) allowed for the assessment of dose-dependent neurotoxicity and the mitigating effects of strawberry extract while maintaining fly viability. These studies could contribute to a better understanding of how dietary compounds could ameliorate the harmful effects of environmental toxins. A graphical abstract to construct this has been illustrated in Fig. 1

Fig. 1.

Graphical abstract representing the study design

In-silico studies

Compound identification and ADME analysis

Active compounds present in strawberry and soot were obtained from literature study which focused on using methodologies such as GC/MS, GC-IMS, HS–SPME–GC–MS and HPLC (Li et al. 2021; Mustafa et al. 2021; Zhang et al. 2023; Urrutia et al. 2017). Several research papers that identified strawberry compounds and soot compounds were analyzed, and their extraction using different solvents was reviewed. Commonly reported compounds were selected. The properties of these compounds were evaluated using the SwissADME tool (Daina et al. 2017). ADME-related properties for compounds of strawberry, namely, oral bioavailability (OB), drug likeness (QED), blood–brain barrier (BBB) penetration, number of Lipinski's rule violations, and gastrointestinal (GI) absorption were evaluated to assess the compounds for their ability to become potential drug candidates. For strawberry compounds, those with OB ≥ 30%, QED ≥ 0.18, high GI absorption, and no Lipinski's rule violations were considered potential drug candidates. For soot compounds, selection criteria focused on the ability of the compound to cross the BBB. Resveratrol and quercetin were selected as positive controls, while formaldehyde was used as the negative control.

Target identification and protein preparation

Target molecules relevant to motor function in neurological conditions were selected based on two neurology-related datasets from DisGeNET (Piñero et al. 2019). C0085084 and C052481. These datasets involve targets that are linked to neurological disorders and their intersection was analyzed to identify common targets which are involved in motor function deficits. Venn diagram (Oliveros et al. 2007) was created to identify the common targets. Orthologs were then identified using the FlyBase database (Öztürk-çolak et al. 2024). 3D structures of human proteins were obtained from Protein Data Bank (PDB) (https://www.rcsb.org/) (Berman et al. 2003). For D.melanogaster proteins, 3D structures were modeled using protein sequences retrieved from UniProt (Bateman et al. 2022). Various tools were employed for 3D modeling, including AlphaFold (Jumper et al. 2021; Varadi et al. 2023), ESMFold (Lin et al. 2023), trRosetta (Du et al. 2021; Su et al. 2021), and SWISS-MODEL (Waterhouse et al. 2018), as the complete PDB structures were not available. The models were then evaluated based on their structural attributes using the SAVESv6.1 metaserver. This evaluation included the compatibility of the 3D model with its amino acid sequence (1D) using VERIFY 3D (Bowie et al. 1991; Lüthy et al. 1992), Ramachandran plot analysis with PROCHECK (Laskowski et al. 1993; Laskowski et al. 1996), and the overall quality score from ERRAT (Colovos et al. 1993), MolProbity scores for steric clashes and rotamer quality (Williams et al. 2017), ProSA Z-scores for energy-based validation (Wiederstein et al. 2007), and QMEAN scores for assessing model reliability (Benkert et al. 2010) which have been included in the supplementary file. Among the generated models, the tool from which the most suitable protein structure was obtained was selected, and the model was downloaded and used for further analysis. All water molecules and heteroatoms were removed from the final protein models using BIOVIA Discovery Studio 2024.

Molecular docking

The 3D structures of compounds from strawberries and soot were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) (Kim et al. 2022a, b). These structures were converted from sdf to pdb format using Discovery Studio. For both single-ligand docking (SLD) and Multiple-ligand simultaneous docking (MLSD), target proteins were prepared using AutoDockTools (ADT) by setting solvation parameters, adding polar hydrogens, assigning Kollman charges, and converting them to pdbqt format. Blind docking was employed to explore potential binding sites without predefining active sites. Grid box dimensions were set with X, Y, and Z coordinates and grid spacing of 1. SLD was conducted using AutoDock Vina (Eberhardt et al. 2021; Trott et al. 2009), with each docking run including eight exhaustive cycles. MLSD was performed using AutoDock 4.2 (Morris et al. 2009), allowing simultaneous docking of multiple ligands. Post-docking, 3D, and 2D interactions of protein–ligand complexes were analyzed using Discovery Studio and LigPlot + (Laskowski et al. 2011). For all the docked molecules, the results are presented as mean ± standard deviation (SD), and the root mean square deviation (RMSD) has also been calculated, which has been included in the supplementary file (Mandal et al. 2021).

In-vitro studies

Materials and reagents

Sugar and corn flour (Parry, India). Agar (Himedia, India), methylparahydroxy benzoate (TEGO), yeast (Gloripan Instant Dry Yeast), and propionic acid (Loba Chemie, India). Orthophosphoric acid (Emparta, India). All reagents were stored at room temperature before use.

Extract preparation

The extraction process was performed for locally obtained fresh strawberries using acetone (Sigma Aldrich, India) as the solvent (Kajdžanoska et al. 2011). A 5 g aliquot of homogenized strawberry was extracted at room temperature with 15 mL of acetone using a GPE Scientific homogenizer (#9.164 666). The resulting supernatant was collected for subsequent analysis (Koraqi et al. 2023). DE soot particles were collected from the exhaust pipes of various heavy-duty engine vehicles in Bangalore (Kangsadalampai et al. 1999). The particulate matter was obtained by carefully scraping the interior surfaces of the exhaust pipes.

Drosophila melanogaster fly line and feeding design

Wild-type Canton Special (CS) flies were utilized for all experiments due to its well-characterized genetic background and consistent behavioral patterns. These flies were raised on basal medium (12 g of corn flour, 75 g of sucrose, 150 g of yeast powder, 12 g of agar powder, 6 mL of propionic acid, 750 µL of Orthophosphoric acid, TEGO, and 1.5 L of distilled water for 1.5 L of food) (Bass et al. 2007). Flies were maintained in an environment with controlled temperature (25 °C). Two to three-day old flies were anesthetized with CO₂ and segregated based on the gender and then transferred to the experimental vials.

Experimental design

Flies of each gender were separated into distinct control groups and nine experimental groups. Experimental groups were exposed to varying concentrations of strawberry extract (2.5, 5, and 10 mg/mL), soot (2.5, 5, and 10 mg/mL), and a combination of strawberry extract and soot (2.5, 5, and 10 mg/mL). For each group, 3–4 male flies and 10 female flies were placed into vials containing 5–6 mL of prepared food medium. Flies were allowed to reproduce and deposit eggs in the food medium. Larvae and flies that emerged from these culture vials were used in the assays and each assay was performed in triplicates. All these assays were conducted separately for each gender to determine if there are any potential motor and behavioral differences between males and females.

Experimental validation

Crawling assay

The larval crawling behavior assay is a crucial technique used to assess rhythmic movement in larvae and identify potential neural defects (Jakubowski et al. 2012). In this study, twelve-third instar larvae were collected from each experimental group. Larvae were then transferred to a solid agarose surface (2% agarose) in a petri dish, where they were allowed to crawl (Nichols et al. 2012). A graph paper was placed beneath the agarose surface to track the path covered by each larva. Crawling behavior was recorded for one minute using a camera, and the total distance traveled was determined by counting the number of grid lines crossed by each larva on the graph paper.

Cold tolerance assay

Cold induced neuropathy has been observed both in humans (Krøigård et al. 2018) as well as D. melanogaster (Himmel et al. 2021). To investigate this, a cold tolerance assay was conducted for all experimental groups. In this assay, petri plates were pre-chilled at 0 °C for 12 h, and subsequently cooling rate was standardized using a calibrated ice bath at 0 °C. Twelve larvae of each gender were separately transferred into these pre-cooled petri dishes, where they underwent cold treatment for 1 min and 30 s. After the treatment, larvae were removed from the cold treatment, and their recovery time was monitored. Recuperation from a chill hypothermia was defined as the point when a larva regained its ability to crawl.

Geotaxis assay

Flies aged 1–2 days old were anesthetized and sorted by gender. Fifteen flies for the experimental groups of each gender were then placed into separate vials and allowed to recover from anesthesia (Jimenez-Del-Rio et al. 2009; Moulin et al. 2021). For the climbing assay, the flies from each vial were transferred into a 10 mL measuring cylinder marked up to 8 cm. Cylinder was sealed with a cotton plug and tapped two to three times to ensure all flies settled at the bottom. Flies were then allowed to ascend the cylinder, and their climbing behavior was recorded for 50 s. Each climbing trial was conducted twice, with a 5-min interval between trials, for three replicates. Number of flies that successfully climbed to the 8 cm mark was noted. At the end of the experiment, the percentage of flies that climbed was calculated by dividing the number of flies that successfully reached the 8 cm mark by the total number of flies placed in the cylindrical tubes.

Vortex assay

Vortex assay is employed to study seizure behavior and susceptibility in adult flies (Mituzaite et al. 2021). Seizure duration is typically assessed by measuring the time required for each fly to regain its posture and mobility. Adult flies, 1–2 days post-eclosion, were collected using CO₂ and placed into empty glass vials. Fifteen flies of each gender from the experimental groups were allowed to recover from anesthesia within the vials. The vials were then subjected to mechanical agitation on a vortexer, operating at maximum speed for 1 min and 30 s. During the seizure event, varied fly behaviors, including paralysis and wing damage, were also observed.

Stress assay

Stress can significantly affect neurological function, with heat being one of the stressors. In this study, a water bath was preheated to a range of temperatures, specifically 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, and 40 °C. Glass vials containing 12 female and 12 male D. melanogaster, aged 3–4 days, of each experimental group were placed into the preheated water bath. Flies were subjected to heat stress for 30 min. Following this exposure, vials were removed from the water bath, and flies were allowed to recover at room temperature for 45 min. Number of surviving flies was then counted, and the survival rate was calculated by dividing the number of flies that survived by the total number of flies tested, then multiplying by 100 to obtain a percentage.

Statistical analysis

GraphPad Prism 10.3.0 (GraphPad Software, San Diego, USA) was utilized for statistical analyses. Differences between experimental groups for each gender were assessed using one-way Analysis of Variance (ANOVA) with a 95% confidence interval (p < 0.05), followed by Tukey's multiple comparisons test. This test compared the mean values of each column with every other column to determine significant differences. For examining discrepancies between genders, an ordinary two-way ANOVA was employed. This analysis compared cell means across both rows and columns. Significance levels were selected as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results and discussion

Compound identification

The dietary compounds chosen were analyzed for their ADME properties. From strawberry, six compounds including 2-Hexenol (Li et al. 2021; Zhang et al. 2023), Cinnamyl acetate (Prat et al. 2013), Ethyl-2-methylbutyrate (Prat et al. 2013; Song et al. 2017), Anthocyanin (Mustafa et al. 2021; Kim et al. 2023; Haugeneder et al. 2018), Ferulic acid (Wu et al. 2023), Alpha terpineol (Teribia et al. 2021; Sheng et al. 2021) exhibited high GI absorption and zero Lipinski’s rules violations. OB of 85% was obtained for Ferulic acid, while for other compounds it was found to be 55% as shown in Table 1.

Table 1.

ADME Related Parameter value of selected compounds of strawberry

Strawberry Compounds	GI absorption	BBB permeation	Lipinski’s rules	Oral Bioavailability (%)
2- Hexenol	High	Yes	Yes, 0 violation	55
Cinnamyl acetate	High	Yes	Yes, 0 violation	55
Ethyl-2-methylbutyrate	High	Yes	Yes, 0 violation	55
Anthocyanin	High	Yes	Yes, 0 violation	55
Ferulic acid	High	Yes	Yes, 0 violation	85
Alpha terpineol	High	Yes	Yes, 0 violation	55

Similarly, the particulate matter was analyzed for their properties. Naphthalene, Acenaphthene, Acenaphthylene, Anthracene, Fluorene, Phenanthrene (Dandajeh et al. 2020) were the six compounds obtained which exhibited low gastrointestinal absorption and the ability to penetrate the BBB as mentioned in Table 2.

Table 2.

Parameter values of selected compounds of soot

Soot Compounds	GI absorption	BBB permeation
Naphthalene	Low	Yes
Acenaphthene	Low	Yes
Acenaphthylene	Low	Yes
Anthracene	Low	Yes
Fluorene	Low	Yes
Phenanthrene	Low	Yes

Target identification

A total of 4 common targets which are involved in motor function deficits were found between the two neurology-related datasets from DisGeNET: C0085084 and C052481.After identifying the shared human targets, further research was conducted to validate their involvement in neurological disorders associated with motor function deficits. Common targets identified from the datasets, namely SOD1 (Namboori et al. 2021; Pahal et al. 2021), TARDBP (Donde et al. 2019; Lee et al. 2022), FUS (Kour et al. 2023; Swetha et al. 2016), and MAPT (Habekost et al. 2021; Zhang et al. 2020), have been extensively studied in the context of diseases such as Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), and other neurodegenerative conditions. These were compared with their orthologs in D. melanogaster, including SOD1 (Kumimoto et al. 2013), Cocoon (Lee et al. 2019), Tau (Sivanantharajah et al. 2019), and Caz (Frickenhaus et al. 2015), using the FlyBase database. These fly orthologs were also confirmed for their involvement in motor function impairment within neurological contexts.

Protein preparation

The following human proteins were retrieved from PDB: SOD1 (PDB ID: 1HL5), TARDBP (PDB ID: 5MDI), FUS (PDB ID: 4FDD), and MAPT (PDB ID: 2ON9). Homology modeling techniques were employed to generate the structural models of the following D. melanogaster orthologous proteins: SOD1 (UniProt ID: P61851), Cocoon (UniProt ID: Q9VUN2), Tau (UniProt ID: Q9VB15), and Caz (UniProt ID: Q27294). Among the various tools used, it was observed that SWISS-MODEL produced the most accurate and reliable models in terms of structural validation. The structural models were assessed using multiple evaluation criteria, and most of the proteins yielded high-quality predictions, however, the Tau model had comparatively lower structural quality and contained a significantly large number of disallowed regions in the Ramachandran plot.

Molecular docking

Molecular docking studies were conducted to investigate the binding affinities of soot-derived and strawberry-derived compounds with target proteins from humans and D. melanogaster. The active site of the target proteins was not predefined, as blind docking was performed. In blind docking, the entire protein surface is considered for ligand binding. This approach allows an unbiased exploration of potential binding sites, which is particularly useful as many natural compounds, including those from strawberries and soot, can interact with multiple regions of a protein.

The SLD studies revealed significant binding affinities across several key neurological proteins. Among the soot-derived compounds- Acenaphthene, Acenaphthylene, Anthracene, Phenanthrene, Naphthalene, and Fluorene, Phenanthrene exhibited the strongest binding affinity with the human gene FUS, achieving a binding energy of − 7.6 kcal/mol (Table 3). The interaction was characterized by multiple hydrophobic contacts with residues such as Leu129(A), Leu130(A), Ile156(A), Val167(A), Ile162(A), and Leu123(A) (Fig. 2a,b). This strong binding suggests that Phenanthrene could potentially influence the protein's function, contributing to neurotoxic effects. When docked with dietary compounds- 2-Hexenol, alpha-Terpineol, Anthocyanin, Cinnamyl acetate, Ethyl 2-methylbutyrate, and Ferulic acid, Anthocyanin showed the highest binding affinity with FUS, achieving a binding energy of -7.1 kcal/mol (Table 4). The interaction involved hydrogen bonds with residues Lys309(A), Arg376(A) and Glu270(A), along with hydrophobic interactions involving Leu274(A) and Asn374(A) (Fig. 3a,b). This indicates that Anthocyanin may competitively bind to the same site as the environmental compound, possibly mitigating its toxic effects.

Table 3.

Binding affinities (kcal/mol) of human target proteins with soot-derived compounds (mean ± SD)

Target	Acenaphthene	Acenaphthylene	Anthracene	Fluorene	Naphthalene	Phenanthrene
Sod1	− 6.07 ± 0.05	− 6.21 ± 0.18	− 6.76 ± 0.13	− 6.06 ± 0.05	− 5.23 ± 0.05	− 6.83 ± 0.12
FUS	− 6.39 ± 0.20	− 6.40 ± 0.22	− 6.80 ± 0.37	− 6.74 ± 0.17	− 5.63 ± 0.28	− 7.60 ± 0.26
TARDBP	− 5.62 ± 0.19	− 5.67 ± 0.13	− 6.14 ± 0.44	− 5.83 ± 0.39	− 4.77 ± 0.32	− 6.01 ± 0.45
MAPT	− 4.06 ± 0.07	− 4.09 ± 0.09	− 4.72 ± 0.07	− 4.41 ± 0.12	− 3.68 ± 0.11	− 4.44 ± 0.09

Fig. 2.

a 3D visualization of interactions between human target FUS and DE compound phenanthrene b: 2D visualization of interactions between human target FUS and DE compound phenanthrene

Table 4.

Binding affinities (kcal/mol) of human target proteins with strawberry-derived compounds (mean ± SD)

Target	2-Hexenol	Alpha-Terpineol	Anthocyanin	Cinnamyl acetate	Ethyl 2-methylbutyrate	Ferulic acid
Sod1	− 4.17 ± 0.10	− 5.31 ± 0.15	− 6.8 ± 0.57	− 5.26 ± 0.17	− 4.32 ± 0.10	− 4.5 ± 0.32
FUS	− 3.82 ± 0.20	− 5.44 ± 0.22	− 7.1 ± 0.26	− 5.3 ± 0.31	− 4.03 ± 0.11	− 5.53 ± 0.20
TARDBP	− 3.46 ± 0.24	− 5.01 ± 0.25	− 6.56 ± 0.25	− 4.88 ± 0.16	− 3.49 ± 0.13	− 5.28 ± 0.44
MAPT	− 2.54 ± 0.22	− 4.09 ± 0.03	− 4.29 ± 0.25	− 3.70 ± 0.23	− 2.58 ± 0.10	− 3.51 ± 0.12

Fig. 3.

a 3D visualization of interactions between human target FUS and dietary compound anthocyanin b: 2D visualization of interactions between human gene target and dietary compound anthocyanin

For the D. melanogaster orthologs, Cocoon demonstrated the strongest binding with Phenanthrene, with a binding score of − 6.7 kcal/mol (Table 5), involving interactions with residues such as Thr260(A), Ser259(A), Phe195(A), Phe232(A) and Asp262(A) (Fig. 4a,b). Similarly, Cocoon exhibited a high binding affinity with Anthocyanin, scoring − 7.8 kcal/mol (Table 6). This interaction was stabilized by hydrogen bonds with Lys167(A), Leu165(A), Arg209(A), Val221(A), Val178(A), Cys176(A) and Arg168(A) along with hydrophobic contacts with Leu165(A) and Val178(A) (Fig. 5a,b). These results highlight the potential for dietary compounds to influence protein behavior in a protective manner against environmental toxins.

Table 5.

Binding affinities (kcal/mol) of D. melanogaster target proteins with soot-derived compounds (mean ± SD)

Target	Acenaphthene	Acenaphthylene	Anthracene	Fluorene	Naphthalene	Phenanthrene
Sod1	− 5.40 ± 0.10	− 5.13 ± 0.18	− 6.54 ± 0.13	− 5.83 ± 0.11	− 4.84 ± 0.05	− 6.20 ± 0.05
Cocoon	− 5.67 ± 0.14	− 5.62 ± 0.19	− 6.09 ± 0.30	− 5.81 ± 0.24	− 5.11 ± 0.24	− 6.5 ± 0.39
Caz	− 5.67 ± 0.30	− 5.83 ± 0.29	− 6.04 ± 0.30	− 5.82 ± 0.49	− 5.12 ± 0.20	− 6.39 ± 0.19
Tau	− 4.64 ± 0.12	− 4.59 ± 0.11	− 5.00 ± 0.17	− 4.78 ± 0.28	− 4.14 ± 0.12	− 5.19 ± 0.34

Fig. 4.

a: 3D visualization of interactions between D. melanogaster target cocoon and DE compound phenanthrene b: 2D visualization of interactions between D. melanogaster target cocoon and DE compound phenanthrene

Table 6.

Binding affinities (kcal/mol) of D. melanogaster target proteins with strawberry-derived compounds (mean ± SD)

Targets	2-Hexenol	Alpha-Terpineol	Anthocyanin	Cinnamyl acetate	Ethyl 2-methylbutyrate	Ferulic acid
Sod1	− 3.76 ± 0.36	− 5.13 ± 0.18	− 7.12 ± 0.17	− 5.23 ± 0.07	− 3.87 ± 0.22	− 5.36 ± 0.18
Cocoon	− 3.10 ± 0.17	− 4.78 ± 0.29	− 7.23 ± 0.45	− 4.76 ± 0.24	− 3.34 ± 0.19	− 5.32 ± 0.51
Caz	− 3.30 ± 0.14	− 4.57 ± 0.34	− 6.16 ± 0.31	− 4.90 ± 0.47	− 3.51 ± 0.29	− 5.10 ± 0.43
Tau	− 2.71 ± 0.11	− 3.80 ± 0.21	− 4.99 ± 0.24	− 3.80 ± 0.17	− 2.59 ± 0.13	− 3.86 ± 0.10

Fig. 5.

a: 3D visualization of interactions between D. melanogaster target cocoon and dietary compound anthocyanin b: 3D visualization of interactions between D. melanogaster target and dietary compound anthocyanin

In the MLSD studies, the human genes exhibited notable interactions when exposed to combinations of soot and strawberry compounds. TARDBP displayed the highest binding affinity with the combination of Acenaphthene and Anthocyanin, resulting in a binding score of − 5.38 kcal/mol. SOD1 also showed significant interactions with Anthracene and Cinnamyl acetate, scoring − 5.37 kcal/mol. For the D. melanogaster genes, Tau demonstrated intermediate interactions with the combination of Acenaphthene and Anthocyanin, achieving a binding score of − 6.26 kcal/mol. Caz also showed notable binding with Acenaphthene and Anthocyanin, with a score of − 6.11 kcal/mol. These results suggest that in complex environmental contexts, these proteins may experience altered activity due to the simultaneous presence of multiple ligands, potentially affecting their roles in neurological pathways.

Further, in the MLSD study involving the FUS gene, both Phenanthrene and Anthocyanin were docked simultaneously, and the combined interaction resulted in a reduced binding score of − 3.39 kcal/mol (Fig. 6). While anthocyanin maintained its interaction with key residues such as Lys309(A), Arg376(A), and Glu270(A) in both SLD and MLSD, there was a reconfiguration of the binding pocket, causing minor shifts in the ligand's orientation. In contrast, phenanthrene showed a significant loss of interacting residues in MLSD, with several hydrophobic contacts-particularly Glu161(A), Val167(A), and Ile162(A) no longer participating in the interaction. Phenanthrene’s remaining interactions were primarily with Leu129(A), Leu130(A), and Leu123(A), resulting in a weakened interaction network in MLSD. This reduction in phenanthrene’s binding affinity indicates a competitive effect of anthocyanin suggesting that anthocyanin modulates phenanthrene’s interaction with FUS.

Fig. 6.

2D visualization of interactions between human target FUS and the combined presence of DE compound phenanthrene and dietary compound anthocyanin

In the MLSD study of the D. melanogaster gene Cocoon with phenanthrene and anthocyanin, a combined binding score of − 3.18 kcal/mol (Fig. 7) was observed, with both ligands interacting through the same residues as in SLD. The key observation is that all amino acids involved in the binding interactions in SLD were also present in MLSD. The primary difference between SLD and MLSD lies in the reconfiguration of the binding pockets, driven by the simultaneous presence of both ligands, without the exclusion of any binding residues.

Fig. 7.

2D visualization of interactions between D. melanogaster target cocoon and the combined presence of DE compound phenanthrene and dietary compound anthocyanin

From the SLD and MLSD results, minimal changes were observed in the residue-level binding interactions. However, a noticeable variation was identified in the docking scores between the SLD and MLSD approaches. Although the docking scores for phytochemicals and soot-derived compounds in the SLD setup showed slight differences, these variations were insufficient to support the hypothesis.

Minimal changes were observed in the residue-level binding interactions. However, a noticeable variation was identified in the docking scores between the SLD and MLSD approaches. Although the docking scores for phytochemicals and soot-derived compounds in the SLD setup showed slight differences, these variations were insufficient to support the hypothesis. The docking scores for the controls have been mentioned in Tables 7 and 8.

Table 7.

Binding affinities (kcal/mol) of human target proteins with controls (mean ± SD)

Target	Formaldehyde (Negative control)	Quercetin (Positive control)	Resveratrol (positive control)
Sod1	− 1.62 ± 0.07	− 7.48 ± 0.30	− 6.81 ± 0.21
FUS	− 1.67 ± 0.07	− 7.11 ± 0.26	− 6.44 ± 0.27
TARDBP	− 1.51 ± 0.15	− 7.37 ± 0.28	− 6.20 ± 0.52
MAPT	− 1.00 ± 0.10	− 4.41 ± 0.21	− 4.37 ± 0.36

Table 8.

Binding affinities (kcal/mol) of D. melanogaster target proteins with controls (mean ± SD)

Targets	Formaldehyde (Negative control)	Quercetin (Positive control)	Resveratrol (positive control)
Sod1	− 1.41 ± 0.11	− 7.06 ± 0.19	− 6.73 ± 0.34
Cocoon	− 1.41 ± 0.09	− 6.79 ± 0.69	− 6.06 ± 0.20
Caz	− 1.33 ± 0.11	− 6.38 ± 0.39	− 5.96 ± 0.25
Tau	− 1.27 ± 0.17	− 5.12 ± 0.15	− 4.69 ± 0.12

Experimental validation

In the experimental study, strawberry extract, particularly the dose of 5.0 mg/mL, significantly improved the motor functions, thermotolerance and the maximal lifespan. This is consistent with an earlier study (Zhang et al. 2022), where a concentration of 5.0 mg/mL of bilberry significantly enhanced life expectancy. The higher concentration that is 10 mg/mL could be causing a metabolic burden, affecting energy levels and muscle function. Visible abdominal coloration changes confirmed ingestion, and images have been included in the Supplementary file.

Crawling assay

Larval movement is facilitated by body contractions, which are controlled directly by the motor neurons in the brain. Therefore, if there is any abnormality in the neurons, it is reflected as a defective crawling pattern of the larvae in flies (Kitamoto et al. 2001; Pulver et al. 2009). Initially, flies exposed to soot displayed a delayed developmental cycle. This delay could be linked to retardation of growth. Furthermore, the larvae crossed fewer grid lines (Fig. 8a,b) and showed an increase in diagonal movement, with the severity of these effects increasing with higher toxicant (diesel exhaust) concentrations. Diagonal movement may indicate neural or muscular impairments, as larvae with no defects tend to exhibit smooth, straight, or slightly curved movements. In contrast, both the flies fed with strawberry extract-supplemented food and the flies grown in normal food media crossed more grid lines, indicating improved locomotor function. There was no significant difference between the food supplemented with strawberry extract and control in females however, in males, the control flies crossed more grid lines. When both toxicants and the dietary supplement (strawberry) were present in the food medium, the flies crossed more grid lines compared to those reared in only the toxicant medium, suggesting that the dietary compounds may ameliorate the effect of the toxicant and these flies crossed lesser grid lines when compared to the flies raised in control and flies fed with strawberry extract-supplemented food. These behaviors were consistent across all concentrations and became more pronounced as toxicant and strawberry extract concentrations were increased. As shown in Fig. 8c, there was no significant difference in locomotor function between male and female flies. Due to the high variation in results, normality was assessed using the Shapiro–Wilk test (p > 0.05) before performing a one-way ANOVA to analyze the effect of treatment on crawling behavior in male and female D. melanogaster flies. For males, a significant effect was observed (F (9,20) = 37.67, p < 0.0001, η² = 0.53), indicating a strong treatment impact. Similarly, for females, the analysis showed a significant effect (F (9,20) = 70.10, p < 0.0001, η² = 0.3103).

Fig. 8.

(a) Comparison of number of grid lines crossed (cm) in males across different groups and concentrations. The p values (log-rank tests) for significant groups are: C vs. D 5% (p = 0.0090), T 5% vs. D 5% (p = 0.0006) and T 10% vs. T + D 10% (p = 0.0003). (b) Comparison of number of grid lines crossed (cm) in females across different groups and concentrations. The p values (log-rank tests) for significant groups are: T 2.5% vs T + D 2.5% (p = 0.0228), and T 2.5% vs. T 10% (p = 0.0228). (c) Gender differences in the number of grid lines crossed (cm) across different groups and concentrations. No significant results were obtained

Cold tolerance assay

Cold-induced peripheral neuropathy has been associated with neurophysiological changes, including reduced motor and sensory conduction velocities in the lower extremities (Song et al. 2017). In D. melanogaster, it has been demonstrated that diuretic neuropeptides have major impacts on the cold tolerance (Terhzaz et al. 2017). In this study, flies exposed to toxicants in their food medium exhibited slower recovery times, with recovery further delayed as the concentration of the toxicant increased. Conversely, flies that were fed food supplemented with strawberry extract recovered more quickly than those in the toxin-fed group. The reason for this might be because of the phytochemicals present in strawberry extract. There was no significant difference (Fig. 9a,b) between the flies grown in food supplemented with strawberry extracts and the control group. When both toxicants and the dietary supplement were included in the food medium, the flies showed a significantly faster recovery as compared to the flies raised in the toxins medium, suggesting that the dietary compounds may enhance cold tolerance. These behaviors remained consistent across all concentrations and became more pronounced as the concentrations of the toxicant and strawberry extract increased. As shown in (Fig. 9c), It was indicated that male flies were more tolerant to cold stress than females, as evidenced by their faster recovery times. This might be because males reduce their expression of sex-specific traits more significantly, allowing them to prioritize survival. This greater shift in metabolic and physiological processes helps them adapt better to cold conditions as seen in a different Drosophila species (Parker et al. 2021).

Fig. 9.

(a) Comparison of recovery time (s) in males across different groups and concentrations. The p values (log -rank tests) for significant groups are: T 2.5% vs. T 10% (p = 0.0048), T 5% vs. T + D 5% (p = 0.0048) (b) Comparison of recovery time (s) in females across different groups and concentrations. The p values (log-rank tests) for significant groups are: T 2.5% vs. T 10% (p = 0.0212), T 5% vs. T + D 5% (p = 0.0009). (c) Gender differences in recovery time (s) across different groups and concentrations. The p values (log-rank tests) for significant groups are: male vs. female C (p = 0.0159), T 2.5% (p = 0.0159), D 2.5% (p = 0.0159) and T + D 5% (p = 0.0159)

Geotaxis assay

The assay is based on negative geotaxis, the innate behavior where flies climb vertically when startled. The negative geotaxis assay in D. melanogaster is a valuable tool for studying locomotor deficits and behavioral changes associated with neurological diseases (Cao et al. 2017). For example, Drosophila models expressing human mutations linked to ALS, such as SOD1, show significant impairments in climbing, reflecting the disease's effect on motor neurons (Madabattula et al. 2015). In this research, adult flies exposed to diesel exhaust had a deleterious impact on locomotor behavior, with reductions in the climbing rate as compared to the control group. The control group’s climbing performance (male: 73.3–93.3%, female: 66.6–80.0%) aligns with previously reported values in wild-type D. melanogaster, where typical climbing rates range from 70 to 95% under ideal conditions (Zhong et al. 2022; Kharat et al. 2019; Garcia et al. 2019). This effect, however, was mitigated by the treatment with strawberry extract (T + D), as the flies in that group showed improved climbing performance. A discrepancy was observed where the control group and the D 5% treatment group exhibited similar climbing performance. In contrast, the D 2.5% and D 10% treatment groups demonstrated significantly reduced climbing abilities compared to the control (Fig. 10a,b). Additionally, no notable differences were observed between both the sexes (Fig. 10c).

Fig. 10.

(a) Comparison of flies climbed (%) in males across different groups and concentrations. The p values (log-rank tests) for significant groups are: T 5% vs. D 5% (p = 0.0032) and T 10% vs. T + D 5% (p < 0.0001) (b) Comparison of flies climbed (%) in females across different groups and concentrations. The p values (log-rank tests) for significant groups are: T 5% vs. D 5% (p = 0.0032), T 10% vs. D 10% (p < 0.0001), T 10% vs. T + D 5% (p < 0.0001) and T 10% vs. T + D 10% (p = 0.0013). (c) Gender differences in climbing ability across different groups and concentrations. No significant differences were observed between males and females at any concentration across the toxicant, dietary and combined toxicant + dietary treatments

Vortex assay

A notable change in the recovery time upon applying a mechanical stimulus (vortexing), for the flies exposed to toxicants and the control group was noticed. Flies exposed to toxicants in their food medium showed paralysis, increased mortality, and a significant increase in recovery time as the concentration of the toxicant increased. Recovery time of the flies exposed to 10% toxicant concentration was found to be the maximum. In contrast, flies that consumed food supplemented with strawberry extract not only recovered faster than those fed the toxic diet but also did not exhibit seizure-like behavior. When both toxicants and the dietary supplement (T + D) were present in the food medium, the flies demonstrated a markedly faster recovery, indicating a potential ameliorative effect of the dietary compounds. These observations were consistent across both male and female flies (Fig. 11a,b). However, it was noted that females generally took longer to recover than males as denoted in (Fig. 11c).

Fig. 11.

(a) Comparison of recovery time (s) in males across different groups and concentrations. The p values (log -rank tests) for significant groups are: T 2.5% vs. T 10% (p = 0.0004), T 5% vs. D 5% (p = 0.0003) and T 10% vs. T + D 10% (p = 0.0295). (b) Comparison of recovery time in females across different groups and concentrations. The p values (log-rank tests) for significant groups are: C vs D 10% (p = 0.0007), T 2.5% vs 10% (p = 0.0043), T 10% vs. D 10% (p < 0.0001), and T 10% vs. T + D 10% (p = 0.0003). (c) Gender differences in recovery time across different groups and concentrations. The p values (log-rank tests) for significant groups are: male vs. female T 2.5% (p < 0.0001), D 2.5% (p = 0.0060) and D 10% (p = 0.0278)

Stress assay

In both male and female flies, survival rates followed similar patterns across control, toxicant (T), dietary compound (D), and combined (T + D) treatments. The optimal survival temperature for flies is 28 °C (Sayeed et al. 1996; Goh et al. 2021). To determine the upper limit, the experiment began with 2 °C increments. No mortality was observed between 30 °C and 35 °C, but complete mortality occurred at 40 °C and above. Both genders maintained 100% survival in the control group up to 39 °C, with a sharp decline at 40 °C. Toxicant exposure led to a rapid drop in survival, especially at 10% toxicant concentration, where both males and females showed significant mortality starting at 37 °C as shown in (Fig. 12a,b). Flies treated with strawberry extract exhibited better thermotolerance than those fed with toxin, showing minor variations between males and females, such as slightly higher survival in females at 10% strawberry extract concentration and 39 °C. In the groups consisting of both toxicant and strawberry extract, partial protection and a similar delay in mortality were observed in the flies of both genders. Overall, the response to stress across treatments was consistent between males and females, indicating no major gender-specific differences in thermotolerance or toxicant sensitivity.

Fig. 12.

(a) Survival rates of male flies under thermal stress (36 °C to 40 °C) in control (C), toxicant (T: 2.5%, 5%, 10%), dietary compound (D: 2.5%, 5%, 10%), and combined treatments (T + D). The control group maintained 100% survival up to 39 °C, with a sharp decline at 40 °C. Toxicant groups exhibited significant mortality starting at 37 °C for higher concentrations, while dietary compound groups showed greater thermotolerance. The combined T + D groups demonstrated intermediate survival patterns, reflecting partial protection from the dietary compound. Data represent mean ± SE (n = 3 replicates, 12 flies per vial). (b) Survival rates of female flies subjected to thermal stress (36 °C to 40 °C) in control (C), toxicant (T: 2.5%, 5%, 10%), dietary compound (D: 2.5%, 5%, 10%), and combined treatments (T + D). Survival declines sharply at 39 °C and 40 °C, particularly in the toxicant (T) groups, with the lowest survival in T 10% at 40 °C. The dietary compound (D) partially mitigates thermal stress, especially at lower toxicant concentrations. Data represents mean ± SE from 3 replicates with 12 flies per vial

Comprising approximately 16,000 genes across four pairs of chromosomes, the D. melanogaster genome, despite its relative simplicity compared to the human genome, shares significant genetic homology with humans, with over 60% of its genes having identifiable human counterparts (Atoki et al. 2024). Broadly, there is evidence in processes generally common to both systems, such as the formation of laminated brain structures, the presence of progenitors with varied modes of neural stem cell division, and the observation that neurogenesis often precedes gliogenesis (El-Danaf et al. 2022). The adult D. melanogaster brain, like the adult mammalian brain, has few proliferative cells. In addition, although the D. melanogaster brain has many fewer neurons, it has many shared complexities with the human brain, including analogous neural cell types, common neurotransmitters (GABA, glutamate, and acetylcholine), similar synapse architecture, and similar physiology and intracellular signaling pathways (Crocker et al. 2021).

In the current study, the Canton-S (CS) strain of D. melanogaster was selected due to its well-characterized genetic background and consistent behavioral responses, making it a widely accepted model in neurotoxicological research. Studies comparing CS, w1118, and Oregon-R strains have shown that genetic background significantly influences neurotoxicity outcomes. For instance, research on cisplatin-induced neurotoxicity demonstrated that CS flies exhibit a distinct response profile compared to w1118 and Oregon-R, highlighting the importance of strain selection in neurotoxicological assessments (Groen et al. 2018). Additionally, w1118 flies have progressive mobility loss, reduced lifespan, and impaired stress resistance, which could confound neurotoxicity studies. Research has also advised against using w1118 as a wild-type control due to potential biological impairments beyond its eye-color mutation (Ferreiro et al. 2018). Oregon-R strains, on the other hand, have shown unexplained lifespan variations, adding another layer of variability (Lints et al. 1989). Furthermore, CS flies have been effectively used in neuroprotection studies, such as the investigation of epigallocatechin-3-gallate (EGCG) against paraquat-induced toxicity, demonstrating their suitability in neurotoxicology research (Martinez-Perez et al. 2018).

The dose selection was based on prior studies to ensure that the chosen concentration effectively highlights the physiological and toxicological effects of the substance. 10 mg/mL concentration was selected to be high enough to induce observable changes while remaining low enough to avoid immediate lethality, allowing for a comprehensive assessment of its impact. At 10 mg/mL, soot exposure in D. melanogaster is expected to cause severe effects, including high mortality, oxidative stress, developmental delays, reproductive decline, and neuromuscular impairments. Previous studies have shown that chronic exposure to microplastics at concentrations up to 10 mg/mL can induce toxic effects in D. melanogaster, with sex-specific differences in response (Kholy et al. 2023; Ranjan et al. 2024). Similarly, research on MagH₂ supplementation in D. melanogaster indicated that lower doses (0.1–1 mg/mL) had beneficial effects on lifespan, whereas higher concentrations (3–10 mg/mL) were toxic and significantly reduced survivorship. Magnesium hydride (MagH₂) itself releases molecular hydrogen (H₂), which has antioxidant and anti-inflammatory properties, but excessive exposure can lead to detrimental effects (Klichko et al. 2019).

Flies exposed to a food medium containing soot demonstrated a decline in motor functions, stress tolerance and thermotolerance, which worsened as the concentration of the toxicant increased. This revealed that exposure to environmental pollutants can impair motor neuronal functions. In contrast, flies treated with both soot compounds and strawberry extract showed improved motor performance, stress tolerance, and thermal resistance as compared to those exposed to soot compounds. The results obtained were consistent in both male and female flies, demonstrating that antioxidants protect against reactive oxygen species (ROS) and neural damage. Moreover, the observation that the treatment of flies with soot reduced eclosion rates in the flies is noteworthy. A study using the comet assay revealed significant genetic damage in flies exposed to urban environments compared to those from rural areas and negative control groups (De et al. 2018). In a study, soil samples from busy roads of Irbid, Jordan, contaminated with Pb, Cd, Cu, and Zn, significantly hindered survival, growth, and metamorphosis in D. melanogaster larvae. Interestingly, while the second generation displayed improved pupa survival, there was a decline in adult survival, suggesting that environmental pollutants continue to exert detrimental effects across generations (Massadeh et al. 2008). Furthermore, research using mice as a model organism revealed that ferulic acid reduces anxiety-like behavior in male rats induced by di-(2-ethylhexyl) (Khalifa et al. 2023). This highlights ferulic acid's anti apoptotic and antioxidant characteristics and emphasizes its neuroprotective effects.

The role and relationship between cold tolerance and neuroprotection are not yet fully established, but existing evidence suggests that neuronal ion homeostasis and synaptic stability play crucial roles. In D. melanogaster, exposure to cold leads to spreading depolarization (SD) in the central nervous system (CNS), where a sudden increase in extracellular K⁺ levels silences neuronal activity, triggering chill coma (Pool et al. 2017). Previous studies have indicated that older insects exhibit slower recovery from SD events, highlighting an age-related decline in neuronal resilience (Andersen et al. 2018). Importantly, the administration of antioxidants has been shown to mitigate oxidative stress, thereby accelerating recovery from SD. This suggests that antioxidants modulate SD effects by reducing oxidative damage, which is associated with the dysregulation of ion homeostasis during these events (Robertson et al. 2024).

Bang-sensitive (BS) mutants, such as bang-sensitive paralytic mutant (bss) and eas ethanol-induced abnormal seizures mutant (eas) and slamdance (sda), a gene involved in synaptic transmission, exhibit seizures, and paralysis and have been implicated in epilepsy. The bss1 mutant (a gain-of-function Na + channel mutation) shows a prolonged recovery time (~ 240 s) compared to other BS mutants like eas (~ 81 s) and (~ 38 s) (Parker et al. 2010). In this study, exposure to the toxicant (T 10%) resulted in recovery times of 50–75 s in males and 98–109 s in females, which are comparable to moderate-severe seizure models (eas: ~ 81 s, bss1: ~ 240 s). However, when dietary compounds were introduced (T + D 10%), recovery times significantly decreased (males: 32–47 s, females: 58–63 s), bringing them closer to sda (~ 38 s). This suggests that dietary supplementation may enhance recovery mechanisms, potentially by modulating oxidative stress or neurotransmitter balance.

While D. melanogaster Parkinson’s disease (PD) models primarily focus on dopaminergic neurodegeneration and motor impairments, seizure-like behaviors are not typically assessed in these models. Although some studies hint at a potential link between PD and seizure activity, this remains an unexplored area, highlighting the need for further investigation into neuroprotective mechanisms across different neurodegenerative and seizure disorders (Sandhu et al. 2017; Basu et al. 2011).

Strawberries are a rich source of polyphenols, including anthocyanins, ellagitannins, and proanthocyanidins, which have been widely studied for their antioxidant and anti-inflammatory properties. However, the precise mechanisms by which these polyphenols influence neurological and metabolic health remain complex and not fully understood (Da et al. 2024). Research suggests that strawberry-derived polyphenols can neutralize ROS, thereby reducing oxidative stress, a key factor in the development of metabolic and neurodegenerative disorders (Sandhu et al. 2017). Studies on the metabolic fate of strawberry polyphenols in healthy older adults have identified various polyphenolic metabolites in plasma, confirming their bioavailability and potential biological activity. Additionally, in obese adults with metabolic syndrome, regular consumption of strawberries, blueberries, and low-calorie cranberry juice has been associated with antioxidant effects, lowered serum cholesterol, and antihypertensive benefits (Basu et al. 2011).

The neuroprotective potential of strawberry polyphenols is further supported by epidemiological studies, which have shown that individuals consuming at least one serving of strawberries per week have a 34% lower risk of developing Alzheimer’s dementia compared to those consuming none or consuming them less than once per month (Agarwal et al. 2019)). Among these bioactive compounds, anthocyanins exhibit strong antioxidant activity, neutralizing ROS and maintaining cellular integrity. This reduction in oxidative stress is critical for preventing metabolic disorders and neurodegeneration. Additionally, anthocyanins can modulate inflammatory pathways by inhibiting nuclear factor kappa B (NF-κB) activation, thereby reducing chronic inflammation associated with metabolic diseases (Mohammadi et al. 2024).

Beyond anthocyanins, ellagitannins present in strawberries undergo microbial metabolism in the gut, producing urolithins. These metabolites are believed to contribute to gut health and brain wellness through the gut-brain axis, further supporting their neuroprotective and metabolic benefits (Banc et al. 2023).

For the in-silico studies, bioactive compounds in strawberries, such as anthocyanins, flavonoids, and tannins, exhibit variable bioavailability, which can influence their therapeutic efficacy (Cervantes et al. 2020). Prior pharmacokinetic studies indicate that strawberry anthocyanins have low and variable bioavailability (Xiao et al. 2017; Azzini et al. 2010), depending on factors such as meal timing and the presence of other dietary components (Sandhu et al. 2016). Flavonoids like quercetin and kaempferol undergo metabolic transformations into conjugated forms, which can be detected in plasma and urine, highlighting their systemic circulation potential (Banaszewski et al. 2013). A ≥ 30% OB and QED ≥ 0.18 threshold was applied to prioritize compounds that are more likely to achieve effective plasma concentrations and exert pharmacological effects. This threshold is consistent with previous studies on plant-based medicines for neurological disorders, where bioavailability played a crucial role in selecting potential therapeutic compounds (Banaszewski et al. 2013; Ye et al. 2022; Chatterjee et al. 2024; Yang et al. 2020).

For the protein models for D. melanogaster, the Swiss Model was selected since it is a comparative (homology) modeling tool that builds 3D protein structures based on experimentally solved homologous templates from the PDB (Biasini et al. 2014). It does not predict structures de novo like AI-based models (ESMFold, AlphaFold) or use energy-based folding like Rosetta.

In our studies, quercetin and resveratrol were used as positive controls, several in-vitro and in-vivo experiments have demonstrated that quercetin has significant anti-inflammatory activities and serves as an effective therapeutic agent against various neurological disorders by reducing stress, inflammatory response and fostering brain growth (Abdullahi et al. 2021; Islam et al. 2021). Studies have found significantly improved behavioral indices, amelioration of oxidative stress, improved antioxidant enzyme activities, and restoration of neurochemical parameters in the treated D. melanogaster flies (Ademiluyi et al. 2022). Resveratrol exhibits neuroprotective properties by promoting dopamine (DA) neuronal survival and activating the neurological pathways, which helps alleviate age-related motor decline (Kawamura et al. 2020). Additionally, it improves motor function and muscular strength in muscular dystrophy patients by reducing oxidative damage and enhancing muscle performance (Andrade et al. 2018). Further, Formaldehyde was included as a negative control because formaldehyde exhibits neurotoxic effects, impacting neuronal morphology, behavior, and biochemical parameters, with potential links to neurological diseases. Exposure to formaldehyde, particularly in anatomists, medical students, and industrial workers, necessitates precautions due to its systemic toxicity (Songur et al. 2010). Additionally, excessive formaldehyde accumulation in muscles and the cerebellum contributes to motor deficits, as demonstrated in hindlimb unloading models and formaldehyde dehydrogenase knockout mice, where formaldehyde buildup led to gait instability and cerebellar ataxia (Yao et al. 2021).

The docking analysis, where quercetin and resveratrol were used as a positive control, revealed that quercetin, resveratrol, and anthocyanin exhibited similar binding affinities with the human gene FUS, with quercetin, resveratrol showed score of − 7.11 ± 0.26 kcal/mol, − 6.44 ± 0.27 kcal/mol, respectively and anthocyanin − 7.1 ± 0.26 kcal/mol. This suggests that the compounds have comparable potential to interact with this protein. These comparable results were seen when the ligands had been docked with D. melanogaster genes as well. However, the negative control, formaldehyde, showed extremely low scores, highlighting the variability compared to the higher binding affinities observed with soot compounds. This might suggest that compounds present in soot form more stable bonds as compared to formaldehyde.

The docking scores in this research depict a different proposition and thereby we think that dietary compounds might be affecting off-target proteins or pathways that are critical for the toxicant's action. These off-target effects could mitigate the toxicant's impact in the organism, which would not be detected in the docking study that focused on a specific target or that the dietary compound may undergo metabolic transformation within the organism, leading to the production of metabolites that either negate or reduce the toxicant's effects. The in-silico model does not account for these metabolic processes, which might explain the discrepancy. Future research could focus on quantifying the protein expression to understand how strawberry extract modulates soot compound metabolism and docking simulations can be performed to study the binding affinity of compounds within strawberry extract and soot with other possible targets. Moreover, research should incorporate molecular validation techniques to further elucidate the neuroprotective effects of dietary compounds and their influence on neuronal function. Western blot and RT-qPCR can quantify neuronal markers and key gene expressions, such as SOD1, cocoon, and tau. These approaches would provide mechanistic insights into how strawberry-derived antioxidants modulate oxidative stress and neurotoxicity at a molecular level, complementing the behavioral findings of this study. Gene expression such as SOD1, cocoon, tau and many more need to be conducted. The applicability of Drosophila research extends beyond basic principles because it provides vital knowledge about neurodegenerative processes in higher-order organisms. Future research employing rodent models has the potential to elucidate the molecular mechanisms of neurodegeneration and facilitate the development of therapeutic strategies translatable to humans (Yin et al. 2022). Based on these results, we infer that there is attenuation by the dietary compounds and that environmental soot particles cause impairment to the motor neuronal functions and that the amelioration is due to certain antioxidant properties present in the strawberry extract.

Conclusion

This study shows that dietary strawberry extract has neuroprotective potential, which reduces motor dysfunction caused by soot exposure in D. melanogaster. The in-vivo tests showed enhanced motor movements along with increased resistance to stress and heat tolerance at strawberry extract doses, with soot-exposed flies exhibiting impaired motor function, reduced stress resilience, and delayed recovery. The crawling assay revealed that soot-exposed flies crossed fewer grid lines than the control (p = 0.0006), however, the addition of strawberry extract led to improved outcomes (p = 0.0003). The recovery time from cold stress was faster among strawberry-treated flies when compared to soot-exposed groups (p = 0.0048). The geotaxis assay indicated reduced climbing performance with soot exposure (p = 0.0032), but adding strawberries enhanced their climbing potential (p < 0.0001). Results from the vortex assay demonstrated that flies fed with strawberries recovered more quickly (p = 0.0003), and the stress assay validated better thermotolerance in the dietary treatment group through delayed death during heat exposure.

The human FUS gene exhibited its highest binding potential to anthocyanin from strawberries at − 7.1 ± 0.26 kcal/mol but phenanthrene from soot showed similar strength at − 7.6 ± 0.26 kcal/mol. For D. melanogaster orthologs, anthocyanin showed a binding strength of − 7.8 kcal/mol, while phenanthrene showed a docking score of − 6.7 kcal/mol. Cocoon and FUS reciprocal interactions decreased their binding scores to − 3.00 ± 0.24 kcal/mol and − 3.12 ± 0.11 kcal/mol, respectively, during MLSD, indicating competitive interactions.

This research contains significant limitations that need consideration. The study lacked measurements of biochemical markers of oxidative stress, such as ROS and antioxidant enzymes. The short lifespan of D. melanogaster may limit the extrapolation of findings to longer-lived organisms like humans. The investigation needs to address the off-target effects of strawberry polyphenols and their by-products. Future studies may incorporate food intake measurements for further validation, while CS was chosen for this study, future investigations could explore the effects of diesel soot exposure on different Drosophila strains to further assess strain-dependent neurotoxicity responses.

Expanding this research to mammalian models, such as rodents, can provide insights into the long-term neuroprotective impacts of strawberry supplementation. Additionally, proteomic and transcriptomic studies have the potential to identify molecular pathways involved in neuroprotection, potentially guiding dietary interventions for mitigating environmental neurotoxicity.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

“Adithi GR, Ananya M & Nidhi N Gambhir contributed to conceptualization, methodology, material preparation, data collection and analysis, original draft preparation. Jhinuk Chatterjee contributed to conceptualization of in silico analysis, methodology, supervision, finalization of manuscript. Professor Dr. Carmen Coelho from Centre of Human Genetics, Bangalore mentored and critically reviewed in vitro assay part of the manuscript. All authors read and approved the final manuscript.”

Funding

“The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.”

Data availability

“Data is provided within the supplementary information files."

Declarations

Competing Interests

The authors declare no competing interests.

Ethics approval

“No ethical approval is required for the current study.”