Fermented Moringa oleifera leaves and Ganoderma lucidum mixtures ameliorate cognitive deficits in scopolamine-induced dementia rats by enhancing brain antioxidant and cholinergic functions

Abstract

Context

Moringa oleifera Lam. (Moringaceae) and Ganoderma lucidum (Curtis) P. Karst. (Ganodermataceae) are two natural resources with established neuroprotective properties. However, whether their combination is safe and has neuroprotective effects against dementia remains unexplored.

Objective

This study aimed to investigate the phytochemical composition, toxicity profile, and neuroprotective activity of fermented M. oleifera and G. lucidum mixture (FMG) in scopolamine-induced dementia model rats.

Methods

FMG was produced by fermentation with Bacillus subtilis. A state of cognitive impairment was induced in rats via daily intraperitoneal administration of scopolamine (4 mg/kg) for 28 days. Following a two-week treatment period, cognitive function was assessed using the Y-maze. Postmortem analyses included biochemical assays to measure brain acetylcholinesterase (AChE) activity and oxidative stress markers, and histological examination of the hippocampus.

Results

LC-MS analysis revealed a rich phytochemical profile. The no-observed-adverse-effect level (NOAEL) was 200 mg/kg/day, while the lowest-observed-adverse-effect level (LOAEL) was 600 mg/kg/day. Treatment at the 200 mg/kg dose significantly reversed memory deficits, restoring spontaneous alternation from 29.1% in scopolamine-treated rats to 82.6% (p < 0.05). This behavioral recovery was correlated with a significant reduction in brain AChE activity, a normalization of lipid peroxidation (TBARS) levels, and the restoration of hippocampal neuronal architecture.

Discussion and conclusions

The restorative effects of FMG are mediated by a dual mechanism involving the enhancement of central cholinergic and antioxidant systems. These results suggest that FMG possesses neuroprotective and antioxidant properties and could be a promising candidate for the management of cognitive deficits.

1. Introduction

Dementia is a progressive neurodegenerative disease that results in memory decline, language impairment, behavioral issues, and the depletion of socialization skills and physical abilities (Cerejeira et al. 2012). Dementia not only adversely affects the quality of life of patients but also has a significant effect on their families and healthcare systems worldwide (Cantarero-Prieto et al. 2020; Mattap et al. 2022). The Global Burden of Disease (GBD) 2019 Dementia Forecasting Collaborators estimated that 57.4 million individuals had dementia in 2019, and that figure will rise to 152.8 million by 2050 (GBD 2019 Dementia Forecasting Collaborators 2022). Surprisingly, 68% of cases are expected to occur in low- and middle-income countries (Rosli et al. 2021). Dementia care is complex and expensive (Nandi et al. 2022). The estimated total cost of lifetime care for a person with dementia is $360 billion in 2024 and nearly $1 trillion in 2050 (Alzheimer’s Dement 2023). Without investment in more effective and accessible treatments and prevention strategies, dementia will impede economic growth and compromise global health and economic equity.

The complex pathology of dementia, involving multiple factors such as cholinergic deficits, oxidative stress, and neuroinflammation, has challenged the efficacy of single-target synthetic drugs. This has spurred the search for multi-target therapeutic strategies. In this context, plant–fungi formulations have garnered significant attention as promising therapeutic approaches for treating a diverse range of diseases. Growing evidence from in vitro and in vivo studies highlights the intricate interplay between plant-derived phytochemicals and fungal secondary metabolites to target multiple biological pathways simultaneously (Vaou et al. 2022). This multitarget property enables plant–fungi formulations to address disease complexity more comprehensively than single-component therapies. It may offer advantages in conditions resistant to conventional treatments, such as dementia. Moringa oleifera Lam. (Moringaceae) and Ganoderma lucidum (Curtis) P. Karst. (Ganodermataceae) are two promising candidates for understanding this interaction, as both have been shown to possess potent neuroprotective properties (Arozal et al. 2022).

Moringa oleifera, often called the ‘miracle tree’, has traditionally been used in Ayurvedic and African medicine for treating inflammation, fatigue, and age-related disorders (Srivastava and Ganjewala 2024). Recent studies show that M. oleifera leaves contain high levels of neuroprotective phytochemicals such as quercetin, kaempferol, genistein, and various phenolic acids (Igado and Olopade 2017; Kou et al. 2018; Abdel-Rahman Mohamed et al. 2019). In vitro studies reveal that M. oleifera extracts protect neuronal cells from oxidative stress, enhance cell viability, and inhibit mitochondrial dysfunction (González-Burgos et al. 2021). The leaf extracts also promote neurogenesis, neuronal survival, and inhibit acetylcholinesterase (AChE), thereby preserving acetylcholine levels crucial for memory and learning (Anwar et al. 2005; Hannan et al. 2014; Khan et al. 2017; Rocchetti et al. 2020). Moringa oleifera leaf has consistently been shown to mitigate neurodegeneration in Alzheimer’s model rats by enhancing antioxidant enzyme activity and preserving hippocampal structure, with notable improvements in spatial learning and memory (Jahn 2013; Sutalangka et al. 2013; Mahaman et al. 2018; Afrin et al. 2022). More importantly, it has recently been reported that fermentation significantly increased the levels of flavonoids, polyphenols, and kaempferol compared to the unfermented extract of M. oleifera (Tran et al. 2023). Fermentation was also shown to reduce plant tannin content, which may contribute to improved palatability and bioavailability (Li et al. 2024). In addition, a higher anti-inflammatory activity was observed in the fermented than in the unfermented M. oleifera (Lee et al. 2020).

Ganoderma lucidum, known as ‘Lingzhi’ in traditional Chinese medicine, has been used for centuries as a longevity tonic and for managing fatigue, insomnia, and memory decline (Wachtel-Galor et al. 2011). It contains polysaccharides, triterpenoids, and phenolic compounds with potent antioxidant, anti-inflammatory, and neurotrophic effects (Saltarelli et al. 2019; Luo et al. 2024). Studies have shown that G. lucidum protects neuronal cells from ROS-induced apoptosis, promotes neurite outgrowth, and supports synaptic integrity (Zhou et al. 2012; Sun et al. 2017). Ganoderma lucidum extracts have also been reported to reduce oxidative stress, modulate neuroinflammation, improve memory performance, and inhibit AChE activity in cognitive deficit animal models, highlighting its therapeutic potential against neurodegenerative disorders (Lee et al. 2011; Lai et al. 2019; Qin et al. 2019; Rahman et al. 2020; Chen et al. 2024). Interestingly, fermentation has also been shown to significantly improve the therapeutic potential of G. lucidum, particularly in terms of its anti-inflammatory and immune response regulation (Yang et al. 2015a, 2015b).

Despite the individual promise of both fermented M. oleifera and G. lucidum in preclinical models, their potential therapeutic synergy has not been explored. To date, no studies have investigated the neuroprotective efficacy of their combination. Furthermore, the safety and toxicological profile of such a mixture, a critical prerequisite for any therapeutic development, remains completely unknown. Therefore, the present study was designed to address this critical gap. We aimed to investigate the neuroprotective effects of a novel, fermented M. oleifera and G. lucidum mixture (FMG) in a scopolamine-induced dementia model. The study evaluated the phytochemical composition, subacute toxicity, and the mixtures ability to ameliorate cognitive deficits through behavioral, biochemical, and histological analyses.

2. Materials and methods

2.1. Plant material

The fine powders of M. oleifera leaves and G. lucidum fruiting body were obtained from Bio Fluid Sdn Bhd, Malaysia. The plant samples were deposited, identified, and verified at the Biodiversity Unit, Institute of Bioscience, Universiti Putra Malaysia (UPM) by Dr. Khairil Mahmud. The voucher numbers for M. oleifera leaves and G. lucidum were KM 0194/25 and KM 0195/25, respectively.

2.2. Preparation of fermented Moringa-Ganoderma (FMG)

The fermentation of M. oleifera and G. lucidum mixtures was conducted following the method described by Tan et al. (2020) with modification. The fine powders of M. oleifera and G. lucidum were homogeneously mixed at a 2:1 ratio. The mixture was soaked in 1% (w/v) glucose solution at a ratio of 1:10 w/v (plant powder: water). The solution was then heated in a water bath (Modern-Lab, Malaysia) at 90 °C for 30 min to increase the bioavailability, yield, and production rate (Zhang et al. 2019). The mixture was then left at room temperature to cool before being inoculated with 5% (v/v, 1 × 10⁵ CFU/mL) Bacillus subtilis ATCC 10876. The inoculated formulation was then placed in a shaker incubator (Innova 4000, Marshall Scientific, USA) and incubated at 37 °C with constant agitation at 200 rpm for 48 h, as B. subtilis has a short fermentation cycle (Chen et al. 2016). The fermented broth was then filtered through filter paper with a pore size of 1 µm (Tisch Scientific, USA) to remove the debris and allow the probiotic to pass through along with the supernatant (Carneiro et al. 2022). The supernatant was then concentrated via freeze–drying methods to preserve microorganisms (Wang et al. 2021), prolong stability (Luo et al. 2021), and lower the pigment content (Janiszewska-Turak et al. 2022).

2.3. Liquid chromatography–mass spectrometry (LC–MS) analysis

For this analysis, the freeze-dried FMG powder was reconstituted in ultrapure water to a final concentration of 5 mg/mL. The solution was filtered with a 0.22 µm syringe filter to remove particulates. The LC–MS analysis of FMG was conducted following a protocol by Jam et al. (2023) via an Agilent 6520 Q-TOF system coupled with a MassHunter workstation (B.02.01). Chromatographic separation was performed on a ZORBAX Eclipse Plus C18 column (1.8 µm, 2.1 × 100 mm) maintained at 40 °C, with a mobile phase of 0.1% formic acid in water (A) and acetonitrile (B) at 0.25 mL/min. A 32-min gradient was applied: 5% B (0–1 min), linear increase to 95% B (1–18 min), hold (18–20 min), and re-equilibration (20–32 min). Positive ESI mode was used with real-time calibration (reference ions: m/z 121.0529 and 922.0098).

For data analysis, the raw files (.d) were converted to .mzML (ProteoWizard) files and processed in MZmine 4.4. Mass features were detected (noise threshold: 1500 counts) with a chromatogram parameter of 0.01 min, 5000 minimum height, and 5 ppm m/z tolerance. The peak alignment used 10 ppm m/z tolerance, 0.3 min RT tolerance, and 70:30 weighting (m/z:RT). Metabolites were annotated against the KEGG and ChemSpider databases.

2.4. Animal ethics and maintenance

The study was conducted on 60 healthy Sprague–Dawley rats (24 females and 36 males) aged 8 weeks, weighing between 200–250 g for males and 160–190 g for females, purchased from Sapphire Enterprise, Selangor, Malaysia. Compared with their male counterparts, female rats were used for the toxicity study, as they are more sensitive to the drug (Pohjanvirta et al. 2012). On the other hand, a neuroprotective study of FMG was conducted on male rats because they are highly inclined toward exploration (Cavigelli et al. 2011). Male rats also have a significantly higher success rate as disease models, particularly in neurology (Bialy et al. 2019). The scopolamine-induced dementia protocol was chosen as it is a well-established and widely used pharmacological model for screening potential anti-amnesic agents, effectively mimicking the cholinergic deficit associated with dementia.

All animals were maintained under standard laboratory conditions (12-h light/dark cycle, 55–60% relative humidity, 23–25 °C) and allowed to acclimatize for one week before the experiment. Considering that rodents need to engage in social behavior, a maximum of three animals were housed in each polypropylene cage with dimensions of 427 × 287 × 198 mm (L × B × H). The animals had free access to food and water. Wood shavings (Pets Dream, Chipsi, Malaysia) were used as bedding materials to remove excess moisture, which can negatively impact their behavior (Tanaka et al. 2014). A pellet (Gold Coin, Malaysia) was used as a food source with the following nutrient contents: crude protein (21%); crude fiber (5%); crude fat (3%); moisture (13%); ash (8%); calcium (0.8%); and phosphorus (0.4%). All study protocols, including the toxicity study, dementia induction and sacrifice technique, were approved on 1st December 2023 by the Committee on Animals for Research and Ethics, Universiti Teknologi MARA, Shah Alam (UiTM CARE: 433/2023), and performed in strict accordance with the institutional guidelines and the Animal Research: Reporting of In vivo Experiments (ARRIVE) guidelines 2.0. Animals were to be euthanized if they exhibited a body weight loss of ≥20%, severe lethargy, unresponsiveness, impaired mobility, or labored breathing. However, none of the animals exhibited clinical signs that met these predefined humane endpoint criteria during the study period.

At the end of the subacute toxicity and efficacy studies, the rats were fasted overnight before being anesthetized with a mixture of xylazine (Sigma-Aldrich, USA) (1.5 mL of 100 mg/mL) and ketamine (Sigma-Aldrich, USA) (10 mL of 100 mg/mL) given intraperitoneally (0.1 mL/100 g). Once the animals exhibited a loss of pedal and corneal reflexes, indicating a surgical plane of anesthesia, they were humanely euthanized via terminal cardiac puncture using a 5 mL syringe fitted with an 18-gauge needle. Death was confirmed according to the AVMA Guidelines for the Euthanasia of Animals, based on the cessation of heartbeat and respiratory movements, followed by exsanguination.

2.5. Subacute toxicity study of FMG

2.5.1. Experimental design

The subacute toxicity study of FMG was conducted according to the OECD Guidelines for the Testing of Chemicals: Repeated Dose 28-day Oral Toxicity Study in Rodents (TG 407) with modifications. In this study, FMG was administered to the animals by oral gavage at a volume of 1 mL/100 g body weight (bwt) once daily for 28 days. A total of 24 nonpregnant female Sprague–Dawley rats were used and assigned to four groups of 6 animals each: Group 1: Control—0.9% saline (NC); Group 2: 200 mg/kg bwt/day of FMG (FMG200); Group 3: 400 mg/kg bwt/day of FMG (FMG400); and Group 4: 600 mg/kg bwt/day of FMG (FMG600). Weekly assessments of body weight were conducted, while daily monitoring was implemented to detect any signs of toxicity, such as breathing, salivation, locomotion, behavior, lacrimation, cyanosis, and death.

On day 29, blood sample were collected (10 mL/150 g) and divided into two tubes for specific analyses. A plain red-top tube containing no anticoagulants (BD VacutainerVR, USA) (subsequently processed for serum and stored at −40 °C) was used for biochemical analysis, and sterile purple tubes with anticoagulant (BD VacutainerVR, USA) were used for hematological analyses. The kidneys, liver, and spleen were collected, rinsed with 0.9% saline solution, and measured to determine the relative organ weight via the following formula:

$$\mathit{\text{Relatives organ weight}}=\frac{\mathit{\text{Organ weight}}\left(g\right)}{\mathit{\text{Body weight}}\left(g\right)}$$ (1)

All the collected organs were immediately preserved in a 10% formalin solution for histopathological examination.

2.5.2. Blood hematological analysis

Hematological analysis was conducted to determine the health status of the animals via blood profiling following 28 days of FMG administration. Hematological measurements, including white blood cell count (WBC), hemoglobin (Hb), red blood cell count (RBC), platelet (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), packed cell volume (PCV), and plasma protein, lymphocyte, monocyte, eosinophil and basophil counts, were performed via an automatic hematological analyzer (MEK 6550, Nihon Kohden, Japan).

2.5.3. Serum biochemical analysis

Biochemical serum analysis was conducted to investigate the impact of FMG on liver and kidney function markers. The liver function markers examined were albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Moreover, the evaluation of kidney function included creatinine (Creat) and urea levels. These assessments were carried out via an automatic biochemical analyzer (Biolis 24i Premium, Biorex, Malaysia).

2.5.4. Histological analysis

Histological analysis of the kidney, liver, and spleen was conducted to observe any morphological changes following 28 days of FMG administration. Following fixation, the organs were processed via automated tissue processing (Leica TP1020, Leica, USA) before paraffin embedding. A semiautomated rotary microtome (HistoCore MULTICUT, Leica, USA) was used to prepare organ sections with a thickness of 5 µm. All hematoxylin and eosin-stained sections were examined via a light microscope (Motic BA410, Wetzlar, Germany) equipped with a digital camera (Moticam Pro 285A, Wetzlar, Germany).

2.6. Neuroprotective study of FMG

2.6.1. Scopolamine injection

All the rats except those in the normal control group were subjected to scopolamine injection (Sigma-Aldrich, USA). Scopolamine was injected intraperitoneally (i.p.) daily (4 mg/kg) for 28 days (Assi et al. 2022). Scopolamine was prepared in a vehicle solution of 0.9% saline.

2.6.2. Experimental design

A total of 36 male Sprague–Dawley rats were randomly assigned to six groups of 6 animals each. Group 1: normal control–0.9% saline (NC); Group 2: dementia control–scopolamine + 0.9% saline (DC); Group 3: dementia–scopolamine + 0.5 mg/kg donepezil hydrochloride (DD); Group 4: scopolamine + 200 mg/kg bwt/day FMG (D-FMG200); Group 5: scopolamine + 400 mg/kg bwt/day FMG (D-FMG400); Group 6: scopolamine + 600 mg/kg bwt/day FMG (D-FMG600). Treatment with donepezil (Eisai, Japan) and FMG was given for 14 days after 28 days of scopolamine injection. Weekly assessments of body weight were conducted throughout the study. In this study, the dose of donepezil in rats was calculated based on the minimal human dose prescribed for a person diagnosed with mild-to-moderate Alzheimer’s disease (5 mg/day). This dose was converted to the Human Equivalent Dose (HED) for a rat by multiplying the human dose by an allometric scaling factor. This calculation was based on an average human weight of 60 kg, resulting in the 0.5 mg/kg dose used in this study. Both donepezil and FMG were prepared in a vehicle solution of 0.9% saline. The administration was conducted via oral gavage, with a standardized volume of 1 mL per 100 g of body weight.

2.6.3. Neurobehavioral assessment

The behavioral test was performed via the Y-maze between 10:00 am and 04:00 pm to assess both hippocampus-dependent spatial working memory and reference memory in the rats. The Y-shaped maze apparatus was made of black opaque acrylic materials with three identical arms (40 cm long × 35 cm high × 12 cm wide) separated by an angle of 120° from one to the other and an equilateral triangular center. Each arm was labeled A, B, and C, as shown in Figure 1(i). The behavioral test was carried out in a quiet and dimly lit room to maximize the protocol for evaluating treatment effects.

Figure 1.

The behavioral test. (i) Y-maze setup, (ii) spatial working memory test protocol, and (iii) spatial reference memory test protocol.

The spatial alteration of the rats was conducted following methods described in the literature (Kraeuter et al. 2019). Each rat was placed in the center of the Y-maze and allowed to freely explore all three arms of the maze for 8 min, as illustrated in Figure 1(ii). Spontaneous alternation is an entry into three different arms on consecutive choices (e.g., ABC, CAB, and BCA) (Galeano et al. 2014). Rats exhibit natural curiosity, which drives them to explore previously unvisited areas, as shown by the high alteration percentages (Lalonde 2002). The arm entry list, time spent in each arm, mobile time, and average speed were collected via video tracking software (ANY-maze, Stoelting Co., Wood Dale, IL, USA). The percentage of spontaneous alternation was calculated according to the following formula:

$$\mathit{\text{Percentage of alternation}}\mathrm{ }\left(\mathrm{\%}\right)=\frac{\mathit{\text{Number of alternation}}}{\left(\mathit{\text{Total number of arm entries}}-2\right)}\times 100$$ (2)

A spatial reference memory test was conducted to determine the ability of the rats to retain their memory. This test was performed according to protocols described in the literature (Swonger and Rech 1972; Kraeuter et al. 2019). This protocol placed three bright distal cues at the respective arms (Figure 1(iii)). This test was divided into ‘training’ and ‘test’ sessions, which were separated by a 4-h interval. During the 15-min training period, the rats were placed in the ‘Start’ arm and allowed to explore both the ‘Start’ and ‘Other’ arms, whereas the ‘Novel’ arm was blocked so that it could not be explored by the rats (Figure 1(iii)). The blockade in the ‘Novel’ arm was opened during the test session. The rats were allowed to explore the Y-maze freely by placing them in the same ‘Start’ arm as in the training session for 5 min. A rat with no preference for any arms during the test session indicated a potential deficit in spatial memory and exploration behavior (Ghafouri et al. 2016). Arm choice and time spent in each arm were recorded via video tracking software (ANY-maze, Stoelting Co., Wood Dale, IL, USA).

2.6.4. Collection of blood and brain tissues

Blood samples were collected (10 mL of blood/150 g rat) (Beeton et al. 2007) and centrifuged at 4000 rpm to obtain the serum. The whole brain was quickly excised, washed with an isotonic ice-cold NaCl (0.9%, w/v) solution, blotted to dryness, and weighed. The brain was divided into two parts: one for histological analysis and the other for biochemical and metabolomic analyses. The serum and brain samples were frozen at −80 °C until further analysis.

2.6.5. Biochemical analysis of the brain

The brain tissue homogenate (10%, w/v) was prepared in 50 mM sodium phosphate buffer (pH 7.4) via a Potter–Elvehjem homogenizer fitted with a Teflon-coated pestle under ice-cold conditions (4–6 °C). The brain homogenates were centrifuged at 9000 × g in a refrigerated centrifuge (4 °C) (Sorvall, Thermo Scientific, USA) for 10 min. The acquired supernatant was assayed via commercial kits, which included an AChE kit to determine cholinergic neurotransmission activity in the brain; a TBARS kit to measure lipid peroxidation in the brain; and GPX-1, catalase, and SOD kits to determine antioxidant activities. All kits were obtained from Qayee-Bio (China). The protein concentration was determined via the Bradford method, with bovine serum albumin as the standard for calibration (Lowry et al. 1951). The enzyme activity was then determined via the following formula:

$$\mathit{\text{Enzyme activity}}=\frac{\mathit{\text{Enzyme concentration}}\left(\frac{\eta g}{\mathit{\text{mL}}}\right)}{\mathit{\text{Protein cocentration}}\left(\frac{\mathit{\text{mg}}}{\mathit{\text{mL}}}\right)}$$ (3)

2.6.6. Histological examination

Histological analysis was conducted according to the protocols described in subheading 2.5.4. Hippocampal changes, especially those in the Cornu Ammonis (CA) 1, CA2, CA3, and dentate gyrus (DG), were examined under a light microscope (Motic BA410, Wetzlar, Germany) equipped with a digital camera (Moticam Pro 285A, Wetzlar, Germany).

2.7. Data analysis

The results are presented as the mean value ± standard deviation (SD). The normality of the data distributions was assessed via the Kolmogorov–Smirnov test. The body weight, relative organ weight, absolute organ weight, hematological data, biochemical data, and behavioral data were analyzed via one-way ANOVA followed by Tukey post hoc analysis. Additionally, the first-arm choice in the reference memory test was analyzed via chi-square analysis to determine the associations between arm choice and treatment. Pearson correlation analysis was used to determine the correlations among all the behavioral parameters. All the statistical analyses were conducted via Prism version 9.0 (GraphPad Software Inc., San Diego, CA, USA) and R version 4.3.2 (R Foundation for Statistical Computing, Vienna, Austria). A 95% confidence level was applied, and results with p < 0.05 were considered statistically significant.

3. Results

3.1. Liquid chromatography–mass spectrometry (LC–MS) analysis

LC–MS analysis revealed that FMG contained 20 unique nonvolatile compounds that can be divided into nine groups (Table 1). Among these, trans-cinnamate was the predominant constituent, with a peak area of 271,554 a.u. (Figure 2).

Table 1.

Nonvolatile compounds identified in FMG extract by LC–MS.

No	Compound name	Compound formula	Retention time (min)	Observed m/z	Peak area (a.u)
1	Raffinose	C18H32O16	0.98	527.15	46,762
2	Anthranilic acid	C7H7NO2	1.00	138.05	18,289
3	L-proline	C5H9NO2	1.10	116.07	25,236
4	Tyramine	C8H11NO	1.12	138.05	18,289
5	N-acetyl-L-leucine	C8H15NO3	1.85	174.11	64,995
6	L-valine	C5H11NO2	2.48	118.08	65,486
7	L-pipecolate	C6H11NO2	2.68	130.08	23,787
8	Aromatic aldehyde	C7H6O	2.88	107.04	23,787
9	Tetraneurin A	C17H22O6	3.01	304.17	97,245
10	Salsoline	C11H15NO2	3.54	194.11	32,809
11	Trans-cinnamate	C9H8O2	4.94	166.08	271,554
12	Vitexin	C21H20O10	6.84	433.11	27,478
13	Quercetin	C15H10O7	7.83	303.05	44,668
14	N-acetylphenethylamine	C10H13NO	7.98	164.10	84,914
15	Luteolin 7-O-(6″-malonylglucoside)	C24H22O14	8.22	535.10	84,839
16	Dodecanoic acid	C12H24O2	8.26	218.21	28,510
17	N-isovalerylphenethylamine	C13H19NO	11.53	206.15	75,625
18	Hexadecasphinganine	C16H35NO2	12.03	274.27	50,780
19	Phytosphingosine	C18H39NO3	12.46	318.30	161,591
20	4-Methylthio-2-oxobutanoic acid	C5H8O3S	18.62	149.02	49,636

Bold values indicate the major compounds identified in the extract.

Figure 2.

LC–MS chromatogram of FMG. The inset chemical structure represents the most abundant compound in the extract, based on the quantitative integrated peak area.

3.2. Subacute toxicity study of FMG

3.2.1. Body weight changes and organ weight analysis

No signs of toxicity or mortality were observed following the 28-day FMG administration period. However, a significant increase in body weight was observed in the FMG600 groups. The total weight gain differed significantly (p < 0.05) between the FMG400 and FMG600 groups and the control group. Postmortem examination further confirmed that there were no significant differences in the absolute or relative organ weights of the kidney, liver, or spleen between the experimental groups, as shown in Table 2.

Table 2.

Effects of FMG on the body weights (g) of Sprague–Dawley female rats in a 28-day subacute toxicity study.

	NC	FMG200	FMG400	FMG600
Initial body weight (g)	221.3 ± 11.980b	221.6 ± 18.610b	226.3 ± 26.580b	216.6 ± 10.120b
Final body weight (g)	235.3 ± 19.090b	238.2 ± 18.190b	250.2 ± 32.920b	275.2 ± 12.580a
Total weight gain (g)	14.0 ± 22.560z	16.5 ± 26.010z	23.7 ± 42.310y	58.4 ± 16.160x
Absolute and relative organ weight
Absolute kidney weight (g)	1.570 ± 0.150b	1.750 ± 0.144b	1.560 ± 0.317b	1.650 ± 0.268b
Relative kidney weight (g)	0.006 ± 0.002b	0.008 ± 0.007b	0.006 ± 0.006b	0.007 ± 0.001b
Absolute liver weight (g)	6.970 ± 0.150b	8.173 ± 0.144b	6.933 ± 0.268b	8.256 ± 0.268b
Relative liver weight(g)	0.030 ± 0.003b	0.037 ± 0.008b	0.030 ± 0.004b	0.037 ± 0.001b
Absolute spleen weight (g)	0.570 ± 0.050b	0.456 ± 0.102b	0.423 ± 0.158b	0.473 ± 0.006b
Relative spleen weight (g)	0.002 ± 0.001b	0.002 ± 0.003b	0.001 ± 0.005b	0.002 ± 0.004b

The data are expressed as the means ± SDs (n = 6). The values with different superscripts ^a and ^b indicate significant differences at p < 0.05 due to the time effect, whereas the values with different superscripts ^x and ^y differ significantly at p < 0.05 due to the treatment effect.

3.2.2. Blood hematological analysis

As shown in Table 3, the 28-day administration of FMG did not cause any significant changes in most of the hematological indices measured, with values in all treatment groups being comparable to those of the normal control group.

Table 3.

Effects of the polyherbal fermented extract mixture of M. oleifera and G. lucidum on the hematological parameters of Sprague–Dawley female rats in a 28-day subacute toxicity study.

Parameters	NC	FMG200	FMG400	FMG600
WBC (109/L)	12.3 ± 0.150b	12.6 ± 0.750b	12.6 ± 1.020b	11.9 ± 1.200b
Hb (g/L)	139.6 ± 7.230b	138.3 ± 5.770b	139.6 ± 1.150b	138.0 ± 6.080b
RBC (1012/L)	7.3 ± 0.070ab	7.0 ± 0.190b	7.5 ± 0.210ab	7.7 ± 0.310a
PLT (109/L)	1082.0 ± 98.700ab	1041.3 ± 48.010ab	1120.3 ± 0.570a	1243.3 ± 22.500a
MCV (fL)	57.6 ± 1.500b	54.6 ± 1.250b	57.0 ± 2.640b	55.0 ± 1.310b
MCHC (g/L)	334.3 ± 8.360b	335.3 ± 5.500b	329.6 ± 6.500b	323.3 ± 5.800b
PCV (L/L)	0.4 ± 0.010b	0.4 ± 0.005b	0.4 ± 0.005b	0.4 ± 0.010b
Plasma protein (g/L)	79.6 ± 3.210b	84.0 ± 5.290b	80.0 ± 2.210b	78.60 ± 8.320b
Lymphocyte (%)	63.3 ± 6.650b	60.3 ± 4.040b	68.0 ± 4.350b	68.60 ± 7.760b
Monocyte (%)	4.0 ± 1.000b	4.0 ± 1.000b	5.0 ± 1.000b	5.0 ± 1.000b
Eosinophils (%)	5.0 ± 1.000b	6.3 ± 1.100b	6.0 ± 1.700b	5.7 ± 1.000b
Basophils (%)	7.0 ± 1.000b	6.3 ± 1.100b	7.3 ± 0.500b	6.0 ± 1.700b

The data are expressed as the means ± SDs (n = 6). Values with different superscripts ^a and ^b indicate significant differences at p < 0.05 between treatment groups.

3.2.3. Serum biochemical analysis

As shown in Table 4, kidney function markers were not significantly different between the control and treatment groups (p > 0.05). However, in the liver function analysis, ALT levels were significantly lower in the FMG400 (−15%) and FMG600 (−42%) groups than in the control group. Conversely, AST levels significantly increased by 26.9% in FMG400 and 6.9% in FMG600 relative to those in the control.

Table 4.

Effects of FMG on the liver and kidney function markers of Sprague–Dawley female rats after 28 days of subacute toxicity.

Parameters	NC	FMG200	FMG400	FMG600
ALB (g/L)	25.6 ± 0.700b	29.2 ± 4.310b	26.8 ± 0.490b	26.5 ± 0.910b
ALP (U/L)	170.5 ± 0.700b	165.5 ± 0.700b	167.0 ± 22.600b	170.5 ± 16.300b
ALT(U/L)	77.0 ± 1.410a	82.5 ± 0.700a	65.0 ± 8.490b	44.5 ± 6.360b
AST (U/L)	137.5 ± 0.700b	138.5 ± 0.700b	174.5 ± 0.700a	147.0 ± 14.800a
Creat (umol/L)	50.5 ± 0.700b	54.5 ± 6.300b	52.0 ± 1.410b	51.5 ± 0.700b
Urea (mmol/L)	9.4 ± 0.070b	8.6 ± 0.910b	8.7 ± 0.140b	7.7 ± 0.910b

The data are expressed as the means ± SDs (n = 6). Values with different superscripts ^a and ^b indicate significant differences at p < 0.05 between treatment groups.

3.2.4. Histopathological analysis

Histopathological examinations were performed on the liver, kidney, and spleen of the rats. The NC group presented a normal histological structure of the liver (Figure 3(a)). It exhibited a typical central vein, well-preserved hepatocytes, and Kupffer cells devoid of any signs of apoptosis. The sinusoids were also found to be normal in size and well-organized. Similar morphological features were observed in the FMG200, FMG400, and FMG600 groups (Figures 3(d,g,j)).

Figure 3.

Photomicrographs representative of H&E-stained livers, kidneys and spleens from the NC- and FMG-treated groups. Normal architecture can be observed in the liver, kidney, and spleen sections of the NC, FMG200, and FMG400 groups, respectively (a–i). This is visualized by the normal size of the central vein (CV) and sinusoid (arrow) with healthy hepatocytes (arrowhead) and Kupffer cells (K) in the liver. Kidney sections revealed a normal structure of glomeruli (Grs) with well-organized and preserved distal (DT) and proximal tubules (PTs). The spleen was observed to have a well-defined white pulp (WP), red pulp (RP), central artery (CA), and splenic septa (black arrow). No structural alterations were observed in the liver or kidney of the FMG600 group (j,k). However, the spleens of the FMG600 group showed distorted WPs and RPs, as well as enlargement of the splenic septa (red arrow) (l).

All treated groups also had a normal glomerular architecture (Figures 3(b,e,h,k)). The distal and proximal tubules looked similar to those of normal tissue. No interstitial or intraglomerular congestion or tubular atrophy was observed. All the nephron cells were normal and presented visible nucleoli without degeneration, hemorrhage, necrosis, or infiltration with lymphocytes.

Examination of the spleen revealed that the normal control group (Figure 3(c)), as well as the FMG200 (Figure 3(f)) and FMG400 (Figure 3(i)) groups, presented a normal architecture with well-defined red and white pulp regions. The central arteries within these groups were also observed to be well organized and to maintain normal-sized splenic septa. However, architectural abnormalities such as distorted areas in both the red and white pulp regions and an enlargement of the splenic septa were observed in the FMG600 group (Figure 3(l)).

3.3. Neuroprotective study of FMG

3.3.1. Body weight changes and absolute and relative brain weights

Table 5 summarizes the body weight changes and brain weight measurements across the experimental groups. Compared with control treatment, repeated scopolamine administration for 28 days significantly reduced body weight gain by 24.7–32.5% (p < 0.05). Treatment with FMG (200–600 mg/kg) significantly attenuated this effect, with dose-dependent increases in final body weight and weight gain (p < 0.05 vs. the scopolamine group). In contrast, treatment with donepezil did not significantly affect the total weight gain when compared to the scopolamine control group (p > 0.05). Neither the absolute nor the relative brain weights differed significantly among the groups (p > 0.05).

Table 5.

Effect of FMG on body weight during the induction and treatment periods (g) and absolute and relative brain weights (g) in the scopolamine-induced dementia model.

		NC	DC	DD	D-FMG200	D-FMG400	D-FMG600
Induction period	Initial body weight (g)	242.7 ± 4.040b	256.3 ± 4.040b	255.3 ± 10.200b	245.0 ± 4.580b	245.7 ± 5.680b	261.3 ± 4.930b
	Final body weight (g)	330.7 ± 1.520a	317.0 ± 8.540a	317.3 ± 4.930a	305.0 ± 11.270a	311.0 ± 9.530a	320.7 ± 14.220a
	Total weight gain (g)	88.0 ± 4.330x	66.3 ± 9.450y	62.0 ± 11.340y	60.0 ± 12.190y	65.3 ± 11.100y	59.4 ± 15.060y
Treatment period	Initial body weight (g)	330.7 ± 1.520b	317.0 ± 8.540b	317.3 ± 4.930b	305.0 ± 11.270b	311.0 ± 9.530b	320.7 ± 14.220b
	Final body weight (g)	366.2 ± 3.580a	333.5 ± 4.130b	335.2 ± 5.290b	332.0 ± 6.120a	339.2 ± 4.040b	350.6 ± 6.260a
	Total weight gain (g)	35.5 ± 3.830x	16.5 ± 8.800y	17.9 ± 8.080y	27.0 ± 6.670x	28.2 ± 5.910x	30.3 ± 9.390x
Absolute and relative brain weight
Absolute brain weight (g)		1.7 ± 0.159x	2.1 ± 0.128x	2.0 ± 0.194x	1.9 ± 0.415x	1.9 ± 0.267x	2.0 ± 0.102x
Relative brain weight (g)		0.006 ± 0.000x	0.006 ± 0.000x	0.006 ± 0.001x	0.005 ± 0.001x	0.005 ± 0.001x	0.005 ± 0.007x

The data are expressed as the means ± SDs (n = 6). Values with different superscripts ^a and ^b indicate significant differences at p < 0.05 due to the time effect, whereas values with different superscripts ^x and ^y differ significantly at p < 0.05 due to the treatment effect.

3.3.2. Neurobehavioral assessment

3.3.2.1. Spontaneous alteration test

Figure 4(a) presents the track plots of the rats in the spontaneous alternation test. The DC group exhibited impaired spatial recognition, as evidenced by limited exploration of arm A (only exploring arms B and C). Notably, FMG treatment, particularly at 200 mg/kg bwt, enhanced spatial navigation, with animals demonstrating balanced exploration of all the maze arms. While the donepezil-treated (DD) group showed similar exploration patterns, their reduced locomotor activity (indicated by fainter tracking lines) distinguished them from the more active D-FMG200 group.

Figure 4.

Effects of FMG on spontaneous alteration tests: (a) track plot, (b) alteration, (c) number of arm entries, (d) total time mobile, and (e) average speed. Normal control (NC), dementia control (DC), donepezil-dementia (DD), dementia-FMG200 (D-FMG200), dementia-FMG 400 (D-FMG400), and dementia-FMG600 (D-FMG600) groups. The data are expressed as the means ± SDs (n = 6). Different superscripts ^a, ^b, and ^c indicate a significant difference (p < 0.05).

Figure 4(b) illustrates the average memory alteration results among the experimental groups. Compared with the NC animals, the DC animals presented a lower spontaneous alteration percentage (74.3 ± 4.040 vs. 29.1 ± 2.280%). Notably, all treatment groups showed a statistically significant improvement in spontaneous alternation compared to the DC group (p < 0.05). The percentage of alternation increased to 84.8 ± 6.201, 82.6 ± 1.929, 63.8 ± 3.440, and 53.5 ± 3.1% in the DD, D-FMG200, D-FMG400, and D-FMG600 groups, respectively.

The effects of FMG treatment on exploratory behavior in scopolamine-injected rats were assessed through arm entries, movement speed, and mobility time (Figures 4(c–e)). Compared with the DC group, all the FMG-treated groups presented significant improvements (p < 0.05) in these parameters. The D-FMG200 group exhibited the most pronounced effects, with a 138.5% increase in arm entries, 62.5% mobility time, and movement speed of 0.05 ± 0.040 m/s, even exceeding the speed of the NC group (0.04 ± 0.002 m/s). Treatment efficacy followed a reverse dose–dependent pattern, with D-FMG400 and D-FMG600 showing intermediate (69.3 and 46.2% more arm entries, respectively) and reduced effects (47.0 and 50.0% mobility time) compared with D-FMG200.

3.3.2.2. Spatial reference memory test

An evaluation of discrimination memory in the spatial reference test revealed that the NC rats consistently chose the novel arm first (6/6 animals), as did the D-FMG200 group (6/6), as shown in Table 6. This preference was slightly reduced in the DD, D-FMG400, and D-FMG600 groups (5/6 animals each). In contrast, only 1/6 of the scopolamine-treated (DC) rats selected the novel arm first. Statistical analysis confirmed that these treatment effects were significant (χ² = 13.85, p = 0.017, df = 5).

Table 6.

Effect of FMG on discrimination memory (first choice) in the scopolamine-induced dementia model.

Groups	Rat first choice novel arm (%)
NC	100.0 (6/6)
DC	17.0 (1/6)
DD	80.0 (5/6)
D-FMG200	100.0 (6/6)
D-FMG400	80.0 (5/6)
D-FMG600	80.0 (5/6)

Track plot analysis further revealed distinct behavioral profiles among the groups in the spatial memory test (Figure 5(a)). Both the DC and D-FMG400 groups exhibited similar behavior patterns, as shown by the greater track density throughout the maze and significantly reduced time in the target arm (Figure 5(b)). While the D-FMG600 and DD groups presented frequent novel arm visits, they presented distributed exploration patterns with considerable time spent in nontarget arms. Notably, the D-FMG200 group demonstrated enhanced spatial memory performance, as evidenced by 36% time spent in the novel arm (p < 0.05 compared to the DC group) and a clear preference for the target arm over other locations.

Figure 5.

Effects of FMG on (a) track plot and (b) total time spent in the novel arm during the spatial reference memory test. Normal control (NC), dementia control (DC), donepezil-dementia (DD), dementia-FMG200 (D-FMG200), dementia-FMG400 (D-FMG400), and dementia-FMG600 (D-FMG600) groups. (A) Other arm, (B) novel arm, (C) start arm. The data are expressed as the means ± SDs (n = 6). Different superscripts ^a, ^b, and ^c indicate a significant difference (p < 0.05).

3.3.3. Serum biochemical analysis

Kidney function marker levels were not significantly different between the control and treatment groups, as depicted in Table 7. However, liver function markers, namely ALB, ALP, and ALT, were significantly lower in all scopolamine-treated animals than in the NC group (p < 0.05). While donepezil treatment restored ALP levels within the normal range, it had no significant effect on the altered ALB or ALT levels. In contrast, treatment with FMG at 200 mg/kg was most effective, restoring all three liver function markers to levels that were not significantly different from the normal control group (p > 0.05).

Table 7.

Effects of FMG on liver and kidney function markers in scopolamine-induced dementia rats.

Parameters	NC	DC	DD	D-FMG200	D-FMG400	D-FMG600
ALB (g/L)	23.1 ± 0.580a	17.4 ± 0.700c	18.1 ± 0.700c	21.4 ± 0.840ab	17.7 ± 0.810c	18.3 ± 0.910bc
ALP (U/L)	303.5 ± 49.300a	204.5 ± 4.950b	254.5 ± 12.020ab	337.5 ± 10.600a	312.6 ± 9.290a	342.0 ± 2.820a
ALT(U/L)	65.6 ± 7.730a	36.3 ± 6.080b	44.3 ± 4.310b	69.3 ± 5.090a	56.1 ± 9.610ab	72.5 ± 9.610a
AST (U/L)	234.6 ± 22.810b	259.0 ± 59.500ab	237.1 ± 20.010ab	212.0 ± 2.820bc	280.0 ± 5.000ab	343.7 ± 59.750a
Creat (umol/L)	58.6 ± 3.2100b	45.5 ± 4.950b	50.0 ± 12.720b	55.0 ± 4.230b	57.6 ± 4.160b	43.5 ± 6.360b
Urea (mmol/L)	11.0 ± 1.170b	9.3 ± 0.420b	11.9 ± 0.210b	9.1 ± 0.560b	8.8 ± 1.320b	10.0 ± 0.560b

The data are expressed as the means ± SDs (n = 6). Different superscripts ^a, ^b, and ^c indicate a significant difference (p < 0.05).

3.3.4. Biochemical analysis of the brain

3.3.4.1. AChE activity and oxidative stress markers

To evaluate the antidementia potential of FMG, we measured brain acetylcholinesterase (AChE) activity across the experimental groups (Figure 6(a)). Compared with NC rats, scopolamine-treated (DC) rats presented significantly elevated AChE levels (p < 0.05), confirming successful dementia induction. Compared with the DC group, all the treatment groups presented significantly reduced AChE activity (p < 0.05), with reverse dose-dependent effects: D-FMG400 (−11.0%) and D-FMG600 (−9.0%) resulted in moderate reductions, whereas D-FMG200 restored AChE levels to near-normal values comparable to those in the NC group.

Figure 6.

Effects of FMG on the brain (a) AChE activity (b), TBARS levels (c) GPx-1, (d) catalase, and (e) SOD activity. Normal control (NC), dementia control (DC), donepezil-dementia (DD), dementia-FMG200 (D-FMG200), dementia-FMG400 (D-FMG400), and dementia-FMG600 (D-FMG600) groups. The data are expressed as the means ± SDs (n = 6). Different superscripts ^a, ^b, and ^c indicate a significant difference (p < 0.05).

Assessment of oxidative stress markers revealed distinct treatment-mediated restoration of antioxidant defenses (Figures 6(b–e)). Compared with the normal control grpup (NC: 34.5 ± 1.9 µg/mg), scopolamine-treated (DC) rats presented significant oxidative damage, with 51.6% higher TBARS levels (52.3 ± 1.9 µg/mg) and suppressed antioxidant capacity (GPx-1: −51.2%; SOD: −30.3%; catalase: −33.6% vs. NC). Compared with DC group (GPx-1: +89.9%; SOD: +85.4%; catalase: +230.7%), donepezil treatment (DD) partially ameliorated these effects; however, lipid peroxidation persisted (+16.4% vs. NC). FMG treatment induced dose-dependent recovery: D-FMG200 achieved near-complete oxidative balance (TBARS: 35.2 ± 1.9 µg/mg; catalase: +192%), whereas D-FMG400 showed preferential catalase and SOD enhancement (+207% and +49.8%, respectively), and D-FMG600 elicited maximal SOD activity (+87.8%) with complete TBARS normalization (p > 0.05 vs. NC).

3.3.5. Histological examination

Histopathological examinations of the hippocampus were conducted to ascertain whether scopolamine and FMG affected tissue structure. At 2 weeks post-scopolamine injection, hippocampal shape distortion was observed in the DC group (Figure 7(b)). The pyramidal layer cells in the subregions of the hippocampus (CA1, CA2, and CA3) migrated into the inner part of the molecular layer. On the other hand, animals in the DD group presented a normal C-shaped hippocampal structure with noticeable enlargement of the hippocampal sulcus (Figure 7(c)). Hippocampal sulcus enlargement with visible swelling, especially in the CA3 area, was also observed in the D-FMG400 and D-FMG600 groups (Figures 7(e,f)). Importantly, the D-FMG200 group (Figure 7(d)) presented a similar hippocampal structure to that of the NC group (Figure 7(a)). Nevertheless, these animals had the largest choroid plexus (CP) dilation, which is responsible for cerebrospinal fluid (CSF) production within the brain ventricular system.

Figure 7.

Representative photomicrographs of H&E-stained hippocampal sections. (a) The normal control (NC) group exhibited an intact hippocampal architecture, consisting of Cornu Ammonis (CA) regions (CA1, CA2, CA3) and the dentate gyrus (DG), separated by the hippocampal sulcus (HS) and choroid plexus (CP). (b) The dementia control (DC) group displayed marked structural disorganization in CA1, CA2, CA3, and the DG. (c) The dementia-donepezil (DD) group showed preserved architecture in the CA1, CA3, and DG, alongside noticeable HS enlargement. (d) The dementia-FMG200 (D-FMG200) group maintained normal CA1, CA2, CA3, and DG structures, with a normal HS size but apparent CP dilation. (e) The dementia-FMG400 (D-FMG400) group exhibited preserved CA1, CA2, CA3, and DG architecture with significant HS enlargement. (f) The dementia-FMG600 (D-FMG600) group demonstrated retained CA1, CA3, and DG structures alongside prominent HS enlargement. Magnification: 40×.

4. Discussion

This study demonstrated that the daily oral administration of FMG extract for 28 days alleviated cognitive dysfunction in scopolamine-induced dementia model rats. This improvement was associated with the modulation of AChE activity and a reduction in oxidative stress levels in the brain. Furthermore, FMG treatment ameliorated histological abnormalities in the hippocampus, particularly in the CA2 and CA3 regions. In addition to its neuroprotective effects, FMG exhibited a favorable safety profile, with no significant adverse effects observed throughout the treatment period, particularly at low doses.

Bioactive compounds have been identified as key factors in counteracting various pathological processes associated with neurodegeneration because of their anti-inflammatory, antioxidant, and neurotransmitter-modulating properties (Nájar et al. 2024). Multiple studies have demonstrated that extracts from M. oleifera and G. lucidum contain a broad spectrum of antioxidant compounds, with phenolics and flavonoids being the most prominent groups (Boh et al. 2007; Al-Owaisi et al. 2014; Andrejč et al. 2022; Ekiz et al. 2023). Our findings align with these reports, demonstrating that the FMG extract yields a high abundance of flavonoids, such as luteolin-malonyl, vitexin, and quercetin, as well as phenolic compounds, including trans-cinnamate and aromatic aldehydes (Table 1). Amino acids such as L-proline, L-valine, and L-acetyl-L-leucine have also been detected. Several studies have reported that acetylated derivatives of leucine exert neuroprotective effects by attenuating neuronal cell death and downregulating neurofibrillary markers associated with neurodegeneration (Hegdekar et al. 2021). Furthermore, we report for the first time the presence of phytosphingosine in M. oleifera and G. lucidum. Phytosphingosine is a sphingoid base compound with potent anti-inflammatory activity. Lee and colleagues (2022) demonstrated that this compound mediated brain inflammatory responses by modulating glial cell activation in a Parkinson’s disease model mouse.

It is also important to note that M. oleifera is a well-known source of glucosinolates and their bioactive isothiocyanate derivatives (Amaglo et al. 2010; Tumer et al. 2015; Amagloh et al. 2017; Chodur et al. 2018). However, these compounds were not detected in the FMG extract under the present analytical conditions. Our LC-MS protocol was intentionally optimized for a broad, non-targeted screening of general nonvolatile metabolites rather than the targeted isolation of specific compound classes. The apparent absence or low abundance of these constituents may be influenced by factors such as the extraction method, the specific plant parts used, or geographical origin (Sukmawaty et al. 2024; Fitri et al. 2025). Therefore, future studies employing targeted analytical protocols specifically designed for glucosinolates and isothiocyanates would be valuable to fully elucidate their potential contribution to FMG’s bioactivity.

Ensuring the safety profile of any new formulation is essential, even for plants and fungi with well-documented therapeutic benefits (Shukla et al. 2016). The 28-day subacute toxicity assessment revealed that FMG has a low toxicity profile, especially at lower doses. However, changes in the levels of serum liver biomarkers, particularly ALT and AST, were observed at medium and high doses (400 and 600 mg/kg, respectively). This observation aligns with previous studies reporting a decrease in ALT and an increase in AST levels following M. oleifera administration, especially at doses higher than 200 mg/kg (Ebrahem et al. 2022; Monraz-Méndez et al. 2022; Younis et al. 2022). Therefore, it is not surprising that M. oleifera extracts are considered promising candidates for treating fatty liver diseases because of their ability to alter liver toxicity markers (Kim et al. 2022; Cortes-Alvarez et al. 2024). Nonetheless, under normal physiological conditions, such fluctuations may also indicate mild hepatic stress. This finding is particularly relevant considering that no histopathological abnormalities were observed in the livers of the FMG400 and FMG600 groups, suggesting that these biochemical changes did not translate into structural damage.

The potential toxicity threshold of FMG may also be reflected in the spleen, where structural alterations were observed in the FMG600 group (Figure 3(l)). Similar findings were reported by Kim et al. (2018), who observed that the oral administration of 1000 mg/kg M. oleifera extract for 28 days resulted in spleen atrophy in both the red and white pulp regions. Additionally, the histological changes in the spleens of FMG600 rats, such as the distortion of the pulp architecture, may indicate an inflammatory or immune response (Szczepanek et al. 2012). These findings imply that FMG at 600 mg/kg may provoke a strong immune reaction in the spleen of rats. Future studies should investigate the underlying mechanisms of FMG-induced splenic toxicity, including cytokine profiling or transcriptomic analysis, to clarify whether the observed effects stem from direct immunomodulation or secondary systemic stress.

The results of the neurobehavioral assessment provide strong evidence for the memory-enhancing properties of FMG. Administration of our extract, particularly at the 200 mg/kg dose, significantly reversed the working memory deficits induced by scopolamine, as shown by the restored spontaneous alternation performance (Figures 4(a,b)). This restoration of working memory that can be defined as a brain function for temporarily storing and manipulating information (Baddeley 1992) is likely attributed to key flavonoids identified in our extract. Research by Afrin et al. (2022) demonstrated that flavonoid compounds, particularly quercetin from M. oleifera, can counteract this disruption by inducing the expression of the NR2A and NR2B subunits of NMDA receptors, which leads to increased working memory. We also observed improvements in rat exploration and locomotor activity, as reflected by increases in arm entries, mobile time, average speed, and movement patterns. These behaviors are often linked to enhanced dopaminergic activity, which is considered a dopamine-related response (Keren-Shaul et al. 2017; Cabañero et al. 2020). Furthermore, the dementia model rats exhibited significant spatial memory impairment, as evidenced by their low prevalence of selecting the novel arm in the spatial memory test and reduced exploration time in the novel arm (Table 6 and Figure 5). Notably, this impairment was reversed following the administration of a low dose of FMG.

Our serum biochemical analysis revealed that untreated dementia rats presented significant liver alterations. The animals in the DC group presented markedly lower levels of ALB, ALP, and ALT (Table 7). This finding contrasts with previous reports suggesting that scopolamine administration does not adversely affect liver enzyme levels or induce hepatic injury (Zhang et al. 2022). This discrepancy may stem from differences in the scopolamine dosage and duration of administration. Nonetheless, our results indicate that repeated scopolamine injections may induce oxidative stress in the liver, leading to enzyme leakage or reduced hepatic enzyme synthesis. Interestingly, the levels of liver function markers in all the FMG-treated groups returned to levels comparable to those observed in the NC group (Table 7). This hepatoprotective effect aligns with previous studies demonstrating similar outcomes when M. oleifera and G. lucidum extracts were used independently (Hamza 2010; Meneses et al. 2023).

The evaluation of acetylcholinesterase (AChE) activity is essential for determining the effect of an extract on cholinergic function, particularly in relation to acetylcholine metabolism and synaptic transmission. Our study revealed that FMG treatment, particularly at 200 mg/kg, repairs AChE dysfunction after scopolamine injection (Figure 6(a)). This result is in parallel with the improvement in the behavioral performance of the D-FMG200 and DD groups, further supporting the idea that a decrease in AChE levels can lead to enhanced learning and memory performance (Jafarian et al. 2019; Anand et al. 2022). These findings also align with previous studies reporting that M. oleifera and G. lucidum effectively regulate AChE activity (Wang, Li et al. 2021). The presence of flavonoids (luteolin-malonyl, vitexin, and quercetin) and phenolic compounds (trans-cinnamate and aromatic aldehydes) is likely to contribute to this activity, as these groups are known for their AChE inhibition properties, as reported by Yu et al. (2015), Ali et al. (2019), and Babaei et al. (2020). According to these studies, these compounds may inhibit AChE activity by binding to the reactive site and altering its catalytic effect, thereby increasing the concentration of acetylcholine in the synapse.

We found that repeated scopolamine injections led to a clear increase in oxidative stress in the brain, as shown by elevated levels of lipid peroxidation (TBARS) in the DC group (Figure 6(b)). This effect was accompanied by a disruption in the activity of key antioxidant enzymes, including SOD, GPx-1, and CAT. Interestingly, FMG treatment particularly at 200 mg/kg bwt, appeared to reverse these effects better than the higher dose (400 and 600 mg/kg bwt), with oxidative stress markers returning to near-normal levels and antioxidant enzyme activity showing notable improvement. A similar result was reported by Azlan et al. (2022), who demonstrated that the neuroprotective potential of the M. oleifera extract was not dose-dependent, with the 250 mg/kg dose exhibiting greater efficacy than the 500 mg/kg dose. In terms of probable mechanisms, several studies have reported that phenolic compounds, flavonoids, and amino acids found in M. oleifera and G. lucidum possess strong antioxidant properties (Pontiki et al. 2014; Xu et al. 2019; Sharma et al. 2024), which could explain the reduction in oxidative stress observed in the FMG-treated groups. In addition, compounds such as tyramine and diethylpropion present in FMG may also increase antioxidant enzyme activity (Garcia-Mijares et al. 2009; Ayanlowo et al. 2020).

Persistent oxidative stress can lead to cumulative neuronal damage and excessive inflammatory responses, resulting in structural brain damage. Our study revealed that the oxidative stress induced by scopolamine injection led to structural alterations in the hippocampus, as shown in the DC group (Figure 7(b)). These observations corroborate the findings from a previous study indicating that repeated scopolamine administration causes neuronal degeneration, characterized by shrinkage, cell loss, and disrupted cytoarchitecture within the hippocampus (Wang et al. 2023). Oral treatment with 200 mg/kg FMG, however, resulted in significant recovery, as the structure of the hippocampus appeared similar to that observed in the normal control group (Figures 7(a,d)). These results further validate the effectiveness of FMG in reducing oxidative stress and regulating the inflammatory response. The presence of phytosphingosine in the extract may contribute to these neuroprotective effects, given its known role in regulating inflammation via the histaminergic system (Kim et al. 2014; Lee et al. 2022). In addition, the L-leucine content of FMG could also prevent cell death due to oxidative stress damage (Hegdekar et al. 2021).

A greater number of immature neurons, greater CP dilation, and closer HS cavities were also observed in the D-FMG200. It has been reported that the regeneration of new neurons can lead to the recovery of hippocampal volume and dendritic structure (Schoenfeld et al. 2023). Although greater separation between the CP and the medial ventricular wall has been linked to abnormal cognitive outcomes (Hertzberg et al. 1994), such enlargement may also reflect an early stage of the recovery process (Egorova et al. 2019). This is because the CP produces cerebrospinal fluid (CSF), which is important for brain development and protection against harmful microbes and toxins (Lee et al. 2015).

While this study provides compelling evidence for the neuroprotective effects of FMG, it is important to acknowledge a key limitation. The 2-week treatment window employed in this study, although adequate for demonstrating acute efficacy in mitigating neurotoxic damage, is relatively short in the context of chronic neurodegenerative conditions. As such, this limited timeframe restricts our ability to draw conclusions about the long-term cognitive benefits of FMG or its potential to alter the course of progressive neurodegenerative diseases. Chronic models and extended treatment durations would be necessary to fully evaluate FMG capacity to sustain neuroprotection, preserve cognitive function, and potentially slow or halt disease progression over time. Additionally, while this study established the functional recovery of antioxidant enzyme activity, future research should employ molecular techniques, such as immunohistochemistry and Western blotting to investigate specific inflammatory signaling pathways, including Glial Fibrillary Acidic Protein (GFAP) and Nuclear Factor kappa B (NF-κB), to further elucidate the cellular mechanisms underlying FMG’s neuroprotective effects.

Furthermore, while the two-paradigm Y-maze protocol effectively demonstrated improvements in spatial working and reference memory, future investigations utilizing a broader behavioral battery, including the Morris Water Maze and Novel Object Recognition test, would provide a more comprehensive profile of the cognitive domains modulated by FMG.

5. Conclusions

In conclusion, this study provides the first comprehensive preclinical evidence for the neuroprotective properties of a novel fermented M. oleifera and G. lucidum mixture (FMG). We have established that FMG is well tolerated at its therapeutically effective dose of 200 mg/kg, which corresponds to its no-observed-adverse-effect level (NOAEL). At this dose, FMG treatment effectively reversed scopolamine-induced deficits in both working and spatial reference memory. These behavioral recoveries were strongly correlated with the restoration of normal hippocampal architecture and were mechanistically linked to the dual enhancement of the central cholinergic system and brain antioxidant defenses.

Funding Statement

This work was supported by the Ministry of Higher Education of Malaysia under Grant 600-RMC/FRGS 5/3 (003/2024).

Ethical approval

All animal procedures were approved on 1st December 2023 by the Committee on Animals for Research and Ethics, Universiti Teknologi MARA, Shah Alam (UiTM CARE: 433/2023).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data are available from the corresponding author upon reasonable request.