Deciphering the mechanisms underlying the dual-target inhibition of carbohydrate-digesting and neurodegenerative enzymes by Syzygium aromaticum (L.) Merr. & L.M. via molecular docking and dynamics simulations

Abstract

Syzygium aromaticum (L.) Merr. & L.M. Perry is a known spice with a high phytochemical content that can be explored in drug discovery. We investigated the in vitro enzyme inhibitory activities of a flavonoid-rich extract of S. aromaticum (FRESA) against type II diabetes (T2D) and Alzheimer’s disease (AD) and identified its anti-T2D and anti-AD phytochemicals via computational prediction. The in vitro enzyme inhibitory activities of a flavonoid-rich extract of Syzygium aromaticum were evaluated via standard protocols following flavonoid-enriched extraction procedures. High-performance liquid chromatography (HPLC) was employed to characterize the constituent bioactive flavonoids. Molecular docking of eight phytochemicals was performed via AutoDock Vina in PyRx 0.8, which identified apigenin, myricetin, and quercetin as hit compounds with high binding affinities and multitarget activities against α-amylase, α-glucosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidase (MAO). Molecular dynamics simulations (100 ns) were conducted via GROMACS 2019.2, and binding free energy calculations were performed via the MM-GBSA approach to validate the stability and interaction integrity of the hit phytochemicals. FRESA (IC₅₀ = 961.943 ± 21.031 μg/mL) exhibited moderate activity against α-amylase compared with that of acarbose (IC₅₀ = 27.104 ± 0.270 μg/mL). Compared with acarbose (IC₅₀ = 17.389 ± 0.436 μg/mL), FRESA had appreciable activity against α-glucosidase (IC₅₀ = 562.045 ± 6.714 μg/mL). FRESA demonstrated significant (p < 0.0001) inhibition of acetylcholinesterase (IC₅₀ = 26.911 ± 0.058 µg/mL), surpassed galantamine (IC₅₀ = 27.950 ± 0.122 µg/mL), and moderately inhibited butyrylcholinesterase (IC₅₀ = 28.168 ± 0.702 µg/mL) to galantamine (IC₅₀ = 23.126 ± 0.683 µg/mL). FRESA also significantly suppressed monoamine oxidase activity in Fe²⁺-induced brain damage in a concentration-dependent manner. HPLC–DAD analysis identified apigenin, caffeic acid, ferulic acid, gallic acid, kaempferol, myricetin, quercetin, and syringic acid as major constituents. Molecular docking revealed apigenin, myricetin, and quercetin as top-ranked multitarget inhibitors, exhibiting strong binding affinities (− 9.0 to − 10.2 kcal/mol) comparable to those of reference inhibitors across α-amylase, α-glucosidase, AChE, BChE, and MAO. Molecular dynamics simulations and MM-GBSA confirmed the binding strength of the hit phytoconstituents in the active pockets of α-amylase, α-glucosidase, AChE, BChE, and MAO, with multitargeting inhibitory activities supporting the in vitro and ex vivo enzyme activities. ADMET profiling indicated favorable drug likeness for apigenin, whereas myricetin and quercetin displayed acceptable pharmacokinetic properties with minimal violations. Our findings provide scientific validation of the anti-T2D and anti-AD properties of S. aromaticum and identify apigenin, myricetin, and quercetin, which could be used for the development of inhibitors of α-amylase, α-glucosidase, AChE, BChE, and MAO as dual therapies to combat T2D and AD. Additional in vivo validation is recommended to ensure a thorough assessment in the present research.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-45482-5.

Introduction

Diabetes and neurodegenerative diseases are prevalent global health issues that impact a significant number of individuals¹. Elevated levels of glucose in the bloodstream, resulting from either insulin resistance or inadequate insulin synthesis, distinguish diabetes as a metabolic condition. It may result in a range of consequences, such as cardiovascular disease, renal impairment, neuropathy, and visual impairment². In contrast, neurodegenerative illnesses include a collection of conditions that impact the nervous system, resulting in a gradual deterioration of cognitive and physical abilities. Examples of neurodegenerative disorders include Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease³. An association between diabetes and neurodegenerative illnesses is often observed since diabetes has been shown to increase susceptibility to the onset of neurodegenerative disorders. Type II diabetes (T2D) is associated with increased susceptibility to Alzheimer’s disease and other types of dementia. The precise processes underlying the association between diabetes and neurodegenerative disorders have not been fully elucidated; nevertheless, prevailing theories suggest that persistent hyperglycemia, insulin resistance, and inflammation are involved in this process⁴. In particular, reductions in the activity of choline acetyltransferase (ChAT) and acetylcholine (ACh) have been strongly associated with cognitive impairment in patients with Alzheimer’s disease. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) cleave acetylcholine and butyryl-choline signaling, respectively, within the synaptic cleft⁵. Therefore, acetylcholine degradation inhibitors such as AChE inhibitors or butyrylcholinesterase inhibit Alzheimer’s disease progression by blocking the scavenging of substrate catabolic products, thus increasing cholinergic signaling in AD patients.

These drugs, such as donepezil, galantamine, and rivastigmine, enhance cognitive function and alleviate symptoms of AD by suppressing AChE and BChE, eventually resulting in greater quantities of acetylcholine in the synaptic cleft. Cholinesterases (AChE and BChE) and monoamine oxidase (MAO) play significant roles in the etiology of neurodegenerative disorders. Accordingly, reversible cholinesterase inhibitors are currently the primary treatment for cognitive decline in neurodegenerative diseases characterized by reduced cholinergic transmission.^6,7

Numerous studies are currently being conducted on T2D and AD; however, there are still no effective therapies available to prevent or cure this disorder. Two medications that have received FDA approval, galantamine and donepezil, are drug treatments for AD that inhibit cholinesterase. However, acarbose and metformin are drug treatments for T2D that inhibit carbohydrate-metabolizing enzymes. α-Amylase and α-glucosidase are key digestive enzymes involved in carbohydrate metabolism and the regulation of postprandial blood glucose levels. α-Amylase, a calcium-dependent hydrolase, catalyzes the breakdown of complex starches into oligosaccharides and smaller sugars, which are subsequently further processed in the gut, whereas α-glucosidase, located in the small intestinal brush border, hydrolyzes oligosaccharides and disaccharides into absorbable glucose units. Inhibiting the activities of these enzymes slows carbohydrate digestion and glucose absorption, thereby reducing postmeal hyperglycemia, a therapeutic strategy widely used in managing type 2 diabetes. Compared with current drugs, natural and synthetic inhibitors of α-amylase and α-glucosidase are actively being investigated to improve glycemic control with fewer side effects⁸. This condition (T2D) is not cured but rather results in temporary and inadequate clinical relief, which is accompanied by side effects such as diarrhea, weight loss, nausea, vomiting and liver damage⁹. Unfortunately, these therapies are reserved for those with mild cognitive disorders and metabolic disorders. They do not halt or reverse the disease, and they offer no cognitive benefits; however, ketamine may slow progression. The concept, therefore, offers a potential route to attack both diseases together, directly targeting multiple pathogenetic factors of T2D and Alzheimer’s disease (AD) with one drug, which may work toward curing the disease as opposed to symptomatic relief or slowing down processes. Researchers are exploring plant-based remedies as potential alternative therapies for T2D and Alzheimer’s disease (AD). These remedies have attracted interest because of their range of properties and distinctive compositions that are safe to use and cost effective with minimal adverse effects while being able to address various factors linked to T2D and AD simultaneously, making them promising candidates for further investigation and innovation in drug development.

The botanical species Syzygium aromaticum, popularly referred to as clove, is indigenous to Africa and Asia. This fragrant spice is recognized for its gastronomic and therapeutic applications, and it has a significant historical background spanning several centuries. The Myrtaceae family includes plants that are distinguished by their unique pink flower buds that transform into fragrant, nail-shaped cloves upon desiccation. The fruit of this particular plant contains numerous phytochemicals, including alkaloids, coumarins, catechins, flavonoids, phenols, saponins, terpenoids, tannins, and steroids¹⁰. The methanol extract of S. aromaticum has demonstrated significant antibacterial activity against Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus¹¹. S. aromaticum has not only antibacterial activity but also potential anticancer action. This study investigated the bioactivity of S. aromaticum extracts and revealed that they were very good at inhibiting a number of different types of cancer cells. The antioxidant activity and protective benefits of S. aromaticum aqueous extract (SAAE) were demonstrated in lipopolysaccharide (LPS)-induced lung inflammation in mice. The SAAE compound inhibited the production of reactive oxygen species (ROS) and the activity of myeloperoxidase (MPO), two processes linked to inflammation and oxidative stress¹². S. aromaticum is a widely recognized herbal remedy that has broad pharmacological efficacy¹³. Several other studies have tested S. aromaticum L. in an animal model. Nassar et al.¹⁴ reported that the alcoholic extract of Syzygium aromaticum had antioxidant properties and protected the liver from damage caused by paracetamol consumption. Recent research has investigated its effects on lipid oxidation and blood glucose levels in rats and revealed an increase in enzyme levels after the addition of S. aromaticum supplements¹⁵. Additionally, a study by Shukri et al.¹⁶ revealed that including Syzygium aromaticum in the diet reduced harm to the liver, eyes, and heart muscles of rats. Ahmad et al.¹⁷ recorded the impact of an extract from Syzygium aromaticum on the behavior of healthy male rats, whereas Issac et al.¹⁸ detailed the safety and effectiveness of a new polyphenol-rich extract derived from Syzygium aromaticum buds for preventing ulcers. Agabje¹⁹ researched how Syzygium aromaticum affects the gastrointestinal system via animal models and emphasized the influence of extracts on digestive health. In a study by Agabje et al.²⁰, they delved deeper into the effects of extracts of Syzygium aromaticum on rodents from a biochemical and toxicological standpoint. Santin et al.²¹ examined the properties of Syzygium aromaticum oil, the main component of which is eugenol, in various animal models. Additionally, Aisha et al.²² scrutinized the effectiveness of S. aromaticum by investigating its antiangiogenic, cytotoxic, and antioxidant properties. Additionally, Adefegha and Oboh²³ examined whether water-extracted phytochemicals from tropical spices, including Syzygium aromaticum, could inhibit enzymes linked to type 2 diabetes and protect the rat pancreas from free radical-induced damage.

Flavonoids, a group of polyphenolic chemicals, are the main bioactive ingredients in Syzygium aromaticum that help control diabetes and protect neurons²⁴. In the field of drug-like molecule discovery and characterization from natural sources, computational prediction and experimental validation are critical²⁵. In silico techniques, such as molecular docking and molecular dynamics simulations, can predict the binding affinity and stability of flavonoid compounds to specific targets implicated in diabetes and neurodegenerative illnesses²⁶. The novelty of our study lies in its comprehensive approach to investigating the dual therapeutic potential of Syzygium aromaticum for diabetes mellitus and Alzheimer’s disease, two conditions that are increasingly recognized as interconnected. Diabetes is a well-established risk factor for dementia, including Alzheimer’s disease, due to shared pathological mechanisms such as insulin resistance, inflammation, and vascular damage. Our study uniquely integrates experimental and bioinformatics methods to explore how the flavonoid-rich extract of Syzygium aromaticum can target these shared pathways, potentially offering a multifaceted therapeutic strategy. By focusing on both conditions simultaneously, our research provides novel insights into the potential of natural compounds to address complex, interrelated diseases, paving the way for more holistic and effective treatment approaches. Our study aimed to comprehensively evaluate the anti-T2D and anti-AD effects and discover bioactive compounds from Syzygium aromaticum as potential drug candidates that might work through a dual therapy strategy via computational prediction and in vitro experimental validation. This study aimed to establish a foundation for subsequent investigations and advancements in naturally derived therapeutic agents for diabetes and neurodegenerative disorders.

Materials and methods

Plant material

Syzygium aromaticum was obtained from a local market in Omu-Aran, Kwara State, Nigeria. The plant was subsequently authenticated by the Forestry Research Institute of Nigeria’s herbarium in Ibadan and assigned the herbarium number FHI 114,106.

Flavonoid-rich extract preparation

Fifty grams of powdered Syzygium aromaticum was macerated for 72 h in 80% methanol to obtain a crude methanolic extract. Twenty grams of the crude methanolic extract was then subjected to flavonoid-rich extraction via a previously described procedure²⁷. The crude methanolic extract was subjected to hydrolysis by refluxing it with 10% sulfuric acid at 100 °C for 30 min. This process produced flavonoid aglycones, which were allowed to precipitate, then redissolved in warm 95% ethanol and filtered, and the volume was adjusted to 100 mL with the same solvent. The resulting solution was concentrated under reduced pressure via a rotary evaporator. Flavonoids were then isolated from the filtrate by precipitation with concentrated ammonium hydroxide and subsequently rinsed with diluted ammonium hydroxide. The final extraction yielded 15.14 g of product.

The decision to use a single extract for the evaluation in this study was based on maintaining specificity and consistency, allowing us to thoroughly investigate the properties and effects of the extract without the confounding variables that might arise from the use of multiple extracts.

High-performance liquid chromatography (HPLC–UV) of flavonoid-rich extracts of Syzygium aromaticum

The flavonoid-rich extract of S. aromaticum was subjected to HPLC to identify the potential bioactive flavonoids present. The procedure described in our previously published article was followed for this process²⁷. Chromatographic analysis was carried out via a gradient elution system consisting of water, methanol, and tetrahydrofuran, following the methods reported above. Phase A was water containing 1% phosphoric acid, while Phase B was a mixture of methanol:tetrahydrofuran (80:20, v/v). The elution schedule was as follows: 0–5 min, 90% A; 5–15 min, a linear decrease to 50% A; 15–20 min, held at 50% A; 20–22 min, linearly increased back to 90% A; and 22–25 min, maintained at 90% A. The mobile phase was pumped at 1.5 mL/min, with an injection volume of 20 µL. The column was flushed and equilibrated for 10 min each before analysis. Flavonoid standards included rutin, quercetin, kaempferol, apigenin, gallic acid, caffeic acid, p-coumaric acid, and ferulic acid. Each sample was run for 40 min, during which the spectra were recorded. The column temperature was maintained at 25 °C, and the detection wavelengths were set at 254 nm and 420 nm.

Enzyme inhibitory assays

α-Amylase and α-glucosidase inhibitory activity

The α-amylase and α-glucosidase inhibitory activities of the flavonoid-rich extract of Syzygium aromaticum (FRESA) were evaluated following previously published methods^27,28.

Determination of AChE and BChE inhibitor activities

The enzyme inhibitory effects of the flavonoid-rich extract of Syzygium aromaticum (FRESA) on AChE and BChE were determined. The method used was as described in earlier studies reported by^28,29. In summary, 100 µL of the tissue homogenates were combined with different concentrations of the flavonoid-enriched extract, followed by the addition of 50 µL of Ellman’s reagent and 250 µL of sodium phosphate buffer (100 mM). The reaction mixture was then incubated at 25 °C for 20 min. After incubation, 50 µL of 50 mM acetylcholine iodide and butyrylcholine iodide were added, and the absorbance was recorded immediately at 412 nm, with readings taken every 3 min over a 15-min period.

Ex vivo studies

Adult male Wistar rats (150–200 g) were obtained from the Department of Biochemistry, Bowen University, Nigeria. The rats were not subjected to surgical anesthesia during the experimental procedures. For humane termination, the animals were euthanized by an overdose of halothane administered via inhalation (≥ 5% v/v) in a closed chamber following an overnight fast. Loss of consciousness and respiration was confirmed prior to sample collection. The brains were subsequently extracted, homogenized in 1% Triton X-100 in 50 mM phosphate buffer, and centrifuged at 3,000 rpm at 4 °C. The resulting supernatant was collected for subsequent ex vivo experiments. The Institutional Research Animal Ethical Committee approved (BUI/BCH/2024/0002) the study protocols and research conducted in accordance with the ARRIVE (Animal Research: Reporting in Vivo Experiments) guidelines. All the experimental methods were performed in accordance with the relevant protocols, guidelines and regulations approved by the Bowen University Research Ethics Committee (BUREC).

Ex vivo induction of brain damage

Ex vivo brain damage was induced via ferrous iron (Fe2+) according to the methodology described by Erukainure et al.³⁰.

Determination of monoamine oxidase inhibitory activity

The inhibitory action of monoamine oxidase was assessed by using a flavonoid-rich extract of Syzygium aromaticum (FRESA), following the methodology outlined by Green and Haughton³¹. The reaction mixture consisted of 0.025 M phosphate buffer (pH 7), 12.5 mM semicarbazide, 10 mM benzylamine (pH adjusted to 7), and 75 μL of rat brain homogenate. Additionally, various quantities of the extract were dissolved in distilled water and added to the mixture. After 30 min, 250 µl of acetic acid were added, and the mixture was boiled for 3 min in a water bath, after which centrifugation was performed. The brain supernatant (1 mL) was combined with an equivalent amount of 0.05% 2,4‐DNPH, and then, 1.25 mL of benzene was added after 10 min. The mixture was then incubated at room temperature. The benzene layer was isolated and combined with an equal amount of sodium hydroxide solution (1 N). The alkaline layer was separated by pouring and then subjected to heating at 80 °C for 10 min. The percentage inhibition was calculated as follows³¹.

Computational study

Protein preparation

The protein structures of human α-amylase (HPA) with PDB ID: 1B2Y, α-glucosidase (PDB ID: 3TOP), human monoamine oxide B (PDBID: 2V5Z), human acetylcholinesterase (hAChE) (PDBID: 4EY7), and butyrylcholinesterase (hBChE) (PDBID: 6EP4)¹⁰ were retrieved from the Protein Data Bank (http://www.rcsb.org). The three-dimensional crystal structure of the target protein was retrieved from the Protein Data Bank (PDB). The structure was inspected for missing residues, incomplete side chains, and structural anomalies prior to docking. Missing atoms were corrected where necessary, and no critical residues were absent within the active site. The existing ligands and water molecules were removed from all the crystal structures, while missing hydrogen atoms were added via MGL-AutoDockTools (ADT, v1.5.6)³².

Ligand preparation

The structural data (SDF) of reference inhibitors (donepezil, galantamine, decamethonium acarbose and safinamide) and the HPLC-identified compounds of Syzygium aromaticum were downloaded from www.pubchem.ncbi.nlm.nih.gov. The ligands were then converted to the pdb format via Open Babel³³.

Validation of the molecular docking protocol

Molecular docking studies were validated following the detailed procedure described by²⁷. The docked poses of the native ligands that were also used as reference standards (acarbose and donepezil) with the lowest docking scores from the initial docking were superimposed with the extracted cocrystallized ligands from both proteins to validate the docking protocol to be used for virtual screening. The RMSD was calculated via Discovery Studio Visualizer, BIOVIA, 2020.

Molecular docking of phytochemicals with targeted active sites

The reference inhibitors and the ligands were docked at the active site against the five protein targets via AutoDock Vina version 1.1.2 in PyRx 0.8³⁴. PyRx 0.8’s Open Babel³³ was used to minimize energy. The energy minimization parameter and conjugate gradient descent used were the universal force field (UFF) and optimization algorithms, respectively. The binding site coordinates of the target proteins were identified by mapping the amino acid residues around the binding site of the native ligand. The dimensions of the grid boxes formed are presented in Table S1. The selected conformer from the docking analysis was further subjected to interactive analysis via Discovery Studio Visualizer version 16.

Molecular dynamics

Molecular dynamics simulation at 100 ns was performed for the hit compounds with 4EY7 and 1B2Y. This was achieved via GROMACS 2019.2 and GROMOS96 43a1 force fields^35–37. The protein and ligand topology files were generated via Charmm GUI^38,39. The enzymes and ligand‒enzyme complex systems were immersed in a cubic box using the TIP4P water model, with periodic boundary conditions applied. The system was set to a physiological concentration of 0.154 M and maintained with neutralized NaCl ions. On the basis of previous research, the parameters employed are as described by^27,40–42.

Binding free energy calculation via MM-GBSA

The protocol described by^27,43,44 was employed for calculating the binding free energy via the Molecular Mechanics Generalized Born Surface Area (MM-GBSA). The MM-GBSA method and decomposition analysis via the gmx MMPBSA package were used to obtain the binding energies of amino acids within 0.5 nm of the ligand to determine the binding free energy of the two top docked phytochemicals from the initial docking analysis.

In silico physicochemical properties and ADMET study

The two top-ranked phytochemicals from the docking analysis for each of the proteins were further subjected to drug-likeness filtering analysis over a wide range of filtering tools. Drug likeness analysis via Lipinski filtering tools was performed on the SwissADME (http://www.swissadme.ch/index.php) webserver.

Data analysis

We used one-way ANOVA to evaluate the data, and the results are expressed as the means ± SDs (n = 3). Tukey’s multiple range post hoc test for the t test was used to establish statistical significance at p < 0.05. GraphPad Prism version 10.4.1 was used to plot the graphs.

Results

HPLC chromatography

High-performance liquid chromatography (HPLC) analysis of the flavonoid-rich extract of Syzygium aromaticum (FRESA) revealed several bioactive phytochemicals with potential therapeutic applications. These compounds include gallic acid, caffeic acid, syringic acid, ferulic acid, myricetin, apigenin, kaempferol, and quercetin (Table 1, Fig. S1).

Table 1.

Bioactive principles identified in flavonoid-rich extracts of Syzygium aromaticum.

Compounds	Conc (mg/ml)
Gallic acid	0.62
Caffeic acid	0.86
Syringic acid	2.12
Ferulic acid	2.67
Myricetin	3.56
Apigenin	6.67
Kaempferol	8.18
Quercetrin	11.29

α-Amylase and α-glucosidase inhibitory activities

On the basis of the results obtained from the α-amylase inhibitory activity, the flavonoid-rich extract of Syzygium aromaticum (FRESA) significantly increased the activity of this enzyme in a concentration-dependent manner, as did the standard acarbose (Fig. 1a). In particular, FRESA exhibited considerable (p < 0.0001) α-amylase inhibitory activity, with an IC₅₀ of 961.943 ± 21.031 µg/mL, albeit weaker than that of the established drug acarbose, with an IC₅₀ of 27.104 ± 0.270 µg/mL (Fig. 1b).

Fig. 1.

α-Amylase inhibitory activity of the Syzygium aromaticum flavonoid-rich extract: (a) α-amylase inhibitory activity; (b) IC₅₀ values. The data are presented as the mean ± standard deviation (SD) (n = 3), with statistical significance determined by t tests (p < 0.0001). S. aromaticum; Acarbose: standard drug.

The α-glucosidase inhibitory activity of FRESA improved in a dose-dependent manner (Fig. 2A) compared with that of the standard drug acarbose, which also increased α-glucosidase activity. Furthermore, the Syzygium aromaticum flavonoid-rich extract inhibited α-glucosidase in a concentration-dependent manner, with an IC₅₀ of 562.045 ± 6.714 µg/mL, in contrast to the IC₅₀ of acarbose (17.389 ± 0.436 µg/mL) for acarbose (Fig. 2A, B).

Fig. 2.

α-Glucosidase inhibitory activity of the flavonoid-rich extract of Syzygium aromaticum: (a) α-glucosidase inhibitory activity; (b) IC₅₀. The data are presented as the mean ± standard deviation (SD) (n = 3), with statistical significance determined by t tests (p < 0.0001). S. aromaticum; acarbose: standard drug.

Acetylcholinesterase and butyrylcholinesterase inhibitory activities

Acetylcholinesterase (AchE) activity was measured, and the percentage inhibition results (Fig. 3A) revealed that FRESA activity significantly increased as the concentration also increased. The flavonoid-rich extract of Syzygium aromaticum exhibited significant inhibitory activity (p < 0.0001) against AChE (IC₅₀ = 26.911 ± 0.058 µg/mL), albeit stronger than that of the standard control, galantamine (IC₅₀ = 27.950 ± 0.122 µg/mL), as shown in Fig. 3B.

Fig. 3.

AChE activity of the Syzygium aromaticum flavonoid-rich extract: (a) AchE inhibitory activity; (b) IC₅₀ values. The data are presented as the mean ± standard deviation (SD) (n = 3), with statistical significance determined by t tests (p < 0.0001). S. aromaticum; Galantamine: standard drug.

Butyrylcholinesterase (BchE) activity was measured, and the percentage inhibition of BchE was determined (Fig. 4A). FRESA activity significantly increased as the concentration increased. Similarly, the flavonoid-rich extract of Syzygium aromaticum leaves had notable (p < 0.0001) inhibitory effects on BChE (IC₅₀ = 28.168 ± 0.702 µg/mL), although the effects were weaker than those of galantamine (IC₅₀ = 23.126 ± 0.683 µg/mL), as indicated in Fig. 4.

Fig. 4.

Butyrylcholinesterase activity of the flavonoid-rich extract of Syzygium aromaticum: (a) AchE inhibitory activity; (b) IC₅₀ values. The data are presented as the mean ± standard deviation (SD) (n = 3), with statistical significance determined by t tests (p < 0.0001). S. aromaticum; Galantamine: standard drug.

Monoamine oxidase activity

The inhibitory effects of flavonoid-rich extracts of Syzygium aromaticum on the activity of monoamine oxidase (MAO) in the oxidized brain were evaluated, and the results are shown in Fig. 5. The untreated rats presented elevated MAO activity (p < 0.0001). Conversely, a significant reduction in MAO activity was observed in the rats treated with varying doses of the plant extract (p < 0.0001). The inhibitory effect of FRESA on MAO activity was concentration dependent, with the strongest inhibition occurring at the 1000 µg/ml dose.

Fig. 5.

Influence of flavonoid-rich extracts of Syzygium aromaticum (FRESA) on monoamine oxidase (MAO) activity in ex vivo brain samples. Compared with the group induced solely with FeSO4, a notable reduction in MAO activity was observed in the range of concentrations of the flavonoid-rich extracts of Syzygium aromaticum. The data are presented as the mean ± standard deviation (SD) (n = 3); ****p < 0.0001; **p < 0.01 according to ANOVA. FRESA: Flavonoid-rich extracts of Syzygium aromaticum.

In silico studies

To confirm the ability of the methodology to be utilized for molecular docking, the produced docked poses of the reference compounds (donepezil and acarbose) with the least energy conformation were superimposed on the native ligand that cocrystallized with the protein targets (4EY7 and 1B2Y) (Fig. S2). After the superimposition, the root mean square deviation (RMSD) was computed. The RDDs for donepezil and safinamide were 0.6211 and 0.1531 Å, respectively. The low RMSD shows that the docking protocol was suitable for the docking of HPLC-identified phytochemicals.

After the validation protocol, with the same docking parameters, the HPLC-identified phytochemicals were docked to the active sites of the proteins. Table S2 displays the binding affinities obtained from the docking study of the compounds discovered by HPLC against the five protein targets. The binding affinities from the docking analysis of the HPLC-identified compounds from the flavonoid-rich extract of Syzygium aromaticum against the five protein targets are shown in Table S2. On the basis of the minimum binding energies, binding poses and interactions in the catalytic site, the top two ranked compounds for each enzyme were selected for interaction analysis. After ranking, the binding energies of the top two compounds that docked to the five targets were close to those of the reference inhibitors (Table S3). To validate the docking protocol, cocrystalized reference compounds (donepezil and acarbose) were docked into the binding site of the cocrystalized proteins with binding energies of − 12.2 and − 12.5 kcal/mol, respectively. The top-ranked phytochemicals were apigenin (− 9.1 kcal/mol) and quercetin (− 9.2 kcal/mol), which are 3TOP protein targets, and quercetin (− 9.8 kcal/mol) and myricetin (− 9.5 kcal/mol), which are 6EP4 protein targets. On the other hand, apigenin and myricetin demonstrated the highest binding tendencies to the remaining three protein targets (1B2Y, 4EY7, and 2V5Z), with binding energies of − 9.1 and − 9.0, − 10.2 and − 10.2, − 9.3 and − 9.4, and − 9.4 kcal/mol, respectively.

Amino acid interactions of the top two docked HPLC-identified phytochemical and reference compounds with the five protein targets

The interactions of the reference compound and two top-ranked HPLC-identified phytochemicals with the catalytic residues of the target proteins are shown in Table S4. The ligand‒enzyme interactions were primarily hydrophobic, with few hydrogen bonds (< 3.40 Å). Like the native ligand, donepezil adopted an extended conformation within the narrow, hydrophobic gorge of 4EY7, forming a single hydrogen bond with Phe295. Additionally, Pi-alkyl interactions occurred between Tyr337, Tyr341, and the piperidine ring of donepezil. The 1-benzyl unit of donepezil engaged in aromatic Pi‒Pi stacking with Trp86 and His447. Additionally, galantamine was stretched in the long, narrow, hydrophobic gorge of 4EY7. It formed 3 hydrogen bonds with Ser203, Glu202 and Ala204, further interacted via amide‒pi stacking with Gly121, and formed pi‒alkyl interactions with Tyr337, Phe295, Phe297, Phe338, and Trp86. The best phytochemical for docking to 4EY7, apigenin and myricetin, was oriented in a similar fashion in the active site of the gorge, forming several hydrogen bonds and hydrophobic contacts (Fig. 6). Decamethonium was docked deeply into the active site gorge in the instance of 6EP4, aligning in the same fashion as the native ligand. Galantamine, a reference inhibitor, was also docked into the active site, where it formed a 3-hydrogen bond with Thr120, a pi‒pi stacking and alkyl contact with Trp82 and an alkyl contact with Leu125. The top-scoring ligands, quercetrin and myricetin, formed multiple hydrogen bonds with the catalytic residues and interacted with all the residues that bound decamethonium (Fig. 7). While acarbose adopted an extended conformation within the 1B2Y binding site, spanning multiple subsites, the top ligands, apigenin and myricetin, primarily occupied the -3 and -1 subsites of 1B2Y (Fig. 8). Both ligands, including Trp-59, Tyr62, His299, Asp197, His305, Glu233, Arg197, and Ala198, formed hydrogen bonds and hydrophobic interactions at the hydrophobic gate of α-amylase. Quercetrin and apigenin bind to the active sites of 3TOP in a manner comparable to that of acarbose, interacting with the same catalytic residues (Fig. 9). In contrast to safinamide, the reference MAO inhibitor, which forms a single hydrogen bond with Gln206 and several hydrophobic interactions, apigenin and myricetin exhibited greater hydrogen interactions with catalytic residues, along with multiple hydrophobic interactions (Fig. 10).

Fig. 6.

Comparison of the docking interactions between top-scoring phytochemicals (apigenin and myricetin) and the reference acetylcholinesterase inhibitor donepezil within the active site of the 4EY7 protein structure. The ligands are depicted as stick models: (a) donepezil, (b) galantamine, (c) apigenin, and (d) myricetin. (i) 3D and (ii) 2D interaction diagrams are provided.

Fig. 7.

Comparison of the docking interactions between top-scoring phytochemicals (myricetin and quercetin) and a reference butyrylcholinesterase inhibitor, decamethonium, within the active site of the 6EP4 protein structure. The ligands are depicted as stick models: (a) decamethonium, (b) myricetin, and (c) quercetin. (i) 3D and (ii) 2D interaction diagrams are provided.

Fig. 8.

Comparison of the docking interactions between top-scoring phytochemicals (apigenin and myricetin) and a reference α-amylase inhibitor, acarbose, within the active site of the 1B2Y protein structure. The ligands are depicted as stick models: (a) acarbose, (b) apigenin, (c) myricetin. (i) 3D and (ii) 2D interaction diagrams are provided.

Fig. 9.

Comparison of the docking interactions between top-scoring phytochemicals (apigenin and quercetin) and a reference α-glucosidase inhibitor, acarbose, within the active site of the 3TOP protein structure. The ligands are depicted as stick models: (a) acarbose, (b) apigenin, and (c) quercetin. (i) 3D and (ii) 2D interaction diagrams are provided.

Fig. 10.

Comparison of the docking interactions between top-scoring phytochemicals (apigenin and myricetin) and a reference monoamine oxidase inhibitor, safinamide, within the active site of the 2V5Z protein structure. The ligands are depicted as stick models: (a) safinamide, (b) apigenin, and (c) myricetin. (i) 3D and (ii) 2D interaction diagrams are provided.

Molecular dynamics

To assess the stability of protein‒ligand complexes, including those with representative proteins, reference inhibitors (acarbose and galantamine), and top-scoring phytochemicals, molecular dynamics (MD) simulations were performed via Tk console scripts. The stability of the complexes was evaluated by analyzing the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (RoG), solvent-accessible surface area (SASA), and hydrogen bond interactions. Figure S3 summarizes the average values and standard deviations of these parameters, while Figs. 11, 12, 13, 14, 15 provide detailed spectral plots. The RMSD plots for the 4EY7 and 1B2Y complexes indicated equilibration within 10 ns, followed by minimal fluctuations throughout the simulation period (Fig. 11). The 4EY7_apigenin complex presented the highest mean RMSD value of 1.70 ± 0.22, suggesting greater flexibility. For the 4EY7 systems, all three complexes presented very similar mean root-mean-square fluctuation (RMSF) values. Similarly, the 1B2Y complex systems presented minimal variations in RMSF values (Fig. 12). The radius of gyration (RoG) plots for both the 4EY7 and 1B2Y complexes indicated equilibration at approximately 10 ns, followed by negligible fluctuations throughout the simulation (Fig. 13). The apigenin, myricetin, and reference compound complexes presented comparable mean RoG values. The solvent-accessible surface area (SASA) plots for the 4EY7 and 1B2Y complexes revealed minimal fluctuations during the simulation period, which was further supported by the close mean SASA values (Fig. 14). The number of hydrogen bonds remained relatively stable throughout the simulations, with minimal fluctuations observed (Fig. 15).

Fig. 11.

Backbone-root mean square deviation (RMSD) plots of the MD simulations of the top docked HPLC-identified phytochemicals and reference compounds complexed to (a) human α-amylase and (b) human acetylcholinesterase.

Fig. 12.

Per residue root mean square fluctuation (RMSF) plots of the MD simulations of the top docked HPLC-identified phytochemicals and reference compounds complexed to (a) human α-amylase and (b) human acetylcholinesterase.

Fig. 13.

RoG plots of MD simulations of top docked HPLC-identified phytochemicals and reference compounds complexed to (a) human α-amylase and (b) human acetylcholinesterase.

Fig. 14.

Solvent-accessible surface area (SASA) plots of molecular dynamics (MD) simulations for complexes of top-docked HPLC-identified phytochemicals and reference compounds with (a) human α-amylase and (b) human acetylcholinesterase.

Fig. 15.

Hydrogen bond fluctuations during molecular dynamics (MD) simulations of complexes formed between top-docked HPLC-identified phytochemicals, reference compounds, and (a) human α-amylase and (b) human acetylcholinesterase.

Molecular mechanics generalized born surface area (MMGBSA) analysis

The binding free energies of the top two ligands docked to the 4EY7 and 1B2Y proteins were calculated via MM-GBSA. The various components that make up the total binding free energy are presented in Table 2. Among the acetylcholinesterase complexes, myricetin demonstrated the strongest binding affinity (− 25.77 ± 4.03 kcal/mol), followed by apigenin (− 22.19 ± 5.17 kcal/mol), both of which outperformed the reference drug donepezil (− 19.89 ± 3.61 kcal/mol). Both ligands displayed stronger binding affinities than did the reference compounds. Acarbose had the smallest interaction (− 10.32 ± 10.61 kcal/mol) with α-amylase, whereas apigenin had the most favorable binding energy (− 19.40 ± 2.76 kcal/mol), followed closely by myricetin (− 17.66 ± 6.00 kcal/mol). van der Waals and electrostatic interactions were the main causes of ligand binding, according to energy decomposition, but polar solvation energies prevented complex formation. Overall, the stability of the complexes was governed by the balance between unfavorable solvation energies and favorable gas‒phase interactions (ΔGGAS). The contributing amino acids that make up the total binding energy were analyzed via decomposition analysis and are presented in Figs. S4–S5. The residues interacting during static docking were observed to be involved primarily in the contribution to the total binding free energy.

Table 2.

Means and SDs of different energy components that determine the binding free energies of the top ligands docked to the target proteins acetylcholinesterase (4ey7) and alpha-amylase (1B2y).

System	ΔVDWAALS	ΔEGB	ΔEEL	ΔGGAS	ΔESURF	ΔGSOLV	ΔTOTAL
4EY7_DONEPEZIL	− 42.78 ± 3.08	38.59 ± 10.65	− 9.71 ± 11.53	− 52.49 ± 12.24	− 5.99 ± 0.37	32.6 ± 10.5	− 19.89 ± 3.61
4EY7_APIGENIN	− 31.28 ± 4.17	28.26 ± 5.26	− 15.09 ± 7.50	− 46.37 ± 9.34	− 4.09 ± 0.53	24.18 ± 5.09	− 22.19 ± 5.17
4EY7_MYRICETIN	− 38.22 ± 3.73	51.66 ± 6.75	− 34.14 ± 10.24	− 72.36 ± 9.55	− 5.08 ± 0.29	46.58 ± 6.65	− 25.77 ± 4.03
1B2Y_ACARBOSE	− 10.53 ± 8.67	18.47 ± 19.76	− 16.01 ± 20.79	− 26.54 ± 27.77	− 2.25 ± 1.82	16.22 ± 18.05	− 10.32 ± 10.61
1B2Y_APIGENIN	− 29.05 ± 2.74	34.03 ± 4.51	− 20.61 ± 5.48	− 49.66 ± 6.07	− 3.77 ± 0.36	30.26 ± 4.29	− 19.40 ± 2.76
1B2Y_MYRICETIN	− 23.47 ± 4.97	35.05 ± 9.59	− 26.11 ± 15.01	− 49.58 ± 14.68	− 3.13 ± 0.64	31.92 ± 9.28	− 17.66 ± 6.00

In silico drug likeness and pharmacokinetic properties of the top docked compounds

The two top-ranked phytochemicals to each of the proteins from the docking analysis were subjected to predictive drug-likeness filtering analyses. The results from these analyses are presented in Table S5. Among the three phytochemicals, apigenin fulfilled the requirements for all the filtering tools (Lipinski, Ghose, Veber and Egan). Myricetin passed the 2-filtering analysis and failed 2 of the filtering analyses with just one violation, whereas quercetin passed one of the filtering analyses.

Discussion

Phytochemicals have various chemical constituents and biological actions with no or few side effects. For this reason, there is increasing interest in investigating it as a prospective candidate for anti-T2D and anti-AD medications in this specific situation⁴⁵. This study investigated the dual therapeutic nature of Syzygium aromaticum and its HPLC-identified compounds. Earlier reports have investigated the potential pharmacological effects of these plant extracts on postprandial blood glucose levels and neuronal damage^10,46–48. Thus, our aim was to assess the anti-T2D and anti-AD properties of flavonoid-rich extracts and phytochemicals, with a specific focus on targeting key factors such as α-amylase, α-glucosidase, AChE, BChE, and MAO, which are fundamental causes of the development of T2D and AD.

HPLC–DAD analysis of FRESA revealed eight bioactive flavonoids, namely, gallic acid, caffeic acid, ferulic acid, syringic acid, apigenin, kaempferol, quercetin and myricetin. Flavonoids such as myricetin, apigenin, kaempferol, and quercetin have a wide range of health advantages, including their ability to combat cancer and neurodegenerative disorders, reduce inflammation, and act as antioxidants. For example, apigenin is present at the pictogram level, whereas apigenin is present at the picogram level; however, recent studies have demonstrated its potent biological activities even at low concentrations^49,50. For example, apigenin has shown significant anti-inflammatory and antioxidant effects, which are relevant to the pathophysiology of both T2D and AD⁵¹. The presence of these phytochemicals in FRESA emphasizes its potential therapeutic use in disease treatment. The large number of phytochemicals found in Syzygium aromaticum support its importance in pharmacology and support its long-standing role as a remarkable source of bioactive substances that have major health benefits.

α-Amylase and α-glucosidase are important enzymes involved in glucose metabolism and diabetes control. Inhibition of these enzymes leads to a decrease in the digestibility of starch. In this study, we demonstrated the significant enzyme inhibitory effects of flavonoid-rich extracts of Syzygium aromaticum on α-glucosidase and α-amylase enzymatic activities, which are crucial for the regulation of postprandial hyperglycemia in individuals with type 2 diabetes mellitus (T2DM). Flavonoids, known for their antioxidant properties, have been found to inhibit these enzymes. The presence of specific chemical groups in flavonoids appears to be responsible for this effect⁴⁹. Our findings indicate that flavonoid-rich extracts of Syzygium aromaticum show potential and warrant further investigation for postprandial hyperglycemia in patients with T2DM. Our study is in agreement with previously published works on S. aromaticum^{10,46–48,52–54}, which showed α-glucosidase and α-amylase activity in diabetic animals.

AChE and BChE are important enzymes involved in cholinergic signaling. They facilitate the degradation of acetylcholine, resulting in decreased neurotransmission levels and gradual cognitive decline. By inhibiting the activities of these enzymes, cholinergic transmission in the brain can be increased, thus assisting in the assessment of AD symptoms⁵⁵. In other words, inhibitors of cholinesterase enzymes are currently the only medications sanctioned by the FDA for the treatment of AD and other neurodegenerative disorders. In AD pathology, abnormal cholinergic functioning is characterized by elevated AChE activity⁵⁶. Our study revealed substantial inhibition of AChE and BChE by the flavonoid-rich extract of S. aromaticum, suggesting its potential neuroprotective efficacy in the treatment of AD. These findings suggest the ability of the flavonoid-rich extract of S. aromaticum to alleviate AD-related enzyme activity. Our study correlates with earlier reports^55,57,58, whose report showed that phenolic extracts of Syzygium aromaticum possess AChE and BChE activities in a mouse model.

Monoamine oxidase A (MAO) is an important enzyme involved in many physiological processes, such as neurotransmitter metabolism and oxidative stress⁴². In our study, Syzygium aromaticum significantly reduced MAO activity in FeSO₄-induced brain damage, suggesting its therapeutic potential in AD. Our findings are in line with earlier research on the neuroprotective ability of Syzygium aromaticum bud extracts^59,60 in hydrogen peroxide-induced oxidative stress in human neuroblastoma SH-SY5Y cell lines as a model.

HPLC–DAD analysis of the flavonoid-rich extracts of Syzygium aromaticum revealed a total of 8 bioactive flavonoids. These bioactive flavonoids were then subjected to molecular docking with proteins associated with T2D and AD (α-amylase, α-glucosidase, AChE, BChE, and MAO) to identify the most promising drug candidates. The docking process identified three promising bioactive flavonids, apigenin, myricetin, and quercetin, which exhibit multitargeting ability with better binding affinity to α-amylase, α-glucosidase, AChE, BChE, and MAO than to acarbose, donepezil, decamethonium, and safinamide. Postdocking MM-GBSA analysis further confirmed these bioactive flavonoids as the most efficacious compounds in accordance with their better binding affinity and binding free energy than those of acarbose and donepezil. Our study also revealed favorable physicochemical properties of these best-hit bioactive flavonoids, with favorable lipophilicity, water permeability, and bioavailability. Thus, these compounds may lead drug candidates for T2D and AD treatment, triggering their selection for MD simulations. In this study, apigenin, myricetin, and quercetin were simulated with α-amylase, α-glucosidase, AChE, BChE, and MAO to confirm the stability and rigidity of the protein‒phytochemical complexes. The three compounds exhibited stable interactions with α-amylase, α-glucosidase, AChE, BChE, and MAO, as demonstrated by the analysis of RMSD, RMSF, ROG, SASA, and H-bonds, confirming their potential as drug agents against T2D and AD. In addition, the analysis of hydrogen bonds revealed significant interactions stabilizing the protein‒phytochemical complex through the elucidation of the best binding nodes. The bioactive flavonoids lead (apigenin, myricetin, and quercetin), which were evaluated through these parameters and compared with those of acarbose, donepezil, decamethonium, and safinamide, support our idea of performing in vitro investigations of flavonoid-rich extracts of Syzygium aromaticum to elucidate their activity in AD.

According to the MM/GBSA results, flavonoids, especially myricetin and apigenin, strongly bind to α-amylase and acetylcholinesterase. The highly hydroxylated structure of myricetin, which encourages hydrogen bonding and electrostatic interactions within the catalytic gorge, may be responsible for its greater binding affinity for acetylcholinesterase. Because polyhydroxylated flavonoids can stabilize enzyme–ligand complexes through a variety of noncovalent interactions, they exhibit improved inhibitory efficacy against acetylcholinesterase, which is consistent with earlier computational and experimental findings⁶¹. The dual binding potential of apigenin and myricetin raises the possibility that they could be used as multitarget medicines, which is a new approach to treating complicated illnesses such as Alzheimer’s disease and metabolic disorders where impaired glucose metabolism and cholinergic dysfunction coexist. Because they can enhance therapeutic efficacy while lowering drug resistance, multitarget-directed ligands are becoming increasingly desirable⁶².

Decomposition of the MM/GBS per residue revealed that most residues identified during molecular docking contributed significantly to ligand stabilization, indicating the reliability of the predicted binding modes. Key aromatic residues in the catalytic gorge of acetylcholinesterase, specifically Trp86, Tyr337, Phe295, and His447, maintained positive energy contributions, highlighting the importance of π-mediated and hydrophobic interactions in maintaining the ligand–enzyme complex. The ligands’ continued correct orientation within the active pocket is further supported by the maintenance of contacts close to the catalytic triad⁶³. Likewise, catalytically significant residues such as Asp197, Glu233, His299, and Trp59 demonstrated significant energy contributions in α-amylase, confirming the theory that the ligands successfully occupied functionally significant subsites. The involvement of these residues supports the anticipated inhibitory potential of these compounds because they align with their known functions in substrate recognition and catalysis⁶⁴. Overall, the evidence suggests that ligand binding is structurally persistent rather than mediated by transitory contacts because these residue-level interactions are preserved following free energy decomposition. This consistency between MM/GBSA analysis and docking predictions offers more proof of the binding mechanism and bolsters the viability of the discovered phytochemicals as viable candidates for additional experimental assessment.

In addition, the prediction of drug likeness revealed that apigenin had the most drug-like features, whereas myricetin and quercetin had fair drug likeness characteristics⁶⁵. Although in silico drug-likeness analysis suggested that myricetin and quercitrin possess promising bioactive profiles, their elevated topological polar surface area (TPSA > 140 Ų) and violations of Lipinski’s criteria signal potential pharmacokinetic liabilities that are important for interpreting their biological relevance. A high TPSA and multiple hydrogen bond donors/acceptors are generally associated with limited passive membrane permeability, which in turn often correlates with reduced oral absorption and bioavailability in vivo. For example, compounds with TPSA values above ~ 140 Ų tend to show poor oral uptake because of inadequate transcellular diffusion across the gastrointestinal epithelium⁶⁶. In the case of myricetin, preclinical pharmacokinetic studies have demonstrated very low absolute oral bioavailability (~ 9–10%) in rodents, which has been attributed to its poor aqueous solubility and slow gastrointestinal absorption, despite dose-dependent plasma exposure⁶⁷. While quercetin, as a glycoside of quercetin, has not been as extensively characterized in pharmacokinetic studies, flavonoid glycosides with high molecular weights and polarities are generally subject to inefficient oral uptake and extensive first-pass metabolism, which can further limit systemic exposure⁶⁸. These ADME limitations imply that, despite promising in vitro efficacy, both myricetin and quercitrin may reach subtherapeutic concentrations in vivo following oral administration, limiting their translational potential unless formulation strategies (e.g., nanoparticle delivery, prodrug design) or structural modification approaches are employed to improve solubility, membrane permeability, and metabolic stability⁶⁹. Favorable Veber and Lipinski properties indicate good permeation or absorption and good oral bioavailability, respectively^70,71. Our findings correlate with those of⁶⁴, whose study focused on the inhibitory potential of bioactive constituents from G. latifolium against important neurotherapeutic targets. This study has limitations that should be acknowledged. In vitro enzyme inhibitory assays were conducted using a flavonoid-rich extract of Syzygium aromaticum, and the observed biological activities cannot be directly attributed to individual phytochemicals or directly correlated with their respective molecular docking scores. In contrast, in silico docking analyses were conducted on identified, quantified compounds, representing a predictive approach. Therefore, direct quantitative correlation between extract-based in vitro inhibition and in silico docking scores is not feasible. Additionally, potential synergistic or antagonistic interactions among extract constituents, which may significantly influence enzyme inhibition, are not captured by molecular docking simulations. Despite these limitations, molecular docking was employed to complement the biochemical findings by providing mechanistic insight into the potential binding modes of the major quantified phytochemicals within the active sites of the target enzymes. The docking results should be interpreted as hypothesis-driven rather than confirmatory, aiding in the identification of candidate compounds that may contribute to the observed multitarget enzyme inhibitory activities. Further studies involving compound isolation, quantification, and enzyme kinetics are needed to validate these in silico predictions.

The findings from our study suggest that incorporating Syzygium aromaticum into the diet could offer therapeutic benefits for managing diabetes mellitus and Alzheimer’s disease because of its bioactive compounds with antioxidant and anti-inflammatory properties. For further research, these findings highlight the need to isolate individual compounds, conduct in vivo studies, and eventually perform clinical trials to validate the therapeutic potential of Syzygium aromaticum. Additionally, exploring the molecular mechanisms and synergistic effects with other dietary components or medications could pave the way for more effective combination therapies.

Additionally, our results have significant implications for the field of drug discovery. We highlight the potential of natural compounds as multitarget therapeutic agents, encouraging a shift toward more holistic and integrative approaches in drug development. Our study underscores the value of combining experimental and computational methods to identify and validate bioactive compounds, paving the way for more efficient and targeted drug discovery processes. Furthermore, the dual therapeutic potential of Syzygium aromaticum suggests that future research should focus on developing multifunctional drugs that address multiple pathways and diseases simultaneously, ultimately leading to more effective and comprehensive treatment options.

Conclusions

Our results revealed that three lead bioactive flavonoids (apigenin, myricetin, and quercetin) reasonably inhibited α-amylase, α-glucosidase, AChE, BChE, MAO receptors and diabetic and neuronal receptors. These enzymes are essential for the breakdown of starch to glucose and the formation of protein fibrils in the neurons of Alzheimer’s disease patients. These findings are supported by the presence of these bioactive flavonoids in the flavonoid-rich extracts of Syzygium aromaticum. The extracts exhibited antidiabetic and anti-AD properties by inhibiting carbohydrate-metabolizing enzymes and cholinesterase enzyme activity. Our results also revealed that bioactive flavonoids regulate carbohydrate-metabolizing enzymes and modulate cholinergic pathways to combat AD. These findings hold promise for developing multitarget-directed bioactive compounds to fight against T2D and AD. We recommend that in vivo experiments be performed to further evaluate our results. Additionally, isolation of these notably important phytochemicals can be further carried out in terms of drug discovery, which could be a way of managing these diseases.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

OAO conceptualized and designed the study; MI, GAG, and OAO wrote the first draft; SD and TD performed the experiment; ABO, OAO, MI, AKO, BEO, BOA, and GAG analyzed and interpreted the data; and OAO supervised the experiment. MI, GAG, ABO, AKO, BEO, BOA, and OAO reviewed the final draft of the manuscript. All authors approved the final version of the manuscript.

Funding

This study did not receive any funding whatsoever.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

The Institutional Research Animal Ethical Committee approved (BUI/BCH/2024/0002) the study protocols and research conducted in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. All the experimental methods were performed in accordance with the relevant protocols, guidelines and regulations approved by the Bowen University Research Ethics Committee (BUREC).

Informed consent

Not applicable.