Alpinia galanga Rhizome Extract Shields Against Noise‐Induced Cardiotoxicity via Antioxidant and Anti‐Inflammatory Actions: Experimental Insights

ABSTRACT

This study evaluated the cardioprotective effects of Alpinia galanga rhizome extract (GRE) against noise‐induced myocardial injury via phytochemical profiling, molecular docking, and in vivo assessment. Male Wistar rats (n = 6/group) were assigned to the following four groups: control (C), control + GRE (100 mg/kg), noise‐exposed (N), and noise‐exposed + GRE (N+GRE, 100 mg/kg). Rats in the N and N+GRE groups were exposed to 90 dB(A) white noise for 2 h/day for 28 days, with GRE administered orally throughout the exposure period. Phytochemical analysis confirmed the presence of flavonoids and phenolic acids with known antioxidant and anti‐inflammatory activities. In vitro, GRE significantly reduced nitric oxide production in lipopolysaccharide‐stimulated RAW264.7 macrophages. In vivo, noise exposure elevated cardiac malondialdehyde levels, impaired antioxidant enzyme activity, and increased circulating tumor necrosis factor‐alpha (TNF‐α) and heme oxygenase‐1 (HO‐1) levels. GRE treatment restored redox balance, suppressed proinflammatory mediator levels, and improved histopathological alterations. Molecular docking analysis indicated strong binding of GRE phytoconstituents to HO‐1 and TNF‐α, supporting the observed in vivo effects. These findings demonstrate that GRE mitigates noise‐induced cardiac injury through its antioxidant and anti‐inflammatory properties, highlighting its therapeutic potential

Alpinia galanga rhizome extract (GRE) was analyzed in vitro to identify its bioactive compounds, tested in vivo in a Wistar rat model of noise‐induced cardiotoxicity, and validated in silico by molecular docking. The combined results highlight the cardioprotective potential of GRE.

1. Introduction

Traditional remedies have gained increasing attention as potential sources of novel compounds for use in alternative therapies. Medicinal plants, known for their bioactive compounds, are increasingly recognized for their therapeutic potential and role in uncovering the mechanisms of various diseases. These compounds are believed to be safer than synthetic drugs for treatment [1]. Among these botanical resources, Alpinia galanga, a prominent member of the Zingiberaceae family, stands out not only for its culinary applications but also for its biologically active constituents, which exhibit significant pharmacological properties [2]. The historical and geographical context of A. galanga underscores its importance as a valuable resource in both traditional and modern medicinal practices, making it a promising candidate for further research and development in the quest for new therapeutic agents. Its phytochemical composition includes a diverse array of bioactive compounds, such as flavonoids, terpenoids, and phenolic acids, which have been extensively studied for their potential health benefits because of their biologically active constituents, which exhibit significant pharmacological properties [3, 4]. The rhizome of A. galanga has been traditionally utilized to address a variety of health concerns, including gastrointestinal disorders, respiratory ailments, circulatory issues, and inflammatory conditions [5]. This botanical gem possesses a broad pharmacological profile that encompasses antioxidant, antidiabetic, antiulcer, antidiarrheal, antiemetic, analgesic, anti‐inflammatory, and anticoagulant properties [3]. Key bioactive components, such as 1'‐acetoxychavicol acetate (ACA), galangin, and kaempferol, are believed to significantly contribute to these therapeutic effects. Recent studies have elucidated the mechanisms through which these compounds exert their benefits, such as modulation of the signaling pathways involved in inflammation and oxidative stress, inhibition of microbial growth, and protection against cellular damage [6, 7, 8, 9]. These findings highlight the immense potential of A. galanga as a source of novel bioactive compounds for biomedical applications.

Although the extensive phytochemical profile of A. galanga indicates its promising therapeutic potential, it is crucial to consider the broader context of environmental stressors, such as occupational noise exposure, which significantly impacts health. In various occupational settings, noise is a major environmental stressor with potential adverse effects on worker health. Industries, such as manufacturing and construction, are particularly prone to high levels of noise exposure, which poses significant challenges in maintaining occupational health and safety.

Noise has been linked to a range of adverse effects, including hearing impairment, stress‐related disorders, and cognitive decline [10, 11]. Exposure to loud noise triggers significant biological stress responses that negatively affect immune function [12] and may result in cardiovascular and metabolic consequences [13, 14]. These consequences include hypertension, ischemic heart disease, and stroke. These cardiovascular effects are mediated via complex mechanisms that involve the activation of the sympathetic nervous system and hypothalamic‒pituitary‒adrenal axis, leading to increased levels of stress hormones, such as cortisol and catecholamines [15]. Noise exposure also contributes to systemic inflammation, oxidative stress, and vascular dysfunction, all of which play roles in the development of cardiovascular diseases. In addition, experimental studies have indicated that chronic noise can provoke cardiotoxicity, leading to histopathological alterations such as myocardial injury, inflammatory cell infiltration, and necrosis [16, 17]. These findings highlight the critical role of noise as an environmental risk factor affecting both cardiovascular structure and function.

This study aimed to investigate the cardioprotective effects of A. galanga rhizome extract on noise‐induced cardiotoxicity. This study focused on evaluating biochemical, inflammatory, and histopathological markers to elucidate the therapeutic potential of the extract.

2. Methods

2.1. Phytochemical and Nutritional Characterization of the A. galanga Rhizome Extract

2.1.1. Sample Preparation and Extraction

Fresh rhizomes of A. galanga were procured from local markets in Tunis and thoroughly washed with tap water. To ensure precise botanical identification, a voucher sample (GR01/22) was identified and deposited in the herbarium of the Botany Department at the Faculty of Sciences of Tunis, with taxonomical confirmation by Dr. Faouzi Horchani.

The extraction process involved the use of dried rhizomes, which were initially crushed and sieved to produce a fine powder. A total of 30 g of A. galanga rhizome powder was then subjected to extraction with 200 mL of 70% ethanol and 30% water for 30 min, followed by vigorous stirring for 5 min. The mixture was subsequently agitated on a hot plate at 37°C for 20 min, a cycle that was repeated three times. The resulting mixture was centrifuged at 6000 × g for 10 min, and the supernatant was filtered through 90 mm filter paper. The ethanol was removed from the filtrate via rotary evaporation, and the final extracts were lyophilized to yield a powder suitable for experimental use, in accordance with the method outlined by Mazaheri and Shahdadi [18].

2.1.2. Proximate and Mineral Composition Analysis

The moisture, crude protein, total lipid, and ash contents of the A. galanga rhizome extract were determined via standardized methodologies established by the Association of Official Analytical Chemists (AOAC, 2018). The elemental composition, specifically the calcium (Ca), iron (Fe), and magnesium (Mg) contents, was quantified in the galangal powder via inductively coupled plasma atomic emission spectroscopy (ICP‒AES).

2.1.3. HPLC Analysis

The A. galanga rhizome extract was passed through a 0.45 µm membrane filter prior to chromatographic assessment. Phenolic constituents were profiled via reverse‐phase high‐performance liquid chromatography (RP‐HPLC) on an Agilent 1100 Series system (Agilent Technologies, Santa Clara, CA, USA) coupled with a diode array detector (DAD). Chromatographic separation was performed at ambient temperature using a Hypersil ODS C18 reversed‐phase column (250 mm × 4.6 mm, 4 µm particle size; Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase comprised solvent A (acetonitrile) and solvent B (water acidified with 0.2% sulfuric acid), which were delivered at a constant flow rate of 0.5 mL/min. The gradient elution program was as follows: 15% A/85% B (0–12 min), 40% A/60% B (12–14 min), 60% A/40% B (14–18 min), 80% A/20% B (18–20 min), 90% A/10% B (20–24 min), and 100% A (24–28 min). A 20 µL aliquot of the sample was injected, and detection was performed at 280 nm. Phenolic compounds were identified by matching retention times and UV spectral data obtained via DAD with those of authenticated reference standards. All analyses were performed in triplicate to ensure reproducibility and methodological robustness, following the protocol outlined by Serairi‐Beji et al. [19].

2.1.4. Analysis of Phenolic Compounds

2.1.4.1. Determination of Total Phenolic Content

The total phenolic content in the A. galanga rhizomes was determined via the Folin–Ciocalteu (FC) assay, as described by Yoo et al. [20]. In this procedure, 1 mL of the galangal rhizome extract was mixed thoroughly with 1 mL of diluted FC reagent and 10 mL of 7.5% sodium carbonate (Na₂CO₃). The mixture was then adjusted to a final volume of 2.5 mL with deionized water and incubated in the dark for 1 h. The absorbance of the resulting solution was measured at 760 nm via a Genesys 10 UV spectrophotometer (Thermo Electron Corporation, France). The phenolic content was quantified and expressed as milligram of gallic acid equivalent (GAE)/g of sample.

2.1.4.2. Determination of Flavonoid Content

The total flavonoid content in the A. galanga rhizome extract was quantified via the method described by Hogan et al. [21]. In this assay, 1 mL of the rhizome extract was combined with 0.3 mL of sodium nitrite (NaNO₂), 0.3 mL of aluminum chloride (AlCl₃), and 2 mL of sodium hydroxide (NaOH). The resulting mixture was incubated in the dark at room temperature for 15 min. Following incubation, the absorbance was measured at 510 nm via a Genesys 10 UV spectrophotometer (Thermo Electron Corporation, France). The flavonoid content was determined by comparing the absorbance to a calibration curve constructed with (+)‐catechin and was expressed as milligram of (+)‐catechin equivalent (CE)/g of sample.

2.1.5. Biological Activities of the Extracts of A. galanga Rhizomes

2.1.5.1. DPPH Free Radical Scavenging Activity

The DPPH free radical scavenging activity was assessed via the method described by Hatano et al. [22]. Serial concentrations of A. galanga rhizome extract, ranging from 5 to 200 µg/mL, were meticulously prepared. Each concentration was subsequently mixed with 1 mL of 0.078 mM DPPH, and the resulting mixture was vigorously shaken before being incubated for 30 min in the dark. Following the incubation period, the absorbance of the mixture was measured at 517 nm. For comparative purposes, ascorbic acid served as the standard antioxidant. DPPH radical inhibition was calculated via the following formula:

$$\begin{array}{ccc}& & \mathbf{Inhibition}\left(\%\right)\hfill \\ & & =\left[\left(\mathbf{Ab}{\mathbf{s}}_{\mathbf{control}}-\mathbf{Ab}{\mathbf{s}}_{\mathbf{sample}}\right)\right]/\left(\mathbf{Ab}{\mathbf{s}}_{\mathbf{control}}\right)]\times \mathbf{100}\hfill \end{array}$$

where Abs_control represents the absorbance of the control solution and Abs_sample denotes the absorbance of the GRE. The determination of the IC₅₀ value, which represents the minimum inhibitory concentration at which 50% of the DPPH radicals are neutralized, was achieved through linear regression analysis. A lower IC₅₀ value is indicative of increased antioxidant power, highlighting the efficacy of A. galanga rhizome extracts in inhibiting DPPH radicals.

2.1.5.2. Radical Cation Scavenging Activity Assay (ABTS)

The ABTS radical cation decolorization assay was conducted on the basis of the methodology outlined by Sharma et al. [23] with minor adaptations. In brief, the ABTS•+ reagent was generated in ethanol and adjusted to an absorbance of 0.70 ± 0.02 nm at 734 nm. Aliquots of 20 µL of the A. galanga rhizome extract were introduced into 200 µL of the ABTS•+ working solution. The mixture was incubated in the dark at 25°C for 4 min before the absorbance was measured. The antioxidant capacity was quantified and expressed as millimoles of Trolox equivalents per gram of dry rhizome extract.

2.1.5.3. Reducing Power Assay

The reducing power was assessed following the method described by Li et al. [24]. Briefly, increasing concentrations of GRE extract (50–250 µg/mL) were combined with 2.5 mL of sodium phosphate buffer (pH 6.6) and 2.5 mL of potassium ferricyanide (164.5 mg, 200 mmol/L, 1%). The mixture was then agitated and incubated at 50°C for 20 min. Following incubation, 2.5 mL of 10% trichloroacetic acid was added, followed by vortexing for 20 s and centrifugation at 1000 rpm for 8 min. Distilled water (2.5 mL) and 1% ferric chloride (0.5 mL) were then added. The absorbances of the samples were measured spectrophotometrically at 700 nm, and the IC₅₀ values of the extracts were determined.

2.1.5.4. Chelating Capacity Assessment of Ferrous Ions

The assessment of the chelating capacity of ferrous ions in the samples followed the experimental protocol proposed by Talaz et al. [25]. In brief, 1.6 mL of sample mixture at different concentrations was meticulously mixed with 1.6 mL of distilled water. Subsequently, 0.4 mL of FeCl₂ solution (0.5 mM) was added to the mixture, which was thoroughly homogenized. Next, 0.4 mL of ferrozine solution (0.5 mM) was added, and the mixture was vigorously shaken for 1 min before being allowed to stand at room temperature for 20 min. The absorbance of the resulting solution was then measured at 562 nm. Blank measurements were taken using the solvent alone, omitting the sample. The percentage of chelation was determined via the following equation:

$$\%\mathbf{Chelation}=\left[\mathbf{1}-\left(\mathbf{Ab}{\mathbf{s}}_{\mathbf{562}}\mathbf{sample}--\mathbf{Ab}{\mathbf{s}}_{\mathbf{562}}\mathbf{control}\right)\right]\times \mathbf{100}$$

To determine the IC₅₀ value of the sample, a sample curve was constructed on the basis of the absorbance measurements at 562 nm. Ethylenediamine tetra acetic acid (EDTA) was used as the standard reference for comparison.

2.1.5.5. Measurement of Nitric Oxide (NO) Production in the LPS‐Induced RAW264.7 Cell Line

The anti‐inflammatory activity of A. galanga rhizome extract (GRE) was assessed in the murine macrophage line RAW 264.7 (American Type Culture Collection, ATCC) by evaluating nitric oxide (NO) production as an indicator of the inflammatory response. The cells were seeded in 24‐well plates at a density of 2 × 10⁵ cells/mL and allowed to adhere for 24 h. After pretreatment with GRE for 1 h, lipopolysaccharide (LPS; 1 µg/mL) was added to stimulate NO production. After a 24‐h incubation period, the culture supernatants were harvested for nitrite quantification.

Nitrite levels, representing NO accumulation, were determined via the Griess reaction, which involves a colorimetric assay based on the diazotization of sulfanilamide in the presence of nitric oxide, followed by coupling with N‐(1‐naphthyl)ethylenediamine dihydrochloride under acidic conditions (5% phosphoric acid). A resazurin reduction assay was performed in parallel to assess the cytotoxicity of the samples. N(G)‐nitro‐L‐arginine methyl ester (L‐NAME) served as a reference inhibitor of NO synthesis. The absorbance was measured at 540 nm via a Varioskan Ascent microplate reader (Thermo Electron, Vantaa, Finland), and nitrite concentrations were calculated via a standard curve constructed from sodium nitrite (NaNO₂) solutions.

2.2. In Silico Study

2.2.1. Target Selection for Molecular Docking

In this molecular docking study, heme oxygenase‐1 (HO‐1) and tumor necrosis factor‐alpha (TNF‐α) were identified as pivotal molecular targets because of their significant roles in the oxidative and inflammatory responses elicited by chronic noise exposure, particularly in cardiac tissue. HO‐1 is a crucial antioxidant enzyme upregulated via the Nrf2 pathway in response to oxidative stress [26]. It contributes to cellular defense by degrading the pro‐oxidant heme into bioactive molecules such as bilirubin and carbon monoxide and plays a protective role in maintaining cardiovascular homeostasis. Its importance is highlighted by its involvement in conditions such as atherosclerosis and neurodegeneration, in which oxidative damage is a major pathological factor [12, 27]. TNF‐α is a proinflammatory cytokine that mediates systemic inflammation and is strongly associated with endothelial dysfunction and tissue injury following acoustic stress [28]. By targeting both HO‐1 and TNF‐α, this study sought to investigate whether the bioactive compounds identified in A. galanga extract, namely, gallic acid, rutin, ferulic acid, and naringenin, can modulate these pathways. This dual‐target approach provides mechanistic insights into the in vivo cardioprotective effects of the extract, potentially through the attenuation of oxidative damage and inflammatory responses.

2.2.2. Molecular Docking Procedure

Molecular docking analyses were conducted via the AutoDock 4.2 software suite [29] to investigate the binding affinities of selected phytoconstituents toward key targets implicated in oxidative stress and inflammation. The three‐dimensional crystal structures of human heme oxygenase‐1 (PDB ID: 3HOK; Rahman et al. [30]) and tumor necrosis factor‐alpha (PDB ID: 2AZ5; He et al. [31]) were retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/).

Prior to docking, the protein structures were curated by removing crystallographic water molecules and adding polar hydrogens and Gasteiger partial charges via AutoDock Tools. The ligand and receptor input files were converted into PDBQT format. The ligand structures were subjected to geometry optimization via ACD/3D Viewer software (https://www.acdlabs.com) to ensure energy minimization and proper conformational stability.

Grid boxes for the docking simulations were defined around the active sites of the target proteins, and interaction maps were generated via AutoGrid to streamline the docking. Docking runs were executed via Lamarckian Genetic Algorithm. Following docking, ligand–target interactions were analyzed and visualized via Discovery Studio Visualizer (BIOVIA, 2017R2; https://www.3ds.com) and PyMOL version 0.99rc6 (https://pymol.org), allowing the identification of key binding residues and interaction types.

2.3. In Vivo Protocol

Forty‐eight male Wistar rats (3–4 months old, 180–220 g) were obtained from the Pasteur Institute (Tunis, Tunisia) for this study. Prior to the experimental procedures, the animals were housed in the institutional animal facility for a 7‐day acclimatization period under controlled environmental conditions (temperature: 23 ± 1°C; 12‐h light/dark cycle). The rats were housed in pairs to reduce environmental and social stress and were provided unrestricted access to standard pellet feed (Industrial Society of Food, Sfax, Tunisia) and potable water.

Following acclimatization, the animals were randomly assigned to four experimental groups (n = 6 per group). During the intervention phase, animals in the noise‐exposed groups (N and N + GRE) were transferred daily to a dedicated sound exposure room, whereas those in the control (C) and GRE‐only groups were placed in a separate, acoustically isolated chamber with ambient noise levels maintained below 40 ± 5 dB (A). All animal procedures were conducted in accordance with the ethical standards of the National School of Veterinary Medicine Ethics Committee, Sidi Thabet (Approval No. CEEA‐ENMV 46/22), and complied with the guidelines of the International Council for Laboratory Animal Science (ICLAS).

2.3.1. Noise Exposure

The rats in the N and N + GRE groups underwent repeated acoustic stimulation over a 28‐day period. Noise exposure was administered via a calibrated audio system consisting of a sound generator (Audacity software, version 2.3.2) configured to emit octave‐band noise within the 8–16 kHz frequency range at an intensity of 90 dB (A). The acoustic signal was broadcast via a centrally positioned loudspeaker to ensure uniform sound distribution across the exposure chamber. The sound pressure levels were continuously monitored via a precision Class 1 integrating sound level meter (Type 2238, Brüel & Kjær, Denmark) to maintain consistency.

Each noise session lasted for 2 h per day, 5 days per week, with automatic playback cycles programmed to repeat at fixed intervals. The exposure windows were scheduled between 09:00 and 11:00 h.

2.3.2. Experimental Design

Twenty‐four male Wistar rats were randomly divided into the following four groups:

Group C: Control rats were housed under normal conditions.

Group GRE: Control rats treated with A. galanga rhizome extract at 100 mg/kg/day for 28 days.

Group N: Rats were exposed to noise.

Group N + GRE: Rats exposed to noise received A. galanga rhizome extract at 100 mg/kg/day for 28 days.

A. galanga rhizome extract was administered once daily by oral gavage at a dose of 100 mg/kg at 8:00 a.m. immediately prior to each noise exposure.

2.3.3. Sample Collection

At the end of the experimental protocol, the animals were anesthetized with ketamine hydrochloride (50 mg/kg, i.p.; KETALAR, Pfizer Inc., USA), obtained from the Central Pharmacy of Tunisia, and subsequently euthanized by decapitation. Blood samples were promptly collected via two separate collection systems: EDTA‐containing tubes for hematological profiling and plain and additive‐free tubes for serum preparation. The latter were allowed to clot at room temperature and subsequently centrifuged at 3000 × g for 10 min at 4°C to isolate the serum for biochemical analyses.

Following dissection, the hearts were rapidly excised, rinsed in chilled phosphate‐buffered saline (PBS; 0.1 M, pH 6.8), and divided into two portions according to standard procedures for organ collection in rats (National Institute of Environmental Health Sciences) [32]. One fraction was fixed in formalin for histopathological evaluation, and the other was flash‐frozen and stored for subsequent assessment of oxidative stress‐related biomarkers.

2.3.4. Assessment of Hematological and Plasma Biochemical Parameters

2.3.4.1. Hematological Parameters

Complete blood counts, including red blood cells, white blood cells, and platelets, were determined via an automated hematology analyzer (I‐Sens i‐Smart 30 Pro Electrolyte Analyzer, UK).

2.3.4.2. Plasma Biochemical Assays

Plasma biochemical parameters, including cholesterol and triglyceride, lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and creatine kinase (CK) levels were quantified via a COBAS INTEGRA 400 Plus automated analyzer (Roche, Switzerland) following standardized protocols.

2.3.5. Measurement of Oxidative Stress Markers

A portion of the excised cardiac tissue was immediately rinsed with ice‐cold isotonic saline, blotted to remove excess fluid, weighed, and stored at −80°C for subsequent biochemical assays. For tissue homogenization, samples were processed on ice via a ULTRA‐TURRAX homogenizer (IKA‐WERK, Germany) in cold phosphate‐buffered saline (PBS; 0.1 M, pH 7.2). The homogenates were subsequently centrifuged at 10 000 rpm for 20 min at 4°C. The resulting supernatants were carefully collected and preserved at −80°C until further biochemical analysis.

2.3.5.1. Lipid Peroxidation Determination

Lipid peroxidation was quantified by determining malondialdehyde (MDA) levels following the established method described by Buege and Aust [33], with minor modifications. Briefly, tissue homogenates were combined with a solution containing 1% butylated hydroxytoluene (BHT) in 20% trichloroacetic acid (TCA, v/v), and the mixture was centrifuged at 1000 × g for 10 min. The supernatant was subsequently reacted with thiobarbituric acid (TBA)–Tris buffer solution (120 mM TBA and 26 mM Tris, v/v) under acidic conditions (0.6 M HCl).

The reaction mixture was incubated at 100°C for 15 min and then cooled to ambient temperature. The absorbance was measured at 532 nm via a UV–visible spectrophotometer (UV‐2650, Labeled, Inc., USA). Lipid peroxidation levels are expressed as nanomoles of MDA per milligram of total protein per sample.

2.3.5.2. Measurement of Catalase Activity

Catalase (CAT) enzymatic activity was determined following the spectrophotometric procedure originally described by Aebi [34], which quantifies the enzymatic decomposition of hydrogen peroxide (H₂O₂). The decrease in absorbance at 240 nm, corresponding to the degradation of H₂O₂, was monitored over time via a UV‒visible spectrophotometer. Enzyme activity was calculated and expressed as micromoles of H₂O₂ decomposed per minute per milligram of protein (µmol/min/mg protein).

2.3.5.3. Measurement of Superoxide Dismutase (SOD) Activity

Superoxide dismutase (SOD) enzymatic activity was determined following the protocol established by Beyer and Fridovich [35], which quantifies the enzyme's ability to inhibit the autoxidation of epinephrine to adrenochrome. The assay was conducted in 50 mM carbonate buffer (pH 10.2) containing epinephrine as the substrate, with bovine catalase (0.4 U/mL) added to eliminate interfering hydrogen peroxide. The rate of adrenochrome formation was monitored spectrophotometrically by measuring the absorbance at 480 nm. SOD activity was calculated on the basis of the degree of inhibition of epinephrine autooxidation and normalized to protein concentration, with results expressed in international units per minute per milligram of protein (IU/min/mg/protein).

2.3.5.4. Measurement of Protein Concentration

The total protein concentration in the supernatant samples was quantified via the Bradford assay [36], which employs bovine serum albumin (BSA) as the reference standard. The absorbance was measured at 595 nm via a spectrophotometer. Protein levels were calculated on the basis of the BSA calibration curve and expressed in milligrams per milliliter (mg/mL).

2.3.6. Quantification of the Serum Levels of Heme Oxygenase‐1 (HO‐1) and Tumor Necrosis Factor‐Alpha (TNF‐α)

Serum concentrations of heme oxygenase‐1 (HO‐1) and tumor necrosis factor‐alpha (TNF‐α) were measured via enzyme‐linked immunosorbent assay (ELISA) kits. HO‐1 levels were quantified via the Rat HO‐1 (Heme Oxygenase‐1), ELISA Kit (ab279414; Abcam, Cambridge, UK), and TNF‐α levels were measured via the Rat TNF‐α ELISA Kit (A78898; Antibodies.com, China). All procedures were performed strictly according to the manufacturers’ protocols to ensure the accuracy, specificity, and reproducibility of the results.

2.3.7. Histological Study

For histopathological evaluation, rat hearts were initially perfused to remove blood residue and subsequently fixed in 10% neutral‐buffered formalin for 48 h to preserve tissue architecture. After fixation, the samples were dehydrated via a graded series of ethanol to gradually remove water. The tissues were then embedded in paraffin wax to provide structural support for sectioning. Using a rotary microtome (Leica Biosystems RM2245, USA), thin sections of 4–5 µm were cut. These sections were mounted on glass slides, deparaffinized with xylene, rehydrated with decreasing ethanol concentrations, and stained with hematoxylin and eosin (H&E) to highlight the cellular and tissue morphology via light microscopic examination [37].

2.3.8. Statistical Analyses

The data are expressed as the means ± standard deviations (SDs). Statistical analyses were performed via Biostat software for Windows. Group comparisons (n = 6 per group) were conducted via the nonparametric Kruskal‒Wallis test, followed by pairwise comparisons via the Mann‒Whitney U test to identify significant differences between groups. Statistical significance was set at p < 0.05.

3. Results

3.1. In Vitro Evaluation of the A. galanga Rhizome Extract

3.1.1. Proximate and Mineral Composition

Table 1 presents the proximate and mineral compositions of the A. galanga rhizome powder. The moisture content was found to be 6.13%, indicating that a relatively low water content may contribute to the stability and shelf‐life of the powder. The crude ash content was 7.10%, reflecting the total mineral content present. The crude fat content was minimal at 0.9%, while the crude protein content reached 23.95%, suggesting a notable protein contribution. Carbohydrates accounted for 47.43%, making them the major macronutrient component.

TABLE 1.

Proximate and mineral composition of the A. galanga rhizome extract.

Component	Amount
Proximate composition
Moisture (%)	6.13
Ash (%)	7.10
Crude fat (%)	0.90
Crude protein (%)	23.95
Carbohydrates (%)	47.43
Mineral composition
Calcium (mg/g dry weight)	3.09
Iron (µg/g dry weight)	13.08
Magnesium (µg/g dry weight)	4.66

In terms of mineral composition, calcium was detected at 3.09 mg/g dry weight, whereas iron and magnesium were present at 13.08 and 4.66 µg/g dry weight, respectively. These values highlight the potential nutritional significance of the A. galanga rhizome powder.

3.1.2. HPLC Analysis

High‐performance liquid chromatography (HPLC) analysis of the A. galanga rhizome extract (Figure 1) revealed the presence of several phenolic and flavonoid compounds with known pharmacological relevance. As shown in Table 2, naringenin was identified as the predominant constituent, with a retention time (RT) of 28.69 min and the highest peak area (722.3, corresponding to 0.31% of the extract). Other identified compounds included rutin (0.20%, RT: 20.92 min), ferulic acid (0.18%, RT: 24.43 min), and gallic acid (0.05%, RT: 3.28 min).

FIGURE 1.

HPLC chromatographic profile of the A. galanga rhizome extract and chemical structures of the identified biomolecules. (A) High‐performance liquid chromatography (HPLC) profile of A. galanga rhizome extract showing the separation and identification of four major bioactive compounds: gallic acid, rutin, ferulic acid, and naringenin. (B) Chemical structures of the four identified biomolecules.

TABLE 2.

Chromatographic characteristics and quantitative analysis of phenolic and flavonoid compounds from A. galangal rhizome extracts.

No.	Retention time (min)	Peak area	Identified compound	Concentration (%)
1	3.528	128.42	−	−
2	3.642	350.96	Unknown	−
3	3.932	53.86	−	−
4	4.168	56.38	−	−
5	4.271	292.15	−	−
6	4.951	101.61	−	−
7	6.018	36.94	−	−
8	9.501	116.36	Gallic acid	0.05
9	11.435	50.84	−	−
10	11.896	73.34	−	−
11	12.659	38.80	−	−
12	13.779	36.38	−	−
13	17.426	57.39	−	−
14	17.984	108.92	Rutin	0.20
15	18.394	99.55	−	−
16	19.416	365.75	Ferulic acid	0.18
17	20.098	40.37	−	−
18	21.274	39.21	−	−
19	23.425	47.78	−	−
20	26.138	95.40	−	−
21	28.699	722.31	Naringenin	0.31

In addition, a prominent unidentified peak was observed at RT = 3.64 min, with a substantial peak area (350.96), suggesting the presence of another major phytochemical. This compound, possibly a phenolic or flavonoid derivative, warrants further structural characterization, potentially via LC‒MS/MS or NMR spectroscopy.

These detected bioactive molecules are widely recognized for their antioxidant, anti‐inflammatory, and cytoprotective activities, which may underlie the neuroprotective potential of A. galanga in oxidative stress‐related pathologies.

3.1.3. Phytochemical Characteristics of the Extracts of A. galanga Rhizomes

The phenolic profile of the A. galanga rhizome extract (GRE) was assessed via the Folin–Ciocalteu method, which is a standard approach for estimating the reducing capacity of plant‐derived compounds. Quantitative analysis, as detailed in Table 3, revealed that GRE contains a considerable concentration of phenolic compounds, with a total content equivalent to 49.05 ± 4.32 mg GAE/g dry extract. In parallel, the flavonoid content, expressed as catechin equivalents, was measured at 17.28 ± 1.08 mg CE/g, suggesting that GRE may be a rich source of antioxidant secondary metabolites.

TABLE 3.

The contents of total polyphenols (TPC) and flavonoids (TFC) in A. galanga rhizome extracts.

Total phenolic content (mg GAE/g)	49.05 ± 4.32
Total flavonoid content (mg CE/g)	17.28 ± 1.08

Note: The TPC is expressed in milligram of gallic acid equivalent (mg GAE/g), and the total flavonoid content (TFC) is expressed in mg of catechin equivalent (mg CE/g). The values represent the means ± SDs of triplicate samples.

3.1.4. Assessment of Antioxidant Activity in A. galanga Rhizome Extract

The antioxidant potential of the A. galanga rhizome extract (GRE) was assessed via multiple in vitro assays, as shown in Table 4. In the DPPH radical scavenging assay, GRE exhibited a half‐maximal inhibitory concentration (IC₅₀) of 39.5 ± 2.38 µg/mL, indicating robust free radical neutralizing capacity. In comparison, the synthetic antioxidant BHA had a lower IC₅₀ value of 10 µg/mL. Additionally, the extract demonstrated metal‐chelating activity, with an IC₅₀ of 13.4 ± 4.08 µg/mL, suggesting its ability to reduce oxidative stress via transition metal ion sequestration. The ABTS assay further confirmed the antioxidant efficacy of GRE, yielding a scavenging activity of 36.23 ± 2.21 mmol Trolox/g of extract. Collectively, these findings underscore the strong antioxidant capacity of GRE and support its potential as a rich source of bioactive phytochemicals with therapeutic relevance to human health.

TABLE 4.

Antioxidant activities of A. galanga rhizome extract.

Contents	GRE
IC50 DPPH (µg/mL)	60.5 ± 2.38
chelating activity (µg/mL)	13.4 ± 4.08
ABTS (mmol Trolox/g)	36.23 ± 2.21

3.1.5. Assessment of Anti‐Inflammatory Activity in A. galanga Rhizome Extracts

The anti‐inflammatory activity of A. galanga rhizome extract (GRE) was assessed by measuring its capacity to suppress nitric oxide (NO) production in lipopolysaccharide (LPS)‐stimulated RAW 264.7 murine macrophages. GRE inhibited NO release in a concentration‐dependent manner, with notable effects observed at 10, 25, and 50 µg/mL GRE. The corresponding IC₅₀ values were 18.51, 35.2, and 51.6 µg/mL, respectively (data not shown), indicating the potent ability of the extract to attenuate LPS‐induced inflammatory responses at relatively low concentrations. These findings suggest that GRE may modulate macrophage activation and exert anti‐inflammatory effects through the suppression of inducible nitric oxide synthase (iNOS)‐mediated NO synthesis.

3.2. Molecular Docking Analysis

Molecular docking is a technique that analyses the conformation and orientation (indicated together as the “pose”) of ligands into the binding site of a macromolecular target. In this study, a possible mechanism of action of the identified phytomolecules (gallic acid, rutin, ferulic acid, and naringenin) toward the receptors (pdb: 3hok) and (pdb: 2az5) of the anti‐human heme oxygenase‐1 and anti‐TNF‐alpha activities, respectively, was identified.

3.2.1. Molecular Docking Study Against “Human Heme Oxygenase‐1” (pdb: 3hok)

The docking results depicted in Table 5 show that among all the docked phytomolecules, the most bioactive compound was rutin, which had the best docking score (binding energy value = −9.2 kcal/mol). The 3D model in Figure 2 shows that this ligand fits well in the binding cavity of “human heme oxygenase‐1.” The interactions depicted in Figure 2, which demonstrate the formation of four hydrogen bonds through its hydroxy groups with His‐B‐25, Glu‐B‐32, Thr‐B‐135, and Asp‐B‐140, reduce the binding energy score and thus stabilize the docking complex. This ligand shows greater potential than the cocrystallized ligands do. In addition, other interactions are formed by rutin, such as carbon hydrogen bonds (Glu‐B‐32), pi‐anions (Glu‐B‐32), pi‐cations (Arg‐B‐35), Pi–Pi stacking (Phe‐B‐214), and pi–alkyl interactions (Val‐B‐50 and Leu‐B‐147). On the other hand, as shown in Figure 3, “naringenin” (−7.9 kcal/mol) was the second most effective phytocompound in the A. galanga rhizome extract, and this docked ligand is involved in H‐bonds with Thr‐B‐135, Pi‐sigma bonds with Ala‐B‐28, Pi–Pi bonds with Phe‐B‐207 and Phe‐B‐214 and Pi–alkyl bonds with Ala‐B‐31 and Ile‐B‐211 (Figure 3C). “Ferulic acid” (−5.9 kcal/mol) is considered the third most bioactive ligand, with H‐bonds, with Ala‐B‐28, Pi‐Anion (Glu‐B‐32), Pi‐Cation (Arg‐B‐35), Pi–Pi Stacked (Phe‐B‐214), and Pi‐Alkyl (Ala‐B‐31) (Figure 3B). “Gallic acid” (−4.4 kcal/mol) was found to be the least active ligand, showing some interactions, as detailed in Figure 3A.

TABLE 5.

Binding energy of the docked compounds in the binding cavity of ‘human heme oxygenase‐1’ (pdb: 3hok).

Compound	Binding energy (kcal/mol)
Gallic acid	−4.4
Rutin	−9.2
Ferulic acid	−5.9
Naringenin	−7.9
Co‐crystallized ligand	−9.0

FIGURE 2.

2D model of different interactions formed by the most active compound, “Rutin,” within the active site of “Human Heme Oxygenase‐1” (pdb: 3hok).

FIGURE 3.

2D model of different interactions: gallic acid (a), ferulic acid (b), naringenin (c), and the cocrystallized ligand (d) within the active site of “human heme oxygenase‐1” (pdb: 3hok).

3.2.2. Molecular Docking Study Against “TNF‐alpha” (pdb: 2az5)

The molecular docking analysis for TNF‐α, as detailed in Table 6, revealed that “rutin” demonstrated the most favorable binding affinity, achieving a docking score of −8.8 kcal/mol, thereby surpassing the cocrystallized ligand, which recorded a score of −8.1 kcal/mol. Among the tested phytoconstituents, rutin was the most potent, displaying strong binding interactions with the target enzyme. It forms four conventional hydrogen bonds with Tyr‐A‐151, Ser‐B‐60, Leu‐B‐121, and Tyr‐B‐151 via its hydroxyl groups, along with additional stabilizing interactions (Figure 4).

TABLE 6.

Binding energy of the docked compounds in the binding cavity of “TNF‐alpha” (pdb: 2az5).

Compound	Binding energy (kcal/mol)
Gallic acid	−5.5
Rutin	−8.8
Ferulic acid	−5.7
Naringenin	−7.5
Co‐crystallized ligand	−8.1

FIGURE 4.

2D model of different interactions formed by the most active compound, “Rutin,” within the active site of “TNF‐alpha” (pdb: 2az5).

In addition, naringenin demonstrated the second‐highest binding affinity (−7.5 kcal/mol) among the docked compounds. Its interaction profile included a hydrogen bond with Tyr‐A‐151, a π‒π stacking interaction with Tyr‐A‐59, and an amide‒π stacking interaction with Leu‐A‐120 (Figure 5C). Ferulic and gallic acids displayed moderate binding affinities and formed relevant contacts with the enzyme (Figure 5A,B).

FIGURE 5.

2D model of different interactions of: gallic acid (a), ferulic acid (b), naringenin (c), and the co‐crystallized ligand (d) within the active site of “TNF‐alpha” (pdb: 2az5).

These findings suggest that several phytocompounds present in A. galanga rhizome extract exhibit promising inhibitory potential against TNF‐α, supporting their possible role in anti‐inflammatory mechanisms.

3.3. In Vivo Assessment

3.3.1. Evaluation of Hematological Parameters

Table 7 presents the hematological parameters of the rats exposed to noise (N), the rats exposed to noise and receiving GRE (N+GRE), the control (C) groups, and the rats that received GRE only (GRE).

TABLE 7.

Complete blood counts.

	Red blood cells (×106 µL)	White blood Cells (×103 µL)	Blood platelets (×103 µL)
C	9.05 ± 0.7	8.20 ± 0.89	746.2 ± 33.37
GRE	9.34 ± 1.3	8.86 ± 1.04	735.33 ± 53.82
N	8,33 ± 1.3	11.86 ± 1.14 *	953.45 ± 47.2 *
N + GRE	10,1 ± 1.7	9.58 ± 0.77	899.5 ± 35.25

Note: The results are expressed as the means ± SDs of six animals. The different pairs of groups were compared via the Mann‒Whitney U test.

^* p < 0.05 versus the control group.

The results revealed a notable increase in white blood cell count in the Noise (N) exposure groups compared with the Control (C) group (11.86 ± 1.14 [103 µL] vs. 8.20 ± 0.89 [10³ µL]). Additionally, there was a significant increase in blood platelets in the rats in the noise (N) group compared with those in the control (C) group (953.45 ± 47.2 [10³ µL] vs. 746.2 ± 53.37 [10³ µL]). The red blood cell count did not significantly differ among the four groups.

The administration of the A. galanga rhizome extract to the N + GRE and G groups did not elicit any significant alterations in hematological parameters compared with those in the control (C) group.

3.3.2. Evaluation of Biochemical Parameters

Table 8 presents the plasma concentrations of cholesterol, triglycerides (TGs), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and creatine kinase (CK).

TABLE 8.

Plasma biochemical parameters of the different groups.

	Cholesterol (mmol/L)	Triglyceride (mmol/L)	Lactate dehydrogenase (U/L)	Aspartate aminotransferase (U/L)	Creatine kinase (U/L)
C	1.20 ± 0.07	0.9 ± 0.08	344.33 ± 40.53	132.33 ± 13.89	385.5 ± 37.31
GRE	1.01 ± 0.07	1.3 ± 0.07 *	323.20 ± 21.93	145.02 ± 21.3	409.5 ± 39.21
N	1.52 ± 0.07 *	1.045 ± 0.08 *	613 ± 64.01 *	261.5 ± 24.24 *	657.16 ± 81.14 *
N + GRE	1.25 ± 0.05	1.4 ± 0.05	344.5 ± 32.65	161.02 ± 12.3	457.83 ± 142.39

Note: The results are expressed as the means ± SDs of six animals. The different pairs of groups were compared via the Mann‒Whitney U test.

^* p < 0.05 versus the control group.

The mean total cholesterol level in the control group was 1.20 ± 0.07 mol/L, which was significantly greater than that in the N groups (1.52 ± 0.07 vs. 1.20 ± 0.07 mol/L, p < 0.05). Similarly, the average level of triglycerides in the control group was 0.9 ± 0.02 mol/L, which was significantly elevated in the N groups (1.29 ± 0.11 vs. 0.9 ± 0.02 mmol/L, p < 0.05). The administration of A. galanga rhizome extract (GRE) during the exposure period resulted in stable levels of both cholesterol and triglycerides.

Our results revealed a significant increase in the LDH plasma concentration following noise exposure compared with that in the control group (613 ± 24.43 vs. 385.66 ± 74.85 U/L, p ≤ 0.01).

Furthermore, compared with control rats, noise‐exposed rats presented significantly increased AST plasma concentrations. The AST levels were 261.5 ± 24.24 versus 261.5 ± 24.24 U/L with p = 0.02.

Additionally, the total concentration of CK significantly increased following noise exposure, from 385.5 ± 37.31 U/L in the control group to 657.16 ± 81.14 (p = 0.02) in the N group.

However, the administration of GRE effectively preserved these cardiac markers at normal levels compared with those in the control group.

3.3.3. Oxidative Stress Parameters

The levels of MDA, SOD, and CAT activities in the heart tissues were measured and are presented in Figure 6.

FIGURE 6.

Effect of exposure to noise (90 dB (A)) and the protective effect of A. galanga rhizome extract (100 mg/kg body weight) on rat heart oxidative stress parameters. (A) Lipid peroxidation, (B) catalase activity, and (C) superoxide dismutase activity. The results are expressed as the means ± SDs of six animals. Different pairs of groups were compared via the Mann‒Whitney U test. * p < 0.01 versus the control group (N = 6), # p < 0.01 versus the NT group (N = 6).

Lipid peroxidation, determined by elevated malondialdehyde (MDA) levels, was significantly greater in the noise‐exposed group (N) than in the control group (0.345 ± 0.14 vs. 0.146 ± 0.061 nmol/mg protein, p < 0.01), as depicted in Figure 6A. Notably, the administration of A. galanga rhizome extract (GRE) significantly ameliorated the N + GRE group relative to the control (0.178 ± 0.02 vs. 0.136 ± 0.061 nmol/mg protein, p < 0.05).

Moreover, the investigation revealed a significant increase in catalase activity in the cardiac tissue of noise‐exposed rats (N) compared with that in the control group (0.076 ± 0.009 vs. 0.049 ± 0.003 µmol/min/mg protein; p < 0.05; Figure 6B). The administration of A. galanga rhizome extract (GRE) significantly improved catalase activity in the N + GRE group compared with that in the noise‐exposed group (0.054 ± 0.006 vs. 0.076 ± 0.009 µmol/min/mg protein, respectively; p < 0.05) (Figure 6B).

Furthermore, the present study demonstrated a significant increase in superoxide dismutase (SOD) activity in the noise‐exposed groups (N) compared with the control group (18.77 ± 0.80 vs. 7.32 ± 1.90 USOD/min/mg protein; p < 0.01; Figure 6C). However, GRE supplementation mitigated this increase in the group of rats that received GRE compared with the control group (8.42 ± 1.20 vs. 7.32 ± 1.90 USOD/min/mg protein).

These findings indicate the potential beneficial effects of GRE in mitigating oxidative stress induced by exposure to noise.

3.3.4. Serum Levels of TNF‐α and HO‐1

The serum levels of TNF‐α and HO‐1 were assessed to evaluate systemic inflammation and antioxidant responses following noise exposure and treatment with A. galanga rhizome extract.

As shown in Table 9, in the noise‐exposed group (N), the TNF‐α concentration increased significantly to 36.22 ± 13.21 pg/mL compared with that in the control group (C), which was 16.53 ± 1.02 pg/mL (p < 0.01). The administration of the extract during noise exposure (N+GRE) significantly reduced TNF‐α levels to 19.12 ± 10.05 pg/mL (p < 0.01 vs. N), which approached control values. No significant change was observed in the GRE‐only group (13.20 ± 5.32 pg/mL) compared with the control. Similarly, HO‐1 levels were elevated in the noise group (7.74 ± 1.07 pg/mL) relative to the control group (4.08 ± 0.02 pg/mL, p < 0.01), indicating oxidative stress response activation. Treatment with A. galanga during noise exposure further increased HO‐1 expression to 11.20 ± 1.11 pg/mL (p < 0.01 vs. N), suggesting enhanced antioxidant defense. The GRE‐only group presented HO‐1 levels similar to those of the control group (4.13 ± 0.05 pg/mL).

TABLE 9.

Serum levels of TNF‐α (pg/mL) and HO‐1 (pg/mL) in the experimental groups.

	TNF‐α pg/mL	HO‐1 pg/mL
C	16.53 ± 1.02#	4.08 ± 0.02#
GRE	13.20 ± 5.32#	4.13 ± 0.05#
N	36.22 ± 13.21*	7.74 ± 1.07*
N + GRE	19.12 ± 10.05#	11.2 ± 1.1#

These results demonstrate that noise exposure triggers systemic inflammation and oxidative stress, which are significantly alleviated by A. galanga extract administration.

3.3.5. Heart Histological Analysis

Histological analysis of myocardial tissue in the control group revealed normal cardiac architecture, with well‐preserved cardiomyocyte striations and a regular myofibrillar arrangement (Figure 7A, a).

FIGURE 7.

Representative light microscopy images of heart tissue sections (stained with H&E ×40 on the left, ×100 on the right) following exposure to noise 90 dB(A) and the protective effect of A. galanga rhizome extract (100 mg/kg body weight).

In contrast, rats exposed to 90 dB(A) noise presented significant histological alterations (Figure 7B, b; B', b'; and B'', b''). Microscopic examination of the myocardial tissue from this group revealed inflammatory lesions localized in the subepicardial region (Figure 3B, b). These lesions are characterized by a notable inflammatory infiltrate consisting of lymphocytes, polynucleires, and histiocytes (Figure 7B', b'). Myocardial fiber necrosis was also observed (Figure 7B', b' and B″, b″). Despite these findings, there was no evidence of myocardial fiber dystrophy, fibrosis, or hypertrophy.

Conversely, the rats that were administered GRE did not exhibit significant histological changes compared with those in the control group (Figure 7C, c), indicating that A. galanga rhizome extract did not induce notable alterations in myocardial architecture.

4. Discussion

Occupational exposure to physical hazards, such as chronic noise, remains a significant public health concern, particularly among industrial workers. Chronic noise has been shown to exert profound systemic effects, including cardiovascular dysfunction, through mechanisms involving stress, oxidative imbalance, and inflammation. This study aimed to evaluate the potential cardioprotective effects of A. galanga rhizome extract (GRE) against noise‐induced cardiotoxicity. A comprehensive investigation was conducted by assessing hematological and biochemical parameters, oxidative stress biomarkers, and histopathological alterations in the myocardial tissue of rats exposed to chronic, high‐intensity noise.

A. galanga rhizome extract (GRE) has attracted considerable scientific interest owing to its diverse bioactive composition and broad pharmacological properties. A. galanga, which is traditionally employed in various ethnomedicinal systems, is known for its potent antioxidant, anti‐inflammatory, antimicrobial, and neuroprotective effects, primarily attributed to its rich array of phytochemicals. In our study, high‐performance liquid chromatography (HPLC) analysis revealed the presence of phenolic and flavonoid compounds, including naringenin, rutin, ferulic acid, and gallic acid. These compounds have been extensively documented for their ability to modulate key molecular pathways associated with oxidative stress, inflammatory signaling, and cellular protection [38, 39, 40]. For example, naringenin is known to inhibit NF‐κB activation and suppress proinflammatory cytokine production [41], whereas gallic acid acts as a potent free radical scavenger and lipid peroxidation inhibitor [42]. The presence of such multifunctional bioactive compounds positions A. galanga as a promising candidate for the development of nutraceuticals and functional therapeutics targeting oxidative stress‐related diseases.

In addition to its pharmacological potential, the phytochemical profile of A. galanga rhizome extract highlights its nutritional relevance. The extract is notably rich in carbohydrates and proteins, providing essential macronutrients that support the energy metabolism and structural integrity of the cells. In addition, its low lipid content is highly important, especially for athletes who are looking for carbohydrate and protein supplements to reduce muscle damage caused by endurance exercise [43].

Its low moisture content contributes to enhanced stability and extended shelf‐life, making it a suitable candidate for pharmaceutical and nutraceutical formulations. Furthermore, the crude ash content reflects a substantial mineral composition, which plays a vital role in maintaining systemic physiological balance.

Among the mineral constituents, calcium, iron, and magnesium play critical roles in fundamental biological processes. Calcium is indispensable for intracellular signal transduction, muscle contraction, and enzymatic activation processes that are particularly relevant to cardiovascular and neuromuscular functions [44]. Iron, a central component of hemoglobin and numerous oxidoreductase enzymes, is essential for oxygen transport, mitochondrial respiration, and cellular energy production [45]. Magnesium acts as a cofactor in more than 300 enzymatic reactions, including those involved in ATP synthesis, ion channel regulation, and antioxidant defense, thereby contributing to cardiovascular homeostasis and stress adaptation [46]. The presence of these bioavailable minerals suggests that A. galanga extract may exert additional protective effects by supporting electrolyte balance, oxygen delivery, and energy metabolism, particularly under physiological stress, such as chronic noise exposure.

Phytochemical analysis of the A. galanga rhizome extract revealed a notably high content of total polyphenols (49.05 ± 4.32 mg GAE/g) and flavonoids (17.28 ± 1.08 mg CE/g), highlighting its rich bioactive compounds and suggesting strong antioxidant potential. These values are in close agreement with those previously reported by Aljobair [4], who reported phenolic and flavonoid contents of 53.18 mg GAE/g and 14.12 mg CE/g, respectively. Similarly, Lu et al. [47] reported similar polyphenol concentrations (58.25 mg GAE/g) in rhizomes collected in China. In contrast, lower concentrations were observed when alternative extraction protocols were used for the same samples. For example, Mahae and Chaiseri [48] reported concentrations of only 8.25 and 5.01 mg GAE/g in water and essential oil extracts, respectively, whereas their ethanolic extracts yielded 31.49 mg GAE/g phenolics and 13.78 mg CE/g flavonoids. The extract from the A. galanga rhizome (GRE) demonstrated significant antioxidant activity, as evidenced by its effective free radical scavenging ability in the DPPH and ABTS assays and its notable metal‐chelating capacity. The IC₅₀ value obtained from the DPPH assay (39.5 ± 2.38 µg/mL) indicates substantial radical‐neutralizing potential, although it is lower than that of the synthetic antioxidant BHA (10 µg/mL). Similarly, the ABTS assay revealed high scavenging activity (36.23 ± 2.21 mmol Trolox/g), further confirming the antioxidant efficacy of the extract. These findings are consistent with those reported by Aljobair [4] and Lu et al. [47], who also documented significant antioxidant capacity in A. galanga extracts via comparable assays.

The variation in polyphenol and flavonoid contents and antioxidant potency across studies can be attributed to differences in extraction methods, plant parts used, solvent polarity, and the geographic origin of the plant material. Nonetheless, the consistent detection of strong antioxidant activity across multiple assays and reports supports the therapeutic potential of A. galanga, particularly in oxidative stress‐related conditions.

To determine the strong antioxidant profile of A. galanga rhizome extract, we investigated its potential to mitigate cardiac oxidative stress resulting from chronic noise exposure. Given the high metabolic activity and constant oxygen demand of the myocardium, it is particularly susceptible to oxidative damage triggered by environmental stressors. In our noise‐exposed rat model, we observed a marked increase in the levels of myocardial malondialdehyde (MDA), which is a well‐established marker of lipid peroxidation. This increase in MDA levels reflects elevated oxidative injury mediated by reactive oxygen species (ROS), including superoxide anions (O₂•−), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH) [49].

Concomitantly, the noise exposure group presented significant upregulation of key endogenous antioxidant enzymes, particularly superoxide dismutase (SOD) and catalase (CAT). This enzymatic activation likely represents a compensatory response to the increased ROS burden, in line with previous findings suggesting that oxidative stress activates intrinsic antioxidant defense mechanisms as a form of cellular adaptation [50, 51].

Supplementation with A. galanga rhizome extract significantly alleviated the oxidative imbalance induced by chronic noise exposure. This was demonstrated by a significant reduction in the levels of MDA, an indicator of lipid peroxidation, and preserved SOD and catalase activities, which remained comparable to those of non‐exposed control animals. The maintenance of antioxidant enzyme activity under stress conditions suggests that GRE exerts cardioprotective effects primarily through the direct scavenging of reactive oxygen species (ROS), which is attributable to its high content of polyphenolic compounds. These findings align with those of previous studies demonstrating the efficacy of plant‐derived antioxidants in mitigating oxidative damage in cardiac tissue exposed to environmental and physiological stressors [52, 53]. Collectively, these results highlight oxidative stress as a key pathogenic mechanism in noise‐induced myocardial injury and reinforce the potential of GRE to preserve redox homeostasis and prevent structural and functional cardiac deterioration under such conditions.

The anti‐inflammatory properties of GRE were further demonstrated by its inhibitory effect on nitric oxide (NO) production in LPS‐stimulated RAW 264.7 macrophages. This suppression of NO synthesis likely reflects the presence of bioactive phenolic constituents in the A. galanga rhizome extract. Polyphenols modulate inflammatory signaling by downregulating inducible nitric oxide synthase (iNOS) expression, primarily via the inhibition of the nuclear factor kappa B (NF‐κB) pathway, a key transcriptional regulator of proinflammatory genes [54]. Among these polyphenols, flavonoids have been extensively studied for their ability to attenuate inflammation by targeting NO generation and upstream mediators involved in the inflammatory cascade [55].

Notably, GRE contains significant levels of naringenin, a flavonoid with well‐documented pharmacokinetics and bioactivity. After oral intake, naringenin is rapidly absorbed in the gastrointestinal tract and undergoes conjugation to form glucuronides and sulfoglucuronides, which are subsequently distributed to peripheral tissues, including the liver, heart, brain, kidneys, and spleen [56]. Recent evidence highlights its cardioprotective potential, which is partly mediated by the activation of mitochondrial large‐conductance calcium‐activated potassium channels, which play crucial roles in preserving mitochondrial function and limiting oxidative damage during inflammatory stress [57, 58].

Molecular docking analyses identified rutin as the most potent bioactive constituent of A. galanga rhizome extract, which exhibited the highest binding affinities toward both heme oxygenase‐1 (HO‐1) and tumor necrosis factor‐alpha (TNF‐α). The strong interaction profile of rutin, characterized by multiple conventional hydrogen bonds and diverse π‐type contacts, suggests a dual modulatory mechanism—the enhancement of antioxidant defenses through HO‐1 activation and the suppression of inflammatory pathways via TNF‐α inhibition. These results align with the well‐established pharmacological properties of rutin, including its ability to inhibit NF‐κB signaling and reduce oxidative damage by limiting lipid peroxidation [59].

Consistent with the in silico data, in vivo serum analysis demonstrated that GRE administration during chronic noise exposure significantly attenuated systemic inflammation, as evidenced by a marked decrease in circulating TNF‐α levels. This anti‐inflammatory effect is consistent with previous findings showing that A. galanga extract reduces TNF‐α expression, while enhancing the expression of anti‐inflammatory cytokines such as IL‐10 and TGF‐β in TNF‐α‐stimulated human peripheral blood mononuclear cells, underscoring its potential to modulate acute inflammatory responses [60].

The anti‐inflammatory efficacy of the extract may also be partially attributed to its rich flavonoid content, including that of galangin, which has been shown to mitigate myocardial ischemia‒reperfusion injury by activating the Nrf2/Gpx4 signaling pathway. This activation reduces ferroptosis, limits lipid peroxidation, and preserves mitochondrial integrity, thereby attenuating oxidative and inflammatory injuries. Furthermore, Song et al. [61] demonstrated that galangin ameliorates severe acute pancreatitis in mice by activating the Nrf2/HO‐1 pathway, leading to reduced oxidative stress and the suppression of proinflammatory cytokine production.

Concurrently, GRE treatment led to a substantial increase in the HO‐1 concentration, reflecting the upregulation of antioxidant defense mechanisms. Rutin emerged as the principal active molecule, while other phytoconstituents, such as naringenin, ferulic acid, and gallic acid, also demonstrated relevant binding affinities and likely contributed to the overall bioactivity of the extract, and their pharmacological roles have been well supported by previous studies [36, 57, 58, 62]. Naringenin, a flavanone widely present in medicinal plants, is known for its anti‐inflammatory effects via the inhibition of proinflammatory cytokines and the modulation of the MAPK and NF‐κB signaling pathways [41, 63]. It has also been shown to increase the activities of antioxidant enzymes, such as SOD and CAT, in oxidative stress models [64]. Ferulic acid exhibits strong free radical scavenging capacity, lipid peroxidation inhibition, and cardioprotective effects [65, 66]. Although gallic acid showed the lowest binding affinity in our docking analysis, it is recognized for its multitarget antioxidant and anti‐inflammatory actions, including the suppression of TNF‐α and IL‐1β expression and the protection of cellular components against oxidative injury [67]. The presence of these compounds, each with distinct yet complementary mechanisms of action, may synergistically enhance GRE therapeutic efficacy.

In addition to oxidative stress markers, plasma biochemical parameters indicative of cardiac injury and lipid metabolism were evaluated to further understand the effects of noise exposure. Compared with control conditions, noise exposure significantly elevated plasma cholesterol and triglyceride levels, highlighting a disturbance in lipid homeostasis that could predispose individuals to cardiovascular risk. Elevated cholesterol and triglyceride levels are well‐known contributors to endothelial dysfunction and atherogenesis, potentially exacerbating noise‐induced cardiac damage [68].

Moreover, the levels of markers of myocardial injury, including lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and creatine kinase (CK), were significantly increased in noise‐exposed rats, indicating membrane damage and leakage of intracellular enzymes into the bloodstream. These findings align with those of previous studies demonstrating the cardiotoxic effects of chronic noise through cellular membrane disruption and inflammation [13, 69]. Importantly, GRE administration modulated plasma lipid profiles and cardiometabolic parameters. These findings are consistent with those of Achuthan and Padikkala [70], who demonstrated that ethanolic extracts of A. galanga significantly reduced the serum levels of total cholesterol, triglycerides, and phospholipids while concurrently increasing HDL concentrations in hypercholesterolemic rats. Such hypolipidemic effects are particularly relevant given the close link between systemic inflammation, oxidative stress, and lipid dysregulation in noise‐induced cardiac dysfunction. Thus, the lipid‐lowering properties of GRE may synergize with its anti‐inflammatory and antioxidant properties, offering multifaceted protection against environmental cardiotoxicity.

Histological examination corroborated the biochemical data, revealing pronounced myocardial tissue alterations in the noise‐exposed rats. Notably, the subepicardial region exhibited dense inflammatory infiltrates, predominantly composed of lymphocytes, polymorphonuclear leukocytes, and histiocytes, accompanied by focal areas of myocardial fiber necrosis. These histopathological features are emblematic of oxidative stress‐induced cellular injury, coupled with a localized inflammatory response. Conversely, cardiac tissue from rats treated with A. galanga extract maintained an intact histological architecture, characterized by preserved cardiomyocyte morphology and orderly myocardial fiber organization, comparable to that observed in the control animals. This morphological preservation underscores the cardioprotective efficacy of the extract, which is likely attributable to its ability to mitigate oxidative damage and inhibit downstream inflammatory cascades.

5. Conclusion

In conclusion, our study confirms that oxidative stress is a key driver of myocardial injury induced by chronic noise exposure, triggering inflammatory cascades, and cardiomyocyte damage. A. galanga rhizome extract effectively counteracts these effects, preserving cardiac structure and function. This protective effect is largely attributed to the high rutin and naringenin content of the extract, which are identified as central mediators of its cardioprotective and anti‐inflammatory activities. These findings underscore the therapeutic potential of A. galanga in stress‐induced cardiac injury.

6. Limitations

While our study provides valuable insights into the cardioprotective effects of A. galanga rhizome extract against noise‐induced cardiotoxicity, certain limitations stemming from the available resources should be acknowledged. Notably, the presence of unidentified peaks suggests the existence of additional major phytochemicals that could not be fully characterized owing to constraints in the available equipment, as only HPLC analysis was performed. Future studies employing advanced analytical techniques, such as LC‒MS‒MS or NMR spectroscopy, will be critical for precisely elucidating their structures. Moreover, although the current study demonstrated significant biological activity, the underlying molecular mechanisms and signaling pathways remain to be comprehensively investigated. A deeper understanding of these mechanisms is essential, as it will provide more detailed insights into the mode of action of the compound and its potential therapeutic applications.

Funding

The authors have nothing to report.

Ethics Statement

This study was approved by the Ethics Committee of the National School of Veterinary Medicine of Sidi Thabet (Ref: CEEA‐ENMV 46/22) and was conducted in accordance with the guidelines of the International Council for Laboratory Animal Science (ICLAS).

Conflicts of Interest

The authors declare no conflicts of interest.

Ben Attia T., Serairi‐Beji R., Horchani M., et al. “ Alpinia galanga Rhizome Extract Shields Against Noise‐Induced Cardiotoxicity via Antioxidant and Anti‐Inflammatory Actions: Experimental Insights.” Molecular Nutrition & Food Research 69, no. 24 (2025): e70320. 10.1002/mnfr.70320

Data Availability Statement

All the data and materials are available upon request.