Alpinia galanga Essential Oil Mitigates Gentamicin-Induced Renal Injury: In silico and in vivo Studies

Abstract

Introduction

Alpinia galanga (L.) rhizome has been widely consumed as a spice and food-flavoring agent and in traditional medicines for the treatment of various diseases such as stomach pain, vomiting, diarrhea, infections, and renal disorders. The clinical use of gentamicin (Genta), an aminoglycoside antibiotic, is constrained due to nephrotoxicity. The existing investigation intended to estimate the mechanistic antioxidant, anti-inflammatory, and anti-apoptotic actions of galangal essential oil (Gal EO) against nephrotoxicity induced by Genta.

Materials and Methods

Gal EO was isolated and subjected to GC-MS for analysis. The oil components were in silico investigated against Genta-induced renal toxicity targets using network pharmacology and molecular docking approaches. In vivo studies involved the alienation of rats into four groups. The control and Genta groups rats received 0.5% CMC orally by gavage for 2 weeks and saline or Genta (100 mg/kg) I.P. injection on the 8th to the 14th day. The Genta + Gal EO (50 mg/kg) and Genta + Gal EO (100 mg/kg) groups received Gal EO (50 or 100 mg/kg, P.O.) daily for 2 weeks and Genta (100 mg/kg) I.P. injection. Renal histopathological and kidney function tests, lipid peroxidation, oxidative, inflammatory mediators, and apoptotic markers were assessed.

Results

Network Pharmacology suggested Toll-like receptors 4 (TLR4) and interleukin-1beta (IL-1β) as potential targets of Gal EO components in Genta-induced renal toxicity. Gal EO significantly decreased Cr, uric acid, BUN, CysC, NGAL, and Kim-1 levels and the urine albumin/creatinine ratio. Gal EO reduced MDA and NO levels with an upsurge in the GSH content, GPx, GSH-R, catalase, and SOD levels. Gal EO lessened the gene expression of TLR4/MYD88/NF-κB/IL-1β with subsequent reduction in ICAM-1 and MCP-1 expression and the levels of MPO, TNF-α, and IL-6 while intensified IL-10. Gal EO diminished caspase 3, caspase 9, and Bax while amplified Bcl2.

Conclusion

Genta-induced nephrotoxicity was mitigated by the anti-inflammatory, antioxidant, and anti-apoptotic effect of Gal EO through decreasing TLR4/MYD88/NF-κB/IL-1β signaling pathway.

Introduction

Galangal typically states two species of the ginger family: greater galangal (Alpinia galanga (L.) Willd.) as well as lesser galangal (Alpinia officinarum Hance) [1]. However, the pharmacologic actions and the composition of these two species are substantial different. A. galanga (L.) Willd. was utilized as a herb in Unani medicine as well as a spice in numerous conventional cookeries. It is a perennial herb that is grew primarily in Asia for cosmetics, medicines, and cookery products [2]. A. galanga rhizome is broadly utilized as a spice and food-flavoring additive, as well as in Chinese, Ayurveda, Thai, and Unani traditional medicines in several ailments, for instance stomach pain, vomiting, diarrhea, infections, bronchitis, high temperature, headache, sore throat, renal disorders, rheumatism, and enteritis [3, 4]. The essential oils and extracts obtained from diverse fragments of A. galanga, have been used as cosmetic ingredients and perfumes [5]. A. galanga rhizome essential oil (Gal EO) exhibited various biological actions, including antibacterial [6], antifungal [7], insecticidal, repellent, antifeedant [8], anti-inflammatory [9], anti-arthritis [10], antitumor, antioxidant [11], and immune-modulator [5] activities. For example, a study concluded that A. galanga extract has strong anti-inflammation activity on the peripheral blood mononuclear cells acute inflammation cells model promoted by TNF-α [12]. Additionally, anti-amnesic and cognitive improvement abilities of A. galanga fractions were established in Alzheimer’s type of amnesia provoked by Aβ [13]. Moreover, the essential oil of A. galanga rhizome displayed acetylcholinesterase inhibition, making it a potential for the treatment of Alzheimer’s disease [14]. For the human use, a randomized triple-blind clinical trial showed that adding A. galanga extract to the SSRIs improved SSRI-associated erectile dysfunction [15]. All these properties make A. galanga a promising for therapeutic option for certain conditions.

Aminoglycoside antibiotics, frequently used for the treatment of infections, are associated with deleterious side effects specifically nephrotoxicity [16]. Gentamicin (Genta) is an aminoglycoside antibiotic frequently consumed in the management of a wide spectrum of bacterial infections. However, its use clinically constrained due to ototoxicity and nephrotoxicity [17]. Genta induce acute kidney failure in about 10–30% of patients. Recent reports proposed several theories for the underlying mechanism of Genta associated acute kidney failure. It has been proposed that Genta accumulates in the proximal convoluted tubules leading to tubular necrosis which increased the production of mitochondrial reactive oxygen species (ROS), pro-inflammatory cytokines, triggered cell apoptosis, and cell death [18]. Genta creates ROS which prompt renal injury via declining the antioxidant defense and augmenting lipid peroxidation causing inflammatory and apoptosis pathways stimulation [19, 20]. The endogenous danger molecules formed during renal tissue injury are the ligands for Toll-like receptors (TLRs). TLRs activated the innate immune system to instruct cell signaling pathways causing pro-inflammatory responses. Numerous reports revealed that TLR4 is a regulator of the inflammatory response in kidney disorders [21]. Accordingly, the existing investigation explored the oxidative stress and TLR4 gene expression variations in the Genta prompted nephrotoxicity. Moreover, Genta promotes inflammatory measures by employing intercellular adhesion molecule (ICAM)-1 and monocyte chemoattractant protein (MCP)-1, which enhances the migration of monocytes and macrophages to the site of injury [22]. Accordingly, diminishing oxidative stress, inflammation, and apoptosis, which can be achieved using natural product, is a promising approach in Genta prompted nephrotoxicity. Consequently, the existing examination was intended to estimate the mechanistic antioxidant, anti-inflammatory, and anti-apoptotic actions of Gal EO against AKI provoked by Genta.

Material and Methods

Plant Material

The dried roots and rhizomes of galangal (A. galanga Family Zingiberaceae) were composed from Markets in Alahsa, Eastern province, Kingdom of Saudi Arabia (KSA), in April 2023. The plant was recognized by taxonomists in King Faisal University. Voucher specimens (No. Z120) were placed in the herbarium of college of Clinical Pharmacy, King Faisal University, KSA.

Separation of Gal EO

The dried roots and rhizomes of A. galanga (750 g) were cut in to pieces (1 cm diameter) and then exposed to hydrodistillation according to Perveen, Bokhari [23]. The retrieved volatile fraction (yield; 0.25% v/dried weight) was dried (anhydrous sodium sulfate) and reserved in brown vials in the refrigerator (4°C) until used.

Gal EO Analysis

The isolated Gal EO was subjected to GC-MS examination according to Perveen, Bokhari [23]. Essential oil components were identified [24] and quantified by relative parentage area calculations, and the relative percentage of the constituents was assessed from the total peak area using percentage area normalization [25–27].

Network Pharmacology

Acquisition of Gal EO Targets

Gal EO identified compounds 3D structures were extracted using PubChem and stored in SDF format. SwissADME (http://www.swissadme.ch/) was used to anticipate the pharmacokinetics, drug-likeness, and physicochemical properties of Gal EO identified compounds using Canonical SMILES obtained from PubChem for each identified compounds [28]. From the Gal EO identified compounds, the compounds which follow Lipinski rule with no violation and exhibited bioavailability score of 0.55 or more were used for subsequent analysis. Swiss Target Prediction (http://www.swisstargetprediction.ch/) as well as PharmMapper (https://www.lilab-ecust.cn/pharmmapper/) databases were inspected to acquire Gal EO identified compounds associated targets. The anticipated targets in Swiss Target Prediction were separated using “probability ≤0.05.” All the targets obtained from Swiss Target Prediction and PharmMapper were collected and verified using the Uniprot (https://www.uniprot.org/).

Acquisition of Genta-Induced AKI Potential Targets

The diseases possible targets were attained using GeneCards (https://www.genecards.org/), and from OMIM (https://omim.org/) using search phrases, “gentamicin induced renal toxicity,” “gentamicin induced renal injury,” and “gentamicin induced acute kidney injury” and from Disgenet (https://disgenet.com/) using the search phrase “Acute kidney injury.” All the targets obtained from GeneCards, OMIM, and Disgenet were collected and verified using the Uniprot.

Recognizing Mutual Genes

The verified Gal EO and Genta-induced AKI potential targets were introduced to Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) for overlapping inquiry. The shared genes were transferred to the STRING (https://www.string-db.org/) to examine the protein-protein interaction (interaction score is 0.70).

Network Construction and Examination

Protein-protein interaction network visualization was mutual using Cytoscape 3.7.2 using “Degree” filter to assess the interaction strength among the core targets. The interconnected genes were imported to DAVID (https://david.ncifcrf.gov/) for Gene Ontology (GO) analysis. GO analysis is utilized to enrich gene associated function comprising biological processes, molecular functions, and cellular component. Furthermore, GO bioprocess and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted using Metascape (https://metascape.org/gp/index.html#/) (p value <0.05 is statistically significant).

Molecular Docking of Major Components in Gal EO against TLR4

Protein Preparation

The crystal structure of the TLR4 protein was retrieved from the Protein Data Bank (PDB), selecting the most appropriate structure based on criteria such as resolution, completeness, and relevance to binding studies (PDB code; 3FXI). In ChimeraX (Version 1.9, UCSF Resource for Biocomputing, University of California, San Francisco, USA), the protein structure underwent preprocessing to prepare it for docking [29], which involved removing heteroatoms, water and solvent molecules, and any bound ligands from the binding site. Additionally, any missing residues were completed, and protonation states were assigned based on physiological pH using ChimeraX’s structure preparation tools.

Ligand Preparation

The 3D structure of the major 11 compounds in Gal EO (more than 1% area percentage) and another 3 compounds showed affinity to TLR4 indicated by Network pharmacology result was obtained from the PubChem database as SMILES and prepared using ChimeraX [29] and SwissDock (http://www.swissdock.ch) [30, 31].

Molecular Docking Using SwissDock

Both the prepared TLR4 protein and ligand structures were uploaded to SwissDock (http://www.swissdock.ch) and docking was implemented using AutoDock Vina (via SwissDock). The Sampling exhaustivity was set to 50, Box center was 11-10-4, the box size was set to 20-20-20, buried cavity prioritization, and the sampling exhaustivity was set to 50. Docking was performed, generating multiple poses and clusters of all ligands within the TLR4 binding site.

Post-Docking Analysis in ChimeraX

The highest-ranked docked poses were downloaded in pdbqt format from SwissDock for further analysis. In ChimeraX, these docked complexes were loaded with the protein pdbqt format to examine binding orientation, interaction distance, and score. The contact analysis tool in ChimeraX was used to identify critical interactions between the ligands and TLR4, including hydrogen bonds, hydrophobic contacts, and potential ionic interactions. The docked pose with the highest binding affinity and favorable orientation was selected for detailed analysis. High-resolution images of the binding interactions were generated using ChimeraX. The binding free energy scores provided by SwissDock were correlated with these visualizations to support the conclusions on binding affinity.

In vivo Studies

Drugs and Chemicals

Gal EO was suspended in 0.5% carboxymethyl cellulose (CMC), while Genta was dissolved in saline. Creatinine (Cr, Cat. No. ab700460), uric acid (Cat. No. ab65344), blood urea nitrogen (BUN, Cat. No. ab83362), cystatin C (CysC, Cat. No. ab201281), albumin (ab108790), neutrophil gelatinase-associated lipocalin (NGAL, Cat. No. ab119597), and kidney injury molecule-1 (Kim-1, Cat. No. ab119597) using kits were procured from Abcam Inc. Also, malondialdehyde (MDA, ab238537), glutathione content (GSH, ab65322) assay kits were purchased from Abcam Inc. Superoxide dismutase (SOD, Cat. No. 706002), glutathione peroxidase (GPx, Cat. No. 703102), and catalase (CAT, Cat. No. 707002) kits were attained from Cayman Chemicals (MI, USA). Glutathione reductase (GSH-R, Cat. No. MBS727973), interleukin-6 (IL-6, Cat. No. MBS269892), 1L-10 (Cat. No. MBS732368), myeloperoxidase (MPO, Cat. No. MBS584547), BCL2-associated X apoptosis regulator (BAX, Cat. No. MBS2512405), and B-cell leukemia/lymphoma 2 protein (Bcl2, Cat. No. MBS2515143) ELISA kits were obtained from MyBioSource (San Diego, USA). Tumor necrosis factor alpha (TNF-α, Cat. No. LS-F23150) was obtained from eBioscience (USA). Cleaved caspase 3 (Cat. No. KHO1091), and cleaved caspase 9 (Cat. No. BMS2025) ELISA kits were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA).

Experiment Design’s Ethical Approval

Male Sprague Dawley rats delivered from the animal house facility of King Saud University, Riyadh, KSA, were used in the existing study. All animal experiments comply with the ARRIVE guidelines and are in agreement with the “Ethical Conduct for Use of Animals in Research” guidelines of King Faisal University, KSA with ethical Approval No. KFU-REC-ETHICS2302.

Experiment Design

Rats (200–240 g) were arbitrarily alienated into four groups after acclimatization for a week, illustrated in Figure 1 as follow: the normal group in which rats received 0.5% CMC orally by gavage for 2 weeks and saline I.P. injection on the 8th to the 14th day; the Genta group in which rats received 0.5% CMC for 2 weeks and Genta (100 mg/kg, I.P.) was injected on the 8th to the 14th day [32, 33]; and the Genta + Gal EO (50 mg/kg) and Genta + Gal EO (100 mg/kg) groups in which rats were given Gal EO (50 or 100 mg/kg, P.O.) daily for 2 weeks Genta (100 mg/kg, I.P.) was injected on the 8th to the 14th day. After the last Genta injection, the animals were weighted and anesthetized with pentobarbital (50 mg/kg) for blood and samples collections. Blood samples were composed, centrifuged for serum isolation which was used for biochemical appraisal of renal function analysis. Rats were euthanized to extract kidneys which weighted and rinsed in ice-cold normal saline divided into two equal parts. The first part was stored at −80°C for subsequent biochemical examination whereas the other part was fixed in 10% buffered formalin for histopathological analyses.

Fig. 1.

Experimental design.

ELISA Biochemical Investigation

Renal function was established by measuring the serum levels of Cr, uric acid, BUN, CysC, albumin, NGAL, and Kim-1 following the ELISA kit’s instructions. The urine albumin/creatinine ratio describes the urine albumin excretion.

Lipid peroxidation estimation was performed by measuring MDA in the renal homogenate. Additionally, oxidative stress indicators including GSH content and GPx, GSH-R, SOD, and CAT levels were assessed using ELISA kits in the renal homogenate. Furthermore, Tissue NO was assessed using the Greiss reagent [34]. Inflammatory mediators including TNF-α, IL-6, MPO, and IL-10 and apoptotic markers comprising Cleaved caspase 3 and 9, Bax, and Bcl2 were assessed in renal homogenate.

Quantitative Real-Time PCR

Real-time PCR was accomplished following the technique defined before [35]. Expression of the target gene was measured and quantitated by the reference gene (β-actin) using 2^−∆∆ct method. The primer sequences used in this study were as follows: TLR4: 5′-CAT​GAC​ATC​CCT​TAT​TCA​ACC​AAG-3′, 5′-GCC​ATG​CCT​TGT​CTT​CAA​TT G-3′; MyD88: 5′-GAG​ATC​CGC​GAG​TTT​GAG​AC-3′, 5′-CTG​TTT​CTG​CTG​GTT​GCG​TA-3′; NF-κB: 5′-ATC​ATC​AAC​ATG​AGA​AAC​GAT​CTG​TA-3′, 5′-CAG​CGG​TCC​AGA​AGA​CTC​AG-3′; IL-1β: 5´-TGA​TGT​TCC​CAT​TAG​ACA​GC-3´, 5´-GAG​GTG​CTG​ATG​TAC​CAG​TT-3´; ICAM-1: 5′-GGG​ATG​GTG​AAG​TCT​GTC​AA-3′, 5′-GGC​GGT​AAT​AGG​TGT​AAA​TGG-3´; MCP-1: 5′-AGC​CCA​GAA​ACC​AGC​CAA​CTC-3′, 5′-GCC​GAC​TCA​TTG​GGA​TCA​TCT​T-3´; β-actin: 5′-TGC​TAT​GTT​GCC​CTA​GAC​TTC​G-3′, 5′-GTTGGCATAGAG GTCTTTACGG-3′

Histopathological Examinations

The kidneys samples were embedded in 10% neutral buffered formalin for 24 h, flushed with tap water. Then, the renal samples were dehydrated, fixed, cut into 5 mm sections, and stained with hematoxylin and eosin to be scrutinized under light microscope by independent pathologist.

Statistical Analysis

Data were exposed as the mean ± SD. Statistical examination was achieved using GraphPad Prism (ISI^®, USA) software. Comparisons were done using one-way ANOVA followed by the Tukey-Kramer post hoc test; #, p < 0.05 compared to the normal animals; ∂, p < 0.05 compared to the Genta-associated AKI animals.

Results

The Essential Oil Analysis

Table 1 and Figure 2 illustrate the GC-MS analysis of the essential oil components of the roots and rhizomes of galangal (A. galanga). The essential oil’s chemical composition is dominated by eucalyptol, accounting for 46.52% of the total area. As the primary constituent, it plays a major role in defining the oil’s chemical identity and characteristics. Eucalyptol’s significant proportion reflects the oil’s emphasis on oxygenated monoterpenes, which are known for their functional versatility. Alongside eucalyptol, 6,10-dimethyl-5,9-undecadien-1-yne contributes 6.67% to the composition, marking it as the most prominent acyclic structure in the oil. This compound provides structural diversity and supports the oil's complex profile. Other oxygenated monoterpenes such as terpinene 4-acetate (4.39%) and α-terpineol (3.1%) also present prominently. These compounds contribute to the overall chemical diversity and are characteristic of essential oils with strong aromatic and functional attributes. Chavibetol, a phenylpropanoid representing 3.49% of the total area, adds another layer of diversity to the oil’s composition. Its presence highlights the role of phenylpropanoids in complementing the dominant monoterpenes. The monoterpene hydrocarbons α-pinene (3.78%) and β-pinene (0.93%) are key components, adding to the oil’s chemical complexity. These bicyclic structures are critical for the oil’s aromatic profile and highlight the balance between oxygenated and non-oxygenated constituents. Additionally, phenylpropanoids such as chavicol (2.79%) and 3-allylguaiacol (1.63%) further contribute to the chemical richness of the oil, demonstrating the inclusion of multiple chemical classes.

Table 1.

GC-MS analysis of the essential oil components of dried roots and rhizomes of galangal (A. galanga)

Number	Rt	Area %	Name
1	5.896	3.78	(1R)-(+)-α-pinene
2	6.602	0.93	β-Pinene
3	7.782	46.52	Eucalyptol
4	8.004	0.73	ɣ-Terpinene
5	8.545	0.6	Linalool
6	8.948	0.6	Cis-p-mentha-2,8-dien-1-ol
7	9.918	4.39	Terpinene 4-acetate
8	10.122	3.1	α-Terpineol
9	10.192	0.64	Cis-carveol acetate
10	10.426	0.72	D-carveol
11	11.347	0.88	Bornyl acetate
12	11.383	0.49	Anethole
13	11.959	0.36	Z-carvyl acetate
14	12.144	2.79	Chavicol
15	12.347	1.63	3-allylguaiacol
16	12.575	2.38	Cis-geranyl acetate
17	12.89	0.67	Eugenol methyl ether
18	13.272	0.4	Caryophyllene
19	13.355	0.28	Trans-α-bergamotene
20	13.616	6.67	6,10-dimethyl-5,9-undecadien-1-yne,
21	13.965	0.26	8-cedren-13-ol
22	14.074	0.36	β-Copaene
23	14.165	2.76	Tetradecane
24	14.225	0.27	α-Farnesene
25	14.314	0.48	β-Bisabolene
26	14.438	3.49	Chavibetol
27	14.534	0.72	Cedrene
28	14.6	0.17	2,4-pentadienoic acid
29	14.871	0.4	Z-α-trans-bergamotol
30	15.344	0.21	Caryophyllene oxide
31	15.573	0.4	Geranyl-α-terpinene
32	15.678	0.54	Cis-Z-α-Bisabolene epoxide
33	16.166	1.09	(Z)-9,17-octadecadienal,
34	16.243	0.55	8-heptadecene
35	16.471	0.24	1-heptatriacotanol
36	19.401	0.76	l-(+)-ascorbic acid 2,6-dihexadecanoate

Area %	Category (compounds, n)
53.16	Monoterpenes (6)
2.51	Sesquiterpenes (6)
10.15	Acyclic structures (4)
50.2	Monocyclic structures (4)
5.2	Bicyclic structures (3)
73.64	Oxygenated compounds (24)
17.62	Non-oxygenated compounds (12)
8.4	Phenylpropanoids (4)
91.26	Total (36)

Rt is retention time and area % is the area percentage of each components related to the total area of the whole oil components (i.e., area normalization). Each category area percentage is calculated according to the area percentage of compounds involved.

Fig. 2.

GC-MS chromatogram of the essential oil components of dried roots and rhizomes of galangal (A. galanga).

The essential oil’s chemical composition, analyzed based on various categories, provides valuable insights into its potential biological activities. Monoterpenes dominate the oil, constituting 53.16% of the total area with 6 compounds identified, Table 1. These compounds, including α-pinene and eucalyptol, are well-documented for their antimicrobial, anti-inflammatory, and antioxidant properties. Their abundance also explains the oil’s high volatility and characteristic aroma, typical of many essential oils. Sesquiterpenes, although minor at 2.51% of the total area, contribute significantly to the oil’s therapeutic potential. With 6 compounds, such as caryophyllene and cedrene, these heavier and less volatile molecules enhance the oil’s anti-inflammatory and analgesic effects, making them crucial for long-lasting activity despite their lower concentration. Analyzing the oil by structural complexity reveals a predominance of monocyclic structures (50.2%) with 4 compounds, such as eucalyptol and chavicol. These compounds strike a balance between pharmacokinetic properties and biological efficacy. Bicyclic structures account for 5.2% with 3 compact and stable molecules like α-pinene and bornyl acetate, known for their respiratory and antimicrobial benefits. Acyclic structures, representing 10.15%, include linear or branched molecules like tetradecane, contributing to the oil's stability and hydrophobic interactions, which can enhance biological activity. The oil’s composition is also characterized by a clear dominance of oxygenated compounds, comprising 73.64% of the total area with 24 compounds identified. These oxygenated compounds, such as linalool, eucalyptol, and anethole, are associated with enhanced solubility, reactivity, and significant therapeutic effects like sedation, antimicrobial action, and antioxidant activity. In contrast, non-oxygenated compounds make up 17.62% of the oil, with 12 simpler hydrocarbon-based molecules like α-pinene and β-pinene, contributing to aroma and general antimicrobial effects. Phenylpropanoids, including chavicol (2.79%), 3-allylguaiacol (1.63%), and chavibetol (3.49%), are secondary metabolites with significant antioxidant and antimicrobial activity. These compounds are particularly effective in neutralizing free radicals and combating microbial pathogens, making the oil potentially beneficial in skin care formulations or as a natural preservative. Minor components like bornyl acetate (0.88%), linalool (0.6%), and anethole (0.49%) are present in trace amounts but remain integral to the oil’s overall complexity. Over a 50 compounds were separated for the essential oil by GC-MS; however, only 36 compound were identified, comprising to a total calculated area percentage of 91.26%, and suggesting that trace unidentified components may still play a synergistic role in the oil's bioactivity. Despite their low concentration, these trace compounds can influence the overall therapeutic potential of the oil.

Network Pharmacology

Targets Collection

From 36 Gal EO identified compounds which were assessed using the SwissADME tool, 30 obeyed Lipinski’s rule with no violation with the predetermined bioavailability Score. Around 329 possible targets for Gal EO identified compounds were gained from SwissTargetPrediction while the PharmMapper provided 2,055 potential genes. All the potential genes were composed; the duplicates were eliminated and then verified using UniPrott which recognized 571 targets. Regarding Genta-induced renal toxicity potential targets, OMIM identified 256, GeneCards recognized 411 and Disgenet showed 158 potential entries. From all Genta-induced renal toxicity targets, Uniprot identified 308 IDs.

Construction of Possible Target Network

Intersection inquiry using Venn diagram displayed 36 goals between Genta-induced renal toxicity and Gal EO identified compounds, thus can be recognized as potential targets of Gal EO identified compounds (529 genes) in Genta-induced renal toxicity (272 genes) (Fig. 3a). These 36 overlapping genes indicate the possible therapeutic targets where Gal EO may modulate pathways concomitant to Genta-associated AKI. Using STRING with an interaction score of 0.07 exhibited number of nodes of 36 and number of edges of 44 (Fig. 3b; online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000548197) in which each node represents a gene or protein whereas the edges (connecting lines) represent interactions. Cytoscape recognized top 10 gene (Fig. 3c). Additionally, bioprocess analysis enrichment analysis showing biological processes, cellular component, molecular function processes in bar plot associated with the 36 overlapping genes (Fig. 3d, e; online suppl. Fig. 2) reveals that higher bars signify a greater statistical significance (lower p values). The key enriched terms comprised transcription regulation, inflammatory response, and cell migration, that are expected the pathways through which Gal EO influenced to counteract Genta-associated AKI.

Fig. 3.

Network pharmacology. a Intersection analysis between Gal EO identified compounds and Genta-induced AKI via Venn diagram displaying 36 targets. b STRING displayed 36 nodes and 44 edges. c Cytoscape acknowledged the top 10 gene. d, e Gene ontology (GO) bioprocess analysis (d) and enrichment analysis (e) showing BP, CC, MF three in one bar plot. f GO, pathway enrichment bubble plot. BP, biological processes; CC, cellular component; MF, molecular function.

Bubble plot was used in GO, KEGG pathway enrichment analysis, in which −log10 of the p values were represented by colors whereas gene counts were shown by bubble size (Fig. 3f; online suppl. Fig. 3). Finally, KEGG pathway enrichment analysis conducted via Metascape (Fig. 3a) as well as the Network of enriched terms colored by cluster ID and by p value, respectively (Fig. 3b, c).

The top 3 genes suggested by the network pharmacology investigation were TLR4, interleukin-1beta (IL-1β), and epidermal growth factor receptor as the common targets of Gal EO identified compounds in Genta-induced renal toxicity and, therefore, were selected as the potential mechanism of action of Gal EO in Genta-induced renal toxicity (Fig. 4).

Fig. 4.

Analysis conducted via Metascape showing KEGG pathway enrichment (a) and network of enriched terms colored by cluster ID and p value, respectively (b, c).

In silico Studies

Table 2 and Figure 5 demonstrate the in silico molecular docking studies of 14 components of the essential oil of galangal root and rhizomes against one of the 3 targets suggested by network pharmacology, TLR4. The combined in silico molecular docking and network pharmacology analyses revealed significant insights into these compounds’ potential activity and pharmacological relevance. The molecular docking outcomes exposed that the binding affinities of the compounds ranged from −3.089 to −4.443 kcal/mol. Among the compounds, α-terpineol displayed the strongest binding affinity (−4.443 kcal/mol), followed by chavicol (−4.368 kcal/mol) and bornyl acetate (−4.312 kcal/mol). Notably, even the less abundant compounds, such as bornyl acetate, anethole, and 2,4-pentadienoic acid, displayed moderate to high binding affinities, despite their minimal representation in the essential oil composition. The GC-MS analysis disclosed that eucalyptol was the utmost abundant compound in the essential oil, contributing 46.52% of the total composition, followed by 6,10-dimethyl-5,9-undecadien-1-yne (6.67%) and terpinene 4-acetate (4.39%). However, the non-abundant compounds (bornyl acetate, anethole, and 2,4-pentadienoic acid) exhibited significant binding affinities to TLR4, suggesting their potential pharmacological importance. Network pharmacology analysis further validated that the majority of these compounds, including the non-abundant ones, demonstrated affinity for TLR4, highlighting their relevance in modulating TLR4-mediated pathways, which are crucial in inflammation and immune responses. The docking studies revealed that many of the compounds formed strong hydrogen bonding and hydrophobic interactions with key residues in the TLR4 binding pocket, such as ARG 460, GLN 436, HIS 458, LYS435, and SER 438. Structural features, such as hydroxyl and aromatic groups, appeared to contribute significantly to the high binding affinities of compounds like α-terpineol and chavicol. These findings emphasize the importance of both abundant and trace components in the essential oil as even minor constituents may play a critical role in modulating biological pathways. The study highlights the therapeutic potential of compounds like α-terpineol, chavicol, bornyl acetate, and eucalyptol, which could serve as promising leads for TLR4-targeted therapies. These compounds hold potential applications in treating inflammatory diseases, infections, and immune disorders where TLR4 is a critical mediator.

Table 2.

Docking results of galangal root and rhizome essential oil with TLR4 protein

Compound	Calculated affinity (kcal/mol) (best pose against TLR4)	Area percentage (GC-MS)
α-Pinene	−3.938	3.78
Eucalyptol	−4.199	46.52
Terpinene 4-acetate	−4.235	4.39
α-Terpineol	−4.443	3.1
Chavicol	−4.368	2.79
3-allylguaiacol	−4.141	1.63
Cis-geranyl acetate	−3.987	2.38
6,10-dimethyl-5,9-undecadien-1-yne,	−3.731	6.67
Tetradecane	−3.089	2.76
Chavibetol	−4.091	3.49
(Z)-9,17-octadecadienal,	−3.716	1.09
Bornyl acetate	−4.312	0.88
Anethole	−3.933	0.49
2,4-pentadienoic acid	−3.300	0.17

The table compares the compound affinity in kcal/mol with the compound abundance in the oil (presented as area percentage extracted from Table 1). The compounds in bold type indicate minor abundance; however, these compounds were detected in network pharmacology to have affinity to TLR4.

Fig. 5.

Docking results of galangal root and rhizome essential oil with TLR4 protein. The docking process was implemented by AutoDock Vina, via SwissDock (http://www.swissdock.ch), and then photos were generated through the manipulation of docking file using ChimeraX. Different hydrophobic interaction are shown in green dotted lines, while H-bonding are shown in orange or red dotted lines.

In vivo Outcomes

The Effect of Gal EO on the Renal Histopathological Examination in Genta-Induced AKI

Kidney tissues obtained from the normal group presented with intact glomeruli and normal tubular structure whereas tissue obtained from Genta alone group exhibited renal damage, including necrotic and anucleate cells in the tubular lumen (black arrow) showing tubular degeneration, possible necrosis, disrupted glomeruli structure (black headed arrow) and inter-tubular hemorrhage (blue arrow) (Fig. 6a, b). On the other hand, management with Gal EO showed noticeable amendment with lessened signs of renal injury and preservation of the glomeruli and tubular structures when compared to the Genta group (Fig. 6c, d).

Fig. 6.

Protective effect of Gal EO (50 or 100 mg/kg for 2 weeks) in Genta-associated AKI on renal histopathological examination in which black arrow indicates necrotic and anucleate cells in the tubular lumen, black arrowheads point to disrupted glomeruli structure and blue arrow shows inter-tubular hemorrhage.

The Effect of Gal EO on the Renal Function in Genta-Induced AKI

Genta administration caused a substantial increase in Cr, uric acid, BUN, CysC, NGAL, and Kim-1 levels as well as the urine albumin/creatinine ratio compared to normal animals. Whereas, the co-administration of Gal EO (50 and 100 mg/kg) with Genta resulted in a significant reduction in the serum levels of Cr, uric acid, BUN, CysC, NGAL, and Kim-1 and the urine albumin/creatinine ratio in comparison with the Genta group (Fig. 7). Nevertheless, no significant difference between the two doses of Gal EO in the urinary albumin/creatinine ratio.

Fig. 7.

Protective effect of Gal EO (50 or 100 mg/kg for 2 weeks) in Genta-associated AKI as reflected in renal function tests including serum level of Cr (a), uric acid (b), BUN (c), CysC (d), NGAL (e), and Kim-1 (f) as well as the urine albumin/creatinine (g). All values were stated as mean ± SD (n = 6). # signifies compared to the normal group; ∂ signifies compared to the Genta group; Φ signifies compared to Genta +Gal EO (50 mg/kg) group. Comparisons done using one-way ANOVA followed by the Tukey-Kramer post hoc test (p < 0.05).

The Effect of Gal EO on Oxidative Stress in Genta-Induced AKI

Genta alone administered animals exhibited elevated levels of MDA and NO levels whereas Gal EO (50 and 100 mg/kg) administration with Genta resulted in a dose dependent decreased MDA by 40.53% and 55.49% and NO levels diminished by 34.62% and 50.69%, respectively (Fig. 8a, b).

Fig. 8.

Protective effect of Gal EO (50 or 100 mg/kg for 2 weeks) in Genta-associated AKI as reflected in lipid peroxidation and oxidative stress including MDA (a), NO (b), GSH content (c), GPx (d), GSH-R (e), catalase (f), and SOD (g) in renal homogenates. All values were stated as mean ± SD (n = 6). # signifies compared to the normal group; ∂ signifies compared to the Genta group; Φ signifies compared to Genta +Gal EO (50 mg/kg) group. Comparisons done using one-way ANOVA followed by the Tukey-Kramer post hoc test (p < 0.05).

Additionally, Genta-depleted GSH content in the renal tissue homogenate reaching 17.15 ± 4.5 nmole/g protein vs. 109.82 ± 17.1 nmole/g protein in normal animals. Alternatively, Gal EO administration intensified GSH content reaching 54.05 ± 6.34 and 91.70 ± 11.06 nmole/g protein in the renal tissue homogenate (Fig. 8c). Similarly, GPx and GSH-R levels were diminished in Genta alone administered animals compared to normal (Fig. 8d, e). Alike, both catalase and SOD both were diminished in Genta alone group showing a percentage reduction of 65.2% and 78% compared to normal group, respectively, whereas the co-administration of Gal EO (50 and 100 mg/kg) with Genta caused a dose dependent increased GPx, GSH-R, catalase, and SOD levels (Fig. 8f, g).

The Effect of Gal EO on Gene Expression of TLR4/MYD88/NF-κB/IL-1β in Genta-Induced AKI

Since the genes recommended by the network pharmacology investigation were TLR4, IL-1β, the gene expressions of TLR4/MYD88/NF-κB/IL-1β pathway were evaluated. As exposed in Figure 9, the gene expression of TLR4/MYD88/NF-κB/IL-1β were elevated in Genta alone group signifying the activation of this pathway. While Gal EO (50 or 100 mg/kg) administration for 2 weeks declined in the gene expression of TLR4/MYD88/NF-κB/IL-1β signifying that Gal EO mitigated this pathway with subsequent proteins. However, no significant difference between the two doses of Gal EO in gene expression of TLR4/MYD88/NF-κB/IL-1β.

Fig. 9.

Protective effect of Gal EO (50 or 100 mg/kg for 2 weeks) in Genta-associated AKI on the gene expressions of TLR4 (a), MYD88 (b), NF-κB (c), and IL-1β (d) in renal homogenates. All values were stated as mean ± SD (n = 6). # signifies compared to the normal group; ∂ signifies compared to the Genta group; Φ signifies compared to Genta +Gal EO (50 mg/kg) group. Comparisons done using one-way ANOVA followed by the Tukey-Kramer post hoc test (p < 0.05).

The Effect of Gal EO on Inflammation in Genta-Induced AKI

Genta-induced tubular injury and necrosis triggers inflammatory reactions by attracting ICAM-1 and MCP-1 to the site of tissue damage, which enhances monocytes and macrophages migration to the injury site and finally results in renal inflammation. In the current study, the gene expression of ICAM-1 and MCP-1 were amplified compared to normal animals (Fig. 10a, b). on the other hand, co-administration of Gal EO (50 or 100 mg/kg) for 2 weeks with Genta resulted in a dose dependent decrease in ICAM-1 gene expression showing a % reduction of 32.79% and 61.38% compared to Genta alone group (Fig. 10a, b). While only the high dose of Gal EO which is 100 mg/kg caused a significant decline in the gene expression of MCP-1, the lower dose of Gal EO exhibited non-significant decrease in the gene expression of MCP-1. For MPO, Genta was associated with significant increase, whereas management with Gal EO (50 or 100 mg/kg) for 2 weeks caused a significant percentage reduction of 30.84% and 54% when related to Genta group (Fig. 10c). Additionally, Genta group exhibited a significant increase reaching 299.3 ± 33.22 vs. 64.83 ± 12.30 pg/mg protein for TNF-α and 153.46 ± 16.91 vs. 63.72 ± 13.07 pg/g protein for IL-6 when compared to normal group (Fig. 10d, e). Also, as illustrated in Figure 10f, IL-10 was significantly decreased in Genta group, while management with Gal EO (50 or 100 mg/kg) caused a dose dependent intensification in IL-10.

Fig. 10.

Protective effect of Gal EO (50 or 100 mg/kg for 2 weeks) in Genta-associated AKI and inflammation as reflected on the gene expressions of ICAM-1 (a), MCP-1 (b), and the levels of MPO (c), TNF-α (d), IL-6 (e), and IL-10 (f) in renal homogenates. All values were stated as mean ± SD (n = 6). # signifies compared to the normal group; ∂ signifies compared to the Genta group; Φ signifies compared to Genta +Gal EO (50 mg/kg) group. Comparisons done using one-way ANOVA followed by the Tukey-Kramer post hoc test (p < 0.05).

The Effect of Gal EO on Apoptosis in Genta-Induced AKI

As revealed in Figure 11, Genta alone administration was associated with renal injury as indicated by the elevation in the apoptotic markers. In Genta group, apoptotic markers elevated as shown in caspase 3 reached 93.51 ± 11.5 nmol/mg protein compared to normal which was 38.15 ± 4.7 nmol/mg protein (Fig. 11a). Similarly, Genta group showed 87.23 ± 14.03 vs. 14.25 ± 4.5 nmol/mg protein for caspase 9 (Fig. 11b), 27.02 ± 5.09 vs. 2.83 ± 0.53 ng/mg for Bax (Fig. 11c), while Bcl2 was reduced to 1.64 ± 0.41 vs. 9.08 ± 2.20 ng/mg protein when related to normal group (Fig. 11d). Conversely, management using Gal EO (50 or 100 mg/kg) for 2 weeks resulted in limitation in apoptosis as evidenced by a dose dependent decrease in caspase 3, caspase 9, and Bax revealing that Gal EO may protect kidney from the injury caused by Genta. Only one dose of Gal EO which is 100 mg/kg caused a significant increase in Bcl2.

Fig. 11.

Protective effect of Gal EO (50 or 100 mg/kg for 2 weeks) in Genta-associated AKI and apoptosis as reflected on caspase 3 (a), caspase 9 (b), Bax (c), and Bcl2 (d) levels in renal homogenates. All values were stated as mean ± SD (n = 6). # signifies compared to the normal group; ∂ signifies compared to the Genta group; Φ signifies compared to Genta +Gal EO (50 mg/kg) group. Comparisons done using one-way ANOVA followed by the Tukey-Kramer post hoc test (p < 0.05).

Discussion

Galangal, the ginger-like spices belong to Zingiberaceae family, was consistently utilized for culinary and medicinal purposes. Galangal rhizomes are used traditionally to treat aches and pains, colds, fever, diarrhea, heartburn or stomach pain, severe thrush, sore throat, cough, and inflammation [36] and immunomodulatory effects [37]. Besides, essential oils of the A. galanga rhizomes exhibited antimicrobial activities [1], antioxidant [38] and anticancer [39]. Subash, Bhaarathi [40] tested the acute oral toxicity of A. galanga rhizome and showed that it is a safe with no death, nor any toxicity to the systems, tissues or organs. The main objective of this research was to assess the anti-nephrotoxic potential of Gal EO in the context of Genta-induced toxicity. Additionally, the study aimed to explore the molecular pharmacological mechanisms through which the oil's components exert their effects, focusing on their antioxidant, anti-inflammatory, and anti-apoptotic properties.

Network pharmacology showed around 36 overlapping genes indicating the possible therapeutic targets where Gal EO may modulate pathways concomitant to AKI associated with Genta. Using STRING and Network Construction by Cytoscape, the current study identified the highest three genes which were TLR4, IL-1β, and epidermal growth factor receptor as the common targets of Gal EO identified active constituents for Genta-induced renal toxicity; therefore, TLR4 and IL-1β were selected as the potential mechanism of action of Gal EO in Genta-induced renal toxicity.

The relationship between compound affinity and abundance in Gal EO provides critical insights into the pharmacological potential of the oil’s constituents. The network pharmacology and the in silico molecular docking results indicated that there is no direct correlation between the abundance of a compound and its binding affinity for TLR4. While abundant compounds like eucalyptol (46.52%) showed moderate binding affinity (−4.199 kcal/mol), some less abundant compounds, such as α-terpineol (3.1%) and chavicol (2.79%), exhibited significantly stronger affinities (−4.443 and −4.368 kcal/mol, respectively). This suggests that the pharmacological activity of essential oils cannot be solely attributed to their major components; minor constituents can also contribute substantially to the biological effects. Interestingly, bornyl acetate, which represents only 0.88% of the oil’s composition, displayed a relatively high binding affinity (−4.312 kcal/mol), comparable to more abundant compounds. Similarly, anethole (0.49%) and 2,4-pentadienoic acid (0.17%) also showed measurable affinity (−3.933 and −3.300 kcal/mol, respectively). These findings highlight the importance of non-abundant compounds, which may interact with biological targets at low concentrations, potentially enhancing or complementing the activity of more abundant constituents. The moderate binding affinity of eucalyptol, despite its high abundance, may be explained by its structural simplicity compared to other compounds. While eucalyptol primarily relies on hydrophobic interactions, highly active compounds like α-terpineol and chavicol feature functional groups (e.g., hydroxyl and aromatic groups) that enable stronger hydrogen bonding and π-π stacking interactions with TLR4. This structural variability underscores the importance of considering both the chemical structure and abundance when evaluating the therapeutic potential of essential oils. From a pharmacological perspective, the activity of an essential oil often results from a combination of its components rather than a single dominant compound. The high abundance of eucalyptol could provide a baseline effect, while potent but less abundant compounds like α-terpineol, chavicol, and bornyl acetate may amplify or diversify the biological activity through synergistic interactions. This synergy could enhance the overall efficacy of the essential oil, even if the individual contributions of minor compounds are not immediately apparent. These findings also suggest that minor constituents, often overlooked in essential oil studies, may have a disproportionate impact on therapeutic outcomes. For instance, while eucalyptol may be the primary contributor to the oil’s bulk chemical properties, minor compounds like bornyl acetate and chavicol may serve as key modulators of specific biological pathways, such as TLR4-mediated inflammation. This highlights the need to explore the pharmacological contributions of both major and minor compounds to fully understand the therapeutic potential of essential oils.

Consistently with previous results [41, 42], Genta group developed AKI, characterized by elevated Cr, uric acid, BUN, CysC, NGAL, and Kim-1 serum levels and the urine albumin/creatinine ratio as well as displayed renal damage in histological examination. In addition, Genta administered animals exhibited elevated levels of MDA and NO levels and depletion in the GSH content, GPx, GSH-R, catalase, and SOD levels in the renal tissue homogenate indicating oxidative stress condition as mentioned before [43, 44]. Furthermore, the activation of TLR4/MYD88/NF-κB/IL-1β pathway followed by augmented ICAM-1 and MCP-1 gene expression and MPO, TNF-α, and IL-6 and reduced IL-10 suggested the inflammation state proceeding within the kidney. Subsequent to inflammation and oxidative stress happening, the apoptosis state was intensified following Genta administration as stated earlier [21, 45, 46]. Taken together, these data evidenced that renal injury resulting from Genta nephrotoxicity is accompanied with intense renal oxidative stress, inflammation, and apoptosis.

In the current study, Gal EO afford a significant protection against glomerular and tubular kidney functional and structural injury as evidenced by the enhanced glomeruli and tubular structures accompanied with a reduction in renal function tests demonstrating renal protection. For renal action of galangal, Kaushik, Kaushik [47] showed that the alcoholic extract of the rhizomes of A. galanga protected against streptozotocin-induced diabetic nephropathy in rats.

Additionally, Gal EO administration with Genta decreased MDA and NO levels and intensified GSH content, GPx, GSH-R, catalase, and SOD levels. Several studies revealed the potent antioxidant ability of galangal. For instance, the extract of the plant rhizomes possessed high antioxidant activity, as assessed by 1.1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power [48]. Moreover, the alcoholic extract of rhizomes of A. galanga revealed antioxidant effect in Japanese quail during oxidative stress induced by hydrogen peroxide [49]. Similarly, the hydroalcoholic extract of rhizome of A. galanga protected against transient forebrain ischemia induced neuronal damage and oxidative injury in the rat brain [50].

Gal EO administration for 2 weeks declined in the gene expression of TLR4/MYD88/NF-κB/IL-1β with subsequent reduction ICAM-1 and MCP-1 gene expressions and MPO, TNF-α, IL-6 levels and intensified IL-10 signifying that Gal EO mitigates this pathway with subsequent inflammatory mediators. George, Shyni [51] studied the mechanism of the anti-inflammatory effect of hydroalcoholic extract of galangal in LPS stimulated murine macrophage cell line (RAW 264.7). The study revealed that pre-treatment with hydroalcoholic extract of galangal downregulated the release of pro-inflammatory mediators (IL-6, TNF-α, NO, and ROS) and stimulated the release of anti-inflammatory mediator IL-10 via blocking LPS induced activation of TLR4/MYD88 and JAK/STAT pathway followed by inhibiting NF-κB in LPS stimulated RAW 264.7 cells [51]. Another study performed by Pothacharoen, Choocheep [10] investigated the effects of A. galanga extracts on the expression of genes involved in catabolic activities in an IL-1β-induced human synovial fibroblast as an inflammatory model. A. galanga extracts downregulated MMP-1, MMP-2 MMP-3, MMP-13, and Cox-2 suggesting that the plant extracts might be a promising therapeutic agent for arthritis. An in vitro study on the anti-psoriatic effect of the A. galanga ethanol extract in HaCaT keratinocyte cells displayed that it modulated NF-κB signaling biomarkers expression to treat psoriasis [52]. As a result of diminishing inflammation and the oxidative stress, apoptosis was deterred in Gal EO as evidenced by the decrease in caspase 3, caspase 9, and Bax and the increase in Bcl2 revealing that Gal EO may protect kidney from the injury caused by Genta.

A limitation of the current study is the absence of protein-level confirmation via Western blotting or immunohistochemistry, which would provide additional mechanistic depth. Future work will include integrated transcriptional and translational analyses to strengthen the observed mechanistic conclusions.

Conclusion

Gal EO, derived from the rhizomes of A. galanga, is well-known for its medicinal properties, including anti-inflammatory, antioxidant, and antimicrobial effects. Its bioactive components, particularly oxygenated compounds, have demonstrated significant therapeutic potential in disease models. The aim of this study was to evaluate the protective effects of Gal EO against Genta-induced nephrotoxicity and elucidate its mechanisms of action through network pharmacology, molecular docking, and in vivo assessments. Network pharmacology identified TLR4 and IL-1β as primary molecular targets of Gal EO in Genta-induced renal toxicity. Molecular docking revealed moderate binding affinities of Gal EO components, especially oxygenated compounds, to TLR4. In vivo findings showed that Gal EO improved renal function and structural integrity, reduced lipid peroxidation, and enhanced antioxidant defenses. It significantly downregulated the TLR4/MYD88/NF-κB/IL-1β signaling pathway, resulting in decreased ICAM-1 and MCP-1 expression and lower levels of inflammatory mediators such as MPO, TNF-α, and IL-6, while increasing the anti-inflammatory cytokine IL-10. Additionally, Gal EO mitigated apoptosis by reducing the expression of caspase 3, caspase 9, and Bax while upregulating Bcl2. Genta-induced nephrotoxicity could have been effectively attenuated by the anti-inflammatory, antioxidant, and anti-apoptotic effects of Gal EO, mediated in part through inhibition of the TLR4/MYD88/NF-κB/IL-1β signaling pathway. This study provides valuable insights into the therapeutic potential of Gal EO and suggests its future application as a natural remedy for managing drug-induced renal injuries, paving the way for further research on its clinical use.

Acknowledgments

The authors extend their appreciation to the Deanship of Scientific Research for Disability Research for funding this work. Molecular graphics and analyses was performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from the National Institutes of Health (R01-GM129325) and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.

Statement of Ethics

The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee in King Faisal University, Approval No. KFU-REC-ETHICS2302. All the experiments were accomplished in agreement with the relevant procedures and regulations of the Ethical Conduct for the Use of Animals in Research at King Faisal University.

Conflict of Interest Statement

The authors declare no conflicts of interest.

Funding Sources

This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Grant No. KFU251734).

Author Contributions

S.A.A., R.S.A., R.K.A., D.M.A., K.S.A., W.A.A., M.E.M., and N.S.Y. contributed to conception, study design, and execution. M.E.M. and N.S.Y. contributed to acquisition, analysis, and interpretation of data. S.A.A., R.S.A., R.K.A., D.M.A., K.S.A., and W.A.A. took part in drafting. M.A.A., M.E.M., and N.S.Y. revised or critically reviewed the article. M.A.A. and M.E.M. gave final approval of the version to be published. S.A.A., R.S.A., R.K.A., D.M.A., K.S.A., W.A.A., M.A.A., M.E.M., and N.S.Y. have agreed on the journal to which the article has been submitted and agree to be accountable for all aspects of the work.

Funding Statement

This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Grant No. KFU251734).

Data Availability Statement

The data that support the findings of this study are not publicly available due to institutional restrictions but are available from the corresponding author upon reasonable request.