INTRODUCTION:

The pace of life has accelerated considerably, affecting people’s lifestyles and daily habits. Nowadays, many individuals dedicate more time to work rather than rest¹. This shift has become a major factor contributing to a restless lifestyle, which in turn leads to stress and disturbances in the circadian rhythm². Excessive activation of the stress system results in a hyperarousal disorder known as insomnia. This condition is marked by difficulties falling asleep, impaired daytime functioning, fatigue, memory problems, and a persistent cycle of anxiety. Current insomnia treatments primarily focus on cognitive behavioral therapy and pharmacological approaches³. However, some studies have reported adverse effects associated with these treatments, including dependence, tolerance, and drowsiness⁴^,⁵.

The sleep-wake cycle is primarily regulated by a pair of neuropeptides known as hypocretins, which are typically produced by orexin-containing neurons located in the lateral hypothalamus (LH). These neuropeptides interact with two types of receptors, orexin-1 (OX-1) and orexin-2 (OX-2), which are connected by small peptides and distributed throughout both the central and peripheral nervous systems⁶^,⁷. When orexins bind to their specific G-protein-coupled receptors (GPCRs), they trigger downstream signaling pathways that carry out various physiological roles, including the promotion and maintenance of wakefulness⁸^,9. Research conducted on freely moving cats revealed that OX-1 levels were higher during active waking periods in the daytime compared to slow-wave sleep (SWS), and these levels were also elevated during rapid eye movement (REM) sleep relative to SWS¹⁰. Another study found that OX-1 concentrations in cerebrospinal fluid (CSF) were low immediately after waking but gradually increased throughout the day’s activity and then declined slowly at night¹¹. However, orexin expression is increased during stress, which can disrupt the circadian rhythm¹². The Food and Drug Administration (FDA) has approved several dual orexin receptor antagonists (DORAs), including Suvorexant, Lemborexant, and Daridorexant, for the treatment of insomnia⁹. Despite the positive therapeutic effects of these drugs, frequent use has been associated with adverse side effects such as drowsiness, sleep paralysis, headaches, dry mouth, hallucinations, and complex sleep behaviors¹³. Therefore, exploring new potential orexin antagonists derived from bioactive compounds could help minimize these drug-related side effects.

Zingiber officinale, commonly known as ginger, is a perennial herbaceous plant renowned for its diverse medicinal properties. The bioactive compounds found in ginger exhibit strong anti-inflammatory and antibacterial effects¹⁴^-¹⁶. More recently, ginger’s potential effects on the central nervous system have been revealed¹⁷^,¹⁸. Its volatile compounds interact with amino butyrate aminotransferase, acting as substitute GABA molecules¹⁹^,²⁰. Specifically, 6-gingerol, a major constituent of ginger, has been shown to effectively reduce seizure-like paroxysmal behavior in zebrafish larvae. This compound is also believed to help restore the balance between GABA and glutamate (GLU) in the epileptic brain¹⁹. Additionally, ginger’s bioactive components are known to cross the blood-brain barrier¹⁸. The neurophysiological activities of ginger have prompted further investigation into its effects on various neural mechanisms. This study aims to evaluate the therapeutic potential of ginger essential oil compounds for treating insomnia by targeting orexin-1 (OX1R) and orexin-2 (OX2R) receptors using molecular docking and molecular dynamics simulations.

MATERIALS AND METHODS:

Preparation of Ginger Essential Oil:

The rhizome of ginger (Zingiber officinale) was first processed into a simple powder. Essential oil was then obtained from 250 grams of this powdered ginger through steam distillation. The oil was extracted using n-hexane and subsequently purified by evaporating the n-hexane solvent²¹.

Gas chromatography and mass spectrometry of Ginger Essential Oils:

Ginger essential oil was analyzed using gas chromatography-mass spectrometry (GC-MS QP2010 Plus) equipped with a non-polar/polar stationary phase column. The oil sample was injected into an Agilent 19091S-433 HP-5MS column. The column temperature was initially set and then increased gradually by 10°C every 28 minutes until it reached a final temperature of 288°C. The chemical components were identified through mass spectrometry (MS).

Receptors and Ligands Retrieval:

The interaction between Zingiber officinale volatile compounds and orexin receptors was investigated through molecular docking and molecular dynamics simulations. The structures of orexin-1 receptor (OX1R, PDB ID: 4zj8)²² and orexin-2 receptor (OX2R, PDB ID: 7xrr)²³ were retrieved from the RCSB Protein Data Bank. Preparation of the receptors involved removing water molecules and native ligands, followed by the addition of hydrogen atoms using AutoDock Tools²⁴^,25. The 3D-structures of 30 selected ligands were downloaded from PubChem in SDF format, converted to PDB files, and subjected to energy minimization using Avogadro ver. 1.2.0.

Molecular Docking and Dynamic:

Molecular docking was conducted using AutoDock Vina version 1.2.3. The docking of ligands to the receptors was performed within a grid box optimized automatically by the software. This grid box was validated by re-docking the native ligand for each receptor to ensure accuracy. The resulting 2D and 3D docking poses were visualized using Discovery Studio²⁶^-²⁹. To further investigate the binding interactions of the best-scoring ligand, alpha-zingiberene (CID92776), with orexin receptors OX1R (PDB ID: 4zj8) and OX2R (PDB ID: 7xrr). Molecular dynamics (MD) simulations were performed using OpenMM on the Google Colab platform. For ligand preparation, the 3D structure of alpha-zingiberene was obtained from the PubChem Open Chemistry Database. The SMILES representations of three different compounds were converted into PDB files using LigParGen, a web-based tool that generates force field parameters for organic molecules and ligands^30–32. Before running the MD simulations, the necessary software libraries and packages were installed, including Anaconda, OpenMM, PyTraj, py3Dmol, ProLIF, NumPy, Matplotlib, and AmberTools. The ligand and protein PDB files were uploaded and managed on Google Drive and Google Colab. The MD simulations followed the protocol outlined by Pablo R. Arantes et al. Protein topology was generated using the FF19SB force field, with TIP3P water molecules. The system was neutralized with NaCl ions at a concentration of 0.15 M using AMBER tleap. Ligand topology was generated using the GAFF2 force field. The equilibration phase included 1,000 steps of energy minimization, followed by a 5-nanosecond simulation with a 2-femtosecond integration time step, at 310°K temperature and 1 bar pressure. Trajectory and log files were recorded every 10 picoseconds. Production MD simulations were then run for 10 nanoseconds with similar parameters: a 2-femtosecond time step, 310°K temperature, 1 bar pressure, and data recorded every 10 picoseconds. Binding free energy calculations were performed using both the MM-GBSA and MM-PBSA methods for comparison. The GB/SA parameters included the OBC model with igb=2 and a salt concentration of 0.15 M. Additional analyses included generating LigPlot images before and after simulation, calculating interaction energies, measuring distances between the ligand and catalytic site residues as well as specific residues, computing the root mean square deviation (RMSD) of the protein’s alpha carbon atoms, plotting RMSD distributions, calculating root mean square fluctuations (RMSF) of the alpha carbons, and other relevant assessments³³.

RESULT:

Phytochemical constituents of ginger essential oil:

The ginger essential oil contained a total of 40 phytochemicals, as shown Figure 1 and Table 1. These phytochemical components were categorized into two groups of secondary metabolites: terpenoids.

Binding affinity and Interaction of ginger bioactive compounds to orexin receptors:

The selective binding of ginger’s bioactive compounds to orexin receptors was evaluated based on their binding affinity scores. These results were compared with those of lemborexant and suvorexant, which served as control compounds. The findings indicated that ginger bioactive compounds exhibited mild selectivity in binding to orexin receptors (figure 2). Generally, the binding affinities of the ginger compounds were higher than those of the controls. Lemborexant and suvorexant showed selective binding to orexin receptors with low binding affinity values, reflecting strong ligand-receptor interactions. According to the binding affinity scores, suvorexant demonstrated a preference for OX1R (-7.112 kcal/mol), while lemborexant was more selective for OX2R (-10.35 kcal/mol). Among the 40 ginger bioactive compounds tested, alpha-zingiberene exhibited the highest selectivity toward orexin receptors, as indicated by a selectivity score of -0.2342. Its binding affinities were -4.231 kcal/mol for OX1R and -7.255 kcal/mol for OX2R. Notably, alpha-zingiberene showed the strongest binding to OX2R among all ginger compounds, having the lowest binding affinity value for this receptor. Additionally, 6-shogaol, another compound found in ginger essential oil, displayed the strongest interaction with OX1R, with the lowest binding affinity (-5.614 kcal/mol) among all identified ginger bioactive constituents.

Figure 1: TIC Ginger Volatile Oil Compounds using GC-MS QP2010 Plus analysis

Table 1: GC-MS analysis of Ginger Volatile Oil Compounds

No.	Peak number	RT (min)	Composition (%)	Code	Compound	Metabolit Secondary
1	27	24.582	18.76	CID521253	I-zingiberene	Sesquiterpenoid
2	26	24.279	17.52	CID92139	alpha-curcumene	Sesquiterpenoid
3	31	25.236	12.92	CID92776	alpha-zingiberene	Sesquiterpenoid
4	29	24.883	7.59	CID10104370	beta.-bisabolene	Sesquiterpenoid
5	28	24.806	6.48	CID5281516	alpha.-Farnesene	Sesquiterpenoid
6	15	18.003	5.49	CID638011	citral/ geranial	Monoterpenoid
7	2	8.168	4.92	CID6616	camphene	Monoterpenoid
8	13	17.143	3.92	CID643779	neral/ cis-citral	Monoterpenoid
9	9	14.935	2.91	CID64685	borneol	Monoterpenoid
10	5	10.611	2.41	CID12304570	sylvestrene	Monoterpenoid
11	6	10.693	2.34	CID2758	eucalyptol/ 1,8-cineol	Monoterpenoid
12	1	7.726	1.03	CID6654	alpha-pinene	Monoterpenoid
13	14	17.498	0.95	CID637566	geraniol	Monoterpenoid
14	35	26.979	0.92	CID13213649	zingiberenol	Sesquiterpenoid
15	19	21.197	0.78	CID12303902	copaene	Sesquiterpenoid
16	10	15.153	0.73	CID35725	tricyclo[4.3.1.1(3,8)]undecan-1-ol	Monoterpenoid
17	3	9.014	0.73	CID14896	beta-pinene	Monoterpenoid
18	40	28.351	0.67	CID5365823	dehydronerolidol	Sesquiterpenoid
19	34	26.559	0.66	CID442793	6-gingerol	Sesquiterpenoid
20	4	9.406	0.64	CID31253	beta.-myrcene	Monoterpenoid
21	20	21.674	0.61	CID6918391	beta-elemene	Sesquitepenoid
22	11	15.667	0.61	CID17100	alpha-terpineol	Monoterpenoid
23	39	28.215	0.58	CID94378	6-paradol	Monoterpenoid
24	8	12.793	0.57	CID6549	linalool	Monoterpenoid
25	12	16.713	0.56	CID8842	citronellol	Monoterpenoid
26	38	27.749	0.47	CID165258	cryptomeridiol	Sesquitepenoid
27	23	23.531	0.45	CID5317319	cis-beta.-farnesene	Sesquitepenoid
28	16	18.509	0.41	CID6448	bornyl acetate	Monoterpenoid
29	37	27.276	0.41	CID6428444	trans-sesquisabinene hydrate	Sesquitepenoid
30	33	25.839	0.40	CID5281794	6-shogaol	Monoterpenoid
31	17	18.619	0.39	CID8163	2-undecanone	Monoterpenoid
32	22	23.056	0.35	CID15094	gamma-muurolene	Sesquitepenoid
33	32	25.405	0.35	CID5352437	(E)-gamma-bisabolene	Sesquitepenoid
34	24	23.596	0.34	CID12315492	beta-sesquiphellandrene	Sesquitepenoid
35	7	12.725	0.32	CID84825	rosefuran/ alpha.-naginatene	Monoterpenoid
36	21	22.052	0.28	CID25147318	sesquithujene	Sesquitepenoid
37	30	25.059	0.28	CID91747213	cis-muurola-4(15),5-diene	Sesquitepenoid
38	18	20.900	0.25	CID519960	(+)-cyclosativene/ cyclosativene	Sesquitepenoid
39	36	27.192	0.19	CID13970960	(+/-)-dihydro-ar-turmerone	Sesquitepenoid
40	25	23.851	0.18	CID10899740	alloaromadendrene	Sesquitepenoid

Figure 2: Selectivities of Binding Affinities Compounds of Ginger Volatile Oil toward Orexin Receptor (OX-1/ OX-2)

Based on binding affinity and selectivity, alpha-zingiberene emerged as the most potent inhibitor of orexin receptors (OXs). The interactions between alpha-zingiberene and the OX1R and OX2R receptors were further examined through 3D visualization (figure 3[c][d]). The binding of alpha-zingiberene to OX1R involved interactions with the amino acid residues Lys1190 and Arg1191 (figure 3[c]) compared to the control compounds suvorexant and lemborexant, alpha-zingiberene exhibited weaker binding, as it engaged fewer amino acid residues in the complex. These results suggest that alpha-zingiberene targets OX1R via a different binding mode than suvorexant and lemborexant (figure 3[a]-[b]), interacting with distinct amino acid residues.

Figure 3: Interaction among suvorexant, lemborexant, and alpha-zingiberene with orexin 1/2 receptor, (a) 4zj8_the human OX-1 receptor with suvorexant (SOV); (b) 7xrr_the human OX2 receptor with lemborexant; (c) 4zj8_the human OX1 receptor with alpha-zingiberene (CID 92776); and (d) 7xrr_the human OX2 receptor with alpha-zingiberene (CID 92776)

Molecular dynamic alpha-zingiberene-OX-2:

DISCUSSION:

According to the GC-MS analysis (figure 1 and table1), I-zingiberene and alpha-zingiberene were identified as the most abundant bioactive compounds in the ginger essential oil, comprising 18.76% and 12.92% of the composition, respectively.

Orexins, comprising OX1 and OX2, are neuropeptides that regulate feeding behavior and various neurological functions. Their mechanisms influence the sleep-wake cycle, neuroendocrine activities, glucose metabolism, energy balance, and stress adaptation⁷^,³⁴. Therefore, targeting orexin receptors to modulate the sleep-wake cycle is considered highly promising¹³^,¹⁹^,³⁵. Suvorexant and emborexant are widely used dual orexin receptor antagonists for treating insomnia⁸^,³⁶. This study, however, demonstrates that the natural bioactive compound alpha-zingiberene from ginger exhibits comparable potency to these commercial orexin receptor antagonists.

[b] RMSD 7xrr_orexin-2-alpha-zingiberene

[c] RMSF 7xrr_orexin-2-alpha-zingeberene

Figure 4: [a] Compare simulation of molecular dynamic 4zj8_orexin-1-alpha-zingiberene and 7xrr_orexin-2-alpha-zingiberene; [b] RMSD parameter of 7xrr/ 4zj8-alpha-zingiberene complexed; and [c] RMSF parameter of 7xrr/ 4zj8-alpha-zingiberene complexed

The identification of potential orexin inhibitors was conducted through in silico analysis by comparing the activity of alpha-zingiberene with that of suvorexant and lemborexant against OX1R and OX2R receptors. Virtual screening via in silico methods serves as a dynamic approach for discovering novel therapeutic agents¹⁰^,²². The effectiveness of ginger’s bioactive compounds against orexin receptors was evaluated based on their binding affinities and binding sites³⁷.

The interactions of alpha-zingiberene with OX1R and OX2R were further examined through 3D visualization (figure 3[c][d]). The interaction between OX1R and alpha-zingiberene is facilitated by alkyl bonds with Lys A:1190 and Lys A:1191, as well as van der Waals forces involving residues ThrA:1151, AsnA:1151, GluA:1153, ThrA:1154, and AsnA:1187 (figure 3[a]-[d]), compared to suvorexant and lemborexant as controls, alpha-zingiberene forms interaction positions with a distinctly different set of amino acid residues in the complex. These results indicate that alpha-zingiberene targets the OX1 receptor in a manner distinct from suvorexant (figure 3[a]). Alpha-zingiberene also interacts more favorably with OX2R through amino acid residues (figure 3[d]): Pi-sigma (HisA:350), Pi-alkyl (PheA:227), Alkyl (ValA:138, TyrA:317, TyrA:354), and van der Waals (ThrA:111, GlnA:134, SerA:321), which are similar to those involved in the interaction with lemborexant (figure 3[b]: conventional hydrogen bond: GlnA:134; Halogen (Fluorine): Ile320, ArgA:1016; Pi-Pi T-shaped: Phe227; Pi-sigma: IleA:320; and van der Waals: ThrA:111, ValA:114, IleA:130, TrpA:120, ProA:131, ThrA:135, SerA:321, AsnA:324, HisA:350). Similarly, other ginger volatile oils, such as dehydronerolidol, alpha-curcumene, alpha-copaene, alpha-pinene, alpha-terpineol, beta-bisabolene, beta-citronellol, geraniol, linalool, and others, also tend to inhibit the human orexin-2 receptor, although their binding affinities are lower than that of alpha-zingiberene, as shown in Figure 2. Therefore, alpha-zingiberene is predicted to be the best ginger oil compound for inhibiting the human orexin-2 receptor.This study found that alpha-zingiberene exhibited the highest selective binding activity toward both OX1R and OX2R among all identified ginger compounds. As an orexin antagonist, alpha-zingiberene likely targets OX1R and OX2R with binding behaviors moderately similar to those of lemborexant. alpha-zingiberene may inhibit the interaction of orexins with their respective G-protein-coupled receptors (GPCRs), specifically OX1R with Hcrtr-1 and OX2R with Hcrtr-2. An orexin antagonist, such as a Dual Orexin Receptor Antagonist (DORA), binds to these receptors without activating them, effectively blocking orexin from binding and initiating its downstream signaling cascade. By acting as an orexin antagonist, it can block the activation of phospholipase C (PLC), phospholipase A (PLA), phospholipase D (PLD), and adenylyl cyclase (AC), leading to reduced cytosolic Ca²⁺ levels and suppression of downstream signaling cascades⁷.

Molecular dynamics simulation analysis is essential because it provides a deep understanding of molecular behavior, aids in material prediction and engineering, and complements and explains experimental data that cannot be obtained by conventional methods alone. A demonstration of molecular dynamics simulation using the alpha-zingiberene-OX2R complex is shown in Figure 4. The stability of the protein–ligand complexes was assessed by calculating the Root Mean Square Deviation (RMSD) and the Root Mean Square Fluctuation (RMSF) of the protein backbone from its initial to final conformation. RMSD (Root Mean Square Deviation) is a fundamental metric in molecular dynamics (MD) simulations used to quantify the average structural deviation of a molecular system, such as a protein or nucleic acid over time relative to a reference structure, often the initial or an experimentally determined conformation. RMSD simulations showed that the alpha-zingiberene-OX2R complex reached overall stability after 2 ns (nanoseconds), with an average stable RMSD of 1.9 Å (figure 4[b]). A lower RMSD value at 2 ns means that the molecule (protein, ligand, or their complex) maintains its initial conformation well and is more dynamically stable. A stable and low RMSD value (around 0.1–0.2 nm or 1–2 Å) during the simulation is a key indicator of molecular stability in molecular dynamics simulations. These results indicate that the alpha-zingiberene-OX2R complex is more stable during the simulation time than other ginger oil compounds. The Root Mean Square Fluctuation (RMSF) measures the flexibility of protein residues by quantifying their deviation from average positions during the simulation. The root mean square fluctuation (RMSF) value is the fluctuation of protein residues from their mean positions during the simulation, which describes the flexibility of the protein structure. Based on the timeline results, alpha-zingiberene interacts effectively with the orexin-2 receptor binding site (figure [4c]. alpha-zingiberene interacts with the active site residues and the A-domain site, and reduces the site fluctuation in orexin-2R. Furthermore, Figure 4a illustrates the timeline interaction of alpha-zingiberene with the active site of orexin-2 at simulation time.

The binding energy of alpha-zingiberene-orexin-2R complex using MMPBSA and MMGBSA solvent model was reported by Open MM on Google Colab. The MMPBSA binding energy (∆G) of alpha-Zingiberene-OX2R complex is 0.0226 ± 3.1674 kcal/ mol and the MMGBSA binding energy (∆G) of alpha-Zingiberene-OX2R complex is -8.9200 ± 2.3693 kcal/ mol. The difference in the binding energy value between MMPBSA and MMGBSA could be due to polar and nonpolar contributions to ΔG, ΔT and ΔS, and type of bonds, angles, dihedral energies, electrostatic energies and van der Waals energies. MMGBSA/PBSA binding energy (∆G) is an estimate of the free energy of binding between ligand (alpha-zingiberene) and receptor (OX1R or OX2R) calculated using the MMGBSA/MMPBS method. A more negative ∆G value indicates a stronger and more stable bond between the ligand and the receptor.

The ∆G value of OX2R is negative, indicating that alpha-zingiberene forms a stronger and more stable bond to OX2R. Several key contributing factors are the characteristics of the binding pocket, the geometric and chemical compatibility between the ligand and the receptor, and the dynamics and flexibility of the protein. A look at the structure of the binding pocket, illustrated in Figure 4[a], shows that critical residues in OX1R (e.g., Val160, Thr161, Arg162, Phe163, Arg164, Ala127 initially, and Leu4, Arg5, Trp8, His13 during the simulation) play a crucial role in stabilizing interactions with ligands such as alpha-zingiberene. These residues engage in hydrophobic interactions and sometimes form hydrogen bonds favorable for certain ligands. Analysis of the geometric and chemical compatibility between the ligand and the receptor indicates that alpha-zingiberene fits better spatially and chemically within the binding pocket of OX2R, resulting in lower (more negative) binding energy and thus greater stability. Conversely, a geometric or polarity mismatch in the OX2R pocket will weaken the interaction, leading to a less negative ∆G value. Furthermore, the dynamics and flexibility of the OX2R protein affect the stability of the ligand-receptor complex during the simulation, as reflected in the MMGBSA binding energy values and their deviations. The standard deviation (±) values indicate that OX2R exhibits a relatively low deviation indicating a more consistent but generally weaker interaction. Overall, the binding energy of the alpha-zingiberene–OX2R complex indicates stability and is consistent with the RMSD, RMSF, and binding affinity analyses³⁸.

Ginger is a famous medicinal herb known for its biological activities. Bioactive compounds of ginger were proven for the activities in neurophysiological mechanisms and in the blood-brain barrier¹⁸^,¹⁹^,³⁹. Gingerols and shogaols, as the major constituents in ginger, performed activities including migraine relief, neuroprotective, monoamine oxidase A (MAO-A), and GABA modulator⁴⁰. Previously, treatment of Z. officinale hydroethanolic extract successfully exerted antiseizure activity in the PTZ-kindling mice model⁴¹. Treatment with ginger also effectively upregulated GABA level in the hippocampus and cortex of the senile female rats¹⁹^,⁴². This study identified the potency of alpha-zingiberene to act as orexin antagonist via modulation of GABA, similarly as lemborexant³⁶. Treatment with alpha-zingiberene via OX1R/OX2R may increase the sleep drive and inhibit wake-promoting neurons, improving total amount of sleep and daytime alertness³⁵. Ginger, as natural herb, may exhibit less adverse effects than drugs¹³^,¹¹⁴³. However, the physiological effect of alpha-zingiberene remains unknown, thus it required further studies to evaluate the potency.

CONCLUSION:

This study identified novel orexin antagonists among the natural bioactive compounds from ginger essential oil. Molecular docking results indicated that these compounds were generally selective for the OX2R receptor. Molecular dynamics simulations also showed that alpha-zingiberene formed a stronger and more stable interaction with OX2R. These findings demonstrate the potential of ginger essential oil compounds, particularly alpha-zingiberene, as promising therapeutic agents for the treatment of insomnia.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors acknowledge to Ministry of Education and Culture for research funding through a national research grant of Indonesia year 2024.

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Received on 13.07.2025 Revised on 24.11.2025

Accepted on 11.02.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3325-3332.

DOI: 10.52711/0974-360X.2026.00473

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