Sedative potential of palmatine chloride in thiopental sodium-induced chicks: evidence from in vivo and in Silico studies

Abstract

The aim of this study is to assess the sedative impact of palmatine chloride (PME) in thiopental sodium-induced sleeping chicks and its underlying molecular mechanism by using in vivo and in silico approaches. Chicks received PME per orally (p.o.) at doses of 1.25, 2.5, and 5 mg/kg per body weight (b.w.), while diazepam (DZP) (2 mg/kg) served as a positive control and vehicle as a negative control. For the purpose of evaluating the experimental compounds synergistic or antagonistic effects, a combination of PME and DZP was administered to the chicks. After thirty minutes, thiopental sodium (40 mg/kg, intraperitoneal (i.p.)) was administered to induce sleep, and latency to sleep onset and sleep duration were measured. In vivo results showed that PME reduced sleep latency and prolonged sleep duration in a dose-dependent manner, with the combination therapy producing a significant enhancement of these effects. In silico docking revealed PME binding to gamma-aminobutyric acid A (GABA_A) receptor α1 and β2 subunits (–7.2 kcal/mol) with shared amino acids. Pharmacokinetic and toxicity analyses suggested favorable drug-like properties. These results indicate PME’s sedative potential, alone or with DZP, likely via GABAergic modulation, warranting further functional validation.

Introduction

Insomnia, characterized by persistent difficulty in initiating or maintaining sleep, is one of the most prevalent neurological disorders worldwide, impairing cognitive performance, emotional stability, and overall quality of life^1,2. The neurobiology of sleep regulation is strongly governed by the GABA system, the principal inhibitory neurotransmitter pathway in the central nervous system. Within this network, the GABA_A receptor plays a pivotal role, serving as the major molecular target of sedative-hypnotic drugs^3,4.

Currently available therapies, including benzodiazepines, barbiturates, and related agents, are effective but remain clinically constrained by rapid tolerance, dependence, and severe adverse reactions such as psychomotor impairment, cardiovascular complications, and cognitive deficits^5,6. These limitations underscore the necessity of identifying safer, mechanism-based alternatives.

Natural products continue to represent a powerful source of therapeutic innovation, offering structurally diverse molecules with central nervous system activity. Phytochemicals such as flavonoids, terpenes, and alkaloids have demonstrated the capacity to modulate GABAergic transmission, thereby exerting anxiolytic and sedative effects^7,8. Among them, palmatine chloride (PME), a protoberberine isoquinoline alkaloid, has attracted growing attention. PME is a quaternary ammonium salt form of palmatine that exhibits superior solubility and stability, making it more suitable for pharmacological evaluation than the free base^9,10. In addition to its wide pharmacological spectrum, including neuroprotective, anti-inflammatory, and antidepressant effects^11,12. PME has been reported to influence neurotransmission by enhancing serotonergic and GABAergic pathways¹³. These findings suggest that PME may serve as a promising candidate for managing sleep disturbances.

Based on this evidence, we hypothesized that PME could exert sedative effects through modulation of the GABA_A receptor, either alone or in combination with DZP, thereby potentiating GABAergic signaling. To test this, the present study investigated the effects of PME on thiopental sodium-induced sleeping chicks and assessed its potential synergism with DZP. Furthermore, to elucidate the molecular basis of its action, in silico docking was performed against GABA_A receptor subunits, and its pharmacokinetic and toxicological profiles were evaluated to establish PME as a viable drug-like candidate.

Methods

In vivo study

Chemicals and reagents

The experimental compound PME (1.25, 2.5, and 5 mg/kg, p.o.) (CAS Number: 10605-02-4, Purity: 98% (HPLC)) was brought from Sigma-Aldrich (USA). Loba Chemie Pvt. Ltd. (India) supplied Tween-80 (0.5%). DZP (2 mg/kg, p.o.) and thiopental sodium (40 mg/kg, i.p.) were purchased from Square Pharmaceuticals Ltd. (Bangladesh).

Dose selection and Preparation

The test doses of PME (1.25, 2.5, and 5 mg/kg) were selected from the previous published literature review¹⁴. We prepared a mother solution at the concentration of 5 mg/kg by adding a sufficient amount of distilled water and Tween-80 (0.5%). At last, we dilute the mother solution at the concentrations of 2.5 and 1.25 mg/kg. On the other hand, DZP, supplied as an injectable solution (5 mg/ml), was diluted with distilled water to achieve a final concentration of 2 mg/kg for oral administration.

Experimental animals

Four-day-old (Grade-A) young broiler chickens (Gallus domesticus) of either sex, with a weight range of 42–48 g, were purchased from Aftab Hatchery Ltd., which is located in Bhagalpur, Bajitpur, Kishoreganj, Bangladesh. Chicks were chosen as a validated alternative neuropharmacological model because thiopental sodium reliably induces sleep in this species, and chicks are highly sensitive to GABAergic modulation, making them suitable for early-phase sedative screening¹⁵. Before our investigation, all of the chicks were kept in the pharmacology lab at Gopalganj Science and Technology University (GSTU) at a consistent temperature (26 ± 3 °C), with 65% relative humidity and under controlled lighting (12-hour dark/light cycle). During this time, they had unlimited access to standard feeds and water ad libitum. All animals were randomly assigned into six groups (n = 5 each). Randomization was performed using a random number generator, and the investigator recording sleep parameters was blinded to treatment groups. Before conducting the sedative test, chicks were fasted for about 12 h. The current experimental test was performed between 8:00 A.M. and 3:00 P.M. All experimental procedures were conducted in accordance with relevant institutional guidelines and regulations. All methods are reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org). The experimental protocols used in this study were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of [Department of Pharmacy, GSTU], under approval number [#gstu-20PHR009-02].

Table 1. In chicks, several treatments and doses were examined.

Treatment Groups	Dose (mg/kg)	Route of Administration	Targeted Receptor
NC (Vehicle): Distilled water containing 0.9% NaCl and 0.5% tween-80	10 ml/kg	p.o.	–
DZP-2	2		GABAA receptor
PME-1.25	1.25		Under Investigation
PME-2.50	2.50
PME-5	5
PME-5 + DZP-2	5 + 2

NC: Negative control; DZP: Diazepam; PME: Palmatine Chloride; GABA_A: Gama-aminobutyric acid type A receptor; p.o.: per orally

Thiopental sodium-induced sleeping test

The test was run following the procedure outlined by Dehar et al. (2012)¹⁶. PME: 1.25, 2.5, and 5 mg/kg; DZP: 2 mg/kg; and vehicle: 10 ml/kg were administered p.o. Then after 30 min of pretreatment, each chick received an i.p. injection of thiopental sodium at the dose of 40 mg/kg to induce sleep before being placed in the observation chamber. After administration of thiopental sodium, we observed two points: first, latency period (LP) and second, duration of sleeping time up to four hours (Table 1). The percentage of sleep incidence, increase in sleep time, and reduction in LP can be determined using the following equations.

% Incidence of sleep= .

% Reduction in LP = .

% Increase in the amount of time spent sleeping = .

Where, A = mean of LP in the NC group, B = mean of LP in the test or standard groups, X = mean of duration in the test or standard groups and Y = Mean of duration in the NC group.

Statistical analysis

Results are presented as mean ± standard deviation (SD) (n = 5). One-way ANOVA was used, followed by t-Student Tukey’s post-hoc test for pairwise comparisons (GraphPad Prism v9.5, San Diego, USA). A p-value < 0.05 was considered statistically significant.

In Silico study

Ligand Preparation

The 3D structures of DZP (Compound ID: 3016), and PME (Compound ID: 19009) were downloaded using the PubChem chemical database (https://pubchem.ncbi.nlm.nih.gov/, obtained on 4 March 2025) and stored in structure-data file (SDF) format. PME was used as palmatine chloride, but the chloride ion was removed for docking. Structures were energy-minimized using Chem3D 21.0.0. The 2D structure of chemical agents is shown in Fig. 1.

Fig. 1 [Images not available. See PDF.]

The two-dimensional chemical structures of diazepam and palmatine.

Protein selection and Preparation

The GABA_A receptor was selected as the molecular target, focusing on α1 and β2 subunits (PDB ID: 6X3X), which are critically involved in sedative and hypnotic drug actions ^3,17,18. The protein structure was prepared by removing heteroatoms, water molecules, and redundant residues using PyMol (v2.4.1). Further energy minimization was conducted using Swiss-PDB Viewer (v4.1.0) with the GROMOS96 43B1 force field ^15,19.

Molecular Docking and visualization

Docking was performed with PyRx (v0.8) using AutoDock Vina. A grid box of 20.01 × 18.54 × 17.29 Å was set along the x-, y-, and z-axes, with 2000 calculation steps. Ligands were converted to PDBQT format, and binding affinities were expressed in kcal/mol. Docking validation was ensured by redocking DZP into the GABA_A receptor binding pocket, which reproduced interactions consistent with previous reports. Visualization of hydrogen bonds and hydrophobic interactions was performed using Discovery Studio Visualizer (v21.1.0).

Prediction of pharmacokinetics and Drug-Likeness

Drug-likeness, absorption, distribution, metabolism, excretion (ADME), and toxicity profiles of PME and DZP were predicted using SwissADME (http://www.swissadme.ch/), pkCSM (https://biosig.lab.uq.edu.au/pkcsm/), and ProTox 3.0 (https://tox.charite.de/protox3/) online servers. Parameters included Lipinski’s rule of five, gastrointestinal absorption, blood–brain barrier penetration, CYP450 enzyme interactions, total clearance, and predicted LD₅₀ values. As these analyses are computationally based, they provide preliminary predictions only, which require experimental validation.

Results

In vivo result

In vivo research, we observed thiopental sodium reliably produced 100% sedation in all tested animal groups. The NC group was characterized by delayed onset of sleep (3.49 ± 0.06 min) and acted as a baseline for comparison with other groups. However, the standard drug DZP-2 decreases LP (1.24 ± 0.14 min) compared to the NC group. On the other hand, PME produced dose-dependent effects on the thiopental sodium-induced animals and significantly (p < 0.05) reduced the LP when compared to the NC group. PME-1.25, PME-2.5, and PME-5 animal groups demonstrated LP at 3.28 ± 0.18, 2.24 ± 0.71, and 2.01 ± 0.44 min. Eventually, with PME-5 when co-treated with DZP-2 (PME-5 + DZP-2), we observed a synergistic effect and produced the lowest LP (1.02 ± 0.28 min) among all tested groups.

On the other hand, in the case of duration of sleep, DZP-2 increased duration of sleep (186.52 ± 7.18 min) compared to the NC (143.93 ± 7.75 min) group. Additionally, PME significantly (p < 0.05) and dose-dependently lengthens animals sleep duration. PME-1.25, PME-2.5, and PME-5 demonstrated durations of sleep at 156.30, 162.61, and 183.02 min, as mean values. When administering combination therapy (PME-5 + DZP-2), sleeping time significantly (p < 0.05) increased compared to the other treatment groups. However, details of in vivo findings are demonstrated in Table 2; Fig. 2.

Table 2. Latency period and duration of sleeping time observed in the palmatine chloride, diazepam, and combination treatment groups.

Treatments	Latency period (min)	Duration of sleep (min)	Sleep incidence (%)
NC (Vehicle)	3.49 ± 0.06	143.93 ± 7.75	100
DZP-2	1.24 ± 0.14*bcd	186.52 ± 7.18*bcd	100
PME-1.25	3.28 ± 0.18*	156.30 ± 6.74*	100
PME-2.5	2.24 ± 0.71*b	162.61 ± 8.16*b	100
PME-5	2.01 ± 0.44*bc	183.02 ± 4.67*bc	100
PME-5 + DZP-2	1.02 ± 0.28*abcd	194.50 ± 4.25*abcd	100

Values are mean ± SD (standard deviation) (n = 5); One-way ANOVA followed by t-Student Tukey’s as post-hoc test; p < 0.05 when compared to the *NC (vehicle), ^aDZP-2, ^bPME-1.25, ^cPME-2.5, ^dPME-5, ^ePME-5 + DZP-2; NC: Negative control (distilled water containing 0.9% NaCl and 0.5% tween 80); DZP: Diazepam; PME: Palmatine Chloride

Fig. 2 [Images not available. See PDF.]

Latency and duration of sleep in different treatment groups of animals. [Values are mean ± SD (standard deviation) (n = 5); one-way ANOVA followed by t-Student Tukey’s as post-hoc test; p < 0.05 when compared to the *NC (vehicle), ^aDZP-2, ^bPME-1.25, ^cPME-2.5, ^dPME-5, ^ePME-5 + DZP-2; NC: Negative control (distilled water containing 0.9% NaCl and 0.5% tween 80); DZP: Diazepam; PME: Palmatine Chloride]

In contrast, compared to the NC group, PME-1.25, PME-2.5, and PME-5 dose-dependently increased the percentage of sleeping time at 7.91, 11.48, and 21.35% (Table 3). However, when chicks received combination therapy (PME-5 + DZP-2), they exhibited the highest sleep duration among all groups at 26%. On the other hand, PME reduced the percentage of LP in a dose-dependent manner, which was 6.01, 35.81, and 42.40% for PME-1.25, PME-2.5, and PME-5, respectively. At last, the PME-5 + DZP-2 groups displayed the highest LP decrease at 70.77%.

Table 3. Percentage increase in duration of sleep and decrease in latency period in thiopental sodium-induced sleeping chicks of the palmatine chloride, diazepam, and combination treatment groups.

Treatments	% Reduction in latency Period	% Increase in the amount of time spent sleeping
NC (Vehicle)	–	–
DZP-2	64.46	22.83
PME-1.25	6.01	7.91
PME-2.5	35.81	11.48
PME-5	42.40	21.35
PME-5 + DZP-2	70.77	26

NC: Negative control (distilled water containing 0.9% NaCl and 0.5% tween 80); DZP: Diazepam; PME: Palmatine chloride

In Silico findings

Interactions of PME and DZP with GABA_A (α1 and β2 subunits) receptor

For the GABA_A receptor, DZP showed the highest binding affinity (− 8.3 kcal/mol) by the synthesis of a single hydrogen bond with LEU A: 285 amino acid residues. Additionally, DZP also formed several hydrophobic bonds, including PHE A: 289, PRO B: 233, MET B: 236, MET A: 286, and LEU B: 232 specific amino acid residues. Despite not forming hydrogen bonds, PME displayed reliable binding affinity (–7.2 kcal/mol) with the GABA_A receptor. This interaction involved several hydrophobic bonds: PHE A: 289, PRO B: 233, MET B: 236, MET A: 286, LEU B: 232, LEU A: 285, MET A: 261, and VAL A: 290 amino acid residues. Diazepam and palmatine 3D and 2D non-bond interactions with the GABA_A receptor (α1 and β2 types of subunits) were shown in Table 4; Fig. 3, together with information on the number of hydrogen bonds.

Table 4. Results of diazepam and palmatine molecular Docking against the GABA_A (α1 and β2 types of subunits) receptor.

Ligands	Receptor (PDB ID)	BA (kcal/mol)	No of HB	Amino acid (AA) residues
				HBs	Others
DZP	GABAA (α1 and β2 types of subunits) receptor (6X3X)	–8.3	1	LEU A: 285	PHE A: 289, MET A: 286, PRO B: 233, MET B: 236, LEU B: 232
PME		–7.2	0	–	PHE A: 289, MET A: 286, LEU A: 285, MET A: 261, VAL A: 290, PRO B: 233, MET B: 236, LEU B: 232

AA: Amino acid; BA: Binding affinity; DZP: Diazepam; GABA_A: Gamma-aminobutyric acid type A receptor; HB: Hydrogen Bond; PDB ID: Protein Data Bank identification code; PME: Palmatine;

Fig. 3 [Images not available. See PDF.]

Diazepam and palmatine 3D and 2D non-bond connections with the GABA_A receptor (α1 and β2 types of subunits).

Pharmacokinetics, drug-likeness properties and toxicological profiles

The pharmacokinetics study is crucial in drug development and discovery due to its evaluation of ADME and toxicity parameters. PME (C₂₁H₂₂NO₄) and DZP (C₁₆H₁₃ClN₂O) have molecular weights (MW) 352.40 g/mol and 284.74 g/mol. However, both compounds (PME and DZP) follow the five Lipinski rules without any violation: H-acceptor < 10, H-donor < 5, logP < 5, and MW < 500, as well as showing high gastrointestinal absorption (97.084 and 97.42%) and good solubility. Molar refractivity (MR) of the PME and DZP are 101.80 and 87.95, respectively, which was within limit (MR ≤ 140) and displayed bioavailability score of 0.55. Compared to DZP (0.294 log ml/min/kg), PME had a higher overall clearance (1.246 log ml/min/kg). In the metabolism part, PME and DZP do not have any effect on CYP2D6 and CYP3A4 substrates or inhibitors.

In this in silico toxicity investigation, PME demonstrates LD₅₀ values of 200 mg/kg with toxicity class 3, whereas DZP exhibited LD₅₀ values of 48 mg/kg with toxicity class 2. However, PME showed no toxic effect in terms of hepatotoxicity, nephrotoxicity, cardiotoxicity, clinical toxicity, and nutritional toxicity, while DZP showed adverse effects in the case of clinical toxicity, as shown in Table 5.

Table 5. Pharmacokinetics, drug likeness properties and toxicological profiles of palmatine and diazepam.

Properties	Factors	PME	DZP
Physicochemical Properties	Formula	C21H22NO4	C16H13ClN2O
	Molecular weight (g/mol)	352.40	284.74
	Number of heavy atoms	26	20
	Number of aromatic heavy atoms	16	12
	Number of H-bond donors	0	0
	Number of H-bond acceptors	4	2
	Molar Refractivity	101.80	87.95
	TPSA (Ų)	40.80	32.67
Lipophilicity	Log Po/w (XLOGP3)	3.75	2.99
Drug-likeness	Lipinski	Yes; 0 violations	Yes; 0 violation
	Bioavailability Score	0.55	0.55
Water Solubility	Log S (ESOL)	−4.58	−3.87
	Class	Moderately soluble	Soluble
Absorption	Caco2 permeability (log Papp in 10 –6 cm/s)	0.863	1.554
	Intestinal absorption (human) Numeric (%) Absorbed	97.084	97.42
	Skin Permeability	−2.554	−2.205
	P-glycoprotein Substrate	Yes	Yes
	P-glycoprotein I Inhibitor	Yes	No
	P-glycoprotein II Inhibitor	Yes	No
Distribution	BBB permeability (log BB)	−0.112	0.331
	VDss (human) (log L/kg)	0.641	0.365
	CNS permeability (log PS)	−1.535	−1.397
Metabolism	CYP2D6 substrate	No	No
	CYP3A4 substrate	Yes	Yes
	CYP1A2 inhibitor	Yes	Yes
	CYP2C19 inhibitor	No	Yes
	CYP2C9 inhibitor	No	Yes
	CYP2D6 inhibitor	Yes	No
	CYP3A4 inhibitor	No	No
Excretion	Total Clearance (log ml/min/kg)	1.246	0.294
	Renal OCT2 substrate	No	Yes
Toxicity	Toxicity class	3	2
	Predicted LD50 (mg/kg)	200	48
	Hepatotoxicity	Inactive	Inactive
	Nephrotoxicity	Inactive	Inactive
	Cardiotoxicity	Inactive	Inactive
	Clinical toxicity	Inactive	Active
	Nutritional toxicity	Inactive	Inactive

BBB: Blood-Brain Barrier; DZP: Diazepam; GI: Gastrointestinal; PME: Palmatine; TPSA: Topological Polar Surface Area; VDss: Volume of Distribution at Steady State

Discussion

The GABA transmitter is crucial to the advancement of contemporary sedation and anesthetic techniques²⁰. Because GABA is the brain’s fundamental inhibitory neurotransmitter. It works by preventing nerve transmission, which lowers neuronal excitability. GABA function ameliorates epilepsy, Parkinson’s disease, insomnia, Alzheimer’s disease, and stable mood ^{21 22}. Hypnotic medications, particularly benzodiazepines like DZP and barbiturates, are well-known for their sedative effects, which are mediated via amplifying GABA_A receptor activity²³. However, regular use of benzodiazepines produces patient tolerance (higher dosages are needed to achieve the same effect), leading to increasing mortality⁶. On the other hand, with long-term use of barbiturates, the body develops a dependency on barbiturates and produces withdrawal symptoms²⁴. As a result, currently available hypnotic medication drugs have limitations in terms of safety. So, there is a need for the discovery of safer alternatives that can effectively address insomnia and other sleep disorders without severe adverse effects.

Thiopental sodium reversibly depresses excitable tissue in the CNS, which was used to provide the sedative effect and induce sleeping time in model²⁵. Indeed, thiopental sodium, a barbiturate anesthetic, induces hypnosis by enhancing GABA inhibitory effects by modifying the GABA_A receptor, leading to increased neuronal activity inhibition²⁶. When thiopental sodium binds to the GABA receptor (part of the Cl^– channel), chloride channels stay open longer, which causes neurons to become hyperpolarized and decrease neuronal activity¹⁷. Any sedative and anxiolytic medication, such as DZP, which lengthened the duration of sleep and reduced the beginning of thiopental sodium-induced sleep, may interact with GABA_A receptor²⁷.

The findings demonstrate that PME exhibits a dose-dependent sedative effect, which aligns with previous reports that palmatine enhances serotonergic and GABAergic signaling to modulate sleep and relaxation¹³. The marked reduction in sleep latency and prolongation of sleep duration observed at higher doses suggest that PME acts through potentiation of the GABA_A receptor pathway, a mechanism shared by classical hypnotics such as benzodiazepines^3,5. However, when a combination of multiple effects is higher than the sum of their individual impacts, this phenomenon is referred to as synergism or synergistic effects²⁸. Notably, the synergistic interaction between PME and DZP highlights the possibility that PME may enhance benzodiazepine efficacy by facilitating chloride ion influx through GABA_A receptor channels, thereby amplifying inhibitory neurotransmission. This synergism is pharmacologically important because it suggests that PME could be used to lower the effective dose of DZP, potentially reducing the adverse effects and dependence risks commonly associated with long-term benzodiazepine use⁶. Thus, PME emerges as an independent sedative agent and also as a potential adjunct therapy in insomnia management.

Computer-assisted drug discovery and design (CADD), it is now feasible to test more chemicals in a shorter time with lower cost. These technologies reduce reliance on animal models and help with the creation of new compounds and the investigation and analysis of the characteristics of natural items²⁹. However, these have several limitations: molecular flexibility, validity and reliability, over-reliance on computational predictions and scoring functions, and algorithms³⁰. Despite their drawbacks, computational approaches are anticipated to be essential to drug discovery and may accelerate the development of life-saving treatments.

Molecular docking is a computer simulation technology used to calculate the binding affinity of drug-receptor complexes in structural molecular biology and drug design³¹. Binding affinity is a crucial metric for evaluating the strength of a small molecule’s binding to its target³². Higher binding affinity indicates a strong drug-receptor interaction, which is crucial for successful drug development and effective cell signaling³³. In this context, previous studies have shown that compounds capable of interacting with key residues in the GABA_A​ receptor can enhance receptor activity and produce sleep-promoting effects. The current observations suggest that PME may occupy the classical benzodiazepine binding site and interact with amino acid residues such as LEU A: 285, PHE A: 289, MET A: 286, PRO B: 233, MET B: 236, and LEU B: 232, which are known to be critical for ligand binding and receptor modulation. These interactions indicate that PME could mimic the effects of standard GABA_A​ modulators like DZP, supporting its potential as a sleep-enhancing agent. Nevertheless, while in silico predictions provide a valuable mechanistic hypothesis, further in vitro and in vivo studies are required to validate these interactions and elucidate the precise pharmacological mechanism of PME. The possible mechanism of sedative effects of PME is depicted in Fig. 4.

Fig. 4 [Images not available. See PDF.]

Proposed mechanism of the sedative effect of palmatine chloride (PME). [Following administration, PME binds to the GABA_A receptor (α1 and β2 subunits) on postsynaptic neurons. This binding is predicted to facilitate chloride ion (Cl⁻) influx through the receptor channel, causing neuronal hyperpolarization. The resulting reduction in neuronal excitability contributes to increased relaxation, delayed sleep onset, and prolonged sleep duration, as observed in vivo. The schematic integrates our docking predictions with the behavioral outcomes, highlighting a possible GABAergic mechanism of action.]

Drug-likeness important guidelines for early drug discovery include drug-likeness, safety, Lipinski’s rule of five, desirable ADMET properties, and oral absorption³⁴. Both compounds adhere to Lipinski’s rules with excellent bioavailability scores and outstanding solubility, indicating PME would be a promising drug candidate for oral bioavailability. Pharmacokinetics is the study of how the body interacts with administered drugs through ADME³⁵. According to the ADME analysis, PME and DZP manifest high absorption at 97.084% and 97.42%, indicating that the GIT effectively absorbs these substances into the bloodstream. When comparing the LD₅₀ of PME (200 mg/kg) and DZP (48 mg/kg) in a toxicity investigation, PME showed a greater lethal dose, suggesting it is less harmful at similar doses. Though, these LD₅₀ values were derived from computational prediction (ProTox 3.0) and therefore are not linked to a specific route of administration. Our docking results suggest that PME interacts with key residues within the GABA_A α1 and β2 subunits that overlap with the DZP-binding pocket, raising the possibility that PME modulates the same receptor site. This mechanistic hypothesis aligns with the observed in vivo sedative effects, where PME reduced sleep latency and prolonged sleep duration. Nevertheless, molecular docking is limited by rigid-receptor assumptions, reliance on scoring functions, and inability to capture dynamic conformational changes. Hence, while the computational evidence is supportive, experimental receptor-binding and electrophysiological studies are required to confirm PME’s precise mechanism of action.

Overall, our study presents several key findings regarding the sedative potential of PME. PME significantly reduced sleep latency and increased sleep duration in experimental animals, indicating strong sedative effects, and further exhibited a synergistic interaction when administered with DZP, although definitive confirmation through isobolographic analysis is needed. While the thiopental sodium-induced sleep test provided preliminary evidence, reliance on this single model limits the conclusiveness of our behavioral findings, and more comprehensive assays, such as the open field test and other locomotor activity paradigms, are warranted to robustly validate PME’s sedative activity. In silico analyses suggested that these effects may result from interactions with specific GABA_A receptor amino acid residues, and computational pharmacokinetic and toxicity predictions indicated a favorable safety and efficacy profile compared to standard drugs. Nevertheless, these pharmacokinetic and toxicity assessments were purely predictive, generated using SwissADME, pkCSM, and ProTox 3.0 servers, which provide hypothesis-generating insights rather than confirmatory evidence, emphasizing that experimental validation of in vivo pharmacokinetics, metabolism, long-term toxicity, and overall safety is essential before clinical translation.

Conclusion

In vivo experiments confirmed that PME exerts significant sedative effects in thiopental sodium-induced sleeping chicks, as evidenced by a dose-dependent reduction in sleep latency and an increase in sleep duration. Importantly, the combination of PME with DZP produced a synergistic effect, suggesting that PME could enhance the efficacy of benzodiazepines while potentially reducing their required dose and associated adverse effects. Complementary in silico analyses provided mechanistic support for these findings by showing favorable interactions of PME with GABA_A​ receptor subunits, consistent with modulation of the GABAergic pathway. Additionally, PME displayed satisfactory drug-likeness, pharmacokinetic, and safety profiles, reinforcing its potential as a drug-like candidate. Taken together, the results provide initial evidence that PME exerts sedative effects alone but may also enhance DZP efficacy, suggesting potential clinical utility as an adjunct therapy for insomnia or anxiety disorders. Such an approach could allow dose reduction of benzodiazepines, thereby mitigating risks of dependence and adverse effects.

Acknowledgements

AcknowledgmentsAuthors extend their appreciation to the University Higher Education Fund for funding this research work under the Research Support Program for Central Labs at King Khalid University through the project number CL/PRI/B/7.

Author contributions

Author ContributionsAll authors including Feroz Khan Nun, Mohammad Y. Alshahrani, Md. Sakib Al Hasan, Emon Mia, Na’il Saleh, Mashael A Almansoori, Mehedi Hasan Bappi, and Muhammad Torequl Islam made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas, that is, revising or critically reviewing the article; giving final approval of the version to be published; agreeing on the journal to which the article has been submitted; and confirming to be accountable for all aspects of the work. All authors have read and agreed to the published version of the manuscript.

Funding

University Higher Education Fund number CL/PRI/B/7.

Data availability

The data that support the findings of this study are available from the corresponding author, Md. Sakib Al Hasan (email: [email protected]), upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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