In the CNS, GABA is the most important inhibitory neurotransmitter. Many medications employed for clinical sedation have the GABA receptor system as their major pharmacological target (Saari et al., 2011). BDZs are also widely employed as CNS depressants. These function via the post-synaptic GABAA receptor interacting route (Ticku et al., 1983). The effectiveness of GABAA receptors appears to decline with prolonged high-dose BDZ usage; this is most likely an accumulation mechanism (Rosenbaum et al., 2007). Low levels of GABA consistently stimulate it, facilitating long-lasting tonic inhibition. Since extrasynaptic GABAA receptors that produce tonic resistance are thought to be extremely susceptible to anesthetics, new research suggests that general anesthetics could be able to distinguish between synaptic as well as tonic GABAA receptors (Miller, 1991). The development of novel, secure, and efficient sedative medications is thus an urgent problem.
The TS-induced sleeping procedure is frequently employed to examine the hypnotic or sedative effects of different test drugs associated with GABAA receptors. When TS binds to GABAA barbiturate sites, the postsynaptic membrane becomes hyperpolarized (Bappi et al., 2024). By acting as a stimulant on GABAA receptors and causing an influx of chloride ions (Cl−) into the cell, the BDZ medication of TS induces sedative activity (Mula, 2016). Therefore, drugs that cause TS in animals may operate on GABAA receptors to reduce the latency period and enhance sleeping time (Mukty et al., 2024). Consequently, the test samples' ability to shorten latency periods and lengthen sleeping time provides evidence of their sedative effects in animals.
According to the in vivo result, we have found that CAF10 significantly (p < 0.05) increased the onset of sleep, which is regarded as a sedative-like activity. In contrast, LIN50 and SCL10 significantly (p < 0.05) reduced the onset of sleep in animals compared to the CAF10 group animal. CAF10 combined with LIN50 and SCL10 significantly (p < 0.05) increased the onset of sleep. An earlier report suggests that SCL glycol has anxiogenic effects in Swiss mice (Georgieva, 1990). The combination group (LIN50 + SCL10 + CAF10) exhibited the lowest latency period compared to all groups, rather than the control group. LIN50 represented a significant (p < 0.05) sleep duration, which was the highest value observed among all groups. SCL10 and LIN50 + CAF10 showed almost similar sleep duration in animals. Conversely, the SCL10 + CAF10 group showed the lowest sleep durations in animals. The triple combination group (LIN50 + SCL10 + CAF10) exhibited the highest sleep duration, compared to the CAF10 and SCL10 + CAF10 groups, but was lower than the other groups. It might be because of the sedative-like yet antidepressant-like effects that have been seen in earlier research (Milanos et al., 2017; Linck et al., 2010), indicating the potential for managing sleep problems and their related side effects, such as anxiety (Hassan et al., 2024) and depression (Lee et al., 2018). The possible mechanism of sedation or its modulatory effects of CAF, SCL, and LIN in comparison to DZP is depicted in Fig. 4.
The in silico analysis reveals that SCL exhibited the strongest binding affinity (‒7.4 kcal/mol) with the GABAA receptor (PDB ID: 6X3X), primarily through pi-sigma and alkyl interactions, despite the absence of hydrogen bonds, indicating its potential as a potent natural modulator. DZP, a well-known benzodiazepine, followed closely with a binding affinity of ‒6.9 kcal/mol, corroborating its established efficacy in enhancing GABAA receptor activity. In drug discovery, HBs enhance the specificity and stability of drug-target interactions, while high binding affinity ensures potent and effective modulation of the target at lower doses, crucial for therapeutic efficacy and safety (Hopkins et al., 2014; Ma et al., 2021). Moreover, CAF and LIN displayed lower binding affinities (‒5.7 kcal/mol and ‒4.7 kcal/mol, respectively), suggesting a less pronounced modulatory effect, though LIN’s sedative properties might still be significant when used in higher concentrations or in combination with other compounds. These results align with existing literature, reinforcing SCL’s potential as a natural alternative to synthetic anxiolytics like DZP, while the roles of CAF and LIN may be more supportive or synergistic in nature. However, the possible mechanism of sedation or the modulatory effects of caffeine, linalool, and sclareol are depicted in Fig. 4.
Drug-likeness is a crucial factor to take into account when selecting substances in the early phases of drug discovery to maximize the likelihood that a chemical will be effective and able to be used in clinical studies (Deore et al., 2019). Drug-likeness is established by examining the compounds' chemical structures and physicochemical qualities. It is commonly used to describe the pharmacokinetics (PK) and efficacy of drugs. It may also be considered a substance with acceptable absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties (Alam et al., 2016). Lipinski's rule of five (RO5) is a commonly used technique for evaluating the drug-like properties and pharmacokinetics of various compounds. RO5 stipulates that a drug candidate should possess a molecular weight of 500 g/mol or lower, not more than five HB donors, not more than ten HB acceptors, and a lipophilicity (LogPo/w) of five or below (Lipinski, 2004). The current investigation demonstrated that CAF, LIN, SCL, and DZP exhibited excellent pharmacokinetic properties and adhered to Lipinski's criteria for drug-like molecules. CAF is very soluble, whereas SCL is moderately soluble; in contrast, LIN and DZP are soluble in nature, suggesting that they can be effectively absorbed by oral administration.
Toxicological screening is critical in ensuring the safety and efficiency of molecules in the formulation of new pharmaceuticals and increasing the therapeutic potential of present substances (Szymański et al., 2011). Toxicity testing is also used to determine the level at which no adverse effects are seen. This information is valuable for conducting clinical research (Parasuraman, 2011). The results of this investigation show that CAF, LIN, and SCL do not possess any hepatotoxicity, immunotoxicity, mutagenicity, cytotoxicity, or carcinogenicity activities. Furthermore, the CAF, LIN, and SCL show a larger fatal dosage (127, 2200, and 5000 mg/kg) than the referral medication 48 mg/kg when comparing their lethal dose (LD50), suggesting that they are less hazardous at a comparable dose as DZP. However, cytotoxicity was one of the adverse effects of DZP. Since our computer analysis could not identify any harmful probability. In current research, we use the web resources SwissADME, pkCSM, and ProTox-3.0 for predicting the drug-like qualities and ADMET features of CAF, LIN, and SCL. Table 4 shows that all computed parameters for CAF, LIN, and SCL (drug-like characteristics and ADMET profile) stayed within permissible ranges.
Taken together, from the in vivo investigation, CAF and SCL reduced the sedative effects of LIN. The in silico study of CAF, LIN, and SCL showed binding affinities (‒5.7, ‒4.7, ‒7.4 kcal/mol) with the α1 and β2 subunits of the GABAA receptor. Additionally, it has acceptable ADME and toxicity criteria. There are several limits in our evaluation, indicating that all mice's sleep cycles are not comparable, BDZ sometimes does not function when altering the physiology of animals, and we couldn't offer therapy to all mice at the exact time, which may impair the findings.