Hydroxycitronellal (HC) is a monoterpene present in essential oils of aromatic plants of different species, obtained from semisynthesis of citronellal, and is widely used as a fragrance in cosmetics. The objective of this work was to evaluate the possible anxiolytic-like activity of HC and its possible mechanism of action using
Anxious behavior is part of the normal spectrum of human experience. In certain situations, all people in life experience anxiety, especially when faced with some warning of imminent danger. This reaction allows us to escape from dangerous situations, as well as to improve our response in the face of adverse conditions when imposed by the environment [
Inherited genetic, neurobiological, environmental, and traumatic experiences can trigger pathologically anxious behavior. Anxious disorders are a clinical condition in which anxiety is the primary symptom and does not originate in other disorders, such as depression [
Pathological anxiety is treated with anxiolytic agents, which are substances that act on the central nervous system (CNS), resulting in changes in behavior, perception, thoughts, and emotions. They are recommended for people who suffer from emotional and psychological disorders that affect the functioning of the mind [
Benzodiazepines (BZDs) are a class of substances presenting anxiolytic effects and, due to their wide therapeutic window, high therapeutic efficacy. These drugs act through GABAA ionotropic receptors, intensifying inhibitory neurotransmission. Clinically, diazepam (DZP) is one of the most frequently used BZDs, and (similar to other BZDs) sedation is one of its more evident adverse reactions; it directly affects locomotor activity and compromises the quality of life of the user [
Alternatives are needed to develop anxiolytics which possess fewer adverse reactions, possibly using medicinal plants, since they are an excellent source of bioactive substances which can present a great influence on health [
Essential oils (EO) are odorous substances [
Hydroxycitronellal (HC) is a monoterpene present in essential oils of the most diverse genera of aromatic plants, yet little reported in the literature regarding its pharmacological effects. This monoterpene can be obtained from another monoterpene and citronellal, a metabolite of plant origin that already has several pharmacological activities proven in the literature, such as analgesic [
Swiss mice (
HC was purchased from Sigma-Aldrich® ChemicalCo (St. Louis, MO, USA). Diazepam and flumazenil were donated by the Brazilian pharmaceutical laboratory, Cristália-Produtos Químicos Farmacêuticos, LTDA (Itapira, SP, BR). All substances were diluted in saline and intraperitoneally administered (i.p.) at a total volume of 0.1 mL/10 g. HC was initially emulsified with Tween 80 (0.5%) in 0.9% saline. The control group received the vehicle (Tween 80-0.5% in 0.9% saline).
The acute toxicity tests were performed according to the OECD “Guidelines for testing of chemicals” n. 423/2001with modifications. Swiss mice, three females per group, including the control, were subjected to single doses of 2000 and 300 mg/kg of HC. The control group was treated with
The rota rod methodology assesses motor impairment in animals after administration of substances with potential CNS activity. The animals were exposed to the apparatus 24 h before the test, and the animals managing to stay on the rotating bar (at 10 rpm) for a period of 1 minute were selected for testing. These preselected animals (
Groups of mice were used (
The animals were submitted to the open field test and evaluated for a period of 5 minutes. For the experimental procedure, groups of 8 mice were used. HC (12.5, 25, 50 mg/kg), vehicle (control group: Tween 80-0.5%), and diazepam (1 mg/kg) were administered intraperitoneally, 30 min before the start of the test. After treatment (30 minutes), each animal was individually submitted to the device. The total number of quadrants covered (crossings), head rearing, and time spent on grooming was recorded [
This test was carried out with the aim of evaluating the possible involvement of GABAA receptors in the anxiolytic effect of the substance. The animals were divided into three pretreated groups (
The HC structure was obtained in (.sdf) format through ChemAxom (
Docking was performed using five different scoring functions (Molegro Virtual Docker (MVD), Gold 5.6.2, AutoDockVina (Vina), AutoDock 4.2.6. (AD4), and Plants). The water molecules were removed from the crystalline structure, and the mean square root deviation (from the “Root Mean Square Deviation” (RMSD)) of the poses was calculated, indicating the degree of docking reliability. The consensus strategy consisted of selecting predicted binding affinity values greater than the crystallographic ligands in at least three docking programs. The results were viewed using the Molegro Virtual Docker v.6.0.1 (MVD) software [
The GROMACS 5.0 software was used to perform molecular dynamics simulations [
Pharmacokinetic predictions (available for free) were obtained from electronic platforms: SwissADME (Swiss Institute of Bioinformatics, Switzerland) and Xenosite (Washington University of Medicine, United States). For predictive models interfacing, it was necessary that the structures were in the smile format, being generated by the Chemdraw Ultra 12.0 software [
The results obtained in the
With administration of HC at a dose of 2000 mg/kg, i.p., all animals died, making it necessary to decrease the dosage, as determined by the OECD (2001). HC was then administered at a dose of 300 mg/kg, i.p., and did not cause the animals to die. In view of the results, it was the estimated LD50 of the tested substance (HC) at 500 mg/kg. Using this result, to conduct pharmacological tests
After administration of HC (i.p.) at doses of 12.5, 25, and 50 mg/kg, there were no significant differences in the length of stay of the animals on the rotating bar (tested after 30, 60, and 120 minutes) in relation to the control group (Figure
Effect of HC (mg/kg, i.p.), the time of stay (s) of the animals on the rota rod. The column represents the
The results presented in Figure
Effect of HC (mg/kg, i.p.) and diazepam, number of entries (a) (
The results obtained for the animals’ permanence in the open arms are shown in Figure
Figure
As for the length of stay in the closed arms, the animals treated with HC at doses of 12.5 (
As to the animal ambulation behavior in the open field tests, the results are shown in Figure
Effect of HC (mg/kg, i.p.), number of crossings (a) (
Figure
The results obtained for the grooming parameter can be seen in Figure
The results of Figure
Influenced the HC after using flumazenil in the number of entries (a) (
Figure
In this study, the results generated using the five scoring functions were validated through redocking of the crystallographic ligand. The RMSD of the positions obtained was calculated. Redocking consists of positioning and predicting the binding affinity of the crystallographic ligand in the region of the active site receiver. The RMSD compares and calculates the mean square root deviation of the positions obtained by redocking, and the ligand structure obtained experimentally. For docking to be considered reliable, the RMSD value must be equal to or less than 2.0 Å.
The programs MVD, Vina, and AD4 select the best binders for the most negative energy values, while the Gold and Plant programs select the best binders for the most positive energy values. HC obtained a higher binding affinity value than the crystallographic ligand (ABU), a cryo-EM structure of the
Results of the binding energy values (kcal/mol) in five different scoring functions.
Molecule | Moldock (MVD) | Goldscore (Gold) | Binding energy (AutoDock Vina) | Binding energy (AutoDock) | Chempl (Plants) |
---|---|---|---|---|---|
Gamma-aminobutyric acid (GABAA) | |||||
HC | -75.40 | 29.52 | -4.8 | -5.38 | 37.94 |
PDB ABU | -88.65 | 26.22 | -5.7 | -4.95 | 20.96 |
Figure
Visualization of interactions 2D and 3D between the binders HC and ABU and the target GABAA. Complex GABAA-HC (a). 3D and 2D interactions between the ligand HC and GABAA amino acids (b, c). 3D and 2D interactions between the ABU ligand and amino acids GABAA (d, e). In green, the interactions of hydrogens are represented; in orange, electrostatic interactions; in red, steric interactions; and in purple, hydrophobic interactions.
The molecular dynamics (MD) simulations were performed taking into account the best results obtained in the consensus docking. The interactions between GABAAR and HC were evaluated, and the crystallographic ligand (ABU) was used to study the flexibility and conformational changes in the complexes during the MD simulations. For this, the mean quadratic deviation (RMSD) was separately calculated for the C
Values of the molecular dynamics. (a) RMSD of C
RMSD analysis of the ligands revealed that HC is more stable than ABU (Figure
To understand the flexibility of the residues and amino acids that contribute to GABAAR conformational changes, the mean square root fluctuation (RMSF) of each receptor amino acid was calculated. Residues with higher RMSF values suggest greater flexibility, while low RMSF values suggest less flexibility. Considering that amino acids with fluctuations above 0.3 nm contribute to the flexibility of the protein structure, we found that of the E and D chain amino acids that interacted with the agonist ligand ABU, not one contributed to a conformational change in the protein (Figure
The docking interactions observed for the GABAAR-HC complex were stable during the molecular dynamics simulations. When analyzed using graphics programs, it was noted that HC loses interactions with the amino acids observed in docking (Figure
Molecular dynamics simulation during 1000 ps. Structure of the GABAAR-HC complex and its active site (a), initial structure (b), 200 ps (c), 400 ps (d), 600 ps (e), 800 ps (f), and 1000 ps (g).
The properties of medicines obtained in the ADMET system are the principal characteristics evaluated during the development phase of a future medicine. In new drug development, the ADMET forecast provides guidance that minimizes failure rates [
Compound | TPSA (Å2)c | Caco-2 permeability (log Papp; log cm/s)d | Int. Abs. (%)e | VDss (log L/kg)f | Fract. Unb.g | Total clearance (log mL/min/kg)h | ||
---|---|---|---|---|---|---|---|---|
HC | 2.15 | -2.04 | 37.30 | 1.49 | 93.84 | -0.04 | 0.50 | 1.36 |
aSwissADME Moriguchi log of octanol-water partition coefficient. bSwissADME Ali log of aqueous solubility. cSwissADME calculation of Topological Polar Surface Area (TPSA). dpkCSM prediction of Caco-2 cell permeability as estimation of absorption in human intestinal mucosa. epkCSM prediction of the proportion of compound absorption thought the human small intestine.fpkCSM prediction of the log of steady state volume of distribution (VDss). gpkCSM prediction of compound fraction unbound in plasma (not bound to serum proteins). hpkCSM prediction of the log of total drug clearance.
HC presented a
Molecules that present TPSA (Topological Polar Surface Area) values of less than 140 Å2 and values above 0.90 (log Papp; log cm/s), for the Caco-2 parameter, suggest that the substance presents a good profile for intestinal absorption. Such
The volume of distribution (VDss) indicates the theoretical volume of a given substance evenly distributed in the blood plasma. Values below 0.45 (log L/kg) indicate a good plasma distribution, with the substance being readily available to interact with its biological target. Elimination of the substance did not increase because of its free form, as was observed for renal clearance (clearance/mL/min/kg). The greater the total clearance, the greater and faster the elimination of the substance will be [
According to a preliminary study, the oral LD50 of 7-HC was 5000 mg/kg [
In the initial toxicity test, during the 14-day observation period, after the administration of 300 mg/kg, i.p., no changes were found that would indicate toxicity, such as changes in the skin, hair, eyes, or respiratory system. The data demonstrated that under the evaluated conditions, HC presents low toxicity, providing an incentive to conduct
The behavioral changes induced upon HC administration (i.p.) were evaluated using the rotating bar (rota rod), elevated plus maze, and open field tests; these tests are well established in the study of substances with central nervous system activity, and except for the rotating bar test, they are models based on the observation of innate animal behavior, that is, on the ethology of rodents.
Rota rod testing is a classic animal model used to assess the influence of unknown substances on an animal’s motor coordination [
To better evaluate the possible HC anxiolytic effects, the animals were subjected to the EPM test, which is considered one of the most well-established tests and the reference standard for detecting anxiolytic and anxiogenic effects [
The open field test is widely used to assess behavioral effects of compound administration, especially in locomotor and anxious parameters [
Finally, to assess whether the effects observed with HC involve interaction with the GABAergic system, the benzodiazepine GABAA receptor antagonist flumazenil was used. The antagonist was administered before the substances under study. If the pharmacological effect was reversed or even decreased significantly, it would mean that the antagonist, when binding to the target, prevented the substance from binding, and consequently, the pathway was involved in the pharmacological effect [
Pretreatment with the antagonist flumazenil reversed the anxiolytic-like effect of HC, as well as DZP, suggesting that HC is capable of reducing anxiety through a mechanism of action similar to that of BZDs (GABAAR). Interactions between GABAAR and other essential oils as well as their isolated compounds, such as citronellal, have been shown to result in CNS depressant activity [
The positive modulation of the GABAA receptor by the enantiomer of the bicyclic monoterpene borneol, found in essential oils of medicinal herbs, such as valerian, was observed using electrophysiology models. Borneol directly activated GABAA receptors, producing 89% and 84%, respectively, of the maximum GABA response indicative of a partial agonist action, being insensitive to flumazenil, indicating that (+)- and (-)-borneol did not act in the classic sites of benzodiazepines [
As discussed by Jiménez-Ferrer et al. [
According to the position of Santos et al. [
Substances that bind to GABAAR, such as benzodiazepines, cause a change in the conformational state of the receptor, which promotes increased affinity for the GABA neurotransmitter, stimulating the opening of GABAAR channels [
According to the data, the interaction of HC with GABAAR can result in greater neuronal hyperpolarization. After HC-GABAAR receptor binding, a greater influx of Cl- into the cell is suggested, regardless of GABA binding. Accumulation of chloride ions inside of the cell increases the threshold of neuronal excitation and prevents nerve impulse conduction [
Using
When compared with the amino acids involved in the BZD-GABAAR interaction, the interaction of HC with GABAAR (through different amino acids) may be responsible for the observed nonimpairment of the test animals’ motor coordination. This revealed the potential of HC for use in formulating a new anxiolytic medication, which might allow patients to perform their daily functions normally, without the risk of accidents.
The hydrogen interactions between the amino acid residues of GABAAR and HC, compared to the ABU crystallographic ligand, presented greater affinity. The interactions between an enzyme and its substrate are complementary and were initially introduced in the famous “key-lock” model, proposed by Fischer [
Previous molecular docking studies between diazepam (DZP) and GABAARs [
Molecular dynamics is important since it assesses structural differences between molecules, as was done for the HC and ABU agonists. The higher the RMSD value, the greater the structural difference between the proteins or structures were compared, as was observed for the HC ligand [
In this study, low RMSD values (>0.3 nm) were observed for the majority of the simulation time, indicating greater HC stability in the system compared to the crystallographic ligand (ABU). Sudden changes in RMSD denote important conformational changes in the molecule, while small RMSD oscillations indicate system equilibrium [
Through the molecular dynamics (MD) results, more stable HC-GABAAR bonding was observed. This stability may be related to the hydrogens of the CHO (aldehyde) functional groups, which can participate in nonclassical hydrogen bonds with certain amino acid residues, and such interactions with GABAAR amino acids can result in chloride ion influx into the cell, decreasing neuronal excitation.
To establish an adequate pharmacokinetic profile for HC, absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters were also evaluated. Absorption and distribution directly influence the bioavailability of the substance, while metabolism and excretion affect the rate of elimination of the substance from the body. Proper disposal of substances decreases the chances of toxic events being triggered [
To present a good bioavailability, a drug must obey at least three of the following parameters: molecular weight less than or equal to 500 Daltons (Da), high lipophilicity (
HC was administered intraperitoneally in
Based on the data obtained in the present work, it is possible to conclude that hydroxycitronellal presents anxiolytic-like effect, which likely occurs through positive modulation of GABAA receptors. Ruling out HC neurotoxicity, the compound’s anxiolytic-like activity did not present adverse effects, such as sedation and neuromuscular impairment commonly observed in benzodiazepines. These findings reinforce the pharmacological potential of the monoterpene citronellal and its derivatives, such as hydroxycitronellal.
Some or all data, models, or code generated or used during the study are available from the corresponding author by request.
The authors declare no conflict of interest.
This study was supported by funds from the Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council for Scientific and Technological Development (CNPq).