An <em>In-Vivo</em> Study on Anticonvulsant, Anxiolytic, and Sedative-Hypnotic Effects of the Polyphenol-Rich <em>Thymus Kotschyanus</em> Extract; Evidence for the Involvement of GABA<sub>A</sub> Receptors
Abstract
Antidepressant-like activity of T. kotschyanus has been recently reported by scientists but insufficient attention has been so far devoted to T. kotschyanus, and there is a lack of information on the other neurobehavioral effects and side effects of this species. In the current study, the anticonvulsant, anxiolytic, and sedative-hypnotic, effects of Thymus kotschyanus extract on male NMRI mice were evaluated using pentylenetetrazole, maximal electroshock, elevated plus maze, and pentobarbital-induced sleeping tests. Since phenolic compounds and flavonoids have main roles in pharmacological effects of most plant extracts, the phenolic and flavonoid contents of the extract were measured with Folin-Ciocalteu and AlCl3 reagents. Acute toxicity, passive avoidance, and open field tests were carried out to assess the toxicity of the extract. To find out the possible mechanism of action, flumazenil as the specific GABAA receptor antagonist was used. Anticonvulsant and hypnotic effects of the extract were observed at 400 and 600 mg/kg. The extract at the dose of 200 mg/kg revealed significant anxiolytic effects, but it did not show any adverse effects on learning and memory at all the tested doses. Results of this study indicate that Thymus kotschyanus extract has anticonvulsant, anxiolytic, and hypnotic effects, which are likely related to the ability of some phenolic compounds to activate α1-containing GABAA receptors but more experiments still need to be carried out in order to find the exact mechanism, active component, and the toxicity of the Thymus kotschyanus extract.
Introduction
Central nervous system impairment causes both mental and behavior disorders. Epilepsy, unipolar depressive disorders, bipolar affective disorder, schizophrenia, post-traumatic stress disorder, obsessive and compulsive disorder, panic disorder, primary insomnia, Alzheimer’s and other dementias are some of the neuropsychiatric conditions. Mental disorders are not exclusive to a special group of people; they can be found in women and men, different stage of the life, rich and poor people, and among people who live either in urban or rural areas (1). Approximately, 1 in 5 adults in the U.S. (44.7 million, or 18.3%) experiences mental illness in a given year, and almost 1 in 25 adults in the U.S. ( 10.4 million, or 4.2%) experiences a serious mental illness in a given year that substantially interferes with or limits one or more major life activities. Recent studies have revealed this fact that psychiatric symptoms instead of being effects of a common cause probably can cause each other. On the other side, these disorders can lead to other health problems and diseases. For example, insomnia can cause fatigue, feelings of guilt, loss of interest or concentration problems, or anxiety can lead to insomnia. Subsequently, the society will face to a more increased burden of disease compared to a spectacular disease (2). Obviously, economic costs to society and the impacts on the quality of lives are the main burdens of mental disorders (3). Therefore, mental disorders have been considered as a growing public health concern and a major social and economic issue. These problems can affect individuals, families, and societies all over the world (4).
Herbal medicines have been used for health and medical purposes for several thousands of years because of their lower toxicity and potent ingredients (5). Thymus kotschyanus belonging to the Thymus genus and Lamiaceae family is originated from the Mediterranean area (6). Several biological and pharmacological properties such as antibacterial, antifungal (7), antiviral (8), anti-helminthic (9), anti-oxidative (7, 10), antispasmodic (11), and sedative (12) effects have been reported for genus Thymus in the literature. Antidepressant-like activity of T. kotschyanus has been recently reported by researchers (13) but insufficient attention has been so far devoted to T. kotschyanus, and there is a lack of information on the other neurobehavioral effects of this species. Therefore, the aim of this study is to evaluate anticonvulsant, anxiolytic, and sedative-hypnotic effects, possible mechanism of action and the toxicity of the Thymus kotschyanus extract.
Experimental
Plants and extraction
Thymus kotschyanus was obtained from Tabriz province of Iran, and it was authenticated in the Department of Pharmacognosy, School of Pharmacy, Shahid Beheshti University of Medical Sciences (Tehran, Iran). The aerial part of the plant was dried at the room temperature and crushed into a very fine powder by a grinder. The methanolic extract of T. kotschyanus was obtained by maceration of the plant powder in 900 mL of methanol 96% on a shaker (Stuart SSL1 shaker, UK) at the room temperature. This procedure was repeated for three consecutive days. Following filtering the solution by a paper filter, the filtrate was concentrated in a rotary evaporator (Heidolph, Germany). The extraction yield (mass of extract/mass of dry matter ×100) was calculated 3.5% w/w.
Drugs and treatments
The extract, pentylenetetrazole (Sigma-Aldrich, St. Louis, MO, USA ), pentobarbital (Sigma-Aldrich, St. Louis, MO, USA ) and midazolam (Darou Pakhsh Pharmaceutical Mfg.co, Iran) were dissolved or suspended in normal saline and were administered with the injection volume of 10 mL/kg while diazepam (Sigma-Aldrich, St. Louis, MO, USA) and flumazenil (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in DMSO 50%. Since the DMSO has some effects on the central nervous system by itself, the injection volume for diazepam and flumazenil was 5 mL/kg. All the drugs used in the present study were injected intraperitoneally (i.p.).
Animals
Male NMRI mice (weighed 18 to 25 g) were used in all experiments. Animals from the Animal House of Shahid Beheshti University of Medical Sciences were caged in groups of ten at a controlled temperature of 22 ± 2 ºC, with free access to water and food. Animal handling in order to adaptation to the laboratory environment was done for three consecutive days before each experiment. All the procedures were conducted according to the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals.
Total phenolic content
Total phenols were determined by colorimetric method using the Folin-Ciocalteu reagent (14, 15). To prepare a linear calibration curve, 1 mL of gallic acid standards at different concentrations (25, 50, 75, 100, 150, and 200 µg/mL; in methanol) were mixed with 5 ml of Folin–Ciocalteu reagent (diluted 1/10 with water). Five minutes later, 4 mL of aqueous Na2CO3 (75 mg/mL) was added, and the mixture was incubated at room temperature for 30 min. The same procedure was carried out on Thymus kotschyanus extract at the concentration of 400µg/mL, and the absorbance was measured at 765 nm using a Shimadzu UV-1601PC UV-Visible spectrophotometer. This procedure was repeated in triplicate.
Total flavonoid content
Flavonoid content was determined using Aluminum chloride reagent (14, 15). To prepare the rutin calibration curve, 2.5 mL of rutin solution at different concentrations (25, 50, 75, 100, and 150 µg/mL; in methanol) was thoroughly mixed with AlCl3 reagent (20 mg/mL). The same procedure was carried out on 400 µg/mL of Thymus kotschyanus extract. Following 40 min incubation at room temperature, the absorbance was measured at 415 nm. This procedure was repeated in triplicate.
Acute toxicity study
Mice in 4 groups were treated with different doses of the extract and were observed within 72 h after each treatment. The maximum non-fatal dose and median lethal dose (LD50) were assumed as the dose that had not induced any mortality and the dose that caused 50% of death, respectively.
Pentylenetetrazole and Maximal electroshock tests
Pentylenetetrazole (PTZ) and Maximal electroshock (MES) models were used to test the anticonvulsant activity of the extract (16, 17). Mice were treated with different doses of the extract (50, 200, 400 and 600 mg/kg body weight) 30 min before induction of seizure in PTZ and MES tests. The ability of the extract to protect mice against the lethal dose of PTZ (100 mg/kg body weight) within 30 min in PTZ test and to reduce the number of hind limb tonic extension (HLTE) in mice following application of electroshock (10 Hz, 37.2 mA and 0.3 s) through the ear electrodes in MES test were assumed as the anticonvulsant activity of the extract. Diazepam (0.25, 0.5, 1 and 2 mg/kg body weight) and normal saline were used as the positive control and vehicle, respectively.
Elevated plus maze test
The elevated plus maze (EPM) apparatus is made out of the black plexiglass and consist of two open arms (length 50 cm, width 10 cm) and two closed arms (length 50 cm, width 10 cm, and height 10 cm) extending from a central platform (10 cm × 10 cm). The plus-maze was located at a height of 50 cm above the floor level. The mice were given different doses of the extract (50, 200, 400 and 600 mg/kg body weight), vehicle and midazolam (2 mg/kg body weight) intraperitoneally and 30 min before their placement on the central platform of the EPM apparatus, facing toward one of the closed arms. Animal’s movement was recorded within 10 minutes, using a digital camera placed above the EPM apparatus. All recorded videos were analyzed by Ethovision XT (Noldus, The Netherlands) software, and the percentage of the spent time in the open arms was measured. Between each trial, the maze environment was completely cleaned by 70 % ethanol (18).
Pentobarbital-induced sleep test
This experiment was performed in 7 groups. Four groups received the T. kotschyanus extract at the doses of 50, 200, 400, and 600 mg/kg body weight, while the other groups received normal saline, diazepam (1 mg/kg body weight) and 600 mg/kg of the extract in combination with flumazenil (10 mg/kg). Thirty minutes afterward, pentobarbital sodium (40 mg/kg, body weight) was administered to each mouse for sleep induction, and the duration between loss and recovery of righting reflex (duration of sleeping) was recorded. All the experiments were performed at the same time every day to avoid the effect of circadian rhythm on the behavior of animals (19).
Step-through passive avoidance test
In this test, the animals learned to avoid an aversive stimulus, represented by a mild foot shock. Step-through passive avoidance test was conducted in a two-compartment (20 × 20 × 20 cm each) apparatus, where one was a dark compartment and preferable to mice, and the other one was a bright compartment. The compartments were divided by a partition which had a sliding door at floor level.
This test was conducted in two consecutive days. On the first day, the mice were treated with the extract at different doses (50, 200, 400 and 600 mg/kg body weight), vehicle and midazolam (1 mg/kg body weight). Thirty minutes later, each mouse was located in the bright compartment, facing away from the sliding door, and was allowed to explore. After 30 seconds, the sliding door was removed and when the mouse entered the dark compartment a foot shock (25 V, 0.5 mA, 2 s) was immediately delivered to the grid floor of the dark room. On the second day, this experiment was repeated while the sliding door was open and the latency to enter the dark compartment was recorded (16, 20).
Open field test
The open field apparatus is a cubic chamber made out of transparent Plexiglas walls (40 cm × 40 cm × 40 cm) with a digital camera placed above of it which can record the locomotion of mice. Thirty minutes and 24 h after administration of the extract, vehicle, and diazepam (2 mg/kg, body weight) each mouse was placed in the open field area for ten minutes. All recorded videos were analysed by an automated tracking system (Ethovision XT software, Noldus, The Netherlands). Ethanol (70%) and water were used to clean the open field area after every test (19).
Statistical analysis
Linear regression method was used for the creation of the calibration curves. Probit-regression method (SPSS software, Chicago, IL; version 17.0) was used to calculate ED50 of the extract. The ED50 value is presented as mean with 95% confidence interval. One way analysis of variance and the Tukey post-test (Graph Pad Prism software, San Diego, CA; version 5.0) were used to compare differences between different groups. In all tests, p < 0.05 was considered as the statistically significant difference.
Results
Acute toxicity study
Following administration of the extract, the maximum non-fatal dose and median lethal dose (LD50) were 1 g/kg and 5683.8 (4763.1- 7243.9) mg/kg, respectively.
Total phenolic and flavonoid contents
As shown in Table 1, total phenolic and flavonoid contents were calculated by using the obtained calibration curves. Results are presented as µg of gallic acid and rutin equivalents in mg of the dry matter of the extract for total phenolic and flavonoid contents, respectively.
Table 1
Total phenolic and flavonoid contents of Thymus kotschyanus
| calibration curves | r2 | Contents (µg/mg) 1 | |
|---|---|---|---|
| Phenolic content determination | y = 0.0028x + 0.0512 | 0.996 | 225.72 ± 12.03 |
| Flavonoid content determination | y = 0.0017x + 0.0006 | 0. 994 | 77.06 ± 4.78 |
1 Presented as µg of gallic acid and rutin equivalents in mg of the dry matter of the extract for total phenolic and flavonoid contents,
respectively. Values are expressed as mean ± S.E.M (n = 3).
Anticonvulsant activity of the extract
Pentylenetetrazole-induced seizures and maximal electroshock tests were conducted to determine the anticonvulsant activity of the extract.
The extract of T. kotschyanus showed anticonvulsant activity by protection against PTZ and reduction of HLTE numbers in PTZ and MES tests. The ED50 values of the extract and diazepam are presented in Table 2.
Table 2
The ED50 of diazepam and the extract of T. kotschyanus in PTZ and MES models
| ED50 (mg/kg) with (95% confidence interval) | ||
|---|---|---|
| PTZ | MES | |
| Diazepam | 0.95 (0.64-1.31) | 1.45 (0.79 - 1.76) |
| Thymus kotschyanus | 424.795 (322.854-543.526) | 345.395 (259.307-436.146) |
Anxiolytic activity of the extract
The anxiolytic activity of the extract was evaluated in the elevated plus maze and open field tests (OFT). In the EPM test, the extract of T. kotschyanus at doses more than 50 mg/kg and midazolam at the dose of 2 mg/kg significantly increased the percentage of open arms spent time comparing to the vehicle group as shown in Figure 1-A. In the OFT, the extract of T. kotschyanus at doses of 200, 400 and 600 mg/kg significantly increased the percentage of central to peripheral zone spent time in the open field area and showed anxiolytic activity comparing to the vehicle group as shown in Figure 1-B.
Effect of T. kotschyanus on the percentage of open arms spent time in EPM test (A). Effect of T. kotschyanus on the percentage of central to peripheral zone spent time in OFT (B). Data are presented as mean ± SEM. * indicates p < 0.05 and *** indicates p < 0.001 compared to the control group; (n = 10) in all groups
Hypnotic effect of the extract
To evaluate the hypnotic effect of the extract, pentobarbital-induced sleep test was conducted. The extract at the dose of 600 mg/kg increased the sleeping time compared to the vehicle group. Flumazenil as the benzodiazepine receptor antagonist was able to significantly reduce the effect of the extract with hypnotic activity (Figure 2).
Effect of T. kotschyanus on potentiation of pentobarbital sleeping time. Data are presented as mean ± SEM. ** indicates p < 0.01 compared to the control group; ## indicates p < 0.01 between two compared groups; (n = 10) in all groups
Effect of the extract on the long-term memory of mice
Passive avoidance test is one of the main tests used to evaluate the effect of different agents on the learning and long-term memory processes. Figure 3 shows the latency in the entrance to the dark compartment of the apparatus. The avoidance latency did not change in any group compared to the vehicle. The midazolam-treated group showed a significant reduction in latency time.
Effect of T. kotschyanus on avoidance latency in passive avoidance test. Data are presented as mean ± SEM. *** indicates p < 0.001 compared to the control group; (n = 10) in all groups
Locomotor activity effect of the extract
The locomotor activity of mice was analyzed in the open field test and presented as total distance movement following administration of the extract at different doses and midazolam. In the open field test, as shown in Figure 4, T. kotschyanus extract and midazolam apparently reduced total distance movement after 30 min (Figure 4-A), but they had no significant effect on the locomotor activity of mice after 24 h (Figure 4-B).
Effect of T. kotschyanus on the total distance movement of mice in the open field test after 30 min (A) and 24 h (B) of treatments. Data are presented as mean ± SEM. ** indicates p < 0.01 and *** indicates p < 0.001 compared to the control group; (n = 10) in all groups
Discussion
Herbal medicines are going to be more important in the treatment of patients as alternative medicines. This is because of increasing resistance to chemical drugs, toxicity, undesirable side effects and the high cost of synthetic medicines (21). Thyme family is well-known as one of the most important species because of its antioxidant, anti-inflammatory (22), anti-bacterial (23), antidepressant (13), anxiolytic (12), properties, and high amount of phenolic compounds (14). Although some useful properties of T. kotschyanus have been reported by the scientists, there is no comprehensive study on the pharmacological effects of T.kotschyanus extract on the central nervous system and its toxicity profile in experimental models.
The total phenolic and flavonoid contents of T. kotschyanus are presented in Table 1. T. kotschyanus was found to have lower phenolic content and higher flavonoid content in comparison to what has been reported by Nickavar et al. (14). It is notable that genetic and environmental factors such as temperature, water status, light condition, nitrogen content, environmental stress, and extraction solvent can affect the total content of phenolic compounds (24).
In order to evaluate the acute toxicity of the extract two factors including maximum non-fatal dose and median lethal dose were estimated. Since some researches (25, 26) indicate that mathematical models such as artificial neural network could be successful in ecological and biological studies, probably more information in the prediction of toxicity of the extract can be obtained from computational toxicology and mathematical modeling (27).
According to the PTZ and MES results, administration of T. kotschyanus showed anticonvulsant activities in both animal models used in the screening of antiepileptic agents. Interestingly, the estimated ED50s are below the maximum non-fatal dose. The results of this investigation support the findings, suggesting anticonvulsant properties of Thyme family (28). Thymol as one of the main component of T. kotschyanus is associated with cessation of convulsion. Thymol is reported as a positive allosteric modulator of the GABAA receptors (29, 30). It has the ability of activation of GABAA receptors even in the absence of the natural agonists (31).
The results of the EPM and the OFT evoked anxiolytic activity of the extract as the mice treated with T. kotschyanus preferred to spend more time in open arms of EPM apparatus and showed a higher tendency to stay in the central zone of open field box. Increased anxiety has been correlated obviously with the increased reactive oxygen species (ROS) (32). Several studies have reported that anxiety-like behavior could be reversed by antioxidants (12, 33, 34). The observed anxiolytic effects could be due to phenolic monoterpenes of T. kotschyanus such as thymol, and carvacrol as they have been reported to possess a high antioxidant activity (35, 36). Moreover, carvacrol shows the anxiolytic effect by interaction with the dopaminergic system (37, 38).
Regarding the result of pentobarbital-induced sleep test, T. kotschyanus extract at the dose of 600 mg/kg showed a significant hypnotic effect compared to the control group which is probably because of interaction between phenolic compounds and α1-containing GABAA receptors. Reversal of the hypnotic effect of the extract by flumazenil, as the GABAA receptor antagonist, is the main evidence of this claim (39). This finding is in agreement with the results of previous studies that have shown the ability of thymol and some other phenolic compounds such as flavonoids to activate GABAA receptors (29-31).
Benzodiazepines are the main class of psychoactive drugs with sedative, hypnotic, anxiolytic, anticonvulsant, and muscle relaxant properties which are commonly used in patients. Since benzodiazepines are indirect GABA modulators, they can enhance the effect of GABA at the GABAA receptor (40). GABAA receptors are found to be pentameric transmembrane receptors which consist of 19 subunits (α1–6, β1–3, γ1–3, δ, ϵ, θ, π, and ρ1–3) (41). Recent studies on memory performance effects of benzodiazepines have revealed that interaction of these compounds with α5-containing GABAA receptors can lead to altered learning performance (42). Considering the avoidance latency results in the passive avoidance test, T. kotschyanus treatment obviously did not impair memory function compared to the control mice. The result of open field test indicates that T. kotschyanus extract has a significant sedative effect by reduction of total distance movement following 30 min of exposure. Since the extract has no significant effect on the locomotor activity of mice following 24 h of exposure, it could be concluded that the results of passive avoidance test are not affected by the sedative activity of the extract. This finding reveals that the active compounds of Thymus kotschyanus extract more likely interact with α1-containing GABAA receptors which are responsible for sedative-hypnotic effects of benzodiazepines, and do not bind to α5-containing GABAA receptors which play a key role in cognitive processes.
Conclusion
The current study showed anticonvulsant, anxiolytic, and sedative-hypnotic effects of Thymus kotschyanus extract in animal models. Since some phenolic compounds and especially flavonoids have been reported as herbal ligands for the benzodiazepine binding site of GABAA receptors (43) and the ability of flumazenil to antagonize the observed effects, involvement of α1-containing GABAA receptors could be reported as the possible mechanism of action, but more experiments should be carried out to find out the exact molecular mechanisms related to observed effects and the main active compound (s) which are responsible for the mentioned activities.
Acknowledgment
The authors wish to thank Mr. Mohammad Kamalinejad from Pharmacognosy Dept., School of Pharmacy, Shahid Beheshti University of Medical Sciences (Tehran, Iran) for his assistance in the plant identification. This study was supported by a grant from Deputy of Research and Technology, Shahid Beheshti University of Medical Sciences [grant number 9216].
References
- 1. Fried EI, van Borkulo CD, Cramer AO, Boschloo L, Schoevers RA, Borsboom DMental disorders as networks of problems: a review of recent insights. Soc. Psychiatry Psychiatr. Epidemiol. 2017;52:1–10.[Google Scholar]
- 2. Borsboom DA network theory of mental disorders. World Psychiatry . 2017;16:5–13.[Google Scholar]
- 3. Sayers JThe world health report 2001-Mental health: new understanding, new hope. Bull. World Health Organ. . 2001;79:1085–6.[PubMed][Google Scholar]
- 4. Organization WHO. Mental Health: A Call to Action by World Health Ministers . 2015. [PubMed]
- 5. Salehi B, Mishra A, Shukla I, Sharifi‐Rad M, Contreras M, Segura‐Carretero A, Fathi H, Nasrabadi N, Kobarfard F, Sharifi‐Rad JThymol, thyme, and other plant sources: Health and potential uses. Phytother. Res. . 2018;32:1688–706.[PubMed][Google Scholar]
- 6. Morales RThe history, botany and taxonomy of the genus Thymus. Thyme: the genus Thymus. 2002:1–43.[PubMed][Google Scholar]
- 7. Kılıcgun H, Korkmaz MDose-dependent Medicinal Effects of Thymus haussknechtii Velen Grown Wild in Turkey. Pak. J. Pharm. Sci. 2016;29:179–83.[PubMed][Google Scholar]
- 8. Nolkemper S, Reichling J, Stintzing FC, Carle R, Schnitzler PAntiviral effect of aqueous extracts from species of the Lamiaceae family against Herpes simplex virus type 1 and type 2 in-vitro. Planta. Med. 2006;72:1378–82.[PubMed][Google Scholar]
- 9. Anthony J-P, Fyfe L, Smith HPlant active components–a resource for antiparasitic agents? Trends Parasitol. 2005;21:462–8.[PubMed][Google Scholar]
- 10. Dogu-Baykut E, Gunes G, Decker EAImpact of shortwave ultraviolet (UV-C) radiation on the antioxidant activity of thyme (Thymus vulgaris L) Food Chem. 2014;157:167–73.[PubMed][Google Scholar]
- 11. Babaei M, Abarghoei ME, Ansari R, Vafaei AA, Taherian AA, Akhavan MM, Toussy G, Mousavi SAntispasmodic effect of hydroalcoholic extract of Thymus vulgaris on the guinea-pig ileum. Nat. Prod. Res. 2008;22:1143–50.[PubMed][Google Scholar]
- 12. Komaki A, Hoseini F, Shahidi S, Baharlouei NStudy of the effect of extract of Thymus vulgaris on anxiety in male rats. J. Tradit. Complement. Med. 2016;6:257–61.[Google Scholar]
- 13. Doosti M-H, Ahmadi K, Fasihi-Ramandi MThe effect of ethanolic extract of Thymus kotschyanus on cancer cell growth in-vitro and depression-like behavior in the mouse. J. Tradit. Complement. Med. 2018;8:89–94.[Google Scholar]
- 14. Nickavar B, Esbati NEvaluation of the antioxidant capacity and phenolic content of three Thymus species. J. Acupunct. Meridian. Stud. 2012;5:119–25.[PubMed][Google Scholar]
- 15. Mahboubi A, Asgarpanah J, Sadaghiyani PN, Faizi MTotal phenolic and flavonoid content and antibacterial activity of Punica granatum L var pleniflora flowers (Golnar) against bacterial strains causing foodborne diseases. BMC Complement. Altern. Med. 2015;15:366.[Google Scholar]
- 16. Faizi M, Jahani R, Ebadi SA, Tabatabai SA, Rezaee E, Lotfaliei M, Amini M, Almasirad ANovel 4-thiazolidinone derivatives as agonists of benzodiazepine receptors: Design, synthesis and pharmacological evaluation. EXCLI J. 2017;16[PubMed][Google Scholar]
- 17. Mohammadi-Khanaposhtani M, Shabani M, Faizi M, Aghaei I, Jahani R, Sharafi Z, Zafarghandi NS, Mahdavi M, Akbarzadeh T, Emami S, Shafiee ADesign, synthesis, pharmacological evaluation, and docking study of new acridone-based 1, 2, 4-oxadiazoles as potential anticonvulsant agents. Eur. J. Med. Chem. 2016;112:91–8.[PubMed][Google Scholar]
- 18. Fajemiroye JO, Zjawiony Jordan K, Alves CE, Aderoju AAEvaluation of Anxiolytic and Antidepressant-like Activity of Aqueous Leaf Extract of Nymphaea Lotus Linn. in Mice. Iran. J. Pharma. Res. 2018;17:613–626.[PubMed][Google Scholar]
- 19. Hajiaghaee R, Faizi M, Shahmohammadi Z, Abdollahnejad F, Naghdibadi H, Najafi F, Razmi AHydroalcoholic extract of Myrtus communis can alter anxiety and sleep parameters: a behavioural and EEG sleep pattern study in mice and rats. Pharm. Biol. 2016;54:2141–8.[PubMed][Google Scholar]
- 20. Faizi M, Dabirian S, Tajali H, Ahmadi F, Zavareh ER, Shahhosseini S, Tabatabai SANovel agonists of benzodiazepine receptors: design, synthesis, binding assay and pharmacological evaluation of 1, 2, 4-triazolo [1, 5-a] pyrimidinone and 3-amino-1, 2, 4-triazole derivatives. Bioorg. Med. Chem. 2015;23:480–7.[PubMed][Google Scholar]
- 21. Atmakuri LR, Dathi SCurrent trends in herbal medicines. J. Pharm. Res. 2010;3:109–13.[PubMed][Google Scholar]
- 22. Habashy NH, Serie MMA, Attia WE, Abdelgaleil SAChemical characterization, antioxidant and anti-inflammatory properties of Greek Thymus vulgaris extracts and their possible synergism with Egyptian Chlorella vulgaris. J. Funct. Foods. 2018;40:317–28.[PubMed][Google Scholar]
- 23. Nabavi SM, Marchese A, Izadi M, Curti V, Daglia M, Nabavi SFPlants belonging to the genus Thymus as antibacterial agents: From farm to pharmacy. Food Chem. 2015;173:339–47.[PubMed][Google Scholar]
- 24. Biesiada A, Tomczak ABiotic and abiotic factors affecting the content of the chosen antioxidant compounds in vegetables. Veg. Crop. Res. Bull. 2012;76:55–78.[PubMed][Google Scholar]
- 25. Jahani AForest landscape aesthetic quality model (FLAQM): A comparative study on landscape modelling using regression analysis and artificial neural networks. JFS . 2019;65:61–9.[PubMed][Google Scholar]
- 26. Jahani AModeling of forest canopy density confusion in environmental assessment using artificial neural network. IJFPR . 2016;24:Pe310–Pe321.[PubMed][Google Scholar]
- 27. Kavlock RJ, Ankley G, Blancato J, Breen M, Conolly R, Dix D, Houck K, Hubal E, Judson R, Rabinowitz J, Richard AComputational toxicology—a state of the science mini review. Toxicol. Sci. . 2007;103:14–27.[PubMed][Google Scholar]
- 28. Skalicka-Woźniak K, Walasek M, Aljarba TM, Stapleton P, Gibbons S, Xiao J, Luszczki JJThe anticonvulsant and anti-plasmid conjugation potential of Thymus vulgaris chemistry: an in-vivo murine and in-vitro study. Food Chem. Toxicol. . 2018;120:472–8.[PubMed][Google Scholar]
- 29. Priestley CM, Williamson EM, Wafford KA, Sattelle DBThymol, a constituent of thyme essential oil, is a positive allosteric modulator of human GABAA receptors and a homo‐oligomeric GABA receptor from Drosophila melanogaster. Br. J. Pharmacol. 2003;140:1363–72.[Google Scholar]
- 30. García DA, Bujons J, Vale C, Sunol CAllosteric positive interaction of thymol with the GABAA receptor in primary cultures of mouse cortical neurons. Neuropharmacology . 2006;50:25–35.[PubMed][Google Scholar]
- 31. Mohammadi B, Haeseler G, Leuwer M, Dengler R, Krampfl K, Bufler JStructural requirements of phenol derivatives for direct activation of chloride currents via GABAA receptors. Eur. J. Pharmacol. 2001;421:85–91.[PubMed][Google Scholar]
- 32. Masood A, Nadeem A, Mustafa SJ, O›Donnell JMReversal of oxidative stress-induced anxiety by inhibition of phosphodiesterase-2 in mice. J. Pharmacol. Exp. Ther. 2008;326:369–79.[Google Scholar]
- 33. Hameed IH, Cotos MRC, Hadi MYAntimicrobial, Antioxidant, Hemolytic, Anti-anxiety, and Antihypertensive activity of Passiflora species. RJPT. 2017;10:4079–84.[PubMed][Google Scholar]
- 34. Sulakhiya K, Patel VK, Saxena R, Dashore J, Srivastava AK, Rathore MEffect of Beta vulgaris Linn leaves extract on anxiety-and depressive-like behavior and oxidative stress in mice after acute restraint stress. Pharmacognosy Res. 2016;8:1.[Google Scholar]
- 35. Achour S, Khelifi E, Attia Y, Ferjani E, Noureddine Hellal AConcentration of antioxidant polyphenols from Thymus capitatus extracts by membrane process technology. J. Food Sci. 2012;77:703–9.[PubMed][Google Scholar]
- 36. Undeger U, Başaran A, Degen G, Basaran NAntioxidant activities of major thyme ingredients and lack of (oxidative) DNA damage in V79 Chinese hamster lung fibroblast cells at low levels of carvacrol and thymol. Food Chem. Toxicol. 2009;47:2037–43.[PubMed][Google Scholar]
- 37. Melo FH, Moura BA, de Sousa DP, de Vasconcelos SM, Macedo DS, Fonteles MM, Viana GS, de Sousa FCAntidepressant‐like effect of carvacrol (5‐Isopropyl‐2‐methylphenol) in mice: involvement of dopaminergic system. Fundam. Clin. Pharmacol. 2011;25:362–7.[PubMed][Google Scholar]
- 38. Sharifi‐Rad M, Varoni EM, Iriti M, Martorell M, Setzer WN, del Mar Contreras M, Salehi B, Soltani‐Nejad A, Rajabi S, Tajbakhsh M, Sharifi‐Rad JCarvacrol and human health: A comprehensive review. Phytother. Res. 2018;32:1675–87.[PubMed][Google Scholar]
- 39. Souza DO, dos Santos Sales V, de Souza Rodrigues CK, de Oliveira LR, Lemos IC, de Araújo Delmondes G, Monteiro ÁB, Nascimento EPPhytochemical Analysis and Central Effects of Annona Muricata Linnaeus: Possible Involvement of the Gabaergic and Monoaminergic Systems. Iran. J. Pharma. Res. 2018;17:1306–17.[PubMed][Google Scholar]
- 40. Page CP, Curtis MJ, Sutter MC, Walker MJ Hoffman BB. Integrated pharmacology. Mosby ; pp. 267–270. [PubMed][Google Scholar]
- 41. Furukawa Y, Tanemura K, Igarashi K, Ideta-Otsuka M, Aisaki KI, Kitajima S, Kitagawa M, Kanno JLearning and memory deficits in male adult mice treated with a benzodiazepine sleep-inducing drug during the juvenile period. Front. Neurosci. 2016;10:339.[Google Scholar]
- 42. Sigel E, Ernst MThe Benzodiazepine Binding Sites of GABA A Receptors. Trends Pharmacol. Sci. 2018;39:659–71.[PubMed][Google Scholar]
- 43. Marder M, Paladini ACGABA (A)-receptor ligands of flavonoid structure. Curr. Top. Med. Chem. 2002;2:853–67.[PubMed][Google Scholar]