Oral administration of crocin significantly alleviated anxiety, and depressive-like behavior following ethanol and nicotine abstinence in adolescent male rats

Abstract

There are increasing concerns about nicotine (Nic) and ethanol (Eth) dependence and more importantly the detrimental effects of Nic abstinence among adolescents. The present study aims to assess the effects of crocin main constituent of saffron on anxiety, and depressive like behavior caused by withdrawal following co-abuse of Eth and Nic by assessing related behavioral and biochemical analysis. To this end, behavioral tests including elevated plus maze, open field and forced swimming tests alongside related biochemical analysis were done in which experienced abstinence or treated with crocin. Nic-Eth abstinence induced anxiety- and depressive-like symptoms, whereas both doses of crocin (20 and 30 mg/kg) decreased anxiety and depression induced by Nic-Eth abstinence. In biochemical analysis, crocin as well as buprenorphine-naloxone (Bup-Nal) improved the both oxidative/antioxidant and inflammatory/anti-inflammatory biomarkers balance. Furthermore, serotonin significantly increased in Nic-Eth group treated with either crocin or Bup-Nal. In addition, MAO activity significantly increased in Nic-Eth group, whereas treatment of that with crocin as well as Bup-Nal notably attenuated MAO activity. BDNF notably increased in Nic-Eth group treated with crocin and Bup-Nal. Considering previous results, crocin have a wide range of pharmacological effects, and the present findings also confirmed that it has strong potential to be effective in anxiety, and depressive-like behavior occurred in withdrawal following Nic and Eth.

Introduction

Considering the large body of clinical and pre-clinical setting, growing rate of co-use of alcohol and cigarette as two of the most popular addictive substances¹, in particular, among adolescents², is well-established which can lead to worsened outcomes, like increase the severity of consequent abstinence³, during which undesirable behaviors including, tremor, memory impairment, concentration disruption, anxiety, and depressive disorders occurred⁴ that ultimately more attempts is required to quit which mostly resulted in quit failure. In fact, nicotine (Nic) is one of the main components of cigarettes involved in addiction phenomena⁵ in addition to enduring and long-term sequels related to smoking⁶. Since adolescence, the vulnerable period by which transition between infancy and adulthood occurred is characterized by brain maturation and cognitive development^7–9, which imply not only the mechanisms involved in drug abuse in adolescents, but also the extent of their effects on brain cells are differ from those proposed in addiction and withdrawal occurred in adulthood¹⁰. Regarding the higher frequency of anxiety and depression due to Eth- Nic withdrawal¹¹, better understanding the underlying mechanism(s) is highly recommended to find new pharmacological option with fewer side effects in comparison with buprenorphine-naloxone (Bup-Nal) as two of the most administered medication for withdrawal of Nic, and Eth in order.

However, there are numerous related studies about co-abuse of Eth and Nic, the precise underlying mechanism(s) is not fully elucidated¹². In this regard, there are several studies demonstrated neurotransmitter changes in both human and animal brain specifically in mesolimbic region, the critical area responsible for both reinforcement/reward effects of psychostimulants along with induction of withdrawal symptoms^13–15.

Additionally, it is well-documented that there are strong association between significant decrease in dopamine level in various part of brain specifically in mesocorticolimbic region and psychoactive disorders occurred during withdrawal¹⁶. In this regard, the activity of monoamine oxidase (MAO), by which the majority of catecholamines like serotonin, and dopamine are metabolized, must be considered more attention in drug therapy for withdrawal^17,18. In support, MAO enzyme activity was notably changed in which treated with addictive substances such as morphine, Nic, ethanol (Eth), cannabis, and benzodiazepines that is addressed by several studies^18–21.

In addition, there are various studies confirming the inevitable role of excessive production of both oxidative stress^22,23 and inflammatory cytokines in not only neurodegenerative disorders, but also in mood instability and anxiety²⁴. In fact, imbalance between oxidative stress/anti-oxidant mediators along with pro-inflammatory/anti-inflammatory cytokines have prominent role in progression of abstinence of numerous addictive substances like Nic or alcohol^25–27. In addition, brain-derived neurotrophic factor (BDNF), as a well-known neuroprotective agent plays a key role in neural plasticity and learning-memory process, demonstrated serum level alteration in abstinence periods of both smokers and alcoholism and regarding available data, BDNF might be related to the pathophysiology of Nic and Eth dependency along with process of deprivation^26,28. Moreover, several evidences confirmed significant decrease in BDNF expression in patients diagnosed with depression or anxiety disorders, and anti-depressant therapy could notably increase BDNF expression, which is implying the prominent role of that in mood stability²⁹. Taken these findings together, various mechanism(s) is involved in anxiety and depression occurred during abstinence following Nic or Eth; however, it needs further investigation. So, finding the new scaffolds to attenuate abstinence caused by Nic and Eth, is highly recommended and needs more attention. In this regard, bupropion, atypical anti-depressant agent, and naloxone, opioid antagonist, are two of the most popular medications approved by food and drug administration, are presented to use in attenuation of abstinence symptoms caused by Nic and alcohol respectively^30,31. Furthermore, some alternative therapies as second-line of treatments like tri-cyclic anti-depressants, clonidine, baclofen, n-acetyl cysteine, especially acetylcholine esterase inhibitors^32,33, display promising results; although more novel therapy is still required.

Considering a large body of pre-clinical and clinical studies, crocin, the main constituent of saffron with strong anti-oxidant, and anti-inflammatory properties and long history in improving anxiety and depression, have potential to assess in abstinence period following Eth or Nic³⁴. So, to better understand the main reason and underling mechanism(s) of co-consumption of alcohol and Nic in adolescents, the present study was designed to assess the effects of crocin on anxiety and depressive like behavior caused by abstinence of alcohol and Nic.

Results

Crocin improved anxiety like behaviors induced by Nic-Eth abstinence in OF test

As shown in Figs. 1A, and 2A, Nic-Eth group demonstrated the less time spent in central (F (6, 63) = 182.4, P < 0.001, Fig. 1A), but the more in peripheral (F (6, 63) = 110.21, P < 0.001, Fig. 1A), which probably implies the animals exposed to Nic and Eth have more anxiety in comparison with vehicle, whereas Nic-Eth group treated with crocin (20, and 30 mg/kg) as well as Bup-Nal improved the time spent in the central and decreased the time in the peripheral as compared to Nic-Eth group. In addition, vehicle received crocin illustrated much better results as compared to vehicle, which means crocin have potential to present as anxiolytic agent; however, it needs more investigation (P < 0.001, for all, Fig. 1A and B). Crocin at dose of 10 mg/kg demonstrated insignificant difference with Nic-Eth group.

Fig. 1.

Assessment of anxiety-like behaviors using the open field test. A The time spent in the central component of the open filed box and B duration of animal exploration across the peripheral sections of the box were recorded in different experimental groups compared to the control (vehicle) subjects. Data are shown as ± SEM. ****P < 0.0001.

Fig. 2.

Assessment of anxiety-like behaviors using the elevated plus maze. A The time spent in the open arms of the platform and B duration of animal exploration across the enclosed spaces were recorded in different experimental groups compared to the control (vehicle) subjects. Data are shown as ± SEM. *P < 0.05 and ****P < 0.0001.

Crocin decreased anxiety induced by Nic-Eth abstinence in EPM test

According to the results, Nic-Eth group spent less time in open arm (F (6, 63) = 88.36, P < 0.001, Fig. 2A), and more time in the close arm (F (6, 63) = 22.43, P < 0.001, Fig. 2B) which implies the animals treated with Nic, and Eth demonstrated the more anxiety versus vehicle. Treatment of this group with both crocin (10, 20, and 30 mg/kg), and Bup-Nal attenuated the negative effect of Nic-Eth as indicated by increasing the time spent in the central, and decreasing in the peripheral zone (P < 0.001, for all except P < 0.001 for crocin at 10 mg/kg, Fig. 2A and B). In addition, vehicle under crocin treatment illustrated much better results as compared to vehicle, which implies anxiolytic effect of crocin (P < 0.001, Fig. 2A and B).

Crocin decreased depression induced by Nic-Eth abstinence in FST test

As shown in Fig. 3A, and B, Nic-Eth group against vehicle demonstrated the less struggling (F (6, 63) = 149.9, P < 0.001, Fig. 3A), and more immobility (F (6, 63) = 52.31, P < 0.001, Fig. 3B), which implies the animals under treatment of Nic-Eth have depressive like behavior. Administration of both crocin, and Bup-Nal significantly improved the struggling, while decreasing the immobility time against Nic-Eth group (P < 0.001, for all, except P < 0.05 for crocin at 20 mg/kg for struggling time, Fig. 3A and B). However, crocin at 10 mg/kg demonstrated insignificant difference with Nic-Eth group. Regarding assessing the effect of crocin in normal animals, the results demonstrated the more struggling and the less immobility of animals who treated with crocin against vehicle, which implies anti-depressant activity of crocin (Fig. 3A, P < 0.001; Fig. 3B, P < 0.05).

Fig. 3.

Assessment of depressive-like behaviors using the forced swimming test. A The time of animal’s struggle for survival in water and B duration of immobility (i.e., the time in which animal dose not swim actively to save itself) were recorded in different experimental groups compared to the control (vehicle) subjects. Data are shown as ± SEM. *P < 0.05 and ****P < 0.0001.

Crocin improved oxidative/anti-oxidative imbalance induced by Nic-Eth abstinence

MDA, as a well-known oxidative stress indicator significantly increased (F (6, 63) = 108.6, P < 0.001, Fig. 4A), whereas thiol content, anti-oxidant mediator, notably decreased (F (6, 63) = 179.5, P < 0.001, Fig. 4B) in which treated with Nic-Eth. Treatment of this group with both crocin at dose of 30 mg/kg as well as Bup-Nal changed MDA, and thiol content through decreasing MDA, and increasing thiol content (Fig. 4A, and B, P < 0.001, for both); however, other doses of crocin demonstrated insignificant difference with Nic-Eth group. SOD, and CAT, two of which have a prominent role in decreasing reactive oxygen species (ROS) were measured and according to Fig. 4C, and D, the Nic-Eth group dramatically decreased both SOD (F (6, 63) = 65.4, P < 0.001, Fig. 4C), and CAT (F (6, 63) = 288.9, P < 0.001, Fig. 4D) activity against vehicle, whereas treatment of this group with both crocin (20, and 30 mg/kg) and Bup-Nal demonstrated notable increase in SOD, and CAT activity (Fig. 4A, P < 0.001 except P < 0.001 for crocin at dose of 20 mg/kg ; and 4B, P < 0.001, for all). However, crocin at dose of 10 mg/kg demonstrated insignificant difference with vehicle. It is notable that vehicle treated with crocin illustrated much better results versus vehicle by decreasing MDA, and increasing thiol content as well as SOD, and CAT activity (Fig. 4A and D, P < 0.001).

Fig. 4.

Measurement of the biochemical markers associated with oxidative stress. Cortical concentrations of MDA (A) and thiol (B) plus the enzymatic activity of SOD and CAT (C, D, respectively) were measured in different experimental groups compared to the control (vehicle) subjects. Data are shown as ± SEM. **P < 0.01 and ****P < 0.0001.

Crocin improved inflammatory/anti- inflammatory mediator imbalance induced by Nic-Eth abstinence

Regarding the anti-inflammatory effect of IL-10, the results demonstrated significant decrease in IL-10 concentration (F (6, 63) = 78.37, P < 0.001, Fig. 5A) in which treated with Nic-Eth, while TNF-α, as an inflammatory mediator dramatically decreased (F (6, 63) = 77.12, P < 0.001, Fig. 5B) versus vehicle. Treatment of Nic-Eth group with crocin as well as Bup-Nal significantly improved IL-10 level (Fig. 5A, P < 0.001 for all except P < 0.01 for crocin at dose of 10 mg/kg), and TNF-α level significantly improved in Nic-Eth group treated with either crocin (20, and 30 mg/kg) or Bup-Nal as compared to Nic-Eth group (Fig. 5B, P < 0.001 for all). Moreover, vehicle treated with crocin demonstrated much better results versus vehicle through increasing IL-10, and decreasing TNF-α (Fig. 5A, P < 0.01; Fig. 5B, P < 0.001). However, crocin at dose of 10 mg/kg demonstrated insignificant difference with Nic-Eth group.

Fig. 5.

Measurement of the biochemical markers associated with the inflammatory response. Cortical concentrations of IL-10 (A) and TNF-α (B) were measured in different experimental groups compared to the control (Vehicle) subjects. Data are shown as ± SEM. **P < 0.01 and ****P < 0.0001.

Crocin altered BDNF reduction induced by Nic-Eth abstinence

Considering the prominent role of BDNF in neuroplasticity, and neurogenesis, the group treated with Nic-Eth demonstrated dramatic decrease in BDNF versus vehicle (F (6, 63) = 78.51, P < 0.001, Fig. 6), whereas not only treatment of this group with crocin (30 mg/kg), and Bup-Nal improved BDNF level, but in vehicle treated with crocin also, the level of BDNF significantly improved (Fig. 6, P < 0.001, for all), which implies neurogenesis activity of crocin; however, it needs further investigation.

Fig. 6.

Measurement of BDNF. Cortical concentrations of TNF-α was measured in different experimental groups compared to the control (vehicle) subjects. Data are shown as ± SEM. ****P < 0.0001.

Crocin improved serotonin levels in Nic-Eth abstinence

Serotonin level dramatically decreased in which treated with Nic-Eth (F (6, 63) = 76.04, P < 0.001, Fig. 7A), whereas treatment of this group with both crocin (30 mg/kg), and Bup-Nal significantly improved serotonin level (Fig. 7A, P < 0.001, for all); however, the other doses of crocin was insignificant difference with vehicle. MAO activity, the key enzyme responsible for dopamine and serotonin metabolism, was measured and according to the results, Nic-Eth group demonstrated the more activity of MAO versus vehicle (F (6, 63) = 91.64, P < 0.001, Fig. 7B), whereas treatment of this group with crocin (30 mg/kg) as well as Bup-Nal significantly attenuated MAO activity resulting in elevation of dopamine levels (Fig. 7B, P < 0.001, for all). It is notable that administration of crocin in vehicle also demonstrated beneficial effect on serotonin level, and decreasing MAO activity (Fig. 7A, and B, P < 0.001, for both), by which leads to dopamine elevation, which probably confirms the anti-depressant activity of crocin on mood statue through modulating serotonin, or dopamine, beside its anti-oxidant and anti-inflammatory effects which is consistent with the present results.

Fig. 7.

Measurement of the cortical serotonin levels and enzymatic activity of MAO-A. Concentration of serotonin (A) and activity of MAO-A enzyme (B) were measured in cortical tissues of different experimental groups compared to the control (vehicle) subjects. Data are shown as ± SEM. ****P < 0.0001.

Discussion

Numerous clinical and pre-clinical studies demonstrated the more anxiety and depression among individuals used Eth or Nic³⁵; however, little is known about underlying mechanism(s) and according to the present findings, the null hypothesis about beneficial effect of crocin in attenuation of abstinence symptoms following co-consumption of Eth and Nic is accepted. In fact, both behavioral and biochemical analysis related to assessing anxiety and depressive like behavior demonstrated beneficial effect of crocin. In behavioral tests, crocin especially at doses of 20, and 30 mg/kg attenuated anxiety caused by abstinence following Nic-Eth through decreasing the time spent in close arm in EPM, increasing the time spent in center/peripheral zone in OF, and increasing struggling over decreasing immobility which is indicated as decreasing depressive like behavior in FST. In biochemical analysis in, crocin mostly at doses of 20, and 30 mg/kg significantly improved the factors related to oxidative stress or inflammatory pathway along with other parameters associated with mood stability including serotonin level and MAO activity and both biochemical and behavioral analysis demonstrated comparable effect of crocin with Bup-Nal as two of approved medications used in abstinence. In the present study, administration of crocin at a dose of 20 mg/kg led to significant improvements in certain parameters, while showing no statistically significant effects on others. These findings suggest a selective pharmacological profile of crocin, which may be attributed to its mechanism of action primarily involving antioxidant and neuroprotective pathways³⁶. One possible explanation for the lack of effect on certain parameters is that the selected dose may have been sub-threshold for modulating some of the more complex or resistant pathways, such as inflammatory cytokine expression or synaptic plasticity. Previous studies have demonstrated that crocin’s effects are dose-dependent, and higher doses may be required to influence broader physiological systems³⁷. Overall, these findings underscore the importance of dose selection and endpoint sensitivity when evaluating crocin’s therapeutic potential. Future studies should incorporate a dose-response design to better elucidate the effective concentration range and mechanistic scope of crocin’s action.

It is notable that vehicle received crocin also demonstrated much better results as compared to vehicle by establishing the balance between inflammatory/anti-inflammatory mediators alongside oxidant/anti-oxidant indicators. Also, it have beneficial effect on serotonin level as well as decreasing MAO activity. As our study shows, a dose of 10 mg of crocin does not have a significant effect on behavioral and biochemical parameters. These results are consistent with the results of our other studies, which show that crocin with a dose of 10 is not always effective³⁸.

The chosen dose of 30 mg/kg crocin, although relatively high in the context of preclinical studies, has scientific justification based on its established pharmacological efficacy in animal models of neuroinflammation, oxidative stress, and depression. This dose is commonly used to elicit robust behavioral and molecular effects. Importantly, its translational relevance is supported by clinical findings. For instance, Talaei et al.³⁹ conducted a double-blind, randomized clinical trial administering up to 30 mg/day of crocin to patients with metabolic syndrome, demonstrating good tolerability and safety over 8 weeks. These findings underscore the feasibility of using crocin at pharmacologically meaningful doses in humans, bridging preclinical data with potential clinical application.

There are numerous studies about various therapeutic effect of crocin on psychoactive disorders, cognition impairment, or other diseases which more attributed to its anti-oxidant, and anti-inflammatory effects; however, little is known about other mechanism(s). In this regard, some studies demonstrated that crocin can inhibit neurodegeneration caused by alcohol via modulation of CREB/BDNF signaling pathway and as stimulator of GLP-1, a neurogenesis factor⁴⁰.

Further, administration of crocin (30 mg/kg) before or along with smoking notably improved memory impairment caused by cigarette through decreasing inflammatory mediators like TNF-α, IL-1β, and apoptotic mediators which is consistent with present findings⁴¹.

Similarly, another study illustrated that 30 mg/kg of crocin demonstrated better anti-depressant effect than escitaloperam, one of the well-known anti-depressant medications, in which exposed to chronic stress^34,42. Consistent with the present results, similar study demonstrated both saffron extract (40, 60, and 80 mg/kg) and crocin (10, 20, and 40 mg/kg) administration during chronic usage of Eth (10%, for 7 days) and abstinence notably improved abstinence symptoms through behavioral tests including open field test, and rotarod test⁴³. Also, another study illustrated significant effect of crocin (150 mg/kg) on biochemical analysis through improving cholinergic system and biomarkers related to oxidative stress and neuro-inflammation by which can delay aging process, and enhancement in cognition performance⁴⁴. In addition, there are various studies about the beneficial effect of crocin on LPS-induced inflammation by reducing production of NO, TNF-α, IL-1β, and ROS, along with reduced expression of iNOS, CD206 which is in agreement with the present results⁴⁵. Furthermore, similar study demonstrated the significant effect of crocin on depression induced by chronic mild stress through increasing sucrose consumption in SPT, increasing struggling time in FST alongside increase in BDNF, CREB, pCREB and SIRT1 in prefrontal cortex⁴⁶, which implies that crocin have various mechanism(s) and it needs further investigation. Crocin illustrated anxiolytic and anti-depressant activity in not only animal studies, but also in clinical setting. In this regard, in clinical trials, patients received crocin (30 mg/twice a day) significantly improved questionnaires and markers related to depression as compared to placebo³⁹.

73As we showed in result section crocin 30 mg/kg as well as Bup-Nal can increase serotonin and decrease MAO in cortical issue. Several studies have demonstrated that crocin modulates the serotonergic system by enhancing serotonin availability and influencing receptor activity. It appears to inhibit serotonin reuptake and upregulate 5-HT1A receptor expression, thereby facilitating serotonergic neurotransmission and contributing to antidepressant-like effects^47,48.

79We acknowledge that the current study does not investigate the long-term effects of crocin following abstinence, nor does it evaluate the potential development of tolerance with prolonged use. These are important considerations that warrant further investigation. Future studies should aim to assess the persistence of crocin’s effects over extended periods post-abstinence and explore whether repeated administration leads to tolerance, which could impact its therapeutic efficacy. Longitudinal behavioral and neurochemical analyses would be valuable in addressing these aspects. In addition, it would be better to study female samples to examine behavioral differences between the two sexes. To the best of author’s knowledge, there is no studies related to potential effects of crocin on anxiety-caused by Nic and Eth and considering the human trend and more interest to use herbal remedies-derived compound instead of medication derived from chemical substances, more attention must be focused about some herbal medicines like crocin, one of the most popular herbal medicine with wide range of therapeutic effects, which can be used as potential treatment in abstinence periods caused by addictive substances. In the present study the best results was acquired by the highest doses of crocin (30 mg/kg), which was comparable with bupropion and naloxone; however, further investigation is required to elucidate the current limitation including entering female gender, considering Nic and Eth groups separately to compare their results with the group treated with both Nic, and Eth and introduce this worthy substance in patient’s bed.

Conclusion

Our findings and a large body of literatures confirmed the potential anxiolytic and anti-depressant effect of crocin with minimum side effects. So, crocin can be introduced as a potential scaffold to use in abstinence following Nic or Eth.

Materials and methods

Animal groups

Seventy adolescent male rats, (21days of ages, weighed about 50 ± 5 g) from animal house of Torbat Heydariyeh university of Medical Science were used in the present study and transferred to standard cages (22 ± 2 °C; 12 h:12 h light-dark cycle, light started on at 7:00 am) in which food and water was freely available. After one week of habituation, the animals were randomly divided into seven groups as follows:

1. Vehicle: the animals received normal saline, intraperitoneally (i.p) from 21 to 42 days of ages following receiving that by oral gavage from 43 to 63 days of ages.

2. Nic-Eth: the animals received Nic 2 mg/kg (i.p) and Eth 20% (cumulative dose 5–20% during 6 days) in drinking water, from 21 to 42 days of ages^49,50 and following receiving normal saline by oral gavage from 43 to 63 days of ages, while receiving Nic, and Eth was stopped.

3. Nic-Eth-C10/20/30 mg/kg: the animals received Nic 2 mg/kg (i.p), and Eth in drinking water from 21 to 42 days of ages and from 43 to 63 days of ages they were stopped receiving Nic, and Eth, while started treated by crocin (C10/20/30 mg/kg).

4. Nic-Eth-Bup-Nal: the animals received Nic 2 mg/kg (i.p), and Eth in drinking water from 21 to 42 days of ages and from 43 to 63 days of ages, they were stopped receiving Nic, and Eth, while they were treated by bupropion (Bup, 20 mg/kg by oral gavage) and naloxone (Nal, 10 mg/kg, i.p).

5. C30mg/kg: the animals received normal saline, intraperitoneally (i.p) from 21 to 42 days of ages following receiving C30mg/kg from 43 to 63 days of ages by oral gavage.

Finally, they got ready for behavioral analysis related to anxiety and depression including elevated plus maze (EPM), open field (OF), and Forced Swimming Test (FST). Our study was based on ARRIVE guidelines and finally animals were sacrificed by carbon dioxide inhalation and then cortical tissues were removed from the brain and kept frozen at -80 °C as fast as possible to analysis biochemical criteria related to oxidative stress (malondialdehyde (MDA)), and anti-oxidant mediators (thiol content, superoxide dismutase (SOD), and catalase along with inflammatory (tumor necrosis factor (TNF-α)) and interlukine-10 (IL-10) as anti-inflammatory mediator. Mono amine oxidase (MAO) activity as well as factors associated with neurogenesis like brain derived-neurotrophic factor (BDNF) were measured.

Crocin powders were purchased from Testa Quality Control Laboratory in Iran and purity was 96.7%. All procedures and protocols applied in the present study were authorized by animal research Ethics Committee of Torbat Heydariyeh University of Medical Sciences (IR.THUMS.AEC.1402.004). In addition, attempt was made to minimize the suffering and the number of animals used in this study.

Behavioral study

Elevated plus maze

One of the most common animal test to assess the anxiety is EPM. The device is consisted of two closed and open arm crossed perpendicularly in the middle to each other. A rat elevated plus maze containing two open and two close arms with 50 cm length and 10 cm width was used. The arms were roughly 100 cm above the floor. In each session the rats were placed in the central area facing the closed arms. The time spent in the open and closed arms were recorded manually during 5 min in 3 trial⁵¹.Regarding the natural exploratory behavior of rodents for new environment, it is expected that the more anxiety leads to the more time spent in close arm in ratio to open arm and these measurements serve as indicator of anxiety⁵².

Open field

One of the most widely used test in animal psychological studies is open field maze test which was firstly developed in 1934 to assess either anxiety behavior or screen medication with anxiolytic properties. Open-field was done as previously reported. The open field equipment was a square box (100 × 100 × 40 cm), and the floor of the apparatus was divided into two smaller segments called central and peripheral zones. The rats were placed in the center of open field. Movements were quantified manually for 5 min in 3 trial and (1) the time spent in in central zone, (2) the time spent in the peripheral zone⁵¹ were calculated.

Forced swimming test

In the case of mice, the first 2 min are considered habituation time and are not counted. In this study, where the animal studied is a rat, the previous day is habituation and its time is not counted. Forced swimming test was also done as previously described⁵¹. In summary, in test day each rat was allowed to swim in a cylindrical plexiglass tank (custom-made, 60 cm in high and 38 cm in diameter) which was filled with water (40 cm depth) at 24 ± 1 °C. The immobility and struggling times were calculated manually by a blind observer. It was in three 5-minute trials, and the average of these 3 trials is presented⁵³.

Biochemical analysis

Assessing total thiol content, and MDA in cortex tissue

Malodialdehyde (MDA), as one of the most product of lipid peroxidation is generated of polyunsaturated fatty acids (PUFAs). According to the protocol described previously, thiobarbituric acid (TBA) as an active reagent was reacted with MDA and this adduct produced a colorful complex, detected at 523 nm. To measure thiol content, Ellmen’s reagent, 5, 5’-dithiobis (2-nitrobenzoic acid), which reacts with cysteine, or thiol groups and after reduction the sulfur bonds, the soluble chromophore was formed and detected at 412 nm.

Assessing SOD, and catalase activity

Catalase and SOD, as two of important enzymes which are responsible for anti-oxidant activity was measured in accordance of Aebi, and Madesh methods. Briefly, the rate constant of decomposition of hydrogen peroxide is associated with CAT enzyme activity, and 50% inhibition of (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide)) caused by enzyme, is correlated with one unite of SOD activity.

Assessing IL-10, TNF-α, BDNF and MAO activity

Rat ELISA kit in accordance to manufacture was used to assess TNF-α, and IL-10, as inflammatory, anti-inflammatory factors, in addition to BDNF, serotonin, and MAO activity.

Statistical analysis

Data were expressed as mean ± SEM. The analysis of data for behavioural tests were done using one-way ANOVA followed by Tukey`s post hoc comparison test in which the statistically significant differences were reported at P < 0.05.

Author contributions

Samaneh Kakhki: Designed experiments, performed experiments and writing - original draft. Elham Fadavi Razdabi: Performed experiments.Faezeh Mohammadzadeh: Performed experiments, Formal analysis.Ali abbaszadeh Cheragheali: Performed experiments.Mahshid Jomehpour: Performed experiments.Farimah Beheshti: Designed and performed experiments, writing - review & editing.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical standards

All procedures and protocols applied in the present study were authorized by animal research Ethics Committee of Torbat Heydariyeh University of Medical Sciences (IR.THUMS.AEC.1402.004).