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ORIGINAL ARTICLE
Year : 2014  |  Volume : 4  |  Issue : 4  |  Page : 246-251

Antidepressant effect of linseed oil on various behavioral and pharmacological models of depression in Swiss albino mice


Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Mumbai, Maharashtra, India

Date of Submission15-Apr-2014
Date of Acceptance24-May-2014
Date of Web Publication22-Aug-2014

Correspondence Address:
Sadhana Sathaye
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga (E), Mumbai 400 019, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.139407

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   Abstract 

Background: Linum usitatissimum Linn. (Linaceae) commonly known as linseed or flaxseed is known to be the richest plant source for alpha-linoleic acid, an omega-3 fatty acid. Aim: The objective of the present study was to evaluate the oil extracted from Linseed (LO) for anti-depressant activity in mice. Materials and Methods: Healthy male Swiss albino mice were randomly divided into five groups as follows: (i) Control, (ii) LO (4 ml/kg b.w./day), (iii) LO (8 ml/kg/day), (iv) Fish oil (FO) equivalent to 300 mg/kg/day of Docosahexaenoic acid (DHA), and (v) Standard (Imipramine). The antidepressant effect was evaluated using behavioral models like the Despair swim test (DST) and Tail suspension test (TST), and pharmacological models like potentiation of norepinephrine toxicity, 5-hydroxytryptophan potentiation, tetrabenazine antagonism, and apomorphine-induced cage climbing. Fluorimetric estimation of norepinephrine, dopamine, and 5-hydroxytryptophan levels in the brain was also carried out. Results: In case of behavioral models, LO at 0.2 ml/mice/day, significantly reduced the immobilization time. A significant antidepressant activity of LO was found in the pharmacological models. This was confirmed by the elevated levels of norepinephrine and dopamine in the brains of LO-treated animals, as compared to control animals. Conclusion: Linseed oil showed a significant antidepressant effect in various experimental models of depression in Swiss mice, which seemed most likely to be mediated through an interaction with the adrenergic and dopaminergic systems.

Keywords: Antidepressant, behavioral, dopamine, linseed oil, mice, norepinephrine


How to cite this article:
Shah RK, Kenjale RD, Ghumatkar P, Sathaye S. Antidepressant effect of linseed oil on various behavioral and pharmacological models of depression in Swiss albino mice. Int J Nutr Pharmacol Neurol Dis 2014;4:246-51

How to cite this URL:
Shah RK, Kenjale RD, Ghumatkar P, Sathaye S. Antidepressant effect of linseed oil on various behavioral and pharmacological models of depression in Swiss albino mice. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2020 Jan 22];4:246-51. Available from: http://www.ijnpnd.com/text.asp?2014/4/4/246/139407


   Introduction Top


Depression is a common and disabling disorder of major public health importance, in terms of its prevalence, and the suffering, dysfunction, morbidity, and economic burden it causes. The report on Global Burden of Disease estimates that the prevalence of unipolar depressive episodes is 1.9% in men and 3.2% in women, and the one-year prevalence has been estimated to be 5.8% for men and 9.5% for women. It is estimated that by the year 2020, the burden of depression will increase to 5.7% of the total burden of disease and it would be the second leading cause of disability-adjusted life years (DALYs), second only to ischemic heart disease. [1]

The biogenic monoamine theory states that depression occurs due to impairment or deregulation of aminergic transmission. [2] Other researchers suggest that although the noradrenaline and serotonergic systems are involved, the specific impairment that underlies depression is unclear and is likely to vary among patients. [3] Medical treatment for depression favors prescription of antidepressant drugs, which work by increasing neurotransmission for one or more of the monoamines-serotonin, norepinephrine, and dopamine systems. Until the early 1980s, antidepressant treatment consisted primarily of the tricyclic antidepressants (TCADs), monoamine oxidase inhibitors (MAOIs), and lithium. The antidepressant properties of these medications are attributed to the modulation of noradrenergic and serotonergic functions. However, these have many side effects, as they bind to multiple unrelated receptors.

The SSRIs (selective serotonin reuptake inhibitors), an important class of antidepressants, were introduced in late 1980s. They have substantial advantages over the TCADs and MAOIs in terms of safety, tolerability, and ease of dosing, however, response failure in many of the most severely affected patients and side effects like gastrointestinal complaints, nervousness-agitation, sexual dysfunction, and weight gain with long-term use of SSRIs, has led to difficulty in long-term treatment and non-compliance. [4] Hence, one of the most important goals in the pharmacological treatment of depression is to provide the patients with efficacious drugs that have few side effects, have low or no toxicity, and a high level of tolerability. Therefore, nutritional foods could be explored as a solution to the above problems and as a supplementary means to fight depression.

Linseed Oil is one of the richest sources of alpha-linoleic acid (ALA). ALA is an essential fatty acid and precursor for Docosahexaenoic acid (DHA), which builds up the brain. ALA and DHA are members of the omega-3 fatty acid family. Fish oil is the richest source of DHA and it has been proved to exhibit antidepressant activity. [5] There is a lack of scientific data regarding the effect of LO on depression.

The present study was conducted to evaluate LO as a potential antidepressant agent, using two behavioral models, namely, the Despair swim test (DST) and Tail suspension test (TST), as well as four pharmacological models namely, potentiation of norepinephrine toxicity, 5-hydroxytryptophan potentiation, tetrabenazine antagonism, and apomorphine-induced cage climbing in mice. The effect of LO on various neurotransmitter levels in the mice brains was also evaluated.


   Materials and methods Top


Chemicals

The LO was obtained from Kamani Oil Industries Pvt. Ltd, Mumbai, India. Fish Oil (FO) or DHA (Mega-3 ® capsules), Tetrabenazine, (Revocon ® ), and Imipramine (Depsonil 25 ® ) were purchased from the local pharmacy in Matunga, Mumbai. 5-hydroxytryptophan was obtained from Kochlight, U.K. Norepinephrine bitartrate was obtained from Norad, Neon Labs Ltd., Mumbai. Dopamine hydrochloride and Apomorphine were purchased from HiMedia Labs, India. All other chemicals were purchased from S.D. Fine Chemicals, Ltd., India. All chemicals and reagents used were of analytical grade.

Animals

The protocol for the present study was approved by the registered Institutional Animal Ethics Committee. Animals were procured from the Haffkine Research Institute, Parel, Mumbai, and housed in polypropylene cages (six per cage), under standard hygienic conditions. Pelleted diet (M/s DS Trading, Mumbai) constituting crude protein (≥20%), crude oil (≥3%), and crude fiber (≥2%) and potable water were provided ad libitum. The animals were acclimatized to the animal house environment for seven days prior to start of the study.

Linseed oil dose selection

The daily dose of LO in mice was extrapolated from the human therapeutic dose of 30 - 60 ml, using the conversion factor of 0.0026, and was found to be 0.1 ml for a 25 g mouse. Thus, two dose levels, 0.1 ml and 0.2 ml, were selected for the present study. [6]

Experimental groups

Swiss Albino male mice (25-30 g) were randomly divided into five groups (n = 6) as follows: (i) Control 0.2 ml distilled water (d.w), (ii) 0.1 ml LO 4 ml/kg b.w./day, (iii) 0.2 ml LO 8 ml/kg b.w./day, (iv) FO equivalent to 300 mg of DHA/kg b.w. per day, and (v) Positive Control 25 mg/kg/b.w. Imipramine (b.w. = body weight).

Behavioral analysis

Despair swim test

This model of behavioral despair is based on the premise that when rodents are forced to swim in a restricted area from which there is no escape, they will, after initial frenzied attempts to escape, adopt a characteristic immobile posture and make no further attempts to escape. This test was carried out according to the method described by Porsolt et al. [7] In brief, the standard drug (Imipramine 25 mg/kg) and test drug treatments were carried out for a period of 21 days. On Day 0 and Day 21 of the study, one hour after dosing, the mice were forced to swim inside a vertical Plexiglas cylinder (45 cm diameter; 25 cm height, containing 15 cm of water, maintained at 25 ± 1°C). The mice were allowed to swim for a maximum time period of 15 minutes and the last five minutes of the test were taken as the total duration of immobility. The total immobility time was defined as the total amount of time in which the mice remained immobile or made only small limb movements necessary for floating. The percent of decrease in immobility was calculated.

Tail suspension test

All treatments were carried out in a similar manner as those for the DST (section 2.5), for 21 days. This test was conducted on Day 0 and Day 21 of the treatment schedule, as described by Steru et al., to screen the antidepressant potential of LO. [8] In this test, the mice were suspended on the edge of a shelf, 60 cm above a table top, by adhesive tape placed approximately 1 cm from the tip of the tail, for five minutes. Immobility time was measured manually and the percent decrease in immobility was calculated. The mice were considered immobile when they hanged passively and completely motionless at least for one minute.

Pharmacological evaluation

Potentiation of norepinephrine toxicity in mice

Antidepressants block the reuptake of biogenic amines into the nervous tissue. In this manner, the toxic effects of norepinephrine are potentiated. [9] All the treatments were carried out on the respective groups one hour prior to the subcutaneous injection of a sub-lethal dose of 3 mg/kg b.w. noradrenaline. The mortality rate was assessed 48 hours post-dosing.

Tetrabenazine antagonism in mice

Tetrabenazine (TBZ) induces a depletion of biogenic amines (noradrenaline, dopamine, and serotonin) without affecting their de novo synthesis. TBZ depletes noradrenaline from nerve terminals and prolongs reuptake into the granula. Noradrenaline is degraded by monoamino-oxidase. Antidepressants inhibit the reuptake of noradrenaline into the nerve terminals, and thereby, increase the noradrenaline concentration at the receptor site. In this manner, the effect of TBZ is antagonized. Therefore, both MAO inhibitors and tricyclic antidepressants are known to prevent or antagonize these effects. The prevention of TBZ-induced ptosis and catalepsy can be used for the evaluation of antidepressants. [10] All groups received the treatment as per the doses mentioned in section 2.4, one hour prior to TBZ (40 mg/kg i.p.) administration. The mice were observed for signs of ptosis and catalepsy at 30, 60, 90, and 120 minutes after TBZ administration. If the cataleptic effect was not antagonized after a limit of 60 seconds the animals were placed into a normal position. Thirty seconds after replacement, the degree of ptosis was scored as follows: Eyes closed = 4, eyes three-fourths closed = 3, eyes half closed = 2, eyes quarter closed = 1, eyes open = 0. Similarly, the cataleptic effect was scored according to the duration of catalepsy - catalepsy more than 60 seconds = 5, between 30 and 60 seconds = 4, between 10 and 30 seconds = 3, between 5 and 10 seconds = 1, less than 5 seconds = 0. The scores of the TBZ controls were taken as 100% and the percentage was calculated for the treated animals.

5-Hydroxytryptophan potentiation in mice

According to the monoamine hypothesis of depression, compounds exert antidepressant activity due to their capacity of enhancing central noradrenergic and/or serotonergic functions. Several antidepressants potentiate the serotonin effects by blocking its reuptake. DL-5-hydroxytryptophan is used as the precursor of serotonin. Characteristic head twitches are observed on administration of DL-5-hydroxytryptophan (5-HTP), which is an indication of serotonergic activity. This test was conducted as described by Martin et al., with minor modifications. [11] All animals received drug and standard treatments as in the behavioral studies (section 2.5). On Day 21, the mice received 150 mg/kg of 5-HTP i.p., 30 minutes prior to treatment, with LO and Imipramine. An animal is considered to be positively influenced if it shows head twitches 15 minutes after the 5-HTP injection, thus indicating serotonergic activity. The total number of head twitches were observed and recorded.

Potentiation of apomorphine-induced cage climbing in mice

This test was performed to evaluate the effect of LO on the dopaminergic system. Administration of apomorphine in mice results in a peculiar climbing behavior characterized initially by rearing followed by a full climbing activity, predominantly mediated by the mesolimbic dopamine system. [12] All animals received drug and standard treatments similar to the behavioral studies (section 2.5). On last day of the study, the mice were injected with apomorphine (10 mg/kg i.p.), 45 minutes after the LO and Imipramine treatment. Fifteen minutes post the apomorphine injection, the time spent in a vertical position (2, 3, or 4 paws clinging to the mesh) was recorded for 30 minutes.

Determination of brain neurotransmitter levels

After 21 days of the treatment as mentioned in section 2.4, mice from different groups were sacrificed, the brains were isolated and washed with ice cold saline, weighed, and immediately used for the estimation of norepinephrine, [13] serotonin, [14] and dopamine [15] levels, using fluorimetry. The standard curve for all the three neurotransmitters was plotted.

Statistical analysis

The data obtained were analyzed using one-way analysis of variance (ANOVA) followed by the Dunnett's test. Data are presented as mean ± standard error of mean (SEM). All data summaries and analyses were performed using the GraphPad Instat version 3.01. A value of P ≤ 0.05 was considered significant.


   Results Top


Behavioral analysis

Despair swim test

In this test, LO at 0.1 ml/mouse significantly decreased the immobilization time (
P < 0.01), which was comparable to that of Imipramine 25 mg/kg and Fish oil 300 mg/kg [Table 1].

Tail suspension test

As shown in [Table 1], similar to DST, a significant reduction was found in the percentage immobility (P < 0.01) in both the LO-treated groups as well as fish oil-treated group.
Table 1: Percentage decrease in immobility in the despair swim and tail suspension tests in Swiss mice after the 21-day treatment (n=6)


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Pharmacological evaluation

Potentiation of norepinephrine toxicity in mice

In this model, pretreatment of animals with LO produced 50% mortality at both the doses, which was considerably higher as compared to the control group (33.33%) suggesting increased adrenergic activity. The mortality rate was highest (66.66%) in mice treated with FO 300 mg/kg and Imipramine 25 mg/kg [Table 2].
Table 2: Percentage mortality in potentiation of norepinephrine toxicity model in Swiss mice (n=6)


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Tetrabenazine antagonism in mice

In this model, tetrabenazine was used to induce catalepsy. Results presented in [Table 3] depict that LO at a dose of 0.1 ml/mouse/day and fish oil 300 mg/kg significantly decreased the catalepsy and ptosis score (P < 0.05). Additionally, LO at a dose of 0.2 ml/mouse/day and Imipramine 25 mg/kg significantly lowered the catalepsy and ptosis score (P < 0.01).
Table 3: Catalepsy and ptosis scores recorded in the 'Tetrabenazine antagonism model' in Swiss mice


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5-Hydroxytryptophan potentiation in mice

In this test model, the number of head twitches 15 minutes after 5-HTP injection were observed and recorded in [Table 4]. The results demonstrated that LO 0.1 ml dose treatment and FO 300 mg/kg significantly increased the number of head twitches (P < 0.01), but was not as effective as fluoxetine 25 mg/kg with P < 0.01. LO 0.2 ml treated group showed few head twitches, which were comparable to the control group of animals.

Potentitation of apomorphine-induced cage climbing in mice
Table 4: Number of head twitches recorded in the 'Hydroxytryptophan potentiation model' in Swiss mice


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In this model, LO at both the doses significantly elevated the cage climbing time in apomorphine-treated mice (P < 0.01) along with FO 300 mg/kg (P < 0.01) and Imipramine 25 mg/kg (P < 0.01).

Determination of brain neurotransmitter levels

A significant rise in norepinephrine levels (P < 0.01) was observed in all the groups namely; LO 0.2 ml/mouse/day, FO 300 mg/kg, and Imipramine 25 mg/kg, indicating a comparable activity of LO at 0.2 ml/mouse/day to both FO and Imipramine.


   Discussion Top


The behavioral models of depression (DST and TST) are relatively specific to all major classes of antidepressant drugs including tricyclics, selective serotonin-reuptake inhibitors, and monoamine oxidase (MAO) inhibitors. [7],[8] The decrease in the immobilization time by LO at 0.1 ml dose was comparable to Imipramine and FO. Also, TST indicated a similar fall in the percentage of immobility in both the LO at 0.1 ml/mouse and the FO group. It was also observed that LO at a lower dose (0.1 ml/mouse/day) produced better antidepressant activity than Imipramine [Table 1]. A preliminary unpublished study from our laboratory showed no significant changes in the locomotor function of mice treated with LO compared to the control, indicating that increased motor activity was not involved in the antidepressant-like effect of LO on the DST and TST models.

The pharmacological models were selected to evaluate the effect of LO on the activity of norepinephrine, dopamine, and serotonin; majorly involved in depressive states. In the potentiation norepinephrine toxicity model, exogenous norepinephrine administration resulted in elevated levels of norepinephrine in the brain, leading to death. [9] LO pretreatment enhanced the mortality in the above model, which proposed elevated adrenergic activity, and thus LO might have antidepressant-like activity.

Catalepsy is defined as a behavioral state in experimental animals, characterized by decreased movement and the ability to sustain induced abnormal postures for a considerable length of time, with an intact righting reflex. Tetrabenazine has been reported to induce catalepsy by depleting striatal dopamine, in a manner similar to that of reserpine. [10] LO significantly antagonized tetrabenazine-induced catalepsy and ptosis [Table 3], indicating action through the striatal dopaminergic system leading to increased availability of dopamine.

The 5-HT induced a head-twitch response in mice, indicative of central serotonergic activity. [11] In the 5-HT potentiation model, intermittent head twitches were observed in mice treated with fluoxetine (positive control), whereas, the LO 0.1 ml dose did not significantly increase the head twitching phenomenon. Furthermore, LO 0.2 ml-treated animals showed a significant rise in head twitches, however, it was not as effective as fluoxetine [Table 4], indicating that LO did not act via the serotonergic system.

The apomorphine-induced cage climbing model was used to evaluate the effect of LO on the dopaminergic system. Exogenous apomorphine induced stereotyped cage climbing behavior in mice by directly stimulating the postsynaptic mesolimbic D 2 dopamine receptors. [12] LO, at both doses, potentiated cage climbing, indicating increased dopaminergic activity. The climbing index was highest in Fluoxetine (25 mg/kg)-treated mice as compared to the controls [Table 5]. Thus, LO could have been acting by stimulating the postsynaptic mesolimbic D2 dopamine receptors or indirectly by releasing dopamine from the mesolimbic dopaminergic neurons, with resultant activation of the postsynaptic mesolimbic D2 dopamine receptors.

To elucidate and confirm the mechanism of the antidepressant action of LO, the brain levels of norepinephrine, serotonin, and dopamine were determined. [13],[14],[15] In the 21-day treatment, LO significantly augmented the norepinephrine levels in a dose-dependent manner, which was similar to that of FO and Imipramine. LO at a 0.1 ml dose produced a significant rise in the dopamine and serotonin levels, however, no such rise was observed with respect to dopamine levels at a higher dose (0.2 ml) of LO. This could be attributed to the saturation effect of LO at a higher dose. In mice pretreated with fish oil (containing DHA), the levels of all the three neurotransmitters were raised significantly as compared to the control [Table 6]. Although ALA is a precursor of DHA, it does not replicate the pharmacological effects of DHA. On the basis of the findings of the present study, it can be postulated that LO may work by a mechanism similar to that of DHA, acting primarily via the adrenergic pathways and probably in a complementary dopaminergic route.
Table 5: Climbing time in the 'Potentiation of apomorphine-induced cage climbing model' in Swiss mice


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Table 6: Levels of norepinephrine, dopamine, and 5-HT in the brains of Swiss mice


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   Conclusion Top


The results of the present study suggest that LO shows a significant antidepressant effect in various behavioral and pharmacological models of depression in Swiss mice, which seems most likely to be mediated through an interaction with the adrenergic and dopaminergic systems. Thus, use of LO can be explored as a nutritional supplement during treatment of clinical depression.


   Acknowledgments Top


The authors are thankful to Kamani Oil Industries, Mumbai for providing LO gift samples and to the Nicholas Piramal Reseach Center, Mumbai, for providing the experimental animals. The authors declare that they have no conflicts of interests.

 
   References Top

1.Grover S, Dutt A, Ajit Avasthi. An overview of Indian research in depression. Indian J Psychiatry 2010;52 Suppl 1:S178-88.  Back to cited text no. 1
    
2.Delgado PL, Moreno FA. Role of norepinephrine in depression. J Clin Psychiatry 2000;61 Suppl 1:5-12.  Back to cited text no. 2
    
3.Kent JM. SNaRIs, NaSSAs, and NaRIs: New agents for the treatment of depression. Lancet 2000;355:911-8.  Back to cited text no. 3
    
4.Parker G, Gibson NA, Brotchie H, Heruc G, Rees AM, Hadzi-Pavlovic D. Omega-3 fatty acids and mood disorders. Am J Psychiatry 2006;163:969-78.  Back to cited text no. 4
    
5.Kinsella JE. Advances in food and nutrition research. 35 th ed. Waltham, Massachusetts: Academic Press; 1991. p. 56.  Back to cited text no. 5
    
6.Ghosh MN. In: Fundamentals of experimental pharmacology. Delhi: Vallabh Prakashan; 2007. p. 167.  Back to cited text no. 6
    
7.Porsolt RD, Bertin A, Jalfre M. Behavioural despair in mice: A primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1977;229:327-36.  Back to cited text no. 7
    
8.Steru L, Chermat R, Thierry B, Simon P. The tail suspension test: A new method for screening antidepressants in mice. Psychopharmacology (Berl) 1985;85:367-70.  Back to cited text no. 8
    
9.Alpermann HG, Schacht U, Usinger P, Hock FJ. Pharmacological effects of Hoe 249: A new potential antidepressant. Drug Dev Res 1992;25:267-82.  Back to cited text no. 9
    
10.Nakagawa T, Ukai K, Kubo S. Antidepressive effects of the stereoisomer cis-dosulepin hydrochloride. Arzneimittelforschung 1993;43:11-5.  Back to cited text no. 10
    
11.Martin P, Frances H, Simon P. Dissociation of head twitches and tremors during the study of interactions with 5-hydroxytryptophan in mice. J Pharmacol Methods 1985;13:193-200.  Back to cited text no. 11
    
12.Costall B, Naylor RJ, Nohria V. Climbing behaviour induced by apomorphine in mice: A potential model for the detection of neuroleptic activity. Eur J Pharmacol 1978;50:39-50.  Back to cited text no. 12
    
13.Shore PA, Olin JS. Identification and chemical assay of norepinephrine in brain and other tissues. J Pharmacol Exp Ther 1958;122:295-300.  Back to cited text no. 13
    
14.Bogdanski DF, Pletscher A, Brodie BB, Undenfriend S. Identification and assay of serotonin in brain. J Pharmacol Exp Ther 1956;117:82-8.  Back to cited text no. 14
    
15.Carlsson A. Detection and assay of dopamine. Pharmacol Rev 1959;11:300-4.  Back to cited text no. 15
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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