|Year : 2013 | Volume
| Issue : 4 | Page : 386-390
Antidepressant-like action of the hydromethanolic flower extract of Tagetes erecta L. in mice and its possible mechanism of action
Aarti Khulbe, Savita Pandey, Sangeeta Pilkhwal Sah
Department of Pharmaceutical Sciences, Kumaun University, Nainital, Uttarakhand, India
|Date of Submission||26-Oct-2012|
|Date of Decision||17-Feb-2013|
|Date of Acceptance||23-Apr-2013|
|Date of Web Publication||15-Jul-2013|
Sangeeta Pilkhwal Sah
Department of Pharmaceutical Sciences, Kumaun University, Nainital, Uttarakhand
Source of Support: None, Conflict of Interest: None
Objective: Tagetes erecta, the marigold, has commercial and ethnomedicinal use; however, reports concerning its efficacy for the treatment of depression are lacking. This study was carried out to elucidate the antidepressant effect of hydromethanolic flower extract of T. erecta.
Materials and Methods: Hydromethanolic extract of flowers of Tagetes erecta was subjected to preliminary phytochemical screening. The extract (12.5, 25, and 50 mg/kg, i.p.) was evaluated for antidepressant effect using forced swim test in mice. The mechanism of antidepressant action was further examined using different drugs and imipramine was used as standard drug.
Results: T. erecta significantly inhibited the immobility period in forced swim test in mice P<0.05). T. erecta (25 mg/kg, i.p.) enhanced the anti-immobility effect of antidepressant drugs like imipramine, fluoxetine, and p-chlorophenylalanine, an inhibitor of serotonin synthesis significantly attenuated its antidepressant effect. The antidepressant effect of T. erecta in the forced swim test was prevented by pretreatment with L-arginine and sildenafil, whereas pretreatment of mice with nitric oxide synthase inhibitors potentiated the action. Pentazocine, a high-affinity sigma receptor agonist, produced synergism with effective dose of T. erecta while progesterone, a sigma receptor antagonist, reversed the antidepressant effect of T. erecta. However, the locomotor activity was not affected at tested doses.
Conclusions: Serotonergic, nitrergic pathway, and sigma receptors are possibly involved in mediating antidepressant action of T. erecta in mouse forced swim test.
Keywords: Antidepressant, forced swim test, nitric oxide pathway, serotonergic pathway, sigma receptors, Tagetes erecta
|How to cite this article:|
Khulbe A, Pandey S, Sah SP. Antidepressant-like action of the hydromethanolic flower extract of Tagetes erecta L. in mice and its possible mechanism of action. Indian J Pharmacol 2013;45:386-90
|How to cite this URL:|
Khulbe A, Pandey S, Sah SP. Antidepressant-like action of the hydromethanolic flower extract of Tagetes erecta L. in mice and its possible mechanism of action. Indian J Pharmacol [serial online] 2013 [cited 2020 Jan 23];45:386-90. Available from: http://www.ijp-online.com/text.asp?2013/45/4/386/115026
| » Introduction|| |
Depression is a global public health issue, associated with substantial disability. According to the most accepted monoamine theory, there is impairment of monoaminergic neurotransmission and decrease in extracellular concentrations of noradrenaline and serotonin in depression.  Besides this, multiple neurotransmitter and receptor systems also play a role in etiology of depression  Nitric oxide (NO) modulates the extracellular levels of various neurotransmitters in the central nervous system, for example, serotonin (5-HT), dopamine (DA), γ-aminobutyric acid (GABA), and glutamate.  Recent evidences have shown that the reduction of nitric oxide (NO) levels within the hippocampus by various nitric oxide inhibitors like 7-nitroindazole, N G -nitro-L- arginine methyl ester, and N G -monomethyl-L-arginine produces antidepressant activity. 
Depression is often associated with chronic pain and few antidepressant drugs afford an integral alleviation of pain. 
Tagetes erecta Linn. (Family-Asteraceae), commonly known as Marigold, leaves are reported to be effective against piles, kidney troubles, muscular pain, ulcers, wounds, and earache. , Antibacterial, antioxidant, hepatoprotective and analgesic properties of T. erecta are documented in the literature. ,,, Antidepressant effect of Tagetes lucida cav. a species of Tagetes has been reported.  Therefore, the aim of this study was to investigate the antidepressant action of another species of Tagetes and to elucidate its mechanism of action.
| » Materials and Methods|| |
Fresh flowers of T. erecta L. were collected from Haldwani, Nainital, India, in the month of November 2009 and identified from Forest Research Institute, Dehradun by Dr. Veena Chandra.
Preparation of the plant extract
The dried and coarsely powdered flowers (20 g) of T. erecta were extracted three times by maceration with hydromethanolic solvent (methanol: water; 4:1) for 7 days at room temperature. The combined extract was filtered and the solvent was evaporated under reduced pressure (40 50°C).
Preliminary phytochemical screening
A preliminary phytochemical analysis was done to assess the presence or absence of various groups of phytochemicals.
Male albino mice (20±5 g) bred in Animal House facility of Department of Pharmaceutical Sciences, Bhimtal campus, Kumaun University, Nainital, India, were housed in cages with food and water ad libitum and maintained on a natural 12 h of light and dark cycle. All the experimental protocols were approved by the Institutional Animal Ethical Committee (# 4/2010) CPCSEA registration number of the IAEC is 490/01/a/CPCSEA.
Drugs and treatment
Imipramine, fluoxetine, and pentazocine (Ranbaxy Co., India); p-chlorophenylalanine (PCPA), L-arginine, L-NAME, and methylene blue (HiMedia); sildenafil and progesterone (Sigma, USA). Imipramine, fluoxetine, L-NAME, L-arginine, methylene blue, and sildenafil were dissolved in distilled water whereas TE, pentazocine, p-chlorophenylalanine, and progesterone were dissolved in 2% w/v Tween 80. The doses of the drugs used were selected based on previous studies. ,, Different doses of TE (12.5, 25, 50 mg/kg) and imipramine (10, 20, 30 mg/kg) were administered intraperitoneally (i.p.) in a fixed volume of 1 mL/100 g body weight. All the treatments were given to different groups of animals each containing six mice, 30 min before the forced swim test (FST) or the locomotor test.
The time-course effect of TE in FST was assessed in an independent group of mice, 15, 30, 60, or 120 min after administration of 25 mg/kg, i.p., of extract.
To study the involvement of mechanisms by which TE causes antidepressant-like action in FST, animals were treated with different drugs. Mice were pretreated with the sub-effective dose of imipramine (10 mg/kg, i.p.) and fluoxetine (5 mg/kg, i.p., a selective serotonin reuptake inhibitor) and 5 min later they received vehicle or extract (25 mg/kg) and 30 min later animals were subjected to forced swim test. In another set of experiments, PCPA (100 mg/kg, i.p., an inhibitor of serotonin synthesis) was administered continuously for four days. On the fourth day, TE (25 mg/kg, i.p.) was administered after 30 min of PCPA and 30 min later mice were subjected to FST.
In a separate series of experiments, the possible participation of sigma receptor in the antidepressant-like effect of TE was investigated. Sigma receptor agonist pentazocine (2.5 mg/kg i.p.) and antagonist progesterone (10 mg/kg s.c.) were injected to mice 5 min before administrating TE (25 mg/kg i.p.) and after 30 min, animals were subjected to FST.
For studying the possible participation of L-arginine nitric oxide-cyclic guanosine monophosphate pathway in the antidepressant-like effect of TE, mice were pretreated with L-arginine, a precursor of nitric oxide (750 mg/kg, i.p., a dose that produces no effect in the forced swim test) and vehicle. Thirty minutes after L-arginine, TE (25 mg/kg, i.p., a dose active in forced swim test and no effect on the locomotor activity) and vehicle was injected and 30 min later animals were subjected to FST. In another set of experiments, the effect of TE (25 mg/kg i.p.) with L-NAME (10 mg/kg, i.p., nitric oxide synthase inhibitor) and methylene blue (10 mg/kg i.p., an inhibitor of nitric oxide synthase and an inhibitor of soluble guanylate cyclase) was studied. These modulators (L-NAME and methylene blue) were administered 5 minutes before TE and 30 min later challenged with forced swim test. To observe the role of cyclic guanosine monophosphate in the antidepressant effect of TE, animals received an injection of sildenafil (5 mg/kg, i.p., phosphodiesterase 5 inhibitor) 30 min before TE (25 mg/kg i.p.). Thirty minutes following TE administration, the animals were subjected to forced swim test.
Forced swim test
Mice were individually forced to swim inside a rectangular glass jar of dimensions 25×12×25 cm  containing 15 cm of water maintained at 23-25°C. After an initial 2-3 min of vigorous activity the animals showed period of immobility by floating with minimum movements. An animal was considered to be immobile whenever it remained floating passively in the water in a slightly hunched but upright position, with nose above the water surface. The total immobility period was recorded with the help of stop watch for a period of 6 min. 
Activity monitoring in animals
The locomotor activity was measured by using actophotometer (Inco, Ambala, India). Mice were allowed to acclimatize to the observation chamber for a period of 2 min. Locomotion was expressed in terms of total photobeams counts per 10 min per animal. 
Results were expressed as mean ± SEM. The intergroup variation was measured by one way analysis of variance (ANOVA) followed by Tukey's test and two way analysis. P<0.05 was considered statistically significant. The statistical analysis was done using the Jandal sigma stat statistical software version 2.0.
| » Results|| |
The yield of the extract was 28.7% w/w and the preliminary phytochemical analysis showed the presence of alkaloids, carbohydrates, glycosides, flavonoids, tannins, proteins, saponins, and polysaccharides.
Effect of acute and chronic administration of TE on immobility period in FST and locomotor activity
TE in a dose range of 12.5, 25, and 50 mg/kg significantly decreased the immobility period as compared to vehicle treated control group (P<0.05); however, the effect was not dose dependent [Figure 1]. Imipramine (20 and 30 mg/kg, i.p.) produced a marked decrease in immobility period (P<0.05). However, the locomotor activity in mice was not altered.
|Figure 1: Effect of acute administration of different doses of T. erecta on immobility period induced by forced swim test in mice. *denotes statistical significance at P<0.05 as compared to vehicle treated control group analyzed by one way ANOVA followed by Tukey's test (n=6). Values are mean±SEM. TE = Tagetes erecta|
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In order to investigate the time interval in which the extract achieves the best performance in FST, a time-response curve was carried out. Results illustrated that 30 and 60 min after the administration of extract (25 mg/kg, i.p.) it was able to produce an antidepressant-like effect in FST (P<0.05) [Figure 2]. Moreover, post-hoc analyses showed that the extract exhibits similar performance in both interval times; however, its antidepressant effect was abolished in 120 min. Considering these data, all experiments were performed using 30 min as interval time to investigate the effect of the extract and its mechanism of action.
|Figure 2: Effect of acute administration of T. erecta (25 mg/kg) on immobility period at different time interval. *denotes statistical significance at P<0.05 as compared to vehicle treated control group analyzed by one way ANOVA followed by Tukey's test (n=6). Values are mean±SEM. TE = Tagetes erecta|
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Investigation of the mechanisms underlying the antidepressant effect of TE
Involvement of the monoaminergic system
Pretreatment with imipramine (10 mg/kg, i.p., serotonin and norepinephrine reuptake inhibitor) enhanced the antidepressant effect of TE (25 mg/kg, i.p.) as shown by significant decrease in immobility period compared to TE and imipramine per se group (P<0.05). Imipramine (10 mg/kg, i.p.) per se did not decrease immobility period [Table 1].
|Table 1: Effect of TE (25 mg/kg i.p.) and its modification by monoaminergic system in mouse forced swim test.|
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Pretreatment with fluoxetine (5 mg/kg, i.p., a selective serotonin reuptake inhibitor) also enhanced the antidepressant effect of TE (25 mg/kg i.p.) as shown by significant decrease in immobility period compared to T. erecta and fluoxetine per se group (P<0.05). Pretreatment with a sub-effective dose of PCPA (100 mg/kg, i.p., an inhibitor of serotonin synthesis) reversed the antidepressant action of TE as shown by significant increase in immobility period compared to TE per se group (P<0.05) [Table 1].
Involvement of the sigma receptor
Pretreatment with pentazocine (2.5 mg/kg, i.p., sigma receptor agonist) enhanced the antidepressant effect of TE (25 mg/kg i.p.) (P<0.05). Pretreatment with a sub-effective dose of progesterone (10 mg/kg s.c., sigma receptor antagonist) blocked the antidepressant action of TE as shown by an increase in immobility period compared to TE (25 mg/kg i.p.) per se group (P<0.05) [Table 2].
|Table 2: Effect of TE pentazocine and progesterone (10 mg/kg, s.c.) on the mean immobility period in mouse forced swim test.|
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Involvement of the L-arginine nitric oxide-cyclic guanosine monophosphate pathway
Pretreatment with sub-effective dose of L-arginine (750 mg/kg, i.p., nitric oxide precursor) blocked the antidepressant action of TE (25 mg/kg i.p.) however subeffective dose of L-NAME (10 mg/kg, i.p., nitric oxide synthase inhibitor) enhanced the antidepressant effect of TE (25 mg/kg, i.p.) (P<0.05). Methylene blue (10 mg/kg i.p.) per se significantly reduced the immobility time (P<0.05). While pretreatment with an effective dose of methylene blue (10 mg/kg i.p., nitric oxide synthase inhibitor) enhanced the antidepressant effect of TE (25 mg/kg i.p.).
Sildenafil (5 mg/kg, i.p., a phosphodiesterase 5 inhibitor) did not affect immobility per se, but pretreatment with a sub-effective dose of sildenafil blocked the antidepressant action of T. erecta (25 mg/kg, i.p.) as shown by an increase in immobility period compared to T. erecta (25 mg/kg i.p.) per se group [Table 3].
|Table 3: Effect of TE L-arginine, L-NAME, methylene blue and sildenafil on the mean immobility period in mouse forced swim test.|
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| » Discussion|| |
This study demonstrated antidepressant-like effect of hydromethanolic extract of T. erecta. The effect was not dose dependent, which may be due to its action on multiple receptors. Higher doses of T. erecta (100, 200, and 400 mg/kg, i.p.) did not affect immobility period in forced swim test as compared to control group; however, these doses were found to have sedative effect when tested in actophotometer (data not shown); therefore, lower doses were evaluated for antidepressant effect. The results demonstrated that lower doses of T. erecta significantly reduced immobility without altering locomotion; thus, it can be stated that antidepressant effect of T. erecta was masked by sedative effect at higher doses.
Phytochemical screening revealed the presence of alkaloids, carbohydrate, flavonoids, glycosides, tannins, proteins, saponins, and polysaccharides in the extract. Alkaloids  and flavonoids  are reported to have antidepressant effect and may contribute to antidepressant like effect of T. erecta.
This study showed that pretreatment with fluoxetine and imipramine enhanced the antidepressant action of T. erecta whereas pretreatment with PCPA reversed its antidepressant effect in forced swim test. PCPA is an inhibitor of the enzyme tryptophan hydroxylase and its administration, for four consecutive days, depletes the endogenous stores of serotonin in mice. The results showed that pretreatment with PCPA significantly reversed the antidepressant effect of T. erecta. From the results, it is concluded that T. erecta might be acting in a way similar to monoamines.
Pentazocine, a selective sigma1 receptor agonist potentiated the antidepressant-like effect of T. erecta while progesterone (10 mg/kg s.c.), a sigma receptor antagonist, abolished its antidepressant action. Further few studies report that SSRI's are known to have higher affinities for sigma1 receptor than tricyclic antidepressants;  hence, it is concluded that T. erecta might be exerting antidepressant action by facilitating serotonergic neurotransmission.
Antidepressant action of T. erecta was attenuated by pretreatment with L-arginine and the action of T. erecta was potentiated by sub-effective dose of L-NAME (nitric oxide synthase inhibitor) and methylene blue (nitric oxide synthase inhibitor and soluble guanylate cyclase inhibitor). Another important observation of the study was the reversal of antidepressant action of T. erecta by sildenafil (a phosphodiesterase 5 inhibitor). This study for the first time demonstrated antidepressant-like effect of T. erecta in the forced swim test, linked to modulating effect on L-arginine-nitric oxide-cyclic guanosine monophosphate pathway.
Sigma receptors are unique proteins that are widely distributed in the body and are found in significant concentration in limbic and endocrine areas that have been implicated in the pathophysiology of depression.  The role of sigma 1 receptors in depression is evident by significant affinity of antidepressants like SSRI's, tricyclic antidepressants, MAOI's, and newer generations of antidepressant for sigma receptors. ,, Antidepressant like effect were observed for several sigma receptor agonists such as di-o-tolylguanidine (DTG), Igmesine, (+) pentazocine, SA4503 in forced swim test and tail suspension test.  Pretreatment with sigma receptor antagonists BD 1047 (N-[2-(3,4-dichlorophenyl) ethyl]-N-methyl-2- (dimethylamino) ethylamine) or NE-100 (N,N- dipropyl-2-[4-methoxy-3-(phenylethoxy) phenyl] ethylamine) abolished antidepressant like action of sigma receptor agonist.  Although, various studies have reported the involvement of sigma receptors in the action of various antidepressants, we hypothesized that sigma receptor modulation may be one of the mechanisms involved in mediating the antidepressant-like action of T. erecta.
In a recent study, the involvement of L-arginine-nitric oxide-cyclic guanosine monophosphate system in mediating the antidepressant effect of venlafaxine, a dual reuptake inhibitor of serotonin and norepinephrine in mouse forced swim test was elucidated.  Various nitric oxide synthase inhibitors have been reported to possess antidepressant-like behavioral properties at doses that are without any effect on locomotor activity. It is further stated that endogenous nitric oxide may exert a negative control over a serotonin and dopamine in the hippocampus.  The results indicate that the inhibition of nitric oxide synthesis may underlie the reduction in the immobility time in the forced swim test elicited by T. erecta. The results of this study indicated that the flower extract of T. erecta was able to exert antidepressant effect through multiple receptor pathways.
Thus, it can be concluded that T. erecta flower extract possess antidepressant action demonstrated by modulation of nitrergic, serotonergic pathway, and sigma receptors in mice.
| » References|| |
|1.||Kiss PJ. Theory of active antidepressants: A nonsynaptic approach to the treatment of depression. Neurochem Int 2008;52:34-9. |
|2.||Kulkarni SK, Dhir A. On the mechanism of antidepressant-like action of berberine chloride. Eur J Pharmacol 2008;589 :163-72. |
|3.||Wegener G, Volke V, Rosenberg R. Endogeneous nitric oxide decreases hippocampal levels of the serotonin and dopamine in vivo. Br J Pharmacol 2000;130:575-80. |
|4.||Joca SR, Guimaraes FS. Inhibition of neuronal nitric oxide synthase in the rat hippocampus induce antidepressant-like effects. Psychopharmacology 2006;185:298-305. |
|5.||Mico JA, Ardid D, Berrocoso E, Eschalier A. Antidepressants and pain. Trends in Pharmacol Sci 2006;27:348-54. |
|6.||Kiritikar KR, Basu BD. Indian Medicinal Plants. 2 nd ed., vol. 2. Dehradun: Bishen Sigh Mahendrapal Singh; 1994. |
|7.||Kurian JC. Plants that Heal. Pune: Oriental Watchman Publishing House; 1995. |
|8.||Rhama S, Madhavan S. Antibacterial Activity of the Flavonoid-patulitrin isolated from the flowers of Tagetes erecta L. Int J Pharm Tech Res 2011;3:1407-9. |
|9.||Chivde BV, Biradar KV, Shiramane RS, Manoj KV. In vitro antioxidant activity studies of the flowers of Tagetes erecta L. (Compositae). Int J Pharm Biol Sci 2011;2:223-9. |
|10.||Giri RK, Bose A, Mishra SK. Hepatoprotective Activity of Tagetes erecta against carbon tetrachloride-induced hepatic damage in rats. Acta Poloniae Pharm Drug Res 2011;68:999-1003. |
|11.||Bashir S, Gilani AH. Studies on the antioxidant and analgesic activities of Aztec marigold (Tagetes erecta) flowers. Phytother Res 2008;22:1692-4. |
|12.||Guadarrama-Cruza G, Alarcon-Aguilarb FJ, Lezama-Velascoc R, Vazquez-Palacios G. Bonilla-Jaimed H. Antidepressant-like effects of Tagetes lucida Cav. in the forced swimming test. J Ethnopharmacol 2008;120:277-81. |
|13.||Dhir A, Kulkarni SK. Involvement of nitric oxide NO signaling pathway in the antidepressant action of buproprion, a dopamine reuptke inhibitor. Eur J Pharmacol 2007a;568:177-85. |
|14.||Dhir A, Kulkarni SK. Involvement of sigma1 receptor modulation in the antidepressant action of venlafaxine. Neurosci Lett 2007b;420;204-8. |
|15.||Dhir A, Kulkarni SK. Involvement of sigma 1 receptors in modulating the antidepressant effects of neurosteroids dehydroepiandrosterone or pregnenolone in mouse tail-suspension test. J Psychopharmacol 2008;564:120-8. |
|16.||Kulkarni SK, Dhir A. Effects of various classes of antidepressants in behavioural paradigms of despair. Prog Neuropsychopharmacol Biol Psychiatry 2007;31:1248-54. |
|17.||Kulkarni SK. Hand Book of Experimental Pharmacology, New Delhi (India): Vallabh Prakashan;1999. |
|18.||Vieyra RP, Venebra MA, Rivas SB, Garcia GF. Nicotine as an antidepressant and regulators of sleep in subjects with depression. Rev Neurol 2009;49:661-7. |
|19.||Dimpfel W. Rat electropharmacograms of the flavonoids rutin and quercetin in comparison to those of moclobemide and clinically used reference drugs suggest antidepressive and/or neuroprotective action. Phytomedicine 2009;16:287-94. |
|20.||Takebayshi M, Hayashi T, Su TP. A perspective on the new mechanism of antidepressants, neuritogenesis through sigma1 receptors. Pharmacopsychiatry 2004;37 Suppl 3:S208-13. |
|21.||Aan het Rot M, Mathew SJ, Charney DS. Neurochemical mechanisms in major depressive disorder. CMAJ 2009;180:305-13. |
|22.||Rao TS, Cler JA, Mick SJ, Dilworth VM, Contreras PC, Iyenger S, et al. Neurochemical characterization of dopaminergic effects of opipramol, a potent sigma receptor ligand in vivo. Neuropharmacology 1990;29:1191-7. |
|23.||Narita N, Hashimoto K, Tomitaka S, Minabe Y. Interactions of selective serotonin re-uptake inhibitors with subtype of sigma receptors in rat brain. Eur J Pharmacol 1996;307:117-9. |
|24.||Itzhak Y, Stein I, Zhang SH, Kassim CO, Cristante D. Binding of sigma ligands to C57BL/6 mouse brain membranes :effects of monoamine oxidase inhibitors and subcellular distribution studies suggest the existence of sigma receptor subtypes. J Pharmacol Exp Ther 1991;57:141-8. |
|25.||Wang J, Mack AL, Coop A, Matsumoto RR. Novel sigma receptor agonists produce antidepressant-like effects in mice. Eur Neuropsychopharmacol 2007;17:708-16. |
|26.||Wenger T, Ledent C, Tramu G. The endogenous cannabinoid, anandamide, activates the hypothalamo-pituitary-adrenal axis in CB1 cannabinoid receptor knockout mice. Neuroendocrinology 2003;78:294-300. |
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]