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Antidepressant-like activity of dehydrozingerone: Involvement of the serotonergic and noradrenergic systems
Pharmacology, Biochemistry and Behavior 127 (2014) 111–117
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Antidepressant-like activity of dehydrozingerone: Involvement of the serotonergic and noradrenergic systems Débora M. Martinez a, Angelita Barcellos b, Angela M. Casaril c, Lucielli Savegnago c,⁎, Eder J. Lernardão b,⁎ a b c
Departamento de Ciência e Tecnologia Agroindustrial (DCTA), Universidade Federal de Pelotas, Pelotas, RS, Brazil Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Laboratório de Síntese Orgânica Limpa (LASOL), Universidade Federal de Pelotas, Pelotas, RS, Brazil Centro de Desenvolvimento Tecnológico, Unidade Biotecnologia, Grupo de Pesquisa em Neurobiotecnologia (GPN), Universidade Federal de Pelotas, Pelotas, RS, Brazil
a r t i c l e
i n f o
Article history: Received 17 June 2014 Received in revised form 7 October 2014 Accepted 25 October 2014 Available online 31 October 2014 Keywords: Dehydrozingerone Antidepressant Monoaminergic system Antioxidant
a b s t r a c t Dehydrozingerone (DHZ) is a phenolic compound isolated from ginger rhizomes (Zingiber officinale). It is known for its diverse spectrum of biological activities as an antioxidant, anti-inflammatory and antitumor compound. The present study was designed to assess the antidepressant effect of DHZ and the involvement of the monoaminergic system and to evaluate its in vitro antioxidant activity in the hippocampus, cortex and cerebellum of mice. For this study, the tail suspension test (TST), forced swim test (FST) and yohimbine lethality test were performed. DHZ administered orally 30 min prior to testing reduced the immobility time in the TST (1–40 mg/kg) and the FST (10–40 mg/kg), with no change in locomotor activity in the open field test. The antidepressant-like effect of DHZ (1 mg/kg) was prevented by ketanserin (1 mg/kg, i.p.; a 5-HT2A/2C receptor antagonist), ondansetron (1 mg/kg, i.p.; a 5-HT3 receptor antagonist), prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist) and yohimbine (1 mg/kg, i.p., an α2-adrenoceptor antagonist) pretreatments. Furthermore, DHZ administered at doses of 10 and 20 mg/kg increased the lethality of yohimbine (35 mg/kg, i.p.). DHZ had antioxidant activity on in vitro lipid peroxidation induced by sodium nitroprusside in all brain regions tested. The results revealed that DHZ has a potent antidepressant effect, which seems to involve the serotonergic and noradrenergic systems. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Depression is one of the most common, episodic and recurrent psychiatric disorders, and it results in a substantial health problem for contemporary society (Nemeroff, 2007; Rosenzweig-Lipson et al., 2007). The World Health Organization predicts that depression may become the second cause of illness-induced disability by the year 2020 (WHO, 2001). In spite of its prevalence and severe impact, the efficacy of currently available antidepressants is often inconsistent; many of them require treatment periods of weeks to months and exert undesirable side effects (Nestler, 1998). Moreover, approximately 30% of patients do not respond to therapy with these drugs (Millan, 2009). The bioprospection of safe and effective agents from herbal medicine and natural products may provide us with antidepressant therapies that have reduced side effects and improved efficacy. In this context, the discovery of new effective natural antidepressants has become an important focus of antidepressant therapy. Recent studies of antidepressant activity of plants and their constituents have revealed the powerful effect of several naturally occurring compounds, including extract of Ginkgo biloba (Rojas et al., 2011), extracts of some species of the Valeriana genus (Hattesohi et al., 2008; Sah et al., 2011; Müller et al., ⁎ Corresponding authors. Tel./fax: +55 53 3275 7517. E-mail address: [email protected] (L. Savegnago).
http://dx.doi.org/10.1016/j.pbb.2014.10.010 0091-3057/© 2014 Elsevier Inc. All rights reserved.
2012) and isolated bioactive compounds, such as the polyphenols fisetin (Ebrahimi and Schuluesener, 2012) and resveratrol (Hurley et al., 2014). St. John's wort (Hypericum perforatum L.) is a well-known herb with antidepressant activity that is used in several countries. This herb has been confirmed to be effective in the treatment of mild to moderate depression and to result in fewer side effects. Dehydrozingerone (4-hydroxy-3-methoxybenzalacetone, DHZ, Fig. 1a) is a well-known phenolic compound isolated from ginger rhizomes (Zingiber officinale). DHZ exhibits a large spectrum of biological properties, such as anti-inflammatory, antioxidant (Parihar et al., 2007; Liu et al., 2008), antifungal (Kubra et al., 2013), antitumor (Tatsuzaki et al., 2007) and antimutagenic attributes (Motohashi et al., 2000; Yamagami and Motohashi, 2002). DHZ is a structural halfanalogue and biosynthetic intermediate of curcumin (Fig. 1b), a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa. Numerous studies have shown the antidepressant effect of curcumin and the mechanism involved using different animal models (Xu et al., 2005a,b; Wang et al., 2008; Kulkarni et al., 2008; Bhutani et al., 2009; Li et al., 2009; Arora et al., 2011). The natural small molecule zingerone (Fig. 1c), a bioactive DHZ analogue extracted from ginger, was able to prevent dopamine reduction in the mouse striatum after induction of oxidative stress (Kabuto et al., 2005). The hydro-alcoholic extract of Z. officinale rhizomes (ginger) possesses antidepressant activity in animal models (Pratap et al., 2012). Additionally, the mood modulation
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A
B
C
Fig. 1. Structures of DHZ, curcumin and zingerone.
effect of ginger has been attributed to molecular interactions of gingerderived secondary metabolites with human serotonin 5HT1A receptors (Nievergelt et al., 2010). Despite the effects of curcumin and zingerone and their structural similarity to DHZ and other natural antioxidants, the antidepressant effect of DHZ has not been investigated. Several studies have shown that clinical antidepressants may decrease oxidative stress in animal models of depression and in humans. Thus, antioxidant activity and monoamine modulation may be among the potential mechanisms of antidepressant action (revised by Lee et al., 2013). In light of the above considerations, we aimed to investigate the antidepressant-like effect of DHZ and the possible involvement of serotonergic and noradrenergic systems in the present study. We also evaluated the in vitro antioxidant activity of DHZ against lipid peroxidation in the hippocampus, cortex and cerebellum of mice. 2. Materials and methods 2.1. Animals The experiments were conducted using male Swiss mice (25–35 g) maintained in groups (3 animals/cage) at 22–25 °C with free access to water and food, under a 12/12 h light/dark cycle. All manipulations were carried out between 08.00 a.m. and 04.00 p.m. All experiments were performed on separate groups of animals and each animal was used only once in each test. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources at the Federal University of Pelotas, Brazil. All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments. All experiments were performed with separate groups of animals (6 animals/group), totaling approximately 202 animals. 2.2. Drugs As dehydrozingerone (DHZ) (4-hydroxy-3-methoxybenzalacetone) is a minor constituent of ginger (Govindarajan, 1982) and we needed a significant amount of DHZ for in vivo assays, DHZ was synthesized using the method described by Ramachandra and Subbaraju (2006). All other chemicals were of analytical grade and obtained from standard commercial suppliers. Ketanserin, WAY100635, ondansetron, prazosin, yohimbine and fluoxetine were purchased from Sigma Aldrich and prepared in saline solution (0.9% NaCl) prior to the tests. For the in vivo experiments, DHZ was dissolved in aqueous solution containing 1% Tween and administered orally (p.o.) at varying doses with a constant volume of 10 ml/kg body weight. For in vitro antioxidant activity, DHZ was dissolved in dimethylsulfoxide. Vehicle-treated animals were also assessed simultaneously. 2.3. Behavioral tests 2.3.1. Tail suspension test (TST) The total duration of immobility induced by tail suspension was measured according to the method described by Steru et al. (1985). Mice were suspended on the edge of a table 50 cm above the floor by
adhesive tape placed approximately 1 cm from the tip of the tail. Immobility time, defined as the absence of escape-oriented behavior, was scored over 6 min as described previously (Rodrigues et al., 2002; Mantovani et al., 2003). To assess the antidepressant effect of DHZ, the compound was administered only once (dose range 0.1–40 mg/kg; 6 animals/group) 30 min before the TST. Fluoxetine (32 mg/kg; 6 animals/group) was used as positive control and was administered 30 min before TST. Another set of experiments was designed to verify the possible involvement of serotonergic and noradrenergic systems in the antidepressant-like effect of DHZ. In these experiments, mice were pretreated with ketanserin (1 mg/kg, intraperitoneal route (i.p.); a 5-HT2A/2c receptor antagonist), WAY100635 (0.1 mg/kg, s.c.; a selective 5-HT1A receptor antagonist), ondansetron (1 mg/kg, i.p.; a 5-HT3 receptor antagonist), prazosin (1 mg/kg, i.p.; an α1-adrenoceptor antagonist) or yohimbine (1 mg/kg, i.p.; an α2-adrenoceptor antagonist), and 15 min later, they received vehicle (1% Tween) or DHZ (1 mg/kg, p.o.). After 30 min of DHZ or vehicle administration, the animals (6 animals/ group) were subjected to the TST. The minimal effective dose of DHZ in the TST (1 mg/kg) was chosen to investigate the mechanism of action of DHZ. All doses of antagonists used in this work, which had no effect in the TST, were chosen according to previously published data (Savegnago et al., 2007; Donato et al., 2013). 2.3.2. Forced swim test (FST) After 30 min of treatment with DHZ (dose range: 0.1–40 mg/kg; 6 animals/group) or vehicle (1% Tween; 6 animals/group), mice were placed in individual open cylinders (45 cm height × 20 cm diameter) filled 23 cm with water at 25 ± 1 °C. The duration of immobility was recorded for 5 min. Each mouse was recorded as immobile when floating motionless or making only those movements necessary to keep its head above water. Fluoxetine (32 mg/kg; 6 animals/group) was used as a positive control and was administered 30 min before the FST (Porsolt et al., 1977). 2.3.3. Measurement of locomotor activity on open field test (OFT) To ensure that results obtained in the TST/FST did not occur due to changes in the motor activity of mice, the animals treated with all doses of DHZ (0.1–40 mg/kg; 6 animals/group) were analyzed in the open-field test (OFT). After 30 min of DHZ administration, each animal was immediately placed at the center of the apparatus and observed for 5 min to record locomotor (number of segments crossed with the four paws) and exploratory activities (expressed by the number of times mice were observed rearing on their hind limbs) (Walsh and Cummins, 1976). 2.3.4. Yohimbine lethality test To reveal whether noradrenergic system was involved in the antidepressant-like effect of DHZ, the yohimbine toxicity potentiation test was performed (Malick, 1983). To this end, mice received a single dose of DHZ (1, 10 and 20 mg/kg) or vehicle (1% Tween as control) orally 30 min before the yohimbine injection (35 mg/kg, i.p.) and were immediately placed in cages (6 animals/group). The number of deaths in each group was recorded 18 h after yohimbine administration.
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2.4. In vitro effect of DHZ against lipid peroxidation induced by sodium nitroprusside (SNP) in brain regions The level of malondialdehyde (MDA) was used as a biomarker to evaluate the effect of DHZ on induced lipid peroxidation in the hippocampus, cortex and cerebellum. The spectrophotometric measurement was carried out according Ohkawa et al. (1979). For these experiments, mice were euthanized by cervical dislocation, and their brains were rapidly removed and placed on ice. The cortex, hippocampus and cerebellum were dissected and kept chilled and homogenized in 50 mM Tris–HCl at pH 7.4 (1/5 w/v). The samples were centrifuged for 10 min at 2400 rpm. The pellet was discarded and a low-speed supernatant from each tissue was used in the lipid peroxidation assay. To induce lipid peroxidation, an aliquot of each tissue sample lysate was incubated for 1 h at 37 °C with DHZ (0.05–500 μM) and sodium nitroprusside (SNP) (100 μM). SNP alone was used as a positive control corresponding to 100% lipid peroxidation, and vehicle that lacked SNP was used as a negative control corresponding to basal lipid peroxidation. The results were expressed as a percentage of lipid peroxidation compared with the SNP-only control. 2.5. Statistical analyses All experimental results are expressed as the mean ± standard error of the mean (S.E.M.). Statistical analysis was performed using one-way or two-way analysis of variance (ANOVA) followed by the Newman– Keuls test or Bonferroni's post hoc test. For the antioxidant analysis, assays were performed in duplicate and repeated four times. Differences were considered statistically significant if p b 0.05. 3. Results 3.1. Effect of DHZ on immobility time in TST, and locomotor and exploratory activities in OFT The effect of DHZ on immobility time in the TST was statistically significant from 1–40 mg/kg when compared with the vehicle-treated group (control), as shown in Fig. 2A. No difference was observed when doses of 1–40 mg/kg were compared (p N 0.05). Fluoxetine, a selective serotonin reuptake inhibitor used as positive control, significantly decreased the immobility time (p b 0.001), and its effect was equivalent to DHZ's effect in doses from 1 to 40 mg/kg. The administration of DHZ at all doses tested (0.1–40 mg/kg) did not significantly alter the locomotor and exploratory behavior of the animal (numbers of crossings and rearing) compared to the control group in the OFT (Table 1).
Fig. 2. Effect of DHZ administered acutely in mice on (A) the tail suspension tail (TST) and (B) the forced swimming test. DHZ (0.1–40 mg/kg) was orally administered 30 min before the test. Values were expressed as the mean ± S.E.M.; (**) p b 0.01; (***) p b 0.001 in comparison to the vehicle treated group (control).
As shown in Fig. 2B, treatment with DHZ at doses from 10 to 40 mg/kg decreased the immobility time compared to treatment with vehicle in the FST. Similar to what had been observed in the TST, fluoxetine (32 mg/kg) decreased the immobility time in the FST and not differ from the DHZ antidepressant-like effect.
ketanserin alone [F (1.14) = 58.79, P b 0.0001] and DHZ plus ketanserin (ketanserin × DHZ) [F (1.14) = 5.98, P = 0.0283]. In contrast, the results depicted in Fig. 3C show that pretreatment with WAY100635 (0.1 mg/kg, s.c.; a selective 5-HT1A receptor antagonist) did not prevent the reduction in immobility time elicited by DHZ (1 mg/kg, p.o.). Two-way ANOVA revealed a statistically significant effect of treatment with DHZ [F (1.18) = 12.76; P = 0.0022] but not of the treatment with WAY100635 [F (1.18) = 0; P = 0.94] or treatment with WAY100635 and DHZ (WAY100635 × DHZ) [F (1.18) = 1.55; P = 0.22]. Prazosin or yohimbine was administered 15 min before DHZ, and the TST was performed 30 min after DHZ administration. The
3.3. The involvement of serotonergic and adrenergic systems on the antidepressant-like effect of DHZ
Table 1 Effect of DHZ administration on behavioral parameters in the open field test in mice.
3.2. Effect of DHZ on immobility time in FST
As shown in Fig. 3A, pretreatment of mice with ondansetron (1 mg/kg, i.p.; a 5-HT3 receptor antagonist) prevented the antiimmobility action of the DHZ (1 mg/kg) in the TST [F (1.17) = 5.36, P = 0.033]. Similarly, pretreatment with ketanserin (1 mg/kg, i.p.; a 5-HT2A/2c receptor antagonist) was able to abolish the antidepressant-like effect of DHZ (1 mg/kg, p.o.) in the TST (Fig. 3B). Two-way ANOVA revealed a significant effect of DHZ alone [F (1.14) = 32.85, P b 0.0063],
Experimental groups
Number of crossings
Number of rearings
Vehicle (Tween 1%) DHZ (mg/kg) 1 10 20 40
108.8 ± 8.7
51.4 ± 1.9
106.3 91.7 108.0 113.0
44.2 41.7 44.8 37.2
± ± ± ±
8.3 9.4 8.6 16.4
± ± ± ±
3.9 5.0 5.8 4.8
The effect of mice behavioral on the open field test was determined by one-way ANOVA followed by Newman–Keuls test. Data presented are mean values ± S.E.M.
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Immobility time (s)
A
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200
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decreased the lipid peroxidation in hippocampus and cortex at higher concentrations (100 and 500 μM), and the maximal antioxidant effect at these concentrations was 35.4 ± 9.1% and 40.3 ± 5.1%, respectively. In the cerebellum, DHZ showed a significant antioxidant effect (0.1–500 μM) with a maximal protective effect of 51.0 ± 3.4%.
*
100 50 0
Vehicle
Fig. 4. Effect of pretreatment of mice with (A) prazosin (1 mg/kg, i.p.) and (B) yohimbine (1 mg/kg, i.p.) on the anti-immobility effect of DHZ (1 mg/kg, p.o) in the TST. Data are presented as the mean ± S.E.M.; (*) p b 0.05 and (**) p b 0.01 in comparison to the vehicletreated control; (##) p b 0.01 and (###) p b 0.001 compared to DHZ pretreated with vehicle.
DHZ
Fig. 3. Effect of pretreatment of mice with (A) ondansetron (1 mg/kg, i.p.); (B) ketanserin (1 mg/kg, i.p.); and (C) WAY100635 (0.1 mg/kg, s.c.) on the anti-immobility effect of DHZ (1 mg/kg, p.o) in the TST. Data are presented as the mean ± S.E.M.; (*) p b 0.05 and (***) p b 0.001 in comparison to the vehicle treated group (control); (#) p b 0.05 compared to DHZ pretreated with vehicle.
4. Discussion Considering the low efficacy and tolerance of most of the currently used medications (50–60%), naturally occurring compounds have been particularly relevant in the search for more effective antidepressant-like therapies with reduced adverse effects (Kiss, 2008; Guadarrama-Cruz et al., 2008).
antidepressant-like effect caused by DHZ (1 mg/kg, p.o.) was not detected in mice pretreated with prazosin (1 mg/kg, i.p.; an α1-adrenoceptor antagonist) (Fig. 4A) or yohimbine (1 mg/kg, i.p.; an α2-adrenoceptor antagonist) (Fig. 4B). A two-way ANOVA revealed a significant effect of DHZ alone [F (1, 22) = 11.53, P = 0.0026], prazosin alone [F (1, 22) = 19.53, P = 0.0002] and DHZ plus prazosin (DHZ × prazosin) [F (1, 22) = 9.23, P = 0.006]. A similar response was also observed with yohimbine pretreatment [F (1, 28) = 17.79, P = 0.0020] DHZ treatment [F (1, 28) = 9.44, P = 0.0047], and DHZ plus yohimbine treatment [F (1, 28) = 40.64, P b 0.0001]. 3.4. Yohimbine lethality test The effect of DHZ (1, 10, and 20 mg/kg, p.o.) on yohimbine-induced lethality is depicted in Fig. 5. DHZ at doses of 10 and 20 mg/kg increased the lethal effect of yohimbine. 3.5. Antioxidant activity of DHZ on the lipid peroxidation induced by SNP The effect of DHZ against induced lipid peroxidation in the hippocampus, cortex and cerebellum of mice is depicted on Fig. 6. DHZ
Fig. 5. Effect of DHZ on yohimbine-induced lethality in mice. Each column represents the mean ± S.E.M.; (*) p b 0.05 in comparison to yohimbine treatment.
D.M. Martinez et al. / Pharmacology, Biochemistry and Behavior 127 (2014) 111–117
A) Hippocampus *
80
**
60 40 20 0
C
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#
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% lipid peroxidation
% lipid peroxidation
B) Cortex
#
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500
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50
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C) Cerebellum #
% lipid peroxidation
100
*
80
*
**
*
60
*** ***
40 20 0
C SNP 0.05 0.1
1
10 50 100 500
DHZ (M) Fig. 6. Effect of DHZ on in vitro lipid peroxidation induced by SNP in (A) hippocampus, (B) cortex and (C) cerebellum. Data are presented as the mean ± S.E.M; (*) p b 0.05; (**) p b 0.01; (***) p b 0.001 compared to SNP sample; (#) p b 0.05 compared to SNP pretreated with Control (C).
In this study, we have shown for the first time that orally administered DHZ significantly decreased the duration of immobility in the TST and FST, suggesting an antidepressant-like profile. FSTs and TSTs both represent behavioral depression models, which are widely used to evaluate antidepressant activity (David et al., 2001; Porsolt et al., 1977; Steru et al., 1985). Many antidepressants have reduced the immobility time of rodents in these models (Connor et al., 2000; Detke et al., 1995). In the present study, we showed that DHZ (1, 10, 20 and 40 mg/kg) administered orally significantly decreased the immobility time of mice in the TST, suggesting an antidepressantlike effect. Additionally, to corroborate the effect observed in TST, we decided to use the FST and observed that DHZ (10, 20 and 40 mg/kg) also decreased the immobility time in this test. The immobility time in both tests can be affected by locomotor activity alterations induced by the studied drug. To determine whether this effect was relevant here, an open field test was conducted, and DHZ at doses that elicited an antidepressant-like effect in the TST and FST did not change the locomotor activity of animals. Oral administration of 150 and 300 mg/kg of ginger hydro-alcoholic extract showed an antidepressant-like effect in rats in the TST and FST, but neither determination of a consistent mechanism, nor identification of the active compound was performed (Pratap et al., 2012). Considering that the monoaminergic system is involved in the pathophysiology and treatment of depression, our study was extended to provide mechanistic evidence relating the antidepressant-like effect of DHZ to an interaction with the serotonergic and adrenergic systems. It is well known that dysregulation of the neurotransmitters noradrenaline, serotonin and dopamine in the central nervous system plays an important role in the pathogenesis of depression. Currently, the most efficacious treatment of major depression is believed to involve an increase in serotonin and/or noradrenaline neurotransmission (Brunello et al., 2002; Elhwuegi, 2004). Our results showed that the anti-immobility effect of DHZ on the TST (1 mg/kg) was prevented by pretreatment of mice with selective antagonists of 5-HT2A/2c (ketanserin) and 5-HT3 (ondansetron), which
suggest the involvement of the serotonergic system in the antidepressant effect of this drug. Our data also showed that the antidepressantlike activity of DHZ was not affected by antagonism of 5HT1A receptors (induced by WAY100635). In contrast, a series of gingerols and shogaols isolated from ginger rhizomes extract, which are more lipophilic than DHZ, have been shown to interact significantly with human 5HT1A in HeLa cells (Nievergelt et al., 2010). Accordingly, the alternative finding that DHZ does not interact with 5HT1A can in part be explained by the difference between the chemical structures of gingerols/shogaols and DHZ and by the in vitro model assay carried out by the authors. In a previous study, Nievergelt et al. (2010) reported that constituents of supercritical CO2 ginger extract (8-gingerol; 10-gingerol; 6-, 8and 10-shogaol; 1-dehydro-6-gingerdione, 1-dehydro-8-gingerdione; 1-dehydro-10-gingerdione and 6-dihydroparadol) interact with the human serotonin 5-HT1A receptor and that 10-shogaol, 1-dehydro-6gingerdione and lipophilic ginger extract partially activate this receptor. Corroborating our findings in relation to 5-HT receptors, previous studies reported that anti-immobility effects in the TST and/or FST brought on by natural compounds with structural similarities to DHZ, such as ferulic acid (administered orally) and curcumin (administered by i.p. injection), were related, at least in part, to those receptors (Zeni et al., 2012; Wang et al., 2008). Notably, curcumin has a poor aqueous solubility, high instability and relatively low in vivo bioavailability, which have been considered major problems for its use as a therapeutic agent. The search for methods to improve the bioavailability of curcumin or alternative molecules with biological potential is a source of constant research. DHZ, as well as ferulic acid and vanillin, is a product of curcumin degradation, and DHZ is more water soluble and stable at neutral pH then curcumin (Anand et al., 2007). The anti-immobility effect of DHZ appears to be modulated by α1 and α2-adrenoreceptor antagonists, prazosin and yohimbine, respectively. The α2-adrenoreceptor antagonist yohimbine also modulates the release of noradrenaline and serotonin. Yohimbine can produce excessive release of noradrenaline by antagonizing presynaptic α2-
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adrenoceptors. The potentiation of the yohimbine-induced lethality test in mice represents a useful screen of antidepressant drugs to evaluate the influence on the serotonergic and noradrenergic systems. Yohimbine, an α2-adrenergic receptor antagonist, promotes an overall increase in the signaling of these systems (Malick, 1983), which induces the release of noradrenaline and serotonin, increases central sympathetic activity and leads to toxic effects (Goldberg and Robertson, 1983). In this study, the lethality effect of yohimbine was increased in doses of 10 and 20 mg/kg of DHZ. The yohimbine lethality test was performed to explore and validate the involvement of noradrenergic system in the mechanism DHZ antidepressant-like action. Similar to DHZ, clinical antidepressants potentiate the lethality caused by yohimbine through the enhancement of serotonergic and noradrenergic transmissions. Considering the link between oxidative damage, neuronal cell death and depressive disorders (Sapolsky, 2000), we have investigated the antioxidant activity of DHZ in the lipid peroxidation of brain regions induced by SNP. SNP is a neurotoxic agent that increases OH• and NO release, which leads to the oxidation of lipids, proteins and DNA and subsequent loss of membrane integrity (Halliwell, 1992; Hue and Padmaja, 1993; Requena et al., 2003). The antioxidant activity of DHZ was evidenced in hippocampus, cortex and cerebellum extracts after induction of in vitro lipid peroxidation by SNP. Abdel-Wahab and Salama (2011) have demonstrated that exposure of animals to stress caused by the FST and TST increases lipid peroxidation and DNA damage levels in the hippocampus, and venlafaxine, an approved antidepressant, had the capacity to antagonize this effect. In this sense, the lipid peroxidation and antioxidant defenses are considered integral components of stress and depression, as reported by Yager et al. (2010). Thus, in the search for new compounds and treatment strategies for depression that could improve conventional therapies by utilizing multiple targets, natural products are an alternative for the development of antidepressants with reduced or no adverse effects (Wang et al., 2008). Our findings suggest that DHZ has an antidepressant-like effect that is related to the modulation of serotonergic and noradrenergic systems, and the involvement of antioxidant activity should be an important avenue for further research. 5. Conclusions In summary, the present study reveals that DHZ has antidepressantlike activity and the mechanisms underlying this effect may involve the serotonergic and noradrenergic systems. The capacity to protect from lipid peroxidation in tissues from brain regions was also demonstrated. More studies are necessary to investigate other mechanisms involved in the DHZ antidepressant-like effect. Acknowledgments The project was supported by CAPES, CNPq (306824/2013-2 and 305132/2013-0) and FAPERGS (2012-2551/13-8 and 11/2026-4). References Abdel-Wahab BA, Salama RH. Venlafaxine protects against stress induced oxidative DNA damage in hippocampus during antidepressant testing in mice. Pharmacol Biochem Behav 2011;100:59–65. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm 2007;4:807–18. Arora V, Kuhad A, Tiwari V, Chopra K. Curcumin ameliorates reserpine-induced paindepression dyad: behavioural, biochemical, neurochemical and molecular evidences. Psychoneuroendocrinology 2011;36:1570–81. Bhutani MK, Bishnoi M, Kulkarni S. Anti-depressant like effect of curcumin and its combination with piperine in unpredictable chronic stress-induced behavioral, biochemical and neurochemical changes. Pharmacol Biochem Behav 2009;92:39–43. Brunello N, Mendlewicz J, Kasper S, Leonard B, Montgomery S, Nelson J, et al. The role of noradrenaline and selective noradrenaline reuptake inhibition in depression. Eur Neuropsychopharmacol 2002;12:461–75.
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Antidepressant-like activity of dehydrozingerone: Involvement of the serotonergic and noradrenergic systems Débora M. Martinez a, Angelita Barcellos b, Angela M. Casaril c, Lucielli Savegnago c,⁎, Eder J. Lernardão b,⁎ a b c
Departamento de Ciência e Tecnologia Agroindustrial (DCTA), Universidade Federal de Pelotas, Pelotas, RS, Brazil Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Laboratório de Síntese Orgânica Limpa (LASOL), Universidade Federal de Pelotas, Pelotas, RS, Brazil Centro de Desenvolvimento Tecnológico, Unidade Biotecnologia, Grupo de Pesquisa em Neurobiotecnologia (GPN), Universidade Federal de Pelotas, Pelotas, RS, Brazil
a r t i c l e
i n f o
Article history: Received 17 June 2014 Received in revised form 7 October 2014 Accepted 25 October 2014 Available online 31 October 2014 Keywords: Dehydrozingerone Antidepressant Monoaminergic system Antioxidant
a b s t r a c t Dehydrozingerone (DHZ) is a phenolic compound isolated from ginger rhizomes (Zingiber officinale). It is known for its diverse spectrum of biological activities as an antioxidant, anti-inflammatory and antitumor compound. The present study was designed to assess the antidepressant effect of DHZ and the involvement of the monoaminergic system and to evaluate its in vitro antioxidant activity in the hippocampus, cortex and cerebellum of mice. For this study, the tail suspension test (TST), forced swim test (FST) and yohimbine lethality test were performed. DHZ administered orally 30 min prior to testing reduced the immobility time in the TST (1–40 mg/kg) and the FST (10–40 mg/kg), with no change in locomotor activity in the open field test. The antidepressant-like effect of DHZ (1 mg/kg) was prevented by ketanserin (1 mg/kg, i.p.; a 5-HT2A/2C receptor antagonist), ondansetron (1 mg/kg, i.p.; a 5-HT3 receptor antagonist), prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist) and yohimbine (1 mg/kg, i.p., an α2-adrenoceptor antagonist) pretreatments. Furthermore, DHZ administered at doses of 10 and 20 mg/kg increased the lethality of yohimbine (35 mg/kg, i.p.). DHZ had antioxidant activity on in vitro lipid peroxidation induced by sodium nitroprusside in all brain regions tested. The results revealed that DHZ has a potent antidepressant effect, which seems to involve the serotonergic and noradrenergic systems. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Depression is one of the most common, episodic and recurrent psychiatric disorders, and it results in a substantial health problem for contemporary society (Nemeroff, 2007; Rosenzweig-Lipson et al., 2007). The World Health Organization predicts that depression may become the second cause of illness-induced disability by the year 2020 (WHO, 2001). In spite of its prevalence and severe impact, the efficacy of currently available antidepressants is often inconsistent; many of them require treatment periods of weeks to months and exert undesirable side effects (Nestler, 1998). Moreover, approximately 30% of patients do not respond to therapy with these drugs (Millan, 2009). The bioprospection of safe and effective agents from herbal medicine and natural products may provide us with antidepressant therapies that have reduced side effects and improved efficacy. In this context, the discovery of new effective natural antidepressants has become an important focus of antidepressant therapy. Recent studies of antidepressant activity of plants and their constituents have revealed the powerful effect of several naturally occurring compounds, including extract of Ginkgo biloba (Rojas et al., 2011), extracts of some species of the Valeriana genus (Hattesohi et al., 2008; Sah et al., 2011; Müller et al., ⁎ Corresponding authors. Tel./fax: +55 53 3275 7517. E-mail address: [email protected] (L. Savegnago).
http://dx.doi.org/10.1016/j.pbb.2014.10.010 0091-3057/© 2014 Elsevier Inc. All rights reserved.
2012) and isolated bioactive compounds, such as the polyphenols fisetin (Ebrahimi and Schuluesener, 2012) and resveratrol (Hurley et al., 2014). St. John's wort (Hypericum perforatum L.) is a well-known herb with antidepressant activity that is used in several countries. This herb has been confirmed to be effective in the treatment of mild to moderate depression and to result in fewer side effects. Dehydrozingerone (4-hydroxy-3-methoxybenzalacetone, DHZ, Fig. 1a) is a well-known phenolic compound isolated from ginger rhizomes (Zingiber officinale). DHZ exhibits a large spectrum of biological properties, such as anti-inflammatory, antioxidant (Parihar et al., 2007; Liu et al., 2008), antifungal (Kubra et al., 2013), antitumor (Tatsuzaki et al., 2007) and antimutagenic attributes (Motohashi et al., 2000; Yamagami and Motohashi, 2002). DHZ is a structural halfanalogue and biosynthetic intermediate of curcumin (Fig. 1b), a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa. Numerous studies have shown the antidepressant effect of curcumin and the mechanism involved using different animal models (Xu et al., 2005a,b; Wang et al., 2008; Kulkarni et al., 2008; Bhutani et al., 2009; Li et al., 2009; Arora et al., 2011). The natural small molecule zingerone (Fig. 1c), a bioactive DHZ analogue extracted from ginger, was able to prevent dopamine reduction in the mouse striatum after induction of oxidative stress (Kabuto et al., 2005). The hydro-alcoholic extract of Z. officinale rhizomes (ginger) possesses antidepressant activity in animal models (Pratap et al., 2012). Additionally, the mood modulation
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A
B
C
Fig. 1. Structures of DHZ, curcumin and zingerone.
effect of ginger has been attributed to molecular interactions of gingerderived secondary metabolites with human serotonin 5HT1A receptors (Nievergelt et al., 2010). Despite the effects of curcumin and zingerone and their structural similarity to DHZ and other natural antioxidants, the antidepressant effect of DHZ has not been investigated. Several studies have shown that clinical antidepressants may decrease oxidative stress in animal models of depression and in humans. Thus, antioxidant activity and monoamine modulation may be among the potential mechanisms of antidepressant action (revised by Lee et al., 2013). In light of the above considerations, we aimed to investigate the antidepressant-like effect of DHZ and the possible involvement of serotonergic and noradrenergic systems in the present study. We also evaluated the in vitro antioxidant activity of DHZ against lipid peroxidation in the hippocampus, cortex and cerebellum of mice. 2. Materials and methods 2.1. Animals The experiments were conducted using male Swiss mice (25–35 g) maintained in groups (3 animals/cage) at 22–25 °C with free access to water and food, under a 12/12 h light/dark cycle. All manipulations were carried out between 08.00 a.m. and 04.00 p.m. All experiments were performed on separate groups of animals and each animal was used only once in each test. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources at the Federal University of Pelotas, Brazil. All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments. All experiments were performed with separate groups of animals (6 animals/group), totaling approximately 202 animals. 2.2. Drugs As dehydrozingerone (DHZ) (4-hydroxy-3-methoxybenzalacetone) is a minor constituent of ginger (Govindarajan, 1982) and we needed a significant amount of DHZ for in vivo assays, DHZ was synthesized using the method described by Ramachandra and Subbaraju (2006). All other chemicals were of analytical grade and obtained from standard commercial suppliers. Ketanserin, WAY100635, ondansetron, prazosin, yohimbine and fluoxetine were purchased from Sigma Aldrich and prepared in saline solution (0.9% NaCl) prior to the tests. For the in vivo experiments, DHZ was dissolved in aqueous solution containing 1% Tween and administered orally (p.o.) at varying doses with a constant volume of 10 ml/kg body weight. For in vitro antioxidant activity, DHZ was dissolved in dimethylsulfoxide. Vehicle-treated animals were also assessed simultaneously. 2.3. Behavioral tests 2.3.1. Tail suspension test (TST) The total duration of immobility induced by tail suspension was measured according to the method described by Steru et al. (1985). Mice were suspended on the edge of a table 50 cm above the floor by
adhesive tape placed approximately 1 cm from the tip of the tail. Immobility time, defined as the absence of escape-oriented behavior, was scored over 6 min as described previously (Rodrigues et al., 2002; Mantovani et al., 2003). To assess the antidepressant effect of DHZ, the compound was administered only once (dose range 0.1–40 mg/kg; 6 animals/group) 30 min before the TST. Fluoxetine (32 mg/kg; 6 animals/group) was used as positive control and was administered 30 min before TST. Another set of experiments was designed to verify the possible involvement of serotonergic and noradrenergic systems in the antidepressant-like effect of DHZ. In these experiments, mice were pretreated with ketanserin (1 mg/kg, intraperitoneal route (i.p.); a 5-HT2A/2c receptor antagonist), WAY100635 (0.1 mg/kg, s.c.; a selective 5-HT1A receptor antagonist), ondansetron (1 mg/kg, i.p.; a 5-HT3 receptor antagonist), prazosin (1 mg/kg, i.p.; an α1-adrenoceptor antagonist) or yohimbine (1 mg/kg, i.p.; an α2-adrenoceptor antagonist), and 15 min later, they received vehicle (1% Tween) or DHZ (1 mg/kg, p.o.). After 30 min of DHZ or vehicle administration, the animals (6 animals/ group) were subjected to the TST. The minimal effective dose of DHZ in the TST (1 mg/kg) was chosen to investigate the mechanism of action of DHZ. All doses of antagonists used in this work, which had no effect in the TST, were chosen according to previously published data (Savegnago et al., 2007; Donato et al., 2013). 2.3.2. Forced swim test (FST) After 30 min of treatment with DHZ (dose range: 0.1–40 mg/kg; 6 animals/group) or vehicle (1% Tween; 6 animals/group), mice were placed in individual open cylinders (45 cm height × 20 cm diameter) filled 23 cm with water at 25 ± 1 °C. The duration of immobility was recorded for 5 min. Each mouse was recorded as immobile when floating motionless or making only those movements necessary to keep its head above water. Fluoxetine (32 mg/kg; 6 animals/group) was used as a positive control and was administered 30 min before the FST (Porsolt et al., 1977). 2.3.3. Measurement of locomotor activity on open field test (OFT) To ensure that results obtained in the TST/FST did not occur due to changes in the motor activity of mice, the animals treated with all doses of DHZ (0.1–40 mg/kg; 6 animals/group) were analyzed in the open-field test (OFT). After 30 min of DHZ administration, each animal was immediately placed at the center of the apparatus and observed for 5 min to record locomotor (number of segments crossed with the four paws) and exploratory activities (expressed by the number of times mice were observed rearing on their hind limbs) (Walsh and Cummins, 1976). 2.3.4. Yohimbine lethality test To reveal whether noradrenergic system was involved in the antidepressant-like effect of DHZ, the yohimbine toxicity potentiation test was performed (Malick, 1983). To this end, mice received a single dose of DHZ (1, 10 and 20 mg/kg) or vehicle (1% Tween as control) orally 30 min before the yohimbine injection (35 mg/kg, i.p.) and were immediately placed in cages (6 animals/group). The number of deaths in each group was recorded 18 h after yohimbine administration.
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2.4. In vitro effect of DHZ against lipid peroxidation induced by sodium nitroprusside (SNP) in brain regions The level of malondialdehyde (MDA) was used as a biomarker to evaluate the effect of DHZ on induced lipid peroxidation in the hippocampus, cortex and cerebellum. The spectrophotometric measurement was carried out according Ohkawa et al. (1979). For these experiments, mice were euthanized by cervical dislocation, and their brains were rapidly removed and placed on ice. The cortex, hippocampus and cerebellum were dissected and kept chilled and homogenized in 50 mM Tris–HCl at pH 7.4 (1/5 w/v). The samples were centrifuged for 10 min at 2400 rpm. The pellet was discarded and a low-speed supernatant from each tissue was used in the lipid peroxidation assay. To induce lipid peroxidation, an aliquot of each tissue sample lysate was incubated for 1 h at 37 °C with DHZ (0.05–500 μM) and sodium nitroprusside (SNP) (100 μM). SNP alone was used as a positive control corresponding to 100% lipid peroxidation, and vehicle that lacked SNP was used as a negative control corresponding to basal lipid peroxidation. The results were expressed as a percentage of lipid peroxidation compared with the SNP-only control. 2.5. Statistical analyses All experimental results are expressed as the mean ± standard error of the mean (S.E.M.). Statistical analysis was performed using one-way or two-way analysis of variance (ANOVA) followed by the Newman– Keuls test or Bonferroni's post hoc test. For the antioxidant analysis, assays were performed in duplicate and repeated four times. Differences were considered statistically significant if p b 0.05. 3. Results 3.1. Effect of DHZ on immobility time in TST, and locomotor and exploratory activities in OFT The effect of DHZ on immobility time in the TST was statistically significant from 1–40 mg/kg when compared with the vehicle-treated group (control), as shown in Fig. 2A. No difference was observed when doses of 1–40 mg/kg were compared (p N 0.05). Fluoxetine, a selective serotonin reuptake inhibitor used as positive control, significantly decreased the immobility time (p b 0.001), and its effect was equivalent to DHZ's effect in doses from 1 to 40 mg/kg. The administration of DHZ at all doses tested (0.1–40 mg/kg) did not significantly alter the locomotor and exploratory behavior of the animal (numbers of crossings and rearing) compared to the control group in the OFT (Table 1).
Fig. 2. Effect of DHZ administered acutely in mice on (A) the tail suspension tail (TST) and (B) the forced swimming test. DHZ (0.1–40 mg/kg) was orally administered 30 min before the test. Values were expressed as the mean ± S.E.M.; (**) p b 0.01; (***) p b 0.001 in comparison to the vehicle treated group (control).
As shown in Fig. 2B, treatment with DHZ at doses from 10 to 40 mg/kg decreased the immobility time compared to treatment with vehicle in the FST. Similar to what had been observed in the TST, fluoxetine (32 mg/kg) decreased the immobility time in the FST and not differ from the DHZ antidepressant-like effect.
ketanserin alone [F (1.14) = 58.79, P b 0.0001] and DHZ plus ketanserin (ketanserin × DHZ) [F (1.14) = 5.98, P = 0.0283]. In contrast, the results depicted in Fig. 3C show that pretreatment with WAY100635 (0.1 mg/kg, s.c.; a selective 5-HT1A receptor antagonist) did not prevent the reduction in immobility time elicited by DHZ (1 mg/kg, p.o.). Two-way ANOVA revealed a statistically significant effect of treatment with DHZ [F (1.18) = 12.76; P = 0.0022] but not of the treatment with WAY100635 [F (1.18) = 0; P = 0.94] or treatment with WAY100635 and DHZ (WAY100635 × DHZ) [F (1.18) = 1.55; P = 0.22]. Prazosin or yohimbine was administered 15 min before DHZ, and the TST was performed 30 min after DHZ administration. The
3.3. The involvement of serotonergic and adrenergic systems on the antidepressant-like effect of DHZ
Table 1 Effect of DHZ administration on behavioral parameters in the open field test in mice.
3.2. Effect of DHZ on immobility time in FST
As shown in Fig. 3A, pretreatment of mice with ondansetron (1 mg/kg, i.p.; a 5-HT3 receptor antagonist) prevented the antiimmobility action of the DHZ (1 mg/kg) in the TST [F (1.17) = 5.36, P = 0.033]. Similarly, pretreatment with ketanserin (1 mg/kg, i.p.; a 5-HT2A/2c receptor antagonist) was able to abolish the antidepressant-like effect of DHZ (1 mg/kg, p.o.) in the TST (Fig. 3B). Two-way ANOVA revealed a significant effect of DHZ alone [F (1.14) = 32.85, P b 0.0063],
Experimental groups
Number of crossings
Number of rearings
Vehicle (Tween 1%) DHZ (mg/kg) 1 10 20 40
108.8 ± 8.7
51.4 ± 1.9
106.3 91.7 108.0 113.0
44.2 41.7 44.8 37.2
± ± ± ±
8.3 9.4 8.6 16.4
± ± ± ±
3.9 5.0 5.8 4.8
The effect of mice behavioral on the open field test was determined by one-way ANOVA followed by Newman–Keuls test. Data presented are mean values ± S.E.M.
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Immobility time (s)
A
250
Vehicle Ondansetron
200
#
150
***
100 50 0
Vehicle
Immobility time (s)
B
DHZ
250
#
200
Vehicle Ketanserin
*
150 100 50 0
Vehicle
Immobility time (s)
C
DHZ
250
Vehicle WAY
200 150
decreased the lipid peroxidation in hippocampus and cortex at higher concentrations (100 and 500 μM), and the maximal antioxidant effect at these concentrations was 35.4 ± 9.1% and 40.3 ± 5.1%, respectively. In the cerebellum, DHZ showed a significant antioxidant effect (0.1–500 μM) with a maximal protective effect of 51.0 ± 3.4%.
*
100 50 0
Vehicle
Fig. 4. Effect of pretreatment of mice with (A) prazosin (1 mg/kg, i.p.) and (B) yohimbine (1 mg/kg, i.p.) on the anti-immobility effect of DHZ (1 mg/kg, p.o) in the TST. Data are presented as the mean ± S.E.M.; (*) p b 0.05 and (**) p b 0.01 in comparison to the vehicletreated control; (##) p b 0.01 and (###) p b 0.001 compared to DHZ pretreated with vehicle.
DHZ
Fig. 3. Effect of pretreatment of mice with (A) ondansetron (1 mg/kg, i.p.); (B) ketanserin (1 mg/kg, i.p.); and (C) WAY100635 (0.1 mg/kg, s.c.) on the anti-immobility effect of DHZ (1 mg/kg, p.o) in the TST. Data are presented as the mean ± S.E.M.; (*) p b 0.05 and (***) p b 0.001 in comparison to the vehicle treated group (control); (#) p b 0.05 compared to DHZ pretreated with vehicle.
4. Discussion Considering the low efficacy and tolerance of most of the currently used medications (50–60%), naturally occurring compounds have been particularly relevant in the search for more effective antidepressant-like therapies with reduced adverse effects (Kiss, 2008; Guadarrama-Cruz et al., 2008).
antidepressant-like effect caused by DHZ (1 mg/kg, p.o.) was not detected in mice pretreated with prazosin (1 mg/kg, i.p.; an α1-adrenoceptor antagonist) (Fig. 4A) or yohimbine (1 mg/kg, i.p.; an α2-adrenoceptor antagonist) (Fig. 4B). A two-way ANOVA revealed a significant effect of DHZ alone [F (1, 22) = 11.53, P = 0.0026], prazosin alone [F (1, 22) = 19.53, P = 0.0002] and DHZ plus prazosin (DHZ × prazosin) [F (1, 22) = 9.23, P = 0.006]. A similar response was also observed with yohimbine pretreatment [F (1, 28) = 17.79, P = 0.0020] DHZ treatment [F (1, 28) = 9.44, P = 0.0047], and DHZ plus yohimbine treatment [F (1, 28) = 40.64, P b 0.0001]. 3.4. Yohimbine lethality test The effect of DHZ (1, 10, and 20 mg/kg, p.o.) on yohimbine-induced lethality is depicted in Fig. 5. DHZ at doses of 10 and 20 mg/kg increased the lethal effect of yohimbine. 3.5. Antioxidant activity of DHZ on the lipid peroxidation induced by SNP The effect of DHZ against induced lipid peroxidation in the hippocampus, cortex and cerebellum of mice is depicted on Fig. 6. DHZ
Fig. 5. Effect of DHZ on yohimbine-induced lethality in mice. Each column represents the mean ± S.E.M.; (*) p b 0.05 in comparison to yohimbine treatment.
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A) Hippocampus *
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DHZ (M) Fig. 6. Effect of DHZ on in vitro lipid peroxidation induced by SNP in (A) hippocampus, (B) cortex and (C) cerebellum. Data are presented as the mean ± S.E.M; (*) p b 0.05; (**) p b 0.01; (***) p b 0.001 compared to SNP sample; (#) p b 0.05 compared to SNP pretreated with Control (C).
In this study, we have shown for the first time that orally administered DHZ significantly decreased the duration of immobility in the TST and FST, suggesting an antidepressant-like profile. FSTs and TSTs both represent behavioral depression models, which are widely used to evaluate antidepressant activity (David et al., 2001; Porsolt et al., 1977; Steru et al., 1985). Many antidepressants have reduced the immobility time of rodents in these models (Connor et al., 2000; Detke et al., 1995). In the present study, we showed that DHZ (1, 10, 20 and 40 mg/kg) administered orally significantly decreased the immobility time of mice in the TST, suggesting an antidepressantlike effect. Additionally, to corroborate the effect observed in TST, we decided to use the FST and observed that DHZ (10, 20 and 40 mg/kg) also decreased the immobility time in this test. The immobility time in both tests can be affected by locomotor activity alterations induced by the studied drug. To determine whether this effect was relevant here, an open field test was conducted, and DHZ at doses that elicited an antidepressant-like effect in the TST and FST did not change the locomotor activity of animals. Oral administration of 150 and 300 mg/kg of ginger hydro-alcoholic extract showed an antidepressant-like effect in rats in the TST and FST, but neither determination of a consistent mechanism, nor identification of the active compound was performed (Pratap et al., 2012). Considering that the monoaminergic system is involved in the pathophysiology and treatment of depression, our study was extended to provide mechanistic evidence relating the antidepressant-like effect of DHZ to an interaction with the serotonergic and adrenergic systems. It is well known that dysregulation of the neurotransmitters noradrenaline, serotonin and dopamine in the central nervous system plays an important role in the pathogenesis of depression. Currently, the most efficacious treatment of major depression is believed to involve an increase in serotonin and/or noradrenaline neurotransmission (Brunello et al., 2002; Elhwuegi, 2004). Our results showed that the anti-immobility effect of DHZ on the TST (1 mg/kg) was prevented by pretreatment of mice with selective antagonists of 5-HT2A/2c (ketanserin) and 5-HT3 (ondansetron), which
suggest the involvement of the serotonergic system in the antidepressant effect of this drug. Our data also showed that the antidepressantlike activity of DHZ was not affected by antagonism of 5HT1A receptors (induced by WAY100635). In contrast, a series of gingerols and shogaols isolated from ginger rhizomes extract, which are more lipophilic than DHZ, have been shown to interact significantly with human 5HT1A in HeLa cells (Nievergelt et al., 2010). Accordingly, the alternative finding that DHZ does not interact with 5HT1A can in part be explained by the difference between the chemical structures of gingerols/shogaols and DHZ and by the in vitro model assay carried out by the authors. In a previous study, Nievergelt et al. (2010) reported that constituents of supercritical CO2 ginger extract (8-gingerol; 10-gingerol; 6-, 8and 10-shogaol; 1-dehydro-6-gingerdione, 1-dehydro-8-gingerdione; 1-dehydro-10-gingerdione and 6-dihydroparadol) interact with the human serotonin 5-HT1A receptor and that 10-shogaol, 1-dehydro-6gingerdione and lipophilic ginger extract partially activate this receptor. Corroborating our findings in relation to 5-HT receptors, previous studies reported that anti-immobility effects in the TST and/or FST brought on by natural compounds with structural similarities to DHZ, such as ferulic acid (administered orally) and curcumin (administered by i.p. injection), were related, at least in part, to those receptors (Zeni et al., 2012; Wang et al., 2008). Notably, curcumin has a poor aqueous solubility, high instability and relatively low in vivo bioavailability, which have been considered major problems for its use as a therapeutic agent. The search for methods to improve the bioavailability of curcumin or alternative molecules with biological potential is a source of constant research. DHZ, as well as ferulic acid and vanillin, is a product of curcumin degradation, and DHZ is more water soluble and stable at neutral pH then curcumin (Anand et al., 2007). The anti-immobility effect of DHZ appears to be modulated by α1 and α2-adrenoreceptor antagonists, prazosin and yohimbine, respectively. The α2-adrenoreceptor antagonist yohimbine also modulates the release of noradrenaline and serotonin. Yohimbine can produce excessive release of noradrenaline by antagonizing presynaptic α2-
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adrenoceptors. The potentiation of the yohimbine-induced lethality test in mice represents a useful screen of antidepressant drugs to evaluate the influence on the serotonergic and noradrenergic systems. Yohimbine, an α2-adrenergic receptor antagonist, promotes an overall increase in the signaling of these systems (Malick, 1983), which induces the release of noradrenaline and serotonin, increases central sympathetic activity and leads to toxic effects (Goldberg and Robertson, 1983). In this study, the lethality effect of yohimbine was increased in doses of 10 and 20 mg/kg of DHZ. The yohimbine lethality test was performed to explore and validate the involvement of noradrenergic system in the mechanism DHZ antidepressant-like action. Similar to DHZ, clinical antidepressants potentiate the lethality caused by yohimbine through the enhancement of serotonergic and noradrenergic transmissions. Considering the link between oxidative damage, neuronal cell death and depressive disorders (Sapolsky, 2000), we have investigated the antioxidant activity of DHZ in the lipid peroxidation of brain regions induced by SNP. SNP is a neurotoxic agent that increases OH• and NO release, which leads to the oxidation of lipids, proteins and DNA and subsequent loss of membrane integrity (Halliwell, 1992; Hue and Padmaja, 1993; Requena et al., 2003). The antioxidant activity of DHZ was evidenced in hippocampus, cortex and cerebellum extracts after induction of in vitro lipid peroxidation by SNP. Abdel-Wahab and Salama (2011) have demonstrated that exposure of animals to stress caused by the FST and TST increases lipid peroxidation and DNA damage levels in the hippocampus, and venlafaxine, an approved antidepressant, had the capacity to antagonize this effect. In this sense, the lipid peroxidation and antioxidant defenses are considered integral components of stress and depression, as reported by Yager et al. (2010). Thus, in the search for new compounds and treatment strategies for depression that could improve conventional therapies by utilizing multiple targets, natural products are an alternative for the development of antidepressants with reduced or no adverse effects (Wang et al., 2008). Our findings suggest that DHZ has an antidepressant-like effect that is related to the modulation of serotonergic and noradrenergic systems, and the involvement of antioxidant activity should be an important avenue for further research. 5. Conclusions In summary, the present study reveals that DHZ has antidepressantlike activity and the mechanisms underlying this effect may involve the serotonergic and noradrenergic systems. The capacity to protect from lipid peroxidation in tissues from brain regions was also demonstrated. More studies are necessary to investigate other mechanisms involved in the DHZ antidepressant-like effect. Acknowledgments The project was supported by CAPES, CNPq (306824/2013-2 and 305132/2013-0) and FAPERGS (2012-2551/13-8 and 11/2026-4). References Abdel-Wahab BA, Salama RH. Venlafaxine protects against stress induced oxidative DNA damage in hippocampus during antidepressant testing in mice. Pharmacol Biochem Behav 2011;100:59–65. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm 2007;4:807–18. Arora V, Kuhad A, Tiwari V, Chopra K. Curcumin ameliorates reserpine-induced paindepression dyad: behavioural, biochemical, neurochemical and molecular evidences. Psychoneuroendocrinology 2011;36:1570–81. Bhutani MK, Bishnoi M, Kulkarni S. Anti-depressant like effect of curcumin and its combination with piperine in unpredictable chronic stress-induced behavioral, biochemical and neurochemical changes. Pharmacol Biochem Behav 2009;92:39–43. Brunello N, Mendlewicz J, Kasper S, Leonard B, Montgomery S, Nelson J, et al. The role of noradrenaline and selective noradrenaline reuptake inhibition in depression. Eur Neuropsychopharmacol 2002;12:461–75.
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