490 Original Article
Authors
M. Aquib, A. K. Najmi, M. Akhtar
Affiliation
Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
Key words ▶ depression ● ▶ thymoquinone ● ▶ modified FST ● ▶ TST ●
Abstract
received 05.06.2014 accepted 21.08.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1389920 Published online: September 10, 2014 Drug Res 2015; 65: 490–494 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Dr. M. Akhtar, Assistant Professor Department of Pharmacology Faculty of Pharmacy Jamia Hamdard (Hamdard University) M. B Road Hamdard Nagar New Delhi-110 062 India Tel.: + 91/011/26059 688 Fax: + 91/011/26059 666 Mobile: + 91/9811777883 makhtar_ph@jamiahamdard. ac.in [email protected]
▼
Objective: The present study was carried out to determine the role of thymoquinone (TQ) in modulating the levels of neurotransmitter and reducing the oxidative stress in animal models of depression. Material and Methods: Mice were divided into 5 groups, each group had 6 animals. TQ (20 mg/ kg) in corn oil and fluoxetine (10 mg/kg) in normal saline were administered intraperitoneally (i.p.) half an hour before performing behavioural tests. Modified forced swim test (MFST) and tail suspension test (TST) were used to assess the antidepressant effect in mice. Animals were sacrificed and their brains were removed for biochemical estimation after performing behavioural tests. Results: TQ treatment showed increased swimming, climbing and decreased immobility times in MFST and TST. Combination of TQ
Introduction
▼
Depression is a psychiatric disorder with a lifetime prevalence approaching 17 % [1]. It is projected by the World Health Organization as the leading cause disease burden for the year 2030 [2, 3]. Worldwide depression is a major cause of disability and premature death in addition to the significant suicide risk [4]. Depression is also associated with the development of heart disease and cancer [5]. The treatment of depression with available medications provides a complete remission just for 50–60 % of the patients [6, 7] and produce side effects that may reduce the patients acceptance for the treatment [8]. Herbal medicines are gaining popularity nowadays as alternative therapies for depression. Thus, developing safe and effective agents from traditional herbs may provide a good way to
Aquib M et al. Role of Thymoquinone in Depression … Drug Res 2015; 65: 490–494
with fluoxetine in MFST and TST showed potentiating effect in the present study. A significant elevation of 5-hydroxytryptamine (5-HT) levels was observed following TQ administration in the behavioural models studied. MFST and TST reduced glutathione and elevated TBARS levels in mice. Pre-treatment of TQ restored glutathione and decreased TBARS levels. TQ combination with fluoxetine also showed reduction of TBARS and increased glutathione levels. Conclusion: TQ demonstrated antidepressant effects in MFST and TST respectively in the present study. It further demonstrated antioxidant effects by reducing thiobarbituric acid reactive substance (TBARS) and increasing reduced glutathione (GSH) levels. Although our results are preliminary, further investigations may be required however, based on afore mentioned results, it may be suggested that TQ could be a potential candidate for the management of depression.
reduce the risk of remission as well as to improve the efficacy of conventional treatment [9–11]. Nigella sativa L. is a member of the Ranunculaceae family; its seeds (black cumin) have been used as additives in food products as well as for medicinal purposes in many countries [12]. Several studies have demonstrated that Nigella sativa and its active constituent thymoquinone (TQ) possess several pharmacological properties such as analgesic [13], inhibit eicosanoid generation and membrane lipid peroxidation [14, 15], antiinflammatory [13, 16], protective effect against chemical-induced carcinogenesis [17], anticonvulsant [18], anti-anxiety-like effect [19] and protective effect in diabetic neuropathy [20]. TQ is a phytochemical, which exhibits antioxidant and anti-inflammatory properties that may be used in the therapy for neurodegenerative diseases. It was demonstrated by Adel A. Alhamdan
Downloaded by: University of Liverpool. Copyrighted material.
Antidepressant Effect of Thymoquinone in Animal Models of Depression
Original Article 491
Materials and Methods
▼
Animals
The experimental study was carried out under controlled condition. Albino male mice 30–40 g were procured from the Central Animal House Facility of Hamdard University, New Delhi and acclimatized accordingly. The mice were maintained on a 12 h light dark cycle with free access to food and water. The study was approved by the Institutional Animal Ethics Committee (Project No.1002) on 30th January 2014, Jamia Hamdard (Hamdard University), New Delhi.
Study design
The animals were divided into 5 different groups and each experimental group had 6 animals. GP I – Normal saline, GP II – Corn oil, GP III – Fluoxetine (10 mg/kg, i.p.), GP IV – Thymoquinone (TQ) per se (20 mg/kg, i.p.), GP V – Fluoxetine (10 mg/kg, i.p.) + TQ (20 mg/kg, i.p.). TQ (20 mg/kg, dissolved in corn oil) and fluoxetine (10 mg/kg, dissolved in normal saline) were administered intraperitoneally half an hour before performing behavioural tests. Immediately after behavioural tests, the animals were sacrificed under light anaesthesia and their brains were removed for biochemical estimations.
Methodology a) Modified forced swim test (MFST) [31, 32]
The mice were forced to swim individually in cylinder (40 cm height, 15 cm diameter) containing fresh water (temperature 22 ± 2º) up to a height of 30 cm for 15 min. This constituted the
‘pre-test’ swim. 24 h later, each mouse was re-exposed to the swimming condition in a similar environment in a 6 min ‘test session’. The total duration of climbing, swimming and immobility in the last 5 min of the 6 min test session was recorded for each animal.
b) Tail suspension test (TST) [33]
The mice were suspended by the tail using adhesive scotch tape to a hook connected to a strain gauge that picked up all the movements of the mouse and transmitted them to a central unit that calculated the total duration of immobility. If antidepressant treatments are given prior to the test, the animals will actively persist engaging in escape-directed behaviours for longer periods of time than after vehicle treatment. The total duration of immobility for 6 min duration was recorded either manually or through an automated device.
c) Thiobarbituric acid reactive substances (TBARS) estimation [34]
One ml of the suspension was taken from the supernatant of the 10 % tissue homogenate and centrifuged at 10 000 rpm. 0.5 ml of 30 % TCA followed by 0.5 ml of 0.8 % TBA was added to it. The tubes were covered with aluminum foils and kept in a shaking water bath for 30 min at 80 °C. After 30 min, the tubes were taken out and kept in ice-cold water for 10 min. They were then centrifuged at 3 000 rpm for 15 min. The absorbance of the supernatant was read at 540 nm at room temperature against an appropriate blank. Blank consisted of 1.0 ml distilled water, 0.5 ml 30 % TCA, 0.5 ml 0.8 % TBA.
d) Tissue glutathione estimation [35, 36]
A known weight of brain tissue was homogenized in 5 ml of 0.02 M EDTA and then 4.0 ml of cold distilled water was added to it. After mixing it well, 1 ml of 50 % trichloroacetic acid (TCA) was added and shaken intermittently for 10 min using a vortex mixer. After 10 min the contents were transferred to centrifuge tubes (rinsed in EDTA) and centrifuged at 6 000 rpm for 15 min. Following centrifugation, 2 ml of the supernatant was mixed with 4.0 ml of 0.4 M Tris buffer (pH 8.9). The whole solution was mixed well and 0.1 ml of 0.01M DTNB was added to it. The absorbance was read within 5 min of the addition of DTNB at 412 nm against a reagent blank with no homogenate.
e) 5-HT estimation [37]
Whole brain was homogenized in 10 volumes of cold acidified n-butanol using an Ultra Turax homogenizer. All other areas (weighing less than 300 mg) were homogenized in 3 ml of acidified butanol. After centrifugation for 5 min at 3 000 rev/min, 2–5 ml of the supernatant was pipette into a 25 ml glass stopper tube and shaken mechanically for 5 min with 5 ml n-heptane and 0.4 ml 0.1 N HCI containing 0.1 % L-cysteine. The phases were separated by centrifugation as before and 5 ml of the organic phase retained for the 5-HIAA determination. To determine 5-HT, 0.1 ml samples of the aqueous phase were pipetted into 12 × 125 mm test tubes and 0.6 ml of 0.004 % OPT in 10 N HCI added. After mixing with a Rota mixer and heating in a boiling water bath for 15 min, the tubes were cooled in water and fluorescence measured in micro-cuvette using spectrophotofluorometer. Activation and fluorescent wavelengths were 360 nm and 470 nm respectively. Standards were prepared as 60 ug/ml solutions in deionized water (stored at − 25 °C), diluted 1:100 for use with 0.1 N HCI containing 0.1 % cysteine and 0.1 ml reacted Aquib M et al. Role of Thymoquinone in Depression … Drug Res 2015; 65: 490–494
Downloaded by: University of Liverpool. Copyrighted material.
that TQ produced neuroprotective effect on repeated immobilization stress-induced oxidative stress in rats [21]. Perveen et al. reported that after 4 weeks daily administration of Nigella sativa oil (active constituent TQ), the rats exhibited increased in concentrations of 5 hydroxy tryptamine (5-HT) and 5- hydroxy indole acetic acid (5-HIAA) in brain [22]. It is well established that depression is caused by a functional deficit of monoamine neurotransmitter concentrations especially 5-HT and noradrenaline in the brain [23]. TQ has anti-inflammatory action too [16] and it is also well-known that inflammation increases the production of free radicals and such an increase was also found in depressed patients [24–26]. The forced swimming test (FST) and tail suspension test (TST) are chosen in the present study as both are non-escapable stressful situations and are widely used for screening substances for potential antidepressant effects [27, 28]. When animals are forced to swim or are hung upside down by the tail in an inescapable situation, they tend to become immobile after initial vigorous activity. Moreover, substances that decrease immobility often have antidepressant properties in humans. This immobility has been described as a symptom of “behavioural despair” [27] and both tests have been suggested as animal models of human depression [27, 28]. Stressful events are considered an important factor in the development of central nervous system disorders including depression [29] and a single experience of a stressful event enhances the vulnerability to stress-related disorders [30]. Thus, the present study was carried out to determine the role of TQ in modulating the levels of neurotransmitters especially 5-HT and reducing the oxidative stress in animal models of depression.
492 Original Article
Table 1 Effect of thymoquinone on modified forced swim test in mice. Group
Treatment (n = 6)
I II III IV V
Normal saline Corn oil Fluoxetine Thymoquinone per se Fluoxetine + thymoquinone
Dose (mg/kg, i.p.) 1 ml 1 ml 10 20 10 + 20
Swimming time (s) 114 ± 3.48 115.5 ± 3.731 177.66 ± 4.89 * * 174.5 ± 3.01 * * 193.16 ± 2.66 * * @
Climbing time (s) 11.33 ± 1.29 12.08 ± 1.03 27.16 ± 2.37 * * * 23.66 ± 1.78 * * 36.66 ± 2.43 * * @
Immobility time (s) 113.5 ± 4.28 111.41 ± 5.47 32.16 ± 2.12 * * 40.16 ± 2.35 * * 10.16 ± 2.85 * * @
n = Number of animals, all values were expressed as mean ± SEM of 6 mice and data were analyzed by one way ANOVA followed by Dunnett Multiple Comparisons Test. * * * p
Authors
M. Aquib, A. K. Najmi, M. Akhtar
Affiliation
Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
Key words ▶ depression ● ▶ thymoquinone ● ▶ modified FST ● ▶ TST ●
Abstract
received 05.06.2014 accepted 21.08.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1389920 Published online: September 10, 2014 Drug Res 2015; 65: 490–494 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Dr. M. Akhtar, Assistant Professor Department of Pharmacology Faculty of Pharmacy Jamia Hamdard (Hamdard University) M. B Road Hamdard Nagar New Delhi-110 062 India Tel.: + 91/011/26059 688 Fax: + 91/011/26059 666 Mobile: + 91/9811777883 makhtar_ph@jamiahamdard. ac.in [email protected]
▼
Objective: The present study was carried out to determine the role of thymoquinone (TQ) in modulating the levels of neurotransmitter and reducing the oxidative stress in animal models of depression. Material and Methods: Mice were divided into 5 groups, each group had 6 animals. TQ (20 mg/ kg) in corn oil and fluoxetine (10 mg/kg) in normal saline were administered intraperitoneally (i.p.) half an hour before performing behavioural tests. Modified forced swim test (MFST) and tail suspension test (TST) were used to assess the antidepressant effect in mice. Animals were sacrificed and their brains were removed for biochemical estimation after performing behavioural tests. Results: TQ treatment showed increased swimming, climbing and decreased immobility times in MFST and TST. Combination of TQ
Introduction
▼
Depression is a psychiatric disorder with a lifetime prevalence approaching 17 % [1]. It is projected by the World Health Organization as the leading cause disease burden for the year 2030 [2, 3]. Worldwide depression is a major cause of disability and premature death in addition to the significant suicide risk [4]. Depression is also associated with the development of heart disease and cancer [5]. The treatment of depression with available medications provides a complete remission just for 50–60 % of the patients [6, 7] and produce side effects that may reduce the patients acceptance for the treatment [8]. Herbal medicines are gaining popularity nowadays as alternative therapies for depression. Thus, developing safe and effective agents from traditional herbs may provide a good way to
Aquib M et al. Role of Thymoquinone in Depression … Drug Res 2015; 65: 490–494
with fluoxetine in MFST and TST showed potentiating effect in the present study. A significant elevation of 5-hydroxytryptamine (5-HT) levels was observed following TQ administration in the behavioural models studied. MFST and TST reduced glutathione and elevated TBARS levels in mice. Pre-treatment of TQ restored glutathione and decreased TBARS levels. TQ combination with fluoxetine also showed reduction of TBARS and increased glutathione levels. Conclusion: TQ demonstrated antidepressant effects in MFST and TST respectively in the present study. It further demonstrated antioxidant effects by reducing thiobarbituric acid reactive substance (TBARS) and increasing reduced glutathione (GSH) levels. Although our results are preliminary, further investigations may be required however, based on afore mentioned results, it may be suggested that TQ could be a potential candidate for the management of depression.
reduce the risk of remission as well as to improve the efficacy of conventional treatment [9–11]. Nigella sativa L. is a member of the Ranunculaceae family; its seeds (black cumin) have been used as additives in food products as well as for medicinal purposes in many countries [12]. Several studies have demonstrated that Nigella sativa and its active constituent thymoquinone (TQ) possess several pharmacological properties such as analgesic [13], inhibit eicosanoid generation and membrane lipid peroxidation [14, 15], antiinflammatory [13, 16], protective effect against chemical-induced carcinogenesis [17], anticonvulsant [18], anti-anxiety-like effect [19] and protective effect in diabetic neuropathy [20]. TQ is a phytochemical, which exhibits antioxidant and anti-inflammatory properties that may be used in the therapy for neurodegenerative diseases. It was demonstrated by Adel A. Alhamdan
Downloaded by: University of Liverpool. Copyrighted material.
Antidepressant Effect of Thymoquinone in Animal Models of Depression
Original Article 491
Materials and Methods
▼
Animals
The experimental study was carried out under controlled condition. Albino male mice 30–40 g were procured from the Central Animal House Facility of Hamdard University, New Delhi and acclimatized accordingly. The mice were maintained on a 12 h light dark cycle with free access to food and water. The study was approved by the Institutional Animal Ethics Committee (Project No.1002) on 30th January 2014, Jamia Hamdard (Hamdard University), New Delhi.
Study design
The animals were divided into 5 different groups and each experimental group had 6 animals. GP I – Normal saline, GP II – Corn oil, GP III – Fluoxetine (10 mg/kg, i.p.), GP IV – Thymoquinone (TQ) per se (20 mg/kg, i.p.), GP V – Fluoxetine (10 mg/kg, i.p.) + TQ (20 mg/kg, i.p.). TQ (20 mg/kg, dissolved in corn oil) and fluoxetine (10 mg/kg, dissolved in normal saline) were administered intraperitoneally half an hour before performing behavioural tests. Immediately after behavioural tests, the animals were sacrificed under light anaesthesia and their brains were removed for biochemical estimations.
Methodology a) Modified forced swim test (MFST) [31, 32]
The mice were forced to swim individually in cylinder (40 cm height, 15 cm diameter) containing fresh water (temperature 22 ± 2º) up to a height of 30 cm for 15 min. This constituted the
‘pre-test’ swim. 24 h later, each mouse was re-exposed to the swimming condition in a similar environment in a 6 min ‘test session’. The total duration of climbing, swimming and immobility in the last 5 min of the 6 min test session was recorded for each animal.
b) Tail suspension test (TST) [33]
The mice were suspended by the tail using adhesive scotch tape to a hook connected to a strain gauge that picked up all the movements of the mouse and transmitted them to a central unit that calculated the total duration of immobility. If antidepressant treatments are given prior to the test, the animals will actively persist engaging in escape-directed behaviours for longer periods of time than after vehicle treatment. The total duration of immobility for 6 min duration was recorded either manually or through an automated device.
c) Thiobarbituric acid reactive substances (TBARS) estimation [34]
One ml of the suspension was taken from the supernatant of the 10 % tissue homogenate and centrifuged at 10 000 rpm. 0.5 ml of 30 % TCA followed by 0.5 ml of 0.8 % TBA was added to it. The tubes were covered with aluminum foils and kept in a shaking water bath for 30 min at 80 °C. After 30 min, the tubes were taken out and kept in ice-cold water for 10 min. They were then centrifuged at 3 000 rpm for 15 min. The absorbance of the supernatant was read at 540 nm at room temperature against an appropriate blank. Blank consisted of 1.0 ml distilled water, 0.5 ml 30 % TCA, 0.5 ml 0.8 % TBA.
d) Tissue glutathione estimation [35, 36]
A known weight of brain tissue was homogenized in 5 ml of 0.02 M EDTA and then 4.0 ml of cold distilled water was added to it. After mixing it well, 1 ml of 50 % trichloroacetic acid (TCA) was added and shaken intermittently for 10 min using a vortex mixer. After 10 min the contents were transferred to centrifuge tubes (rinsed in EDTA) and centrifuged at 6 000 rpm for 15 min. Following centrifugation, 2 ml of the supernatant was mixed with 4.0 ml of 0.4 M Tris buffer (pH 8.9). The whole solution was mixed well and 0.1 ml of 0.01M DTNB was added to it. The absorbance was read within 5 min of the addition of DTNB at 412 nm against a reagent blank with no homogenate.
e) 5-HT estimation [37]
Whole brain was homogenized in 10 volumes of cold acidified n-butanol using an Ultra Turax homogenizer. All other areas (weighing less than 300 mg) were homogenized in 3 ml of acidified butanol. After centrifugation for 5 min at 3 000 rev/min, 2–5 ml of the supernatant was pipette into a 25 ml glass stopper tube and shaken mechanically for 5 min with 5 ml n-heptane and 0.4 ml 0.1 N HCI containing 0.1 % L-cysteine. The phases were separated by centrifugation as before and 5 ml of the organic phase retained for the 5-HIAA determination. To determine 5-HT, 0.1 ml samples of the aqueous phase were pipetted into 12 × 125 mm test tubes and 0.6 ml of 0.004 % OPT in 10 N HCI added. After mixing with a Rota mixer and heating in a boiling water bath for 15 min, the tubes were cooled in water and fluorescence measured in micro-cuvette using spectrophotofluorometer. Activation and fluorescent wavelengths were 360 nm and 470 nm respectively. Standards were prepared as 60 ug/ml solutions in deionized water (stored at − 25 °C), diluted 1:100 for use with 0.1 N HCI containing 0.1 % cysteine and 0.1 ml reacted Aquib M et al. Role of Thymoquinone in Depression … Drug Res 2015; 65: 490–494
Downloaded by: University of Liverpool. Copyrighted material.
that TQ produced neuroprotective effect on repeated immobilization stress-induced oxidative stress in rats [21]. Perveen et al. reported that after 4 weeks daily administration of Nigella sativa oil (active constituent TQ), the rats exhibited increased in concentrations of 5 hydroxy tryptamine (5-HT) and 5- hydroxy indole acetic acid (5-HIAA) in brain [22]. It is well established that depression is caused by a functional deficit of monoamine neurotransmitter concentrations especially 5-HT and noradrenaline in the brain [23]. TQ has anti-inflammatory action too [16] and it is also well-known that inflammation increases the production of free radicals and such an increase was also found in depressed patients [24–26]. The forced swimming test (FST) and tail suspension test (TST) are chosen in the present study as both are non-escapable stressful situations and are widely used for screening substances for potential antidepressant effects [27, 28]. When animals are forced to swim or are hung upside down by the tail in an inescapable situation, they tend to become immobile after initial vigorous activity. Moreover, substances that decrease immobility often have antidepressant properties in humans. This immobility has been described as a symptom of “behavioural despair” [27] and both tests have been suggested as animal models of human depression [27, 28]. Stressful events are considered an important factor in the development of central nervous system disorders including depression [29] and a single experience of a stressful event enhances the vulnerability to stress-related disorders [30]. Thus, the present study was carried out to determine the role of TQ in modulating the levels of neurotransmitters especially 5-HT and reducing the oxidative stress in animal models of depression.
492 Original Article
Table 1 Effect of thymoquinone on modified forced swim test in mice. Group
Treatment (n = 6)
I II III IV V
Normal saline Corn oil Fluoxetine Thymoquinone per se Fluoxetine + thymoquinone
Dose (mg/kg, i.p.) 1 ml 1 ml 10 20 10 + 20
Swimming time (s) 114 ± 3.48 115.5 ± 3.731 177.66 ± 4.89 * * 174.5 ± 3.01 * * 193.16 ± 2.66 * * @
Climbing time (s) 11.33 ± 1.29 12.08 ± 1.03 27.16 ± 2.37 * * * 23.66 ± 1.78 * * 36.66 ± 2.43 * * @
Immobility time (s) 113.5 ± 4.28 111.41 ± 5.47 32.16 ± 2.12 * * 40.16 ± 2.35 * * 10.16 ± 2.85 * * @
n = Number of animals, all values were expressed as mean ± SEM of 6 mice and data were analyzed by one way ANOVA followed by Dunnett Multiple Comparisons Test. * * * p
