Antidepressant effect of Nigella sativa in stress-induced depression

 

Dheeraj Ahirwar¹, Bharti Ahirwar²

 

¹School of Pharmacy, Chouksey Engineering College, Bilaspur, (CG) India 495006

²Institute of Pharmacy, Guru Ghasidas Central University, Bilaspur (CG) India 495001

 

ABSTRACT:

Nigella sativa (NS) and its seeds have a long history of folkloric uses in all traditional systems of medicines globally for the treatment of various ailments. Therefore, the exploration of the antidepressant activity of this plant is to be the main aim. Nigella sativa seed powder was extracted with alcohol for 3 h in soxhlet. The extract was concentrated in the rotatory evaporator under reduced pressure. DPPH, superoxide radical scavenging, nitric oxide radical scavenging assay, and ABTS assay were carried out for antioxidant activity. The antidepressant activity was performed in stressed mice followed by tail suspension, locomotor activity, and forced swim test. Results indicated the potential antidepressant effect of NS extract in stressed mice, exposed to 6 h immobilizations. Methylene blue and SB-203580 potentiated the effect of sub-doses of NS in stressed mice along with significant antioxidant properties.  NS extract also significantly decreased plasma nitric oxide.Seed extract of NS may consider as a new herbal antioxidant and antidepressant agent.

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INTRODUCTION:

The mood disorder is commonly known as depression which is prevalent with multi symptoms and contributors to an encumbrance of disease globally. Impaired enzymatic function and excessive production of reactive oxygen species are due to depression or stress. Extreme stress leads to damage or imbalance neurotransmitters which affect brain functioning suggested neuropsychiatric disorders [1-3]. Stress disturbs the metabolism of lipids as free radical produced by lipid peroxidation and it also affects the function of proteins, nucleic acids and other cell structures which may lead to apoptosis [4].  Lipid peroxidation also produced well known Malondialdehyde (MDA) is a highly toxic aldehyde molecule that interacts with DNA and proteins may cause mutagenic [5]. In depression, increased MDA levels and excess production of free radicals affect glutaminergic transduction leads to increased glutamate concentration which causes neuron damage [6]. Glutamate stimulates the intracellular production of superoxide radicals [7]. In depression activated inducible nitric oxide synthase (iNOS) convert L-arginine to nitric oxide which is one of the free radicals in neutrophils, monocytes and macrophages [8].

 

Recently, excessive nitric oxide production is characterized in depressed patients and iNOS inhibitors are effective in depression treatment [9].  Currently, various therapeutic drugs have been used to treat depression, but due to their severe side effects such as psychomotor impairment, potentiation of other central depressant drugs and dependence liability, their clinical uses are still limited. Therefore, there is a need to consider herbal therapies as an alternative therapy and search for new herbal therapeutic drugs for the treatment of depression has progressed constantly across the world. Traditionally, many medicinal plants have been used in the treatment of depression and yet many herbal drugs are unexplored. Therefore, in this research, our aim was to explore Nigella sativa to treat stress-mediated depression as well as to elucidate the underlying mechanism in laboratory animals.

 

MATERIALS AND METHODS:

Nigella sativa seeds were procured from Local market, Bilaspur (CG) India. A taxonomist at Central Council for Research in Ayurveda and Siddha, Bangalore, India has confirmed its authentication. Specimen (RRCBI/NS-11-17) was stored in the departmental herbarium for future reference and stored. Dried seed powder (80 g) was extracted with ethanol for 3hours in a Soxhlet apparatus. The extract (oily fraction) was concentrated under reduced pressure and stored for further study.

Antioxidant screening of extract

Various in vitro antioxidant models were selected for the study such as DPPH free radical scavenging assay [10], superoxide radical scavenging activity [11], nitric oxide radical scavenging assay [12], and ABTS radical scavenging assay [13].

 

Animals

Swiss albino mice (22–30 g) were employed in the study. Animals were housed under laboratory conditions with alternating light and dark cycles of 12 h each. They had free access to food and water ad libidum. The animals were acclimatized to the laboratory conditions before behavioral experiments. Animal Experiment was carried according to CPCSEA-IAEC standard and after their approval (SOP/IAEC/21/2017).

 

Experimental protocol

Animals were divided into different groups. All the animals were stressed before the commencement of the experiment and after treatment, all the animals were subjected to tail suspension test, forced swim test and locomotor activity (actophotometer).  Animals of group I administered normal saline and treated as a control group. Group II animals were administered with normal saline at the onset of immobilization (6 h) and treated as immobilization group. Rats of group III were administered with Fluoxetine (15mg/kg, i.p) at the onset of immobilization. Group IV animals were treated with  NS extract (50 mg/kg). Group V animals were treated with  NS extract (100 mg /kg). Group VI animals were administered  NS extract (200 mg/kg). Group VII was administered with MB (15mg/kg). Group VIII animals were treated with MB + NS extract (15+100). Rats of group IX were treated with MB + NS extract (15+200). Group X animals were administered  SB (1mg/kg). Group XI animals administered SB + NS extract (1+100). Group XII was treated with SB +  NS extract (1+200).

 

Statistical analysis

All statistical analysis has been done using one-way analysis of variance (ANOVA) followed by Tukey’s test in the Graph Pad Instat (GPIS) package, version 3.05.

 

RESULTS AND DISCUSSION:

Data on table 1 revealed significant antioxidant activity of NS extract.

 

The potential antidepressant effect of Nigella sativa extract was found in stressed mice, exposed to 6 h immobilization. cGMP inhibitor MB is a downstream component of nitric oxide (NO) signaling pathway potentiated the sub-effective dose of  NS extract. Further, p38MAPkinase inhibitor SB-203580 was also observed to enhance the effect of NS extract (Table 2).

 

Table 1 Antioxidant activity of NS extract

Concentration (mg/mL)	% Inhibition on DPPH Radical	% Inhibition on Superoxide Radical	(% Inhibition) on Nitric oxide Radical	(% Inhibition) on ABTS Radical
	NS	NS	NS	NS
50	10.00	14.03	13.63	19.37
100	12.12	15.26	21.51	27.68
200	16.18	46.18	39.76	46.69
300	66.16	58.88	53.24	59.31
400	71.12	65.28	62.82	71.25
500	89.26	70.17	72.54	84.18
IC 50  (mg/mL)	226.72	254.75	281.74	214.78

NS = Nigella sativa

Table 2: The effect of different treatments on Immobility.

Treatment	Dose (mg/kg, i.p.)	Immobility time (sec)
		FST	TST	LMA
IM	6h	215 ± 11.3	234.2 ± 12.3	156.1 ± 13.0
FLU	15	125.4 ± 8.8a	154.4 ± 8.8a	137.1 ± 16
Extract	5	182.0 ± 10.5	192.3 ± 16.5	154.3 ± 15
EXTRACT	10	164.0 ± 7.8	185.2 ± 10.8	137.5 ± 11
EXTRACT	20	121.2 ± 9.3b	148.0 ± 15.3b	119.2 ± 10
MB	15	202.5 ± 13.9	236.7 ± 11.9	149.3 ± 19.6
MB + EXTRACT	15+10	104.3 ± 11.8c	125.4 ± 14.2c	138.5 ± 16.7
MB + EXTRACT	15+20	64.8 ± 15.3d	87.0 ± 7.3d	159.3 ± 15.8
SB	1	128.9 ± 10.4e	164.2 ± 13.4 e	141.6 ± 10
SB + EXTRACT	1+10	98.7 ± 12.8f	119.2 ± 8.1f	155.3 ± 14
SB + EXTRACT	1+20	59.7 ± 14.7g	74.1 ± 11.2g	143.8 ± 18.5

IM = Immobilization, FLU = Fluoxetine, MB = Methylene blue, SB = SB - 203580.

Values are expressed as Mean ± S.E, p < 0.0001, a = p < 0.001 significant difference from immobilized group, b = p < 0.001 significant difference from immobilized group, c = p < 0.05 significant difference from EXTRACT (10 mg/kg) treated group, d = p < 0.05 significant difference from extract (20 mg/kg) treated group, e = p < 0.001 significant from immobilized group, f = p < 0.05 significant from extract (10 mg/kg) treated group, g = p < 0.05significant from extract (20 mg/kg) treated group.

 

 

It is a well-known fact that SB-203580 is an upstream element of iNOS formation in and after stress. Immobilization is the combination of emotional and physiological stress and directly involve in stress while painful stimuli is not directly involved [14]. Stress induces the release of a series of macromolecules cascades and elevated nitric oxide is one of the major contributors in stress-induced depression [15]. It is well documented that within 6 h immobilization activates the release of TNF- α via activation of NF-kappa B [16]. TNF-α plays a crucial role to activate the mitogen, and protein kinase pathways (MAPK) p42/p44 MAPK, JNK/SAPK, and p38 activated by mitogen, and p38 is responsible for interleukin-6 production [17]. Thus, it is hypothesized that immobilization-induced stress or depression involved inactivation of p38 MAPkinase and as a result increased the immobility duration and induce the symptoms of depression in the behavioral model [18]. Fluoxetine is selective serotonin reuptake inhibitors that blocked the reuptake of serotonin, resulted in increased extracellular serotonin concentrations in brain which exert an antidepressant effect. In comparison with the stress control group, fluoxetine significantly decreased the attack of depression frequency support the hypothesis that NOS inhibitors can be a new class of antidepressant drugs that may act on neuronal NOS [19]. Further, NOS inhibitors may exert their effect on enzyme level without affecting any changes in cellular biochemistry and dependency. Within 2 h stress has been reported to activate NOS which produced anxiety in rodents [18]. In the nitric oxide overproduction along with enhancing cytokine, glutamate level in stress induced by so many factors through NFκB activation [20]. NS extract significantly decreased plasma nitrite in immobilization-induced stressed mice group may be inhibiting iNOS mRNA expression. Similarly, SB-203580 treated group showed decreased plasma nitrite levels and produced an antidepressant effect (Table 3).

 

cGMP is a second messenger in neuronal cell–cell communication and in cell–cell signaling from between presynaptic fibers as well as between postsynaptic structures [21]. In the present study, MB significantly enhanced the antidepressant effect of Nigella sativa extract (NS) in stressed mice by influencing the NO–cGMP signaling pathway, thereby, preventing the further downstream signaling of nitrergic stimulus induced by immobilization stress [22].  None of the treatments produced any significant effect on locomotor activity.

 

 

Table 3: The effect of different treatments on plasma nitrite levels (µmol/L).

IM = Immobilization, FLU = Fluoxetine, MB = Methylene blue, SB = SB-203580.

Values are expressed as Mean ± S.E, p < 0.0001, a = p < 0.001 significant difference from immobilized group, b = p < 0.01 significant difference from immobilized group, c = p < 0.05 significant difference from extract (10 mg/kg) treated group, d = p < 0.05 significant difference from extract (20 mg/kg) treated group, e = p < 0.001 significant from immobilized group, f = p < 0.05 significant from extract (10 mg/kg) treated group, g = p < 0.05significant from extract (20 mg/kg) treated group.

 

Table 4: The effect of vehicle and immobilization on total immobility time. Values expressed in mean ± S.E.    p = 0.0003. Veh: vehicle, IMMO: immobilization.

Dose	Immobility time (sec) in FST	Immobility time (sec) in TST	Immobility time (sec) in LMA	Plasma nitrite level (µmol/L
Veh 10 ml/kg	144.75 ± 7.2	188.5 ± 10.6	327.6 ± 15.3	14.3 ± 1.1
IM  6h	215.9 ± 11.3	234.2 ± 12.3	156.1 ± 13.0	24.2 ± 1.8

 

Further, NS significantly influence plasma nitrite levels (Table 4), an indicator of NO production in depression, which further exerts their action on depression symptoms rather than on locomotor aspect involved in the behavior of mice in both the behavioral paradigms used in the present study.

 

CONCLUSION:

The present study was designed to investigate the possible effect of Nigella sativa, in mice under stressed conditions. Administration of Nigella sativa extract decreases the immobility time as well as plasma nitrite levels. MB and SB-203580 enhance the effect of Nigella sativa extract in stressed mice suggested NS is a good herbal antidepressant and antioxidant drug.

 

ACKNOWLEDGMENT:

The authors are thankful to Science and Engineering Research Board, New Delhi, India (SB/EMEQ-325/2014) for financial support.

 

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Received on 18.12.2019         Modified on 10.02.2020

Accepted on 20.03.2020         © RJPT All right reserved