INTRODUCTION:

Depression is a common mental disorder that presents with depressed mood, loss of interest or pleasure, decreased energy, feelings of guilt or low self-worth, disturbed sleep or appetite, and poor concentration. It is a potentially life-threatening disorder that affects hundreds of millions of people all over the world. It can occur at any age from childhood to late life and is a prodigious cost to society as this disorder causes severe distress and disruption of life and, if left untreated, can be fatal¹. The World Health Organization (WHO) has ranked depression as the 4^th leading cause of disability worldwide and projects that by 2020, it will be the second leading cause of premature death^2,3. In conventional system of medicine, treatment outcome of depression is suboptimal.

The use of currently available antidepressants i.e Flouxetine, Sertraline, Paroxetine (Selective serotonin reuptake inhibitors), Imipramine, Desipramine (Tricyclic antidepressant), Selegiline (Monoamine oxidase inhibitors) and many more drugs are limited by their side effects such as nausea, dry mouth, hypertention⁴, tremor³, insomnia¹, mania², sexual dysfunction etc.⁵, slow response, and inadequate treatment efficacy⁶ Full remission is difficult to be achieved.

Several drugs have been reported to have beneficial effects in psychiatric (mental) disorders. The ancient Unani system of medicine, mentions a number of single and compound drug formulations of plant origin that are used for the treatment of psychiatric disorders.⁷ Sharbat Ahmed Shahi (SAS) is one of them. It is a compound formulation usually prescribed for the patients of depression in Unani system of medicine since long time due to its minimum side effects as compared to its counter synthetic antidepressants. SAS is herbal formulation, strengthens heart and brain. It is effective in melancholia, mania, psychoneurosis and palpitation. It is beneficial in cerebral insufficiency^8,9 and has been scientifically proved to possess various pharmacological activities like anxiolytic activity¹⁰ and schizophrenia¹¹.

Sharbat Ahmad Shahi traditionally used in depression so the present study was undertaken to evaluate the antidepressant activity of SAS by employing forced swimming test and tail suspension test in swiss albino mice and hence oxidative stress is considered to be an essential predisposing factor in the aetiology of depressive illness, we deliberated the cerebral levels of glutathione (GSH) and thiobarbituric acid reactive substance (TBARS) in mice brain.

METHODS:

Animals:

Swiss albino mice of either sex weighing 25-30g bred in Central Animal House Facility, Jamia Hamdard, New Delhi were used. Before performing the experiment all the animals were maintained under standard laboratory conditions throughout the experimental period and administered with standard rodent diet and water ad libitum. The study was approved by the Institutional Animal Ethics Committee (IAEC) (1501/CPCSEA, 2018) and was carried out in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Drug and doses:

All the drug solutions were freshly prepared prior to the experiment. Distilled water, Fluoxetine (Standard drug) was given orally in doses of 10ml/kg and 20mg/kg body weight, respectively. In each experiment the test drugs (SAS, HAESAS, AqESAS) and standard drug ‘Fluoxetine’ were diluted with distilled water to the desired concentration for dose administration to the mice. All the drugs were administered once orally via gauge needle.

The dose of the test drugs for mice was calculated by multiplying the human dose with conversion factor of 12 by the method of Freireich et al. The human therapeutic dose of SAS is 25-35 ml⁸ as mention in the classical Unani literature. The higher dose of SAS was given to the mice in present study. The human dose of SAS was converted to mice dose as 6ml/kg. The hydroalcoholic and aqueous extract of SAS was calculated on the yield percentage of extract with reference to the dose of crude drug which was found to be 17.00% and 32.00%, respectively. So the dose of hydroalcoholic and aqueous extract was found to be 0.34mg/kg and 0.64mg/kg, respectively.

Treatment schedule:

Table 1: Treatment schedule

S. No	Group	Treatment	Dosage (p.o)	No of Animals	Duration
Ι	Control	Distilled water	10ml/kg	6	14 Days
ΙΙ	Standard	Fluoxetine	20mg/kg	6	14 Days
ΙΙΙ	Test Group A	SAS	6ml/kg	6	14 Days
ΙV	Test Group B	HAE	0.34 mg/kg	6	14 Days
V	Test Group C	AqE	0.64 mg/kg	6	14 Days

SAS= Sharbat Ahmad Shahi, HAE= Hydroalcoholic Extract, AqE=Aqueous extract

Behavioural tests:

Forced swimming test:

FST or behaviour despair by¹² was proposed as a model to test for antidepressant like activity. Depression was produced by forcing the animal to swim individually in a cylindrical shaped container (height 25cm and 20cm in diameter, containing fresh water of 15cm height and maintained at 25±1ºC). This constituted pre-test session. After 15 min they were removed from water and blow dried with a hair dryer (set in warm mode) and returned to their cages. The mice were divided into five groups (n=6) and all the drugs will be administered. 23.30 hours after pre-test, the mice of first group were given distilled water in the dose 10ml/kg (control group) for 14 days, group ΙΙ were given Fluoxetine (Standard drug) in a dose of 20mg/kg orally, while Group ΙΙΙ, ΙV and V were given Test drugs i.e 6ml/kg of SAS, hydro alcoholic and aqueous extract (0.34mg/kg and 0.64mg/kg) orally, respectively. After 30 mins each mouse was left in the container with water as above for 6 mins and in the last 4 mins, the following behaviours were recorded:

· Immobility: Floating in water without escape behaviour.

· Swimming: Active movements and circling in water.

· Climbing: Active movements of forelimbs on the wall of the container.

Water was changed after testing each animal as used water could show behavioural alarm signals. After the test, the mice were blow dried and returned to their cages.

Tail suspension test:

Tail suspension test was performed according to the method described by¹³. Depression was produced by suspending the animal from the edge of a table 60cm above the floor by an adhesive tape placed approximately 1cm from the tip of the tail. Immobility time was recorded during a 6mins period. Mice were considered immobile only when they hung passively and were completely motionless. The changes in the immobility duration were studied after administering the drugs 1hr prior to test in separate groups of animals. The antidepressant activity was expressed as reduction in the immobility duration between the control, standard and animal treated with test drugs.

Biochemical test:

After the behavioural testing, animals were decapitated and the brains were quickly removed and rinsed with ice-cold saline and then homogenized in chilled phosphate buffer (0.1M; pH-7.4) 10 times (w/v) and centrifuged at 800 × g at 4ºC for 5 min. The obtained supernatant was again centrifuged at 10,000g at 4ºC for 10min to collect the post mitochondrial supernatant (PMS) for estimation of oxidative stress markers. Thiobarbituric acid reactive substance (TBARS) and glutathione (GSH) were measured according to the methods of^14,15.

STATISTICAL ANALYSIS:

After performing all the experiments data are presented as mean±SEM. The data was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test for comparing control and the various groups using Graph Pad software. Statistical significance was estimated at the 5% level.

RESULTS:

Forced swimming test:

After administration of all the test drugs and standard drug for 14 days in mice, it was found that after treatment of mice with Fluoxetine (FXT), Sharbat Ahmad Shahi (SAS), hydroalcoholic extract of SAS (HAESAS) and aqueous extract of SAS (AqESAS), showed a significant decrease in immobility time in comparison to control. It was also observed that SAS (p<0.001), HAESAS (p<0.001) and AqESAS (p<0.001) treated mice showed significant decrease in immobility time as compared to FXT.

All the treated groups viz FXT (P<0.001), SAS (p<0.001), HAESAS (p<0.01) and AqESAS (p<0.05) increased the swimming duration in comparison to control. AqESAS increase the swimming time as compared to FXT treated group but the difference was statistically significant (p<0.05). While in case of SAS and HAESAS treated group the difference were statistically not significant.

In climbing time it was also observed that FXT (p<0.001), SAS (p<0.001), HAESAS (p<0.01) and AqESAS (p<0.05) increased the duration of climbing time as compared to control group.

Fig 1: The data was represented after analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. ***p<0.001 as compared vs control,^$$$p<0.001 as compared vs FXT.

Fig 2: The data was represented after analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests.*p<0.05, **p<0.01,***p<0.001 as compared vs control ^$p<0.05 as compared vs FXT.

SAS also increased the climbing time as compared to FXT treated group, but the difference was not statistically significant. While the increament produced by HAESAS (p<0.01) and AqESAS (p<0.001) were statistically significant as compared to FXT treated group. It was also observed that AqESAS showed significant increase in climbing time (p<0.05) as compared to SAS as shown in Table-1, Fig- 1, 2 and 3.

Tail suspension test:

It was found that pre-treatment of mice with Fluoxetine (FXT), Sharbat Ahmad Shahi (SAS), hydroalcoholic extract of SAS (HAESAS) and aqueous extract of SAS (AqESAS), showed a significant decrease in immobility time in comparison to control. It was also observed that SAS treated group decrease the immobility time as compared to standard group i.e Fluoxetine but the difference was not statistically significant. While decreament produced by HAESAS (p<0.001) and AqESAS (p<0.001) was statistically significant. Inter-comparison of HAESAS and AqESAS group, it was observed that decrease in immobility time was more in HAESAS group, but the difference was not statistically significant as shown in Table-2, Fig-4.

Fig 3: The data was represented after analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. *p<0.05, **p<0.01, ***p<0.001, as compared vs control ^$$p<0.01, ^$$$p<0.001as compared vs FXT and ^#p<0.05 as compared vs SAS.

Tail suspension test:

Table 3: Effect of Fluoxetine, Sharbat Ahmad Shahi, HAESAS and AqESAS on Tail Suspension Test (TST)

S. No	Group	Mean±SEM
1	Control	189±5.504
2	Fluoxetine (20mg/kg/day, oral)	21.00±2.671^***
3	SAS (6ml/kg/day, oral)	56.00±1.807^***
4	HAESAS (0.34mg/kg/day, oral)	130.5±14.17^***$$$###
5	AqESAS (0.64mg/kg/day, oral)	141.2±10.91^**$$$###

Fig 4: The data was represented after analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. **p<0.01, ***p<0.001 as compared vs control ^$$$p<0.001 as compared vs FXT and ^###p<0.001 as compared vs SAS.

Biochemical test:

Effect of Fluoxetine, Sharbat Ahmad Shahi, HAESAS and AqESAS on Stress marker LPO (TBARS) in brain tissue of mice:

After behavioural parameters, we estimated oxidative stress markers in mice by oral administration of drugs such as Fluoxetine (FXT), Sharbat Ahmad Shahi (SAS), Hydroalcoholic extract of SAS (HAESAS), Aqueous extract of SAS (AqESAS). The results depicted a significant decrease in TBARS level in mice brain treated with standard drug FXT (p<0.001) and test drugs SAS (p<0.001), HAESAS (p<0.01) and AqESAS (p<0.05) as compared to control. These outcomes show a potential effect of standard and test drug against oxidative stress. It was observed that all the test groups decrease the TBARS level as compared to standard group, but the differences were not statistically significant. Similarly in inter-comparison of test drugs, it was seen that decrease was slightly more in SAS group as compared to HAESAS and AqESAS group but difference were not significant statistically as shown in Table-3, Fig-5.

Table 4: Effect of Fluoxetine, Sharbat Ahmad Shahi, HAESAS and AqESAS on Stress marker LPO (TBARS) in brain tissue of mice

S. No	Group	LPO µmoles TBARS formed/h/g tissue Mean±SEM
1	Control	0.214753 ± 0.009976
2	Fluoxetine (20mg/kg/day, oral)	0.141733 ± 0.005749***
3	SAS (6ml/kg/day, oral)	0.135633 ± 0.018765***
4	HAESAS (0.34mg/kg/day, oral)	0.1515 ± 0.0042^**
5	AqESAS (0.64mg/kg/day, oral)	0.174833±0.022715 *

Fig 5 All bars represent mean values with vertical lines indicating S.E.M. Asterisks indicates groups that differentiate significantly from control.* p<0.05; **p<0.01; ***p<0.001; versus control.

Effect of Fluoxetine, Sharbat Ahmad Shahi, HAESAS and AqESAS on Stress marker (GSH) in brain tissue of mice:

It was found that after treatment of mice with Fluoxetine (FXT), Sharbat Ahmad Shahi (SAS), Hydroalcoholic extract of SAS (HAESAS), Aqueous extract of SAS (AqESAS). There was a significant increase in GSH level in mice brain treated with standard drug FXT (p<0.01) and test drug SAS (p<0.01), HAESAS (p<0.05) and AqESAS (p<0.05). These outcomes shows a potential effect of standard and test drug against oxidative stress. It was observed that the increase in GSH level produced by FXT (p<0.01), SAS (p<0.01), HAESAS (p<0.05) and AqESAS (p<0.05) was statistically significant as compared to control. Similarly, the increase was noticed in SAS, HAESAS and AqESAS group but the difference were statistically not significant. While in inter-comparison of SAS, HAESAS and AqESAS group, it was seen that the increase was noticed more in SAS treated group as compared to HAESAS and AqESAS group, but again difference was not significant statistically as shown in Table-4, Fig-6.

Fig 6 All bars represent mean values with vertical lines indicating S.E.M. Asterisks indicates groups that differentiate significantly from control.* p<0.05; **p<0.01 versus control.

Table 5: Effect of Fluoxetine, Sharbat Ahmad Shahi, HAESAS and AqESAS on Stress marker (GSH) in brain tissue of mice

S. No	Group	µmoles of GSH /g tissue Mean±SEM
1	Control	4.528667 ± 0.028915
2	Fluoxetine (20mg/kg/day, oral)	5.993667 ± 0.027817**
3	SAS (6ml/kg/day, oral)	5.984333 ± 0.36681**
4	HAESAS (0.34mg/kg/day, oral)	5.946333 ± 0.55137^*
5	AqESAS (0.64mg/kg/day, oral)	5.849667±0.007334*

DISCUSSION:

In the present study, for the evaluation of antidepressant activity of classical polyherbal formulation i.e. Sharbat Ahmad Shahi (SAS), its hydroalcoholic extract (HAESAS) and aqueous extract (AqESAS), two widely used behavioural paradigms Forced Swimming Test (FST) and Tail Suspension Test (TST) were selected, both are considered to be a relevant model for screening antidepressant activity. Prolonged immobility time in FST is an attestation of stress and depressed states¹². Tail suspension test assesses immobility after animals were subjected to inescapable stress of being suspended by their tail and is useful test for assessing the behavioural effects of antidepressant compounds¹⁶. We observed significantly increased immobility time in animals exposed to FST and TST. Fluoxetine, a SSRI extensively used in the treatment of depression, compared to test drugs viz SAS, HAESAS and AqESAS.

In Forced Swimming Test (FST), it was found that after treatment of mice with fluoxetine (FXT), Sharbat Ahmad Shahi (SAS), hydroalcoholic extract of SAS (HAESAS) and aqueous extract of SAS (AqESAS), significant decrease in immobility time was observed in comparison to control. It was also observed that SAS, HAESAS, and AqESAS treated mice showed significant decrease in immobility time as compared to FXT.

All the treated groups viz FXT, SAS, HAESAS and AqESAS increased the swimming duration in comparison to control. In comparison to FXT, treated group AqESAS significantly increased the swimming time, while it was not significant in case of SAS and HAESAS treated group. In climbing time it was also observed that FXT, SAS, HAESAS and AqESAS increased the duration of climbing time as compared to control group. SAS also increased the climbing time as compared to FXT treated group, but the difference was not statistically significant. While the increament produced by HAESAS and AqESAS were statistically significant as compared to FXT treated group. Table No-2, Fig-1, 2 and 3.

In Tail Suspension Test (TST), it was found that pre-treatment of mice with Fluoxetine (FXT) and the test drugs significantly decreased the immobility time as compared to control It was also observed that SAS treated group decrease the immobility time as compared to standard group i.e Fluoxetine but the difference was not statistically significant. While decreament produced by HAESAS and AqESAS was statistically significant. Inter-comparison of HAESAS and AqESAS group, it was observed that decrease in immobility time was more in HAESAS group, but the difference was not statistically significant as shown in Table No-3, Fig-4.

After behavioural parameters, we have estimated oxidative stress markers in mice by oral administration of drugs such as Fluoxetine (FXT), sharbat ahmad shahi (SAS), Hydroalcoholic extract of SAS (HAESAS), Aqueous extract of SAS (AqESAS). The results depicted a significant decrease in TBARS level in mice brain treated with standard drug FXT and test drugs SAS, HAESAS and AqESAS as compared to control. These outcomes show a potential effect of standard and test drug against oxidative stress. It was observed that all the test groups decrease the TBARS level as compared to standard group, but the differences were not statistically significant. Similarly in inter-comparison of test drugs, it was seen that decrease was slightly more in SAS group as compared to HAESAS and AqESAS group but difference were not significant statistically as shown in Table No-4, Fig-5.

It was found that after treatment of mice with Fluoxetine (FXT), Sharbat Ahmad Shahi (SAS), Hydroalcoholic extract of SAS (HAESAS), Aqueous extract of SAS (AqESAS), there was a significant increase in GSH level. These outcomes show a potential effect of standard and test drug against oxidative stress. It was observed that the increase in GSH level produced by FXT, SAS, HAESAS and AqESAS was statistically significant as compared to control. Similarly, the increase was noticed in SAS, HAESAS and AqESAS group when compared with FXT but the difference was statistically not significant. While in inter-comparison of SAS, HAESAS and AqESAS group, it was seen that the increase was noticed more in SAS treated group as compared to HAESAS and AqESAS group, but again difference was not significant statistically as shown in Table No-5, Fig-6.

Hence, from biochemical estimation of brain tissue we observed that there is a significant reduction in antioxidant enzyme TBARS and increased GSH level in whole brain of mice. Treatment with standard drug (FXT) and test drugs (SAS, HAESAS and AqESAS) restored the increased GSH and reduced TBARS levels, our findings thus indicate the antioxidant potential of the standard and test drugs against oxidative stress as shown in table No- 4and5, fig- 5 and 6.

Thus, in the present study, fluoxetine, a SSRI significantly increased the swimming and climbing time where as reduced the immobility time. Further, SAS, HAESAS and AqESAS of the ingredient of SAS reduced the immobility time with a concurrent increase in the swimming and climbing time. We, however, provide the first evidence for its effect on forced swimming test and Tail suspension test, a possible role for serotonergic mechanism in its antidepressant effect. Recent evidence indicates that oxidative stress plays an important role in the pathogenesis of depressive illness. Experimental data from several studies indicate a lower antioxidant defense against lipid peroxidation, a lower blood GSH levels in patients with depression and a therapeutic benefit from antioxidant supplementation (Altschuleet al1952; Ozcan et al. 2004). In our study, mice exposed to forced swimming test and tail suspension test exhibited oxidative stress and increase of free radical generation as evidenced by a significant reduction in TBARS levels and an increase in GSH levels. Our results, thus, provide further evidence for the involvement of oxidative stress in the biochemical mechanisms underlying depression.

CONCLUSION:

The present study thus proves that the test drugs such as Sharbat Ahmad Shahi (SAS), hydroalcoholic extract of Sharbat Ahmad Shahi (HAESAS) and aqueous extract of Sharbat Ahmad Shahi (AqESAS) possess significant antidepressant activity as depicted by their decreased in immobility time and increased in the duration of swimming and climbing time in FST and also due to the reduction in immobility time in TST. The results of oxidative stress markers showed a significant decreased in TBARS and increased in GSH level in mice brain treated with above test durgs. The study though supports the traditional claim, further studies are essential to isolate the chemical constituents that are responsible for the antidepressant activity of these drugs.

ACKNOWLEDGEMENT:

The authors are highly thankful to the Department of Ilmul Advia, School of Unani Medical Education and Research; Jamia Hamdard, New Delhi, for providing necessary facilities for completing the experimental work.

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Received on 03.08.2019 Modified on 27.03.2021

Research J. Pharm. and Tech. 2022; 15(8):3674-3680.

DOI: 10.52711/0974-360X.2022.00616

S. No	Group	Mean±SEM
S. No	Group	Immobility Time (sec)	Swimming Time (sec)	Climbing time (sec)
1	Control	27.50±1.47	173.7±4.104	30.50±3.394
2	Fluoxetine (20mg/kg/day, oral)	2.833±0.307***	197.5±2.335***	65.83±2.880***
3	SAS (6ml/kg/day, oral)	9.167±0.792***$$$	194.2±1.447***	59.50±4.380***
4	HAESAS (0.34mg/kg/day, oral)	9.500±0.763***$$$	189.5±2.754**	47.67±2.404**$$
5	AqESAS (0.64mg/kg/day, oral)	13.00±0.894***$$$	185.2±1.537*$	44.50±1.68*$$$#

Pre-Clinical Evaluation of Antidepressant and Antioxidant Activities of a Renowned Polyherbal Formulation