INTRODUCTION:

Depression is a state of low mood and aversion to activity that can affect a person's thoughts, behavior, feelings and sense of well-being¹.When mild, depression can be treated without medicines but, when moderate or severe, people may need medication and professional talking treatments.

Clinical depression is one of the most common complications associated with chronic illnesses. In some cases, a chronic illness may actually lead to depression. The rate for depression occurring with other medical illnesses is quite high².

People with depressed mood can feel sad, anxious, empty, hopeless, helpless, worthless, guilty, irritable, ashamed or restless.

They may lose interest in activities that were once pleasurable, experience loss of appetite or overeating, have problems concentrating, remembering details or making decisions, and may contemplate, attempt or commit suicide. Insomnia, excessive sleeping, fatigue, aches, pains, digestive problems or reduced energy may also be present³.

Herbal remedies which were used traditionally now significantly documented for the safety profile and as a therapy for some of the pathological conditions. From the last decade synthetic drugs have been combined with herbs which show some promising results. Medha gulika, a licensed polyherbal formulation comprising of plants Acorus calamus, Clitoriaternatea, Glycyrrhiza glabra, Elaeocarpus sphaericus, Bacopa monnieri, Centella asiatica and Sarcostemmaacidum, been marketed with the claim of antidepressant activity. But till now there is no study has been carried out which indicates the effect of Medhagulikaon antidepressant activity. So the present study has been designed to evaluate the antidepressant activity of Medha gulika using different experimental models.

MATERIALS AND METHOD:

Polyherbal ‘Medha gulika’ tablets manufactured by Velingil Oushadhasala, Cheranelloor P.O, Kochi, Kerala. It is preserved in the departmental library, KCP Manglore for future reference.

Animal selection:

Swiss albino mice weighing 18-30 gm, were used for the study. The mice were inbred in the central animal house of the Department of Pharmacology, Karavali College of Pharmacy, Mangalore, under suitable conditions of housing, temperature, ventilation and nutrition were used for antidepressant activity. They were kept in clean dry cages week before the beginning of the experiment to acclimatize with the experimental conditions. The animals were fed with standard pelleted diet (Lipton India Ltd., Mumbai) and distilled water ad libitum was maintained at 21°C-23°C under a constant 12hrs light and dark cycle. The animal care and experimental protocols were in accordance with CPCSEA /IAEC.

Preparation of test solution:

The tablets are crushed in to powder, a small quantity of water is added to make a smooth suspension, triturated with mortar and pestle, a pinch of Tween 80 is added to make a uniform suspension. Required quantity of water is added.

Dose selection:

The doses which were administered to rats were calculated accordingly from human dose. The human dose was converted to rat dose by using Human equivalent dose method (HED)⁴. The low, moderate and high doses of Medhagulika were selected as 53.85mg/kg, 102.77mg/kg and 154.16mg/kg respectively.

A. TEST FOR ANTIDEPRESSANT ACTIVITY

1. Forced Swim Test^5,6

Method: Adult Swiss albino mice (young mice) divided in to five groups and each group containing six animals are fasted overnight, prior to the test but water was supplied ad libitum. The animals were divided as follows.

Group I – Received 0.05ml/10g of Normal saline intra peritoneally.

Group II – Received 15 mg/kg Imipramine intra peritoneally.

Group III – Received 53.85 mg/kg Medhagulika orally.

Group IV – Received 102.77 mg/kg Medhagulika orally.

Group V – Received 154.16 mg/kg Medhagulika orally.

Experimental Procedure:

Mice were individually placed into a glass cylinder filled with 15 cm of water for 6 min. As a measure of depression-like behavior, the total duration of immobility and the number of immobility episodes were recorded. Immobility is defined as the absence of movement, unless they are necessary for the animal to stay afloat (head above water). Maintained the temperature of water at 26 ± 1°C. At this height of water, animals were not able to support themselves by touching the bottom or the side walls of the chamber with their hind-paws or tail. The duration of immobility was manually recorded during after 2 min of total 6 min testing period.

2. Tail Suspension Test:

Group I – Received 0.05ml/10g of Normal saline intra peritoneally.

Group II – Received 15 mg/kg Imipramine intra peritoneally.

Group III – Received 53.85 mg/kg Medhagulikaorally.

Group IV – Received 102.77 mg/kg Medhagulika orally.

Group V – Received 154.16 mg/kg Medhagulika orally.

Procedure:

Each mice was individually suspended to the edge of a table, 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail. Total period of immobility was recorded manually for 6 min. Animal was considered to be immobile when it didn’t show any body movement, hung passively and completely motionless. The observer, recording the immobility of animals was blind to the drug treatment given to the animals under study^6,7.

3. Learned Helplessness Test :

Animals exposed to inescapable and unavoidable electric shocks in one situation later fail to escape shock in a different situation when escape is possible. (Overmier and Seligman 1967; Maier and Seligman 1976). This phenomenon was evaluated as a potential animal model of depression⁵. The animals were divided as follows.

Group I – Received 0.05ml/10g of Normal saline intra peritoneally.

Group II – Received 15 mg/kg Imipramine intra peritoneally.

Group III – Received 53.85 mg/kg Medhagulika orally.

Group IV – Received 102.77 mg/kg Medhagulika orally.

Group V – Received 154.16 mg/kg Medhagulika orally.

Procedure:

After the appropriate treatment, the animals were tested for acquisition of jump-up escape in the same apparatus. At the beginning of a trial, the platform was pushed into the box and 0.2 mA shock initiated. Shock was terminated in 10 s if the animal has not escaped onto the platform by this time. If an escape response occurred, the animal was allowed to remain on the platform for the duration of 10 s, then returned to the grid floor. Ten such trails with an inter-trial interval of 20 s were given. In control group of mice, this training resulted in 80% acquiring learned helplessness behavior. Drugs were given before the training and test period.

4. Open Field Test :

The animals were divided as follows.

Group I – Received 0.05ml/10g of Normal saline intra peritoneally.

Group II – Received 1 mg/kg Diazepam intra peritoneally.

Group III – Received 53.85 mg/kg Medhagulika orally.

Group IV – Received 102.77 mg/kg Medhagulika orally.

Group V – Received 154.16 mg/kg Medhagulika orally.

Procedure:

This test utilizes behavioral changes in rodents exposed to novel environments and was used to confirm that the observed antidepressant effect was not due to stimulation of general motor activity. Various types of open field apparatus have been used to test the mice.

Mice were carried to the test room in their home cages and were handled by the base of their tails at all times. Mice were placed into one of the four corners of the open field and allowed to explore the apparatus for 5 minutes. After the 5 minutes test, mice were returned in their home cages and open field was cleaned with 70 % ethyl alcohol and permitted to dry between tests. To assess the process of habituation to the novelty of the arena, mice were exposed to the apparatus for 5 minutes on 2 consecutive days. Parameters such as Activity in the centre, Number of squares crossed at periphery and Rearing (No. of times the animal stand on the rear paws)^5,8.

Statistical analysis:

Data were presented as mean ± Standard Error of Mean (SEM). One-way Analysis of Variance (ANOVA), followed by Dunnet’s multiple comparison test. For all test probability 0.05 or less was accepted as significance.

ANOVA (Analysis of variance).

In statistics, analysis of variance is a collection of statistical models and their associated procedures, in which the observed variance is partitioned into components due to different explanatory variables. In its simplest form ANOVA gives a statistical test of whether the means of several groups are all equal and therefore generalize Dunnett‟s multiple comparison tests to more than two groups.

RESULT:

I) ANTIDEPRESSANT ACTIVITY OF MEDHA GULIKA

1. Forced Swim Test :

In FST, Table No 1 shows that animals treated with three doses of Medhagulika (53.85,102.77 and 154.16 mg/kg) showed decrease in their immobility times, which was significant (136.50±0.65; p<0.01 and 131.42±0.56 , 123.26±0.72 ; p<0.001) when compared with control (139.41±0.68). Similarly, animals treated with imipramine (15 mg/kg), as expected, showed a significant decrease in the immobility time (61.36±0.88; p<0.001). Animals treated with high dose (154.16 mg/kg) and moderate dose (102.77 mg/kg) shows more significant decrease in immobility time when compared with low dose (53.85 mg/kg).

Table No 1: Effect of Medhagulika on Immobility time in FST.

Group No.	Drug Treatment	Dose (mg/kg)	Immobility period, mean ± S.E.M [n=6]
I	Control	0.05 ml/10 g	139.41±0.68
II	Imipramine	15	61.36±0.88***
III	MGLD	53.85	136.50±0.65**
IV	MGMD	102.77	131.42±0.56***
V	MGHD	154.16	123.26±0.72***

Values were mean ± S.E.M. for (n=6) expressed as the time (in sec) of 6 animals each group. Data analysis was performed using Dunnett’s test.^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Figure 1: Effect of Medhagulika on Immobility time in FST

Values were mean ± S.E.M. for (n=6) expressed as the time (in sec) of 6 animals each group. Data analysis was performed using Dunnett’s test.^*P < 0.05, ^**P < 0.01, ^***P < 0.001

2. Tail Suspension Test :

Animals treated with three doses of Medhagulika showed decrease in their immobility times, which was significant (159.14±0.78; p<0.05, 157.88±1.14; p<0.01 and 138.24±1.08; p<0.001) when compared with control (162.35±0.85). Similarly, animals treated with imipramine (15 mg/kg) as expected, showed a significant decrease in the immobility time(72.22±068 ; p<0.001). Animals treated with high dose(154.16 mg/kg) showed more significant decrease in immobility time (Table No 2).

Table No 2: Effect of Medhagulika on Immobility time in TST.

Group No.	Drug Treatment	Dose (mg/kg)	Immobility period, mean ± S.E.M [n=6]
I	Control	0.05 ml/10 g	162.35±0.85
II	Imipramine	15	72.22±0.68***
III	MGLD	53.85	159.14±0.78*
IV	MGMD	102.77	157.88±1.14**
V	MGHD	154.16	138.24±1.08***

Values were mean ± S.E.M. for (n=6) expressed as the time (in sec) of 6 animals each group. Data analysis was performed using Dunnett’s test.^*P<0.05,^*^*P<0.01,^***P <0.001

Figure 2: Effect of Medhagulika on Immobility time in TST

Values were mean ± S.E.M. for (n=6) expressed as the time (in sec) of 6 animals in each group. Data analysis was performed using Dunnett’s test.^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. control.

Table No 3: Effect of Medhagulika on Learned helplessness test in albino rats.

Group No.	Drug Treatment	Dose mg/kg	Day I		Day II				Day III
Group No.	Drug Treatment	Dose mg/kg	EF	AR	EF		AR		EF	AR
I	Control	0.05ml/10g	20.41±0.85	4.26±0.66	19.56±0.64		4.17±0.48		19.86±0.58	5.08±0.36
II	Imipramine	15	11.48±0.72***	19.34±0.86***		7.36±0.86***		20.48±0.96***	3.48±0.64***	21.58±0.76***
III	MGLD	53.85	19.22±0.62*	7.26±0.72**		16.42±0.62**		6.82±0.64**	17.12±0.96**	7.68±0.48**
IV	MGMD	102.77	17.64±0.82**	9.48±0.76***		14.68±0.72***		7.28±0.48**	14.36±0.86***	11.24±0.52***
V	MGHD	154.16	16.24±0.88***	11.64±0.86***	11.68±0.88***		10.72±0.86***		10.68±0.76***	14.18±0.62***

Values were mean ± S.E.M. for (n=6) expressed as the time (in sec) of 6 animals each group. Data analysis was performed using Dunnett’s test.^*P < 0.05, ^**P < 0.01, ^***P < 0.001

DAY I

Figure. 3: Comparative profile of escape failure (I) and avoidance response (II) in LHT afteroral administration of 53.87,102.77and 154.16 mg/kg of Medhagulika at day 1.

DAY II

Figure 4: Comparative profile of escape failure (I) and avoidance response (II) in LHT afteroral administration of 53.87,102.77and 154.16 mg/kg of Medhagulika at day 2.

DAY III

Figure 5: Comparative profile of escape failure (I) and avoidance response (II) in LHT afteroral administration of 53.87,102.77and 154.16 mg/kg of Medhagulika at day 3.

Table No 4: Effect of Medhagulika on open field test in mice.

Group No.	Drug Treatment	Dose (mg/kg)	No. of squares crossed (Mean ± SEM )		No Of Rearings (Mean± SEM)
Group No.	Drug Treatment	Dose (mg/kg)	Centre	Periphery	No Of Rearings (Mean± SEM)
I	Control	0.05ml/10g	10.20±0.68	76.12±0.38	3.64±0.58
II	Diazepam	1	28.42±0.56***	114.36±0.42***	8.64±0.68***
III	MGLD	53.85	10.48±0.54	76.28±0.36	3.18±0.36
IV	MGMD	102.77	10.64±0.74	76.46±0.54	3.26±0.32
V	MGHD	154.16	10.26±0.64	76.14±0.64	3.28±0.42

Values were mean ± S.E.M. for (n=6) expressed as the time (in sec) of 6 animals in each group. Data analysi test.^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. control.

3. Learned Helplessness Test

Animals treated with three doses of Medhagulika at day 1, day 2 and day 3 showed the significant increase in avoidance response and decrease in escape failure (Table No 3).

4. Open field test

The open field test was performed to confirm the property to alter general motor activity by Medhagulika, because any alteration in general motor activity may give false positive/ negative results in Forced swim test. There was a slight increase in the number of squares crossed (peripheral) by mice in Medhagulika treated groups but it was not statistically significant compared to control. There was a significant increase in no. of crossings in diazepam group as compared to control group. There was significant increase in the rearing of animals with diazepam in comparison to the control group. There was no increased number of rearing in test drug treated groups (Table No 4).

DISCUSSION:

Depression is happened due to the alteration in neurotransmitters in our brain. So the aim of an antidepressant is to stabilize and normalize the neurotransmitters in our brain (naturally occurring brain chemicals), such as serotonin, dopamine, and norepeniphrine. According to various studies, these neurotransmitters play a vital role in regulating mood.

The incidence of depression in the community is very high and is associated with lot of morbidity. Hence, it is very important to address these problems and find effective remedies. Though several drugs are available, all are associated with some limitations, modern antidepressants do affect some people with undesirable side-effects and there is an urgent need for alternative medications for these disorders. Despite the use of Medhagulika for treating nervous disorders, there is no scientific reports about the evaluation of its pharmacological effects. In this work, it was demonstrated that the administration of different doses of Medhagulika in mice was able to induce antidepressant effects.

Unfortunately, it is not fully known what exactly causes clinical depression for a particular individual. There are many theories about causes such as biological and genetic factors, environmental influences, and childhood or developmental events.

The monoamine hypothesis of depression predicts that the underlying pathophysiological basis of depression is, at least in part, a depletion in the levels of 5-HT, NE, and/or DA in the CNS. This hypothesis appears to be supported by the mechanism of action of antidepressant drugs, which elevate the levels of these neurotransmitters in the brain. Because the FST also provides a useful model for investigating neurobiological mechanisms underlying antidepressant-like responses⁹. Most antidepressant drugs in clinical use promote an increase in 5-HT availability by directly inhibiting serotonin reuptake, affecting serotonin turnover in the brain, and also interacting with 5-HT_1A, 5-HT₂, and 5-HT_3Areceptors¹⁰.

In parallel with the serotonergic system, the noradrenergic system is also strongly implicated in the pathophysiology of depression¹¹. At least in part, depression seems to also be associated with a hypofunction of the noradrenergic system, so that some antidepressants act by increasing the synaptic availability of NE¹⁰. In this context, the α1- and α2-adrenoceptors have been shown to underlie some of the antidepressant-like responses of drugs in behavioral models of depression¹¹.

Studies show that the dopaminergic system, in part, may also be implicated in the regulation of mood. Currently, there is evidence from several reports regarding the efficacy of antidepressants related to the potentiation of dopaminergic neurotransmission in the treatment of depression¹².

The behavioural despair test centered on a rodent's response to the threat of drowning, whose result has been interpreted as measuring susceptibility to negative mood, representing a kind of hopelessness in the animal. It is commonly used to measure the effectiveness of antidepressants¹³.

The FST and TST are the most common animal models used for screening potential antidepressant agents, which induce a state of immobility in animals facing an inescapable situation. Such immobility behavior has been hypothesized to reflect behavioral despair, which in turn may reflect depressive disorders in humans. Therefore, the antidepressant-like activity of a compound is expressed by a decrease in the immobility of animals submitted to forced swimming and tail suspension. This behavioral change is sensitive to major classes of antidepressant drugs including monoamine oxidase inhibitors, tricyclics, selective 5-HT reuptake inhibitors, and atypical¹⁴. Thus, our results suggested an antidepressant-like profile from Medhagulika.

In learned helplessness studies, an animal is repeatedly exposed to an aversive stimulus which it cannot escape. Eventually, the animal stops trying to avoid the stimulus and behaves as if it is helpless to change the situation. When opportunities to escape become available, learned helplessness means the animal does not take any action. A drug is considered to be effective if the learned helplessness is reduced and the number of failures to escape is decreased¹⁵. In this test, animals treated with three doses of Medhagulika (53.85,102.77and 154.16mg/kg ) at day 1, day 2 and day 3 showed the significant increase in avoidance response and decrease in escape failure. The Medhagulika showed dose dependent activity.

In contrast to antidepressants, stimulant drugs cause marked motor stimulation in tests, this behavior profile is consistent with a false positive effect in the FST model and is often distinguished from the antidepressant-like reduction in immobility by assessing locomotor activity in the OFT^14,16. Thus, to investigate the possibility that Medhagulika reduced immobility by a stimulant action, mice were assessed for the ability to increase motor activity in the OFT.

As stated earlier, “MEDHA GULIKA” is a poly herbal compound comprising of various plant extracts,the potential effect of MEDHA GULIKA polyherbal formulation may be attributed to one or more bioactive principles present in these extracts such as steroidal saponins, flavonoids, alkaloids,glycosides and free amino acids. There may be synergistic herb-herb interactions enhancing the total efficacy of the formulation. The exact mechanism of action of the drug needs to be evaluated by further extensive studies.

CONCLUSION:

The present study was aimed to expose the antidepressant activity of Medhagulika in swiss albino mice using four animal models. The data obtained was satisfactory and conclusive and so as to accomplish our objectives. In conclusion the present data indicate that the administration of Medhagulika to mice has shown significant dose dependant antidepressant activity supporting folk information regarding antidepressant activity of the formulation, relatively sub-chronic study may be necessary to arrive at a better picture.

The exact mechanism underlying antidepressant effect is not clear but it may be apparently related to active components present in them. Hence further studies would be necessary to evaluate the contribution of active chemical constituents for the observed antidepressant activity, as it still remains to be determined which components were responsible for these effects. In our study we have made an attempt to prove its efficacy in experimental animals. Further study can be done in human subjects.

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Received on 16.04.2015 Accepted on 15.05.2015

Asian J. Pharm. Tech. 2015; Vol. 5: Issue 2, Pg 115-121

DOI: 10.5958/2231-5713.2015.00017.3