Evidences for the involvement of monoaminergic and GABAergic systems in antidepressant-like activity of garlic extract in mice

Objectives: The present study was undertaken to investigate the effect of the ethanolic extract of Allium sativum L. (Family: Lilliaceae), commonly known as garlic, on depression


Introduction
Depression is a common mood disorder that affects a person's life, affecting his/her mood, thoughts, thinking, behavior, feelings etc.The main biochemical theory of depression is the monoamine hypothesis, which states that depression is caused by a functional deficit of monoamines (norepinephrine, serotonin and dopamine) at certain sites in brain. [1]Reduced monoaminergic signaling has long been thought to underlie depressive disorders.Since all the synthetic drugs available for the treatment of depression have various adverse effects and problematic interactions, our aim was to explore the potential of plants in the management of depression.Allium sativum Linn.(Family: Lilliaceae) is commonly known as garlic.Dried bulbs of garlic are incorporated in day to day practice as condiment/spice in the food items.Garlic is endowed with several medicinal properties. [11]It has been reported to possess anti-stress, [12] anti-ageing, memory improving properties and has the potential for preventing the progression of Alzheimer's disease. [13]The antidepressant property of this plant has not been reported scientifically.Therefore, our study was focused on evaluation of the antidepressant potential of Allium sativum in mice and to also study probable mechanisms responsible for the antidepressant activity.

Collection of Plant material
Dried bulbs of Allium sativum Linn.were purchased from the local market in Hisar, Haryana (India).

Preparation of extract of Allium sativum
About 200 g of chopped garlic slices were imbibed in 20% v/v ethanol for 10 days, at ambient temperature (37 0 C) and filtered.The crude extract was dried on a water bath and kept in a refrigerator till further use.The yield of the extract was 20% w/w.Garlic extract has been reported to contain active components like allicin, alliin, allyl methyl thiosulfinate, γ-glutamyl-S-allyl cysteine, γ-glutamyl-S-cis-1-propenyl cysteine. [14]

Animals
Swiss albino mice of either sex, three to four months of age and weighing around 20-30 g were procured from the Disease Free Small Animal House, CCS Haryana Agricultural University, Hisar (Haryana), India.The animals had free access to food and water.However, food was withdrawn 1 h before and 2 h after the administration of the drugs.The animals were housed in an animal room, with alternating light-dark cycle of 12 hr each.The animals were acclimatized to the laboratory conditions for at least five days prior to the behavioral experiments.The experiments were carried out between 0900 h and 1800 h.The Institutional Animal Ethics Committee (IAEC) approved the experimental protocol.Care of laboratory animals was in adherence with the guidelines specified by the CPCSEA, Ministry of Forests and Environment, Government of India (Registration number 0436).

Vehicles
The extract was dissolved in distilled water.Fluoxetine, imipramine, prazosin and baclofen were dissolved separately in normal saline (0.9%).Sulpiride was dissolved in normal saline, followed by the addition of one drop of glacial acetic acid.p-Chlorophenylalanine (p-CPA) was dissolved in a minimum quantity of 0.1 N sodium hydroxide and the pH was adjusted to 7 with 0.1 N hydrochloric acid.The volume for oral administration and intraperitoneal injection was 1 ml/100 g of mouse.

Laboratory Models employed for Testing Antidepressant activity
Forced-swim test: Forced swim test was proposed as a model to test antidepressant activity by Porsolt et al. [15] The method was the same as described by Dhingra and Sharma [16] Mice were forced to swim individually in a glass jar (25 x 12 x 25 cm 3 ) containing fresh water up to 15 cm height and maintained at 25 0 C (± 3 o C). [17] After an initial 2 min period of vigorous activity, each animal assumed a typical immobile posture.A mouse was considered to be immobile when it remained floating in the water without struggling, making only minimum movements of its limbs, necessary to keep its head above the water.The total duration of immobility was recorded during the next 4 min of the total test duration of six minutes.The changes in immobility duration were studied after administrating the drugs in separate groups of animals.Each animal was used only once.
Tail-suspension test: The total duration of immobility induced by tail suspension was measured according to the method described as a means of evaluating potential antidepressants. [18]Mice were suspended on the edge of a table, 50 cm above the floor, with the help of an adhesive tape placed approximately 1 cm from the tip of the tail.Immobility time was recorded during a 6 min period.The animal was considered to be immobile when it did not show any movement of the body and hanged passively.

Measurement of MAO-A and MAO-B activities
On the 14 th day, the mice were sacrificed after 6 min exposure to FST, and the brain samples were collected immediately on an ice plate.The collected brain samples were washed with cold 0.25M Sucrose-0.1MTris-0.02MEDTA buffer (pH 7.4) and weighed.Mouse brain mitochondrial fractions were prepared following the procedure of Schurr and Livne. [19]22] The protein concentration was estimated by Lowry method, using bovine serum albumin as the standard. [23]

Measurement of Locomotor activity
The locomotor activity of the control group and the animals treated with the extract was evaluated with the help of Photoactometer (INCO, Ambala, India).The differences in the locomotor activity scores were noted before and after the drug treatment.

Experimental protocols
The animals were divided into 24 groups and each group comprised a minimum of six mice.

Groups for Tail Suspension Test (TST)
Group 1: Control group (vehicle for garlic extract): Distilled water was administered orally for 14 successive days.At 90 min after administration on the 14 th day, immobility period was recorded.Group 2: Fluoxetine (20 mg/kg) was administered orally for 14 successive days.At 90 min after administration on the 14 th day, immobility period was recorded.
Group 3: Imipramine (15 mg/kg) was administered orally for 14 successive days.At 90 min after administration on the 14 th day, immobility period was recorded.
Group 4, 5 and 6: Garlic extract (25, 50 and 100 mg/kg respectively) was administered orally for 14 successive days.At 90 min after administration on the 14 th day, immobility period was recorded.

Groups for Forced Swim Test (FST)
Groups 7 to 12 are the same as groups 1 to 6 mentioned in the groups for TST, except that immobility period was recorded using FST.

Groups for studying the mechanism of action of garlic extract
Group 13 (Control Sulpiride): Distilled water was administered orally for 14 successive days and then sulpiride (50 mg/kg) was injected on the14 th day, after 45 min of last oral administration of vehicle.The animals were subjected to TST, after 45 min of sulpiride injection.
Group 14: Garlic extract (100 mg/kg) was administered orally for 14 successive days and then sulpiride (50 mg/kg) was injected on the 14 th day, after 45 min of last oral administration of extract.The animals were subjected to TST, after 45 min of sulpiride injection.
Group 15 (Control Prazosin): Distilled water was administered orally for 14 successive days and then prazosin (62.5 µg/kg) was injected on the 14 th day, after 45 min of last oral administration of vehicle.The animals were subjected to TST, after 45 min of prazosin injection.
Group 16: Garlic extract (100 mg/kg) was administered orally for 14 successive days and then prazosin (62.5 µg/kg) was injected on the 14 th day, after 45 min of last oral administration of extract.The animals were subjected to TST, after 45 min of Prazosin injection.
Group 17 (Control p-CPA): Distilled water was administered orally for 14 successive days and then p-CPA (100 mg/kg) was injected from the 11 th day to the 14 th day, after 45 min of last oral administration of vehicle.The animals were subjected to TST, after 45 min of p-CPA injection.
Group 18: Garlic extract (100 mg/kg) was administered orally for 14 successive days and then p-CPA (100 mg/kg) was injected from the 11 th day to the 14 th day, after 45 min of last oral administration of extract.The animals were subjected to TST, after 45 min of p-CPA injection.
Group 19 (Control Baclofen): Distilled water was administered orally for 14 successive days and then baclofen (10 mg/kg) was injected on the 14 th day, after 45 min of last oral administration of vehicle.The animals were subjected to TST after 45 min of baclofen injection.
Group 20: Garlic extract (100 mg/kg) was administered orally for 14 successive days and then baclofen (10 mg/kg) was injected on the 14 th day, after 45 min of last oral administration of extract.The animals were subjected to TST, after 45 min of baclofen injection.

Groups for biochemical estimations
Group 21: Distilled water was administered orally for 14 successive days.The mice were sacrificed under light anesthesia with chloroform and the brain was dissected and used for the estimation of monoamine-oxidase A and B levels.
Group 22: Imipramine (15 mg/kg) was administered orally for 14 successive days.The mice were sacrificed under light anesthesia with chloroform.The brain was dissected and used for the estimation of monoamine-oxidase A and B levels.
Group 23: Garlic extract (100 mg/kg) was administered orally for 14 successive days.The mice were sacrificed under light anesthesia with chloroform.The brain was dissected and used for the estimation of monoamine-oxidase A and B levels.

Groups for locomotor activity
Group 24: The effect of garlic extract (100 mg/kg) on the locomotor function of mice was studied using Photoactometer (INCO, Ambala, India), to rule out the increase in the locomotor performance of mice due to the extract.The difference in the locomotor activity scores was noted before and after administration of the extract.

Statistical analysis
All the results were expressed as Mean ± Standard Error (SEM).Data was analyzed using one-way ANOVA, followed by Dunnett's t-test.The data for locomotor activity scores was subjected to paired t-test.P < 0.05 was considered as statistically significant.

Effect of garlic extract on immobility periods in TST and FST
Garlic extract (25, 50 and 100 mg/kg, p.o.) administered to mice for 14 successive days decreased immobility periods significantly in a dose-dependent manner, as compared to control in both TST and FST, indicating significant antidepressant-like activity.A dose of 100 mg/kg p.o. of the extract showed the most potent antidepressant effect, as indicated by the highest decrease in the immobility period.The efficacy of the extract was found to be comparable to fluoxetine (20 mg/kg) and imipramine (15 mg/kg) administered for two successive weeks in both FST and TST [Tables 1 and 2].Effect of combination of garlic extract with sulpiride, prazosin, p-CPA and baclofen on immobility period in TST Sulpiride (50 mg/kg i.p.), prazosin (62.5 mg/kg i.p.) and p-CPA (100 mg/kg i.p.) alone significantly increased the immobility period, as compared to the control group.Pretreatment of animals with sulpiride or p-CPA or prazosin or baclofen significantly blocked the decrease of immobility time elicited by garlic extract (100 mg/kg p.o.) in TST [Table 3].

Effect of Garlic extract on brain monoamine oxidase (MAO) activity
Garlic extract (100 mg/kg p.o.) administered to mice, for 14 successive days, significantly decreased brain MAO-A and MAO-B activity, as compared to the control group.MAO inhibition was comparable to imipramine [Table 4].

Effect of garlic extract on locomotor activity
Garlic extract (100 mg/kg p.o.) administered for 14 successive days did not show any significant change in the locomotor function of mice (870.1 ± 99.8), as compared to the control (805 ± 121.5) group.

Discussion
In the present study, 20% ethanolic extract of garlic (25, 50 and 100 mg/kg p.o.) administered to mice, for 14 successive days, showed significant antidepressant-like activity in Forced Swim Test (FST) and Tail Suspension Test (TST).The efficacy of garlic extract was found to be comparable to fluoxetine and imipramine.FST and TST are two commonly used behavioral despair models of depression.These tests are quite sensitive and widely employed in rodents to predict antidepressant potential by decrease of immobility period produced by several different classes of antidepressant drugs. [15,17]It has been reported that TST is less stressful and has higher pharmacological sensitivity than FST. [23]arlic extract did not show significant change in the locomotor activity of mice, as compared to the control group; so this extract did not produce any motor effects.It confirms the assumption that the antidepressant-like effect of the extracts is specific.According to our results, the antidepressant-like effect of garlic extract was significantly reversed by the treatment of animals with prazosin (α 1adrenoceptor antagonist), sulpiride (a selective dopamine D 2receptor antagonist), p-CPA (serotonin synthesis inhibitor) and baclofen (GABA B agonist) when tested in TST.This suggests that garlic extract might produce antidepressant-like effect through interaction with α 1 -adrenoceptors, dopamine D 2 receptors, serotonergic and GABAergic receptors, thereby increasing the levels of norepinephrine, dopamine, serotonin and decreasing GABA levels in the brain of mice.Garlic extract (100 mg/kg p.o.) administered to mice, for 14 successive days, significantly decreased brain MAO-A and MAO-B activities as compared to the control group.MAO inhibiting activity by the extract was comparable to imipramine.Thus, antidepressant-like activity of the extract might also be due to inhibition of MAO, resulting in increase in the brain levels of monoamines.
In conclusion, garlic extract showed significant antidepressant-like activity probably by inhibiting MAO-A and MAO-B levels, and through interaction with adrenergic,    dopaminergic, serotonergic and GABAergic systems.The antidepressant-like action of the extract was comparable to imipramine and fluoxetine.Hence, garlic extract may have potential therapeutic value for the management of depressive disorders.
Dhingra and Kumar: Antidepressant-like activity of garlic

Table 1 Effect of ethanolic extract of Allium sativum on immobility period in tail suspension test
GE = garlic extract, Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's t-test as compared to control.*P<0.05 as compared to control F (5, 28) = 21.48 (P < 0.0001) Indian J Pharmacol | Aug 2008 | Vol 40 | Issue 4 | 175-179

Table 3 Effect of combination of ethanolic extract of Allium sativum with sulpiride, baclofen, p-CPA and prazosin Group Treatment for Number of Dose Immobility time No. 14 days p.o. animals (kg-1) (Sec) (Mean ± SEM)
GE = Garlic extract, Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's t-test.F (9, 50) = 28.966(P < 0.0001).a P < 0.05 as compared to control, b P < 0.05 as compared to Garlic extract