Ayurveda, the Indian traditional system of medicine, mentions a number of single and compound drug formulations of plant origin that are used in the treatment of psychiatric disorders. On one hand these agents have less adverse effects, and on the other hand they have been shown to be comparable in efficacy to their synthetic counterparts [Sudhakar Pemminati et al., 2010].

Synthetic antidepressants are often associated with their anticipated side effects like dry mouth, inability in driving skills, constipation and sexual dysfunction and majority of patients are reluctant to take this treatment [Singh Rudra Pratap et al., 2012].

Nature plants, such as Hypericum perforatum, Cissampelos sympodialis, Terminalia bellirica Roxb, Bacopa monniera, Ginkgo biloba, Pueraria lobata may be an important source of new antidepressant drugs and the safety of nature plant extracts maybe better than that of synthetic antidepressants [Zhiyu Zhao et al., 2008]

Avena sativa commonly known as oats belongs to the family of poaceae. The primary chemical constituents are saponins (Avenacosides A and B), flavonoids, starch, alkaloids (trigonelline, avenine, gramine), steroids, calcium, B-vitamins, lysine, methionine and alkaloids such as gramine. They also contain iron, manganese and zinc [Danielle Ryan et al., 2007]. In tredicinal medicine of Avena sativa used as nervine tonic, cardiac tonic, stimulant, antispasmodic, thymoleptic, antidepressant (used in menopausal phase) [Khare, 2007].

Several studies on angiotensin-I converting enzyme inhibitory [Cheung et al., 2009], anti-inflammatory, anti-itchy [Sur et al., 2009], anti-HIV [Shun C.W et al., 2008] and cardioprotective activities [Ryan et al., 2007] of Avena sativa have been reported.

The Anti depressant activity of Avena sativa is mentioned in Indian system of traditional medicine but there is no scientific evidence to prove its activity. Hence, the present study is designed to evaluate the antidepressant activity of Avena sativa using different animal models in mice.

2. MATERIALS AND METHODS:

Plant material collection and authentication

The seeds of Avena sativa were collected from a local distributor in Tirupati in the month of February. The plant was identified and authenticated by K. Madhava Chetty, Assistant Professor, Department of Botany, Sri Venkateswara University, Tirupati, Chittoor district, Andhra Pradesh.

Preparation of extract:

The collected seeds of Avena sativa were shade dried at room temperature and grinded coarsely. The seeds were extracted by Soxhelet apparatus using methanol. The resulting extract was concentrated in vacuum under reduced pressure and dried in desiccators. Thus, the prepared extract was used for further pharmacological evaluation.

MATERIALS:

Apomorphine, Desipramine was procured from Sigma life sciences, Bangalore; Fluoxetine procured from Fludac, Cadila and Rectal Thermometer from UGO BASILE.

Preliminary Phytochemical analysis:

Methanolic extract of Avena sativa seeds was subjected to preliminary Phytochemical analysis to test for presence of various Phytoconstituents viz., Alkaloids, Carbohydrate and Reducing sugar, Steroids, Proteins, Tannins, Flavonoids, Flavanone, Glycosides, Saponins, Triterpenoids [Trease G E and Evans M C, 1983; Khandelwal KR, 2000; Kokate CK, 1994] .

Animals:

Albino mice of either sex weighing between 18-25 gm were used in this study. All animals were procured from Sainath Agency, Hubsiguda. After procuring, the animals were acclimatised for 7 days in quarantine room and housed in groups of six under standard husbandry conditions like room temperature (23±2°C), relative humidity (30-70%) and 12/12 h light/dark cycle. All the animals were fed with synthetic standard diet (Amrut Laboratories Pranava Agro Industries Ltd. Hyderabad) and water was supplied ad libitum under strict hygienic conditions. All the experimental protocols were approved by Institutional Animal Ethical Committee (IAEC).All the animal studies were performed as per rules and regulations in accordance to guideline of CPCSEA registration number: 1236/c/08/CPCSEA.

All experiments were carried out during the light period (9:00 to 17:00 h) to avoid circadian rhythm.

Acute Oral Toxicity study:

OECD guidelines (425) state that, before establishing pharmacological activity of the New Chemical Entity is mandatory to establish maximum tolerated dose in mice [OECD 2001]. The purpose of the sighting study is to allow selection of appropriate starting dose for the main study. The starting dose for a sighting study was selected from the fixed dose levels of 5, 50, 300, 2000mg/kg as a dose expected to produce evident toxicity.

In vivo Models for Antidepressant activity

2.7.1

Forced Swim Test:

Animals were divided into 4 groups of 5 animals in each, weighing between 18-25gms

Group I – Control (Distilled water 10ml/kg, p.o)

Group II – Standard (Fluoxetine 25mg/kg p.o)

Group III –Low dose (MSEAS 100 mg/kg, p.o)

Group IV – High dose (MSEAS 200mg/kg, p.o)

Experiment was carried out in narrow glass cylinder (13 cm in diameter × 24 cm high containing water (25°C) to a depth of 10 cm, from which they cannot escape. All the animals were fasted for 3hrs prior to the oral administration of vehicle/standard/test compounds. Thirty minutes later, the animals were subjected to swim for 6 minutes; the first two minutes the animal is allowed to adjust to the new conditions; the next four minutes the immobility time was measured with a stopwatch at 30, 60, 120 and 240 minutes after oral administration. Immobility time was the time during which the animals will be necessary to keep afloat [Kulkarni and Ashish Dhir, 2007].

Tail Suspension Test:

Animals were divided into 5 groups of 4 animals in each weighing between 18-25gms

Group I – Control (Distilled water 10ml/kg, p.o)

Group II – Standard (Fluoxetine 25mg/kg p.o)

Group III – Low dose (MSEAS 100 mg/kg, p.o)

Group IV – High dose (MSEAS 200mg/kg, p.o)

The control and test compounds were administered p.o, and standard drug was administered p.o route, 60 minutes prior to testing. The mice were suspended on the edge of a shelf 58cm above the table top by adhesive tape placed approx. 1cm from the tip of tail. The duration of immobility was recorded for the period of 6minutes by using stopwatch. After the initial period of vigorous motor activity, the mice became still. Mice were considered immobile when they hanged passively and completely motionless. The duration of immobility time was recorded before the treatment and 60 minutes after the treatment [Kulkarni and Ashish Dhir, 2007].

Apomorphine Induced Hypothermia:

Animals were divided into 4 groups of 5 animals in each weighing between 18-25gms

Group I – Control (Distilled water 10ml/kg, p.o)

Group II –Standard (Desipramine 20mg/kg p.o)

Group III- Low dose (MSEAS 100 mg/kg, p.o)

Group IV – High dose (MSEAS 200mg/kg, p.o)

All the animals were fasted for 3hrs prior to oral administration of vehicle/standard/test compounds. One hour after oral administration of the test compounds or the vehicle, 16mg/kg apomorphine was injected s.c. to the animals.The rectal temperature of each mouse was measured by an electronic thermometer at 10, 20, 30, 60 and 120 minutes after apomorphine treatment and the degree of hypothermia was determined [Sanchez-Mateo et al., 2002].

Statistical Analysis:

Results will be presented as mean ± SEM. The data will be subjected for statistical analysis by One way analysis of variance (ANOVA) followed by Dunnet’s t test and P<0.05*, 0.01** and 0.001*** were considered as significant.

3. RESULTS:

Preliminary Phytochemical screening:

The methanolic seed extract of Avena sativa was subjected to Preliminary Phytochemical tests and showed the presence of carbohydrates, alkaloids, flavanoids, steroids, glycosides, saponins, amino acids, gums and mucilage.

S.NO	PHYTOCHEMICAL CONSTITUENTS	INFERENCE
1	Test for Carbohydrates Molisch’s test Fehling’s test Barfoed’s test Benedict’s test	+ + + +
2	Test for Alkaloids Dragendorff’s test Wagner’s test Mayer’s test Hager’s test	+ + + +
3	Test for Anthraquinone glycosides	-
4	Test for steroids Salkowski test Libermann Burchard	+ +
5	Test for Flavonoids Shinoda test	+
6	Test for Saponins Foam test	+
7	Test for tannins	-
8	Test for glycosides	+
9	Test for triterpinoids	-
10	Test for gums	+
11	Test for mucilage	+

+ indicates presence;

- indicates absence

Acute Oral Toxicity study:

The methanolic seed extract of Avena sativa was found to be safe up to the dose level of 2000mg/kg, po, and did not produce any toxic symptoms. The survived animals were sacrificed and complete absorption of drug through GIT was observed. Hence 1/20th and 1/10th of Maximum Therapeutic Dose (2000mg/kg) were selected for the pharmacological models.

Forced Swim Test:

The result of the effect of methanolic seed extract of Avena sativa on the duration and % inhibition of immobility is shown in Table 1 The animals treated with 100mg/kg, p.o of MSEAS and Fluoxetine 25mg/kg, p.o showed significant decrease in immobility time was observed up to 60 min but not 200mg/kg, p.o of MSEAS when compared with control.

Table 1: Percentage inhibition of immobility time in Forced swim test

S.No	Treatment	30 min	60 min	120 min	240min
1.	Fluoxetine (25mg/kg)	33.16	36.52	48.2	35.52
2.	MSEAS(100mg/kg)	8.69	32.87	17.95	4.62
3.	MSE(200mg/kg)	13.78	7.30	5.56	13.74

n = 5 in each group. Significance at p < 0.05*, p <0.01** and ns –not significant v_scontrol group

Figure 1: Effect of MSEAS on immobility time in Forced swim test in mice

Tail suspension test:

The results were presented in Table 2 revealed that the immobility time was significantly decreased in animals treated with 100mg/kg , p.o of MSEAS and Fluoxetine 25mg/kg ,p.o but not 200mg/kg ,p.o of MSEAS when compared with control.

Table 2 Percentage inhibition of immobility time in Tail suspension test

S.No	Treatment group	% Inhibition
1.	Standard	34.87
2.	MSEAS (100mg/kg)	36.48
3.	MSEAS (200mg/kg)	15.45

n = 5 in each group. Significance at p < 0.05*, p <0.01** and ns –not significant v_scontrol group.

Figure 2. Effect of MSEAS on immobility time in Tail suspension test in mice

Antagonism of Apomorphine induced Hypothermia:

In this test animals treated with Desipramine (20mg/kg, po) but not two doses of MSEAS (100 and 200mg/kg, po) showed significant Antagonism of Hypothermia when compared with control.

Table 3 : Effect of MSEAS on % Inhibition of temperature in Apomorphine induced hypothermia

S.No	Treatment	10 min	20 min	30 min	60min
1.	Desipramine(20mg/kg)	1.42	5.97	8.92	7.65
2.	MSEAS(100mg/kg)	7.00	5.70	3.66	2.02
3.	MSEAS(200mg/kg)	5.74	4.14	2.24	0.50

n = 5 in each group. Significance at p < 0.05*, p <0.01** and ns –not significant v_scontrol group.

Figure 3: Effect of MSEAS on temperature in Apomorphine induced hypothermia

Figure 4. Effect of MSEAS on degree of Hypothermia in apomorphine induced Hypothermia

4. DISCUSSION:

Depression is a heterogenous mood disorder characterized with regular negative moods, decreased physical activity, feelings of helplessness and is caused by decreased brain levels of monoamines like noradranline, dopamine and serotonin. Therefore, drugs restoring the reduced levels of these monoamines in the brain either by inhibiting monoamine oxidase or by inhibiting reuptake of these neurotransmitters might be fruitful in the treatment of depression that has been classified and treated in a verity of ways. Although a number of synthetic drugs are being used as standard treatment for clinically depressed patients, they have adverse effects that can compromise the therapeutic treatment. Thus, it is worthwhile to look for antidepressants from plants with proven advantage and favourable benefits-to-risk ratio.

On the basis of the above information, methanolic seed extract of Avena sativa was selected for evaluating its antidepressant activity due to its traditional use in treatment of depression.

In Acute Oral Toxicity study, MSEAS did not show any lethal effect even up to the doses of 2000mg/kg, po and test doses of 100 and 200mg/kg, po were used for the Pharmacological activity.

On the basis of the clinical association of depressive episodes and stressful life events, many of the animal models for the evaluation of antidepressant drug activity assess stress-precipitated behaviours. The two most widely used animal models for antidepressant screening are the forced swimming and tail suspension tests. These tests are quite sensitive and relatively specific to all major classes of antidepressants. In TST, immobility reflects a state of despair which can be reduced by several agents which are therapeutically effective in human depression. Similarly in the FST, mice are forced to swim in restricted space from which they cannot escape. This induces a state of behavioral despair in animals, which is claimed to reproduce a condition similar to human depression. It has been seen that the TST is less stressful and has higher pharmacological sensitivity than FST [Santosh P et al., 2011].

Results showed that the administration of the MSEAS produced a diminution of duration of immobility time of mice exposed to the both FST and TST. In the present study, the MSEAS (100mg/kg, po) administered to mice produced significant anti depressant effect in both FST and TST and their efficacies were found to be comparable to Fluoxetine (25mg/kg, po).

For the assessment of mechanism of action of MSEAS, Antagonism of Apomorphine induced Hypothermia model was used. This model can be regarded as a hint for Anti depressant activity through Noradrenaline uptake. Compounds with a marked Noradrenaline or Dopaminergic components are active against Apomorphine induced Hypothermia but not through seratonergic system [Sanchez-Mateo et al., 2002]. In this model, Desipramine (20mg/kg, po) but not two doses of MSEAS (100 and 200mg/kg, po) significantly antagonized the Apomorphine induced Hypothermia when compared with control group.

From all the above, the Anti depressant activity of methanolic seed extract of Avena sativa was found to be significant at 100mg/kg, po. The flavanoid components of MSEAS might be interacting with 5-HT in mediating the anti depressant effect of Avena sativa.

5. CONCLUSION:

The MSEAS contained carbohydrates, alkaloids, flavanoids, steroids, glycosides, saponins, amino acids, gums and mucilage. From the above findings, the Anti depressant activity of MSEAS was significant at 100mg/kg , p.o in Forced swim test, Tail suspension test and not in Antagonism of Apomorphine induced Hypothermia. Shortening of immobility time in the forced swimming and tail suspension tests indicating MSEAS acting either by enhancement of central 5-HT and catecholamine neurotransmission and in Antagonism of Apomorphine induced Hypothermia indicating MSEAS was not acting through Adrenergic system.

However, more extensive Pharmacological studies of this plant are required for complete understanding of the Anti depressant activity of methanolic seed extract of Avena sativa.

6. REFERENCES:

1 Cheung, I.W.Y., Nakayama, S., Hsu, M.N.K., Samaranayaka, A.G.P., Li-Chan, E.C.Y. (2009). Angiotensin-I converting enzyme inhibitory activity of hydrolysates from oat (A. sativa) proteins by in silico and in vitro analyses. Journal of Agricultural and Food Chemistry, 57, 9234-9242.

2 Danielle Ryan, Megan Kendall and Kevin Robards. (2007). Bioactivity of oats as it relates to cardiovascular disease. Nutrition Research Reviews, 20, 147–162. Dinesh Dhingra and Amandeep Sharma. (2006). “A review on antidepressant plants”. Natural Product Radiance, 5(2), 144-152.

3. Galdino P M, Nascimento M.V.M, Sampaio B.L, Ferreira R.N, Paula J.R, Costa E.A. (2009). Antidepressant-like effect of Lafoensia pacari A. St.-Hil. ethanolic extract and fractions in mice. Journal of Ethnopharmacology, 124, 581–585.

4 .Khandelwal KR. (2000). “Practical Pharmacognosy. Techniques and Experiments” Pune, Nirali Prakashan, 2, 149-155.

5. Khare C P. (2007). Indian Medicinal Plants an Illustrated Dictionary. Springer, 73-74.Kokate CK. (1994). “Practical Pharmacognosy”, New Delhi, Vallabh Prakashan, 4,110-111.

6. Kulkarni S.K and Ashish Dhir. (2007). Effect of various classes of antidepressants in behavioral paradigms of despair. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 31, 1248–1254.

7. OECD 2001-Guideline on acute oral toxicity (AOT) Environmental health safety monograph series on testing and adjustment No.425.

8. Ryan, D., Kendall, M., Robards, K. (2007). Bioactivity of oats as it relates to cardiovascular disease. Nutrition Research Reviews, 20, 147-162.

9. Sanchez-Mateo C.C, Prado B, Rabanal R.M. (2002). Antidepressant effects of the methanol extract of several Hypericum species from the Canary Islands. Journal of Ethnopharmacology, 79, 119–127.

10. Santosh P, Venugopl R, Nilakash A S, Kunjbihari S, Dr. Mangala L. (2011). Antidepressant Activity of methanolic extract of Passiflora foetida leaves in mice. Int J Pharm Pharm Sci, 3(1), 112-115.

11. Santosh P, Venugopl R, Nilakash A S, Kunjbihari S, Dr. Mangala L. (2011). “Antidepressant activity of methanolic extract of Passiflora foetida leaves in mice”. Int J Pharm Pharm Sci, 3(1), 112-115.

12. Shun C.W., Bligh, S.W.A., Chang, L.Z., Song, S.S., Zheng, T.W., Zhi, B.H., Crowder, J., Branford- White, C., Vella, C. (2008). Sulfated b-glucan derived from oat bran with potent anti-HIV activity. Journal of Agricultural and Food Chemistry, 56, 2624-2629.

13. Singh Rudra Pratap, Jain Ritesh, Mishra Rahul, Tiwari Prasant. (2012). Anti depressant activity of Hydro alcoholic Extract of Zingiber officinale. International Research Journal of Pharmacy, 3 (2).

14. Sudhakar Pemminati, Gopalakrishna H N, Ashok K Shenoy, Sudhanshu Sekhar Sahu, Shishir Mishra, Vinayak Meti, Nair Vinod. (2010). “Antidepressant activity of aqueous extract of fruits of Emblica officinalis in mice. Int J App Bio and Pharma Tech, 1(2), 449-454.

15. Sur, R., Nigam, A., Grote, D., Liebel, F., Southall, M.D. (2009). Avenanthramides, polyphenols from oats, exhibit anti-inflammatory and anti-itch activity. Archives of Dermatological Research, 300, 569-574.

16. Trease G E, Evans MC. (1983). “Text book of Pharmacognosy” London, BailliareTindall, 12, 336.

Received on 22.05.2013

Modified on 09.06.2013

Accepted on 12.06.2013

Research J. Pharmacology and Pharmacodynamics. 5(4): July–August 2013, 212-217