1. INTRODUCTION:

Depression is one of the major mental disorders characterized with symptoms such as regular negative moods, decreased physical activity, feelings of helplessness, sluggish thought and cognitive function [Galdino et al., 2009]. According to the World Health report, approximately 450 million people suffer from a mental or behavioral disorder. This amounts to 12.3% of the global burden of disease, and will rise to 15% by 2020.⁽¹⁾

Depression is caused by chemical imbalances in the brain which may be hereditary, stressful life changes, stroke, Parkinson's disease, or multiple sclerosis, stroke, social isolation, medical conditions such as hypothyroidism (underactive thyroid), medications (such as sedatives and high blood pressure medications), cancer, major illness, or prolonged pain and sleeping problems.⁽²⁾

Despite the development of new molecules for pharmacotherapy of depression, it is unfortunate that this disorder goes undiagnosed and untreated in many patients. Although the currently prescribed molecules provide some improvement in the clinical condition of patients, it is at a cost of having to bear the burden of their adverse effects.

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.⁽^{3, 4)}

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.⁽^5,6)

Eicchornia crassipes commonly known as water hyacinth is a free-floating perennial aquatic plant belongs to the family of Pontederiaceae. The primary chemical constituents are carbohydrates, alkaloids, flavonoid, tannins, saponin, terpenoid, alkaloids, proteins, phenols they also contain iron, manganese and zinc [Danielle Ryan et al., 2007]. In traditional medicine of Eicchornia crassipes used as nervine tonic, stimulant, antispasmodic,Anti oxidant, antidepressant (used in menopausal phase)⁷

Several studies on Anti Inflammatory activity [Cheung et al., 2009], anti-oxidant activity, [Sur et al., 2009] of Eicchornia crassipes have been reported.

The Anti depressant activity of Eicchornia crassipes 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 Eicchornia crassipes using different animal models in mice.

2. MATERIALS AND METHODS:

2.1 Plant material collection and authentication

The leaves and shoots of Eicchornia crassipes were collected from pond near Parvathipuram Town, Vizianagaram Dist., Andhra Pradesh. It was authenticated by Dr. M. Vasubabu, Sr. Lecturer, Dept of Botany, Govt. Degree College, Vizianagaram. The botanical nomenclature of the plants was duly identified by using standard floras and also cross-checked with Herbarium records. The Plant material was shade dried for 10 days and pulverized.

Preparation of extract

The collected leaves and shoots of Eicchornia crassipes were shade dried at room temperature and grinded coarsely. The leaves and shoots were extracted by percolator using water as solvent and by Soxhelet apparatus using chloroform. The resulting extract was concentrated in vacuum under reduced pressure and dried in desiccators. Thus, the prepared extract was used for further pharmacological evaluation.⁽^8,9)

2.2 Materials

Imipramine was procured from Sigma life sciences, Bangalore.

2.3 Preliminary Phytochemical analysis

The both aqueous and chloroform extracts of leaves and shoots of Eicchornia crasspies was subjected for phytochemical screening and found that carbohydrates, alkaloids, flavonoid, tannins, saponin, terpenoid, alkaloids, proteins, phenols were present

Animals

Albino mice of either sex weighing between 20-30gm were used in this study. All the animals were acclimatized in the quarantine room a NIN Animal house (National Institute of Nutrition, Hubsiguda, Hyderabad), for 7 days and housed in groups of 5 under standard husbandry conditions like room temperature 23±2°C, relative humidity 30-70% and light/ dark cycle of 12 hours.

All the animals were fed with synthetic standard diet (National Institute of Nutrition, Hubsiguda, and Hyderabad) and water under still be supplied ad libitum under strict hygienic conditions. All the experimental protocols were approved by Institutional Animal Ethical Committee (IAEC) of Andhra University. All the animals’ studies were performed as per the rules and regulations in accordance to the guidelines of CPCSEA with registration number.

All animals were fasted 3h prior to oral administration of vehicle/standard/test compounds during the experiment were carried out during the light period (9:00 to 17:30h) to avoid circadian rhythm.

2.4 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. ¹⁰

2.5 In vivo Models for Antidepressant activity

2.5.1 Forced Swim Test

Animals were divided into 4 groups of 5 animals in each, weighing between 20-30 gms

The extracts of both AEEC and CEEC

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

Group II - Standard (Imipramine 10mg/kg,p.o)

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

Group IV- High dose (EEC 200mg/kg,p.o)

For the forced swim test (FST), mice of the either sex were individually forced to swim in an open cylindrical container (diameter 10cm, height 25cm) containing 19cm of water at 25±1°C. Treatment was given 60min prior to study as described by study design all animals were forced to swim for 6min and the duration of immobility was observed and measured during the final 4min interval of the test. Each mice was judged to the immobile when it ceased struggling and remained floating motionless in the water, making only those movements to keep its head above water. A decrease in the duration of immobility is indicative of an antidepressant like effect.⁽¹¹⁾

Figure 1: Representation of mice in FST

TAIL SUSPENSION TEST:

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

The extracts of both AEEC and CEEC

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

Group II - Standard (Imipramine 10mg/kg,p.o)

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

Group IV- High dose (EEC 200mg/kg,p.o)

Figure 2: Representation of mice in TST

The tail suspension method used in this study was similar to those described by steru et al., (1985). Treatment was given 6min prior to study as described by study design. Mice were suspended on the edge of the table, 50cm above the floor, with the help of adhesive tape placed approximately 1cm from the tip of tail. The total duration of immobility induced by tail suspension was recorded during 6min of the 10min period. Animal was considered to be immobile when it did not show any movement of the body, hanged passively and completely motionless.⁽¹²⁾

2.6 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 extract of leaves and shoots of Eicchornia crasspies was subjected for phytochemical screening and found that carbohydrates, alkaloids, flavonoid, tannins, saponin, terpenoid, alkaloids, proteins, phenols were present. The results were shown below in table:1

S. NO	PHYTOCHEMICAL CONSTITUENTS	INFERENCE
S. NO	PHYTOCHEMICAL CONSTITUENTS	AEEC	CEEC
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’ test	- - - -	+ + + +
3	Test for Tannins	+	-
4	Test for Flavonoids · Schinoda test	+	+
5	Test for Terpenoid	+	-
6	Test for Proteins · Biuret test	-	+
7	Test for phenols	-	+
8	Test for saponins	+	+

+ indicates presence; - indicates absence

Acute Oral Toxicity study

The aqueous and chloroform leaf and shoot extract of Eicchornia crassipes 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 aqueous and chloroform extracts of Eicchornia crassipes on the duration and % inhibition of immobility is shown in Table The animals were treated with distilled water 10ml/kg p.o as control,100mg/kg, p.o of AEEC and CEEC and 200mg/kg, p.o of AEEC and CEEC, Imipramine 10mg/kg, p.o.as standard.

Table 2: Percentage inhibition of immobility time in Forced swim test - Aqueous extract

S. no	Treatment	% of immobility
S. no	Treatment	30 min	60 min	120 min	240 min
1.	Control (Distilled water10ml/kg)	50.5	52.4	51.3	50.2
2.	Standard (Imipramine 10mg/kg)	25.6	28.5	26.3	27.5
3.	Low dose (100mg/kg)	13.5	14.8	16.3	17.2
4.	High dose (200mg/kg)	9.3	8.2	7.6	5.9

n=5 in each group. Significance at p <0.005*, p<0.001** and ns-not significant Vs control group

Table 3 Percentage inhibition of immobility time Forced swim test chloroform extract

S. no	Treatment	% of Immobility
S. no	Treatment	30min	60min	120min	240min
1.	Control (Distilled water 10ml/kg)	50.5	51.4	52.3	50.1
2.	Standard(Imipramine 10mg/kg)	26.6	27.5	28.3	28.5
3.	Low dose (100mg/kg)	14.5	15.8	15.3	16.2
4.	High dose (200mg/kg)	8.3	8.2	6.6	5.9

n=5 in each group. Significance at p <0.005*, p<0.001** and ns-not significant Vs control group

Tail suspension test

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.⁽¹³⁾

On the basis of the above information, both aqueous and non aqueous (chloroform) leaf and shoot extract of Eicchornia crassipes was selected for evaluating its antidepressant activity due to its traditional use in treatment of depression.

In Acute Oral Toxicity study, both AEEC and CEEC 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. ^(14,15)

Results showed that the administration of the CEEC produced a diminution of duration of immobility time of mice exposed to the both FST and TST than AEEC. In the present study, the CEEC (200mg/kg, po) administered to mice produced significant anti depressant effect in both FST and TST than CEEC (100mg/kg,po) and AEEC and their efficacies were found to be comparable to Imipramine (10mg/kg, po).

From all the above, the Anti depressant activity of chloroform extract of leaf and shoot of Eicchornia crassipes was found to be significant at 200mg/kg, po. The flavanoid components of CEEC might be interacting with 5-HT in mediating the anti depressant effect of Eicchornia crassipes.

5. CONCLUSION:

The AEEC and CEEC contains carbohydrates, alkaloids, flavanoids, steroids, glycosides, saponins, amino acids, gums and mucilage. From the above findings, the Anti depressant activity of CEEC was significant at 200mg/kg , p.o in Forced swim test, Tail suspension test. Shortening of immobility time in the forced swimming and tail suspension tests indicating CEEC acting either by enhancement of central 5-HT and catecholamine neurotransmission. However, more extensive Pharmacological studies of this plant are required for complete understanding of the Anti depressant activity of chloroform extract of leaf and shoot extract of Eicchornia crassipes.

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.

1. Dinesh Dhingra and Amandeep Sharma. (2006). “A review on antidepressant plants”. Natural Product Radiance, 5(2), 144-152.

2. 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.

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

4. Khare C P. (2007). Indian Medicinal Plants an Illustrated Dictionary. Springer, 73-74.

5. 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-gudeline 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. Trease G E, Evans MC. (1983). “Text book of Pharmacognosy” London, Bailliare Tindall, 12, 336.

Received on 22.05.2015 Accepted on 30.06.2015

Asian J. Res. Pharm. Sci. 5(3): July-Sept.; Page 139-144

DOI: 10.5958/2231-5659.2015.00022.3

S. no	Treatment	% of inhibition
1.	Control (Distilled water 10ml/kg)	50.57
2.	Standard (Imipramine 10mg/kg)	35.65
3.	Low dose (100mg/kg)	28.32
4.	High dose (200mg/kg)	15.85