Experimental Evaluation of Antidepressant activity of Aqueous and Chloroform Leaf and Shoot Extracts of Eicchornia crassipes Linn in Mice

 

Praveen Kumar Uppala1*, Dr. K. Atchuta Kumar2, Sujit Kumar Patro3, Murali Krishna. B1

1Bhaskara Institute of Pharmacy, Affiliated to Andhra University, Visakhapatnam

2Principal, Bhaskara Institute of Pharmacy, Affiliated to Andhra University, Visakhapatnam

3Associate Professor, Bhaskara Institute of Pharmacy, Affiliated to Andhra University, Visakhapatnam

*Corresponding Author E-mail: praveen.chintu32@gmail.com

 

ABSTRACT:

The main objective of the study to evaluate the Anti-depressant activity of aqueous and chloroform extract of Eicchornia crassipes in Forced swim test (FST), Tail Suspension test (TST) in mice. Phytochemical screening showed presence of carbohydrates, alkaloids, flavanoids, steroids, saponins, amino acids, gums and mucilage. AEEC and CEEC did not produce any lethal effect even upto 2000mg/kg, p.o during Acute Oral Toxicity study. In FST and TST, CEEC showed diminution of duration of immobility time in 200mg/kg but not in 100mg/kg. From the above finding concluding that, shortening of immobility time in the FST and TST indicating, CEEC showed more antidepressant activity acting either by the enhancement of central 5-HT or catecholamine neurotransmission compared to AEEC in mice.

 

KEYWORDS: Eicchornia crassipes, Aqueous extract of Eicchornia crassipes, Chloroform extract of Eicchornia crassipes, Forced swim test, Tail suspension test

 

 


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

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

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

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


 


 

Figure 3 :  Effect of AEEC on Immobility time in Forced swim test in mice

 

Figure 4 : Effect of CEEC on Immobility time in Forced swim test in mice


 

Table 4 Percentage inhibition of immobility time in Tail suspension test of Aqueous extract

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

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

 

Table 5 Percentage inhibition of immobility time in Tail suspension test of chloroform extract

S.

no

Treatment

% of

inhibition

1.

Control (Distilled water 10ml/kg)

51.57

2.

Standard (Imipramine 10mg/kg)

33.65

3.

Low dose (100mg/kg)

27.32

4.

High dose (200mg/kg)

14.85

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

 


 

Figure 5: Effect of AEEC on Immobility time in Tail Suspension test in mice

A-control,       B- Imipramine 10mg/kg ,    C- AEEC 100 mg/kg,    D- AEEC 200 mg/kg

 

 

Figure 6 : Effect of CEEC on Immobility time in Tail Suspension test in mice

A-control        B- Imipramine 10mg/kg      C- CEEC 100 mg/kg      D- CEEC 200 mg/kg

 


Tail suspension 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

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:

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Received on 22.05.2015          Accepted on 30.06.2015        

© Asian Pharma Press All Right Reserved

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

DOI: 10.5958/2231-5659.2015.00022.3