Antidepressant activity of ethanolic extract of leaves of Centella asiatica. Linn by In vivo methods.

 

P. Thamarai Selvi*, M. Senthil Kumar, R. Rajesh, T. Kathiravan

Department of Pharmacology, Annai Veilankanni’s Pharmacy College, Chennai-15, India

*Corresponding Author E-mail: tthamarai_pharma@yahoo.com

 

ABSTRACT:

The antidepressant activity of ethanolic extract of Centella asiatica .Linn (Umbelliferae) were studied at doses of 100mg and 200mg/kg against forced swimming test in rodents. Imipramine (30mg/kg, i.p) was selected as reference standard and it showed significant antidepressant activity in rodents. A dose dependent reduction in immobility time was observed in FST. Whereas the extract significantly increases the exploratory behaviour in mice. The finding from the present investigation indicates that extract has significant antidepressant activity as shown by its effects on different experimentally induced different models.

 

KEYWORDS: Centella asiatica, Antidepressant, Forced swimming, Hole board test.

 

 


INTRODUCTION:

Mental depression is a chronic illness that affects a person’s mood, thoughts, physical health and behaviour. Symptoms of depression include biological and emotional components. Biological symptoms include retardation of thought, action and appetite. Emotional symptoms include mystery, apathy and pessimism, low self- esteem consisting of feeling of guilt, inadequacy and ugliness, indecisiveness and loss of motivation1. Antidepressant drugs such as tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRI) are used to treat depression showing various side effect and thus, the search for a new antidepressant herb without side effects is important2. Herbal drug used in depression are Centella asiatica , Hypericum perforatum, Rhodiola rosea,  Pfaffia paniculata, Rauwolfia serpentine, Rhododendron molle3. Centella asiatica (CA), a clonal, perennial herbaceous creeper belongs to the family umbellifere (Apiceae) and is found throughout India in moist places up to an altitude of 1800m in tropical and subtropical countries in swampy areas, including parts of India, Pakistan, Sri Lanka, Madagascar, South Africa, South Pacific and Eastern Europe. The plant possess cardiotonic, nerve tonic, sedative  to  nerves , stomachic ,carminative, improves  appetite, antileprotic , memory and febrifuge4. Preliminary phytochemical studies  showed the presence of Saponins, Terpenoids, Alkaloids, Phenols5.

 

Several investigations have proposed that this plant possess sedative and anxiolytic6, antiepileptic7, memory enhancing8, cardioprotective9 and immuno modulating activity10. On the basis of above information the leaves of Centella asiatica was selected for evaluating its antidepressant activity have been raised. Therefore the present study was aimed to explore this indigenous plant for antidepressant activity. 

 

MATERIALS AND METHODS:

Collection and Authentication of plant material: The plant materials were collected from Chennai, Tamil Nadu plant material was identified and authenticated by resident botanist, Prof. Dr. Jayaraman, Plant Anatomy Research Centre [PARC Chennai]. A voucher specimen was submitted at Annai Veilankanni’s Pharmacy College Chennai. Reg. No: PARC/2011/824.

 

Preparation of ethanolic extract:

The dried plant material was crushed into fine particles (powder) using a mixer. The powdered plant material (500gm) was packed in a soxhlet apparatus and subjected to continuous hot percolation for 8hrs using ethanol (1:4) as solvent. Extract obtained was passed through the Whatman filter paper no.1 and the ethanol was evaporated (at 400c) with the help of heating mantle and dried in a desiccators.

 

Preparation of crude extract11:

The crude extract of Centella asiatica was freshly prepared every day by dissolving in distilled water in order to obtain the desired concentration before the oral administration via intra gastric tube once daily. Each animal should receive the same volume of substance in order to avoid from the confounding error due to different in volume.

 

Drugs and chemicals:

Imipramine hydrochloride (Sigma-Aldrich, St Louis, USA) was used as reference standards for antidepressant activity.  Diazepam hydrochloride used as a reference standard diluted with saline to 3mg/10ml before use and administered intraperitoneally in a volume of 10ml/kg(3mg/kg)

 

Animals:

Young adult male Wister rats, 8 weeks old were used as experimental model. The weights of the animals on the first day of experiment were 180-220grams.They were randomly housed 5 per kg and maintained in 10:14 light: dark cycle and given access to food and water ad libitum. All injections in this study were performed once daily between 8.00-9.00am.IAEC.NO:793/03/C/CPCSEA

 

Preliminary phytochemical screening12:

The extract was subjected to preliminary phytochemical screening by the methods previously described by Kokate and Jayaraman. J.

 

Acute toxicity study13:

The procedure was followed as per OECD 423 guidelines. The extract was administered orally at a dose 2000mg/kg body weight to different groups of mice and observed for signs of behavioural, Neurological toxicity and mortality 14 days.

 

Forced swimming test (FST):

 Rats of either sex were individually forced to swim in an open cylindrical container (diameter 10cm,height 25cm), containing 19cm of water at 25±1°c. All the rats of either sex were divided in six different groups. The first group assigned as control receiving only vehicle (Nacl 5ml/kg). The other four groups received acute dose of aqueous, ethanolic extract of CA (100,200mg/kg). The sixth group received standard drug Imipramine (30mg/kg). The total duration of immobility was recorded during the last 6min of the 10min period. Each mouse was judged to be 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(14-17).

 

Hole Board Test (HBT):

Experiment was conducted, 30mins after injection of control vehicle, AECA, EECA and diazepam (4mg/kg) by placing mouse on a wooden board. The number of head dips and time spend in each dips was recorded during 3mins trial18.

Statistical analysis:

The statistical analysis of various studies were carried out using paired ‘t’ test and analysis of variance (ANOVA) followed by Dunnet’s ‘t’ test, p<0.05 were considered as significant.

 

RESULTS:

Preliminary phytochemical screening:

The extracts showed the presence of Saponins, Terpenoids, Alkaloids, Phenols. But Steroids, Gums and Mucilage, Flavonoids, Tannins, Protiens, Carbohydrates, Glycosides were absent.   

 

Acute toxicity study:

The EECA was studied for acute toxicity at doses of 2000mg/kg. The extract was found devoid of mortality of all animals. So, the dose selected for the antidepressant activity were 100, 200mg/kg.

 

Evaluation of Antidepressant activity:

Forced swimming test:

It was observed that EECA at doses of 100mg and 200mg/kg exhibited significant reduction in immobility time when compared to control in dose dependent manner. Similarly the animals treated with Imipramine (30mg/kg) as expected showed significant decrease in immobility time.

 

Table-1

Group

Dose (i.p; mg/kg)

Time of immobility in seconds

Control

5ml/kg

149 ± 2.469

Imipramine

30mg/kg

117 ± 2.875**

EECA 100

100mg/kg

134 ± 3.276*

EECA 200

200mg/kg

125 ± 3.055**

EECA-Ethanolic extract of Centella asiatica. One way ANOVA followed by Dunnet’s test. Values are mean ± S.E.M ( n = 6), in each group *p < 0.05, **p < 0.01 when compared to control.

 

Fig-1-Forced swimming test

 

Table-2


Group

Dose(i.p; mg/kg)

No. of head dippings per 3 min

No. of line crossings per 3min

Control

5ml/kg

22 ± 1.43

18 ± 0.9

Diazepam

4mg/kg

8 ± 0.71**

10 ± 1.24***

EECA 100

100mg/kg

18 ± 1.04*

15 ± 0.55*

EECA 200

200mg/kg

13 ± 1.01**

14  ± 0.55**

One way ANOVA followed by Dunnet’s test. Values are Mean ± S.E.M ;(n = 5), in each group *p<0.05,**p<0.01,***P<0.001, when compared to control

 


Hole board test:

There was a significant reduction in the number of head dippings and line crosses by mice in EECA treated groups(100mg, 200mg/kg, i.p) as compared to control. There was a significant decrease in exploratory behaviour in diazepam treated group as compared to control group.

 

Fig-2- Hole board test

 

DISCUSSION:

The prevention and management of stress disorders remains a major clinical problem. Hence it is very important to address these problems and find effective remedies. Though several drugs are available, all are associated with some limitations and there is an urgent need for alternative medications for these disorders19. To treat a wide variety of nervous disorders, Centella asiatica is a known choice for more than hundred years. In this work, it was demonstrated that the administration of different doses of the ethanolic extract of Centella asiatica in rats and mice was able to induce antidepressant effects. In forced swimming test, the extract can decrease the immobility time in rats with mild sedative effect. It was found that Centella asiatica can produce antidepressant like activity at a dose of 100mg and 200mg/kg body weight in a dose dependent manner. The decrease in the immobility time is accompanied with the increase in swimming time. Previous demonstrated that many neurotransmitters were involved in the pathophysiology of depression. Numerous studies have demonstrated that antidepressant drugs such as Thioxetine, Imipramine stimulated the action of serotonin and act by inhibiting the reuptake of biogenic amines in CNS. These drugs were widely used as antidepressant drugs and agreed with studies in animal models, such as forced swimming test20. An antidepressant drugs reduce the exploratory behaviour depending upon the concentration. At present, the study revealed that the ethanolic extract of EECA significantly reduces the number of head dippings and number of line crossings were the indicator of exploratory behaviour21. The findings from the present investigation indicate that EECA possesses significant antidepressant activity as shown by its mitigating effects on different experimentally induced stress models in rats and mice.

 

CONCLUSION:

This means that the forced swimming test and hole board test is more sensitive and better reflects the state of depression. Our present study confirmed that the Centella asiatica ethanolic extract has the antidepressant activity as its significantly reduces the immobility time and increases the exploratory behaviour during depression in animal models.

 

ACKNOWLEDGEMENT:

The authors are thankful to Dr. M. Senthil Kumar, Principal and Head of the Department of Pharmaceutics, Annai Veilankanni’s Pharmacy, for his encouragement in carrying out this work.

 

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Received on 18.04.2012          Accepted on 23.05.2012        

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Asian J. Res. Pharm. Sci. 2(2): April-June 2012; Page 76-79