INTRODUCTION:

At present, anxiety and depression are the most frequent psychiatric conditions commonly found. A number of the population suffers from these conditions at some time during their life. To date, the efficacy of the drugs for these conditions are very limited so the need for newer, better-tolerated and more efficacious treatments is remaining high. Therefore, herbal therapies should be considered as alternative/complementary medicines. Recently, the search for novel pharmacotherapy from medicinal plants for psychiatric illnesses has progressed significantly. This has been reflected in the large number of herbal medicines whose psychotherapeutic potential has been assessed in a variety of animal models¹. Several herbal drugs have been introduced during the recent past for decreasing anxiety and stress in many emotional and physical disorders. The use of adaptogenic plants have been suggested in the traditional system of medicine, for the cure of emotional disorders, depression, anxiety and gastric ulcers. Moreover, a number of plants such as Cnestis ferruginea, Alchornea cordifolia, green tea and Sida cardifolia have been reported to possess antistress properties^2-5. Centella asiatica linn commonly known as Vallarai is found throughout marshy areas of India and in middle East.

All the parts of plants were used for medicinal purpose. Centella asiatica is also known as gotukola. In Ayurvedic system of medicine it is commonly used to treat various diseases such as cardiotonic, nervetonic, sedative to nerves, insomnia and epilepsy. The primary active constituent of Centella asiatica are saponins also called as triterpenoids including asiaticoside. This constituent is mainly responsible for this activity. Several research workers have investigated that the plant possess cognition and antioxidant properties⁶, gastric ulcer healing activity⁷, cytotoxic and antitumor activity⁸, memory enhancing⁹ and cardioprotective activity¹⁰. The present study has been undertaken to investigate the antistress and hypnotic effect of aqueous extract of Centella asiatica in rodents.

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 aqueous extract:

450gm of leaf extracted with chloroform water by double maceration for 48hrs. The extract was filtered through muselin cloth. The filtrate was evaporated to dryness in vaccum and kept in a refrigerator.

Preparation of crude extract:

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 volume¹¹.

Drugs and chemicals:

Imipramine hydrochloride (Sigma-Aldrich, St Louis, USA) was used as reference standards for antistress activity. Phenobarbitone sodium (Sigma, U.K) dissolved in saline used accordingly.

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:

An acute toxicity study of AECA was carried out in female wister rat (150-200gm). The animals were fasted overnight prior to the experiment. Study conducted to acute oral toxicity as per OECD guidelines 423.

Evaluation of antistress activity:

Tail Suspension Test (TST):

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 induced by tail suspension was measured by placing the mice both acoustically and visually isolated were suspended 50cm above the floor by adhesive tape placed approximately 1cm from the tip of tail. Immobility time was recorded during a 6min period. Mice were considered immobile only when they hung passively and were motionless¹⁴.

Phenobarbitone Induced Sleeping Time in mice:

Groups of 5 mice were used and Phenobarbitone sodium (14mg/kg) was injected 15min, after administration of control vehicle and AECA (100,200mg/kg) . Duration between the time of injection to loss of righting reflex measured as onset of action and time interval between the loss and regaining of righting reflex was measured as sleeping time¹⁵.

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 AECA 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 antistress activity were 100, 200mg/kg.

Tail Suspension Test (TST):

The animals treated with aqueous extract at an acute dose of 100mg/kg and 200mg/kg of body weight significantly reduced the immobility time (p<0.05) by 40 sec and 50 sec as compared to the immobility time of control (i.e;) 130 sec. The standard drug Imipramine when administered at an acute dose of 30mg/kg body weight significantly reduced the immobility time (p<0.01) by 62 sec when compared to control.

Group	Dose (i.p; mg/kg)	Time of immobility in seconds
Control	5ml/kg	130 ± 1.50
Imipramine	30mg/kg	68 ± 2.33**
AECA 100	100mg/kg	90 ± 1.65*
AECA- Aqueous 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.

Effect of AECA on Phenobarbitone Induced Sleeping Time:

The aqueous extract when administered at an acute dose of 100mg/kg and 200mg/kg of body weight reduces (p<0.01), P<0.001) the onset of sleeping time in Phenobarbitone treated animals by 0.15min and 0.146min and potentiate the duration of sleep by 0.031min and 0.04min when compared to control.

Group	Dose (i.p;mg/kg)	Mean onset of sleep ± S.E.M(mins)	Mean duration of sleep ± S.E.M(mins)
Control	5ml/kg	0.794 ± 0.017	0.078 ± 0.003
AECA 100	100mg/kg	0.644 ± 0.014**	0.109 ± 0.001**
AECA 200	200mg/kg	0.548 ± 0.017***	0.118 ± 0.001***

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

DISCUSSION:

For the purpose of investigation of antistress activity of this plant, we used one animal model, tail suspension test (TST) was developed by Steru et al., 1985. The immobility displayed by rodents when subjected to unavoidable stress such as forced swimming is thought to reflect a state of despair or lowered mood¹⁶, which are thought to reflect depressive disorders in humans. In addition, the immobility time has been shown to be reduced by treatment with antidepressant drugs. Moreover, a significant correlation was found between the clinical efficacy of antidepressant drugs and their potency in both models. It has been recently shown that the regulation of adrenergic receptor may be the major mechanism of this model. The results indicate that Centella asiatica may have an antistress-like effect¹⁷. However, further experiments evaluating the levels of noradrenaline and serotonin in different brain regions are necessary to confirm this hypothesis. Porsolt et al. proposed this behavioral model for the screening of new antidepressant compounds, concluded that the immobility time observed in the test reflected a state of lowered mood or hopelessness in animals, thus, this animal model is the most widely used tool for preclinical screening of 15 putative antidepressant agents. The TST shows a strong sensitivity to monoamine alterations and is a very specific cluster of stress induced behaviours that are not related to depression symptoms in humans, but which are nonetheless exquisitely sensitive to monoaminergic manipulations. It also provides a useful model to study neurobiological and genetic mechanisms underlying stress and antidepressant responses¹⁸. In this study the aqueous extract significantly reduced the immobility time by 90sec and 80sec at a dose of 100mg, 200mg/kg which was comparable to that of standard drug Imipramine. The present study demonstrated the hypnotic activity of Centella asiatica at a dose of 100mg/kg, 200mg/kg body weight significantly increases the duration of sleep induced by phenobarbitone in animals in a dose dependent manner, this discrepancy about the dose that found hypnotic activity between this study and previous study just mention might probably due to different concentration of active ingredients existing in the plant extracts used in both studies, and different in extraction procedure. Although the mechanism underlying this increased duration of sleep may occur via the increase in GABA nergic activity. Recently it was reported that Centella asiatica could increase the GABA level in the brain. The present study showed that the aqueous extract significantly potentiate the Phenobarbitone induced sleep by 0.109 sec, 0.118 sec in a concentration dependent manner. In summary the aqueous extract of Centella asiatica has an antistress and hypnotic activity¹⁹.

CONCLUSION:

Our present study confirmed that the Centella asiatica aqueous extract has the antistress activity as its significantly reduces the immobility time and increases the duration of sleep due to its hypnotic property.

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 07.07.2012 Accepted on 09.08.2012

Asian J. Res. Pharm. Sci. 2(3): July-Sept. 2012; Page 91-94