Abstract

The aim of the study was to evaluate the effect of ethanolic extract of roots of Withania somnifera (23 mg/kg, p.o) on acute stress-induced biochemical and immunological perturbations in mice. The standard group was administered water soluble root powder of Panax ginseng (100 mg/kg, p.o) while the stress control group was administered distilled water orally. After 7 days of pretreatment with the extract, the animals were concomitantly exposed to swim endurance test and cold restraint stress (4oC for 1 hour). Cold restraint stress resulted in significant increase in adrenal gland weight with concomitant decrease in spleen weight in stress control group which was significantly reverted by pretreatment with extract of Withania somnifera. The activation of HPA system results in secretion of corticotropin hormone, adrenocorticotropic HORMONE(ACTH), BENDRROPHIN and glucocorticoids into the circulation. Pretreatment of animals with Withania somnifera extract (23 mg/kg, p.o) improved the swim duration in mice and significantly restored back the stress-induced alterations in plasma cortisol, blood glucose and triglyceride levels.

Key words: HPA system, Withania somnifera, Adrenocorticotropic hormone, Cortisol, Swim endurance test, Cold restraint stress.

Introduction

Adaptogens are naturally occurring substances found in plants. It is absolutely safe and non-toxic to the human body and increases THE BO0DY'Ss NON_SPECIFIK resistance to internal and external stimuli and brings the dysfunctioning body's system back into balance. Adaptogens can successfully combat the negative effects of stress, improve health and well-being, and enhance body's performance [1-10, 13].

Withania somnifera (Ashwagandha) has been used for thousands of years as a popular remedy for many conditions. Perhaps its main use, as described in Ayurvedic literature, is as a "rasayana" or rejuvenating drug. The word Ashwagandha indicates the equine (of horses) odour of the plant. Another name Avarada suggests the application of this plant for enhancing

longevity. The root drug is considerd a tonic and roborant. The root of Withania somnifera is used to make the Ayurvedic sedative and diuretic "Ashwagandha", which is also considered an adaptogen. It is said to "protect the organism from illness" through maintaining the healthy balance of the physical energies [16, 31]. The root contains the steroid lactone withaferin A and related withanolides, besides various alkaloids [38, 39]. The sitoindosides IX and X represent C-27-glycowithanolides, the sitoindosides VII and VIII, acyl-esteryl glucosides. The sitoindosides VII, VIII, IX and X represent the adaptogenic active substances of Withania somnifera, in spite of diverse steroidal structures [11, 15].

Ashwagandha is one of the main herbs for promoting ojas and rejuvenating the body. It is well- known semen promoter and it treats impotency and infertility. It increases physical endurance and improves sexual function [17, 19-26]. It is a rejuvenative general tonic, which stimulates immune system. Ashwagandha has adaptogenic, immunomodulatory and anti-inflammatory effects [11, 12, 28]. It regenerates hormonal system and has anti- stress properties. It is used in many general tonics and preparations, such as chayavana prash [14, 15]. The present study has been undertaken to find out the mechanism of anti-stress activity of Withania somnifera in stress- induced mice.

Materials and Methods

Plant material roots of Withania somnifera were collected, dried in shade, and finely powdered. The powder was soaked in absolute ethanol (95%) and left for 48 hours. The supernatant was collected and the residue was further soaked in absolute ethanol (95%) for 24 hours. The supernatant was collected and filtered. The filtrate was subjected to Rota vapour extraction at a temperature below 60oC for 24 hours. The concentrated form of the extract was obtained and freeze-dried.

The study was conducted on healthy, adult, male albino mice having a body weight of 35 + 5 g. They were acclimatized to laboratory condition for 2 weeks prior to experimentation. Animals were housed in propylene cages (6 mice/cage) in a mice

experimentation laboratory at a temperature of 25oC + 2oC with 12 - 12 h dark -light cycle. They were provided with standard food and water ad libitum. Institutional animal ethical committee (I.A.E.C) approval was obtained before the experiment and care was taken to handle the mice in humane manner. All the chemicals used in the present study were obtained from Euro Diagnostics (Mumbai, India), India Scientific Company (Patna, Bihar) and Bihar Scientific Corporation (Patna, Bihar).

Experimental Design

The adult animals (8 weeks old) were divided into 4 groups (n = 6 in each group) as follows: Group I consisted of Normal control (NC), these mice remained undisturbed in the home cage throughout the experimental period. Group II consisted of Stress control (SC), which were fed with equivolume of distilled water orally for 7 days. Group III (Stress+P.ginseng) consisted the standard group, these mice were fed with aqueous root powder of Panax ginseng, (p.o) for 7 days. Group IV consisted of (Stress+W.somnifera), treatment group which were fed with ethanolic extract of

Withania somnifera, (p.o) for 7 days.

Stress Procedure

Swim Endurance Test: The mice in group IV were given ethanolic extract of Withania somnifera, 23 mg/kg, (p.o), using oral gauge for 7 days. The standard group (III) was administered water soluble root powder of Panax ginseng 100 mg/kg, (p.o), while the stress control group (II) was administered distilled water for 7 days orally. On the 8th day, the animals were allowed to swim till exhausted in a propylene tank of dimension 24 cm* 17 cm* 14 cm, filled with water to a height of 10 cm. The end point was taken when the animals drowned and 'swimming time' for each animal was noted. The mean swimming time for each group was calculated and the data was statistically analyzed.

Cold Restraint Stress: The mice in group IV were given ethanolic extract of Withania somnifera 23 mg/kg, (p.o), using oral gauge for 7 days. The standard group (III) was administered water soluble root powder of Panax ginseng 100 mg/kg, (p.o), while the stress control group (II) was administered distilled water for 7 days orally.

On the 8th day, the animals were individually placed in plastic containers of capacity 350 ml. They were immobilized in their normal position, using adhesive tape. The containers were placed in a cold chamber maintained at 4oC for 1 hour. The blood was collected by orbital sinus veinpuncture method in a heparinised tube and the following investigations were carried out. Total WBC count was done using

Neubauer's chamber, blood glucose was determined by GOD/POD method, plasma cortisol was determined by Enzyme Linked Immunosorbent Assay (ELISA) [32], serum triglyceride was determined by GPO-POD method [27], total cholesterol was determined by CHOD-POD method and HDL cholesterol was determined by CHOD-PAP method.

Statistical Analysis

Data was analyzed by application of one way analysis of variance (ANOVA) using Graph pad in stat software. P<0.01 was considered to be significant.

Results and Discussion

Acute toxicity studies with the extract revealed that LD50 is 1750 mg/kg body weight, (p.o). As shown in figure 1, the extract of Withania somnifera improves swim duration in mice. Mice pretreated with ethanolic extract of Withania somnifera 23 mg/kg, (p.o), and water soluble root powder of Panax ginseng 100mg/kg, (p.o), show significant improvement in the swimming time (P<0.01), as compared to control. (n = 6 in all groups, SC vs S+W.somnifera, P<0.01; SC vs S+P.ginseng, P<0.01; One way ANOVA, P<0.01, F = 41.336; Fig. 1).

The induction of cold restraint stress led to a rise in total WBC count, blood glucose, plasma cortisol and serum triglyceride levels. All the two treatments produced a significant reduction in total WBC count (P<0.01), as compared to controls. (n = 6 in all groups, NC vs SC, P<0.01; SC vs S+W.somnifera, P<0.01; SC vs S+P.ginseng, P<0.01; One way ANOVA, P<0.01, F = 6.006; Fig. 2).

The blood glucose was significantly increased, when the animals were subjected to cold restraint stress compared to control (P<0.01). Pretreatment of animals with the extract of Withania somnifera 23 mg/kg, (p.o), or water soluble root powder of Panax ginseng 100 mg/kg, (p.o), prevented this (P<0.01). (n = 6 in all groups, NC vs SC, P<0.01; SC vs S+W.somnifera, P<0.01; SC vs S+P.ginseng, P<0.01; One way ANOVA, P<0.01, F = 60.373; Fig. 3).

The plasma cortisol level which was found to be elevated in the animals subjected to cold restraint stress was significantly reduced by all the four treatments (P<0.01), compared to controls. (n = 6 in all groups, NC vs SC, P<0.01; SC vs S+W.somnifera, P<0.01; SC vs S+P.ginseng, P<0.01; One way ANOVA, P<0.01, F = 92.616; Fig. 4).

The triglyceride level was increased in the animals subjected to cold restraint stress compared to control (P<0.01). However, no significant change in the serum cholesterol level was observed. Treatment of animals with the extract of Withania somnifera 23 mg/kg, (p.o), or water soluble root powder of Panax ginseng 100

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