Anti-stress effect of ethyl acetate soluble fraction of Morus alba in chronic restraint stress

Abstract

Context: Restraint stress is a well-known method to induce chronic stress which leads to alterations in various behavioral and biochemical parameters.

Objective: The present work was designed to study anti-stress effects of Morus alba in chronic restraint stress (RS)-induced perturbations in behavioral, biochemical and brain oxidative stress status.

Materials and methods: The stress was produced by restraining the animals inside an adjustable cylindrical plastic tube for 3 h once daily for ten consecutive days. The ethyl acetate soluble fraction of Morus alba (EASF) 25, 50, 100 mg/kg and diazepam (1 mg/kg) per day was administered 60 min prior to the stress procedure. The behavioral and biochemical parameters such as open field, cognitive dysfunction; leucocytes count; blood glucose and corticosteroid levels were determined. On day 10, the rats were sacrificed and biochemical assessment of superoxide dismutase (SOD), lipid peroxidation (LPO), catalase (CAT), and glutathione reductase (GSH) in whole rat brain were performed.

Results: Chronic restraint stress produced cognitive dysfunction, altered behavioral parameters, increased leucocytes count, SOD, LPO, glucose and corticosterone levels, with concomitant decrease in CAT and GSH activities. Gastric ulceration, adrenal gland and spleen weights were also used as the stress indices. All these RS induced perturbations were attenuated by EASF of Morus alba.

Discussion: The results of the study suggest that in addition to its classically established pharmacological activities, the plant also has immense potential as an anti-stress agent of great therapeutic relevance.

Conclusion: This study indicates the beneficial role of Morus alba for the treatment of oxidative stress-induced disorders.

Keywords::

• Morus alba
• restraint stress
• anti-stress
• lipid peroxidation
• serotonin

Introduction

Stress is a state of threatened homeostasis provoked by psychological, physiological or environmental stressors. Stressor is a stimulus either internal or external, which activates the hypothalamic pituitary adrenal axis and the sympathetic nervous system resulting in physiological changes (Nirmal et al., 2008). During stressful situations, the energy requirement of an organism is increased, resulting in enhanced generation of free radicals (Kenjale et al., 2007). This increase in production of free radicals has been reported to cause damage to cell membranes, enzymes, DNA, lipids and proteins. Oxidative stress is a disparity between the rates of free radical production and elimination through endogenous antioxidant mechanisms (El-Sherbiny et al., 2003). Several clinical research findings implicated oxidative stress in the pathophysiology of chronic stress. Various types of stress such as mild electric foot shock, restraint, food deprivation and anticipation stress cause a selective increase in oxidative stress (Dunn, 1998). Glucocorticoids (GCs) are hormones secreted by the adrenals in response to both physical and psychological types of stress. GCs increase the toxicity of oxygen radical generators, and may increase the basal level of ROS in the cells (Mcintosh et al., 1998). Chronic stress leads to several physiological, biochemical and behavioral perturbations, which include glucose intolerance, ulceration, immunosuppression, cognitive deficits and male sexual dysfunction. These pathological states may be at least partly induced by oxidative stress (Kumar et al., 2001).

Restraint stress is an easy and well-known method to induce chronic physical and emotional stress (Glavin et al., 1994). Two major types of experimental restraint stress models have been confirmed. The first model includes confinement, where the animal’s movement is limited by restricted space. The second model utilizes immobilization, where the limbs and body of the animal are held immobile. Among the various methods employed, immobilization has been used extensively and is a widely accepted model for studying stress-induced physical as well as psychological alterations and consequences of the stress. During stress induction various biochemical enzymes and immunological systems are affected. Some of the stress–induced alterations have been attributed to an imbalance in the neuro-endocrine system (Kenjale et al., 2007). Hence, measurement of the some of the biochemical, endocrinal and behavioral parameters will serve as an important basis for the evaluation of anti-stress activity (Rai et al., 2003).

Morus alba Linn. (mulberry) is one of the best known and most widely distributed tree or shrub of the family Moraceae, which is extensively cultivated throughout the plains of India. A number of earlier studies have indicated that Morus alba exhibits an activity profile which may reflect putative pharmacological effects. Scientific studies have established the multifarious utility of this plant in a wide array of pharmacological activities, including hypotensive (Fukai et al., 1985), hypoglycemic (Singab et al., 2005), hypolipidemic, cardioprotective (Byambaa et al., 2005), hepatoprotective (Hyuncheol et al., 2002), hypouricemic (Cheyl, 2006), antihyperglycemic, antioxidant, antiglycation (Naowaboot et al., 2009) and anxiolytic (Yadav et al., 2008) effects. M. alba contains flavonoids such as isoquercetin, quercetin, which demonstrated a free radical scavenging activity (Kim et al., 1999). On the other hand, the antioxidant potency of some phenolic compounds from Morus alba has been reported (Fukai et al., 2003). The naturally occurring flavonoids, especially those of Morus alba have shown antioxidant activity in different model systems (Kim et al., 1999; Byambaa et al., 2005; Fukai et al., 1985; El-Beshbishy et al., 2006). Anthocyanin of Morus alba extracts inhibited LDL oxidation and macrophage-derived foam cell formation induced by oxidative LDL (Liu et al., 2008). The plant is a very good source of ascorbic acid, of which over 90% is present in reduced form. It also contains carotene, vitamin B₁, folic acid, folinic acid, isoquercetin, quercetin, tannins, flavonoids, anthocyanin and saponins which act as a good source of natural antioxidants (CSIR, 1956); which provided a way to study molecular mechanisms underlying the anti-stress role of Morus alba.

In this study, we have investigated the effect of EASF of Morus alba in chronic restraint stress (CS)-induced perturbations in behavioral, biochemical and brain oxidative stress status.

Methods

Plant material

The roots of M. alba were collected in the month of October (2007) from the local area in Nashik, India and authenticated by P.S.N. Rao (Director, Botanical Survey of India, Pune). A voucher specimen of the plant has been deposited at the Botanical Survey of India, Pune (voucher specimen no. NVMA2). The roots were shade-dried and reduced to coarse powder. The powdered plant material was defatted using petroleum ether (60–80°C) by Soxhlet extractor and then extracted by methanol for 72 h to obtain the extract. The extract was filtered and concentrated under reduced pressure. The yield of methanol extract of M. alba roots was found to be 18.25% w/w. The methanolic extract was exhaustively extracted with ethyl acetate to obtain ethyl acetate soluble (EASF, 10.3% w/w) and ethyl acetate insoluble fractions (EAIF, 6.1% w/w). Suspensions of EASF were prepared in distilled water using Tween 80 (0.2% v/v) as suspending agent. The extract was administered in doses of 25, 50 and 100 mg/kg per day orally (p.o.), 60 min before restraining. The control group was given only vehicle (0.2% v/v, Tween 80) in volumes equivalent to that of the plant extracts.

Animals

Male Wistar rats (150–180 g) were used for the study. The animals were housed in polypropylene cages and maintained at 25° ± 2°C, 12:12 h light:dark cycle and 50% ± 5% Relative Humidity (RH) with free access to food and water ad libitum. Animals were acclimatized to laboratory conditions before the test. All the experiments were carried out during the light period (08:00–16:00 h). The studies were carried out in accordance with the guidelines given by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), New Delhi (India). The Institutional Animal Ethical Committee of M.V.P.S. College of Pharmacy, Nashik (India) approved the protocol of the study (IAEC/2008/01).

Drugs and chemicals

Diazepam injection i.p. (Ranbaxy Laboratory, Delhi, India) was used as standard drug. thiobarbituric acid (TBA) (Research Lab Fine Chem Industries, Mumbai, India), nitroblue tetrazolium (NBT) (Himedia Laboratories, Mumbai, India), 5, 5′- dithiobis (2-nitro benzoic acid) (DTNB) (Alfa Aesar, Johnson Mathey, Chennai, India). All chemicals used were of analytical grade and purchased from standard manufacturers.

Experimental procedure

Treatment

Animals were divided into 6 groups having 6 animals in each group. Group I – control non-stress (0.2% v/v, Tween 80); Group II – vehicle (chronic restraint stress); Group III – diazepam (1 mg/kg per day, p.o.) + Chronic restraint stress (CS); Group IV – EASF (25 mg/kg per day, p.o.) + CS; Group V – EASF (50 mg/kg per day, p.o.) + CS; Group VI – EASF (100 mg/kg per day, p.o.) + CS.

Induction of chronic restraint stress

The stress was produced by restraining the animals inside an adjustable restraint cage. The rats were confined individually and exposed continuously to restraint stress for 3 h once daily for ten consecutive days. The EASF 25, 50, 100 mg/kg and diazepam (1 mg/kg) were administered 60 min prior to the stress procedure up to ten consecutive days. The behavioral parameters during stress such as open field and cognitive dysfunctioning were performed. Blood was removed from retro-orbital plexus and centrifuged at 2000 rpm for 10 min. Serum was separated and taken for the measurement of blood glucose and corticosteroid levels on day 0, 1, 5 and 10 respectively. On day 10 rats were sacrificed; brain, adrenal gland and spleen were isolated and weighed. Brain was homogenized and superoxide dismutase (SOD), catalase activity (CAT), reduced glutathione (GSH) and lipid peroxidation (LPO) levels were determined in whole rat brain.

Behavioral study

Effect of EASF on cognitive deficits-induced by chronic restraint stress in elevated plus maze

Rats were placed individually at the end of either of the open arms facing away from the central platform and transfer latency (TL) to enter the closed arm was noted. The time taken by each animal to move from open arm to either of the closed arms was recorded. This duration of time was called transfer latency (TL). Each rat explored the maze for 5 min. TL was noted on test day 1, 2, 5 and 10 after induction of stress (Jaiswal & Bhattacharya, 1992).

Effect of EASF on cognitive deficits-induced by chronic restraint stress in passive shock avoidance test (PSAT)

The term passive avoidance is usually employed to describe experiments in which the animal learns to avoid a noxious event by suppressing a particular behavior. Rats were placed in a rectangular box (50 × 50 cm) with electrifiable grid floor. The grid floor was connected to a shock device which delivers scrambled foot shocks. Each rat was placed on a platform and the latency to step down on the grid floor was measured (familiarization). After 10 s of exploration, it was returned to the home cage. Immediately after the animal has descended from the platform an unavoidable foot shock (50 Hz; 1.5 mA; 1s) was applied (learning, Day 0). After 24 h of learning trial, the rat was again placed on the platform and the step-down latency was measured (acquisition, day 1). The test was finished when the animal stepped down or remained on the platform (cut-off time: 300 s). Step-down latency was measured on days 2, 5 and 10 (Vogel & Vogel, 2002).

Effect of EASF on locomotion in open field test

Locomotor activity was evaluated in an open field paradigm. The open field was made of plywood and consisted of a floor (96 × 96 cm) with high walls (61 × 61 cm). The floor was divided into 16 squares. Each animal was placed at one corner of the apparatus and observed for next 5 min. It was observed for the ambulations (number of squares crossed), number of rearing, and latency to come to the center of the open field. The test was performed on day 0, 1, 5 and 10 (Lister, 1990).

Biochemical analysis

Dissection and homogenization

On day 10 immediately after chronic restraint stress the animals were killed by decapitation. The brain was removed, rinsed with isotonic saline and weighted. A 10% (w/v) tissue homogenate was prepared in 0.1 M phosphate buffer (pH 7.4). The post nuclear fraction for catalase assay was obtained by centrifugation of the homogenate at 1,000 g for 20 min at 4°C in a Remi C–30 (Remi Industries, Mumbai); for other enzyme assays, centrifugation was at 12,000 g for 60 min at 4°C. A Shimadzu 160A spectrophotometer was used for subsequent assays (Naidu et al., 2003).

Catalase activity

Catalase activity was assessed by the method of Luck (1971), where the breakdown of H₂O₂ was measured at 240 nm. Briefly, the assay mixture consisted of 3 mL H₂O₂ phosphate buffer (0.0125 M H₂O₂) and 0.05 mL supernatant of brain homogenate (10%) and the change in the absorbance was measured at 240 nm. The enzyme activity was calculated using the mmol extension coefficient of H₂O₂ 0.07. The results were expressed as μmol H₂O₂ decomposed/min/mg protein (Luck, 1971).

Superoxide dismutase activity

Superoxide dismutase activity was assayed according to the method of Kono (1978), wherein the reduction of nitroblue tetrazolium chloride was inhibited by the superoxide dismutase which was measured at 560 nm spectrophotometrically. Briefly, the reaction was initiated by the addition of hydroxylamine hydrochloride to the reaction mixture containing NBT and post nuclear fraction of brain homogenate. The results were expressed as units/mg protein, with one unit of enzyme defined as the amount of SOD required to inhibit the rate of reaction by 50% (Kono, 1978).

Lipid peroxidation assay (LPO)

The quantitative measurement of lipid peroxidation in brain was done by the method of Wills (1966). The amount of malondialdehyde (MDA) formed was measured by reaction with thiobarbituric acid at 532 nm. The results were expressed as nmol MDA/mg protein, using the molar extension coefficient of chromophore (1.56 × 10⁵ M⁻¹ cm⁻¹) (Wills, 1966).

Estimation of reduced glutathione

Reduced glutathione in the brain was estimated according to the method of Ellman (1959). A 0.1 mL sample of homogenate was precipitated with 0.75 mL 4% sulfosalicylic acid. The assay mixture contained 0.5 mL supernatant and 4.5 mL DTNB in 0.1 M phosphate buffer, pH 8. The yellow color developed was read immediately at 412 nm. The results were expressed as nmol GSH/mg protein (Ellman, 1959).

Protein estimation

The protein content was measured according to the method of Lowry et al. (1951), using bovine serum albumin as standard and expressed as µg protein/mg tissue (Lowry et al., 1951).

Estimation of blood serum glucose level

Serum glucose levels were determined using a standard glucose oxidase-peroxidase kit.

Estimation of blood serum corticosterone level

The quantitative measurement of serum corticosterone in brain was done by the method of Bartos and Pesez (1979).

Effect of EASF of on milk-induced leucocytosis

Subcutaneous (s.c.) injection of milk is known to produce an infection-like condition by acting as an antigen and increasing the leucocyte count. Anti-stress activity can be evaluated on the basis of the capacity of the drug to prevent this stress-induced increase in white blood cell count. Rats were anesthetized under light ether and blood was collected from the retro-orbital plexus. Total leucocyte count was measured before drug administration and 24 h after milk injection (boiled and cooled, 4 ml/kg s.c.) on days 1, 5 and 10. Difference in total leucocyte count before and 24 h after drug administration was calculated (Thakur & Mengi, 2005).

Measurement of adrenal gland and spleen weights

After sacrifice, the adrenal gland and spleen were removed and weighed (Rai et al., 2003).

Gastric ulceration

After sacrificing rats on day 10, the stomachs were removed, opened along the greater curvature; and pinned on a cork plate. The number of ulcers and their severity was registered with a stereo-microscope. The severity of ulcer was scored as 0 = no ulcer, 1 = superficial ulcers, 2 = deep ulcers, 3 = perforations. The ulcer index (UI) was calculated (Vogel & Vogel, 2002).

Statistical analysis

Results are expressed as mean ± SEM, and the statistical analysis of data was done using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Probability level less than 0.05 was considered statistically significant.

Results

Behavioral study

Effect of EASF on cognitive deficits induced by chronic restraint stress in the elevated-plus maze

The transfer latency (TL) to enter the closed arms was significantly increased in vehicle (CS) treated group as compared to control on all days. Pretreatment with EASF (25, 50 and 100 mg/kg) did not show any significant change in transfer latency on day 0. The transfer latency was significantly (p < 0.01) decreased in diazepam and the EASF (100 mg/kg) treated group on days 1 and 2. The transfer latency was significantly (p < 0.01) decreased on days 5 and 10 by all doses of EASF and diazepam as compared to the vehicle group (Figure 1).

Figure 1.  Effect of EASF on chronic restraint stress induced alterations of transfer latency (TL) in elevated plus maze. Each column represents mean ± SEM (n = 6). Compared with vehicle (chronic stress) group ^*p <0.05; ^**p <0.01 (one-way ANOVA followed by Dunnett’s test.

Effect of EASF on cognitive deficits induced by chronic restraint stress in the passive shock avoidance test

The step-down latency was decreased in the vehicle (CS) treated group as compared to control on all days in the passive shock avoidance test. No change was found on day 0 in step-down latency. Pretreatment with EASF (25, 50 and 100 mg/kg) significantly (p < 0.05; p < 0.01) increased step-down latency at all doses on days 2, 5 and 10. The diazepam-treated group showed significant increase in step-down latency only on day 1 (Figure 2).

Figure 2.  Effect of EASF on chronic restraint stress induced alterations of step down latency in passive shock avoidance test. Each column represents mean ± SEM (n = 6). Compared with vehicle (chronic stress) group ^*p <0.05; ^**p <0.01 (one-way ANOVA followed by Dunnett’s test).

Effect of EASF on locomotion in the open field test

The number of ambulations in the open field test was significantly decreased in the vehicle-treated group as compared to control on all days. Administration of EASF (50 and 100 mg/kg) showed significant (p < 0.05; p < 0.01) increase in number of ambulations on day 2. On day 5 only the dose 100 mg/kg was found to be effective in increasing the number of ambulations. All the treatments significantly (p <0.05; p <0.01) increased number of ambulations on day 10 (Figure 3).

Figure 3.  Effect of EASF on chronic restraint stress-induced alterations in number of ambulations in open field test. Each column represents mean ± SEM (n = 6). Compared with vehicle (chronic stress) group. ^*p <0.05; ^**p <0.01 (one-way ANOVA followed by Dunnett’s test).

The latency to enter into the main arena of the open field test was significantly increased in the vehicle treated group as compared to the control group on all days. Administration of EASF (25 mg/kg) showed significant (p <0.05) decrease in latency to enter into the main arena only on day 10. The dose 50 mg/kg showed significant (p <0.01) decrease in latency to enter into the main arena on day 5 and 10, whereas the dose 100 mg/kg significantly (p <0.01) decreased latency to enter into the main arena of open field apparatus on all days. The latency to enter into the main arena was significantly decreased in the diazepam-treated group on days 5 and 10 (p < 0.01) (Data not shown).

The number of rearings was significantly decreased in the vehicle-treated group on all days as compared to the control group in the open field test. Pretreatment with EASF (50 and 100 mg/kg) significantly increased the number of rearings on days 5 and 10 (p < 0.05; p < 0.01). The number of rearings was also significantly increased (p < 0.05) in the diazepam-treated group (data not shown).

Effects of EASF on chronic restraint stress-induced alterations in rat brain LPO, SOD, CAT, and GSH

Effect on brain CAT and GSH level

Chronic stress decreased the levels of CAT and GSH, whereas pretreatment with EASF and diazepam increased these levels (Table 1).

Table 1.  Effects of EASF of Morus alba on chronic restraint stress-induced alterations in rat brain CAT, SOD, LPO and GSH.

Treatment (mg/kg, p.o.)	CAT	SOD	LPO	GSH
Control	4.3 ± 1.1	11.7 ± 3.4^**	6.3 ± 1.7^**	4.6 ± 0.9
Vehicle (CS)	6.6 ± 1.3	30.5 ± 4.6	29.2 ± 3.2	1.2 ± 0.8
Diazepam (1)	15.2 ± 0.7	28.0 ± 3.6	11.9 ± 1.3^**	2.7 ± 1.2
EASF (25)	7.8 ± 1.2	15.4 ± 1.3 ^**	13.7 ± 1.1^**	3.0 ± 0.3
EASF (50)	9.6 ± 0.8	17.5 ± 2.0^*	11.3 ± 1.8^**	2.3 ± 1.0
EASF (100)	11.4 ± 5.5	14.3 ± 1.6^**	8.7 ± 0.9^**	2.4 ± 1.1

Values are expressed as mean ± SEM (n = 6).

Compared with vehicle (chronic stress) group *p <0.05, **p <0.01 (one-way ANOVA followed by Dunnett’s test).

CAT, µmol of H₂O₂ decomposed/min/mg protein; SOD, U/mg protein; LPO, nmol of MDA/mg protein; GSH, µmol of GSH/mg protein.

Effect on brain SOD

Chronic stress significantly increased the SOD activity. Pretreatment with EASF significantly (p < 0.05; p < 0.01) reduced elevated levels of SOD at all doses (Table 1).

Lipid peroxidation assay (LPO)

Chronic stress led to significant increase in LPO level. Pretreatment with EASF significantly (p < 0.01) reduced elevated level of LPO at all doses. Diazepam also significantly decreased LPO level (p < 0.01) (Table 1).

Serum glucose level

Exposure to chronic stress significantly increased the serum glucose level. Pretreatment with EASF (25, 50 and 100 mg/kg) significantly (p < 0.01) decreased glucose level as compared to vehicle-treated group. The serum glucose level was also significantly decreased in the diazepam-treated group (p < 0.01) (Figure 4).

Figure 4.  Effect of EASF on chronic restraint stress-induced changes in blood glucose level. Each column represents mean ± SEM (n = 6). Compared with vehicle (chronic stress) group ^*p <0.05; ^**p <0.01 (one-way ANOVA followed by Dunnett’s test).

Serum corticosterone level

The serum corticosterone level was significantly increased in the vehicle-treated group after exposure to chronic stress as compared to the control group on days 5 and 10 of the test. Administration of EASF (25, 50 and 100 mg/kg) significantly decreased (p <0.05; p <0.01) corticosterone level on days 5 and 10. The serum corticosterone level was also significantly decreased in the diazepam-treated group (Figure 5).

Figure 5.  Effect of EASF on chronic restraint stress-induced changes in blood corticosterone level. Each column represents mean ± SEM (n = 6). Compared with vehicle (chronic stress) group. ^*p <0.05; ^**p <0.01 (One-way ANOVA followed by Dunnett’s test).

Milk-induced leucocytosis

The milk-induced leucocytosis was significantly increased in the vehicle-treated group as compared to the control on all days of the test. Administration of EASF (25, 50 and 100 mg/kg) significantly (p < 0.01) decreased milk-induced leucocytosis. The milk-induced leucocytosis was also significantly (p < 0.01) reduced in the diazepam-treated group (Figure 6).

Figure 6.  Effect of EASF on chronic restraint stress-induced alterations in leucocytes count in experimental animals. Each column represents mean ± SEM (n = 6). Compared with vehicle (chronic stress) group ^*p <0.05; ^**p <0.01 (one-way ANOVA followed by Dunnett’s test).

Adrenal gland and spleen weights

The adrenal gland weights of experimental animals were significantly increased in the vehicle (CS)-treated group as compared to the control group. Treatment with EASF (25, 50 and 100 mg/kg) significantly (p <0.01) decreased the adrenal gland weights. The adrenal gland weights were also significantly (p < 0.05) decreased in the diazepam-treated group.

The spleen weights of experimental animals were significantly decreased in the vehicle (CS)-treated group as compared to the control group. Pretreatment with EASF (100 mg/kg) significantly (p < 0.05) increased the spleen weight. The doses 25 and 50 mg/kg of EASF and diazepam were not found to be effective to restore chronic stress-induced decreased spleen weight to a significant extent (Table 2).

Table 2.  Effect of EASF on chronic restraint stress-induced changes in adrenal gland and spleen weights.

Treatment (mg/kg p.o.)	Adrenal gland wt (mg/100 g)	Spleen wt (mg/100 g)
Control	19.6 ± 1.4^**	653.8 ± 35.8^**
Vehicle (CS)	36.5 ± 1.7	485.3 ± 19.6
Diazepam (1)	26.2 ± 1.5^*	535.5 ± 20.8
EASF (25)	24.4 ± 2.4^**	540.3 ± 27.3
EASF (50)	22.0 ± 3.1^**	559.0 ± 20.8
EASF (100)	23.0 ± 1.8^**	686.6 ± 65.9^*

Values are expressed as mean ± SEM (n = 6).

Compared with vehicle (chronic stress) group *p <0.05, **p <0.01 (one-way ANOVA followed by Dunnett’s test).

Gastric ulceration

Chronic stress exposure induced a significant increase in the ulcer index scores. Pretreatment with EASF (25, 50 and 100 mg/kg) significantly decreased the ulcer index (p <0.05; p <0.01). Administration of diazepam significantly (p <0.01) decreased the ulcer index (Table 3).

Table 3.  Effect of EASF on chronic stress-induced gastric ulcerations.

Treatment(mg/kg p.o.)	No. of ulcers	Severity of ulcers	Ulcer Index
Control	0	0	0
Vehicle (CS)	20.0 ± 1.6	36.6	6.6
Diazepam (1)	11.3 ± 1.3	25.1	3.7^**
EASF (25)	7.8 ± 0.7	20.4	2.6^*
EASF (50)	7.5 ± 0.5	20.0	2.5^**
EASF (100)	6.6 ± 0.6	18.8	2.2^**

Values are expressed as mean ± SEM (n = 6).

Compared with vehicle (chronic stress) group *p <0.05, **p <0.01 (one-way ANOVA followed by Dunnett’s test).

Discussion

Today stress is one of the major mental disorders affecting a large number of the population, which disturbs normal physiological equilibrium of the body by producing adverse effects on the nervous, endocrine, biochemical and immune systems (Kausik et al., 2003). When stress is applied for a short duration of time, i.e., acute stress (AS), the physiological changes are self-limiting and adaptive in nature. Acute or short duration stress appears to have limited aversive effects on the individual since the body sets in motion an array of physiological, biochemical and endocrine responses to counter stress effects. However, chronicity and excessiveness of the stressor leads to inability of the organism to cope with the stress and results in stress-related perturbations (Chrousos & Gold, 1992) such as cognitive dysfunction, behavioral depression, anxiety, increase in glucose, corticosterone level, gastric ulcerations, immunosuppression, increased oxidative stress, and significant changes in monoamine levels. This indicates that chronicity of stressor is a major factor in stress-induced ill effects (Bhattacharya & Muruganandam, 2003). A variety of stress situations have been employed to investigate the consequences of stress and to evaluate anti-stress agents, and lack of consistency of stress protocols and their biological consequences is astounding.

Among the various methods employed, immobilization has been used extensively and widely accepted as a model for studying stress-induced physical as well as psychological alterations. The effect of EASF was evaluated on the chronic restraint stress model. During chronic restraint stress various biochemical enzymes and immunological systems are affected. Some of the stress-induced alterations have been attributed to an imbalance in neuro-endocrine system. Hence, measurement of some of the endocrine parameters will serve as an important basis for the evaluation of anti-stress activity (Kenjale et al., 2007; Rai et al., 2003). Restraint stress causes increase in brain 5-hydroxytryptamine metabolism, stimulates corticosterone secretion by adrenals and precipitates behavioral deficits (Sur & Bhattacharya, 1997).

Chronic restraint stress is known to interfere with cognitive functions, leading to retarded memory. Stress decreases brain monoaminergic content which may be responsible for cognitive dysfunctioning. EASF significantly improved memory at all doses on days 2, 5 and 10. Diazepam significantly improved memory on days 1 and 2.

The passive shock avoidance test was also used for testing learning and memory. Pretreatment with EASF (25, 50 and 100 mg/kg) significantly increased step-down latency at all doses on days 2, 5 and 10, indicating retention of memory. Diazepam significantly increased the step-down latency on days 1 and 2.

The open field model was used for testing locomotor activity (Vogel & Vogel, 2002). Chronic stress decreased the number of ambulations and rearings and increased latency to enter main arena, indicating its effect on normal locomotor activities of animals. In the open field paradigm, all the treatments significantly increased the number of ambulations on day 10. EASF (100 mg/kg) significantly decreased latency to enter into the main arena of the open field apparatus on all days. EASF (50 and 100 mg/kg) significantly increased the number of rearings on days 5 and 10. The ambulations, and number of rearings were significantly increased by diazepam, whereas latency to enter into the main arena was decreased.

Chronic restraint stress causes oxidative stress by generation of superoxide anion, hydrogen peroxide and hydroxyl radicals. The toxic peroxidative product causes widespread cellular injury. The natural cellular antioxidant enzyme SOD scavenges the superoxide ion by speeding up its dismutation. CAT and GSH scavenge hydrogen peroxide and other peroxides. The ROS scavenging activity of SOD is effective only when it is followed by the actions of CAT and GSH because the dismutase activity of SOD generates hydrogen peroxide from the superoxide ion, which is more toxic than oxygen-derived free radicals and requires to be scavenged further by CAT and GSH. Apart from its own toxicity, hydrogen peroxide in the presence of iron leads to the generation of toxic hydroxyl ions (Blake et al., 1987). Excess of SOD is known to induce deleterious tissue effects.

The chronic stress increased the SOD activity with concomitant reduction in CAT and GSH activities, resulting in increased generation of hydrogen peroxide and hydroxyl ions which are not effectively scavenged and accumulate to increase LPO level. Pretreatment with EASF significantly reduced elevated levels of LPO at all doses. Diazepam also significantly decreased LPO levels. For effective antioxidant activity, increased levels of CAT and GSH in proportion to increased SOD concentration is necessary. Pretreatment with EASF significantly reduced elevated levels of SOD at all doses. As discussed earlier, CAT and GSH enzymes play an important role in antioxidant activity. Due to chronic stress there is decrease in the levels of these two antioxidant enzymes. Pretreatment with EASF increased the brain CAT and GSH levels. Diazepam also increased CAT and GSH levels. Thus, the observed anti-stress effect of EASF may be at least partly due to its antioxidant activity.

During stress, release of various hormones such as catecholamines and glucocorticoids results in elevated plasma glucose levels because excess of cortisol causes insulin resistance leading to increased gluconeogenesis and eventually hyperglycemia (Kausik et al., 2003). EASF at all doses significantly decreased glucose and corticosterone levels. Diazepam also significantly decreased glucose and corticosterone levels.

Subcutaneous injection of milk produces an increase in leucocyte count (Thakur & Mengi, 2005). EASF significantly reduced leucocyte count, which suggests that the plant may possess adaptogenic activity. Diazepam also decreased leucocyte count.

Chronic stress resulted in adrenal hypertrophy indicating the active involvement of the HPA axis, which is highly responsible for stress (Rai et al., 2003). The adrenal hypertrophy takes place in response to secretion of ACTH from the pituitary. EASF significantly decreased adrenal gland weight at all doses. Diazepam also significantly decreased adrenal gland weight. The spleen weights were also normalized by EASF.

Chronic stress ulcers in rats have been correlated with an increase in gastric mucosal SOD activity. Antioxidants reduced this increase and incidence of ulceration (Das & Banerjee, 2003). It is observed that in chronic stress there is excess increase in levels of SOD, which may cause deleterious effects. Administration of EASF reversed stress-induced gastric ulceration.

Conclusion

The present investigation indicates that EASF of M. alba has significant antistress and antioxidant activities as shown by its mitigating effects on several chronic stress-induced physiological, neurochemical and behavioral perturbations.

Therefore M. alba can be used as an adaptogenic agent for treatment of several mental disorders. Thus, M. alba may provide a alternative to conventional antistress agents.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.