Chemicals
All chemicals and solvents used were of analytical grade and were obtained from SISCO Research Laboratories, Mumbai, India.
Plant material
CS collected from Nilgiris Biosphere, Tamil Nadu, India was authenticated by Botanist, Dr. S. Rajan, Survey of Indian medicinal plants, Govt. Arts College, Ootacamund. For future references voucher specimen of CS was preserved as herbarium in department of Pharmacognosy, JSS College of Pharmacy, Ooty, India.
Preparation of extract
Shade dried aerial parts of CS was coarsely powdered and macerated with 60% methanol at room temperature for 72 h. The filtrate was dried in a rotary vacuum evaporator under reduced pressure at 50°C. The extract was stored in desiccator for future use.
Estimation of total phenolic content
Total phenolic content of CS was estimated by employing Follin-Ciocalteau method [18] as stated here. Extract solution of 0.1 ml CS (containing 1000 μg) was taken in a volumetric flask and diluted with distilled water to 46 ml. About 1 ml of Folin – Ciocalteu reagent was added to the contents of the flask and mixed thoroughly. After 3 min, 3 ml of Na2CO3 (2%) was added and the mixture was allowed to stand for 2 h with intermittent shaking. The absorbance was measured at 760 nm. Phenolic content was calculated using pyrocatechol as standard.
Animals
Thirty adult male Wistar strain rats (160–175 g) were procured from the central animal facility and divided into five groups of six animals each. They were housed in colony cages at an ambient temperature of 25 ± 2°C and 40–65% relative humidity, with 12-h light: dark cycle. The animals had free access to standard pellet chow and drinking water. Institutional Animal Ethics Committee (IAEC) approved the study and all the experiments were carried out by following the guidelines of CPCSEA, India.
Groups and treatment
Four groups of rats were exposed to CMS and received p.o.: vehicle (0.3% Carboxy methyl cellulose CMC, 1 ml/kg), diazepam (DZM, 2 mg/kg), CS (125 and 250 mg/kg) respectively. Drugs (CS and DZM) were prepared as fine suspension in 0.3% CMC and administered one hour before the stress exposure for 21 days (Figure 1).
Induction of stress
Stress was induced by employing the Chronic Unpredictable Mild Stress (CMS) protocol. It was based on the modus operandi originally used by Pal and Dandiya (1993) [19] with minor modifications. Each stress regimen was carried out for 2 periods with the following stressors: food deprivation for 24 h, day-night reversal, soiled bedding (~150 ml water per cage) for 22 h, cage tilting (~45 degree inclined) for 22 h, crowded housing (10 animals per cage) for 12 h, exposure to a novel odor (household air freshener) for 12 h, restraint stress for 20 min, cold stress 4–8°C and heat stress 38–39°C for 20 min and intermittent white noise (80 dB) for 5 h for 3 periods.
Behavioural analysis
Open field exploratory behaviour test
Open field test was used to study the exploratory and anxiety behaviour of rats [20]. The open field apparatus consisted of a square arena 60 × 60 cm with 40 cm high wall. The entire apparatus was painted black except for 6 mm white lines that divided the floor into 16 equal size squares. The apparatus was illuminated with a low intensity diffuse light (45 W) situated 45 cm above the floor level. Entire room, except the open field was kept dark during the experiment. Each animal was placed in the central square and observed for 5 min and the following behaviours were recorded. Ambulation – the number of grid lines it crossed with all the four paws; rearing – by counting the number of times the animal stood on its hind limbs; grooming-number of times the animal made these responses viz. grooming of the face, licking/cleaning and scratching the various parts of the body, defecation – the number of fecal boli excreted during the period and immobility period. Between tests, the apparatus was cleaned with 5% alcohol.
Behavioural despair test
Behaviour despair model used by Porsolt et al., 1978 was followed to test antidepressant activity [21]. Rats were forced to swim individually in a glass jar (45 × 12 × 45 cm3) containing fresh water of height 35 cm and maintained at 25 ± 3°C. After an initial 2–3 min period of vigorous activity, each animal assumed a typical immobile posture. A rat was considered to be immobile when it remained floating in the water without struggling, making only minimum movements of its limbs necessary to keep its head above water. The total duration of immobility was recorded during the next 4 min of a total 6 min test. Rats were then allowed to dry in a pre-warmed enclosure (~32°C) before being returned to their home cage. All the behavioural experiments were carried out between 0900–1400 h.
Blood and organs collection
Twenty four hours after the last stress administration the blood was collected from overnight fasted rats through retro orbital puncture for biochemical analysis. Plasma was separated by centrifuging the blood at 4000 rpm for 10 min. Plasma glucose and total lipids were estimated using diagnostic kits immediately (Ecoline, Merck, India).
During conditions of stress, kidney synthesizes ascorbic acid to compensate glutathione loss. Hence, the endogenous antioxidants such as SOD, CAT; non-enzymic antioxidant – ascorbic acid and TBARS levels in were studied in brain, kidneys and adrenals. On 23rd day rats were sacrificed and organs were isolated, weighed and homogenized immediately with ice cold 10% KCl. The supernatant was separated by centrifugation at 5000 rpm for 10 min and stored at -80°C until the assay was done.
Superoxide dismutase (SOD)
SOD estimation was performed based on its ability to spontaneously inhibit oxidation of adrenaline to adrenochrome [22]. 2.78 ml of sodium carbonate buffer (0.05 mM; pH 10.2), 100 μl of EDTA (1.0 mM) and 20 μl of the supernatant or sucrose (blank) were incubated at 30°C for 45 min. Thereafter, the reaction was initiated by adding 100 μl of adrenaline solution (9.0 mM). The change in the absorbance was recorded at 480 nm for 8 min. Throughout the assay procedure temperature was maintained at 30°C. One unit of SOD produced approximately 50% of auto-oxidation of adrenaline. Results were expressed as Units/mg protein.
Catalase (CAT)
CAT measurement was done based on its ability to decompose hydrogen peroxide (H2O2) [23]. Briefly, 2.25 ml of potassium phosphate buffer (65 mM, pH 7.8) and 100 μl of the supernatant or sucrose (0.32 M) were incubated at 25°C for 30 min. H2O2 (7.5 mM; 650 μl) was added to initiate the reaction. The change in absorbance was measured for 3 min at 240 nm and the results were expressed as U/mg protein.
Ascorbic acid
Ascorbic acid was estimated by spectroscopy method [24]. One ml of 4% trichloroacetic acid was added to 250 μl of supernatant. This mixture was kept at 4°C for 1 h and centrifuged at 4000 rpm for 10 min. A 0.5 ml aliquot of the supernatant was taken and 125 μl of dinitrophenyl hydrazine was added. This mixture was heated in water bath at 85°C for 30 min. Then 875 μl of 65% sulphuric acid was added slowly under ice cold water. After complete mixing, the tubes were allowed to stand for 30 min at room temperature, and the OD was read at 540 nm. The calibration curve was prepared by using ultrapure ascorbic acid as standard (1 mg/ml in sodium acetate buffer). The blank and the standard solution were processed similarly. The ascorbic acid levels were expressed as μg/mg of adrenals.
Lipid peroxide
The lipid peroxidation in terms of thiobarbituric acid reactive substances (TBARS) was measured using the method of Ohkawa et al., (1979) [25]. Mixture of 500 μl of the supernatant, 200 μl of 8% sodium dodecyl sulphate, 1.5 ml of 20% acetic acid solution, 1.5 ml of 0.9% aqueous solution of thiobarbituric acid and 1.3 ml of distilled water were heated in boiling water bath for 30 min. After cooling, the red chromogen was extracted into 5 ml mixture of n-butanol and pyridine (15:1v/v). The organic layer was separated and the absorbance was measured at 532 nm. Lipid peroxide content was expressed as nmole of malondialdehyde (MDA)/mg tissue. The calibration curve was prepared by using 1, 1, 3, 3-tetra ethoxypropane (TEP) as standard.
Protein estimation
Protein content of the samples was estimated by the method of Lowry et al., using bovine serum albumin as standard [26].
Statistical analysis
Results were expressed as mean ± SE. Data were analyzed by one-way ANOVA followed by Dunnet's multiple comparisons using GraphPad Prism 4.0, statistics software. In all the tests, the criterion for statistical significance was p < 0.05.
