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PREVENTION OF COLD STRESS INDUCED ADRENAL HYPERTROPHY BY SPIRULINA PLATENSIS

Spirulina platensis (Spirulina) was investigated on an acute stress (4h cold stress, 4±1°C) induced biochemical alteration in albino rats. Gastric ulcerations, adrenal gland weight, adrenal gland ascorbic acid content, and histopathology were also used as stress indices. Panax ginseng (ginseng) was used as standard adaptogenic agent for comparison. Acute cold stress resulted in elevation of blood glucose levels, increase in adrenal gland weight, and reduction in adrenal gland ascorbic acid content and histological changes. Spirulina (100 mg/kg, 200 mg/kg and 500 mg/kg p.o.) and ginseng (100 mg/kg p.o.) attenuated all these cold stress induced perturbations. Results demonstrated the anti-stress activity of Spirulina, qualitatively comparable to ginseng, against a variety of biochemical and histological perturbations induced by acute cold stress. INTRODUCTION Stress is known to alter the physiological homeostasis of the organism and complex mechanisms contributing to the breakdown in adaptational processes (George et al., 1992). It has been postulated that stress is involved in etiopathogenesis of a variety of diseases like depression and anxiety, immunosupression, endocrine disorders, male potency and cognitive dysfunctions to the diseases like peptic ulcers, hypertension and ulcerative colitis (Goel, 1991). Using agents, which could include a state of nonspecific increase to resistance to affect the internal homeostasis, can provide the answer to this problem. Spirulina is a rich blend of proteins, lipids and carbohydrates (Sheshadri and Umesh, 1992; Johnson and Shubert, 1986), minerals (Zinc, Magnesium, Manganese, Selenium) vitamins (β-carotene, riboflavin, cyanocobalamine, α-tocopherol) and αlinoleic acids (Denise, 1993; Takeuchi, 1978). Spirulina is known to support the human immune system and promote cellular health that is primarily affected in stress (Fukino et al., 1990). The objective of the study was to explore the usefulness of Spirulina as an antistress and adaptogenic agent. MATERIALS AND METHODS Animals Male Swiss albino rats (200-250 g) were obtained from registered breeders (Haffkine Institute, Parel, Mumbai, India). Drugs, Reagents, Solutions and Biochemical Kits Spirulina spray dried powder (M/S Parry Neutraceuticals, Chennai, India), ginseng (Glenmark Ltd., Mumbai, India), sodium carboxy methyl cellulose (Loba Chemie, Mumbai, India), glacial acetic acid, formalin, trichloroacetic acid, hydrochloric acid, sulfuric acid and 2,4-dinitrophenyl hydrazine (S.D. Fine chemicals, Mumbai, India.), heparin, thiourea (Biological E. Ltd., Mumbai, India), ascorbic acid (Glaxo Ltd. Mumbai, India) glucose diagnostic kit (E. Merck India Ltd). Stress Induction The rats were divided into 6 groups, each group containing six rats. All animals Proc. WOCMAP III, Vol.6: Traditional Medicine & Nutraceuticals Eds. U.R. Palaniswamy, L.E. Craker and Z.E. Gardner Acta Hort. 680, ISHS 2005 102 were pre-treated for 15 d with the respective test drugs, after which the rats were exposed to cold stress at 4±1°C for 4h. Group I Unstressed rats received 1 mL/kg p.o. of 0.3% Na. CMC in saline; served as normal group. Group II Stressed rats received 1 mL/kg p.o. of 0.3% Na. CMC in saline; served as negative control. Group III Stressed rats were treated with standard oral drug (ginseng); served as positive control. Group IV-VI Stressed rats in groups IV, V, and VI received Spirulina suspended in 0.3% Na. CMC solution. The doses of Spirulina used for pretreatment were 100, 250 and 500 mg/kg p.o. respectively. Animals were sacrificed following stress induction, under the influence of ether anesthesia. 5 mL of blood was collected in heparinised tubes from each animal and plasma obtained by centrifugation (Superspin R-V/FM, Plastocraft, India) for estimation of glucose and ascorbic acid levels. Adrenal glands from each rat were decapsulated for determination of adrenal gland weight, adrenal gland ascorbic acid content and histopathological studies. Anti-ulcerogenic effect of Spirulina was also evaluated by isolating the stomach from the rats. The isolated stomachs were cut open along the greater curvature, washed with cold water and examined microscopically (X 10). The mean percent ulcer and the severity of gastric mucosal lesions (cumulative length in mm) were determined (Sairam et al., 2001). Determination of Plasma Glucose Levels The plasma glucose was estimated as described earlier (Philip, 1994). 10 μL of plasma was mixed with 1 mL of Glucose oxidase-Peroxidase (GOD-POD) reagent. The resulting mixture was incubated at 37°C for 15 min. Absorbance of test and standard was measured at 540 nm (Merck biochemical analyzer, Microlab 200, Vital Scientific, Netherlands). Determination of Plasma Ascorbic Acid Levels In a centrifuge tube containing 6 mL of 6% trichloroaceticacid, 2 mL of plasma was added drop by drop to form fine suspension. It was allowed to stand for 5 min and centrifuged (2500 rpm, 15 min). To the supernatant fluid, 0.5 g of acid washed norit (activated charcoal) was added, stirred vigorously and then filtered through a filter paper. To each 1 mL of norit filtrate one drop of 10% thiourea and 0.25 mL of 2,4dinitrophenylhydrazine reagent were added. The mixture was incubated for 3 h at 37°C and placed in a beaker containing ice. To each of these tubes, while in ice bath, 1 mL of 85% H2SO4 was added drop by drop. Tubes were shaken thoroughly to ensure complete mixing. After 30 min, tubes were wiped and read in UV-Visible Spectrophotometer (Jasco V530, Japan) at 540 nm. Appropriate standards were run along with the unknown tubes. Standard solutions of ascorbic acid varying from 1-25 μg/mL in 4% trichloroacetic acid were prepared for standard curve of ascorbic acid. The ascorbic acid content per 100 g of adrenal gland for each group was then calculated (Roe and Kuether, 1949). Adrenal Gland Histopathology The separated adrenal glands were preserved in saline formalin. Microscopic sections of these adrenal glands were prepared and sent to Bombay Veterinary College, Parel, Mumbai, India for histopathological studies. Data Analysis The data were analyzed using two-tailed students t-test.