Restraint Stress Induced Changes and Their Modification by Spirulina platensis in Albino Rats: an Experimental Study

Spirulina platensis is a blue green algae containing vital nutrients (proteins, lipids and carbohydrates), minerals (zinc, magnesium, manganese, selenium), vitamins (β–carotene, riboflavin, cyanocobalamine, α-tocopherol), and α–linoleic acid, reported to promote physical health, improve defense mechanisms of the human body and enhance longevity of life. These attributes are similar to the modern concept of adaptogenic agents, which are known to afford protection of the human physiological system against diverse stressors. Present study investigated the adaptogenic activity of S. platensis against chronic restraint stress induced perturbations in glucose metabolism and immunosupression. Adrenal gland weight, corticosterone content of adrenal gland, plasma corticosterone levels, and histopathological studies of adrenal gland were used as stress indices. Ginseng (Panax ginseng) was used as the standard adaptogenic agent for comparison. Chronic restraint stress induced marked increase in plasma glucose levels, significant increase in adrenal gland weight and plasma corticosterone levels with concomitant decrease in adrenal gland corticosterone content. Restraint stress resulted in distortion of cords, loss of architecture and formation of lesions in the cortex of adrenal gland. These effects were attenuated by S. platensis (100, 200 and 500 mg/kg, p.o.) and ginseng (100 mg/kg, p.o.) administered once daily over a period of 14 d (prior stress period) and continued for next 7 d (during the period of stress induction). Results indicate that S. platensis has adaptogenic activity, qualitatively comparable to ginseng, against a variety of biochemical, physiological and histological perturbations induced by restraint stress. INTRODUCTION Blue green algae (Spirulina platensis) is rich in proteins, lipids and carbohydrates, minerals (zinc, magnesium, manganese, selenium), vitamins (β–carotene, riboflavin, cyanocobalamine, α-tocopherol), and α–linoleic acid (Sheshadri and Umesh, 1992), reputed to promote physical health and improve resistance of the body and other external factors tending to perturb the homeostasis of humans and promote revival of physiological functions after debilitating diseases (Brekhman and Dardymov, 1969). There is comprehensive experimental and clinical evidence that stress alters the physiological homeostasis of an organism. Complex mechanisms contribute to the breakdown in adaptational processes resulting in various visceral, behavioral and endocrinological changes. Hypothalamic-pituitary-adrenal (HPA) axis and adrenal glands are particularly crucial for the regulation of stress physiology (Munck et al., 1995). Restraint stress (RS) is widely used to study the stress-induced changes in experimental animals. Preliminary studies have shown that chronic RS significantly increased the weight of adrenal glands (Bhattacharya et al., 2000). Therefore the effect of S. platensis pretreatment on chronic RS induced changes in adrenal gland was studied extensively. The parameters such as plasma glucose, cholesterol and corticosterone levels, adrenal gland corticosterone content and histological changes in adrenal gland cellular structure were used to determine the stress indices in this study. Proc. WOCMAP III, Vol.6: Traditional Medicine & Nutraceuticals Eds. U.R. Palaniswamy, L.E. Craker and Z.E. Gardner Acta Hort. 680, ISHS 2005 50 MATERIALS AND METHODS Animals Male Swiss albino rats (200-250 g) were obtained from registered breeder (Haffkine Institute, Parel, Mumbai, India). Three animals were housed per cage under standard conditions (23 ± 1.0°C; relative humidity 55 ± 10%; 12h/12h light/dark cycle), fed standard pellet diet (M/S Amrut Ind. Ltd. Pune, India) and purified water, ad libitum. Drug Treatment Spray-dried blue green alga (S. platensis) (M/S Parry Neutraceuticals, Chennai, India) was orally administered as 0.3% carboxy methylcellulose (CMC) suspension, in the doses of 100, 200, 500 mg/kg/d (p.o.) for 14 consecutive days. Stress induction was initiated on 15 d and continued for next 7 days. Treatment was continued during the stress period. Ginseng was used as standard adaptogenic agent for comparison. Control animals were treated with vehicle (0.3% CMC suspension). Induction of Stress The rats were randomly assigned to the unstressed control, stress and drug treated stress groups. Those assigned to vehicle or drug treated groups were subjected to immobilization, done by stretching and firmly tying the limbs to iron rod with the help of thread and keeping the animals in inverted position for half an hour. Animals were sacrificed on day 7, immediately on completion of stress procedure. Assessment of Stress Intensity 1. Estimation of Blood Glucose Levels (Philip, 1994). The plasma was analyzed for estimation of glucose; in brief 10 μL of plasma was mixed with 1 mL Glucose OxidasePeroxidase (GOD-POD) reagent. The resulting mixture was incubated at 37°C for 15 min. Absorbances of test and standard were measured at 540 nm (Merck biochemical analyzer, microlab 200, Vital Scientific, Netherlands). Glucose + O2 + H2O GOD Gluconic acid + H2O2 2 H2O2 + 4-Aminoantipyrine + Phenol Peroxidase Quinoneimine + 4 H2O 2. Estimation of Plasma Cholesterol. The cholesterol levels on first day and on seventh day, after completion of stress period, were determined by using CHOD-PAP method (Richmond, 1974); 10 μL plasma was mixed with 1 mL reagent containing peroxidase, cholesterol oxidase, cholesterol esterase and 4-aminoantipyrine. Cholesterol and its esters are released from lipoproteins by detergents. Cholesterol esterase hydrolyzes the esters. In the subsequent enzymatic oxidation by cholesterol oxidase, H2O2 is formed. This is converted into colored quinonimine, which was read at 546 nm (Merck biochemical analyzer, microlab 200, Vital Scientific, Netherlands). 3. Estimation of Plasma Corticosterone Levels. On seventh day after completion of stress period the rats were anesthetized and sacrificed. Blood was collected in tubes containing EDTA-Na2, centrifuged and the separated plasma stored at -20°C. The concentrations of corticosterone in plasma were estimated by RIA method (Yalow and Berson, 1971). 4. Adrenal Gland Function Test. On seventh day both the adrenals of each rat were isolated and weighed. Mean weight of adrenals per 100 g of body weight for each group was calculated. One of the two adrenals was preserved for histopathological studies in formalin, while the other was used for estimation of corticosterone content. Corticosterone was estimated by using RIA method (Yalow and Berson, 1971). One adrenal gland, of each rat, preserved in formalin was sent to micron laboratory for histopathological studies.