Vol. 44, No. 4, April 1998 Pages 787-793 Spirulina maxima PREVENTS INDUCTION OF FATTY LIVER BY CARBON TETRACHLORIDE IN THE RAT P.V. Torres-Duran, R. Miranda-Zamora, M.C. Paredes-Carbaial*, D. Mascher*, J.C. Diaz-Zagoya, and M.A. Ju&rez-Oropeza Departamento de Bioquimica y *Departamento de Fisiologia, Facultad de Medicina, UNAM. P.O. Box. 70-159. M6xico, D. F. 04510, MEXICO. Received July 28, 1997 Summary The aim of the present work was to assess the capacity of Spirulina maxima to prevent fatty liver development induced in rats by an intraperitoneal single dose (lml/kg) of carbon tetrachloride. Liver and serum lipids were quantified two or four days after treatment with this agent. Liver lipid concentration did not differ in rats fed on a purified diet with or without Spirulina. However, after carbon tetrachloride treatment, liver triacylglycerois were significantly lower in rats fed on a diet with Spirulina 5% than in rats without Spirulina in their diet (P < 0.05). Furthermore, the increased liver cholesterol values, induced by carbon tetrachloride treatment, were not observed in rats that received Spirulina. These results support the potential hepatoprotective role of Spirulina. Key Words: Spirulina maxima, fatty liver, carbon tetrachloride, triacylglycerols, cholesterol. Introduction Spirulinas belong to the family Oscillatoriaceae and form the Algae of Cyanophyta; they usually grow in alkaline waters of Africa, Asia, North and South America (1). Spirulina maxima is rich in protein (60-70 %), vitamins, carotenoids, minerals, chlorophyll, and gamma-linolenic acid (2). Spirulina maxima was used as a food source by the Aztecs, who named it tecuiuatl. Nowadays, this alga is consumed by some human groups, in general as a food supplement. Toxicological studies of Spirulina in several species have not revealed any toxic effect after using different acute, chronic and reproductive tests (3-8). ~Corresponding author. M.A. Jufirez-Oropeza. Department of Biochemistry, School of Medicine, UNAM. P.O. Box 70-159, Mexico, D.F. 04510, MEXICO. E-mail majo@servidor.unam.mx 787 1039-9712/98/040787-07505.00/0 Copyright 9 1998 by Academic Press Australia. All rights of reproduction in any form reserued.
It has been reported that in humans Spirulina reduces the body weight in obese subjects (9), as well as their total cholesterol plasma levels (10). Furthermore, Spirulina maxima has been shown to decrease vascular tone of aortic rings from rats fed on a normal purified diet (11), as well as to prevent the development of fatty liver in rats fed on a fructose-rich purified diet (12). Because a fructose-rich diet is associated with increased triacylglycerol plasma levels (13, 14), the aim of this study was to evaluate the preventive effect of Spirulina maxima on a different and acute model of fatty liver. In this model, fatty liver was induced in rats by a sublethal single dose of carbon tetrachloride. Materials and Methods Reagents. All reagents and chemicals used were of analytical grade, except corn oil and corn starch, which were commercial products for culinary use. Glucose and organic solvents were from Merck (Mexico). The rest of the diet components were from Sigma (St. Louis Mo.). The spray-dried powder of Spirulina maxima employed in the experimental diet was a generous gift from Spirulina Mexicana, S.A. de C.V. (Mexico). This powder was mixed to homogeneity with the rest of the diet ingredients. Animals and diets. Fifty male Wistar rats, weighing 190-240 g, were maintained in a room with controlled temperature (20-25 ~ and light exposure (7:00-19:00 h). Two groups of twenty-five rats each were formed (diets A and B), selected at random and caged by lots of two or three animals. Each group was fed on one of the following purified diets: A. B. Control- Normal diet, without Spirulina. Experimental- Normal diet, with Spirulina (5%). The diets were prepared according to the guidelines recommended by the American Institute of Nutrition (15) and they were adjusted in their protein and carbohydrate contents (Table I). Water was supplied ad libitum and the amount of diet provided was 20 g/day/rat. After an acclimatization period of five days during which each group was fed on its respective purified diet, the animals were treated with a single intraperitoneal injection of either carbon tetrachloride (1.0 ml/kg of body weight, diluted 1:2 with corn oil as vehicle) in order to induce fatty liver (groups AC and BC) or of an equivalent volume of the vehicle (groups A and B). The latter groups were included in order to determine if diets could produce some effects on hepatic and serum lipid levels. 788
BIOCHEMISTRYcind MOLECULAR BIOLOGY INTERNATIONAL TABLE I Diet Composition Ingredients 1 Diets A B % Control Experimental Casein 20.0 17.0 Corn starch 7.0 5.0 Corn oil 5.0 5.0 Cellulose 3.0 3.0 Vitamin mixture 1.0 1.0 Mineral mixture 3.5 3.5 Methionine 0.3 0.3 Choline chlorjde 0.2 0.2 Glucose 60.0 60.0 Spirulina -- 5.0 maxima ~Composition of diets A and B was adjusted to the same content of protein and carbohydrate, (Spirulina maxima contains 60-70 % of protein). Lots of five rats from each group were killed at zero, two or four days (0, 48 or 96 hours, respectively) after receiving an intraperitoneal injection of carbon tetrachloride or vehicle. Before the animals were killed by cervical dislocation, they were starved 12 hours and anesthetized with diethylether. The serum was separated by blood centrifugation and stored at -78 ~ Livers were excised, weighed and stored at -78 ~ until lipid analyses were performed. Preparation of the liver lipid extracts. Total lipids were extracted with chloroformmethanol (3:1 v/v) by a modified Folch's method. For liver samples, 1 g of tissue was homogenized in 4 volumes of 0.05 M phosphate buffer, ph 7.2. Then, the ph was adjusted to 6.0 by the addition of 2 N HCI solution and this suspension was extracted three times with 4 volumes each of a chloroform-methanol mixture (3:1 v/v). The extract was washed with 10 ml of water, the organic fractjon was evaporated under a nitrogen stream, then weighed (for total lipids), and stored at -78 ~ until cholesterol and triacylglycerols analyses were performed. Analytical Methods. Serum and liver concentrations of cholesterol or triacylglycerols were measured using the commercial enzymatic-colorimetric kits from Lakeside, Mexico (CHOD-PAP and GPO-PAP, respectively). Total liver lipids were measured gravimetrically. Statistical analysis. Results were evaluated by analysis of variance (ANOVA) and Duncan's test using SPSS software. A P value less than 0.05 was considered significant.?89
Results and Discussion Livers from rats of group B (experimental-normal diet, with Spirulina 5% and treated with vehicle) showed minor modifications in total lipids (on day 2), triacylglycerols (on day 0), and total cholesterol (on day 0) relative to livers from rats of group A (Table II). However, since the observed differences were either not maintained or increased at the length of the experiment, and because the levels of lipids fluctuated in the range of values observed at the other two time periods, it can be assumed that the addition of Spirulina to purified diet does not induce significant changes on the liver lipids of the rat. Livers from animals treated with carbon tetrachloride and fed on a diet without Spirulina (group AC) showed an important increase in triacylglycerol concentration at day 4 after treatment relative to livers from rats of group BC. Livers from this group showed also higher levels of cholesterol than those observed in livers from rats fed on a diet containing Spirulina (group BC). However, although groups treated with carbon Days after treatment TABLE II Effects of Spirulina on Changes Induced by Carbon Tetrachloride on Liver Lipids Total Total Tri- Total Group Lipids acylglycerols Cholesterol (mg/g, wet weight) A 37.5 1.06 0.34 B 28.2 "0.58 "1.05 A 25.2 + 5.5 0.48 + 0.10 0.96 + 0.46 B *36.9 + 1.9 0.74 + 0.22 1.06 + 0.15 AC "47.1 + 4.4 1.78 + 1.60 1.13 + 0.43 BC *44.7 + 7.8 *0.95 + 0.34 0.75 + 0.41 A 35.6 + 0.4 0.83 + 0.79 0.68 + 0.23 B 38.4 + 4.7 1.65 + 0.50 0.80 + 0.23 AC 41.5 + 5.5 *8.87 + 1.67 "1.76 + 0.50 BC 37.2 + 6.1 ~.1.82 + 0.66 1:0.93 + 0.20 Animals receiving purified diets, A (control) or B (experimental, 5% Spirulina), were treated with a single dose of vehicle (groups A and B) or carbon tetrachloride (groups AC and BC). Values are expressed as mean + SD of n = 5. *Significantly different from group A or.1:group AC at the corresponding time. 790
tetrachloride showed higher values of total lipids (on day 2) than corresponding rats treated with the vehicle (groups A and B), the differences were not maintained (Table II). These results are in agreement with those from a previous report indicating that total lipid concentrations of the liver did not increase in fatty liver, but differ regarding the cholesterol changes (12). However, a small increase in liver cholesterol levels has also been observed previously in fatty liver induced by carbon tetrachloride (16). Regarding the serum lipids, triacylglycerol and total cholesterol levels were similar at any time in rats fed on the purified diet with or without Spirulina receiving only the vehicle (i.e., rats from group A or B, Table III). Thus, under the present experimental conditions, it can be assumed that Spirulina maxima does not modify lipid metabolism in the animals treated with the vehicle. However, carbon tetrachloride induced a small increase in serum triacylglycerol levels in both groups (on day 2), but the differences were not observed at the end of the experimental period. TABLE III Effects of Spirulina maxima on Changes Induced by Carbon Tetrachloride on Serum Lipids Days Total Tri- Total after Group acylglycerols Cholesterol treatment (mg/dl) 0 A 63.1 + 7.8 60.9 + 14.8 B 67.7 + 27.0 75.3 + 7.2 A 49.3 + 13.2 40.5 + 10.5 B 57.9 + 13.8 43.7 + 7.5 AC *75.7 + 4.1 57.9 + 6.8 BC *78.7 + 7.2 45.4 + 8.2 A 111.0 + 28.9 66.4 + 11.0 B 144.2 + 16.8 68.3 + 9.4 AC 98.7 + 20.7 79.7 + 12.9 BC 95.7 + 27.7 87.6 + 15.3 Animals receiving purified diets, A (control) or B (experimental, 5% Spirulina), were treated with a single dose of vehicle (groups A and B) or carbon tetrachloride (groups AC and BC). Values are expressed as mean + SD of n = 5. *Significantly different from group A at the corresponding time. 791
It has been suggested that carbon tetrachloride administration induces an increased synthesis of fatty acids as well as a decreased release of hepatic lipoproteins (16, 17). The results in the present study showed that dietary Spirulina prevents the changes induced by carbon tetrachloride on liver lipids. Since the hepatotoxic effect of carbon tetrachloride is related to free radical generation, it is possible that the potential hepatoprotective role of Spirulina maxima is associated with its antioxidant constituents, such as gamma-linolenic acid, selenium, chlorophyll, carotene, and vitamins E and C (2). However, further studies are required to test this hypothesis. Conclusions The addition of Spirulina maxima (5%) to a purified diet did not modify liver or serum lipid concentrations in animals which received only the vehicle. However, the increase of liver triacylglycerol and cholesterol concentrations, induced by carbon tetrachloride treatment, was prevented by the inclusion of Spirulina maxima in the purified diet. No differences were observed in serum total cholesterol or triacylglycerol concentrations between animals of groups with the same treatment (i.e., A vs B, AC vs BC) at any time. These results support the potential hepatoprotective role of Spirulina. Acknowledgments This work was supported in part by grant IN-201296 from DGAPA-UNAM, Mexico. References 1 2. 3., 5... 8. Ciferri, O. (1983) Microbiol. Rev. 47, 551-578. Kay, R.A. (1991) Crit. Rev. Food Sci. Nutr. 30, 555-573. Krishnakumari, M.K., Ramesh H.P., and Venkataram, L. (1982) J. Food Prot. 44, 934-935. Chamorro, G., Salazar M., and Pages, N. (1996) Phytother. Res. 10, 28-32. Chamorro, G., Herrera, G., Salazar, M., Salazar, S., and UIIoa, V. (1988) Med. Nutr. 24, 104-106. Yoshino, Y., Hirai, Y., Takahashi, H., Yamamoto, N, and Yamazaki, N. (1980) Jpn. J. Nutr. 38, 221-225. Chamorro, G., and Salazar, M. (1990) Arch. Latinoamer. Nutr. 40, 86-94. Salazar, M., Chamorro, G.A., Salazar, S., and Steele, C.E. (1996) Fd. Chem. Toxic. 34, 353-359. 792
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