Effects of Protein Isolates from Radish and Spinach Leaves on Serum Lipids Levels in Rats. Tsutomu SATOH, Masahiko GOTO, and Kiharu IGARASHI

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1 J. Nutr. Sci. Vitaminol., 39, , 1993 Note Effects of Protein Isolates from Radish and Spinach Leaves on Serum Lipids Levels in Rats Tsutomu SATOH, Masahiko GOTO, and Kiharu IGARASHI Department of Bioproduction, Faculty of Agriculture, Yamagata University, Tsuruoka 997, Japan (Received August 6, 1993) Summary Radish and spinach leaf protein isolates (RLP and SLP, respectively) were prepared from chilled aqueous 0.2% sodium hydroxide extract of their leaves. The RLP and SLP, and those supplemented with methionine (RLP+Met and SLP+Met, respectively) to become equal to casein in methionine content, were compared with casein for their effects on serum cholesterol level in rats fed with a cholesterol-enriched diet for 14 days. Each protein isolate was incorporated into the cholesterol enriched diet to provide a 15% protein level. RLP was extremely inferior to SLP and casein for body weight gain of rats, but that of rats fed with RLP+Met diet was almost equal to that of casein and SLP groups. The serum cholesterol level in rats fed with SLP and SLP+Met diets was significantly lower as compared with that of the casein-fed rats. Both the amounts of excreted cholesterol and bile acids were significantly higher in rats fed with the SLP and SLP+Met diets than that of the casein-fed rats. These results suggest that the hypocholesterolemic action of SLP may in part have been due to the inhibition of intestinal absorption of both cholesterol and bile acids. RLP+Met diet tended to decrease the serum cholesterol level as compared to casein diet, but the difference was not significant. Key Words leaf protein isolate, radish leaf protein, spinach leaf protein, serum cholesterol It is well known that plasma cholesterol level in human and several experimen tal animals is affected by dietary proteins, and especially, plant proteins such as soybean protein isolate (1-5), sunflower seed protein isolate (6), potato protein (7), and wheat gluten (8, 9) exert hypocholesterolemic effect as compared with animal protein such as casein. Cho et al. reported that leaf protein isolates, prepared by heating green juice from Italian ryegrass and alfalfa, exert hypocholesterolemic effect in rats (10). However, the effects of other leaf protein isolates, prepared by a simiple method, on the serum cholesterol levels have not been examined. This paper describes the effects of protein isolates from radish and spinach 627

2 628 T. SATOH et al. leaves on the serum cholesterol levels in rats fed with a cholesterol -enriched diet. Materials and methods. Preparation of protein isolates: Fresh leaves of radish and spinach were ground with approximately equal amounts of aqueous 0.2% sodium hydroxide in weight at 5 Ž in a Waring blender obtained was filtered through two pieces of gauze. The homogenate, and the filtrate was centrifuged at 15,000 ~g for 20 min at 5 Ž. The supernatant filtered on No. 2 filter paper was adjusted to around ph 4 with 5% acetic acid to produce a protein precipitate precipitate obtained was continuously washed with acetone, ethanol, and diethyl ether on a glass filter until the filtrate became colorless. The, then dried in a vacuum desiccator, and used as protein isolate. The yields of protein isolates were 12 and 5 g, respectively, from 1 kg of fresh spinach and radish leaves. Nitrogen content and amino acids composition of each protein isolate was determined by the Kjeldahl method and by a method described elsewhere (11), respectively. Animals and diets: The composition of the experimental diets is given in Table 1. Five kinds of diets consisted of casein (C) diet, radish leaf protein isolate (RLP) diet, RLP diet supplemented with L-methionine (RLP+Met), spinach leaf protein isolate (SLP) diet, and SLP diet supplemented with L-methionine (SLP+ Met). In the RLP+Met and SLP+Met diets, methionine was added to the RLP and SLP diets at the expense of sucrose to become equal to the casein diet in methionine content. Four-week-old male weanling Wistar rats (Japan SLC, Hamamatsu, Japan), each weighing 60 g, were randomly divided into 5 groups of 6 rats each, respectivel y. The rats were individually housed in stainless-steel cages with screen bottoms and kept under controlled conditions with a 12h light-12h dark cycle (06:00, Table 1. Composition of diets (%). C, casein; RLP, radish leaf protein isolate; SLP, spinach leaf protein isolate; Met, methionine % protein. 2Mineral and vitamin mixtures (Harper's mixture (20)) were obtained from Oriental Yeast Co., Tokyo, Japan. J. Nutr. Sci. Vitaminol.

3 LEAF PROTEIN AND SERUM CHOLESTEROL :00 light), a temperature range of Ž, and humidity of about 55%. Food and water were provided ad libitum for 14 days. The feces of each rat were collected during the last 4 days of the experimental period, and freeze-dried prior to lipid extraction. Analytical procedures: After the 14th day of the feeding period, the rats were anesthetized with Nembutal (Dainippon Pharmaceutical Co., Osaka, Japan) after 12 h of starvation, and bled by cardiac puncture. The liver were immediately removed, weighed, and stored at -20 Ž until lipid analysis. The serum was separated by centrifuging the blood at 3,000 rpm for 15 min. Liver lipids were extracted and purified by the method of Folch et al. (12). Total cholesterol, free cholesterol, HDL-cholesterol, and triacylglycerol in the serum were measured enzymically by using commercial kits (Cholesterol E-Test, Free-cholesterol C-Test, HDL-cholesterol-Test, and Triglyceride E-Test, respec tively, Wako Pure Chemical Ind., Osaka, Japan). Free fatty acid and phospholipids in the serum were also measured by using commercial kits (NEFA-Test and Phospholipid B-Test, respectively, Wako Pure Chemical Ind.). For the determina tion of total cholesterol in the liver, a commercial kit, Mono-test Cholesterol (Boehringer Mannheim Yamanouchi Co., Tokyo, Japan) was used. Fecal choles terol and bile acids were extracted with ethanol under reflux as described by Sautier et al. (13), and the respective amounts were measured using commercial kits, Mono-test Cholesterol and Bile acids-test (Wako Pure Chemical Ind.), respective ly. Statistical analysis: The data were statistically analyzed by Duncan's multi ple range test after an analysis of variance (ANOVA), and significant differences of the means were inspected at p <0.05. Results and discussion. Nitrogen content of protein isolates from radish and spinach leaves was 12.1 and 12.8% on dry matter basis, respectively. The amino acid composition, and Arg/Lys and (Met+Cys)/Gly ratios of RLP and SLP, in which both ratios are considered to be important factor in the regulation of cholesterol metabolism (10, 14-19), are shown in Table 2. Sulfur-containing amino acids were higher in RLP and SLP than in soybean protein isolate (Ajipron-S2, Ajinomoto Co., Tokyo), suggesting that nutritive value of these leaf protein isolates is superior to that of soybean protein isolate. However, the reason for less body weight gain in the RLP group, compared with that in the SLP group, remains to be clarified. The Arg/Lys and (Met+Cys)/Gly ratios of these protein isolates were both intermediate values between casein and soybean protein isolate, indicating the possibility that protein isolates prepared by extracting from spinach and radish leaves with chilled 0.2% sodium hydroxide may have hypocholesterolemic action. Table 3 shows the effects of dietary leaf protein isolates on food intake, body weight gain, and serum and liver lipids concentrations. Both food intake and body weight gain were significantly lower on the RLP group compared with the casein, RLP+Met, SLP, and SLP+Met groups, but these were almost recovered by adding methionine to the RLP diet. Body weight gain of the SLP group was almost Vol. 39, No. 6, 1993

4 630 T. SATOH et al. Table 2. Amino acid composition of casein, soybean protein isolate, and radish and spinach leaf protein isolates (g/16g N). equal to that of the casein group. These results indicate the possibility that the nutritive value of SLP is superior to RLP and comparable to that of casein, and further indicate that methionine supplementation to the RLP is effective to improve its nutritive value. However, as the 15% protein level in the diet is not enough for the maximum gain in body weight in the growing rats, it may be necessary to examine with a higher protein level. Rats fed with the RLP, SLP, and SLP+Met diets showed a significantly lower serum cholesterol level compared with that of rats fed with the casein diet. The RLP+Met diet tended to decrease the serum cholesterol level compared to the casein diet, but not to a statistically significant degree. Atherogenic index of the rats fed with the RLP, RLP+Met, SLP, and SLP+Met diets was significantly lower than that of the rats fed with the casein diet, suggesting the possibility that RLP and SLP, and those supplemented with methionine, are superior to casein to prevent atherosclerosis. The concentration of liver total cholesterol of rats fed with the RLP, SLP, and SLP+Met diets, especially the latter two groups, was signi fi cantly lower than that of the casein-fed rats. However, the concentration of liver total cholesterol of rats fed with the RLP+Met diet was not different from that of J. Nutr. Sci. Vitaminol.

5 Values are M }SEM of 5 to 6 rats per group. Values within the same row and not sharing a common superscript letter are significantly different at p<0.05. C casein; RLP radish leaf protein isolate; SLP spinach leaf protein isolate; Met, methionine. ROL 631 Table 3. Effects of protein isolates from radish and spinach leaves on the growth food intake liver weight serum and liver lipid concentrations, and fecal excretion of steroids in rats fed with the choesterol-enriched diet. 1( Total chol-hdl-chol)/hdl-chol (i.e. VLDL-chol+LDL-chol/HDL-chol.

6 632 T. SATOH et al. the casein-fed rats, suggesting that SLP has a higher activity to lower liver cholesterol level compared with RLP. Liver triacylglycerol level of rats fed with the SLP and SLP+Met diets was not different from that of the casein-fed rats. On the other hand, liver triacylglycerol level of the RLP and RLP+Met groups was significantly higher than that of the casein, SLP, and SLP+Met groups. The reason for this remains to be clarified. Rats fed with the RLP+Met, SLP, and SLP+Met diets excreted a greater amount of feces, cholesterol, and bile acids than those fed with the casein diet. These results indicate that inhibition of intestinal absorption of cholesterol and bile acids may result in a lower serum cholesterol level. The present results indicated that protein isolates from radish and spinach leaves have an activity to lower serum cholesterol level and atherogenic index in rats fed with the cholesterol-enriched diet. Furthermore, the supplementation of methionine to radish leaf protein isolate improves its nutritive value. REFERENCES 1) Woodward, C. J. H., and Carrol, K. K. (1985): Digestibilities of casein and soya-bean protein in relation to their effects on serum cholesterol in rabbits. Br. J. Nutr., 54, ) Sautier, C., Flament, C., Doucet, C., and Suquet, J. P. (1986): Effects of eight dietary proteins and their amino acid content on serum, hepatic and fecal steroids in rats. Nutr. Rep. Int., 34, ) Sugano, M., Yamada, Y., Yoshida, K., Hashimoto, Y., Matsuo, T., and Kimoto, M. (1988): The hypocholesterolemic action of the undigested fractions of soybean protein in rats. Atherosclerosis, 72, ) Nagata, Y., Tanaka, K., and Sugano, M. (1981): Further studies on the hypocholest erolemic effect of soya-bean protein in rats. Br. J. Nutr., 45, ) Sautier, C., Dieng, K., Flament, C., Doucet, C., Suquet, J. P., and Lemonnier, D. (1983): Effects of whey protein, casein, Soya-bean and sunflower proteins on the serum, tissue and faecal steroids in rats. Br. J. Nutr., 49, ) Huff, M. W., and Carrol, K. K. (1980): Effects of dietary proteins and amino acid mixtures on plasma cholesterol levels in rats. J. Nutr., 110, ) Schrijver, R. D. (1990): Cholesterol metabolism in mature and immature rats fed animal and plant proteins. J. Nutr.,120, ) Kuyvenhoven, M. W., West, C. E., Hakkart, B. C., Mensink, G. B. M., and Beynen, A. C. (1987): Digestibility of dietary proteins and serum cholesterol in rats. Nutr. Rep. Int., 36, ) Mokady, S., and Liener, I. E. (1982): Effect of plant proteins on cholesterol metabolism in growning rats fed atherogenic diets. Ann. Nutr. Metab., 26, ) Cho, Y.-S., Horigome, T., Sakaguchi, E., and Uchida, S. (1988): Effect of feeding of leaf proteins on serum cholesterol concentration in rats. Nippon Eiyo Shokuryo Gakkaishi (J. Jpn. Soc. Nutr. Food Sci.), 41, (in Japanese). 11) Igarashi, K., Kamiyama, H., Tezuka, M., and Yasui, T. (1985): Changes in amino acid composition during sun-curing of leaves and stems of red clover. Jpn. J. Zootech. J. Nutr. Sci. Vitaminol.

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