Hypolipidemic and antiperoxidative effect of coconut protein in hypercholesterolemic rats

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1 Indian Journal of Experimental Biology Vol. 39, October 2001, pp Hypolipidemic and antiperoxidative effect of coconut protein in hypercholesterolemic rats G Salil & T Rajamohan* Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram , India Fax dlcampus@vsnl.net.in Received 20 September 2000; revised 8 May 2001 Effect of coconut protein in rats fed high fat cholesterol containing diet on the metabolism of lipids and lipid peroxides was studied. In addition, effect of coconut protein were compared with rats fed L- arginine. The results indicate that those fed coconut protein and those fed L-arginine showed significantly lower levels of total cholesterol, LDL+ VLDL cholesterol, Triglycerides and Phospholipids in the serum and higher levels of serum HDL cholesterol. The concentration of total cholesterol, triglycerides and phospholipids in the tissues were lower in these groups. There was increased hepatic cholesterogenesis which is evident from the higher rate of incorporation of labeled acetate into free cholesterol. Increased conversion of cholesterol to bile acids and increased fecal excretion of bile acids were observed. Feeding coconut protein results in decreased levels of Malondialdehyde in the heart and increased activity of Superoxide dismutase and Catalase. Supplementation of coconut protein causes increased excretion of urinary nitrate which implies higher rate of conversion of arginine into nitric oxide. In the present study, the arginine supplemented group and the coconut protein fed group produced similar effects. These studies clearly demonstrate that coconut protein is able to reduce hyperlipidemia and peroxidative effect induced by high fat cholesterol containing diet and these effects are mainly mediated by the L-arginine present in it. One characteristic dietary feature of Kerala population is the use of fresh coconut kernel in most culinary preparations. Coconut kernel contains apart from coconut oil, 5-6% protein. Our earlier studies in humans and animals has shown that coconut kernel contains cholesterol lowering effect l. Further studies revealed that one of the factor responsible for the cholesterol lowering action is coconut kernel protein 2. Studies from this laborator/ and elsewhere 4 suggest that lysine/arginine ratio of a protein influences its effect on cholesterol metabolism. Animal protein with higher lysine/arginine ratio were hypercholesterolemic while many plant proteins with their lower lysine/arginine ratio had hypocholesterolemic effect. Among the several plant proteins studied in this laboratory, coconut kernel protein contains 2.13% lysine and 24.5% arginine giving a very low lysine/ arginine ratio and it also shows very significant cholesterol lowering action 2 The objective of the present investigation was to examine whether coconut kernel protein is able to reduce the hyperlipidemic and peroxidative effect induced by high fat cholesterol containing diet. In *Correspondent author view of the very high content of L-arginine, the effect of coconut protein were compared with L-arginine. The results of these studies are presented in this paper. Materials and Methods Preparation of coconut protein--coconut cake obtained after expelling of the oil was used for the experiment. The remaining oil in the cake was removed by extracting with petroleum ether (60-80 C). The defatted cake was stirred with 10% NaCI solution in large containers using mechanical stirrer for 24 he. The saline extract was collected and by adjusting the ph with dilute HCI solution, the proteins were precipitated. The precipitated globulin fraction was collected by centrifugation and redissolved in minimum volume of 10% NaCI solution. After centrifugation the protein was pre.cipitated and the globulin fraction was collected, washed with distilled water and dried in a vacuum oven at 60 C and was used for feeding purpose. Animal experiments-male albino. rats (Sprague Dawley strain, weight gm) bred in the animal house of the laboratory were divided into 4 groups and fed as follows:-

SAUL & RAJAMOHAN: HYPOUPIDEMIC & ANTI PEROXIDATION EFFECT OF COCONUT PROTEIN 1029 Group I - Control rats fed casein alone Group II - Rats fed choiesterol + casein Group III - Rats fed cholesterol +

2 SAUL & RAJAMOHAN: HYPOUPIDEMIC & ANTI PEROXIDATION EFFECT OF COCONUT PROTEIN 1029 Group I - Control rats fed casein alone Group II - Rats fed choiesterol + casein Group III - Rats fed cholesterol + casein +coconut kernel protein - Rats fed cholesterol + casein + L-arginine. Casein contains 4.1 % arginine and coconut protein contains 24.5% arginine. 1kg diet of group III contains SOg of casein and SOg of coconut protein which provides 22.SS g of arginine. Therefore in group IV diet g of arginine was included, so that the arginine content of group III and group IV were similar (Table 1). The Vitamin mixture and Salt mixture used had the same composition as described before 5. The rats were housed individually in polypropylene cages in a room maintained at 25 ±1 C with 12hr light and 12 hr dark. Food intake was recorded daily and gain in weight was recorded weekly. The experimental period was 60 days. The animals were given food and water ad libitum. At the end of this period they were deprived of food overnight, stunned by a blow at the back of the neck and sacrificed by decapitation. Blood and tissues were removed to ice cold containers for various estimations. Before sacrifice 24 hr stool samples were collected twice and used for the estimation of neutral sterols and bile acids and urine samples were collected for nitrate estimation. Analytical methods-total cholesterol, triglycerides and phospholipids were estimated using the procedure described earlier 5. Separation of serum lipoprotein into high density lipoprotein and very low density lipoprotein+low density lipoprotein was carried out as described by Warnick et al 6. Activity of glucose-6- phosphate dehydrogenase (EC ) and malic enzyme (EC ) of the liver was. determined by the procedure of Kornberg and Horecker 7 and Severo Ochoa 8 respectively. HMG CoA reductase (EC ) activity of liver was determined as described by Venugopala Rao and Ramakrishnan 9 Malondialdehyde was estimated by the thiobarbituric acid method of Nichans and Samuelson 10. Activity of catalase (EC ) and Superoxide dismutase (EC ) was determined by the method of Maehly and Chancel I and Kakkar et al. 12 respectively. Protein in the enzyme extract was determined after TCA precipitation by the method of Lowry et al 13 For the extraction of fecal bile acids, the stool samples were homogenised with equal weight of water and lyophilized to a fine powder. The lyophilized samples were extracted with IN NaOH in 95% ethanol at 80 C for 2hr and the residue reextracted with hexane and estimated by the method of Snell and Sne1l 14 For radiolabeled studies the lipids were extracted by Radins procedure l5. Urinary nitrate was estimated by the method of Trivedi and Goe1 16 Statistical analysis was carried out by Student's test 17 Results The diet consumption and gain in body weight was more or less similar in the four groups. The food Table I-Composition of the diet (g/ioo g) Diet Group I Group II Group III Corn starch Casein Coconut kernel protein Ground nut oil Salt mixture Vitamin mixture I I I Cholesterol 0 I I L-arginine Table 2-Food intake and body weight gain in rats fed the respective diets for 60 days Food intake (g) Body weight gain (g) Group I 1l.l5 ± ± 2.46 Group II ± ± Group III ± ± 3.52 II. 57 ± ± I. 74

3 1030 INDIAN J EXP BIOL, OCTOBER 2001 intake (g/day) of the rats of group I and II were 11.15±0.59 and 12.18±0.65 and for groups III and IV were ± 0.42 and 11.57±0.39. The gain in body weight for the duration of the experiment for group I and II were 58 ± 2.46 g and 63.5 ± 2.95 g and group III and IV were 58.5 ± 3.52 g and 64 ± l.74 g (Table 2). Concentration of total cholesterol, VLDL + LDL cholesterol in the serum, cholesterol in the liver, heart and kidney were significantly increased, while HDL cholesterol showed decrease in rats fed high fat cholesterol diet when compared to rats fed normal diet. Inclusion of coconut protein and L-arginine into high fat cholesterol containing diet produced significantly lower levels of total cholesterol and VLDL + LDL cholesterol, while HDL cholesterol showed increase. In addition, tissue cholesterol levels were also lower in these groups (Table 3). Concentration of triglyceride and phospholipids in the serum and tissues showed significantly higher level, in rats fed high fat cholesterol diet while inclusion of coconut protein and L-arginine resulted in significant decrease in serum and tissue triglycerides & phospholipids. Similar results were observed in rats fed coconut protein and those fed L-arginine (Table 4). The activity of HMG CoA reductase in liver showed decrease in rats fed high fat cholesterol containing diet when compared to rats fed normal diet. Feeding coconut protein and L-arginine results in higher activity of HMG CoA reductase (Table 5). The activity of malic enzyme and glucose-6-phosphate Serum (mgllooml) Total cholesterol VLDL+LDL HDL Tissues (mg/joog) Heart Kidney Liver Table }----Concentration of cholesterol in the serum and tissues Group I Group II Group III ± S±2.8S" ±2.36 b SS.97±1.S ±3.0S" S2.38±1.47b 39.26± ±0.69" S2.37±1.41 b ±3.78 3S2.62±8.11 " ±3.20 b ± S.69±9.71 a 28S.70±5.99 b 4l4.90± S3±20. 71" ±11.36 b 96.04±1.92 c 48.80±1.32 c S2.18± 1.48 c ±3.70 c 284.S0±6.26 c ±9.1lc P<O.OI : "Group II significantly different from group I. hgroup 1II significantly different from group II. c significantly different from group II. Triglycerides Table 4,--{:oncentration of triglycerides and phospholipids in the serum and tissues Group I Group II Group III Serum (mg/joo ml) 6.9S±O.14 1O.43±0.20' 6.90±0.ISb Tissues (mg/joo g) Heart 43.64± S6±2.08" 48.46± 1.06 b Kidney 48.S±O ±2. 18" S7.S±1.20 b Liver 24.2S±O ±0.72" 30.31±0.64 b Phospholipids Serum (mg/jooml) ± ±3.86" ±2.63 b Tissues (mg/joog) Heart ±3S ±S2.S7 a ±38.57 b Kidney 147S.76± ±SO.13 a ±40.69 b Liver ± S±60.97 a ±44.19 b 6.9S±O.ISc 47.24±1.l3 c 60.63±1.33 c 31.44±O.69 c ±2.S9 c IS14.12±40.87c 14Sl.S8±43.SS c 2l04.77±46.30 c P<O.Ol. 'Group II significantly different from group I. bgroup III significantly different from group II. c significantly different from group II.

SAUL & RAJAMOHAN: HYPOLWIDEMIC & ANTI PEROXIDA TION EFFECT OF COCONUT PROTEIN 1031 Table 5--Activitics of HMG-CoA reductase, glucose-6-phosphate dehydrogenase and malic enzyme in the liver Groups

4 SAUL & RAJAMOHAN: HYPOLWIDEMIC & ANTI PEROXIDA TION EFFECT OF COCONUT PROTEIN 1031 Table 5--Activitics of HMG-CoA reductase, glucose-6-phosphate dehydrogenase and malic enzyme in the liver Groups HMGCoA Glucose-6-Phosphate Malic Enzyme Reductase* Dehydrogenase (units/g proteins) Group I 2.16±O.O ±O ±8.60 Group II 3.71±O.09 a 14.93±O.29 a 238±4.75' Group III 3.24±O.08 b 12.58±O.26 b 186±3.91 b 3.35±O.09 c 11.90±0.35 c 190±4.18 c *Ratio of HMG CoA to mevalonate, lower ratio indicates higher enzyme activity. P<O.Ol: IGroup II significantly different from group I. bgroup III significantly different from group II. c significantly different from group II. Table 6-ln vivo incorporation of [1,2_14C] acetate into liver lipids Groups Free Cholesterol Phospholipids Triglyceride Cholesterol ester (countslminutelg tissue) Group I Group II Group III 133± ± ± ± ±l.lS8 727± ±1.62 a 331±9.93 a 115±2.42 b 569±11.94b 37±1.23 b I 245±37.36 b 105±3.l5 c 535±11.77c 34±l.02 c P<O.Ol: 'Group II significantly different from group I. bgroup III significantly different from group II. c significantly different from group II. 1385±41.55 c Table 7-Concentration of fecal bile acids and urinary nitrate Group I Fecal Bile acids Urinary Nitrate (mg/loog) (mg/loo ml) Group I 5.07±O.1O 19.50±0.58 Group II 6.70± ±O.37 GROUP III 7.98±<l.16 b 22.95±<l.67 b GROUP IV 8.0S±<l.18 c 27.65±<l.64 c P<O.Ol: aoroup II significantly different from group I, ~roup III significantly different from group II, < significantly different from group II dehydrogenase showed decrease in the liver in rats fed high fat cholesterol containing diet. While inclusion of coconut protein and L-arginine results in decreased activity of these enzymes (Table 5). Incorporation of 14C acetate into free cholesterol, ester cholesterol, triglycerides and phospholipids in the liver were decreased in rats fed high fat cholesterol diet. Inclusion of coconut protein and L-arginine results in significant increase in the incorporation of 14C acetate into free cholesterol and ester cholesterol and decreased incorporation into triglycerides and phospholipids (Table 6) as compared to those of rats fed high cholesterol diet. Excretion of bile acids were higher in rats fed high fat cholesterol containing diet when compared to normal rats. Inclusion of coconut protein and L-arginine also results in significant increase in the fecal excretion of bile acids (Table 7). The excretion of nitrate in the urine showed decrease, in rats fed cholesterol containing diet when compared to normal rats. Supplementation of L-arginine and coconut protein resulted in increased excretion of nitrate in the urine (Table 7). Concentration of malondialdehyde in the heart showed increase in rats fed high fat cholesterol containing diet, while inclusion of coconut protein and L-arginine results in significant decrease in the concentration of malondialdehyde (Table 8). The activity of superoxide dismutase and catalase were higher in the heart in rats fed high fat cholesterol containing diet when compared to rats fed normal diet. Inclusion of coconut protein and L-arginine also results in increased activity of these enzymes (Table 8).

1032 INDIAN J EXP BIOL, OCTOBER 2001 Table &-Concentration of Malondialdehyde (mm/100 g tissue) & activities of super oxide dismutase and catalase in the heart Groups MDA SOD Catalase Group I I.4S±O.

5 1032 INDIAN J EXP BIOL, OCTOBER 2001 Table &-Concentration of Malondialdehyde (mm/100 g tissue) & activities of super oxide dismutase and catalase in the heart Groups MDA SOD Catalase Group I I.4S±O ± ±O.S4 Group II 2.2S±O.OSa 2S.83±O.S2" 26.9S±O.62" Group IIJ 0.78±0.02b 30.33±0.88 h 29.69±0. 71 b 0.7S±O.0I c 33.38±I.OOc 41.73±I.04 c Catalase : (Values x units/mg protein). SOD : (Units/mg protein) P<O.OI : agroup II significantly different from group I. bgroup III significantly different from group II. c significantly different from group II. Discussion In the present study the diets of the rats of group 1 contained 8% groundnut oil, while diets of group II, III & IV contained 15% groundnut oil and 1 % cholesterol. Compared to rats fed normal diet, rats fed high fat cholesterol diet showed significantly higher levels of cholesterol, triglycerides and phospholipids in the serum and tissues. In addition the LDL+ VLDL concentration were also higher. Inclusion of coconut protein into high fat cholesterol diet results in significant lowering of lipids in the serum and tissues. There was lower levels of serum total cholesterol, LDL+ VLDL cholesterol and decreased levels of cholesterol in the liver, heart and kidney. Triglycerides and phospholipids were also lower in rats fed coconut protein. The hypocholesterolemic effect observed in the present study were similar to those observed in the earlier study in this laboratory in rats fed normal diet 2 Supplementation of coconut protein results in marginally higher activity of HMG CoA reductase in the liver as compared to that seen in rats fed high,fat cholesterol diet. This observation indicate that there was increased cholesterogenesis in the liver in rats fed coconut protein. HMG CoA reductase is the enzyme which catalyses the conversion of HMG CoA to mevalonate using NADPH as reducing equivalent and is the major rate limiting step in cholesterol biosynthesis. Incorporation of labeled acetate into free cholesterol and ester cholesterol were increased in rats fed coconut protein which correlates with the increased cholesterogenesis. On the other hand the cholesterol level in the serum and tissues were lower in rats fed coconut protein as compared to those in high fat cholesterol fed animal. This may be due to the fact that the rate of degradation of cholesterol to bile acid is more than the rate of synthesis of cholesterol. In this connection it has been reported that endogeneously synthesized cholesterol is the prefered substrate for bile acid synthesis 18. In the present study the fecal excretion of bile acids were higher which indicates higher rate of catabolism of cholesterol. Glucose-6-phosphate dehydrogenase and malic enzyme are two key enzymes which provides NADPH for fatty acid synthesis. The decreased activity of these enzymes leads to decrease in lipid synthesis. The lower rate of incorporation of 14C acetate into iriglycerides and phospholipids in the liver supports this observation. Feeding coconut protein results in decreased lipid peroxide (malondialdehyde) in the heart. The activity of superoxide dismutase and catalase were higher in rats fed coconut protein. These enzymes play an important role in the defence mechanism against the harmful toxic oxygen free radicals in the biological system. The increased activity of these. antioxidant enzymes results in decreased accumulation of superoxide and H These effects indicate that administration of coconut protein caused reduction in lipid peroxides in the heart. Earlier studies in this laboratory indicate that administration of coconut protein results in decreased lipid peroxidation in rats fed sunflower oil diet l. All these effects were similar in rats fed coconut kernel protein and L-arginine which indicate that L-arginine is the major factor which is responsible for the observed beneficial effects. The increased excretion of nitrate observed in the present study implies higher rate of arginine metabolism. In the endothelial cells, the precursor for the biosynthesis of Nitric Oxide (NO) is L-arginine. Nitric oxide is a key cell signalling molecule in physiological and pathological conditions and is

SAUL & RAJAMOHAN: HYPOUPIDEMIC & ANTI PEROXIDATION EFFECT OF COCONUT PROTEIN 1033 generated by a family of enzymes termed nitric oxide synthase (NOS) which catalyse the oxidation of one of the

6 SAUL & RAJAMOHAN: HYPOUPIDEMIC & ANTI PEROXIDATION EFFECT OF COCONUT PROTEIN 1033 generated by a family of enzymes termed nitric oxide synthase (NOS) which catalyse the oxidation of one of the terminal guanidino-nitrogens of L-arginine to yield nitric oxide and citrulline. The nitric oxide further converting to nitrate. Thus the higher rate of excretion of nitrate indicate increased rate of conversion of arginine into nitric oxide. Nitric oxide has anti proliferating effect on smooth muscle cells 19,20. It has also been demonstrated that nitric oxide inhibits platelet function through the elevation of cyclic guanosine monophosphate (cyclic GMP) or formation of S-nitrozothiols Nitric oxide is known to inhibit chemotaxis and adhesion of monocytes and neutrophils 23 and play a protective role against oxidation of LDL by mouse macrophages 24 In vivo studies, administration of L-arginine inhibited intimal thickening of aorta of hypercholesterolemic rabbit. These reports implies the hypothesis that nitric oxide may alter the initiation and progression of atherosclerosis 25. In the present study arginine supplemented casein diet and the coconut protein diet produced similar effects. Thus the correlation between the arginine content of the protein and its effect on cholesterol metabolism is true in the case of coconut protein. Antiatherogenic and hypolipidemic effect of the L arginine have been reported by serveral others This study clearly demonstrates that dietary coconut protein is able to reduce the hyperlipidemia and peroxidative effect induced by high fat cholesterol containing diet and these effects are mainly mediated by the L-arginine present in it. Acknowledgement The authors thank Mr. Anurag P and Ms. Shalini A Nair for their co-operation and support throughout the work. 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7 1034 INDIAN J EXP BIOL, OCTOBER Yates M T, Lambert L E & Whitten J P, A protective role for nitric oxide in the oxidative modification of low density lipoproteins by mouse macrophages, FEBS Lett, 309 (1992) Cooke IP, Singer A H, Tsao P, Zera P, Rowan R A & Billinghan M P, Antiatherogenic effects of L-arginine in the hypocholesterolemic rabbit, J Clin Invest, 90 (1992) Hurson M, Regan M C; Kirk S J, Wasserkrug H L & Barbul A, Metabolic effects of arginine in a healthy elderly population, J Parenter Entemal Nutr, 19 (1995) Rainer H Boger, Stefanic M, Ralf P Brundes & Andreas Mugge, Dietary L-arginine reduces the progression of atherosclerosis in cholesterol fed rabbits - comparison with lovastatin, Circulation, 96 (1997) 1282

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