181
Effects of Polyphenolic Natural Products on the Lipid Profiles of Rats Fed High Fat Diets T. Yugarani a, B.K,H, Tan b. M. Teh c and N.P. Das*, a Departments of aBiochemistry, bPharmacology and Cpathology, Faculty of Medicine, National University of Singapore, Singapore 0511, Republic of Singapore
Male Wistar rats were fed a high fat diet (HFD) containing 2.5% cholesterol and 16% lard supplemented with polyphenolic natural products namely quercetin, morin or tannic acid (100 mgfrat/day) for 4, 7 and 10 wk. Rats fed H F D without the supplements served as control. The effects of these compounds on blood lipid profiles, enzymes, fiver fat and aorta of the rat were studied. In rats fed H F D containing tannic acid, plasma total cholesterol (TC), low density lipoprotein cholesterol (LDLC) and triglyceride (TG) were reduced by 33.3%, 29.6% and 65.1%, respectively, at week 10. High density lipoprotein cholesterol (HDLC) concentration was not altered. Fat deposition was also decreased in the fiver of these rats. Morin significantly reduced plasma TG (65.1%) and fiver fat only at week 7 while at week 10 it reduced plasma TC and LDLC by 30.9% and 29.3% respectively. The plasma HDLC concentration was increased by 47.3% at week 4 but no effect was seen at weeks 7 and 10. In the rats fed H F D containing quercetin, plasma HDLC was increased by 28.6% at week 7 but at week 10, plasma LDLC was increased by 21.2%. Quercetin did not cause any significant changes on the plasma TC, TG and liver fat at weeks 4, 7 and 10. Plasma alanine aminotransferase, alkaline phosphatase and biliruhin in control and treated groups were not significantly different. However, hepatic lipase activity in rats fed tannic acid was significantly lower. Aortae of all groups of rats showed no abnormalities. The present report indicates that tannic acid and morin are effective in reducing plasma and liver fipids when supplemented with a high fat diet in rats. Lipids 27, 181-186 (1992).
Coronary heart disease (CHD) is one of the most prevalent causes of death in the United States (1). Hypercholesterolemia is an important etiological factor in CHD. Studies have shown that the risk of developing CHD is linearly related to serum cholesterol concentration (2) and low density lipoprotein cholesterol (LDLC; ref. 3), while high density lipoprotein cholesterol (HDLC) exerts a protective effect (4). Other complications of hypercholesterolemia include the development of premature atherosclerosis and atherothrombotic brain infarction (1). Dietary lipids have been reported to cause atherosclerosis in various animal models (5-8). In rats, cholesterol feeding increased low density lipoprotein (LDL) and decreased high density lipoprotein (HDL; ref. 9). *To whom correspondence should be addressed. Abbreviations: ALAT,alanineaminotransferase;ANOVA,analysis of variance;AP, alkalinephosphatase; ATP, adenosinetriphosphate; BF3, borontrifluoride;BHT, butylated hydroxytoluene;CHD, coronary heart disease; EDTA, ethylenediaminetetraacetate;GL group 1; G2, group 2; G3, group 3; G4, group 4; G5, group 5; HDLC,high density lipoproteincholesterol;HFD, high fat diet; HFD + M, high fat diet + morin;HFD + T, high fat diet + tannic acid; HFD + Q, high fat diet + quercetin;LDLC,low densitylipoproteincholesterol; NADH, reducednicotinamideadeninedinucleotide;ND, normaldiet; TC, total cholesterol; TG, triglyceride.
The polyphenolic natural products namely flavonoids and tannic acid (hydrolyzable tannin; Fig. 1) are ubiquitous in plants and are easily accessible to animals and man through their diets (10). It is estimated that the average American (USA} adult dietary intake of these natural products is about 1 g/day comprised of mixed flavonoids (11). These polyphenolic substances have been effectively used to treat certain human diseases (12,13). Flavonoids increased the survival time of rats when fed together with either a thrombogenic or atherogenic diet (14}. In in vitro studies, they have exhibited antithrombotic effects (15). Quercetin, found commonly in vascular plants (16), reduced plasma total cholesterol (TC) when maintained in colloidal suspension in vitro (17) but not plasma total cholesterol (TC) in rats (18). It is also non-toxic (19) and non-carcinogenic (20). Morin is structurally related to quercetin (a flavonol; Fig. 1). Tannins are commonly found in food (21) and in herbal medicine (22). Tannic acid (a hydrolyzable tannin) occurs in the bark and fruits of many plants (23) and is non-toxic when fed to rats at dietary levels of less than 5% (24). In view of the limited reports on the biological effects of polyphenolic natural products on rats fed atherogenic diets (14), it was felt useful to carry out a study in order to evaluate the effects of some of these compounds namely quercetin, morin and tannic acid when supplied as food supplements (100 mg/rat/day) to rats fed high cholesterol and lard diets. The single dose was selected because our preliminary investigation (data not shown} had indicated it to be the most ideal dosage to use Single dosage studies
OH Ho~O~
~OH HO~o
OH
0
OH
QUERCETIN
H 0
MORIN
HCOH HO"'--HO\
HCOH H~O
"o-~coo~. 2 HO"
TANNIC ACID FIG. 1. Structure of polyphenolic compounds used in the present study. LIPIDS, VoI. 27, no: 3 (1992)
182
T. YUGARANI ET AL. have been used to study the effects of other compounds on serum and liver cholesterol (25,26). Any beneficial effects of these natural products may provide a basis for future investigations for the possible treatment of lipid disorder resulting from high fat dietary intake Their reported non-toxicity in animals rendered the compounds useful for the present study. MATERIALS AND METHODS
Chemicals. Cholesterol was purchased from E. Merck (Darmstadt, Germany). Morin and tannic acid were obtained from Extrasarsyntex (Ganey, France). Quercetin was supplied by Sigma Chemical Co. (St. Louis, MO). Animals and treatment. Male Wistar rats, body weight approximately 180 g were used. The rats were divided into five groups, each group comprising nine rats as follows: Group 1 (G1)--normal diet; Group 2 (G2)--high fat diet (HFD) without supplement; Group 3 (G3)--HFD with quercetin; Group 4 (G4)--HFD with morin; and Group 5 (G5)--HFD with tannic acid. The compositions of the diets and their fatty acid contents are given in Tables 1 and 2, respectively. The normal diet was adopted from Griffith's (27) earlier report because it was necessary to use a natural diet which contained a low level of flavonoids and other phenolic compounds. During the first week of the experiment, each rat consumed about 7 g/day of the powdered diet. This amount was increased by 1 g weekly. Each polyphenol (100 mg/rat/day) was mixed thoroughly into the diet prior to feeding. At the end of 4, 7 and 10 wk, 3 rats were taken from each group, weighed and then decapitated. Whole blood was collected into tubes containing ethlenediaminetetraacetate (EDTA) and centrifuged TABLE 1 Compositions of the Diets
Contents
Normal diet (g/100 g)
High fat diet (g/100 g)
70.7 23.6 3.5 1.2 1.0 0 0
52.6 23.2 3.5 1.2 1.0 2.5 16.0
Wheat flour Milk powder Dried yeast powder Sodium chloride Multivitaminsa Cholesterol powder Pork lard
aVitamin compositionis given in our earlier report (29). TABLE 2 F a t t y Acid Composition of Experimental Diets and Lard (% of total fatty acids)
Fatty acid
Normal diet
High fat diet
Lard
10:0 12:0 14:0 16:0 18:0 18:111-9 18:2n-6
4.0 4.8 12.3 27.3 10.3 18.2 9.2
2.0 2.2 6.3 27.9 12.6 29.6 9.7
--2.1 28.3 37.4 10.6
18:3n-6
5.7
3.5
--
LIPIDS, Vol. 27, no. 3 (1992)
14.9
to obtain plasma. The plasma was either analyzed immediately or stored at - 7 0 ~ until analyses were carried out. Tissue preparation. The fresh liver from each rat was partly cut into 2-mm sections and preserved in 10% formalin. Further 10-~m sections were made and stained with Oil Red 'O' for examination of fat distribution. A sample of liver (1 g) was used for the determination of TC and triglyceride (TG). TC and TG were extracted according to the method of Haug and Hostmark (28). Hepatic lipase was extracted from the remaining fresh liver according to our earlier reported method (29). Thoracic aorta was removed from each rat and preserved in 10% formalin. Each aorta was subsequently cut into 10 equal sized sections, dehydrated and paraffin embedded. Using a microtome. each section was further cut into 8-~n slices. The slices were stained with hemotoxylin and eosin (H and E) and examined under a light microscope for evidence of atherosclerotic changes. Lipid analysis. The concentrations of plasma LDLC, HDLC, TC, and TG and of liver TG and TC were determined using commercially available diagnostic kits from Boehringer Mannheim GmbH (Singapore) according to the protocol supplied. Briefly, cholesteryl esters were enzymatically cleaved by cholesterol esterase to liberate cholesterol and free fatty acids. The enzyme-liberated cholesterol and the endogenous free cholesterol were oxidized by cholesterol oxidase to form A4 cholestenone and hydrogen peroxide (H202). The H202 generated was reacted with 4-aminophenazone and phenol (catalyzed by peroxidase) to form a pink complex. The color intensity was read at 500 nm. LDL in plasma was reacted with polyvinyl sulfate which resulted in the formation of the LDLC precipitate. The supernatant was taken to determine the total cholesterol concentration. From the difference between plasma TC and TC in the supernatant after centrifugation, the LDLC value was calculated. For the determination of HDLC, phosphotungstic acid and MgC12 were added to the sample to precipitate chylomicrons, very low density lipoprotein (VLDL) and LDL. The HDL remained in the supernatant after centrifugation and the cholesterol content was determined as described above. TG was hydrolyzed by lipase to form free fatty acids and glycerol. The glycerol was reacted with glycerol kinase in the presence of adenosine triphosphate (ATP) to form glycerol-3-phosphate The glycerol-3-phosphate was oxidized by glycerol phosphate oxidase to form dihydroxyacetonephosphate and H202. The H202 was complexed with 4-aminophenazone and phenol, and the pink color formed was read at 500 nm. Enzyme and bilirubin analysis. Activities of the enzymes alkaline phosphatase (AP) and alanine aminotransferase (ALAT) in plasma, hepatic lipase, and plasma total bilirubin concentrations were determined using diagnostic kits from Boehringer Mannheim GmbH. Briefly, in the determination of AP, the substrate p-nitrophenyl phosphate was hydrolyzed to phosphate and p-nitrophenol by the enzyme The formation of p-nitrophenol was then measured at 405 nm. In the determination of ALAT, the sample was added to the test reagent which contained a mixture of a-oxoglutarate and L-alanine as substrate a-Oxoglutarate and L-alanine were acted upon by ALAT to form glutamate and pyruvate"
183 EFFECTS OF POLYPHENOLS ON HIGH FAT DIETS respectively. The p y r u v a t e was further converted to lact a t e in an irreversible reaction b y lactate dehydrogenase in the presence of reduced nicotinamide adenine dinucleotide (NADH) and the decrease in absorbance due to the utilization of N A D H was followed at 340 nm. H e p a t i c lipase hydrolyzed triolein (in test reagent) to form monoglyceride and oleic acid. The decrease in turbidity due to the hydrolysis of triolein was also measured at 340 nm. Total bilirubin in p l a s m a was coupled with diazotized sulfanilic acid in the presence of caffeine to give an azo dye which absorbed at 580 nm. Diet fatty acid analysis. The lipids in the rat diets were extracted using chloroform/methanol (2:1, v/v) containing 0.02% butylated hydroxytoluene (BHT) as an antioxidant. The chloroform layer, which contained the lipids, was removed and saponified with methanolic K O H and concentrated HC1. The free f a t t y acids were extracted into n-hexane and then m e t h y l a t e d using borontrifluoride (BF3)/methanol (30). The f a t t y acid m e t h y l esters were analyzed on a Varian model 3400 gas c h r o m a t o g r a p h equipped with a flame ionization detector and a fused silica capillary column (DB-1; from J & W Scientific, Folsom, CA). A p r o g r a m m e d run over the t e m p e r a t u r e range of 150~176 on-column injection, and oxygen-free nitrogen as carrier gas were used. Statistical analysis. Values of the biochemical parameters in each group are expressed as m e a n +_SEM. One way analysis of variance (ANOVA; ref. 31) followed by Duncan's Multiple-Range Test (32) were used to evaluate the significance of differences found between mean values. P values < 0.05 were considered to be statistically significant.
WEEK 4
~ A 12o-~ ~ ~ o 80~ o ~= 40 u
/
t
2040-
ui.oE R ~ ~oe 8o-
/r
u , o ..a O ~ ~
2
240J ~ = ~
/
O
u o _~ ~, 80 ~ ~
~;;~JG1
RESULTS AND DISCUSSION
Food intake in all the five groups of r a t s were unaffected by the experimental diet. The rats in each group consumed all the allotted a m o u n t of food daily. Since hypercholesterolemia was induced in the experimental group of r a t s by feeding cholesterol and lard, the G1 normal diet (ND) r a t s were used as a comparison to G2 (HFD) in order to determine at which t i m e interval (week 4, 7 or 10) blood cholesterol was significantly increased. Feeding of the H F D for 4 wk did not significantly induce hypercholesterolemia in G2 (Fig. 2). However, at week 7 the p l a s m a TC in G2 was significantly increased in comparison to G1. The p l a s m a cholesterol concentration was p r e s u m a b l y stabilized in G2 after 7 wk because there was no further increase at week 10. In c o n t r a s t to this, p l a s m a TC in G1 did not fluctuate significantly from week 4 to 10 with an average concentration of 76.4 _+ 2.1 mg/100 mL. The G2 ( H F D without supplement) r a t s were used as control for the test groups G3-G5. In G5, p l a s m a TC was significantly reduced by 26.4% and 33.3% at week 7 and 10, respectively (Fig. 2). In G4, p l a s m a TC was only significantly reduced at week 10 (30.9%), thus indicating t h a t morin is able to reduce p l a s m a cholesterol upon prolonged intake. All the rats showed progressive increase in body weight from weeks 4-10. However, body weights did not differ significantly a m o n g s t groups (G1-G5) during the 10 wk period (Table 3). Therefore. the reduction in p l a s m a TC in G4 and G5 cannot be a t t r i b u t e d to weight loss or lower
WEEK 10
WEEK 7
~G2
~G3
~-1G4
~G5
FIG. 2. Concentration of plasma total cholesterol, LDL-C, HDLC-C and triglyceride. Values are expressed as mg/100 mL plasma. denotes a statistically significant difference between G2 and other groups (p < 0.05).
TABLE 3 Average Body Weight Increases (measured as percentage) a
% Increase in body weight Animal groups G1 G2 G3 G4 G5
(ND) (HFD) (HFD + Q) (HFD + M) (HFD + T)
Week 4
Week 7
Week 10
36.3 +_+_1.6a 48.3 + 5.3a 38.4 _ 6.3 a 47.6 + 11.0a 46.5 +_ 5.6a
58.6 +- 14.1a,b 73.0 + 5.4a 74.8 +- 1.8a 61.7 - 4.6 a 73.0 + 8.2a
89.2 _+ 9.5a 85.6 +_ 7.8a 100.0 +_ 3.1 a 100.0 _ 8.2a 88.5 _+ 5.7a
aIn each experiment, values in the same column without common superscript letters denote significant difference (p < 0.05). Values are mean _+ SEM of three animals.
weight gain of the r a t s in these two groups. In contrast, large reductions in body weight have been associated with reduced blood lipids {33}. Quercetin (G3) did not cause any significant changes on p l a s m a TC concentration at weeks 4, 7 or 10 (Fig. 2). B a s a r k a r and H a t w a l n e (18) have also reported t h a t feeding of quercetin did not lower p l a s m a
LIPIDS, Vol. 27, no. 3 (1992)
184
T. YUGARANI E T AL. TC in rats. However, in in vitro experiments, quercetin reduced p l a s m a cholesterol when maintained in colloidal suspension (17}. The p l a s m a L D L C concentration in G1 was significantly lower than in G2 at weeks 7 and 10 (Fig. 2). At week 10, L D L C in G4 and G5 were significantly reduced by 29.3% and 29.6%, respectively while in G3 a significant increase occurred (21.2%; Fig. 2). No significant differences were observed at weeks 4 and 7. P l a s m a concentration of L D L C is largely dependent on the rates of its p r o duction and removal from the circulation (34). L D L C is produced from very low density lipoprotein (VLDL) which is secreted by the fiver and m o s t of the p l a s m a L D L C is removed via a hepatic receptor mediated process (35,36). A reduction in p l a s m a L D L C by morin or tannic acid as observed in the present s t u d y suggests t h a t these compounds m a y interfere with either one of these processes or both. P l a s m a H D L C concentration in G1 and G3 were significantly higher (50.0% and 28.6%, respectively) t h a n in G2 at week 7, b u t not at weeks 4 or 10 (Fig. 2). In G4 t h o u g h H D L C was significantly increased by 47.3% at week 4, this effect was not sustained in the subsequent weeks (7 and 10). No significant difference was observed in p l a s m a H D L C concentration in G5 when compared to control (G2) at weeks 4, 7 or 10. Mahley and Holcombe (9) have reported an increase in L D L and a decrease in H D L following cholesterol feeding in the rats. Cholesterol t r a n s p o r t to extrahepatic tissues is primarily ensured by L D L while H D L has an i m p o r t a n t role in reversing the cholesterol t r a n s p o r t process, whereby excess cholesterol is removed from peripheral tissues to the fiver for excretion (37}. I t is interesting to note t h a t tannic acid is able to reduce L D L C and TC without affecting the H D L C level. Thus, there is a significant increase in the H D L C to TC ratio for this group of rats when compared to the control group (G2; Table 4). This ratio is suggested to be a more useful index for determining the quality and effects of fat on health. We have also recently reported a high ratio value on rats fed refined, bleached and deodorized p a l m oil (29). Joslyn and Glick (24) have shown t h a t tannic acid is non-toxic at dietary concentration of 5% or less. They also observed t h a t tannic acid toxicity depended on the initial b o d y weight of the r a t s used. Rats with a higher initial body weight showed greater resistance to tannic acid toxicity. In the present investigation we have fed less t h a n 1.5% dietary concentration of tannic acid to r a t s with an initial b o d y weight of a b o u t 180 g. We have observed no
weight loss nor death throughout the experimental period and all the rats appeared to be normal and healthy. No significant differences were observed in p l a s m a TG concentration between all groups at week 4. A t week 7, p l a s m a TG concentration in G2 was significantly higher t h a n in G1 (52.4%) indicating t h a t the increase of TG in G2 is of dietary origin and the TG level appeared to be stabilized from week 7 onward because no significant difference was observed at week 10. The p l a s m a TG of G4 and G5 were lower t h a n G2 b y 65.1% and 56.6%, respectively, at week 7. At week 10, p l a s m a TG in G5 was further reduced (65.1%). I t has been reported t h a t enrichment of diet with n-3 f a t t y acids also reduces p l a s m a TG concentrations in the r a t (38). Perhaps morin or tannic acid m a y have acted in the similar process as the n-3 f a t t y acids. The significant increase in liver weight (Table 5) without a significant increase in b o d y weight (Table 3) of r a t s in G 2 - G 5 in comparison to G1 (normal diet fed rats) is probably due to higher fat deposition resulting from a higher fat i n t a k e Microscopic examination of the fiver s u p p o r t s this conclusion for there was a minimal a m o u n t of fat deposited in the liver of normal diet fed rats (Fig. 3a). The tannic acid fed group (G5) showed less fat deposition (but higher t h a n G1) in the fiver when compared to G 2 - 4 (Fig. 3b & c). This indicates t h a t tannic acid is able to reduce the infiltration of fat into the liver cells of r a t s fed high fat diet. Analysis of the liver TG and TC content at week 7 (Table 6) showed fiver TC in G1 and G5 to be significantly lower t h a n G2. I n G1 and G4 the fiver TG content was significantly lower t h a n in G2 (Table 6). A l t h o u g h there was a significant decrease in fiver TG of G4, this effect was not manifested in liver weight or liver fat distribution. D a t a for liver cholesterol and TG contents at weeks 4 and 10 were not obtained. Sugano et al. (39) observed reductions in fiver cholesterol concentration of r a t s when fed with chitosan and cholesterol, and they have explained the reduction as a result of chitosan interfering with cholesterol absorption. I t m a y be pertinent to suggest t h a t tannic acid m a y also interfere with cholesterol absorption. The fiver plays an i m p o r t a n t role in the catabolism of cholesterol. Conversion of cholesterol to bile acids occurs exclusively in the liver and represents the major pathway for the elimination of cholesterol from the b o d y (40). Retention of biliary constituents due to bile duct obstruction was reported to show an initial rise in p l a s m a bilirubin accompanied by an increase in A P and A L A T (41) as
TABLE 4
TABLE 5
Ratio of HDL Cholesterol to Total Cholesterol a
Liver Weight (g/100 body weight)a
Animal groups G1 G2 G3 G4 G5
(ND) (HFD) (HFD + Q) (HFD + M) (HFD + T)
Week 4
Week 7
Week 10
0.35 _ 0.06a 0.28 _+ 0.04a 0.29 _+ 0.04a 0.27 +__0.03 a 0.30 +_ 0.05a
0.67 +__0.12b 0.24 +__0.05a 0.30 +_ 0.05a 0.22 ___0.02 a 0.35 ___0.06b
0.68 +_ 0.14b 0.34 ___0.09 a 0.23 + 0.04 a 0.25 ___0.05a 0.45 + 0.06b
aIn each experiment, values in the same column without common superscript letters denote significant difference (p < 0.05). Values are mean +_ SEM of three animals. LIPIDS, VoI. 27, no. 3 (1992)
Animal group G1 G2 G3 G4 G5
(ND) (HFD) (HFD + Q) (HFD + M) (HFD + T)
Week 4 2.9 3.5 3.5 3.3 3.3
___0.1b ___0.1 a + 0.2a +_ 0.3a, b +_ 0.1a, b
Week 7 2.9 3.3 3.3 3.3 2.9
+__0.1 a +_ 0.2a + 0.1 a +_ 0.3 a __ 0.1 a
Week 10 2.6 3.3 3.2 3.3 3.3
__. 0.1b __. 0.2 a +_ 0.1 a __ 0.1 a +_ 0.1 a
aIn each experiment, values in the same column without common superscript letters denote significant difference (p < 0.05}. Values are mean _ SEM of three animals.
185
EFFECTS OF POLYPHENOLS ON HIGH FAT DIETS
FIG. 3. a) Liver of a rat fed normal diet for 10 wk shows minimal deposition of fat in the hepatocytes. (Oil Red O stain, 75X). b) Liver of a rat fed high fat diet supplemented with tannic acid (100 mg/ rat/day) for 10wk shows moderate deposition of fat in the hepatocytes with sparing of the centrilobular hepatocytes. (Oil Red O stain, 80X). c) Liver of a rat fed high fat diet without polyphenolic compounds supplement for 10 wk shows severe and panlobular involvement of hepatocytes. (Oil Red O stain, 75X). Liver of rats fed high fat diet with quercetin (100 mg/rat/day) and morin (100 mg/rat/day) for 10 wk also showed similar appearance.
well as cirrhosis of the liver (42). Others have shown increases in tissue levels of A P due to diet induced hyperlipidemia (43). In contrast, the present s t u d y showed no significant differences in the activities of p l a s m a AP, A L A T and bilirubin concentration in all groups of rats. The activities ranged from 47.2 • 5.2 to 52.8 • 3.2 U/L and 40.3 • 4.8 to 45.1 • 4.1 U/L for ALAT and AP, respectively. The total bilirubin concentration ranged from 11.0 • 2.5 to 15.2 • 2.7 rag/100 mL. Thus, these results indicate t h a t feeding of a diet high in cholesterol and lard content or a cholesterol and lard diet supplemented with polyphenolic compounds does not cause bile retention or liver cirrhosis. H e p a t i c lipase activity in G1 and G5 were significantly lower t h a n in G2 at weeks 7 and 10 (Fig. 4). J a n s e n and H u l s m a n n (44) have suggested an involvement of hepatic lipase in the delivery of cholesterol to the liver. They have proposed t h a t cholesterol is taken up from peripheral cells b y a phospholipid rich H D L and subsequently delivered to the fiver via a m e c h a n i s m involving the depletion of phospholipid from H D L by hepatic lipase Along this line others have proposed t h a t the increase in hepatic fipase activity could contribute to increases in liver cholesterol (45). Our findings are in agreement with these reports as increased hepatic lipase activity in G2, G3 and G4 correlates well with increased fiver cholesterol concentration at week 7 (correlation coefficient, r = +0.632). Atherosclerotic plaques were n o t observed in the a o r t a of the five groups of rats. Microscopic examination of the H and E stained paraffin sections of the thoracic a o r t a showed smooth, intact intimal fining with no evidence of raised f a t t y plaques or proliferation of s m o o t h muscle cells, thereby demonstrating the absence of atherosclerosis in all the groups of experimental rats. In summary, the present s t u d y has shown t h a t both tannic acid and morin can cause favorable changes in p l a s m a lipid profiles of the t y p e t h a t have been correlated with CHD. Tannic acid reduced fat deposits in liver cells at nontoxic concentrations. Thus, tannic acid has a potential to avert complications arising from high fat intake. Further studies need to be carried out to unravel the m e c h a n i s m of the observed hypolipidemic effect of tannic acid.
WEEK
7
WEEK
10
A
TABLE 6 Liver Total Cholesterol and Triglyceride Concentration at Week 7a
Animal group G1 G2 G3 G4 G5
(ND) (HFD) (HFD + Q) (HFD + M) (HFD + T)
Total cholesterol (mg/g liver) 2.2 _ 0.1b 26.6 _ 0.9a 18.6 +_ 2.6a 22.3 _ 2.2a 15.9 _ 0.9b
Triglyceride (mg/g liver) 15.9 42.9 45.1 29.5 33.1
___ 1.5b _ 10.9a _ 3.6 a __. 2.0b _ 5.9a
aIn each experiment, values in the same column without common superscript letters denote significant difference (p < 0.05). Values are mean _ SEM of three animals.
,'(XXN 4"
/-,XXX] ~X)(XI ,1XYJq KX'X~
1 o,
Bo, V1o,
FIG. 4. Hepatic lipase activity of rats at weeks 7 and 10. Values are expressed as mU/mg protein. * denotes a statistically significant difference between G2 and other groups (p < 0.05).
LIPIDS, Vol. 27, no. 3 (1992)
186
T. YUGARANI E T AL. REFERENCES
1. Lewis, L.A., and Nait~ H.K. (1978) Clit~ Chem. 12, 2081-2098. 2. Kannel, W.B., CasteUi, W.P., Gordon, T., and McNamara, P.M. (1971) Ann. Intern. Med. 74, 1-13. 3. Goldstein, J.L., Hazzard, W.R., Schrott, H.G., Bierman, E.L., and Motulsky, A.G. (1973)J. Clin. Invest. 52, 1533-1543. 4. Mattson, F.H., and Grundy, S.M. (1985) J. LipidRes. 26, 194-202. 5. Vles, R.O., BuUer, J., Gottenbos, J.J., and Thomasson, H.J. (1964) J. Atheroscler. Res. 4, 170-183. 6. Kritchevsky, D. (1969) Ann. N.Y. Acad Sci. 162, 80-88. 7. Malmros, H. (1969)Lancet 2, 479-484. 8. Wissler, R.W., and Vesselinovitch, D. (1975)Adv. Exp. MecL Biol. 60, 65-76. 9. Mahley, R.W., and Holcombe, K.S. (1977) J. Lipid Res. 18, 314-324. 10. Harborne, J.B. (1975) in The Flavonoids (Mabry, T.J., and Mabry, H., eds.) 2nd edn., pp. 88-95, Chapman and Hall, London. 11. Kuhnau, J. (1976) Wld. Rev. Nutr. Diet 24, 117-121. 12. Havsteen, B. (1983) Biochem. Pharmacol. 32, 1141-1148. 13. Wagner, H. (1979) in Recent Advances in Phytochernistry (Swain, T., Harborne, J.B., and Sumere, V., eds.) Vol. 2, pp. 589-616, Plenum Press, New York. 14. Robbins, R.C. (1967)J. Atheroscler. Res. 7, 3-10. 15. Gryglewski, R.J., Korbut, R., Robak, J., and Swies, J. (1987) Biochem. PharrnacoL 36, 310-322. 16. Robinson, T. (1967) in The Organic Constituents of Higher Plant~ Their Chemistry and Interrelationships, pp. 178-209, Burgess, Minneapolis. 17. Di Maggio, G. (1959) Intern. Z. Vitaminforsch. 229, 223-226. 18. Basarkar, P.W, and Hatwalne, V.G. (1975} Baroda J. Nutr. 12, 99-103. 19. Booth, A.N., and De Eds, F. {1958) J. Am. Pharrn. Assoa 47, 183-184. 20. Hiron~ I., Uen~ I., Hosaka, S., Takanashi, H., Matsushima, T., Sugimura, T., and Natori, S. (1981) Cancer Lett. 13, 15-21. 21. Narasinga Rao~B.K, and Prabhavathi, T. (1982)J. ScL FoodAgria 33, 89-95. 22. Haslam, E., Lilley, T.H., Ya Cai, Martin, R., and Magnolat~ D. (1989) Planta Med. 55, 1-8.
LIPIDS, Vol. 27, no. 3 (1992)
23. The Merck Index (1989) (Budavari, S., ed.) 11th edn., p. 9027, Merck & Ca Inc, Rahway. 24. Joslyn, M.A., and Glick, Z. (1969) J. Nutr. 98, 119-126. 25. Tanaka, M., Iio, T., and Tabata, T. (1987) Lipids 22, 1016-1019. 26. Assis, S.R, and Basu, T.K. (1990) Nutr. Res. 10, 99-108. 27. Griffiths, L.A. (1964) Biochem. J. 92, 173-179. 28. Haug, A., and Hostmark, A.T. (1987) J. Nutr. 117, 1011-1017. 29. Pereira, T.A., Sinniah, R., and Das, N.R (1990) Biochem. Meal Metab. Biol. 44, 207-217. 30. Bannon, C.D., Craske, J.D., Ngo, T.H., Harper, N.L., and O'Reurke, K.L. (1982)J. Chromatogr. 247, 63-69. 31. Armitage, P., in Statistical Methods in Medical Research, 2rid edn., pp. 36-48, BlackweU Scientific, Oxford. 32. Duncan, D.B. (1975) Biometrics 31, 339-359. 33. Olefsky, J., Reaven, G.M., and Farquhar, J.W. (1974) J. Clin. Invest. 53, 64-69. 34. Brown, M.S., Kovanen, P.J., and Goldstein, J.L. (1981) Science 212, 628-635. 35. Spady, D.K., Bilheimer, D.W.,and Dietschy, J.M. (1983) Proc NatL Acad. Sci. USA 80, 3499-3509. 36. Brown, M.S., and Goldstein, J.L. (1986) Science 232, 34-47. 37. Gurr, M.I., Borlak, N., and Ganatra, S. (1989) Nutr. Res. Rev. 2, 63-86. 38. Balasubramaniam, S., Simons, L.A., Chang, S., and Hickie, J.B. (1985) J. Lipid Res. 26, 684-689. 39. Sugano, M., Watanabe, S., Kishi, A., Izume, M., and Ohtakara, A. (1988) Lipids 23, 187-191. 40. Siperstein, M.D., and Chaikoff, I.L. (1952) J. BIOL Chem. 198, 93-104. 41. Putzki, H., Reichert, B. and Heymann, H. (1989) Cli~ Chitta Acta 181, 81-86. 42. Kountouras, J., Billing, B.H., and Scheuer, RJ. (1984) Br. J. Exp. Path. 65, 305-311. 43. Adoga, G.I., and Osuji, J. (1986) Biochem. Int. 13, 614-624. 44. Jansen, H., and Hulsmann, W.C. (1980) Trends Biochem. Sci. 5, 265-270. 45. De Pury, G.G., and Collins, F.D. (1972) Lipids 7, 225-228. [Received June 13, 1991; Revision accepted January 27, 1992]
Effects of Polyphenolic Natural Products on the Lipid Profiles of Rats Fed High Fat Diets T. Yugarani a, B.K,H, Tan b. M. Teh c and N.P. Das*, a Departments of aBiochemistry, bPharmacology and Cpathology, Faculty of Medicine, National University of Singapore, Singapore 0511, Republic of Singapore
Male Wistar rats were fed a high fat diet (HFD) containing 2.5% cholesterol and 16% lard supplemented with polyphenolic natural products namely quercetin, morin or tannic acid (100 mgfrat/day) for 4, 7 and 10 wk. Rats fed H F D without the supplements served as control. The effects of these compounds on blood lipid profiles, enzymes, fiver fat and aorta of the rat were studied. In rats fed H F D containing tannic acid, plasma total cholesterol (TC), low density lipoprotein cholesterol (LDLC) and triglyceride (TG) were reduced by 33.3%, 29.6% and 65.1%, respectively, at week 10. High density lipoprotein cholesterol (HDLC) concentration was not altered. Fat deposition was also decreased in the fiver of these rats. Morin significantly reduced plasma TG (65.1%) and fiver fat only at week 7 while at week 10 it reduced plasma TC and LDLC by 30.9% and 29.3% respectively. The plasma HDLC concentration was increased by 47.3% at week 4 but no effect was seen at weeks 7 and 10. In the rats fed H F D containing quercetin, plasma HDLC was increased by 28.6% at week 7 but at week 10, plasma LDLC was increased by 21.2%. Quercetin did not cause any significant changes on the plasma TC, TG and liver fat at weeks 4, 7 and 10. Plasma alanine aminotransferase, alkaline phosphatase and biliruhin in control and treated groups were not significantly different. However, hepatic lipase activity in rats fed tannic acid was significantly lower. Aortae of all groups of rats showed no abnormalities. The present report indicates that tannic acid and morin are effective in reducing plasma and liver fipids when supplemented with a high fat diet in rats. Lipids 27, 181-186 (1992).
Coronary heart disease (CHD) is one of the most prevalent causes of death in the United States (1). Hypercholesterolemia is an important etiological factor in CHD. Studies have shown that the risk of developing CHD is linearly related to serum cholesterol concentration (2) and low density lipoprotein cholesterol (LDLC; ref. 3), while high density lipoprotein cholesterol (HDLC) exerts a protective effect (4). Other complications of hypercholesterolemia include the development of premature atherosclerosis and atherothrombotic brain infarction (1). Dietary lipids have been reported to cause atherosclerosis in various animal models (5-8). In rats, cholesterol feeding increased low density lipoprotein (LDL) and decreased high density lipoprotein (HDL; ref. 9). *To whom correspondence should be addressed. Abbreviations: ALAT,alanineaminotransferase;ANOVA,analysis of variance;AP, alkalinephosphatase; ATP, adenosinetriphosphate; BF3, borontrifluoride;BHT, butylated hydroxytoluene;CHD, coronary heart disease; EDTA, ethylenediaminetetraacetate;GL group 1; G2, group 2; G3, group 3; G4, group 4; G5, group 5; HDLC,high density lipoproteincholesterol;HFD, high fat diet; HFD + M, high fat diet + morin;HFD + T, high fat diet + tannic acid; HFD + Q, high fat diet + quercetin;LDLC,low densitylipoproteincholesterol; NADH, reducednicotinamideadeninedinucleotide;ND, normaldiet; TC, total cholesterol; TG, triglyceride.
The polyphenolic natural products namely flavonoids and tannic acid (hydrolyzable tannin; Fig. 1) are ubiquitous in plants and are easily accessible to animals and man through their diets (10). It is estimated that the average American (USA} adult dietary intake of these natural products is about 1 g/day comprised of mixed flavonoids (11). These polyphenolic substances have been effectively used to treat certain human diseases (12,13). Flavonoids increased the survival time of rats when fed together with either a thrombogenic or atherogenic diet (14}. In in vitro studies, they have exhibited antithrombotic effects (15). Quercetin, found commonly in vascular plants (16), reduced plasma total cholesterol (TC) when maintained in colloidal suspension in vitro (17) but not plasma total cholesterol (TC) in rats (18). It is also non-toxic (19) and non-carcinogenic (20). Morin is structurally related to quercetin (a flavonol; Fig. 1). Tannins are commonly found in food (21) and in herbal medicine (22). Tannic acid (a hydrolyzable tannin) occurs in the bark and fruits of many plants (23) and is non-toxic when fed to rats at dietary levels of less than 5% (24). In view of the limited reports on the biological effects of polyphenolic natural products on rats fed atherogenic diets (14), it was felt useful to carry out a study in order to evaluate the effects of some of these compounds namely quercetin, morin and tannic acid when supplied as food supplements (100 mg/rat/day) to rats fed high cholesterol and lard diets. The single dose was selected because our preliminary investigation (data not shown} had indicated it to be the most ideal dosage to use Single dosage studies
OH Ho~O~
~OH HO~o
OH
0
OH
QUERCETIN
H 0
MORIN
HCOH HO"'--HO\
HCOH H~O
"o-~coo~. 2 HO"
TANNIC ACID FIG. 1. Structure of polyphenolic compounds used in the present study. LIPIDS, VoI. 27, no: 3 (1992)
182
T. YUGARANI ET AL. have been used to study the effects of other compounds on serum and liver cholesterol (25,26). Any beneficial effects of these natural products may provide a basis for future investigations for the possible treatment of lipid disorder resulting from high fat dietary intake Their reported non-toxicity in animals rendered the compounds useful for the present study. MATERIALS AND METHODS
Chemicals. Cholesterol was purchased from E. Merck (Darmstadt, Germany). Morin and tannic acid were obtained from Extrasarsyntex (Ganey, France). Quercetin was supplied by Sigma Chemical Co. (St. Louis, MO). Animals and treatment. Male Wistar rats, body weight approximately 180 g were used. The rats were divided into five groups, each group comprising nine rats as follows: Group 1 (G1)--normal diet; Group 2 (G2)--high fat diet (HFD) without supplement; Group 3 (G3)--HFD with quercetin; Group 4 (G4)--HFD with morin; and Group 5 (G5)--HFD with tannic acid. The compositions of the diets and their fatty acid contents are given in Tables 1 and 2, respectively. The normal diet was adopted from Griffith's (27) earlier report because it was necessary to use a natural diet which contained a low level of flavonoids and other phenolic compounds. During the first week of the experiment, each rat consumed about 7 g/day of the powdered diet. This amount was increased by 1 g weekly. Each polyphenol (100 mg/rat/day) was mixed thoroughly into the diet prior to feeding. At the end of 4, 7 and 10 wk, 3 rats were taken from each group, weighed and then decapitated. Whole blood was collected into tubes containing ethlenediaminetetraacetate (EDTA) and centrifuged TABLE 1 Compositions of the Diets
Contents
Normal diet (g/100 g)
High fat diet (g/100 g)
70.7 23.6 3.5 1.2 1.0 0 0
52.6 23.2 3.5 1.2 1.0 2.5 16.0
Wheat flour Milk powder Dried yeast powder Sodium chloride Multivitaminsa Cholesterol powder Pork lard
aVitamin compositionis given in our earlier report (29). TABLE 2 F a t t y Acid Composition of Experimental Diets and Lard (% of total fatty acids)
Fatty acid
Normal diet
High fat diet
Lard
10:0 12:0 14:0 16:0 18:0 18:111-9 18:2n-6
4.0 4.8 12.3 27.3 10.3 18.2 9.2
2.0 2.2 6.3 27.9 12.6 29.6 9.7
--2.1 28.3 37.4 10.6
18:3n-6
5.7
3.5
--
LIPIDS, Vol. 27, no. 3 (1992)
14.9
to obtain plasma. The plasma was either analyzed immediately or stored at - 7 0 ~ until analyses were carried out. Tissue preparation. The fresh liver from each rat was partly cut into 2-mm sections and preserved in 10% formalin. Further 10-~m sections were made and stained with Oil Red 'O' for examination of fat distribution. A sample of liver (1 g) was used for the determination of TC and triglyceride (TG). TC and TG were extracted according to the method of Haug and Hostmark (28). Hepatic lipase was extracted from the remaining fresh liver according to our earlier reported method (29). Thoracic aorta was removed from each rat and preserved in 10% formalin. Each aorta was subsequently cut into 10 equal sized sections, dehydrated and paraffin embedded. Using a microtome. each section was further cut into 8-~n slices. The slices were stained with hemotoxylin and eosin (H and E) and examined under a light microscope for evidence of atherosclerotic changes. Lipid analysis. The concentrations of plasma LDLC, HDLC, TC, and TG and of liver TG and TC were determined using commercially available diagnostic kits from Boehringer Mannheim GmbH (Singapore) according to the protocol supplied. Briefly, cholesteryl esters were enzymatically cleaved by cholesterol esterase to liberate cholesterol and free fatty acids. The enzyme-liberated cholesterol and the endogenous free cholesterol were oxidized by cholesterol oxidase to form A4 cholestenone and hydrogen peroxide (H202). The H202 generated was reacted with 4-aminophenazone and phenol (catalyzed by peroxidase) to form a pink complex. The color intensity was read at 500 nm. LDL in plasma was reacted with polyvinyl sulfate which resulted in the formation of the LDLC precipitate. The supernatant was taken to determine the total cholesterol concentration. From the difference between plasma TC and TC in the supernatant after centrifugation, the LDLC value was calculated. For the determination of HDLC, phosphotungstic acid and MgC12 were added to the sample to precipitate chylomicrons, very low density lipoprotein (VLDL) and LDL. The HDL remained in the supernatant after centrifugation and the cholesterol content was determined as described above. TG was hydrolyzed by lipase to form free fatty acids and glycerol. The glycerol was reacted with glycerol kinase in the presence of adenosine triphosphate (ATP) to form glycerol-3-phosphate The glycerol-3-phosphate was oxidized by glycerol phosphate oxidase to form dihydroxyacetonephosphate and H202. The H202 was complexed with 4-aminophenazone and phenol, and the pink color formed was read at 500 nm. Enzyme and bilirubin analysis. Activities of the enzymes alkaline phosphatase (AP) and alanine aminotransferase (ALAT) in plasma, hepatic lipase, and plasma total bilirubin concentrations were determined using diagnostic kits from Boehringer Mannheim GmbH. Briefly, in the determination of AP, the substrate p-nitrophenyl phosphate was hydrolyzed to phosphate and p-nitrophenol by the enzyme The formation of p-nitrophenol was then measured at 405 nm. In the determination of ALAT, the sample was added to the test reagent which contained a mixture of a-oxoglutarate and L-alanine as substrate a-Oxoglutarate and L-alanine were acted upon by ALAT to form glutamate and pyruvate"
183 EFFECTS OF POLYPHENOLS ON HIGH FAT DIETS respectively. The p y r u v a t e was further converted to lact a t e in an irreversible reaction b y lactate dehydrogenase in the presence of reduced nicotinamide adenine dinucleotide (NADH) and the decrease in absorbance due to the utilization of N A D H was followed at 340 nm. H e p a t i c lipase hydrolyzed triolein (in test reagent) to form monoglyceride and oleic acid. The decrease in turbidity due to the hydrolysis of triolein was also measured at 340 nm. Total bilirubin in p l a s m a was coupled with diazotized sulfanilic acid in the presence of caffeine to give an azo dye which absorbed at 580 nm. Diet fatty acid analysis. The lipids in the rat diets were extracted using chloroform/methanol (2:1, v/v) containing 0.02% butylated hydroxytoluene (BHT) as an antioxidant. The chloroform layer, which contained the lipids, was removed and saponified with methanolic K O H and concentrated HC1. The free f a t t y acids were extracted into n-hexane and then m e t h y l a t e d using borontrifluoride (BF3)/methanol (30). The f a t t y acid m e t h y l esters were analyzed on a Varian model 3400 gas c h r o m a t o g r a p h equipped with a flame ionization detector and a fused silica capillary column (DB-1; from J & W Scientific, Folsom, CA). A p r o g r a m m e d run over the t e m p e r a t u r e range of 150~176 on-column injection, and oxygen-free nitrogen as carrier gas were used. Statistical analysis. Values of the biochemical parameters in each group are expressed as m e a n +_SEM. One way analysis of variance (ANOVA; ref. 31) followed by Duncan's Multiple-Range Test (32) were used to evaluate the significance of differences found between mean values. P values < 0.05 were considered to be statistically significant.
WEEK 4
~ A 12o-~ ~ ~ o 80~ o ~= 40 u
/
t
2040-
ui.oE R ~ ~oe 8o-
/r
u , o ..a O ~ ~
2
240J ~ = ~
/
O
u o _~ ~, 80 ~ ~
~;;~JG1
RESULTS AND DISCUSSION
Food intake in all the five groups of r a t s were unaffected by the experimental diet. The rats in each group consumed all the allotted a m o u n t of food daily. Since hypercholesterolemia was induced in the experimental group of r a t s by feeding cholesterol and lard, the G1 normal diet (ND) r a t s were used as a comparison to G2 (HFD) in order to determine at which t i m e interval (week 4, 7 or 10) blood cholesterol was significantly increased. Feeding of the H F D for 4 wk did not significantly induce hypercholesterolemia in G2 (Fig. 2). However, at week 7 the p l a s m a TC in G2 was significantly increased in comparison to G1. The p l a s m a cholesterol concentration was p r e s u m a b l y stabilized in G2 after 7 wk because there was no further increase at week 10. In c o n t r a s t to this, p l a s m a TC in G1 did not fluctuate significantly from week 4 to 10 with an average concentration of 76.4 _+ 2.1 mg/100 mL. The G2 ( H F D without supplement) r a t s were used as control for the test groups G3-G5. In G5, p l a s m a TC was significantly reduced by 26.4% and 33.3% at week 7 and 10, respectively (Fig. 2). In G4, p l a s m a TC was only significantly reduced at week 10 (30.9%), thus indicating t h a t morin is able to reduce p l a s m a cholesterol upon prolonged intake. All the rats showed progressive increase in body weight from weeks 4-10. However, body weights did not differ significantly a m o n g s t groups (G1-G5) during the 10 wk period (Table 3). Therefore. the reduction in p l a s m a TC in G4 and G5 cannot be a t t r i b u t e d to weight loss or lower
WEEK 10
WEEK 7
~G2
~G3
~-1G4
~G5
FIG. 2. Concentration of plasma total cholesterol, LDL-C, HDLC-C and triglyceride. Values are expressed as mg/100 mL plasma. denotes a statistically significant difference between G2 and other groups (p < 0.05).
TABLE 3 Average Body Weight Increases (measured as percentage) a
% Increase in body weight Animal groups G1 G2 G3 G4 G5
(ND) (HFD) (HFD + Q) (HFD + M) (HFD + T)
Week 4
Week 7
Week 10
36.3 +_+_1.6a 48.3 + 5.3a 38.4 _ 6.3 a 47.6 + 11.0a 46.5 +_ 5.6a
58.6 +- 14.1a,b 73.0 + 5.4a 74.8 +- 1.8a 61.7 - 4.6 a 73.0 + 8.2a
89.2 _+ 9.5a 85.6 +_ 7.8a 100.0 +_ 3.1 a 100.0 _ 8.2a 88.5 _+ 5.7a
aIn each experiment, values in the same column without common superscript letters denote significant difference (p < 0.05). Values are mean _+ SEM of three animals.
weight gain of the r a t s in these two groups. In contrast, large reductions in body weight have been associated with reduced blood lipids {33}. Quercetin (G3) did not cause any significant changes on p l a s m a TC concentration at weeks 4, 7 or 10 (Fig. 2). B a s a r k a r and H a t w a l n e (18) have also reported t h a t feeding of quercetin did not lower p l a s m a
LIPIDS, Vol. 27, no. 3 (1992)
184
T. YUGARANI E T AL. TC in rats. However, in in vitro experiments, quercetin reduced p l a s m a cholesterol when maintained in colloidal suspension (17}. The p l a s m a L D L C concentration in G1 was significantly lower than in G2 at weeks 7 and 10 (Fig. 2). At week 10, L D L C in G4 and G5 were significantly reduced by 29.3% and 29.6%, respectively while in G3 a significant increase occurred (21.2%; Fig. 2). No significant differences were observed at weeks 4 and 7. P l a s m a concentration of L D L C is largely dependent on the rates of its p r o duction and removal from the circulation (34). L D L C is produced from very low density lipoprotein (VLDL) which is secreted by the fiver and m o s t of the p l a s m a L D L C is removed via a hepatic receptor mediated process (35,36). A reduction in p l a s m a L D L C by morin or tannic acid as observed in the present s t u d y suggests t h a t these compounds m a y interfere with either one of these processes or both. P l a s m a H D L C concentration in G1 and G3 were significantly higher (50.0% and 28.6%, respectively) t h a n in G2 at week 7, b u t not at weeks 4 or 10 (Fig. 2). In G4 t h o u g h H D L C was significantly increased by 47.3% at week 4, this effect was not sustained in the subsequent weeks (7 and 10). No significant difference was observed in p l a s m a H D L C concentration in G5 when compared to control (G2) at weeks 4, 7 or 10. Mahley and Holcombe (9) have reported an increase in L D L and a decrease in H D L following cholesterol feeding in the rats. Cholesterol t r a n s p o r t to extrahepatic tissues is primarily ensured by L D L while H D L has an i m p o r t a n t role in reversing the cholesterol t r a n s p o r t process, whereby excess cholesterol is removed from peripheral tissues to the fiver for excretion (37}. I t is interesting to note t h a t tannic acid is able to reduce L D L C and TC without affecting the H D L C level. Thus, there is a significant increase in the H D L C to TC ratio for this group of rats when compared to the control group (G2; Table 4). This ratio is suggested to be a more useful index for determining the quality and effects of fat on health. We have also recently reported a high ratio value on rats fed refined, bleached and deodorized p a l m oil (29). Joslyn and Glick (24) have shown t h a t tannic acid is non-toxic at dietary concentration of 5% or less. They also observed t h a t tannic acid toxicity depended on the initial b o d y weight of the r a t s used. Rats with a higher initial body weight showed greater resistance to tannic acid toxicity. In the present investigation we have fed less t h a n 1.5% dietary concentration of tannic acid to r a t s with an initial b o d y weight of a b o u t 180 g. We have observed no
weight loss nor death throughout the experimental period and all the rats appeared to be normal and healthy. No significant differences were observed in p l a s m a TG concentration between all groups at week 4. A t week 7, p l a s m a TG concentration in G2 was significantly higher t h a n in G1 (52.4%) indicating t h a t the increase of TG in G2 is of dietary origin and the TG level appeared to be stabilized from week 7 onward because no significant difference was observed at week 10. The p l a s m a TG of G4 and G5 were lower t h a n G2 b y 65.1% and 56.6%, respectively, at week 7. At week 10, p l a s m a TG in G5 was further reduced (65.1%). I t has been reported t h a t enrichment of diet with n-3 f a t t y acids also reduces p l a s m a TG concentrations in the r a t (38). Perhaps morin or tannic acid m a y have acted in the similar process as the n-3 f a t t y acids. The significant increase in liver weight (Table 5) without a significant increase in b o d y weight (Table 3) of r a t s in G 2 - G 5 in comparison to G1 (normal diet fed rats) is probably due to higher fat deposition resulting from a higher fat i n t a k e Microscopic examination of the fiver s u p p o r t s this conclusion for there was a minimal a m o u n t of fat deposited in the liver of normal diet fed rats (Fig. 3a). The tannic acid fed group (G5) showed less fat deposition (but higher t h a n G1) in the fiver when compared to G 2 - 4 (Fig. 3b & c). This indicates t h a t tannic acid is able to reduce the infiltration of fat into the liver cells of r a t s fed high fat diet. Analysis of the liver TG and TC content at week 7 (Table 6) showed fiver TC in G1 and G5 to be significantly lower t h a n G2. I n G1 and G4 the fiver TG content was significantly lower t h a n in G2 (Table 6). A l t h o u g h there was a significant decrease in fiver TG of G4, this effect was not manifested in liver weight or liver fat distribution. D a t a for liver cholesterol and TG contents at weeks 4 and 10 were not obtained. Sugano et al. (39) observed reductions in fiver cholesterol concentration of r a t s when fed with chitosan and cholesterol, and they have explained the reduction as a result of chitosan interfering with cholesterol absorption. I t m a y be pertinent to suggest t h a t tannic acid m a y also interfere with cholesterol absorption. The fiver plays an i m p o r t a n t role in the catabolism of cholesterol. Conversion of cholesterol to bile acids occurs exclusively in the liver and represents the major pathway for the elimination of cholesterol from the b o d y (40). Retention of biliary constituents due to bile duct obstruction was reported to show an initial rise in p l a s m a bilirubin accompanied by an increase in A P and A L A T (41) as
TABLE 4
TABLE 5
Ratio of HDL Cholesterol to Total Cholesterol a
Liver Weight (g/100 body weight)a
Animal groups G1 G2 G3 G4 G5
(ND) (HFD) (HFD + Q) (HFD + M) (HFD + T)
Week 4
Week 7
Week 10
0.35 _ 0.06a 0.28 _+ 0.04a 0.29 _+ 0.04a 0.27 +__0.03 a 0.30 +_ 0.05a
0.67 +__0.12b 0.24 +__0.05a 0.30 +_ 0.05a 0.22 ___0.02 a 0.35 ___0.06b
0.68 +_ 0.14b 0.34 ___0.09 a 0.23 + 0.04 a 0.25 ___0.05a 0.45 + 0.06b
aIn each experiment, values in the same column without common superscript letters denote significant difference (p < 0.05). Values are mean +_ SEM of three animals. LIPIDS, VoI. 27, no. 3 (1992)
Animal group G1 G2 G3 G4 G5
(ND) (HFD) (HFD + Q) (HFD + M) (HFD + T)
Week 4 2.9 3.5 3.5 3.3 3.3
___0.1b ___0.1 a + 0.2a +_ 0.3a, b +_ 0.1a, b
Week 7 2.9 3.3 3.3 3.3 2.9
+__0.1 a +_ 0.2a + 0.1 a +_ 0.3 a __ 0.1 a
Week 10 2.6 3.3 3.2 3.3 3.3
__. 0.1b __. 0.2 a +_ 0.1 a __ 0.1 a +_ 0.1 a
aIn each experiment, values in the same column without common superscript letters denote significant difference (p < 0.05}. Values are mean _ SEM of three animals.
185
EFFECTS OF POLYPHENOLS ON HIGH FAT DIETS
FIG. 3. a) Liver of a rat fed normal diet for 10 wk shows minimal deposition of fat in the hepatocytes. (Oil Red O stain, 75X). b) Liver of a rat fed high fat diet supplemented with tannic acid (100 mg/ rat/day) for 10wk shows moderate deposition of fat in the hepatocytes with sparing of the centrilobular hepatocytes. (Oil Red O stain, 80X). c) Liver of a rat fed high fat diet without polyphenolic compounds supplement for 10 wk shows severe and panlobular involvement of hepatocytes. (Oil Red O stain, 75X). Liver of rats fed high fat diet with quercetin (100 mg/rat/day) and morin (100 mg/rat/day) for 10 wk also showed similar appearance.
well as cirrhosis of the liver (42). Others have shown increases in tissue levels of A P due to diet induced hyperlipidemia (43). In contrast, the present s t u d y showed no significant differences in the activities of p l a s m a AP, A L A T and bilirubin concentration in all groups of rats. The activities ranged from 47.2 • 5.2 to 52.8 • 3.2 U/L and 40.3 • 4.8 to 45.1 • 4.1 U/L for ALAT and AP, respectively. The total bilirubin concentration ranged from 11.0 • 2.5 to 15.2 • 2.7 rag/100 mL. Thus, these results indicate t h a t feeding of a diet high in cholesterol and lard content or a cholesterol and lard diet supplemented with polyphenolic compounds does not cause bile retention or liver cirrhosis. H e p a t i c lipase activity in G1 and G5 were significantly lower t h a n in G2 at weeks 7 and 10 (Fig. 4). J a n s e n and H u l s m a n n (44) have suggested an involvement of hepatic lipase in the delivery of cholesterol to the liver. They have proposed t h a t cholesterol is taken up from peripheral cells b y a phospholipid rich H D L and subsequently delivered to the fiver via a m e c h a n i s m involving the depletion of phospholipid from H D L by hepatic lipase Along this line others have proposed t h a t the increase in hepatic fipase activity could contribute to increases in liver cholesterol (45). Our findings are in agreement with these reports as increased hepatic lipase activity in G2, G3 and G4 correlates well with increased fiver cholesterol concentration at week 7 (correlation coefficient, r = +0.632). Atherosclerotic plaques were n o t observed in the a o r t a of the five groups of rats. Microscopic examination of the H and E stained paraffin sections of the thoracic a o r t a showed smooth, intact intimal fining with no evidence of raised f a t t y plaques or proliferation of s m o o t h muscle cells, thereby demonstrating the absence of atherosclerosis in all the groups of experimental rats. In summary, the present s t u d y has shown t h a t both tannic acid and morin can cause favorable changes in p l a s m a lipid profiles of the t y p e t h a t have been correlated with CHD. Tannic acid reduced fat deposits in liver cells at nontoxic concentrations. Thus, tannic acid has a potential to avert complications arising from high fat intake. Further studies need to be carried out to unravel the m e c h a n i s m of the observed hypolipidemic effect of tannic acid.
WEEK
7
WEEK
10
A
TABLE 6 Liver Total Cholesterol and Triglyceride Concentration at Week 7a
Animal group G1 G2 G3 G4 G5
(ND) (HFD) (HFD + Q) (HFD + M) (HFD + T)
Total cholesterol (mg/g liver) 2.2 _ 0.1b 26.6 _ 0.9a 18.6 +_ 2.6a 22.3 _ 2.2a 15.9 _ 0.9b
Triglyceride (mg/g liver) 15.9 42.9 45.1 29.5 33.1
___ 1.5b _ 10.9a _ 3.6 a __. 2.0b _ 5.9a
aIn each experiment, values in the same column without common superscript letters denote significant difference (p < 0.05). Values are mean _ SEM of three animals.
,'(XXN 4"
/-,XXX] ~X)(XI ,1XYJq KX'X~
1 o,
Bo, V1o,
FIG. 4. Hepatic lipase activity of rats at weeks 7 and 10. Values are expressed as mU/mg protein. * denotes a statistically significant difference between G2 and other groups (p < 0.05).
LIPIDS, Vol. 27, no. 3 (1992)
186
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