Beruflich Dokumente
Kultur Dokumente
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).
OH
Ho~O~
~OH
HO~o
OH
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
Contents
Normal 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
Fatty acid
Normal 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
--
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
2040-
ui.oE
R ~ ~oe 8o-
/r
u
, o
..a O
~ ~
240J
~ =
~
u o
_~ ~, 80
~ ~
~;;~JG1
WEEK 10
WEEK 7
~G2
~G3
~-1G4
~G5
TABLE 3
Average Body Weight Increases (measured as percentage) a
(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
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
TABLE 4
TABLE 5
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
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
185
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
WEEK
10
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
,'(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).
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.
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]