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GREEN TEA LOWERSCHOLESTEROLLEVELTHROUGH
AN INCREASEIN FECAL LIPID EXCRETION
Teddy T.C. Yang and Marcel W.L. Koo
Department of Pharmacology, Faculty of Medicine
The University of Hong Kong, l/F, Li Shu Fan Building
5 Sassoon Road, Hong Kong
(Received in final form September 9, 1999)
Summary
Lung Chen Tea, a Chinese green tea, has been found to lower serum and liver
cholesterol. In this study, its dose response and mechanisms of action on
cholesterol lowering in diet-induced hypercholesterolemic
Sprague-Dawley rats
were investigated. The activities of three major lipid metabolizing enzymes, 3hydroxy-3-methylglutaryl-coenzyme
A (HMG-Co A) reductase, cholesterol 7ahydroxylase and fatty acid synthase (FAS), as well as fecal excretion of bile acids
and cholesterol were examined. Lung Chen Tea administration for eight weeks
significantly
lowered the serum cholesterol in the 2% and 4% groups. The
activities of the three enzymes were not affected by Lung Chen Tea, but the fecal
bile acids and cholesterol excretions were significantly increased. These results
demonstrated that Lung Chen Tea lowered plasma cholesterol by increasing fecal
bile acids and cholesterol excretion. Further investigation is required to evaluate
the exact mechanisms of action of Lung Chen Tea.
Key Worak Lung Chen tea, green tea, cholesterol, HMG-CoA reductase, cholesterol 7a-hydroxylase, fatty acid
synthase, bile acids
Hypercholesterolemia
is a major risk factor in coronary heart disease while maintaining the
blood cholesterol level within normal range can reduce the risk of having coronary heart disease.
Current drug treatments of hypercholesterolemia
involve the use of statins, fibrates and bile acids
binding resins. They lower cholesterol level by decreasing de nova synthesis of cholesterol
through inhibition of HMG-CoA reductase, affecting lipoproteins metabolism and reducing
absorption of cholesterol respectively.
Corresponding
The University
address: Dr. M.W.L. Koo, Department of Pharmacology, Faculty of Medicine,
of Hong Kong, l/F Li Shu Fan Building, 5 Sassoon Road, Hong Kong.
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Tea Lowers Cholesterolby Lipid Excretion
Vol. 66, No. 5, 2000
It has been shown that Japanese green tea and Lung Chen Tea, a Chinese green tea, lowered
plasma cholesterol (1,2). Tea catechins, the most abundant components in green tea, have been
implicated for its hypocholesterolemic
effect. In this study, the effect of Lung Chen Tea on
hepatic enzymes that regulate cholesterol synthesis, degradation,
and lipogenesis were
investigated. Diet-induced hypercholesterolemic
rats were treated with different concentrations
of Lung Chen Tea for eight weeks and their effects on HMG-CoA reductase, cholesterol 7ahydroxylase and FAS were examined.
Materials
and Methods
Chemicals
dithiothreitol
(DTT),
Acetyl
CoA,
ammonium
formate,
cholesterol
oxidase,
ethylenediaminetetraacetate
(EDTA), ethylene glycol-bis
(P-aminoethyl
ether) N’N’NWtetraacetic acid (EGTA), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin
(EGC), (-)-epigallocatechin
gallate (EGCG), glucose 6-phosphate,
glucose 6-phosphate
dehydrogenase, 7P-hydroxycholesterol,
hydroxysteriod dehydrogenase, leupeptin, malonyl CoA,
l3-nicotinamide adenine dinucleotide (NAD+), P-nicotinamide adenine dinucleotide phosphate
(NADP’),
P-nicotinamide
adenine
dinucleotide
phosphate,
reduced
form (NADPH),
phenylmethylsulfonyl
fluoride (PMSF), and sodium formate were purchased from Sigma
Chemical Co. (St Louis, MO). 7a-Hydroxycholesterol
was obtained from Steraloids, Inc.
(Pauling, NV). QAE-Sephadex A25 (chloride form) was ordered from Pharmacia (Upsala,
Sweden). [14C] HMG-CoA was from DuPont NEN (USA). Beckman ready gel liquid
scintillation cocktail was from Beckman (Beckman Instruments Inc., USA).
Animals and treatment
Male Sprague-Dawley
(SD) rats of 1 lo-130 g were used. They were housed in an airconditioned room at 20°C and 6570% relative humidity with a 12-h light-dark cycle. They were
given free access to Purina rat chow (Ralston Purina, USA) and tap water for 1 week before the
experiment. Afier 1 week of acclimatization, rats were randomly divided into groups and were
assigned to different dietary treatments. Rats of the normal control group were fed with standard
Purina rat chow and tap water ad libitum throughout the experimental period. Others were given
cholesterol-enriched
diet containing 1% cholesterol and 0.5% cholic acid (2) for 9 weeks after
acclimatization. Rats of the disease model group were given free access to tap water for the 9
weeks period while the other three groups were given tap water for 1 week followed by 1%, 2%
and 4% Lung Chen Tea for the following eight weeks. One percent, 2% and 4% tea solutions
were prepared by soaking 5 g, 10 g and 20 g of tea leaves in 500 ml of boiled water for 30
minutes, and the tea solution was filtered before supplied to the rats (3).
At the end of the experimental period, blood was collected from tail vein of the rats after 12-h of
fasting. The animal was then killed by decapitation and the liver was perfused with cold normal
saline before excision. It was blotted dry, and weighed before frozen in liquid nitrogen. The
tissue samples were stored at -70°C until used.
Vol. 66, No. 5,200O
Tea Lowers Cholesterol by Lipid Excre.tion
413
Blood lipids determination
Serum total cholesterol and triglyceride levels were assayed enzymatically
(Cholesterol
liquicolor and Triglycerides
GPO liquicolor, Human Gesellschafi
tir Biochemica
und
Diagnostica mbH, Germany) with the methods described by Roeschlau et al. (4) and Jacobs and
VanDmark (5) respectively. Serum HDL-cholesterol level was determined after precipitation of
apolipoprotein B containing lipoproteins by phosphotungstic acid and magnesium chloride (6).
Measurement
of tea catechins
Lung Chen Tea was imported from West Lake of Hangzhou, China. It was prepared from the
youngling buds of the tenderest leaves sprouting in the spring season. Catechins in tea solutions
were analyzed with high performance liquid chromatography (HPLC) as described by Matsuda
et al. (7) with minor modification. A HPLC solvent delivery system (ConstraMetric 3200,
Waters) and Beckman Ultrasphere ODS column (5 urn, 4.6 x 250 mm) were used to separate the
catechins in the tea solutions. Twenty microliters of tea sample were injected into the column
and methanol:acetic acid (25%:0.1%) was used as the mobile phase. The flow rate was adjusted
to 1 mYmin and the absorbance was set at 280 nm with a sensitivity of 0.01 absorbance units full
scale (aufs).
Determination
of liver weight and lipids content
Relative liver weight was represented as the ratio of liver to body weight. Liver cholesterol and
triglyceride were extracted with chloroformmethanol
(2:l) mixture (8). Cholesterol and
triglyceride contents in the liver were measured with diagnostic kits and expressed as mg
cholesterol or mg triglyceride/g liver.
Determination
of hepatic enzymes activities
Hepatic FAS activity was determined with the method described by Nepokroeff et al. (9). Liver
was homogenized in 1.5 volume of ice-cold phosphate-bicarbonate
buffer (70 mM NaHC03, 85
mM KzHP04, 9 mM KHzPO~, 1 mM EDTA and 1 mM DTT at pH 8.0). The homogenate was
centrifuged
at 11,500 rpm (Beckman
52-21 Centrimge,
USA) fot 10 minutes
and
ultracentrifuged at 40,000 rpm for 60 minutes at 4°C (Beckman L8-M ultracentrifuge, USA).
One hundred microliters of the supematant and 1 ml of 500 FM potassium phosphate buffer at
pH 7.0 containing 33 nmole acetyl CoA; 100 nmole malonyl CoA; 100 nmole NADPH and 1
umole EDTA were pre-incubated at 30°C for 10 minutes. One hundred micromoles NADPH in
10 pl of normal saline was added to initiate the reaction and the change in absorbance at 340 run
was recorded for 5 minutes. Correction for auto-oxidation and other enzymatic oxidation of
NADPH was made by measuring the absorbance in the reaction mixture with the omission of
malonyl CoA. The activity of FAS was expressed as nmol NADPH oxidized/mg protein/minute.
HMG-CoA reductase activity was measured by homogenizing
the liver in 9 volumes of
homogenizing buffer (50 mM Tris, 0.4 mM NaCl, 0.25 M sucrose, 1 mM EDTA, 10 mM DTT at
pH 7.4) and centrifuged to obtain the microsomes, which was then re-suspended in the buffer
(10). One hundred microliters of 100 mM sodium phosphate buffer containing 30 pM [14C]
HMG-CoA (0.01 uCi), 10 mM imidazole, 5 mM DTT, 10 mM EDTA, 12 mM glucose 6-
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Tea Lowers Cholesterol by Lipid Excretion
Vol. 66, No. 5, 2000
phosphate, 0.5 unit glucose 6-phosphate dehydrogenase, 3 mM NADP+, at pH 7.4, was mixed
with 25 ul of microsomal preparation in a 1.5 ml microcentrifuge tube. They were incubated at
30°C for 10 minutes and the reaction was terminated by the addition of 25 pl of 2 M HC1 and 10
mM mevalonolactone.
The mixture was further incubated at 37’C for 30 minutes to allow
lactonization
of mevalonate,
the enzymatic
product
of HMG-CoA
reductase,
to
mevalonolactone. Five hundred microliters of 20 mM ammonium formate was added and then
centrifuged at 10,000 rpm for 15 minutes at room temperature to pellet the denatured protein.
Five hundred microliters of the supernatant was applied to the QAE-Sephadex A25 (formate
form) column while the eluant was discarded. The QAE-Sephadex A25 (formate form) was
prepared by soaking the QAE-Sephadex A25 (chloride form) overnight in 1 M sodium formate
and then in 200 mM ammonium formate with several changes. A further 3 x 1 ml of 20 mM
ammonium formate was loaded into the column and the eluant was collected into counting vials.
Ten milliliters of scintillant (Beckman ready gel liquid scintillation cocktail, USA) was added to
each counting vial and the radioactivity was determined with a scintillation counter (Beckman
LS6500, USA). Blank samples were prepared in the same way except HCl was added before the
addition of the microsomal preparation. The activity of HMG-CoA reductase was expressed as
pm01 mevalonate/mg protein/minute.
The activity of cholesterol 7a-hydroxylase was measured with reverse-phase HPLC (11). Liver
was homogenized in 9 volumes of 100 mM potassium phosphate buffer at pH 7.2, consisting of
100 mM sucrose, 50 mM potassium chloride, 50 mM NaF, 5 mM EGTA, 3 mM DTT, 1 mM
EDTA, 1 mM PMSF and 100 pM leupeptin. Microsomal preparation was obtained by
centrifugation
and it was homogenized in the original isolation buffer before the protein
concentration was adjusted to 50 mg/ml. One milliliter of the reaction mixture contained 0.1 M
potassium phosphate buffer, 50 mM NaF, 5 mM DTT, 1 mM EDTA, 20% glycerol, 0.015%
Chaps, 1 mg microsomal preparation and 10 mM NADPH at pH 7.4. The mixture was preincubated at 37°C for 5 minutes and the reaction was initiated by the addition of 100 pl of 100
mM NADPH and incubated at 37°C for 20 minutes. The reaction was terminated by adding 30
pl of 20% sodium cholate and 1 pg 7P-hydroxycholesterol
was added as an internal recovery
standard. The conversion of 7a-hydroxycholesterol
to 7a-hydroxy-4-cholesten-3-one
was
induced by the addition of 44 pl of 0.1% cholesterol oxidase in potassium phosphate buffer at
pH 7.4, containing 1 mM DTT and 20% glycerol and incubated at 37°C for another 10 minutes.
At the end of the incubation, the reaction was terminated by the addition of 2 ml of 95% ethanol.
The products of the enzymatic reactions, 7a- and 7P-hydroxy-4-cholesten-3-one,
were extracted
three times with petroleum ether and re-suspended in 100 pl of acetonitrile:methano1(7:3).
They
were injected into a reverse-phase Beckman Ultrasphere-ODS column (5 pm, 4.6 x 250 mm) of
a HPLC (Waters 600s Controller & 616 Pump, USA). The mobile phase used was 70%
acetonitrile:30% methanol and the absorbance was monitored at 240 nm with a photodiode array
detector (Waters 996, USA). The products were eluted at a flow rate of 0.8 ml/min for 18
minutes, thereafter, the rate was increased to 2.0 ml/min for a further 14 minutes to elute all
cholesterol from the column. The activity of the cholesterol 7a-hydroxylase
was expressed as
pmol 7ct-hydroxycholesterolimg
protein/minute.
Determination
ofprotein
The protein concentration of the microsomal preparation for the assays of HMG-CoA reductase,
cholesterol 7a-hydroxylase and FAS activities were measured by the Lowry method (12).
Vol. 66. No. $2000
Determination
Tea Lowers Cholesterolby Lipid Exation
415
offecal cholesterol and bile acids
Feces of the last two days of the eight-week treatment period were collected. They were freezedried for 48 hours and stored at -7O’C until used. The feces were ground into fine powder and
extracted with a chlorofotmmethanol
(2:l) mixture in accordance to the method described by
Folch et al. (8). The amount of fecal bile acids was determined enzymatically with the method of
Turley and Dietschy (13). The reaction mixture that contained 750 pl Tris-HCl (0.133 M Tris,
0.666 mM EDTA, pH 9.5), 300 ~1 hydrazine sulphate (pH 9.5), 100 pl NAD+ (7 mM, pH 7.0),
and 30 pl samples was pre-incubated at 30°C before the addition of 30 ul hydroxysteriod
dehydrogenase preparation. It was incubated for another 60 minutes at 30°C in a shaking water
bath and the absorbance of the mixture was recorded at 340 mn against reagent blank. Fecal
cholesterol was determined using commercially available enzymatic kit (Cholesterol Liquicolor,
Human Gesellschaft ftir Biochemica und Diagnostica mbH, Germany).
Statistical analysis
Data were expressed as the meansfSEM and the statistical significance was first analyzed with
one-way ANOVA and the difference between groups were analzyed with post hoc test Tukey
HSD. Difference of the catechins contents between different concentrations of green tea were
analyzed with the Student’s unpaired two-tailed t-test.
Results
Body weight, food andfluid consumption during experimental period
Table I shows the food and fluid consumption during the experimental period. There was no
difference in food consumption among all rats in various treatment groups and fluid intake in the
1 % Lung Chen Tea group was similar to the tap water controls. Rats however consumed less
2% and 4% Lung Chen Tea @<O.OOl). No significant difference in the percentage increase in
body weight was observed among all groups of rats (Table I).
Quantities of catechins in the Lung Chen Tea solutions
Figure 1 shows the concentration of catechins in l%, 2% and 4% Lung Chen Tea solutions. Four
percent Lung Chen Tea contained the highest amount of all four major catechins @<O.OOl),
while the catechin contents in 1% Lung Chen Tea were the lowest (p<O.OOl).
Eflect on serum lipid levels
There was no significant change in serum triglyceride level in all rats (Table II). Cholesterolenriched diet significantly increased the total cholesterol level (p<O.OOl). Rats treated with 2%
and 4% Lung Chen Tea had lower serum total cholesterol than the 1% Lung Chen Tea group
(~~0.05 and ~~0.02 respectively) and the Disease Model (~~0.05, and ~~0.02 respectively).
Serum HDL-cholesterol level was increased significantly only in the 4% Lung Chen Tea treated
group (pcO.05).
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Tea Lowers Cholesterol by Lipid Excretion
Vol. 66, No. 5, 2000
TABLE I
Food, Fluid Consumptton
Experimental Period
Treatment Group
n
and Increase
m Body
9
Cholesterol-enriched dret
10
Lung Chen Tea 1%
6
Lung Chen Tea 2%
9
Lung Chen Tea 4%
7
Disease Model
during
the
lo-week
Increase in Body
Fluid Consumption
Food Consumption
(g/rat/day)
Standard diet
Normal Control
Weight
Weight (%)
(g/rat/day)
27.9kO.7
35.9+0.7
233.7f10.4
30.4kO.7
32.751.6
30.3-fo.7
30.6k1.2
36.9kO.7
38.0&1.4
30.lf0.7* +
31.7*0.9* -
241.9k6.3
234.3k15.7
229.5+8.7
230.559.6
Values are expressed as means+SEM; * p<O.OOl when compared with the Disease
Model; + p<O.OOl when compared with the 1% Lung Chen Tea group; n = no. of rats
in each group.
*
0.5
0.2
0 I
I%
2%
Lung
Chen
F1g.
4%
Tea
I
Quantities of the four major tea catechins namely (-)-epicatechin (EC), (-)-epicatechin
gallate (ECG), (-)-epigallocatechin
(EGC) and (-)-epigallocatechin
gallate (EGCG),
were determined in I%, 2% and 4% Lung Chen Tea solutions. Values are expressed
as means+SEM of 6 determinants; * p<O.OOl when compared with the 1% and 2%
Lung Chen Tea solutions; + p<O.OOl when compared with the 1% Lung Chen Tea
solution.
Effect on liver weight and liver lipid content
Rats
fed with
cholesterol-enriched
diet
had
a significant
increase
in relative
liver weight
Vol. 66, No. 5, 2000
Tea Lowers Cholesterol by Lipid Excretion
417
(p<O.OOl, Table III). Two percent and 4% Lung Chen Tea effectively reduced the elevation
when compared with the 1% Lung Chen Tea group and the Disease Model groups (Table III).
The liver cholesterol and triglyceride contents were increased by cholesterol feeding @<O.OOl,
Table III). Two percent and 4% Lung Chen Tea suppressed the accumulation of liver cholesterol
but the liver triglyceride level was not affected.
TABLE II
Serum Total Cholesterol,
with Cholesterol-enriched
HDL-Cholesterol and Triglyceride Levels after Treatment
Diet and Lung Chen Tea for Eight Weeks
Treatment Group
n
Serum Total
Cholesterol
Serum HDL
(mg/dll
Standard diet
Normal Control
9
44.6k4.9
33.97U.9
60.5227.9
117.3+7.7**
121.5k8.7
97.8+5.2++
90.3f2.9” ++
21.74*2.7*
26.05t5.9
26.63f1.6
34.58+2.4+#
63.48f5.0
57.69f4.0
60.80f6.4
53.33k7.2
Cholesterol-enriched diet
Disease Model
IO
Lung Chen Tea 1%
6
Lung Chen Tea 2%
9
Lung Chen Tea 4%
7
Serum Triglyceride
(mg/dU
Values are expressed as meansfSEM; * pcO.05, ** p<O.OOl when compared with the
Normal Control; + p<O.OS, ++ ~~0.02 when compared with the Disease Model;
‘p<O.O5, “p<O.O2 when compared with the 1% Lung Chen Tea group, # pcO.05 when
compared with the 2% Lung Chen Tea group; n = no. of rats in each group.
EApeCton hepatic lipid metabolizing
enzymes
Figure 2 shows the activities of HMG-CoA reductase after 8 weeks treatment with Lung Chen
Tea. Cholesterol-enriched
diet significantly lowered the activity of HMG-CoA reductase when
compared with the Normal Control @<O.OOl). There was no significantly difference in HMGCoA reductase activities in rats treated with Lung Chen Tea.
A decreasing trend in FAS activity was observed in hypercholesterolemic
rats but there was no
statistical difference in enzyme activity when compared with the Normal Control (Figure 3).
The activity of cholesterol 7a-hydroxylase
is shown in Figure 4. Cholesterol 7ct-hydroxylase
was not affected by the increase in cholesterol intake. However, there was a increasing trend of
cholesterol 7a-hydroxylase activity in rats consuming 4% Lung Chen Tea (Figure 4).
E#ect on fecal lipid excretion
Fecal bile acids were significantly elevated in hypercholesterolemic
rats (p<O.OOl, Figure 5).
Four percent Lung Chen Tea significantly increased bile acids excretion when compared with
the Disease Model (p~O.05, Figure 5).
Cholesterol
feeding significantly
increased fecal cholesterol excretion when compared with rats
Tea Lowers Cholesterolby Lipid Excretion
418
Vol. 66, No. 5, 2000
receiving standard diet (p<O.OO1, Figure 6). Four percent Lung Chen Tea increased excretion of
cholesterol when compared with the Disease Model (~~0.05, Figure 6).
FABLE 111
Relative Liver Weight, Liver Cholesterol
with Lung Chen Tea for Eight Weeks
Treatment Group
n
and Trlglycerlde
Content after Treatment
Liver Cholesterol
(mg/g liver)
Relative Liver Weight
(g/lOOg body weight)
Liver Triglyceride
(mgig liver)
Standard diet
Normal Control
9
3.lkO.l
3.3kO.5
8.SkO.8
6.0f0.2*
6.3+_0.I
5.6kO.l’ +’
5.4kO.2’ ++
25.812.1*
25.0+1.7
18.5+1.7+‘+
17.3fl.2” ++
20.2?0.7*
20.5+3.3
21.1f0.8
18.8k1.3
Cholesterol-enriched dret
Disease Model
Lung Chen Tea 1%
Lung Chen Tea 2%
Lung Chen Tea 4%
10
6
9
7
Values are expressed as means+SEM; * p<O.OOl when compared with the Normal
Control; * pco.05, ++ pcO.02 when compared with the Disease Model; ’ pcO.05,
‘+p<0.02 when compared with the 1% Lung Chen Tea group; n = no. of rats in each
group.
7
FKIII
Fed with standard
diet
K?ZZB Fed with cholesterol-enriched
diet
L
Normal
Control
Disease
I y.
2%
Lung Chen
Model
4%
Tea
Fig. 2
Effect of Lung Chen Tea on hepatic HMG-CoA reductase activity (nmol/min/mg
protein). Values are expressed as means+SEM; T p~O.001 when compared with the
Normal Control.
Tea Lowers Choleskrol by Lipid Excretion
Vol. 66, No. 5,200O
419
0
Fed with
standard
EZZ
Fed with
cholesterol-enriched
diet
diet
”
Normal
Control
Disease
Model
1%
Lung
4%
2%
Chen
Tea
Fig. 3
Effect of Lung Chen Tea on hepatic fatty acid synthase
NADPWmin/mg
protein). Values are expressed as meansGEM.
significant difference between all groups.
activity (nmol
There was no
x
.‘1:
>
.:
sm
80
2,
rn,
A.j
70
;
i;
r0
5‘
z
4J
z
_c
0
I
Fed
@ZZZ?A Fed
with
with
standard
diet
cholesterol-enriched
diet
60
50
.r
E
0
E
2
40
30
20
2
2
10
t
z
0
Normal
Control
Disease
M ode1
1%
4%
2%
Lung
Chen
Tea
Fig. 4
Effect of Lung Chen Tea on hepatic cholesterol
protein). Values are expressed as means*SEM.
7a-hydroxylase
activity (pmol/min/mg
420
Tea Lowers Cholesterol by Lipid Excretion
CIIIIEX
EZZZ
Fed
Fed
with
with
Normal
Control
Vol. 66, No. 5, 2000
standard
diet
cholesterol-enriched
diet
1y.
Disease
M ode1
4%
2%
Lung
Chen
Tea
Effect of Lung Chen Tea on fecal bile acids excretion. Feces were collected on the last
two days of experimental period. Values are expressed as means?SEM; ’ p<O.OOl
when compared with the Normal Control; * ~~0.05 when compared with the Disease
Model.
I
BZZI
Fed
Fed
with
with
standard
diet
cholesterol-enriched
diet
*
T
+
z
P)
=;
b)
0
_sS
0
m
h:
Lr,
12
IO
8
6
4
2
Normal
Control
Disease
model
I%
Lung
2%
Chen
4%
Tea
Fig. 6
Effect of Lung Chen Tea on fecal cholesterol excretion. Feces were collected on the
last two days of experimental period. Values are expressed as means&EM; ’ p<O.OOl
when compared with the Normal Control; * pcO.05 when compared with the Disease
Model.
Vol. 66, No. 5, 2000
Tea Lowers Cholesterolby Lipid Excretion
421
Discussion
Green tea and tea catechins have been shown to be hypolipidemic (1,2), however, the effect of
green tea on the major lipid metabolizing enzymes has not been investigated. In this study,
hypercholesterolemic
rats were treated with Lung Chen Tea and its effects on HMG-CoA
reductase, cholesterol 7ol-hydroxylase, fatty acid synthase (FAS), bile acids and cholesterol
excretion were examined. Due to the bitter taste of Lung Chen Tea, rats drank significantly less
2% and 4% Lung Chen Tea. However, it did not affect the apparent growth of the rats, since the
percentage increase in body weight was similar over the experimental period (Table I). Analysis
of the tea catechins content showed that the amount of catechins increased with the concentration
of Lung Chen Tea (Figure 1). Epigallocatechin gallate (EGCG) which accounted for almost 70%
(w/w) of the total catechins (Figure 1) was most abundant in Lung Chen Tea. Even though the
consumption of 2% and 4% Lung Chen Tea was lower than that of I%, the intake of catechins
was still higher than that of 1% Lung Chen Tea.
Lung Chen Tea dose dependently lowered serum total cholesterol level (Table II). However, 1%
Lung Chen Tea that contained lower content of catechins did not prevent the elevation (Table II).
Lung Chen Tea (4%) also reduced the HDL lowering effect of excessive cholesterol intake
@<0.05, Table II). Since HDL facilitates translocation of cholesterol from peripheral tissue; like
arterial walls to liver for catabolism, thus a relative increase in HDL may slow down the
atherogenic process (14). Cholesterol-enriched
diet has no effect on serum triglyceride level and
rat consuming Lung Chen Tea has similar serum triglyceride level when compared with control
animal. As plasma triglyceride level is determined by hcpatic triglyceride synthesis, release from
the liver, and the activity of lipoprotein lipase, it thus showed that Lung Chen Tea has little
effect on these processes (15).
The relative liver weight in cholesterol fed rats was significantly increased due to excessive
accumulation of cholesterol and triglyceride (Table III). As described by Fungwe et al. (16),
cholesterol has an stimulatory effect on hepatic fatty acid biosynthesis and the incorporation of
newly synthesized fatty acid into hepatic triglyceride. Thus, liver triglyceride content was
significantly
increased in hypercholesterolemic
rats. Lung Chen Tea lowered the liver
cholesterol content and thus reduced the relative liver weight (Table III), however, it has no
effect on liver triglyceride level. It has been reported that tea catechins inhibited intestinal
absorption of cholesterol (1) and this may be one of the mechanisms by which Lung Chen Tea
lowered plasma cholesterol levels. A reduction in intestinal cholesterol absorption prevents the
accumulation of cholesterol in the liver. Since the expression of LDL receptor is controlled by
feedback inhibition of intracellular cholesterol, reduction in hepatic cholesterol accumulation in
turn stimulates the production of more high affinity LDL receptors (16). This results in an
increase in clearance of cholesterol from the circulation by LDL receptor and thus lowers blood
cholesterol (17).
In the present study, the three major lipid metabolizing enzymes in the liver were investigated.
HMG-CoA reductase activities in cholesterol-enriched
diet groups were lower than that of the
standard diet group (Figure 2). HMG-CoA reductase is the rate-determining
enzyme for
cholesterol synthesis and its activity is regulated by the feedback inhibition of cholesterol and
sterols (18). Increase in cholesterol intake resulted in an accumulation of cholesterol and
triglyceride in the liver (Table III) and thus suppressed the transcription of HMG-CoA reductase
gene, In this study no effect of Lung Chen Tea on HMG-CoA reductase activity was observed
422
Tea Lowers Cholesterolby Lipid Excretion
Vol. 66, No. 5, 2000
(Figure 2), therefore the hypocholesterolemic effect of Lung Chen Tea may not be explained by
the suppression of cholesterol synthesis. This result supported the findings of Chisaka et al. (19)
that (-)-epigallocatechin
gallate (EGCG) isolated from Japanese Green Tea, did not affect the in
vitro incorporation of j4C-acetate into cholesterol in liver slices obtained from normal or
hypercholesterolemic rats.
Hepatic FAS is the key enzyme in fatty acid synthesis and its level is regulated by hormones and
nutrition intake of lipids and carbohydrates (20). Fatty acids synthesized by FAS will be
incorporated with glycerol to form triglyceride. From the present experiment, an increase in
cholesterol intake has led to a slight but insignificant decrease in FAS activity (Figure 3). Rats
consuming Lung Chen Tea has no inhibitory effect on the activity FAS. These are in line with
the absence of effect of Lung Chen Tea on serum and liver triglyceride levels (Table II).
Conversion of cholesterol to bile acids is the major pathway of cholesterol elimination and it
accounts for about 50% of daily cholesterol excretion (21). Cholesterol 7a_hydroxylase, the rate
determining enzyme in the conversion of cholesterol to bile acids, is mainly regulated by the
feedback inhibition of bile acids re-absorbed from the intestine (22). It was found that
cholesterol-enriched
diet did not affect the activity of cholesterol ‘la-hydroxylase.
Rats
consuming 4% Lung Chen Tea showed a rise in cholesterol 7a-hydroxylase
activity but not
reached significant level (Figure 4). Fecal excretion of bile acids and cholesterol were found to
be increased in cholesterol-fed rats, while 4% Lung Chen Tea further enhanced their excretion
(Figures 5 and 6). The increase in the excretion of bile acids and cholesterol seems to activate
cholesterol 7a-hydroxylase.
The increase in cholesterol 7a-hydroxylase
can enhance the
conversion of liver cholesterol to bile acids for excretion. This leads to a decrease in hepatic
cholesterol content which in turn stimulated LDL receptor expression and lowered blood
cholesterol level (23). Two percent Lung Chen Tea reduced serum and liver cholesterol levels
without significantly increased the fecal cholesterol and bile acids excretion, thus implying that
other mechanisms may contribute to the hypocholesterolemic effect of Lung Chen Tea.
The present study demonstrated that Lung Chen Tea has hypocholesterolemic
effect. It lowered
serum total cholesterol and elevated HDL-cholesterol level. It has no inhibitory effect on the de
novo synthesis of cholesterol and lipogenesis. One of its mechanisms of hypocholesterolemic
action may be due to the promotion of cholesterol and bile acids excretion.
Acknowledgments
The authors would like to thank Mr. H.C. Leung for his excellent technical assistance. The
present study was partly supported by the Hsin Chong K.N. Godfrey Yeh Education Fund of the
University of Hong Kong.
Vol. 66, No. $2000
Tea LowersCholesterolby Lipid Excretion
423
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