NUTRITION
ELSEVIER
Nutntion 23 (2007) 687-695
www elsevier coni/locate/nut
Basic nutritional investigation
Influence of green tea on enzymes of carbohydrate metabolism,
antioxidant defense, and plasma membrane in rat tissues
Sara Anees Khan, M.Sc, Shubha Priyaravada, M.Sc, Natarajan A. Arivarasu, M.Sc,
Sheeba Khan, M.Sc, and Ahad Noor Khan Yusufi, Ph.D.*
Department oj BwLhemistr\. hacult\ of Life Sciences Ahgmh Muslim Unwersity Aligarh Ultni Piadesh, India
Manuscript received January 11. 2007, accepted June 12, 2007
Abstract
Objective: Green tea, consumed worldwide since ancient times, is considered beneficial to human
health. We hypothesized that green tea would enhance antioxidant defenses and specific metabolic
activities of rat intestine, liver, and kidney to improve their fimctions
Methods: The effect of green tea given to rats in the diet or drinking water for 25 d was determined
on blood chemistry and on activities of enzymes of carbohydrate metabolism, brush border
membrane, and antioxidant defense
Results: Senim glucose, cholesterol, phosphate, and body weight decreased, whereas the activities
of lactate and malate dehydrogenases and glucose-6- and fructose 1,6-bis-phosphatases increased m
the intestine and kidney but slightly changed m the liver Activity of glucosc-6-phosphatc dehydrogenase profoundly increased m the renal cortex but decreased in other tissues Lipid peroxidation
increased in the intestine and renal medulla and decreased in the renal cortex and liver, catalase
increased in all tissues but the medulla. Superoxide dismutase activity decreased in the intestine but
increased in renal tissues Activities of brush border membrane enzymes in general increased in the
intestine and kidney.
Conclusion: Green tea consumption resulted m enhanced enzyme activities of carbohydrate
metabolism and antioxidant defenses, which may lead to improved health. © 2007 Elsevier Inc
All rights reserved.
Kewvords
Green lea (Camellia sinensis): Carbohydrate metabolism. Antioxidant enzymes. Intestine, Liver, Kidney
Introduction
Tea in the form of green tea (GT) or black tea is one of
the most widely consumed beverages in the world today
second only to water [1]. Since ancient times GT consumption has been known to maintain and improve health. PolyThis work was supported by a Junior Research Fellowship from the
Council of Scientific and Industrial Research, New Delhi, India to Sara
Anees Khan, M Sc , and a Junior Research Fellowship/Senior Research
Fellowship to Shubha Pnyamvada, M.Sc, from the Indian Council of
Medical Research, New Delhi, India. Financial support to the department
from the Umversity Grant Commission (UGC-DRF), the Department of
Science and Technology (DST-FIST), and research grant 58/2I/2001-BMS
from the Indian Council of Medical Research to Ahad Noor Khan Yusufi,
Ph.D , IS gratefully acknowledged
* Corresponding author Tel +91-571 270-0741, fax -1-91-571-2706002
E mail address yusufi@lycos com (A N K Yusufi)
0899-9007/07/$ - see front matter ( ' 2007 Elsevier Inc. All rights reserved
dor.lO 1016/j nut 2007 06 007
phenols are plant metabolites occurring widely in plant food
and exhibit outstanding antioxidant and free radical scavenging properties [2]. GT is an excellent source of polyphenols such as catechins [3], orgallotannins, flavonols, flavandiols, and phenolic acids [4]. In pailicular GT catechins and
their derivatives are known to contribute beneficial health
effects ascribed to tea by their antioxidant [5], antimutagenic, and anticarcinogenic properties [6]. GT consumption
has been linked to lowering of various forms of cancers
[7,8]. GT constituents also have been shown to have cardioprotective, neuroprotective, antidiabetic, and antimicrobial properties [9,10]. In addition, GT has been found to be
useful in the treatment of arthritis, high cholesterol levels,
infection, and impaired immune function [11]. GT consumption also has resulted in improved kidney functions in
animal models of renal failure [12,13]. We hypothesized
that GT enhances specific metabolic activities and anti-
688
S. A. Khan et al. / Nutrition 23 (2001) 687-695
oxidant defenses in various rat tissues due to its intrinsic
free radical scavenging properties to improve their functioning. To address this hypothesis the effect of GT
consumption was investigated on the activities of certain
enzymes of carbohydrate metabolism, brush border membranes (BBMs), lysosomes, and antioxidant defense system
in the rat model.
Green tea consumption resulted in decreases in serum
gluco.se, cholesterol, and inorganic phosphate (Pi) and an
increase in semm phospholipids. The activities of enzymes
of glucose oxidation and its production selectively but variably increased in intestine and kidney tissues. Lipid peroxidation (LPO), an indicator of cell injury, and enzyme activities of the antioxidant defense system, e.g., superoxide
dismutase (SOD) and catalase, and BBM were also altered
but differentially in different tissues by GT consumption.
The results indicate an overall improvement in metabolic
activities in various tissues most likely by GT-polyphenolmediated reduction of oxidative damage under normal physiologic conditions in the rat model.
Materials and methods
Materials
Green tea (Kangra, Himanchal Pradesh, India and Lipton-Unilever, Englewood Cliffs, NJ, USA) was purchased
from commercial sources (Jain Pan House, New Delhi,
India). All other chemicals used were of analytical grade
and were purchased from Sigma Chemical Co. (St. Louis,
MO, USA) or Sisco Research Laboratory (Mumbai, India).
Experimental design
The animal experiments were conducted according to the
guidelines of the Committee for Purpose of Control and
Supervision of Experiments on Animals, Ministry of Environment and Forests, Government of India. Adult male
Wistar rats, weighing 150-180 g, fed with a standard pellet
diet (Aashirwad Industries, Chandigarh, India) and water ad
libitum, were conditioned for 1 wk before the start of the
experiment. All animals were kept under conditions that
prevented them from experiencing unnecessary pain and
discomfort according to guidelines approved by the ethical
committee. A known amount of diet (250 g) and waler/GT
extract (2 X 125 mL = 250 mL/d) was provided in two
servings to each group of rats and was found to be sufficient.
Three groups of rats (six to eight rats/group/cage) were
studied. The rats of group 1 (control) received the standard
diet and water for 25 d. The rats of group 2 (GT extract)
were given the standard diet and GT extract (3% w/v) in
drinking water, whereas the rats of group 3 (GT diet) received GT in the diet (3% w/w) and 250 mL of water as
mentioned above also for 25 d. After 25 d of tea administration, the rats were sacrificed under light ether anesthesia.
Blood samples were collected from non-fasted rats and the
liver, kidney, and entire intestine (starting from the ligament
of Trietz to the end of ileum) were extracted. The intestines
were washed by flushing them with ice-cold buffered saline
(1 niM Tris-HCl, 9 g/L of NaCI, pH 7.4). The liver and
kidney were placed in Tris buffered saline. All preparations
and analyses of various parameters were carried out simultaneously under similar experimental conditions to avoid
any day-to-day variations.
Preparation of GT extract and GT diet
Green tea (30 g ) was added to 500 mL of boiling water
and was steeped for 15-20 min. Infusion was cooled to
room temperature and then filtered. The tea leaves were
extracted a second time with 500 mL of boiling water and
filtered, and the two filtrates were combined to obtain 3%
GT extract (3 g of tea leaves/100 mL of H^O). The resulting
clear solution is similar to tea brews consumed by humans.
The GT diet was prepared by adding GT (3 g/100 g of diet)
in powdered rat diet, mixed well with small amounts of
water, and then made in the form of cookies, after drying in
an oven (50-60°C) for 30 min. GT polyphenols were detected by thin layer chromatography as reported by Malik
et al. [14]. According to the manufacturer's information, the
antioxidant content was 95 mg/g of GT.
Preparation of homogenates
The washed intestines were slit in the middle and the
entire mucosa was gently scraped with a glass slide and
weighed. A 6.5% homogenate of this mucosa was prepared
in 50 mM mannitol, pH 7.0, in a glass Teflon homogenizer
(Remi Motors, Mumbai, India) with five complete strokes.
The homogenate was then subjected to a high-speed UltraTurrex homogenizer (Type T-25, Janke & Kunkel GMBH
& Co. KG., Staufen, Germany) for three pulses of 30 s each
with an interval of 30 s between each stroke.
The kidneys were decapsulated and kept in ice-cold 154
mM NaCl and 5 mM Tris-HEPES buffer, pH 7,5. The
cortical and medullary regions were carefully separated and
homogenized (as mentioned above) in 50 mM mannitol
buffer to obtain 10% (w/v) homogenate. The 10% liver
homogenate was similarly prepared in 10 mM Tris-HCl
buffer, pH 7.5.
One part of the homogenates (of intestine, kidney, and
liver) was centrifuged at 2000g for 10 min at 4°C and the
supernatant was saved for assaying the enzymes of carbohydrate metabolism; the second part was centrifuged at
3000g for 15 min at 4°C and the supernatant was used for
assay of free radical scavenging enzymes; and the third
part was used for estimation of total thiol (-SH) and
LPO.
689
S A Khan el al /Nutrition 23 (2007) 687-695
Preparation of BBM
Intestinal BBM was prepared at 4°C using dvfferential
precipitation by CaC^ [15]. Mucosa scraped from four to
five washed intestines was used for each BBM preparation.
CaClj was added to the homogenate to a final concentration
of 10 mM and the mixture was stirred for 20 min on ice. The
final membrane preparations were suspended in 50 mM
sodium maleate buffer, pH 6.8.
Kidney BBM was prepared from whole cortex homogenate using the MgClj precipitation method as described by
Yusufi and Dousa [16]. The final membrane preparations
were suspended in 300 mM mannitol, pH 7.4, and the
BBMs thus prepared were saved and stored at -20°C until
further analysis for BBM enzymes. Each sample of BBM
was prepared by pooling tissues from two to three rats.
Serum chemistries
Serum samples were deproteinated with 3% trichloroacetic acid at a ratio of 1.3, left for 10 min, and then centrifuged
at 2000g for 10 min. The protein-free supernatant was used
to determine Pi. The precipitate was used to quantitate total
phospholipids. Serum urea nitrogen and cholesterol levels
were determined directly in serum samples. Glucose was
estimated by an o-toluidine method using a kit from Span
Diagnostics (Mumbai, India). These parameters were determined by standard procedures as mentioned in a previous
study [17].
Assay of carbohydrate metabolism enzymes
The activities of the enzymes involving oxidation of reduced nicotinamide adenine dinucleotide or reduction of nicotinamide adenine dinucleotide phosphate were determined
spectrophotometrically on a Cintra 5 (GBC Scientific Equipment, Pty, Victoria, Australia) fixed for 340 nm using 3 mL of
assay in a 1-cm cuvette at room temperature (28-30°C). The
enzyme assays of lactate dehydrogenase (LDH; E.C. 1.1.1.27),
malate dehydrogenase {E.C. 1.1.1.37), malic enzyme (ME;
E.C. 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PDH;
E.G. 1.1.1.49), glucose-6-phosphatase (G6Pase; EC.3.1.3.3),
and fmctose-l, 6-bisphosphatase (FBPase; E.C.3.1.3.11) activities
were studied as described by Khundmiri et al. [18]. Hexokinase
was estimated by the method of Crane and Sols [19] and the
remaining glucose was measured by method of Nelson [20].
Assay of BBM marker enzymes and lysosomal marker
enzymes
The activities of alkaline phosphatase (ALP), leucine
amino peptidase (LAP), y-glutamyl transferase (GGTase),
sucrase, and acid phosphatase (ACPase) were determined as
described by Farooq et al. [21].
Assay of enzymes involved in free radical scavenging
Superoxide dismutase (B.C. 1.15.1.1) was assayed by the
method of Marklund and Marklund [22]. Catalase
(B.C. 1.11.1. 6) activity was assayed by the method of Giri
et al. [23].
LPO and total -SH group estimation
Total SH groups were determined by the method of
Sedlak and Lindsay [24] and LPO by the method of Ohkawa
et al. [25].
Definition of unit
One unit of enzyme activity is the amount of enzyme
required for the formation of 1 fi-mol of product per hour
under specified experimental conditions. Specific activity is
enzyme units per milligram of protein.
Statistical analysis
All results are expressed as mean ± SEM for at least
three to four separate preparations. The data were analyzed
for statistical significance by Student's t test for group
comparisons or by analysis of variance. P < 0.05 was
considered statistically significant.
Results
Considering reported numerous health benefits of GT,
the effect of GT given in the diet (GT diet) or as tea extract
(GT extract) in drinking water was determined on certain
enzymes involved in various pathways of carbohydrate metabolism, biomarkers of renal and intestinal BBMs, and
various parameters of antioxidant defense mechanism were
determined in different rat tissues used as a model for
humans.
Effect of GT on body weight and serum parameters
In general, the rats remained active and alert throughout
the study. The daily food and fluid intakes were similar in
Table 1
Effect of GT consumption via drinking fluid or diet on body weight
(grams) of rats*
Groups
Before treatment
After treatment
% Change
Control
GT extract
GT diet
1667 + 5 3
168 8 ± 6 3
164 3 ± 7 4
162 5 ± 8 53
150 ± 7 9
145 ± 10*
-2 5
-11 I
-11 7
GT, green tea
* Results are expressed as mean ± SEM
* Significantly different at P < 0 05 from control
S A Khan el al I Numtion 23 (2007) 687-695
690
Table 2
Effects of GT consumption via drinking fluid or diet on serum parameters*
Groups
BUN (mg/dU
Glucose (mg/dL)
Cholesterol (mg/dL)
Inorganic PO^ (/xmol/mL)
Phospholipid (/j,gs/mL)
Control
GT extract
GTdiet
25 43 ± 0 36
20 99 ± 0 61* (-17%)
2 1 0 9 + 1 17* (-17%)
141 09 ± 4 64
116 11 ± 5 62* (-18%)
107 78 ± 6 89* (-24%)
137 60 + 30 20
103 97 ± 2 03 (-24%)
98 35 ± 2 80 (-29%)
164 ± 0 04
1 29 + 0 04* (-21%)
1 07 + 0 14* (-35%)
109 4 0 = I 96
162 0 0 = 13 8" (+48%)
14190 = 9 30' (+30%)
BUN, serum urea nitrogen, GT, green tea
* Results are mean + SEM of six different samples Values in parentheses represent percent change from control
* Significantly different at P < 0 05 from control
control and GT rats (data not shown). The amount of GT
ingested m the diet or by drinking was approximately the
same. As shown in Table 1, GT consumption resulted in
slight loss of body weight (—11%) compared with control
rats. Serum glucose, cholesterol, and Pi significantly declined, whereas phospholipids significantly increased by
both forms of GT consumption. Serum urea nitrogen was
slightly lowered (Table 2). The changes caused by the GT
diet or GT extract on various parameters varied in magnitude, but the changes were always in the same direction.
renal cortical homogenates. The activities of ALP, GGTase,
and ACPase also slightly increased, whereas LAP decreased
in the renal medulla. In liver homogenates ALP and
GGTase decreased, whereas LAP and ACPase were not
affected. Similar to homogenates, the activity of ALP decreased, whereas GGTase, LAP, and sucrase activities significantly increased in intestinal BBM by GT consumption
(Table 4). The activities of ALP and GGTase increased,
whereas LAP activity significantly lowered in BBM vesicles isolated from the renal cortex (Table 4)
Effect of GT on biomarkers of BBM and lysosomes in
different tissues
Effect of GT on enzymes of carbohydrate metabolism
The influence of GT consumption was determined on
BBM and lysosomal enzymes in the liver, intestine, and
renal cortical and medullary homogenates and in BBM
vesicles isolated from the renal cortex and intestinal mucosa. The results summarized in Table 3 show that the
activities of ALP, GGTase, LAP, sucrase, and ACPase
significantly increased in intestinal homogenates m GT
compared with control rats. The activities of ALP and
ACPase increased, whereas GGTase and LAP decreased in
The effect of GT consumption was determined on the
activities of carbohydrate metabolism enzymes in the intestine, liver, and kidney. The activity of hexokinase slightly
increased in the liver (-^25%) and renal medulla (+20%)
when GT was given in the diet and in the intestine (+18%)
when given in the drinking water but did not change in the
renal cortex with either method. However, the activity of
LDH markedly increased m the intestine and kidney tissues
but only slightly increased m the liver. The effect on LDH
activity was more expressive in GT extract than in GT diet
Table 3
Effects of GT consumption via dnnking fluid or diet on brush border membrane enzymes in different tissue homogenates*
Tissue
Intestine
Control
GT extract
GT diet
Liver
Control
GT extract
GT diet
Cortex
Control
GT extract
GT diet
Medulla
Control
GT extract
GT diet
ALP
(/xmol/mg protein/h)
GGTase
(/i.mol/mg protein/h)
LAP
(;j.mol/mg protein/h)
ACPase
(^imol/mg protein/h)
Sucrase
(/xmol/mg protein/h)
7 26 ± 0 15
3 50 ± 0 08
8 45 ± 0
3 93±0
22 53 ± 0 88
24 46 ± 1 45 (+9%)
25 99 ± 1 36 (+15%)
1 30 ± 0 04
161 ± 0 0 2 t ( + 2 4 % )
1 38 ± 0 06 (+6%)
4 46 ± 0 14
6 63 ± 0 26t (+49%)
4 9 4 ± 0 15* (+11%)
9 07 ± 0 09* (+25%)
4 97 ± 0 15* (+42%)
124 ± 0 0 3
103 ± 0 0 6 * ( - 1 7 % )
105 ± 0 06* (-15%)
2 25 ± 0 0 8
1 12 ± 0 13* (-50%)
1 48 ± 0 08* (-34%)
0 59 ± 0 02
063 ± 0 02 (+7%)
0 59 ± 0 03
7 33 ± O 24
7 07 ± 0 35 (-4%)
6 89 ± 0 50 ( - 6 0%)
2 93 ± 0 14
3 5 6 ± 0 11* (+22%)
3 35 ± 0 08* (+14%)
40 52 ± 0 56
37 42 ± 0 65* (-8%)
33 54 ± 129* (-17%)
4 75 ± 0 32
3 18 ± 0 27* (-33%)
2 91 ± 0 19" (-39%)
604 ± 0 14
6 40 ± 0 22 (+6%)
7 93 ± 0 33" (+31%)
1 71 ± 0 1
2 07 ± 0 18* (+21%)
171 ± 0 08
10 59 ± 0 41
1264 ± 0 7 2 * ( + 19%)
10 65 ± 0 54 (+0 6%)
1304±081
7 19 ± 0 22* (-45%)
7 44 ± 0 22* (-43%)
6 29 ± 0 26
7 53 ± 0 2'(+20%)
8 06 ± 0 39* (+28%)
14T(+16%)
15T(+12%)
ACPase, acid phosphatase, ALP, alkaline phosphatase, GGTase, y-glutamyl transferase, GT, green tea, LAP, leucine amino peptidase
* Results are mean ± SEM of three to four different preparations Values in parentheses represent percent change from control
* Significantly different at P < 0 05 from control
5 A Khan el al /Nutrition 23 (2007) 687-695
I
compared with control rats in the intestine (+672%) and
renal cortex (+111%) The activity of maldte dehydrogenase, an en/yme of the Tricarboxylic acid cycle (TCA)
cycle, was similarly enhanced in the intestine ( + 31%) and
renal cortex (+67%) to a greater extent by GT extiact than
by GT diet but was slightly lowered in the renal medulla
(-18%; Table 5).
The effect of GT was also determined on the activities of
enzymes involved in gluconeogenesis and the hexose monophosphate (HMP)-shunt pathway (Table 6) The activities
of G6Pase, FBPase, G6PDH, and ME changed similarly
with the GT extract and the GT diet in all tissues. The
activity of G6Pase significantly increased in the intestine
(+34% to +42%), renal cortex (+13% to +16%), and
medulla (+27% to +43%) but decreased in the liver
(-13% to -20%). The activity of FBPase also increased
but to a much lesser extent than G6Pase in these tissues. The
activity of FBPase similarly to G6Pase was lowered in the
hver. The activity of G6PDH (HMP-shunt) significantly
decreased in the intestine (-40% to -48%), liver ( - 7 3 % to
-79%), and renal medulla (-32% to -58%) but profoundly increased in the renal cortex (+121% to +146%)
The activity of ME, which is a source of cellular reduced
nicotinamide adenine dinucleotide phosphate, together with
G6PDH significantly decreased in all tissues except the
renal cortex. It should be noted that the activities of various
metabolic enzymes studied in the liver altered in a narrow
range except G6PDH, which was significantly lowered by
GT consumption.
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Because GT has been reported to exert its biological
effects primarily by perturbation of the antioxidant defense
system, the activities of SOD and catalase and related parameters of oxidative stress were determined in control and
GT rats. It was observed that GT given in the diet or in
drinking water caused similar alterations in these parameters. LPO measured as the level of malondialdehyde was
significantly lowered in the liver and renal cortex but significantly enhanced in the intestine and renal medulla by GT
consumption. Total SH levels were significantly decreased
in the liver and renal cortex but significantly increased in the
renal medulla in GT compared with control rats. The activity of SOD significantly decreased in the intestine and liver
but significantly increased in the renal cortex and medulla
(Table 7). In contrast, catalase activity markedly increased
in the intestine and liver but did not change in renal tissues
by GT ingestion.
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Discussion
-K
Tea is the most consumed beverage in the world, aside
from water. Studies carried out in laboratories and on
S. A. Khan el al. / Nutrition 23 (2007) 687-695
692
Table 5
Effects of GT consumption via drinking fluid or diet on the activity of metabolic enzymes in different tissue homogenates
Tissue
Intestine
Control
GT extract
GT diet
Liver
Control
GT extract
GT diet
Cortex
Control
GT extract
GT diet
Medulla
Control
GT extract
GT diet
Hexokinase (\x.mo\lm% protein/h)
LDH (pimol/mg protein/h)
1.13 ±0.27
8.72 ± 0.26* (+672%)
4.98 ± 0.34* ( + 341%)
116.92 ±3.58
138.02 ±6.36^ (+18%)
117.68 ± 5.25 ( + 0.65%)
35.85 ± 2.41
40.41 ±0.99 (+13%)
43.13 ± 1.76* (+20%)
30.08 ± 1.03
30.71 ± 1.13 ( + 2%)
37.68 ± 1.72* (+25%)
6.46 ± 0.56
13.6 ± 1.22* (+111%)
7.23 ±0.46 (+12%)
86.88 ± 2.09
82.39 ± 2.34 (-5%)
80.92 ± 1.46* (-7%)
11.69 ±0.60
15.89 ±0.86* (+36%)
15,83 ± 0.96* (+35%)
66.60 ±1.191
70.13 ± 1.80 (+5%)
80.18 ± 1.87* (+20%)
MDH (/umol/mg protein/h)
21.52 ± 1.29
28.11 ±4.24 ( + 31%)
22.41 ± 1.38 (^ 4%)
3.78 ± 0.24
3.97 ± 0.38 ( + 5%)
4.58 ±0.14* ( + 21%)
23.26 ± 0.90
38.92 ± 5.35* (-f 67%)
33.43 ± 2.66* (+44%)
26.99 ± 0.65
22.24 ± 1.85* (-18%)
26.17 ± 1.25 (-3%)
GT, green tea; LDH, lactate dehydrogenase; MDH, malate dehydrogenase
* Results are mean ± SEM of three to four different preparations. Values in parentheses represent percent change from control.
* Significandy different at P < 0.05 from control.
animaKs have suggested thai GT in particular has extensive health benefits. Most of these effects are considered
to be due to the presence of chiefly tea catechins, polyphenolic compounds [8] that exhibit antioxidative, antiinflammatory, anticarcinogenic, anti-arteriosclerotic, and
antibacterial effects [9]. We propose that GT polyphenols
and other constituents cause specific adaptive alterations
in the cellular metabolism of certain tissues and thus
improve their functioning. To address this proposal, the
rats were given GT in the diet or in drinking water and
the activities of various enzymes involved in carbohydrate metabolism, antioxidant defense system, and BBM
were determined in the intestine, liver, and renal tissues.
In general, GT consumption resulted in slight loss of
body weight that was associated with lowering of blood
glucose, cholesterol, and Pi. These observations are in
partial agreement with some previous reports in humans
and animals [26]. Oral administration of GT has been
shown to decrease plasma total cholesterol and blood
triacylglycerols and serum glucose in fasted and nonfasted human subjects [27,28]. The suppressive effects of
GT catechins on the postprandial levels of triacylglycerols and cholesterol have also been reported in humans
[29]. Taken together, these observations may be respon-
Table 6
Effects of GT consumption via drinking fluid or diet on the activity of metabolic enzymes in different tissue homogenates*
Tissue
Intestine
Control
GT extract
GT diet
Liver
Control
GT extxact.
GT diet
Cortex
Control
GT extract
GT diet
Medulla
Control
GT extract
GT diet
G6Pase
(/xmol/mg protein/h)
FBPase
(/bimol/mg protein/h)
G6PDH
(/xmol/mg protein/h)
ME
((xmol/mg protein/h)
1.96 ± 0.05
2.79 ±0.17* (+42%)
2.62 ±0.13* (+34%)
2.70 ± 0.09
2.89 ±0.09 (+7%)
2.81 ±0.06 (+4%)
1.08 ±0.05
0,56 ±0.13* (-48%)
0.65 ± 0.08* (-40%)
0.68 i 0.09
0.35 - 0.06* (-49%)
0.42 ± 0.05* (-38%)
0.15 ± 0.002
0.12 ±0.002* (-20%)
0.13 ±0.005* (-13%)
0.85 ± 0.02
0.77 ± 0.02* (-9%)
0.75 ±0.03* (-12%)
3.23 ±0.21
0.87 ±0.11* (-73%)
0.69 ±0 . 1 1 ' ( - 7 9 % )
0.69 ± 0.05
0.60 ± 0.06 (-13%)
0.58 ±0.07 (-16%)
0.56 ± 0.01
0.65 ±0,02* (+16%)
0.63 ±0.01* (+13%)
2.57 ± 0.05
2.85 ±0.06* ( + 11%)
2.72 ±0.15 (+6%)
0.24 ± 0.02
0.53 ±0.03* (+121%)
0.59 ±0.01* (+146%)
0.45 ± 0.08
0.44 r 0.06 (-2%)
0.42 ± 0.09 (-7%)
0.45 ± 0,01
0.57 ± 0,02* ( + 27%)
0.64 ±0,02* (+43%)
1.76 ±0.04
2.08 ±0.12* (+18%)
2.05 ±0.06* ( + 17%)
0.57 ± 0.05
0.24 ± 0.04* (-58%)
0.39 ±0.01 (-32%)
1.53 ± 0.11
1.19 ± a i 5 ( - 2 2 % )
0.86 = 0.09* (-44%)
FBPase, fructose-1,, 6-bisphosphatase; G6Pase, glucose-6-phosphatase; G6PDH, glucose -6-phosphate dehydrogenase; GT, green tea; ME. malic enzyme
* Results are mean ± SEM of three to four different preparations. Values in parentheses represent percent change from control.
* Significantly different at P < 0.05 from control.
693
S A Khan ei al / Numiion 23 (2007) 687-695
Table?
Effects of GT consumption via dnnking fluid or diet on enzymic and non enzymic antioxidant parameteri, in different tissue homogenates*
Tissue
Intestine
Control
GT extract
GT diet
Liver
Control
GT extract
GT diet
Cortex
Control
GT extract
GT diet
Medulla
Control
GT extract
GT diet
LPO (nmol/gm tissue)
Total - S H (/imol/gm tissue)
SOD (Units/mg protein)
Catalase (jxmol/ing protein/mm)
6142 ± 2 60
87 20 ± 6 75" (+42%)
105 60 ± 7 04* (+72%)
1 88 ± 0 05
2 00 ± 0 08 (+6%)
2 45 ± 0 08* (+30%)
7 04 ± 0 63
4 73 ± 0 40" (-33%)
3 40 ± 0 30* (-52%)
2 90 ± 0 55
6 30 ± 0 45 (+117%)
10 79 ± I 23* ( + 272%)
408 20 ± 43 20
265 4 0 + 14 4 0 ' ( - 3 5 % )
21601 ± 13 80* (-47%)
10 67 ± 0 40
7 60 ± 0 25* (-29%)
6 89 ± 0 59" (-35%)
82 90 ± 1 94
72 9 0 ± 140* (-12%)
75 80 ± I 30" (-9%)
1342 ± 2 15
36 75 ± 5 90 (+174%)
42 88 ± 2 37 ( + 220%)
272 60 ± 20 43
228 00 ± 16 00 (-16%)
171 70 ± 21 90" (-37%)
7 49 ± 0 38
5 73 ± 0 22* (-24%)
6 42 ± 0 75 (-14%)
12 31 ± 1 24
18 20 ± 1 60* (+48%)
21 31 ± 1 50 (+73%)
108 40 ± 9 80
129 90 ± 5 20 (+20%)
135 9 ± 6 90 ( + 25%)
73 26 ± 5 58
131 70 ± 10 55* (+80%)
89 31 ± 2 00* (+22%)
2 90 ± 0 04
3 64 ± 0 01" (+26%)
4 66 ± 0 2 3 (+61%)
22 92 ± 0 65
28 6 ± 1 82* (+25%)
36 90 ± 0 75* (+61%)
139 40 ± 7 34
1045 ± 3 88 (-25%)
132 03 ± 5 42* (-5%)
GT green tea LPO, lipid peroxidation, - S H thiol, SOD, superoxide dismutase
* Results are mean ± SEM of three to four different preparations Values in parentheses represent percent change from control
* Significantly different at f < 0 05 from control
sible for body weight loss and in lowering the incidence
of cardiovascular diseases [29,30]
The present results further demonstrate that GT causes
selective adaptive alterations in the activities of certain
enzymes involved in glycolysis, TCA cycle, gluconeogenesis, and HMP-shunt pathway in the intestine, liver, and
renal cortex and medulla The activities of hexokinase except m the renal cortex and that of LDH variably increased
The acUviUes of malale dehydrogenase, a TCA cycle enzyme, except in the renal medulla, were also increased in
the same manner These observations suggest that GT consumption enhanced glucose degradation in the intestine,
liver, and kidney albeit to different extents m these tissues
The profound increase of LDH activity in the intestine and
renal medulla are suggestive of an increase in anaerobic
glycolysis as a major source of energy because these tissues
function in a low oxygen environment [17] Further, GT
consumption resulted in an increase in the activities of
G6Pase and FBPase in the intestine and renal tissues, indicating that the production of glucose by gluconeogenesis
was also enhanced in these tissues However, the activities
of these enzymes significantly decreased in the liver Thus
the effect of GT on glucose degradation and its production
appear to be tissue specific
The effect of GT on carbohydrate metabolism has not
been studied in detail in major tissues except m the liver and
adipocytes GT consumption increased glucose metabolism
in adipocytes [30,31], whereas hepatic glucose production
was reported to be inhibited by GT, leading lo lower blood
glucose levels [32] A GT polyphenol, epigallocatechin-3gallate, was reported to be an insulin mimetic in that it
lowered blood glucose in obese Zucker rats [33,34] Moreover, Waltner-Law et al [32] reported a significant decrease
in the expression of genes that control gluconeogenesis such
as poshhoenol pyruvate carboxykmase and G6Pase genes m
liver cells The observed decrease m the activities of glu
coneogenic enzymes (G6Pase and FBPase) in liver homogenates IS compatible with these observations
In contrast to the enzymes of glycolysis, TCA cycle, and
gluconeogenesis, the activity of G6PDH (HMP shunt) and
ME were differentially altered by GT The activity of
G6PDH significantly declined in the intestine, liver, and
renal medulla but profoundly increased in ihe renal cortex
The activity of ME was selectively decreased m the intes
tine and renal medulla but not affected in the liver and renal
cortex These enzymes act to produce cellular reduced nic
otinamide adenine dmocleotide phosphates that play an im
portant role in reducing anabolic pathways and the antiox
idant defense mechanism The increased activity of G6PDH
in the renal cortex might support reducing anabolic reac
tions, e g , lipid biosynthesis, and improve the antioxidant
defense mechanism to lower oxidative damage In contrast,
lower activities of G6PDH and ME in the intestine, liver,
and renal medulla may have resulted in lower cholesterol
synthesis and thus lower blood cholesterol GT has been
shown to increase energy expenditure and fat oxidation in
humans [35,36] The observed increase in oxidative metabolism (mitochondrial enzymes) and increased glucose pro
duction by gluconeogenesis is in partial agreement with
these reports Pi is an essential component of intermediary
metabolism, energy conservation as adenosine triphosphate,
and biosynthesis of cellular membranes (as phospholipids)
and other important biomolecules such as nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and vanous RNAs The decrease m serum Pi may
suggest Its overutilization in these processes Moreover, Pi
may have been converted to phospholipids as indicated by
higher serum phospholipids levels needed for much required synthesis of membranes such as the endoplasmic
694
S. A. Khan et al. /Nutrition 23 (2007) 687-695
reticulum, mitochondria, and plasma membrane to support
higher metabolic activities.
Most beneficial health effects ascribed to GT are considered to be mediated by potential antioxidant properties of its
constituents that scavenge free radicals and reduce oxidative
damage [37]. Several lines of evidence suggest that prooxidant and antioxidant actions of plant polyphenols may be
important mechanisms for their anticancer properties [38].
Reactive oxygen species (ROS) are normal byproducts of
aerobic metabolism. Most intracellular ROS are generated
via mitochondrial electron transport, although other normal
biological processes contribute. It has been reported that GT
exerts its biological effects on the basis of the redox state of
a particular cell/tissue [39,40] and according to the level of
GT polyphenols accumulated in the tissues [41]. To maintain proper redox balance, many defense systems have
evolved. A major cellular defense against ROS is provided
by SOD and catalase, which together convert superoxide
radicals first to HjOj and then to water and molecular
oxygen. Other enzymes, e.g., glutathione (GSH) peroxidase,
use the thiol reducing power of glutathione to reduce oxidized lipids and protein targets of ROS. It has been reported
that GT causes increases in the activities of catalase, SOD,
GSH-peroxidase, and GSH-transferase and reduces LPO in
the liver and kidney [42]. The present results demonstrated
that GT consumption increased LPO in the intestine and
renal medulla but markedly decreased in the liver and renal
cortex. The activities of SOD and catalase were also affected largely by GT consumption in these tissues. The
activity of SOD was significantly lower in the intestine and
liver but increased in the renal cortex and medulla. In
contrast, the activity of catalase profoundly increased in the
intestine and liver and to a lesser extent in the renal cortex
but decreased in the renal medulla.
The present results imply that the biological defense
system was perturbed by GT consumption as a result of free
radical scavenging properties of its polyphenols and other
active constituents. The profound lowering of LPO in the
renal cortex and liver in GT compared with control rats
suggests that oxidative damage even under normal physiologic conditions was significantly lowered by GT constituents. The reduction in LPO in the liver was associated with
a profound increase in catalase activity, whereas in the renal
cortex it appeared to be due to increases in catalase and
SOD activities. In contrast, an increase in LPO in the intestine was associated with a marked decrease of SOD activity,
although there was a significant increase in catalase activity.
However, in the renal medulla enhanced LPO can be attributed to decreased catalase activity. The cellular response to
oxidative stress in the intestine and renal medulla compared
with the liver and renal cortex might have been due to the
nature of metabolic activities in these tissues. Although the
liver and renal cortex have higher oxidative metabolism, anaerobic metabolism is more prevalent in the intestine and renal
medulla due to lower oxygen tension in these tissues. The
significant effects of GT consumption on various parameters in
different tissues can also be attributed to the presence of active
tea components after digestion/absorption/extraction or due to
accumulation in various tissues. However, these observations
clearly demonstrate that GT consumption activates the antioxidant defense system in all tissues. GT-induced antioxidant
defense mechanism in the intestine and liver appeared to be
largely catalase mediated, whereas in renal tissues it was predominantly SOD mediated. The activities of certain marker
enzymes except LAP in the kidney and ALP in the intestine
were also increased significantly in BBMs, which is major
target of oxidative damage. It is very likely that the increase in
activities of these enzymes was due to protection provided by
GT polyphenol-induced lowering of oxidative damage to
membranes compared with non-GT-consuming control rats.
Conclusions
We conclude that the ingestion of GT produced a significant increase in the activities of certain enzymes of glucose
degradation and its synthesis in the intestine, liver, and
kidney. The effects seem in part to be mediated by GT
polyphenols having antioxidant free radical scavenging
properties that lower oxidative damage. The results strongly
support the notion that GT consumption helps maintain and
improve health by increasing antioxidant defense and cellular metabolic activities in various tissues of normal rats, as
people have benefited by GT consumption since ancient
times.
Acknowledgments
The authors gratefully acknowledge the help extended by
Dr. Neelam Farooq in the critical evaluation of the manuscript.
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