Aqueous extract of black tea ( Camellia sinensis) prevents ethanol + cholecystokinin-induced...

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Life Sciences 78 (200

Aqueous extract of black tea (Camellia sinensis) prevents

ethanol+cholecystokinin-induced pancreatitis in a rat model

Dolan Das, Sandip Mukherjee, Asankur S. Das, Maitrayee Mukherjee, Chandan Mitra *

Department of Physiology, Presidency College, Calcutta, India

Received 17 June 2005; accepted 7 September 2005

Abstract

Black Tea Extract (BTE), a phytocompound has been attributed with a plethora of health-promoting actions. We have previously demonstrated

that BTE inhibits chronic hepatitis in a rat model induced with high-fat and ethanol (EtOH). This study reports that BTE prevents altered

pancreatic acinar cell functions, oxidative stress, inflammatory changes and DNA damage in the EtOH+cholecystokinin (CCK)-induced model of

pancreatitis. The EtOH+CCK model rats were administered with BTE, and were examined the activity of pancreatic digestive enzymes (amylase

and lipase), proinflammatory cytokines (IL-6 and TNF-a), oxidative and antioxidative enzymes (nitric oxide, NO; malondialdehyde, MDA;

superoxide dismutase, SOD; catalase, CAT), antioxidant level (glutathione, GSH), histopathological changes and the integrity of genomic DNA.

Results show that because of chronic EtOH treatment, serum level of amylase and lipase (two biomarkers for pancreatitis) and pancreatic levels of

MDA and NO (two biomarkers of oxidative stress) increased significantly, which could be effectively blunted by BTE. BTE could normalize

EtOH+CCK-induced suppressed activities of SOD and CAT, and GSH content of pancreatic tissue. Also, histopathological and inflammatory

changes during EtOH+CCK-induced pancreatitis could be blunted by BTE. Furthermore, BTE could effectively reduce EtOH+CCK-induced

increase in DNA fragmentation and damage. These findings suggest that BTE prevents pancreatitis caused by chronic EtOH+CCK toxicity

presumably by enhancing antioxidant, anti-inflammatory and antiapoptotic activity in rats.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Ethanol+CCK-induced pancreatitis; Oxidative stress; Proinflammatory cytokines; DNA fragmentation; Apoptosis; Black Tea Extract (BTE)

Introduction

Alcohol abuse is the main cause of pancreatitis in about

80% of cases (Aleynik et al., 1999). In developed countries,

60–70% of patients with chronic pancreatitis have a long

history (6–12 years) of heavy consumption of alcohol (190–

220 ml/day) before the onset of clinically apparent disease

(Hunger et al., 1997). Although the mechanisms by which

ethanol leads to pancreatic damage remain unknown, there is

growing evidence that ethanol exerts direct toxic effect on the

pancreatic acinar cells (Rydzewska et al., 2001). Ethanol can

change systemic and pancreatic lipid metabolism with accu-

mulation of lipid droplets within the acinar cells, alter

membrane fluidity and integrity. Ethanol even sensitizes the

acinar cells to cholecystokinin (CCK)-stimulated intracellular

0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.lfs.2005.09.020

* Corresponding author. 14/17A Golf Club Road, Calcutta—700 033, India.

Tel.: +91 33 2413 1383/2417 8645 (R), +91 33 2219 8149 (O).

E-mail address: [email protected] (C. Mitra).

zymogen proteolysis. In presence of combined-stimulation of

CCK and alcohol, it is easier to induce acute pancreatitis (Wu,

2000). Mounting evidence has accumulated that oxygen

radicals and other reactive oxygen species (ROS) may play a

pivotal role in the pathogenesis of acute and chronic

pancreatitis (Tsai et al., 1998; Norton et al., 1998; Casini et

al., 2000; Andican et al., 2005). Evidence further suggests that

chronic alcoholic pancreatitis may develop as a result of

repeated necro-inflammatory episodes of acute pancreatitis

(Rydzewska et al., 2001).

It has been reported that the combination of an ethanol

(15%) containing diet and infusion of a low dose of CCK-

8 causes pancreatitis in experimental animal (rat) with

increased levels of amylase and lipase, infiltration of neutro-

phils and macrophages, and the number of apoptotic acinar

cells (Pandol et al., 1999, 2003). All these observations have

led to the concept that the development of alcoholic pancre-

atitis requires cofactors (Jerrells et al., 2003). In our present

study we selected an animal model of alcoholic pancreatitis

6) 2194 – 2203

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D. Das et al. / Life Sciences 78 (2006) 2194–2203 2195

induced by a combination of chronic ethanol and low-dose of

CCK in rat.

During the last decade, numerous in vitro and in vivo

studies have suggested that tea has numerous potentially

beneficial medicinal properties (Chen et al., 2002). The

chemicals that make tea a potential protector of health are

called polyphenols and these polyphenolic compounds are

collectively known as the tea flavonoids. Tea flavonoids

possess antioxidant properties in vitro and have been proposed

as a protective dietary component, reducing the risk of various

cancers and other diseases (Langley-Evans, 2000; Chen et al.,

2002). Further, it has been reported that antioxidants in tea are

more potent than those found in many fruits and vegetables and

neutralize highly reactive molecules, called free radicals, which

damage cells (Luczaj and Skrzydlewska, 2004). It has also

been reported that consumption of black tea resulted in a

significant increase in plasma antioxidant activity (Leenan et

al., 2000; Rizvi et al., 2005). Earlier it was observed that green

tea catechins have a protective effect on the pathogenesis of

cerulein-induced pancreatitis (Takabayashi and Harada, 1997).

The aim of the present study was to examine the efficacy of

black tea extract (BTE) in preventing chronic EtOH+CCK-

induced pancreatitis in a rat model.

Materials and methods

Plant material

The black tea extract was prepared from CTC (Curl, Tear

and Crush) BOP (Broken Orange Pickoe) grade black clonal

tea. Preparation of aqueous extract of black tea was done by

following the method as described elsewhere (Wei et al., 1999).

Briefly, 1.25 g of tea leaves were added to 25 ml of boiling

water and was steeped for 15 min. The infusion was cooled to

room temperature and then filtered. The tea leaves were

extracted a second time with 25 ml of boiling water and

filtered, and the two filtrates were combined to obtain a 2.5%

aqueous-tea extract. The resulting clear solution was similar to

tea brews consumed by human. This black tea extract (BTE)

was fed to animals by gavage technique at a dose of 1 ml/100 g

body weight/day at a temperature of 37 -C (Wei et al., 1999).

Animal model and study protocol

Male albino rats, weighing 145–165 g, were used in the

experiment. They were housed in a temperature controlled

room (25T2 -C) with 12-h light–dark cycles for at least 1

week. After the stabilization period, rats were randomly

divided into four groups: Control (Gr. A), CCK (Gr. B),

EtOH+CCK (Gr. C) and EtOH+CCK+BTE (Gr. D). All rats

were pair-fed with a normal laboratory diet containing 7% of

total energy as fat, 18% as protein and 75% as carbohydrates,

part of which, in the ethanol formula, are isocalorically

replaced by ethanol (to the extent of 36% of total energy)

(Lieber and Decarli, 1994) and free access to drinking water.

Animals of EtOH+CCK and EtOH+CCK+BTE groups were

pair-fed with 15% (v/v) ethanol (Luma and Vorne, 1976)

(volume that contributed isocalorically 36% of total energy)

once daily for 30 days by gavage technique. Animals of

EtOH+CCK+BTE were fed with 2.5% (w/v) BTE at a single

dose of 1 ml/100 g body weight/day for that same period (30

days) by gavage technique (Wei et al., 1999).

All ethical care and treatment of animals were undertaken by

following the guidelines of the Animal Care and Ethics

Committee of Presidency College, Calcutta. After the treatment

period (30 days) was over, rats were fasted and anesthetized by

intramuscular injection of urethane (500 mg/kg body weight)

(Miura et al., 1997). A polyethylene cannula (PE 50) was placed

in the right jugular vein for CCK infusion. Animals of CCK,

EtOH+CCK and EtOH+CCK+BTE groups were infused with

CCK in physiological saline solution at a dose of 50 ng/kg body

weight/h (Pandol et al., 1999) for 1 h. Animals of control group

were infused with physiological saline solution alone. A

midline celiotomy was performed and, the common pancreat-

ic-bile duct was cannulated extraduodenally with a PE 10

catheter through which pancreatic secretions were collected.

The bile duct was ligated on the liver side of the pancreas to

prevent the flow of bile into the common pancreatic-bile duct.

Core temperature was maintained by using a heating pad and

overhead lamp. The animals were allowed to stabilize for 30

min before the onset of infusion. After surgery, the abdomen

was covered with a gauze soaked in liquid Vaseline to avoid

tissue dryness. Animals were killed at the end of the experiment

with an overdose of anesthesia (Saluja et al., 1997). Blood

samples were taken from the heart before the animals were

killed in order to measure TNF-a and IL-6.

Preparation of tissue extracts

The abdomen was opened, pancreas was quickly removed

and placed in a beaker containing ice-cold Tris–HCl buffer

(pH 7.4). It was minced into small pieces on ice and

homogenized immediately in a glass homogenizing tube

equipped with a Teflon pestle. The homogenate was processed

according to the method of Koyama et al. (1983) for the

estimation of NO, MDA and SOD. For CAT estimation, the

tissue was homogenized in ice-cold isotonic phosphate buffer

(pH 7.4) and processed according to the method of Cohen et al.

(1970). For GSH estimation, pancreatic tissue was homoge-

nized according to the method of Ellman (1959) in ice-cold

phosphate buffer (pH 8).

Measurement of amylase and lipase

Biochemical assay of amylase and lipase were performed

from collected pancreatic secretions by using kit methods.

Amylase and lipase kits were obtained from E. Merck (India)

Ltd. and Dyasis (Germany), respectively.

Measurement of tumor necrosis factor-a (TNF-a) and inter-

leukin-6 (IL-6)

Serum TNF-a level was estimated by using the CYTELISA

rat TNF-a kit obtained from CYTIMMUNE Sciences Inc.,

D. Das et al. / Life Sciences 78 (2006) 2194–22032196

Maryland, USA. All samples were assayed in duplicate. The

intra assay variation was 6.7%. To avoid inter assay variation

all samples were run at one time. Optical density of each well

was determined by using a microplate reader (Thermo

Labsystems, Finland).

Serum IL-6 was estimated by using the ELISA Quantikine

rat IL-6 immunoassay kit obtained from R & D Systems Inc.,

Minneapolis, MN, USA. All samples were assayed in

duplicate. The intra assay variation was 5.5%. To avoid inter

assay variation all samples were run at one time. Optical

density of each well was determined by using a microplate

reader (Thermo Labsystems, Finland).

Measurement of NO production

NO production was estimated by Griess reaction (Giusep-

pina et al., 1999) which was expressed in the form of nitrite

accumulation. In brief, 100 Al of tissue extract was mixed with

100 Al of Griess reagent [equal volumes of 1% (w/v)

sulphanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v)

napthylethylenediamine hydrochloride (NEDH)] and incubated

at room temperature for 10 min, and then the absorbance at 550

nm was measured in a UV-double beam spectrophotometer

(Shimadzu 160A). The amount of nitrite in the sample (AMunit) was calculated from a sodium nitrite standard curve. The

results were expressed as AM nitrite/mg of protein.

Measurement of lipid peroxidation

The quantitative measurement of lipid peroxidation was

performed following the thiobarbituric acid (TBA) test. The

amount of MDA formed was quantitated by reaction with TBA

and used as an index of lipid peroxidation. In brief, 2 ml of

sample was added with 1 ml of 20% trichloroacetic acid and 2

ml of 0.67% (w/w) TBA and heated in a lightly stoppered tube

for 10 min in a boiling water bath. The tubes were cooled and

centrifuged and the absorbance was taken at 530 nm. The

results were expressed as nM MDA/mg of protein using molar

extinction co-efficient of the chromophore (1.56�105 cm2/

mM) (Wills, 1987).

Measurement of antioxidant enzymes: SOD and CAT

SOD was assayed according to the method of Misra and

Fridovich (1972) by comparing the adrenochrome formation in

control, CCK, EtOH+CCK, and EtOH+CCK+BTE-supple-

mented groups of animals. In brief, 0.5 ml of 1.8 mM

epinephrine (freshly prepared), 0.5 ml of 0.6 mM EDTA and

0.5 ml of 0.3 M sodium carbonate (pH 10.2) was added with

the sample which contained 100 Ag of protein determined by

trial and error. The reaction was initiated by addition of

epinephrine and the increase in absorbance at 480 nm was

observed.

CAT was assayed by the method described by Cohen et al.

(1970). The enzyme-catalyzed decomposition of H2O2 was

measured. In brief, 0.5 ml aliquot of cold CAT sample and a

blank consisting of 0.5 ml distilled water was taken in test

tubes and the enzymatic reactions was initiated by adding 5 ml

of cold 6 mM H2O2 and mixed thoroughly. After exactly 3 min

the reaction was stopped by rapidly adding 1 ml 6 N H2SO4

and mixed thoroughly. Then 7 ml of 0.01 N KMnO4 reagent

was added, mixed thoroughly and the reading was taken at 480

nm within 30–60 s.

Measurement of GSH

GSH was estimated in the pancreas samples according to the

method of Ellman (1959) and the results were expressed as AMGSH/mg of protein. In brief, 20 Al DTNB (5,5V-dithiobis 2-

nitrobenzoic acid) was added to 3 ml of reagent mixture

(containing 3 ml sample, 2 ml buffer and 5 ml water) in a

cuvette. The colour developed rapidly and the absorbance was

read at 412 nm after 2 min in a UV-double beam spectropho-

tometer (Shimadzu 160A).

Measurement of protein

Protein in the homogenate of pancreas was estimated by the

method of Lowry et al. (1951) using BSA as standard.

Quantification of fragmented DNA by diphenylamine (DPA)

Fresh pancreas samples were collected in phosphate buffer

(pH 8) and DNA damage was quantified in each sample

individually according to the method described earlier by

Kurita-Ochiai et al. (1997). To measure pancreatic DNA

fragmentation by spectrophotometry, a portion of the pancreas

was homogenized in chilled phosphate buffer and the

homogenates were centrifuged at 400 �g for 10 min and

the supernatant was discarded. Pellet was then resuspended in

400 Al of hypotonic lysis buffer (0.2% Triton X-100, 10 mM

Tris, 1 mM EDTA, pH 8.0) and centrifuged for 15 min at

13,800 �g to separate intact chromatin in the pellet from

fragmented/damaged DNA in the supernatant. Pellets were

resuspended in 400 Al hypotonic lysis buffer and then 400

Al 0.5 N perchloric acid was mixed with both pellets and

supernatants. Then, 800 Al diphenylamine (DPA) reagent

[0.088 M DPA, 98% (v/v) glacial acetic acid, 1.5% (v/v) conc.

H2SO4 and 0.5% of 1.6% acetaldehyde solution] was added

and the samples were kept at 4 -C for 48 h. Absorbance was

measured at 575 nm using a UV-double beam spectropho-

tometer (Shimadzu 160A). DNA fragmentation in samples

[(frag. DNA in sup.) / (frag. DNA in sup.+ intact DNA in

pellet)] were expressed as percentage of total DNA appearing

in the supernatant fraction.

Light microscopy and histopathological evaluation

Histological studies of the pancreas were conducted by

methods as described earlier (Bancroft et al., 1996). Briefly, the

pancreas from rats of all groups were carefully isolated,

trimmed of fat, and fixed in 10% buffered formalin. The

formalin-fixed pancreas were then dehydrated with ethanol and

embedded in paraffin. Sections of 4–5 Am were cut and stained

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Pan

crea

tic

Am

ylas

e (U

/L)

#

Co

ntr

ol

CC

K

CC

K+E

tOH

CC

K+E

tOH

+BT

E

*

**

**

0

500

1000

1500

2000

2500

3000

Pan

crea

tic

Lip

ase

(U/L

) #

Co

ntr

ol

CC

K

CC

K+E

tOH

CC

K+E

tOH

+BT

E

*

**

**

A B

Fig. 1. Effects of BTE supplementation (2.5% at a dose of 1 ml/100 g body weight/day for 30 days) on EtOH (15% [v/v]/100 g body weight/day for 30 days)+CCK

(50 ng/kg body weight/h for 1 h)-induced pancreatic amylase and lipase activity of rat. Error bars represent meansTSEM (n =6). # Denotes significance based on

Kruskal–Wallis nonparametric ANOVA test at p <0.01; ** denotes significance based on Mann–Whitney U multiple comparison test at p <0.01; * denotes not

significant ( p >0.05).


with hematoxylin and eosin. All slides were evaluated in a

blinded manner by one of the authors. Pathological changes in

the pancreas were scored as described by Tsukamoto et al.

(1988) as follows: steatosis (the percentage of acinar cells

containing fat droplets): <25%=1+, <50%=2+, <75%=3+,

75%�4+; inflammation and necrosis: 1 focus per low-power

view=1+; >1 focus=2+.

Statistical analysis

Values are expressed as meanTSEM. Statistical significance

was tested by the Mann–Whitney U multiple comparison test

for comparison of data between groups, and the Kruskal–

Wallis nonparametric ANOVA test for the comparison of

sequential data within a group. SPSS 10 software was used for

0

10

20

30

40

50

60

70

80

Ser

um

TN

F-a

lph

a (p

g/m

l) #

Co

ntr

ol

CC

K

EtO

H+C

CK

EtO

H+C

CK

+BT

E

**

***

Ser

um

IL-6

(p

g/m

l) #

A B

Fig. 2. Effects of BTE supplementation (2.5% at a dose of 1 ml/100 g body weight/d

(50 ng/kg body weight/h for 1 h)-induced serum cytokine levels (TNF-a and IL-6) o

Kruskal–Wallis nonparametric ANOVA test at p <0.01; ** denotes significance b


statistical analysis. Differences were considered significant if

p <0.05.

Results

Effects of administration of chronic EtOH+CCK on pancreatic

amylase and lipase: protective effects of BTE

Fig. 1A and B depict the effect of administration of

EtOH+CCK on pancreatic amylase and lipase activity. Fig.

1A shows that, compared to control, there was no significant

change in the amylase activity level after infusion of CCK alone.

However, in the EtOH+CCK-infused group, this pancreatic

amylase level was found significantly ( p <0.01) higher com-

pared to both control and CCK groups. BTE supplementation

0

50

100

150

200

250

300

350

Co

ntr

ol

EtO

H+C

CK

EtO

H+C

CK

+BT

E

CC

K

*

**

**

ay for 30 days) on EtOH (15% [v/v]/100 g body weight/day for 30 days)+CCK

f rat. Error bars represent meansTSEM (n =6). # Denotes significance based on

ased on Mann–Whitney U multiple comparison test ( p <0.01); * denotes not

0

0.5

1

1.5

2

2.5

3

nM

MD

A /m

g o

f p

rote

in #

*

**

**

Co

ntr

ol

CC

K

EtO

H+C

CK

EtO

H+C

CK

+BT

E

Fig. 4. Effects of BTE supplementation (2.5% at a dose of 1 ml/100 g/body

weight/day for 30 days) on EtOH (15% [v/v]/100 g body weight/day for 30

days)+CCK (50 ng/kg body weight/h for 1 h)-induced MDA production in rat

pancreatic tissue. Error bars represent meansTSEM (n =6). # Denotes

significance based on Kruskal–Wallis test ( p <0.01); ** denotes significance

based on Mann–Whitney U multiple comparison test ( p <0.01); * denotes not



was found effective in reducing such EtOH+CCK-induced

increase level of amylase activity ( p <0.01) near to control level.

Similar to amylase, lipase activity was found increased in

EtOH+CCK-infused group, compared to both control and CCK

groups. Like amylase, BTE supplementation in an identical way

reduced EtOH+CCK-induced increased lipase activity near to

control level ( p <0.01) (Fig. 1B).

Effects of administration of chronic EtOH+CCK on TNF-aand IL-6: protective effects of BTE

To ascertain that inflammatory responses occur due to

EtOH+CCK administration and BTE supplementation may

recover this, the serum concentration of pro-inflammatory

cytokines TNF-a and IL-6 were assayed. Both the cytokines,

TNF-a and IL-6 levels in serum (pg/ml) were significantly

higher ( p <0.01) in EtOH+CCK group, compared to CCK and

control groups of animals. Such increased levels of TNF-a and

IL-6 were significantly restored ( p <0.01) after BTE supple-

mentation (Fig. 2A and B).

Effects of administration of chronic EtOH+CCK on NO and

MDA production: protective effects of BTE

Figs. 3 and 4 show the effect of administration of

EtOH+CCK on nitrite accumulation, an indicator of NO

synthesis, and MDA production, an index of lipid peroxidation,

in pancreatic tissue of different groups of rats. Results show

that compared to control, there was no significant change in

NO and MDA production in the CCK group. However, in the

EtOH+CCK-infused group, both NO and MDA production

were found significantly ( p <0.01) higher compared to both

control and CCK groups. BTE supplementation was found

effective in reducing such EtOH+CCK-induced increase level

of NO and MDA production ( p<0.01).

0

1

2

3

4

5

6

7

µM N

itri

te /m

g o

f p

rote

in #

#

Co

ntr

ol

CC

K

EtO

H+C

CK

EtO

H+C

CK

+BT

E

*

**

**



days)+CCK (50 ng/kg body weight/h for 1 h)-induced NO production in rat

pancreatic tissue. Error bars represent meansTSEM (n =6). ## Denotes




Effects of administration of chronic EtOH+CCK on SOD and

CAT: protective effects of BTE

Figs. 5 and 6 represent the result of administration of

CCK+EtOH on antioxidant enzymes SOD and CAT in

pancreatic tissue of different groups of rats. Results indicate

that, compared to control, rats infused with CCK showed no

significant ( p >0.05) change in both SOD and CAT activity.

However, EtOH+CCK group of rats showed marked increase

in the activity of both SOD and CAT ( p <0.01) compared to

control and CCK. When BTE was supplemented, this increased

0

1

2

3

4

5

6

IU S

OD

/ m

g o

f p

rote

in #

#

Co

ntr

ol

CC

K

EtO

H+C

CK

EtO

H+C

CK

+BT

E

**

**

*



days)+CCK (50 ng/kg body weight/h for 1 h)-induced SOD activity in ra



based on Mann–Whitney U multiple comparison test ( p <0.01); * denotes no


t

t

0

0.5

1

1.5

2

2.5

3

1st

ord

er r

eact

ion

rat

e co

nst

ant

of

CA

T /

mg

of

pro

tein

##

Co

ntr

ol

CC

K

EtO

H+C

CK

EtO

H+C

CK

+BT

E

**

**

*



days)+CCK (50 ng/kg body weight/h for 1 h)-induced CAT activity in rat






production of SOD and CAT levels were found to be reduced

significantly ( p <0.01).

Effects of administration of chronic EtOH+CCK on GSH:

protective effects of BTE

Fig. 7 depicts the result of administration of EtOH+CCK on

GSH content of pancreas of different groups of rats. Figure

shows that there is no significant change in GSH content in the

0

20

40

60

80

100

µM G

SH

/mg

of

pro

tein

##

Co

ntr

ol

CC

K

EtO

H+C

CK

+BT

E

EtO

H+C

CK

*

**

**



days)+CCK (50 ng/kg body weight/h for 1 h)-induced GSH content in whole

pancreas of rat. Error bars represent meansTSEM (n =6). ## Denotes




CCK group, compared to control. In EtOH+CCK group, GSH

level was found significantly ( p <0.01) lower as compared to

control and CCK. On BTE supplementation, this lower level of

GSH was recovered significantly ( p <0.01).

Effects of administration of chronic EtOH+CCK on genomic

DNA damage: protective effects of BTE

The effect of EtOH+CCK treatment on the integrity of

genomic DNA is presented in Fig. 8. Compared to physiolog-

ical saline infused control group (44.8%), there was no

significant ( p >0.05) change in pancreatic genomic DNA

fragmentation in CCK group (46.5%). However, infusion of

EtOH+CCK caused an increase in genomic DNA fragmenta-

tion (70.2%) which was significantly ( p <0.01) higher com-

pared to control and CCK. BTE-supplementation was found

effective in blunting such increased fragmentation to as low as

45% ( p <0.01).

Histopathological observation

No histological alterations were detected in the pancreas

of control (Fig. 9A) and CCK infused groups of rats.

However, the pancreas of rats of EtOH+CCK showed

extensive acinar vacuolization, steatosis, inflammatory

changes and also changes in normal histoarchitecture of the

pancreatic acini (Fig. 9B). All these histopathological

changes, viz., steatosis, acinar vacuolization and inflamma-

tion were markedly reduced after BTE supplementation, and

histoarchitecture of pancreatic acini was returned near to

normal (Fig. 9C). Total pathology scores of pancreas from

different groups of rats are summarized in Fig. 10. Results

show that, compared to control, different pathological

changes, viz., inflammation, necrosis and steatosis were

significantly aggravated in EtOH+CCK-administered groups.

0

10

20

30

40

50

60

70

80

% D

NA

Fra

gm

enta

tio

n #

Co

ntr

ol

CC

K

EtO

H+C

CK

EtO

H+C

CK

+BT

E

*

**

**



days)+CCK (50 ng/kg body weight/h for 1 h)-induced genomic DNA

fragmentation in whole pancreas of rat. Error bars represent meansTSEM

(n =6). # Denotes significance based on Kruskal–Wallis test ( p <0.01); **

denotes significance based on Mann–Whitney U multiple comparison test

( p <0.01); * denotes not significant ( p >0.05).

Fig. 9. Representative light photomicrographs (�400) of paraffin -embedded sections of rat pancreas showing effects of BTE supplementation (2.5% at a dose of 1

ml/100 g body weight/day for 30 days) on EtOH (15% [v/v]/100 g body weight/day for 30 days)+CCK (50 ng/kg body weight/h for 1 h)-induced histopathological

changes. All tissues were stained with hematoxylin–eosin. (A) Section from control rat with no histopathological changes. (B) Section from rat treated with

EtOH+CCK; typical characteristics of pancreatitis are prominent: (i) extensive acinar vacuolization (6), (ii) steatosis (@), (iii) signs of inflammation associated with

changes in normal histoarchitecture of pancreatic acini. (C) Section from rat treated with EtOH+CCK+BTE; a complete protection is observed.


BTE supplementation could significantly blunt such aggra-

vated response caused by EtOH+CCK administration (Fig.

10A). Histopathological examination further revealed that,

compared to control, total pathological score in EtOH+CCK

group was 837%, and in EtOH+CCK+BTE group it was

295% (Fig. 10B).

00.20.40.60.8

11.21.41.61.8

Co

ntr

ol

EtO

H+C

CK

EtO

H+C

CK

+BT

E

Co

ntr

ol

INFLAMMATION # NECR

Pat

ho

log

ical

Sco

res **

†

0100200

300400

500600700800

900

% In

crea

se in

Tot

al P

atho

logy

Sco

reC

ompa

red

to C

ontr

ol

A

B

Fig. 10. Effects of BTE supplementation (2.5% at a dose of 1 ml/100 g body weight

CCK (50 ng/kg body weight/h for 1 h)-induced pancreatic Pathology Scores in rats. E

denotes significance based on Kruskal–Wallis test at p <0.01 and ## in case of steat

significance based on Mann–Whitney U multiple comparison test at p <0.01 and

Discussion

This study has revealed that BTE is an effective chemopre-

ventive agent against an experimental rat model of pancreatitis

with EtOH+CCK (Schoenberg et al., 1995; Whittakar et al.,

1996). It is well known that elevation of pancreatic digestive

EtO

H+C

CK

EtO

H+C

CK

+BT

E

Co

ntr

ol

EtO

H+C

CK

EtO

H+C

CK

+BT

E

OSIS # STEATOSIS ##

**

†

**

**

EtO

H+C

CK

EtO

H+C

CK

+BT

E

/day for 30 days) on EtOH (15% [v/v]/100 g body weight/day for 30 days) and

rror bars represent meansTSEM (n =6). # In case of inflammation and necrosis

osis denotes significance based on Kruskal–Wallis test at p <0.001; ** denotes

. at p <0.05.


enzymes lipase and amylase in the blood or pancreatic juice

suggests pancreatitis (Kono et al., 2001; Pfefferkorn et al.,

2002). Significant elevation in the activities of both of these

enzymes in our studies by chronic EtOH+CCK confirmed that

pancreatitis was developed in our experimental rat model. BTE

supplementation could significantly reduce such elevated

activities of these enzymes suggesting that BTE possibly has

a certain degree of protective influence against EtOH+CCK-

induced pancreatitis and altered pancreatic acinar cell func-

tions. Earlier, we observed similar chemopreventive action of

BTE against EtOH-induced chronic hepatitis in a rat model

(Das et al., 2004). Such regulatory influence of BTE against

pancreatitis-induced altered enzymatic function are in quite

agreement with plethora of medicinal effects attributed to BTE

earlier (Krishnamoorthy, 1991; Matsumoto et al., 1991; Chen

et al., 2002).

The role of various cytokines, such as IL-1, IL-6, and TNF-

a in the development of local inflammatory changes and

release of pro-inflammatory cytokines (Grewal et al., 1994),

and also in the development of acute and chronic pancreatitis

(Rongione et al., 1997; Gukovsky et al., 1998; Kuldip et al.,

2003) are well documented. In the present study, combined

EtOH+CCK administration significantly raised both serum IL-

6 and TNF-a levels together with increased activities of serum

amylase and lipase, and thus confirmed that local inflammatory

changes and release of pro-inflammatory cytokines did occur in

our experimental model which possibly caused pancreatitis and

changes in acinar cell functions. All these experimentally

induced pathological changes were blunted significantly by

BTE supplementation, indicating that BTE possibly has an

ameliorative role against the deleterious effects on the pancreas

caused by combined EtOH+CCK administration.

There is growing evidence that ethanol exerts direct toxic

effects on pancreatic acinar cells. Our results from the

pathologic findings showed that chronic EtOH+CCK caused

significant histological changes in the exocrine pancreas as

evidenced by the appearance of steatosis, extensive acinar

vacuolization, inflammatory changes and also changes in

normal histoarchitecture of the pancreatic acini. The appear-

ance of fat droplets in the acinar cells has been used as an

indicative criterion for pancreatitis development in rats (Kono

et al., 2001). Because the exocrine pancreas of rats after BTE

supplementation appeared to be normal in contrast to that of

rats on combined EtOH+CCK, the results indicated that

supplementing rats with BTE may have significant recovery

effects on the histological changes of pancreas.

It was reported earlier that reactive oxygen species (ROS)

generated in pancreatic acinar cells play an important role in

chronic ethanol-induced pancreatitis (Norton et al., 1998;

Szuster-Ciesielska et al., 2001; Wittel et al., 2003;). Further,

ethanol-induced augmented ROS production has been impli-

cated with lipid peroxidation (Takabayashi et al., 1995; Kono et

al., 2001) which induces either apoptosis or necrosis depending

on its extent (Andican et al., 2005). An earlier report also

suggest that ethanol sensitizes the pancreas in inflammatory

responses, cell death and fibrosis caused by CCK (Pandol et al.,

2003). We observed identical results after chronic (30 days)

ethanol ingestion. MDA formation, the index of lipid perox-

idation, was significantly increased after ethanol treatment and

BTE supplementation was potentially effective in reducing

lipid peroxidation, suggesting that BTE might have antioxidant

principles to produce such response.

NO is a cytotoxic agent involved as mediator in inflamma-

tory disorders (Kalf et al., 2000), and because of its

cytotoxicity, overproduction is deleterious to cells (Krippeit-

Drews et al., 1995). It has also been reported that NO can

induce oxidative stress (Lynn et al., 1998; Andican et al.,

2005). In our studies, EtOH+CCK administration was found to

cause a significant increase in NO production which, we

speculate, might be responsible for pancreatic injury and

inflammatory changes. BTE supplementation could reduce this

enhanced NO production, even lower than the control value.

Earlier, it was reported that black tea scavenges NO and

peroxynitrite, and also inhibits the excessive production of NO

by the inducible form of NO synthase (iNOS) (Paquay et al.,

2000). Therefore, it seems logical to infer that BTE, because of

its antioxidant principles, might be capable of protecting the

pancreas from EtOH+CCK-induced injury and inflammatory

changes.

Our study further revealed that chronic (30 days) EtOH

exposure decreased the activities of the ROS scavenging

enzymes, SOD and CAT. This is in line with an assumption

suggested earlier by Sandhir and Gill (1999) that a decrease in

the activity of antioxidant enzymes SOD, glutathione perox-

idase (GPx) and glutathione following ethanol exposure may

be due to the damaging effects of free radicals or alternatively

could be due to a direct effect of acetaldehyde, formed from

oxidation of ethanol, on these enzymes. BTE supplementation,

in our studies, could restore the activity of both of these

antioxidant enzymes and possibly could reduce generation of

free radicals and pancreatic damage. This was found to be

similar with that observed earlier that tea flavonoids have anti-

oxidant properties (Langley-Evans, 2000; Chen et al., 2002)

and is a powerful chemopreventive agent against oxidative

damage caused by free radicals (Sarkar and Bhaduri, 2001).

Thus, results of these studies together with those of earlier

findings, suggest that BTE possibly because of its rich content

of antioxidative active principles has an ability to protect the

pancreas from EtOH+CCK-induced damage by its direct

antioxidative effect.

GSH is a naturally occurring antioxidant important in the

antioxidant defence of the body. Increased lipid peroxidation

and depletion of intracellular GSH in pancreatic tissue in

alcohol-induced acute pancreatitis have been reported (Andican

et al., 2005). Recently it has been reported that tea polyphenols

function as antioxidants through the induction of antioxidant

enzymes, such as glutathione S-transferases and superoxide

dismutases (Frei and Higdon, 2003). This observation is well in

line with our results that BTE supplementation could effec-

tively restore EtOH+CCK-induced reduction in GSH content

of pancreas.

Earlier, it has been reported that DNA fragmentation and

damage signify lipid peroxidation and increased NO produc-

tion which might lead to cell death (Heller et al., 1995; Lynn et


al., 1998; Ray et al., 2001). Measurement of DNA fragmen-

tation also has been used for quantification of apoptotic events

in cells (Suenobu et al., 1999). Indeed, our quantitative study

has revealed that EtOH+CCK-induced increased fragmenta-

tion and damage of DNA are correlated with increased lipid

peroxidation and NO production. BTE supplemented reduction

in DNA fragmentation and damage along with the reduction of

lipid peroxidation and NO production therefore suggest that

BTE has potentiality to sustain integrity of genomic DNA by

reducing oxidative stress factors. It is well documented that

green tea is an efficient radical scavenger, activates enzymes

important for detoxification and prevent in vivo DNA damage

and lipid peroxidation (Cheng et al., 1991). Our results indeed

support such notion because BTE supplementation could

effectively reduce EtOH+CCK-induced increase in DNA

fragmentation and damage, as well as NO and MDA formation.

In summary, it may be proposed that BTE prevents

EtOH+CCK-induced alterations in pancreatic acinar cell

functions, oxidative stress, inflammatory changes, DNA

damage and apoptotic changes. Since this model of

EtOH+CCK-induced pancreatic damage in rat simulates many

of the characteristic features of human alcoholic pancreatitis,

we suggest that natural antioxidants and scavenging principles

present in BTE might be equally effective in reducing human

pancreatic damage. Thus, BTE may have some obvious

therapeutic implications against chronic EtOH-induced pancre-

atitis which are to be explored.

Acknowledgements

This work was financially sponsored by the National Tea

Research Foundation (N.T.R.F.), Calcutta, India. The authors

thank Dr. J. R. Vedasiromoni, Drug Development Division,

Indian Institute of Chemical Biology, Calcutta, India for the

generous gift of black tea samples.

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