See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/259943341

Protective effect of catechin from black tea in an in-vitro model of myocardial

ischemic reperfusion injury

Article  in  Journal of Pharmacy Research · January 2014

CITATIONS READS

0 105

5 authors, including:

Mohamed Saleem Thattakudian Sheikuduman Lalit Kumar

Riyadh ELM University Manipal Academy of Higher Education, Manipal, India
79 PUBLICATIONS   924 CITATIONS    46 PUBLICATIONS   200 CITATIONS   

SEE PROFILE SEE PROFILE

Gauthaman Karunakaran

59 PUBLICATIONS   947 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Antitubercular drug design and synthesis View project

M.Phil View project

All content following this page was uploaded by Gauthaman Karunakaran on 08 December 2016.

The user has requested enhancement of the downloaded file.

 Gauthaman K et al. / Journal of Pharmacy Research 2014,8(1),1-6
Research Article Available online through
ISSN: 0974-6943 www.jpronline.info

Protective effect of catechin from black tea in an in-vitro

model of myocardial ischemic reperfusion injury
Verma R1,2, Das M 1, Mohamed Saleem T.S 1,4, Kumar L1,5 , Gauthaman K1,3*
1
Himalayan Pharmacy Institute, Majhitar, East Sikkim, India.
2
Department of Pharmaceutical Chemistry, 5Department of Pharmaceutics,
Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India.
3
Department of pharmacology, College of pharmacy, King Khalid University, Abha Kingdom of Saudi Arabia.
4
Department of Pharmacology, Annamacharya College of Pharmacy, New Boyanapalli, Rajampet-516126, A.P., India.

Received on:21-11-2013; Revised on: 08-12-2013; Accepted on:11-12-2013

ABSTRACT
Aim: Present study was designed to compare cardio-protective activity of crude aqueous extract and catechin fractions of black tea on the
oxidative stress model of myocardial ischemic reperfusion injury in rat. Method: Black tea extract and catechin fraction were administered to
male Wister rats weighing between 200-250 g in three different doses (n=6), by gastric gavage at a dose of 500 mg/kg body weight (BT1), 1000
mg/kg body weight (BT2), 1500 mg/kg body weight (BT3) and catechin fraction at a dose of 10 mg/kg body weight (CT1), 20 mg/kg body
weight (CT2), 30 mg/kg body weight (CT3) daily for thirty days. Rat’s heart was subjected to in vitro global ischemic reperfusion injury (5 min
perfusion, 9 min no flow and 12 min reperfusion). Biochemical parameters like TBARS, SOD, GSH and catalase were measured in treated
groups at basal level and in in vitro ischemic reperfusion injury of the treated groups. Results: Pre-treatment with the black tea crude extract
on the dose of both 500 & 1000 mg/kg and catechin 10, 20 & 30 mg/kg has a definite, dose-dependent modulatory effect on the antioxidant
milieu of heart. CT2IR treated group offered better cardio-protection when subjected to IR injury. No definite pattern of change in the level of
TBARS and endogenous antioxidants was observed. Hence these modulatory actions seem to be enzyme-specific. Conclusion: From these
findings it has been concluded that Catechin fraction in a smaller dose (20 mg/kg) shows significant cardioprotective activity when compared
with other doses and parent black tea extract. Further research is required to find out the mechanism of action of Catechin for cardioprotection.

KEYWORDS: Antioxidants, Ischemic reperfusion injury, Catechin, Tea Polyphenol

1. INTRODUCTION
Myocardial ischemia-reperfusion (IR) injury aggravates myocardial ral antioxidants have aroused huge interest. 3 Tea (Camellia sinensis,
damage and chiefly oxidative stress has been shown to play an im- family Theaceae) is the most widely drunk beverage throughout the
portant role. Oxidative stress plays a major role in the development ofworld, after water. 4 Black tea contains more amounts of polyphenols
IR injury through the generation of oxygen free radicals (OFR) and in comparison to other tea. Tea consumption has been associated
also depletion of endogenous antioxidants e.g. superoxide dismutase with various health benefits which are antioxidative, antimutagenic,
(SOD), reduced glutathione (GSH) and catalase (CAT). 1 anticarcinogenic, antibacterial, antidiabetic, anti-inflammatory, cardio-
protective properties chiefly because of tea flavanols.5,6 The present
Many plants based sources have been found to be a good source of research was undertaken to compare the cardioprotective potential of
phytochemical antioxidants and shows cardiac health improving prop- black tea aqueous extract and catechin fraction of black tea by oxida-
erties by scavenging free radicals. 2 Oxidative stress have been found tive stress induced myocardial IR injury.
during myocardial ischemia-reperfusion while antioxidants guard the
heart from ischemia-reperfusion injury. Therefore, for dealing with 2. MATERIALS AND METHODS
the management of heart disease antioxidants usage, particularly natu-
2.1 Drugs and Chemicals
*Corresponding author. Black tea was collected from the source (Brooke bond, Darjeeling). All
Karunakaran Gauthaman the reagents and chemicals were of analytical grade and purchased
Associate Professor, from Sigma Chemicals, USA; S.D. Fine Chemical Ltd., Mumbai, India.
Department of Pharmacology,
College of Pharmacy, 2.2 Preparation of crude aqueous extract of black tea
King Khalid University, Tea sample (500 g) was infused with freshly boiled distilled water for
Abha Kingdom of Saudi Arabia.
Journal of Pharmacy Research Vol.8 Issue 1.January 2014 1-6
 Gauthaman K et al. / Journal of Pharmacy Research 2014,8(1),1-6
10 min in a flask. The infusion was filtered through a plug of cotton once a day for 30 days. After 48 hours of the last dose rats were
wool and rapidly cooled under tap water. The extract was freeze-dried randomly selected, heparinised 375/200gms i. p, and half an hour later
and weighed to calculate the yield. 7 rats were anesthetized with anesthetic ether as per treatment proto-
col.
2.3 Preparation of Catechins enriched fraction from black tea
Black tea (500 g) was extracted with 80% ethanol and the obtained 2.7 Baseline changes in the myocardial endogenous antioxidant
ethanolic extract further fractionalted with chloroform to obtain aque- enzyme
ous decaffeinated extract. The aqueous decaffeinated extract was The heparinized, anesthetized rats were sacrificed and hearts were
fractionated with ethyl acetate to obtain catechin enriched fraction. harvested and stored in liquid nitrogen for the estimation of endog-
The catechin enriched fraction was lyophilized and purified to obtain
enous antioxidants.
brownish red catechin.8,9 Method of fractionation of catechin from
black tea present in Figure I.
The group studied for crude extract of black tea
Group BL: Vehicle (saline) treated rats.
Extract with Extract Group BT1 BL: Rats treated with 500mg/kg of crude black tea extract
80% Ethanol with in saline.
Heat chloroform Group BT2 BL: Rats treated with 1000mg/kg of crude black tea ex-
Alcohol Aqaeous
Black tea decaffeinated tract in saline.
Extract
extract Group BT3 BL: Rats treated with 1500mg/kg of crude black tea ex-
tract in saline.
Extract
with
Evaporate The group studied for the catechin fraction of black tea
EtOAC
solvent Group BL: Vehicle (water) treated rats.
and Group CT1 BL: Rats treated with 10mg/kg of catechin fraction in
Purified dark lyophilize water.
Catechin Group CT2 BL: Rats treated with 20mg/kg of catechin fraction in
brownish red catechin enriched
fraction obtained water.
fraction
Group CT3 BL: Rats treated with 30mg/kg of catechin fraction in
water.
Figure 1. Extraction of catechin fraction from black tea.
2.8 Production of in-vitro myocardial ischemic-reperfusion injury
2.4 Preliminary phytochemical screening Rats were anaesthetized with ether, hearts rapidly excised and washed
Qualitative Phytochemical screening of the crude aqueous extract of in ice – cold saline and then perfused by the non-recirculating
black tea and ethyl acetate extract (catechin fraction) was performed Langendorff’s technique (AD instruments, Australia), using a modi-
as per the standard procedures described in order to identify the fied Kreb’s Henseleits solution pH 7.4 containing (in mM): glucose
chemical constituents. 10 11.1; NaCl 118.5; NaHCO3 25; KCl 2.8; KH2PO4 1.2; CaCl2 1.2; MgSO4
0.6, with a pH of 7.4. The buffer solution equilibrated with 95% O2 +
2.5 Experimental animals 5% CO2 was delivered to the aortic cannula at 37°C less than 65 mm
Male Wistar albino rats 200–250 g body weight, were obtained from Hg pressure. An initial 5 min equilibration period was followed by 9
min. of zero flow (ischemia) and thereafter 12 min of re-flow
the Institute’s Animal House and were kept in a well-ventilated animal
(reperfusion) in order to produce IR injury. 11-13
house with 12 hours light and dark cycle, maintained in compliance
with the Guidelines for Animal experimentation. The research proto- Six rats from each group were subjected to oxidant stress arising out
col was approved by the Institute Animal Ethical Committee (HPI/08/ of IR injury of heart in this protocol and the following groups were
60/IAEC/0050). studied.

2.6 Experimental methods For crude aqueous extract of black tea

Rats were divided into four groups (n = 6) and fed either with the
crude aqueous extract of black tea in three doses 500mg/kg (BT1),
1000mg/kg (BT2), 1500mg/kg (BT3) (dose has been selected based
on previous study) or vehicle (normal saline) administered orally
through gavages once a day for 30 days along with standard rat
chow (Hindustan Lever, Mumbai, India) and water ad libitum.
Similarly for catechins enriched fraction of black tea rats were divided
into four groups (n=6) and were given catechin fractions in three
doses 10mg/kg (CT1), 20mg/kg (CT2), 30mg/kg (CT3) (dose has been
selected based on previous study) or vehicle (normal saline) daily
Journal of Pharmacy Research Vol.8 Issue 1.January 2014
Group BL: Vehicle (saline) treated rats.
Group C: vehicle- treated rat hearts subjected to 26 min. flow.
Group I: Vehicle-treated rat hearts subjected to 5 min perfusion + 9
min ischemia.
Group IR: vehicle- treated rat hearts subjected to 5 min flow + 9 min.
no-flow + 12 min. reflow.
Group BT1 IR: 500mg/kg treated rat hearts subjected to 5 min flow +
9 min. no-flow + 12 min. reflow.
Group BT2 IR: 1000mg/kg treated rat hearts subjected to 5 min flow +
9 min. no-flow + 12 min. reflow.
1-6
 Gauthaman K et al. / Journal of Pharmacy Research 2014,8(1),1-6
Group BT3 IR: 1500mg/kg treated rat hearts subjected to 5 min flow +
9 min. no-flow + 12 min. reflow.
For the catechin enriched extract of black tea
Group BL: Vehicle (saline) treated rats
Group C: vehicle- treated rat hearts subjected to 26 min. flow
Group I: Vehicle-treated rat hearts subjected to 5 min perfusion + 9
min ischemia
Group IR: vehicle- treated rat hearts subjected to 5 min flow + 9 min.
no-flow + 12 min. reflow.
Group CT1 IR: 10mg/kg treated rat hearts subjected to 5 min flow + 9
min. no-flow + 12 min. reflow.
Group CT2 IR: 20mg/kg treated rat hearts subjected to 5 min flow + 9
min. no-flow + 12 min. reflow.
Group CT3 IR: 30mg/kg treated rat hearts subjected to 5 min flow + 9
min. no-flow + 12 min. reflow.
At the end of each experiment, cardiac tissue samples were stored in
a frozen condition in nitrogen container for biochemical estimations.
2.9 Biochemical parameters
In the heart homogenate the following biochemical parameters were
studied:
Myocardial thiobarbituric acid reactive substances TBARS 14
Myocardial thiobarbituric acid reactive substances (TBARS) were
determined by a modified version of the method described by Okhawa
et al. (1979). First homogenization of the rat hearts was done in 10%
trichloroacetic acid (TCA) at 4°C. Then in a test tube homogenate
(0.2ml) was pipetted out, followed by the addition of 0.2ml of 8.1%
sodium dodecyl sulfate (SDS), 1.5ml of 20% acetic acid (pH 3.5) and
1.5ml of 0.8% thiobarbituric acid (TBA). Boiling of the tubes was
done for 60min at 95°C and then was cooled on ice. In the tubes
double distilled water (1.0ml) and 5.0ml of n-butanol–pyridine (15:1 v/
v) mixture were added and centrifuged at 4000 x g for 10min. The
absorbance of the colour which was developed in the organic layer
was measured at 532 nm. Data are expressed as nmol of TBARS/ gm
wet weight.
Myocardial reduced glutathione GSH 15
Myocardial reduced glutathione (GSH) was estimated by the method
of Ellman et al. (1959). The rat hearts were homogenized with 10%
TCA and centrifuged at 3000 x g for 10 min. The reaction mixture
constituted of 0.1mL of supernatant, 2.0ml of 0.3M phosphate buffer
(pH- 8.4), 0.4 ml of double-distilled water and 0.5ml of 5, 5 dithio bis 2-
nitrobenzoic acid (DTNB). Incubation of reaction mixture was done
for 10 minutes and then absorbance was measured at 412 nm. Data are
expressed as mole per gram wet weight.
Myocardial SOD 16
Superoxide dismutase (SOD) levels in the hearts were determined by
the method of McCord & Firdovich method (1969) and modified by
Kakkar et al., 1984. The sample (0.6ml) was added to sodium pyro-
phosphate buffer (pH-8.3), followed by the addition of 0.1ml of 186 M
phenazine methosulfate, 0.3ml of 300mM nitroblue tetrazolium and
Journal of Pharmacy Research Vol.8 Issue 1.January 2014
0.2ml of 780M NADH. For 90 seconds the reaction mixture was incu-
bated at 30°C and then the reaction was stopped by adding 1.0ml of
acetic acid, further 4.0ml of n-butanol was added and then the reac-
tion mixture was centrifuged at 3000 x g for 10min. The absorbance of
the organic layer was measured at 560 nm. Data are expressed as units
per mg protein.
Myocardial catalase 17
Catalase was estimated by the method described by Aebi and
Bergmeyer (1974). The sample was added to a 3.0-ml cuvette contain-
ing 1.95ml of 50mM phosphate buffer (pH 7.0). Then after adding1.0ml
of 30mM hydrogen peroxide, changes in absorbance were followed
for 30 s at 240nm at an interval of 15 s. Catalase levels are expressed as
units per mg protein.
2.10 Statistical analysis
All values are expressed as mean ± SEM. Data for various biochemi-
cal parameters were analyzed using analysis of variance (ANOVA) -
one way. Significance is set at p<0.05.
3. RESULTS
3.1 Preliminary phytochemical screening
A dark brownish red shiny crystal like residue of 2.0% w/w yield was
obtained for catechin fraction. Preliminary phytochemical screening
for the black tea crude extract and catechin fraction was performed.
Crude extract showed positive test for alkaloids, glycosides, phe-
nolic compounds, tannins whereas catechin fraction showed the pres-
ence of phenolic compounds and flavonoids group, catechins, tannins.
3.2 Biochemical parameters
The results of biochemical parameters are shown in Table 1 and 2.
3.2.1 Myocardial TBARS
In a crude black tea extract treated groups, a significant increase in
basal levels of myocardial TBARS on administration of black tea, at a
dose of 500 mg/kg (BT1 BL) was observed. While in the, 1000mg/kg
and 1500 mg/kg groups, the baseline TBARS was not significantly
altered. In in vitro ischemic reperfusion injury of the treated groups,
there was a significant decrease in TBARS in BT1 IR (500mg/kg) and
BT2 IR (1000mg/kg) groups.
Administration of catechin fraction shows, no change in basal levels
of myocardial TBARS was seen. In in vitro IR injury of the catechins
fraction pre-treated groups there was a significant decrease in myo-
cardial TBARS levels with all the three doses.
3.2.2 Myocardial GSH
In group treated with crude black tea extract a significant rise in basal
levels of GSH was observed in all groups. In in vitro ischemic
reperfusion injury of the treated groups a significant rise in levels of
GSH was observed in all groups.
1-6
 Gauthaman K et al. / Journal of Pharmacy Research 2014,8(1),1-6
Table 1. Observation table for the Myocardial TBARS, GSH, SOD and catalase in crude aqueous extract
(black tea) pre-treated groups.

Groups Parameters
TBARS nmole/g wet wt GSH µg/g wet wt SOD I.U/mg protein Catalase I.U/mg protein

BL 45.94 ± 1.57 327.08 ± 7.4 3.8 ± 0.1 45.0 ± 7.0

C 49.10 ± 1.9 337.65 ± 4.6 3.2 ± 0.1 44.5 ± 2.9
I 48.8 ± 1.31 323.55 ± 6.06 1.8 ± 0.3* 25.1 ± 6.1*
IR 59.73 ± 1.62*** 279.41 ± 6.05*** 1.7 ± 0.2* 33.4 ± 0.9*
BT1BL 64.64 ± 7.88 450.82 ± 24.37 * 6.5± 0.6* 35.6 ± 0.18*
BT1IR 44.18 ± 6.41 363.64 ± 16.54** 8.2 ± 0.5? 32.4 ± 0.43?
BT2BL 95.55 ± 16.09** 571.28 ± 80.05 18.8 ± 0.4* 94.6 ± 0.13
BT2IR 74.28 ± 14.06** 544.77 ± 123.05 * 15.9 ± 0.1* 82.4± 8.1
BT3BL 61.52 ± 5.17* 406.77 ± 38.26 15.4 ± 0.7* 69.6 ± 6.6*
BT3IR 71.51 ± 4.63* 401.89 ± 36.35 * 13.7 ± 0.1 ? 70.8 ± 4.2 ?

All values are expressed as Mean ± SEM (n=6) ***p < 0.001 vs BL & I **p < 0.01 vs IR & BL *p < 0.05 vs IR & BL (One way ANOVA)

Table 2 Observation table for the Myocardial TBARS, GSH, SOD and catalase in catechins fraction (black tea) pre-treated groups.
Groups Parameters
TBARS nmole/g wet wt GSH µg/g wet wt SOD I.U/mg protein Catalase I.U/mg protein

BL 45.94 ± 1.57 327.08 ± 7.4 3.4 ± 0.1 43.0 ± 7.0

C 49.10 ± 1.9 337.65 ± 4.6 3.2 ± 0.1 44.5 ± 2.9
I 48.8 ± 1.31 323.55 ± 6.06 1.8 ± 0.3* 25.1 ± 6.1*
IR 59.73 ± 1.62*** 279.41 ± 6.05*** 2.7 ± 0.2* 33.3 ± 1.1*
T1BL 58.4 ± 3.9* 849.6 ± 18.0* 11.7 ± 1.0* 62.4 ± 3.3*
T1IR 52.1 ± 3.8 ? 436.2 ± 16.5? 8.4 ± 0.9? 43.5 ± 3.3 ?
T2BL 48.3 ± 5.7 1059.5 ± 18.1* 18.2 ± 1.1* 47.0 ± 1.8
T2IR 53.8 ± 3.4 ? 519.4 ± 12.1? 9.0 ± 0.7* 40.3 ± 5.9
T3BL 54.2 ± 3.3 636.6 ± 8.7* 10.3 ± 0.6* 66.3 ± 6.3*
T3IR 55.2 ± 4.6 463.0 ± 3.9? 8.2 ± 0.7? 50.8 ± 4.8 ?

All values are expressed as Mean ±SEM (n=6) *p < 0.001 vs BL ?p < 0.001 vs IR (One way ANOVA)

Administration of catechin fraction shows a significant increase in 4. DISCUSSION

the levels of antioxidants GSH in T1 BL and T2 BL groups was seen.
In both CT2IR and CT3IR groups, a significant rise in the levels of
GSH was observed, and they showed a better recovery profile than
the T1IR groups subjected to in vitro IR injury.
3.2.3 Myocardial SOD
In crude black tea extract treated group a significant rise in basal
levels of SOD was observed in all groups. In group BT2 IR, there was
no significant increase in SOD levels.
Administration of catechin fraction shows significant increase in the
levels of antioxidants SOD in T1 BL and T2 BL groups. A significant
rise in SOD level was observed in both CT2IR and CT3IR groups.
3.2.4 Myocardial Catalase
Crude black tea extract: A Significant rise in basal levels of catalase
was observed in all groups. In group BT2 IR, there was no significant
increase catalase level.
In our study, the antioxidant activity of black tea in in vitro model of
myocardial ischemic-reperfusion injury was evaluated. Interestingly,
there was a significant increase in basal levels of myocardial TBARS
on administration of black tea, at a dose of 500 mg/kg (BT1 BL). It is
generally accepted that oxygen free radicals are key mediators of
myocardial oxidative stress-induced injury. 18,19 The increase in TBARS
level is an indicator of enhanced oxidative stress in ischemia which
might have stimulated the protective mechanism by enhancing en-
dogenous antioxidants.20,21 While in the, 1000mg/kg and 1500 mg/kg
groups, the baseline TBARS was not significantly altered. The rea-
son why a lower dose of 500 mg/kg caused a significant TBARS
accumulation is not forthcoming from the present study. Also, sig-
nificant rise in baseline GSH, SOD and Catalase was noted in black
tea treated groups. These are clear indications that oral administra-
tion of black tea can augment the endogenous cardiac antioxidant
defense system, along with an apparent lipid peroxidation, occurring
during this phase. The exact mechanism of this effect is not clear and
needs further investigation.

Administration of catechin fraction shows a significant increase in In in vitro ischemic reperfusion injury of the treated groups, a signifi-
the levels of catalase was observed only in the T2 BL group. A signifi- cant decrease in TBARS in BT1 IR (500mg/kg) and BT2 IR (1000mg/
cant rise in SOD level was observed in both CT2IR and CT3IR groups. kg) groups was seen. A significant rise in levels of GSH was observed

Journal of Pharmacy Research Vol.8 Issue 1.January 2014 1-6

 Gauthaman K et al. / Journal of Pharmacy Research 2014,8(1),1-6
in all groups. In group BT2 IR, there was no significant increase in
SOD and catalase levels. It has been proven that if cellular SOD
increases without an associated increase in catalase cause more harm
by favouring the production of H2O2.22-24
In the present study, treatment with catechin fraction showed that
there was no change in basal levels of myocardial TBARS, while
there was a significant increase in the levels of antioxidants (GSH and
SOD) in CT1 BL and CT2 BL groups. A significant increase in the
levels of catalase was observed only in the CT2 BL group. Similarly,
catechin pre-treated rats showed no significant changes in the spe-
cific activities of antioxidant enzymes. The increased activities of
endogenous antioxidant enzyme levels should protect the myocar-
dium when subjected to oxidative stress.25 In the present study,
reperfusion of the ischemic myocardium was reported to cause de-
pression in the biochemical recovery (antioxidant status), as reported
earlier.26 The widely reported oxidant stress induced by IR injury was
evidenced by enhanced lipid peroxidation and deterioration of en-
dogenous antioxidant status (SOD, catalase and glutathione).
In in vitro IR injury of the catechins fraction pre-treated groups there
was a significant decrease in myocardial TBARS levels with all the
three doses. In both CT2IR and CT3IR groups, a significant rise in the
levels of GSH, SOD and catalase were observed, and they showed a
better recovery profile than the CT1IR group subjected to in vitro IR
injury. This indicates that this dosage withstands the oxidative stress
associated with in vitro IR injury. On the other hand, in CT1IR group,
the GSH and SOD show significant protection but not the catalase.
Previous studies have shown that changes in tissue antioxidant en-
zyme activities in response to internal or external stimuli, do not al-
ways parallel each other. The exact stimulus for the altered activity of
the catalase is not known; however during oxidation stress radical
formation is increased which they may act as a signal. 27, 20
5. CONCLUSION
The results from pharmacological activities demonstrate that pre-
treatment with the Black tea crude extract at the dose of both 500 &
1000 mg/kg and administration of catechin at a dose of 20 mg/kg has
a positive, dose-dependent variable effect on the antioxidant status
of the heart. Thus catechins fraction from black tea can provide post-
ischemic myocardial preservation through endogenous antioxidant
potential mechanism. The CT2IR treated group offered better Cardio-
protection when subjected to IR injury. There was also no certain
pattern of change in the level of TBARS and the endogenous antioxi-
dants. Therefore these varying actions appear to be enzyme-specific.
Further study is required to explore mechanisms causing these
changes in antioxidant enzyme expression due to oxidant stress and
drug administration also, it will be more fruitful if isolation of the
compounds is done which are responsible for the particular enzyme
action.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Journal of Pharmacy Research Vol.8 Issue 1.January 2014
Funding
This research was supported by AICTE, New Delhi under Research
Promotion Scheme.
REFERENCES
1. Gauthaman K, Saleem TSM, Ravi V, Patel SS, Devaraj SN.
Alcoholic Extract of Terminalia Arjuna Protects Rabbit Heart
against Ischemic-Reperfusion Injury: Role of Antioxidant
Enzymes and Heat Shock Protein. World Academy of Science,
Engineering and Technology. 2008; 42: 488-98.
2. Sunan W, John PM, Rong T, Massimo FM. How natural
dietary antioxidants in fruits, vegetables and legumes pro-
mote vascular health. Food Research International. 2011;
44: 14–22.
3. Zhao Y, Zhao B. Protective Effect of Natural Antioxidants on
Heart against Ischemia- Reperfusion Damage. Current Phar-
maceutical Biotechnology. 2010; 11(8): 868-74.
4. Labbe D, Tremblay A, Bazinet L. Effect of brewing tempera-
ture and duration on green tea catechin solubilization: basis
for production of EGC and EGCG enriched fractions. Sepa-
ration and Purification Technology. 2006; 49: 1-9.
5. Sajilata MG, Bajaj RP, Singhal RS. Tea polyphenols as
nutraceuticals. Comprehens Rev Food Sci & Food Saf. 2008;
7: 229-54.
6. Cabrera C, Gimenez R, Lopez CM. Determination of Tea Com-
ponents with Antioxidant Activity. J Agric Food Chem. 2003;
51: 4427-35.
7. Erol TN, Sari F, Polat G, Velioglu SY. Antioxidant and Anti-
bacterial Activities of Various Extracts and Fractions of Fresh
Tea Leaves and Green Tea. Tarim Bilimleri Dergisi. 2009;
15(4): 371-8.
8. Cutler SJ, Cutler HG. Naturally occurring antimutagenic and
cytoprotective agents. In: Biologically active natural prod-
ucts: Pharmaceutics. USA: CRC press Boca Raton; 2000. p.
136.
9. Rangari V. General methods of extraction, isolation and puri-
fication. In: Pharmacognosy and Phytochemistry. 1st Ed.,
Pune: Career publication (India); 2007: p. 131.
10. Raaman N. Qualitative phytochemical screening. In: Phy-
tochemical techniques-A guide to modern techniques of
plant analysis. New Delhi: New India Publishing Agency
(India); 2006: p. 19 -24.
11. Borchgrevink PC, Schie R, Bergan AS, Bakoy OE, Jynge P.
Magnesium and reperfusion of ischemic rat heart as accessed
by 31P NMR. Am J Physio. 1989; 256: 11195-204.
12. Maulik M, Maulik SK, Kumari R. Importance of dosage and
timing of magnesium administration: A study on the Sesame
oil enhances endogenous antioxidants in ischemic myocar-
dium of rat isolated ischemic-reperfused heart. Mag Res.
1999; 12: 37-42.
13. Gauthaman K, Maulik M, Kumari R, Manchanda SC, Dinda
AK, Maulik SK. Effect of chronic treatment with bark of
Terminalia arjuna: A study on the isolated ischemic-
reperfused rat heart. J Ethanopharmacol. 2001; 75: 197-201.
1-6
 Gauthaman K et al. / Journal of Pharmacy Research 2014,8(1),1-6

14. Okhawa H, Oohishi N, Yagi K. Assay for lipid peroxides in
animal tissues by thiobarbituric acid reaction. Ann Bichem.
1979; 95: 351-58.
15. Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophy.
1959; 82: 70-77.
16. Kakkar P, Das B, Viswanathan PN. A modified spectrophoto-
metric assay of superoxide dismutase. Ind J Biochem Biophy.
1984; 21: 130-32.
17. Aebi H, Bergmeyer HU. Catalase. In: Methods of enzymatic
analysis. Vol 2. 2nd Ed. In: H.U. Bergmeyer HU, editor. Verlag
Chemie, Weinheim: Academic Press. 1974: p. 673-85.
18. Maxwell SRJ. Reperfusion injury: a review of the pathophysi-
ology clinical manifestation and therapeutic option. Int J
Cardiol. 1997; 58: 95-117.
19. Jennings RB. Myocardial Ischemia: observations, definitions
and speculation’s. Mol Cell Cardiol. 1970; 1(4): 345-49.
20. Singal PK, Dhalla AK, Hill M, Thomas TP. Endogenous an-
tioxidant changes in the myocardium in response to acute
and chronic stress conditions. Mol Cell Biochem. 1993; 129:
179-86.
21. Maulik G, Mukherjee S, Das DK. Evaluation of antioxidant
effectiveness of a new selected vegetable. Environ Nutr
Interac. 1997; 1: 287- 97.
22. Yim MB, Chock PB, Stadtman ER. Copper, Zinc super oxide
Dismutase catalyzes hydroxyl radical production from hy-
drogen peroxide. National Academic Science. 1990; 87(13):
5006–10.
23. Michiels C, Raes M, Toussaint O, Remacle J. Importance of
Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell
survival against oxidative stress. Free Rad Biol Med. 1994;
17(3): 235-48.
24. Gardner R, Salvador A, Moradas-Ferreira P. Why does SOD
overexpression sometimes enhance, sometimes decrease,
hydrogen peroxide production? A minimalist explanation.
Free Rad Biol Med. 2002; 32(12): 1351–57.
25. Dhalla SN, Elmoselhia BA, Hatac T, Makinoc N. Status of
myocardial antioxidants in ischemia–reperfusion injury.
Cardio Res. 2000; 47: 446–56.
26. Banerjee SK, Dinda AK, Manchanda SC, Maulik SK. Chronic
garlic administration protects rat heart against oxidative
stress induced by ischemic reperfusion injury. BMC
Pharmacol. 2002; 2: 16.
27. Fuji H, Kuzuya T, Hoshida S, Yamashita N, Oe H, Taniguchi
N, et al. Reoxygenation-induced synthesis of mitochondrial
Manganese-superoxide dismutase in rat neonatal myocar-
dial cells. J Mol Cell Cardiol. 1992; 24: S168.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.8 Issue 1.January 2014 1-6

View publication stats