Chemico-Biological Interactions 181 (2009) 472479

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Chemico-Biological Interactions
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The potential for interaction of hydrochlorothiazide with garlic in rats
Syed Mohammed Basheeruddin Asdaq
a,
, Mohammed Naseeruddin Inamdar
b
a
Department of Pharmacology, Krupanidhi College of Pharmacy, Varthur Hobli, Chikkabellandur Village, Carmalaram Post, Bangalore 560035, India
b
Department of Pharmacology, Al-Ameen College of Pharmacy, Bangalore 560027, India
a r t i c l e i n f o
Article history:
Received 2 April 2009
Received in revised form 24 July 2009
Accepted 27 July 2009
Available online 4 August 2009
Keywords:
Garlic
Hydrochlorothiazide
Interaction
Isoproterenol
a b s t r a c t
The present study was undertaken to determine the pharmacokinetic and pharmacodynamic interaction
of hydrochlorothiazide (HCTZ) with garlic homogenate (GH), in rats. The inuence of garlic on pharma-
cokinetics of HCTZ was studied by HPLC method, while pharmacodynamic interaction was studied using
diuretic activity, ECGand BP changes and isoproterenol (ISO) induced myocardial injury. HCTZ was given
orally at 10mg/kg and GH was administered at three different doses of 125, 250 and 500mg/kg, p.o. The
CK-MB, LDH, SOD, catalase and histopathological studies were carried out. The administration of HCTZ
in GH pretreated rats found to decrease the QRS duration, RR interval, QT segment, systolic blood pres-
sure, heart rate, serumpotassiumlevel, serumLDHand serumCK-MB activities signicantly. The diuretic
effect of HCTZwas signicantlyincreasedinpresenceof GH; however, kaliuresis was signicantlyreduced
in presence of GH 250mg/kg. Histopathological studies of heart tissue reveal the protective effect of GH
250mg/kg in presence or absence of HCTZ during ISOstress to myocardium. The pharmacokinetic studies
showthat GHincreases the bioavailability and half-life, along with decrease in clearance and elimination
rate of HCTZ when administered orally. It was concluded that careful addition of garlic in moderate doses
might result in benecial effect during treatment of hypertension in patients with myocardial stress
as garlic causes substantial fall in excretion of potassium when compared to HCTZ alone treatment in
rats. This could be important in reducing the dose of HCTZ to achieve enhanced therapeutic effect with
minimal adverse effect.
2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
During the recent past, a dramatic rise in the use of herbs and
herbal remedies has been witnessed in many parts of the world [1].
While such products had been used with apparent safety in tradi-
tional societies for many centuries, when they are being combined
with pharmacological agents, posses the possibility of potential
interaction between the two groups of substances. Reports indi-
cate that about 1520% of individuals on prescription medications
also use herbal supplements and less than 40% of patients disclose
to their physicians the usage of herbal remedies, even if they expe-
rience severe side effectsbecause of the fear of censure or rebuke
[2]. The problem is further compounded by the fact that many
physicians are themselves not always familiar with the potential
for herbdrug interactions [3]. Hence, it is imperative to promote
credible research on the safety and efcacy of combined herbdrug
treatment for variety of ailments including cardiovascular diseases
[4].
Diuretics, in particular hydrochlorothiazide (HCTZ), are often
avoided as monotherapy in the management of hypertension in

Corresponding author. Tel.: +91 80 65973260; fax: +91 80 51309161.

E-mail addresses: basheer 1@rediffmail.com, sasdaq@gmail.com(S.M.B. Asdaq).
patients with ischemic heart diseases. Thiazides affect the renal
tubular mechanisms of electrolyte reabsorption, directly increas-
ing excretion of sodium and chloride in approximately equivalent
amounts. Indirectly, the diuretic action of hydrochlorothiazide
reduces plasma volume, withconsequent increase inurinarypotas-
siumloss, plasma reninactivity, aldosteronesecretionanddecrease
in serum potassium [5,6]. In patients with cardiac ischemia, heart
failure or left ventricular hypertrophy, even mild-to-moderate
hypokalemia increases the likelihood of cardiac arrhythmias [79].
The combination of HCTZ with ACE-I, aldosterone antagonist or
angiotensin II type 1 receptor blocker is found to minimize the
potassium loss characteristically induced by the thiazide compo-
nent.
Traditionally, garlic (Allium sativum) and its preparations have
been widely recognized as agents for the prevention and treat-
ment of cardiovascular and other metabolic diseases, such as
atherosclerosis, hyperlipidemia, thrombosis, hypertensionanddia-
betes. Garlic contains biologically active compounds that exert
multiple benecial effects on human organism. We previously
reported improved survival and cardiac function by add-on gar-
lic therapy with propranolol [10] and captopril [11] in rats with
myocardial infarction. However, there is noscientic report toindi-
cate the effect of combinedtherapy of garlic withHCTZ. The present
study was undertaken to evaluate the pharmacokinetic and phar-
0009-2797/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2009.07.022
S.M.B. Asdaq, M.N. Inamdar / Chemico-Biological Interactions 181 (2009) 472479 473
macodynamic interaction of HCTZ with garlic using experimental
models in rats.
2. Materials and methods
2.1. Experimental animals
Laboratory bred female Wistar albino rats (200250g) were
housedat 255

Cina well-ventilatedanimal house under 12:12h

light dark cycle. The rats had free access to standard rat chow
(Amrut Laboratory Animal feed, Maharashtra, India) containing (%,
w/w) protein 22.10, oil 4.13, bre 3.15, ash 5.15, sand (silica) 1.12,
and water ad libitum. The animals were maintained under stan-
dard conditions in an animal house approved by Committee for
the Purpose of Control and Supervision on Experiments on Animals
(CPCSEA).
2.2. Chemical
All chemicals used were of analytical grade and purchased
fromstandard companies. Pure sample of hydrochlorothiazide was
gifted by Bangalore Test House (Bangalore, India). Biochemical kits
were procured from Crest Biosystems (Goa, India).
2.3. Preparation of garlic homogenate
Garlic (A. sativum, family: Liliaceae) bulbs were purchased from
the local market. The cloves were peeled, sliced and ground into a
paste and suspended in distilled water. Three different concentra-
tions of the garlic homogenate (GH) were prepared, 0.05, 0.1 and
0.2g/ml, corresponding to 125, 250 and 500mg/kg body weight of
animal [12]. GH was administered within 30min of preparation.
2.4. Determination of diuretic activity
The method of Wiebelhaus et al. [13] was used, with mod-
ication, for the determination of diuretic effect. Adult female
albino rats weighing 200250g were divided into following
eight groups consisting of eight animals each: group I: vehi-
cle (1ml/kg, p.o. for 30 days); groups IIIV: garlic homogenate
125, 250 and 500mg/kg respectively for 30 days orally; group
V: hydrochlorothiazide 10mg/kg on the day of experiment, p.o.;
groups VIVIII: garlic homogenate 125, 250 and 500mg/kg respec-
tively for 30 days +HCTZ. Prophylactically treated animals were
fasted overnight with water allowed ad libitum. The following
morning rats were given orally 25ml/kg of normal saline solution,
and immediately after normal saline administration, the rats were
placedindividually ina modiedfunnel having a wire meshandt-
ted with a graduated test tube. In HCTZ incorporated groups, HCTZ
was given orally as a ne homogenized suspension in a volume of
25ml/kg of normal saline solution. Urine excreted for the next 5h
was collected and the total volume of urine for each rat was com-
paredwiththevolumeof urineproducedafter theadministrationof
normal saline. The volume of urine excretedduring 5hfor eachani-
mal in the group is expressed as the percent of the liquid (normal
saline) administered. This percentage gives a measure of urinary
excretion independent of the animal weight. The ratio of urinary
excretion in the test group to urinary excretion in the control group
is used as a measure of the diuretic action for the given dose of
the drug. As the diuretic action is prone to variability, a param-
eter known as diuretic activity was calculated instead. To obtain
the diuretic activity, the diuretic action of the test groups (gar-
lic homogenate) was compared with that of the standard (HCTZ)
[14]. Percentage of saline load excreted=volume of urine/volume
of saline load100. Urinary excretion=total urinary output/total
liquid administered100. Diuretic action=urinary excretion of
treated group/urinary excretion of control group. Diuretic activ-
ity=diuretic action of test drug/diuretic action of standard drug.
Urinary Na
+
and K
+
contents were analyzed by ame photometer,
while Cl

content was measured by auto analyzer.

2.5. Isoproterenol (ISO) induced myocardial damage
The animals were divided into seven groups consisting of
eight animals each: group I: vehicle (1ml/kg, p.o. for 30 days);
group II: isoproterenol (175mg/kg, s.c); group III: hydrochloroth-
iazide 10mg/kg [14] for 7 days, p.o.; group IV: garlic homogenate
250mg/kg for 30 days orally; groups VVII: garlic homogenate 125,
250 and 500mg/kg respectively for 30 days and HCTZ for last 7
days p.o. At the end of treatment period, animals of all groups
excluding group I were administered ISO (175mg/kgs.c) for 2 con-
secutive days. Blood was withdrawn from retro orbital vein 48h
after therst doseof ISOunder anesthesiaandserumwas separated
by centrifugation for lactate dehydrogenase (LDH) and creatine
phosphokinase-MB(CK-MB) measurement. Thebloodpressureand
ECG changes were recorded under appropriate conditions. The
heart was immediately isolated from each animal under ketamine
(70mg/kg, i.p) and xylazine (10mg/kg, i.p) anesthesia. In each
group consisting of eight animals, four excised hearts were homog-
enized to prepare heart tissue homogenate (HTH) using sucrose
(0.25M) [15]. The activity of LDH, CK-MB, superoxide dismutase
(SOD) [16] and catalase [17] were measured in HTH. Microscopic
slides of myocardium were prepared for histopathological studies
fromthe hearts of remaining four animals. The myocardial damage
was determined by giving scores depending on the intensity as fol-
lows [18]; no changes score 00; mild score 01 (focal myocytes
damage or small multifocal degeneration with slight degree of
inammatory process); moderate score 02 (extensive myobril-
lar degeneration and/or diffuse inammatory process); marked
score 03 (necrosis with diffuse inammatory process).
2.6. Blood pressure and electrocardiograms measurement
As discussed above, 48h after rst dose ISO administration just
prior to collection of blood samples, mean arterial blood pres-
sure was measured in awaked animals by the non-invasive blood
pressure module (NIBP pressure meter, LE 5001, V02/0402L, Pan-
lab, Hardvard apparatus, Barcelona, Spain) and ECG was recorded
in anesthetised animals [ketamine (70mg/kg, i.p) and xylazine
(10mg/kg, i.p)] by subcutaneous lead II method (Physiograph, EKG
coupler, SO-02, INCO, India), QRS duration, RR interval and QT seg-
ment was measured.
2.7. Statistical analysis
Results of pharmacodynamic parameters are expressed as
meanSEM. Thestatistical signicancewas determinedusingone-
wayanalysis of variance(ANOVA) followedbyBonferronis test. The
results were considered statistically signicant when P<0.05.
2.8. Pharmacokinetic interaction
Both high and moderate doses of garlic homogenate (500 and
250mg/kg, p.o.) were selected for this interaction study. Animals
were divided into three groups consisting of eight animals each:
group I: hydrochlorothiazide 10mg/kg p.o. (single dose), group II:
GH 250mg/kg for 30 days (p.o.) +HCTZ (single dose) and group III:
GH 500mg/kg for 30 days (p.o.) +HCTZ (single dose). Immediately
after HCTZ administration, 0.5ml of blood samples was withdrawn
at each time intervals over 24h (0, 1, 2, 4, 8, 16 and 24h) by
puncturing retro orbital vein under partial ether anesthesia and
474 S.M.B. Asdaq, M.N. Inamdar / Chemico-Biological Interactions 181 (2009) 472479
subjected to analysis. The hypovolaemia is prevented by intraperi-
toneal administration of 0.5ml of normal saline immediately after
each withdrawal of blood.
2.9. Extraction procedure [19]
Solid sodiumbicarbonate (200mg) and ethyl acetate (2ml) was
added to 1ml serum. After mixing on a Vortex mixer for 1min
and centrifuging for 5min, 1ml of the organic layer was trans-
ferredtoanother tube and0.5ml of a 1Nsodiumhydroxide solution
added. This solution was mixed and allowed to stand for 1min at
roomtemperature, after whichtheupper layer was discarded. Ether
(1ml) was then added to the sodium hydroxide solution, followed
by mixing. After standing for 1min, the ether layer was discarded
and traces of ether were removed with a gentle stream of air. An
aliquot of the aqueous layer was injected into the liquid chromato-
graph. Since initial studies indicated that hydrochlorothiazide was
unstable instronglyalkaline media for prolongedperiods, the back-
extraction step into sodiumhydroxide solution was performed just
prior to the liquid chromatographic analysis.
Liquid chromatography: a Waters 6000 pumping system
(Waters Associates) coupledto a SpectroowSF 770 variable wave-
length detector (Schoeffel Instrument Corp.) was used for these
studies. Areverse phase systemconsisting of a 30cm4mmFBon-
dapak CIS column (Waters Associates) was utilized with methanol
0.01Msodiumdihydrogenphosphate solution(1:4) as the eluent,
at a ow rate of 0.6ml/min. The detector was operated at 271nm.
For the lower levels present in serum, a sensitivity of 0.01A (full
scale deection) was required. Peak heights were used for quan-
titation. All standard curves were linear and passed through the
origin.
2.10. Pharmacokinetic determination [20]
For the generated data on hydrochlorothiazide and garlic inter-
action to be analyzed, we assume that the kinetics of HCTZ
elimination was linear. The data was represented in a plasma
level-time curve fromwhere the area under time curve (AUC
024h
)
was calculated using Trapezoid rule. The maximum concentra-
tion (C
max
) and maximum time (T
max
) were obtained directly
from generated data. The elimination constant (K
e
) and half-
life (T
1/2
) were determined from the semi-log plot of the data.
The clearance (CL) and apparent volume of distribution (V
d
) of
the drug in the animals were calculated from the equations:
CL =V
d
K
e
, V
d
: the administered dose of drug/initial plasma
concentration of drug obtained at intercept of semi-log plot
of plasma drug sample. AUC
total
=AUC
024h
+C
24h
/K
e
. The mean
plasma concentrationtime curve for HCTZ (10mg/kg) alone and
HCTZ+ once a day administration of oral garlic homogenate
(250mg/kg) for 30 days was determined. The study was done for
Fig. 1. Effect of garlic on plasma concentration of HCTZ.
24h since the half-life of HCTZ is 5.68h [21]. The results were
analyzed statistically using students t-test.
3. Results
3.1. Effect on electrolyte excretion in urine (Table 1)
As shown in Table 1, there was signicant increase in excretion
of Na
+
and Cl

electrolytes in urine of animals previously treated

with GH125, GH250 and GH500mg/kg. Further, the extent of Na
+
,
K
+
and Cl

excretion is remarkably high in animals treated with

HCTZ, indicating its enhanced potency. Moreover, upon addition of
HCTZinGHtreatedanimals, there was further increase ininhibition
of reabsorption of Na
+
and Cl

. However, there was no signicant

change in the excretion of K
+
in groups pretreated with GH 125,
GH 250 and GH 500mg/kg as compared to control. The GH treated
groups remained unaltered upon addition of HCTZ in excretion of
K
+
when compared to control. The maximum effect was seen with
combination of HCTZ and GH (250mg/kg, p.o.).
3.2. Effect on percentage of saline load excreted, diuretic action
and diuretic activity (Table 2)
As evident from Table 2, there was signicant increase in per-
centage of saline load excretion in animals pretreated with GH250
when compared to control group. It is noted that HCTZ signicantly
enhances saline load excretion that is further augmented signi-
cantly in animals pretreated with moderate dose of GH (GH 250).
As indicated by Table 2, the diuretic action and diuretic activity was
signicantly elevated when GH and HCTZ were used concurrently.
The maximum diuretic activity was seen at moderate doses of GH
in presence of HCTZ.
Table 1
Effect on electrolyte excretion in urine.
Groups Electrolyte concentration (mmol/l)
Na
+
K
+
Cl

Control 54.35 1.19 45.15 2.23 110.48 1.96

GH 125mg/kg, p.o. 65.12 3.67
**
41.41 1.49 120.51 1.53
*
GH 250mg/kg, p.o. 87.73 2.01
***
39.49 1.43 135.98 3.68
**
GH 500mg/kg, p.o. 72.29 2.81
***
40.21 0.98 113.03 4.34
*
HCTZ (10mg/kg, p.o.) 109.20 2.01
***
59.42 1.71
**
137.98 1.58
***
GH 125mg/kg, p.o. +HCTZ 127.97 4.10
***,aa,bb
45.44 0.82
a,b
151.75 3.22
***,a,b
GH 250mg/kg, p.o. +HCTZ 138.07 2.07
***,aaa,bbb
40.68 1.31
bbb
163.40 0.78
***,aa,bb
GH 500mg/kg, p.o. +HCTZ 123.02 2.91
***,aa,bb
51.23 0.88
*,aa
151.70 1.30
***,a,b
All values are meanSEM, n=8;
*
P<0.05,
**
P<0.01,
***
P<0.001 when compared to control;
a
P<0.05,
aa
P<0.01,
aaa
P< 0.001 when compared to corresponding dose of GH
alone;
b
P<0.05,
bb
P<0.01,
bbb
P<0.001 when compared to HCTZ (comparison between HCTZ vs HCTZ+GH).
In GH groups 30 days of GH p.o.; in HCTZ group single dose of HCTZ p.o. and in interactive groups 30 days of GH treatment p.o. +single dose of HCTZ p.o.
S.M.B. Asdaq, M.N. Inamdar / Chemico-Biological Interactions 181 (2009) 472479 475
Table 2
Effect on percentage of saline load excreted, diuretic action and diuretic activity.
Groups Percentage of saline load excreted Diuretic action Diuretic activity
Control 46.70 0.63 1
GH 125mg/kg, p.o. 68.16 2.12
*
1.45
*
0.53
GH 250mg/kg, p.o. 75.59 13.5
***
1.61
*
0.59
GH 500mg/kg, p.o. 64.50 1.32
*
1.38
*
0.51
HCTZ (10mg/kg, p.o.) 128.74 1.54
***
2.7
**

GH 125mg/kg, p.o. +HCTZ 134.11 1.82

***,aaa
2.87
**,aaa
1.06
GH 250mg/kg, p.o. +HCTZ 166.45 1.36
***,aaa,bb
3.56
***,aaa,b
1.31
GH 500mg/kg, p.o. +HCTZ 137.23 2.57
***,aaa
2.93
**,aaa
1.08
All values are meanSEM, n=8;
*
P<0.05,
**
P<0.01,
***
P<0.001 when compared to control;
a
P<0.05,
aa
P<0.01,
aaa
P<0.001 when compared to corresponding dose of GH
alone;
b
P<0.05,
bb
P<0.01,
bbb
P<0.001 when compared to HCTZ (comparison between HCTZ vs HCTZ+GH).
In GH groups 30 days of GH p.o.; in HCTZ group single dose of HCTZ p.o. and in interactive groups 30 days of GH treatment p.o. +single dose of HCTZ p.o.
Table 3
Effect on electrocardiographic parameters and hemodynamic ndings in rats.
Groups sBP (mmHg) HR (beats/min) Body weight (g) HW/BW (mg/g) QRS duration (ms) QT segment (ms) RR interval (ms)
Control 125 2 345 25 241 3 3.42 0.12 15.211.11 55.11 4.43 14.31 0.87
Isoproterenol control 112 4
*
386 32
*
195 2
***
4.52 0.32
*
18.58 2.10
*
59.03 5.32 16.21 1.81
HCTZ (10mg/kg, p.o.) 118 3
*
357 21

221 4
*,
4.01 0.22
*,
19.22 2.19
**
54.10 4.92 15.21 1.21
GH 250mg/kg, p.o. 113 4
*
34132

232 6

3.54 0.31
b,
14.84 1.47
*
53.19 2.23

14.21 1.43

GH 125mg/kg, p.o. +HCTZ 108 5

*,b
329 18
*,b,
224 8
*,
3.87 0.43
*,b,
15.33 1.87
*
54.15 2.18 15.21 1.44
GH 250mg/kg, p.o. +HCTZ 104 4
*,b
315 22
*,bb,
242 5
b,
3.43 0.51
b,
14.322.16
*,bb,
53.17 1.19

14.13 1.21

GH 500mg/kg, p.o. +HCTZ 111 8

*
331 21
*,b,
210 7
*
3.97 0.37
*,
16.98 1.98 58.43 1.13 15.43 1.54
All values are meanSEM, n=8;
*
P<0.05,
**
P<0.01,
***
P<0.001 when compared to control; P<0.05,

P<0.01,

P<0.001 when compared to isoproterenol control;
b
P<0.05,
bb
P<0.01,
bbb
P<0.001 when compared to HCTZ (comparison between HCTZ vs HCTZ+GH), sBP: systolic blood pressure, HR: heart rate, HW/BW: heart weight/body weight.
In GH groups 30 days of GH p.o.; in HCTZ group 7 days of HCTZ p.o. and in interactive groups 30 days of GH treatment p.o. +7 days of HCTZ p.o. At the end of treatment,
all groups except control, were subjected to two dose of ISO 175mg/kgs.c.
3.3. Effect on electrocardiographic parameters and hemodynamic
ndings (Table 3)
All the treatments showed signicant decrease in systolic blood
pressure compared to control. The combination of GH 250mg/kg
with HCTZ showed more reduction in systolic blood pressure
compared to their individual treatments. The heart rate was signif-
icantly elevated in isoproterenol (ISO) control compared to normal
control. However, the chronotropic effect was signicantly reduced
inall treatment groups comparedtoISOcontrol. Thecombinationof
GH250mg/kg with HCTZ was more effective than individual treat-
ment in reducing inclined heart rate as compared to ISO control.
The body weight of animal was signicantly reduced in iso-
proterenol administered group when compared to normal control.
Similarly, fall inbodyweight was alsofoundinHCTZ, GH125+HCTZ
as well as in GH 500+HCTZ groups. However, pretreatment of ani-
mals with GH 250mg/kg either in presence or absence of HCTZ
found to revert back the normal weight. Heart weight/body weight
ratio was augmented in ISO rat which was signicantly reduced
in all treatment groups. Administration of HCTZ in GH (250mg/kg,
p.o.) pretreated animals demonstrated maximum fall in elevated
HW/BW ratio compared to their individual treatment.
Signicant changes in the ECG conguration were observed in
the rats with ISO treatment such as signicant prolongation of QRS
durationas well as non-signicant longer QT segment andRRinter-
val. The HCTZ treated animals showed signicant prolongation of
QRS complex compared to normal control. The combination of GH
250mg/kg with HCTZ was found to signicantly decrease the QRS
complex compared to their individual treatment. There was fall in
QT segment and RR interval in GH 250mg/kg alone or in presence
of HCTZ compared to ISO control.
3.4. Effect on biochemical parameters, antioxidants and
histological scores (Table 4)
The LDH, CK-MB, SOD and catalase activities were signicantly
decreased in heart tissue homogenate (HTH) by ISO treatment
compared to normal control. Prior treatment of rats with HCTZ
(P<0.05) and GH250mg/kg (P<0.001) showed signicant increase
in these parameters compared to ISO control. Addition of HCTZ
during the last 7 days treatment of GH 250mg/kg provided signif-
icant (P<0.05) rise in LDH and CK-MB activities in HTH compared
to their individual treatment. Similarly, histological examinations
(Figs. 24) of slides prepared from myocardium of experimen-
tal animals treated with HCTZ, GH 250, GH 125+HCTZ and GH
250+HCTZ indicated a decrease in scores compared to ISO con-
trol. However, administrationof HCTZ to GH500mg/kg treatedrats
was unable to show any signicant change when compared to ISO
control and GH 500mg/kg alone.
3.5. Effect on pharmacokinetic prole (Table 5)
The C
max
, AUC
024h
and AUC
total
showed signicant differ-
ence between the HCTZ alone and HCTZ+GH (250mg/kg) treated
groups. The time to reach peak (T
max
) in the plasma concentration
of HCTZ occurred at the same time in both groups, whereas, C
max
Fig. 2. H&E (400) stained microscopic section of isoproterenol (ISO) control. There
is loss of cellular architecture, nuclear duplication and increased inltration of leu-
cocytes with prominent hyperchromasia.
476 S.M.B. Asdaq, M.N. Inamdar / Chemico-Biological Interactions 181 (2009) 472479
Fig. 3. H&E (400) stained microscopic section of heart tissue of animals pretreated
with GH 250mg/kg for 30 days orally and subsequently two doses of isoproterenol
(ISO) 175mg/kg, s.c. Normal architecture restored with focal inltration as evident
from the gure.
was remarkably highinpresence of moderate dose of GHindicating
enhanced extent of absorption (Fig. 1). This was also demonstrated
by a signicant difference between AUC
024h
of HCTZ alone and in
presence of GH (250mg/kg). There was signicant prolongation of
elimination half-life T
1/2
(h) in presence of moderate dose of GH
(5.68h in human) from 8.8 to 12.7. The HCTZ clearance was also
reduced signicantly from 1.328 to 0.870 (ml/kgh) in presence of
GH(250mg/kg). It is alsoimportant tonote that there was nosignif-
icant difference between the two groups on the rate of absorption
(K
a
). However, the elimination rate of HCTZ is drastically reduced
in presence of GH (250mg/kg).
Fig. 4. H&E (400) stained microscopic section of heart tissue of animals pretreated
withGH250mg/kgfor 30days andHCTZ10mg/kgfor 7days orallyandsubsequently
two doses of isoproterenol (ISO) 175mg/kg, s.c. There is protection frommyocardial
damage as revealed by restoration of normal architecture with reduced interstitial
space.
High dose of GH (500mg/kg) was not able to alter any of the
pharmacokinetic parameters measured for HCTZ except slight sig-
nicant incline in AUC
total
and decline in CL.
4. Discussion
The present study was undertaken to evaluate the pharma-
cokinetic and pharmacodynamic interaction of garlic with HCTZ
using experimental models in rats. The results observed suggest
that GH (250mg/kg, p.o.) when combined with HCTZ enhances
the cardioprotective activity of latter during myocardial damage
induced by ISO in rats. The kaliuretic activity of HCTZ was found to
Table 4
Effect on biochemical parameters, antioxidants and histological scores in rats.
Groups LDH activity CK-MB activity Heart tissue homogenate Histological
scores
Serum (U/l) Heart tissue
homogenate (U/g)
Serum (U/l) Heart tissue
homogenate (U/g)
SOD (Units/mg
protein)
Catalase
(Units/mg protein)
Control 432.38 11.10 716 16.80 25.912.73 46.082.19 2.51 0.08 3.26 0.05 0.5 0.22
Isoproterenol control 556.86 11.90
*
286.25 14.18
***
39.51 1.82
**
21.21 0.36
***
1.53 0.03
***,
1.66 0.03
***
2.66 0.21
***
HCTZ (10mg/kg, p.o.) 503.21 7.65
*,
333.09 12.43
***
35.28 1.32
**
26.22 2.11
***,
1.91 0.17
**,
1.91 0.27
***,
1.5 0.34
**,
GH 250mg/kg, p.o. 412.28 13.36
*,
522.61 14.00
***,
29.33 1.97
*,
42.22 2.21

2.24 0.10

3.16 0.04

0.5 0.22

GH 125mg/kg,
p.o. +HCTZ
452.21 9.21
,b
488.11 4.32
***,,b
29.29 2.11
*,,b
38.66 0.98
*,,b
2.28 0.05
,b
2.29 0.04
*,,b
1.5 0.22
**,
GH 250mg/kg,
p.o. +HCTZ
429.20 5.79
,bb
569.09 22.43
**,,bbb
25.87 1.32
,bb
46.87 0.98
,bbb
2.55 0.06
,bb
3.38 0.67
,bbb
0.5 0.22
,bb
GH 500mg/kg,
p.o. +HCTZ
588.20 10.01
***,b
302.12 9.22
***
34.21 3.77
***
23.99 2.21
***
1.51 0.13
***
1.65 0.43
***
2.33 0.33
***,b
All values are meanSEM, n=8;
*
P<0.05,
**
P<0.01,
***
P<0.001 when compared to control; P<0.05,

P<0.01,

P<0.001 when compared to isoproterenol control;
b
P<0.05,
bb
P<0.01,
bbb
P<0.001 when compared to HCTZ (comparison between HCTZ vs HCTZ+GH).
In GH groups 30 days of GH p.o.; in HCTZ group 7 days of HCTZ p.o. and in interactive groups 30 days of GH treatment p.o. +7 days of HCTZ p.o. At the end of treatment,
all groups except control, were subjected to two dose of ISO 175mg/kgs.c.
Table 5
Pharmacokinetic parameters of hydrochlorothiazide (HCTZ).
Parameters HCTZ alone HCTZ+GH 250mg/kg HCTZ+GH 500mg/kg
Cmax (g/ml) 3.09 0.10 7.50 0.29
***
4.10 0.21
Tmax (h) 4.00 0.00 4.00 0.00 4.00 0.00
AUC
024h
(g/hml) 39.26 0.99 93.24 6.02
***
53.02 2.32
AUC
total
(g/hml) 46.86 0.32 131.94 13.48
***
76.32 2.11
*
Ke (h
1
) 0.079 0.0054 0.055 0.0058
**
0.072 0.0044
CL (ml/kgh) 1.328 0.054 0.870 0.096
***
1.088 0.076
*
T
1/2
(h) 8.814 0.607 12.725 1.118
***
9.625 0.512
V
d
(ml/kg) 16.832 0.623 15.622 1.067 15.119 1.072
Ka (h
1
) 0.563 0.077 0.427 0.0528 0.512 0.056
Values are meanSEM, n=8;
*
P<0.05,
**
P<0.01,
***
P<0.001 when compared to HCTZ alone; HCTZ: hydrochlorothiazide 10mg/kg; GH: garlic homogenate.
In HCTZ group single dose of HCTZ p.o. and in interactive groups 30 days of GH treatment p.o. +single dose of HCTZ p.o.
S.M.B. Asdaq, M.N. Inamdar / Chemico-Biological Interactions 181 (2009) 472479 477
be substantially reduced in presence of GH (250mg/kg, p.o.). The
bioavailability of HCTZ was signicantly increased when given to
animals previously treated with GH (250mg/kg, p.o.).
Many medicinal herbs and pharmacological drugs are known
to produce therapeutic effect at one dose while being toxic at
higher dose. Interactions between herbs and drugs may increase
or decrease the pharmacological or toxicological effects of either
component. Herbal medicines are ubiquitous: the dearth of reports
of adverse events and interactions probably reects a combina-
tion of under-reporting and the benign nature of most herbs used.
Experimental dataintheeldof herbdruginteractions arelimited;
case reports scarce and case series rare [22]. Nevertheless, recent
data indicate that potentially serious interactions exist between
some common herbal remedies and widely used conventional
drugs [2325], including those used in the therapy of cardiovas-
cular diseases [2628]. Hence, it is widely accepted that in-depth
and appropriate studies on drugherb interactions should be car-
ried out to conrmthe efcacy of combined drugherb treatments
[4].
Garlic (A. sativum L., family: Liliaceae) has been considered
as a valuable healing agent by people of many different cul-
tures for thousands of years. Garlic for the current study was
purchased from the local vegetable market which is the most
widely used form of garlic. The same garlic and its preparations
are commonly employed as spice and condiment as well as phy-
totherapeutic agent. Similar quality garlic was used by us and
others for our earlier research purposes [10,11,2931]. The dose
of garlic was selected based on dose-dependent study reported in
earlier literature whichwas further conrmedbyour studies. Garlic
preparations contain a wide variety of organosulfuric compounds,
S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC), which
are mainly derived from alliin. When garlic tissue is disrupted, the
enzyme alliinase comes into contact with alliin and catalyzes its
breakdown into allicin [3235]. Fresh garlic homogenate is known
to possess the highest concentration of active constituent, allicin
with half-life upto 2.4 days when compared to normal half-life of
allicin, 216h [36]. The various preparations of garlic have been
described as antibacterial, antifungal and anticarcinogenic agent
and have been reported to inhibit platelet aggregation [37]. Allicin
(allyl 2-propenethiosulnate) was earlier thought to be the princi-
ple bioactive compound responsible for the cardioprotective effect.
However, recent studies suggest that allicin is an unstable and
transient compound with oxidant activity [38] that is virtually
undetectable in blood circulation after garlic ingestion and decom-
poses to form the SAC and SAMC [39] by reacting with an enzyme
allinase or alliin lyase, which is located only in the vascular bundle
sheath cells [36]. GH was administered orally for 30 days to avail
the bioactivity of SAC and SAMC at highest possible level. Most of
pharmacologicallyactive preparations obtainedfromgarlic contain
active ingredients and are devoid of the specic smell. Their ef-
cacy is, however, an arguable issue and depends on the presence of
specic active ingredients and the manufacturing method.
Hydrochlorothiazide affects the renal tubular mechanisms of
electrolyte reabsorption, directly increasing excretion of sodium
and chloride in approximately equivalent amounts. Indirectly,
the diuretic action of HCTZ reduces plasma volume, with conse-
quent increase in urinary potassium loss, plasma renin activity,
and aldosterone secretion, and decrease in serum potassium [6].
The clinical manifestations of K
+
depletion vary greatly between
individual patients, and the severity depends on the degree of
hypokalemia. In patients without underlying heart disease, abnor-
malities in cardiac conduction are extremely unusual. In patients
with cardiac ischemia, heart failure or left ventricular hypertro-
phy, however, even mild-to-moderate hypokalemia increases the
likelihood of cardiac arrhythmias [79]. Therefore, it is proved that
their undesirable metabolic consequences have been suspected of
contributing to increase in cardiovascular morbidity and mortal-
ity. Hence, search for concurrently administered safe therapeutic
medicament continues which can ameliorate the hypokalemia in
patients with ischemic heart diseases.
Previous investigations of diuretic agents have foundit advanta-
geous to pretreat or prime the test animal with various uids [40].
As diuretics are employed clinically in the treatment of oedema, it
would seem to be most important to demonstrate effectiveness in
the presence of electrolyte and water. Thus, excess water and elec-
trolyte was given to stimulate oedema. The result of the current
investigations showed increase in diuretic activity of HCTZ in pres-
ence of GH which is best at moderate dose of GH (250mg/kg). The
increased efcacy could be because of enzyme inducing capacity of
garlic. Thus it is speculated that the enhanced activity of HCTZ in
presence of GH(especially GH250) is due to decreased metabolism
of HCTZ inliver leading to prolonged natriuretic effect of HCTZ. This
information was later conrmed by pharmacokinetic interactive
studies. As the co administration of garlic and HCTZ reduced the
elimination rate constant and the clearance of the drug especially
in linear kinetics, it invariably caused prolongation of the half-life.
The liver is the main site of metabolism of HCTZ and about 50%
of the HCTZ is cleared from the systemic circulation by the liver;
since garlic decreasedthe clearance of the drug, it may be garlic that
altered the metabolismof the HCTZ in the liver. These pharmacoki-
netic interactive effects of garlic must be cautiously considered if
patient consuming garlic must use the HCTZ, as the peak plasma
concentration of HCTZ in presence of garlic is more than two fold.
One of the important nding of the present studywas signicant
decreaseinkaliuretic activityof HCTZinpresenceof GH(250mg/kg,
p.o.). It was also interesting to note the induction of hypochloremia
with hypotension by HCTZ in presence of different doses of GH
without any prominent signs of hypochloremia such as muscle
spasm, shallow respiration and tetany [41]. This led to exploring
the effect of combined therapy in animals subjected to myocar-
dial damage induced by ISO. The myocardial damage was produced
by administration of isoproterenol [1-(3,4-dihydroxyphenyl)-2-
isopropylamino-ethanolhydrochloride], which [42] is a synthetic
catecholamine and -adrenergic agonist that induces severe stress
in the cardiac muscle leading to development of myocardial necro-
sis. Isoproterenol induced myocardial necrosis showed membrane
permeability alterations, whichbring about the loss of functionand
integrity of myocardial membrane. The administration of two high
doses of ISOwas found induce myocardial damage of severe nature
which is evident from our observation. ISO induces myocardial
damage by various mechanisms such as myocardial hypoperfu-
sion[38], glycogendepletion[43,44], electrolyte imbalance [45,46],
lipid accumulation [47], lipid peroxidation [48] and free radical
damage [49]. Stimulation of
3
receptors induced thermogenesis
[50]. Hence, after administration of ISO, the animals were main-
tained under cold conditions to prevent death of the animals due
to hyperthermia and respiratory failure. The combination of effec-
tive diuretic dose of GH (250mg/kg, p.o.) and HCTZ was found to
decrease systolic blood pressure, regulates heart rate and congure
electrocardiographic parameters. The HCTZ induced prolongation
of QRS complex was substantially declined in presence of GH, espe-
cially with 250mg/kg, p.o. in rats subjected to myocardial damage.
The ST segment elevation was also reduced in animals treated with
combination of HCTZ and GH (250mg/kg, p.o.).
A number of studies are available that suggest the crucial role of
free radicals in pathogenesis of ISO-induced myocardial damage.
The pathophysiological changes following ISO administration are
comparable to those taking place in human myocardial alterations
[18]. ISO-induced myocardial damage is associated with decreased
endogenous antioxidants such as superoxide dismutase and cata-
lase in serum which are structurally and functionally impaired by
free radicals resulting in damage to myocardium. Inclination in
478 S.M.B. Asdaq, M.N. Inamdar / Chemico-Biological Interactions 181 (2009) 472479
endogenous antioxidant activities in HTH is indication for struc-
tural integrity and protection to the myocardium that is achieved
by prior administration of GH. It is interesting to note the alter-
ation in SOD is with concomitant uctuation in catalase after prior
treatment of animals withGH. Elevatedactivityof catalaseinHTHis
morebenecial thanincreaseinSODactivityalonebecausewithout
a simultaneous increase in catalase activity, increased SOD activity
may lead to intracellular accumulation of H
2
O
2
with detrimental
effects [51]. There was no interference of HCTZ on the exhibi-
tion of antioxidant and oxidant properties of moderate doses and
high doses of GH respectively. The membrane of myocardium was
kept intact in animals pretreated with GH (250mg/kg, p.o.) and
HCTZ as evident from elevated LDH and CK-MB activities in HTH
with depleted activities in serum. Damage to cardiac muscula-
ture was also demonstrated and conrmed by histopathological
scores. An increase in score is indicative of myocardial damage
[52]. Pretreatment with GH at doses of 250mg/kg alone or with
HCTZ substantially decreased the pathological scores and kept the
myocardial integrity during ISOdamage. This effect might be due to
augmentation of endogenous antioxidant enzyme synthesis. These
results suggest the stabilization of GH mediated protection during
HCTZ administration. It was also interesting to note some cardio-
protectiveeffect of HCTZinhistological slides. As HCTZis apotential
antihypertensive agent. Hypertension increases vascular ROS pro-
duction [53]. Agents that can suppress hypertension might also be
able to scavenge free radicals. This scavenging of free radicals could
be responsible for cardioprotective effect of HCTZ.
Higher doses of garlic might be containing more amount of
allicin. Normally, upon administration, allicin is metabolically con-
verted into safe active substances, SAC and SAMC, which are found
to be antioxidant. However, at high concentration, allicin might not
completely get converted into these safe substances and hence we
found marked disturbance in biochemical and histological param-
eters at higher doses. Allicin is known to be a transient compound
which is practically untraceable in blood after ingestion of very
high doses of garlic [54,55]. Therefore free radical scavenging
action of fresh garlic homogenate is attributed to SAC, SAMC and
other organosulfur compounds which are readily formed upon its
administration. However, at high concentration, allicin might not
completely get converted into these safe substances. There was no
signicant alteration in blood concentration of HCTZ in presence
of GH 500mg/kg. This is in line with number of studies on garlic
juice and garlic homogenate demonstrating injurious effect of high
dose of garlic on various tissues like intestinal lining and stomach
[56]. This couldbe due toenhancedlevel of allicininsteadof protec-
tive SAC and SAMC. The potential bioactive constituents might be
responsible for enzyme inhibitory role of garlic, thereby enhancing
the bioavailability of HCTZ in the body.
The results of the present study indicate that combining HCTZ
with GH (250mg/kg, p.o.) could provide an opportunity to reduce
the dose of HCTZ, which may help in minimizing the hypokalemia
as well as achieve enhanced therapeutic effect. At the same time,
proper precaution and care should be exercised to avoid hyper-
kalemia. Therefore, patients on this type of combination should be
carefully followed for electrolyte imbalance such as hypokalemia,
hypochloremic alkalosis and hyponatremia periodically and when
symptoms (i.e., dry mouth, thirst, lethargy, restlessness, confu-
sion, muscle pain, cramps, muscle weakness, oliguria, tachycardia,
nausea, vomiting) occur, serum electrolytes should be urgently
measured and necessary treatment instituted.
5. Conclusion
In conclusion, this study revealed that garlic could cause
increase in the bioavailability and half-life along with decrease in
the clearance and elimination rate constant of hydrochlorothiazide
per oral. This may pose a negative implication in clinical practice as
toxicity of HCTZ may easily be reached especially during multiple
dosing because of the possibility of drug accumulation. However,
careful addition of garlic in moderate doses might result in ben-
ecial effect during treatment of hypertension in patients with
myocardial stress as garlic causes substantial fall in excretion of
potassiumwhen compared to HCTZ alone treatment in rats. Hence,
further studies should be carried out to determine the inuence of
specic active constituent of GHwhen combined with HCTZ in ani-
mals subjected to myocardial damage. We hope that this type of
study will open new areas of research for interaction and counter-
action between herb and conventional drugs when they are taken
concurrently.
Conict of interest
None.
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