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Chemico-Biological Interactions 181 (2009) 472–479

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

he potential for interaction of hydrochlorothiazide with garlic in rats

yed Mohammed Basheeruddin Asdaqa,∗, Mohammed Naseeruddin Inamdarb

Department of Pharmacology, Krupanidhi College of Pharmacy, Varthur Hobli, Chikkabellandur Village, Carmalaram Post, Bangalore 560035, IndiaDepartment of Pharmacology, Al-Ameen College of Pharmacy, Bangalore 560027, India

r t i c l e i n f o

rticle history:eceived 2 April 2009eceived in revised form 24 July 2009ccepted 27 July 2009vailable online 4 August 2009

eywords:arlicydrochlorothiazide

nteractionsoproterenol

a b s t r a c t

The present study was undertaken to determine the pharmacokinetic and pharmacodynamic interactionof hydrochlorothiazide (HCTZ) with garlic homogenate (GH), in rats. The influence of garlic on pharma-cokinetics of HCTZ was studied by HPLC method, while pharmacodynamic interaction was studied usingdiuretic activity, ECG and BP changes and isoproterenol (ISO) induced myocardial injury. HCTZ was givenorally at 10 mg/kg and GH was administered at three different doses of 125, 250 and 500 mg/kg, p.o. TheCK-MB, LDH, SOD, catalase and histopathological studies were carried out. The administration of HCTZin GH pretreated rats found to decrease the QRS duration, RR interval, QT segment, systolic blood pres-sure, heart rate, serum potassium level, serum LDH and serum CK-MB activities significantly. The diureticeffect of HCTZ was significantly increased in presence of GH; however, kaliuresis was significantly reducedin presence of GH 250 mg/kg. Histopathological studies of heart tissue reveal the protective effect of GH

250 mg/kg in presence or absence of HCTZ during ISO stress to myocardium. The pharmacokinetic studiesshow that GH increases the bioavailability and half-life, along with decrease in clearance and eliminationrate of HCTZ when administered orally. It was concluded that careful addition of garlic in moderate dosesmight result in beneficial effect during treatment of hypertension in patients with myocardial stressas garlic causes substantial fall in excretion of potassium when compared to HCTZ alone treatment inrats. This could be important in reducing the dose of HCTZ to achieve enhanced therapeutic effect with minimal adverse effect.

. Introduction

During the recent past, a dramatic rise in the use of herbs anderbal remedies has been witnessed in many parts of the world [1].hile such products had been used with apparent safety in tradi-

ional societies for many centuries, when they are being combinedith pharmacological agents, posses the possibility of potential

nteraction between the two groups of substances. Reports indi-ate that about 15–20% of individuals on prescription medicationslso use herbal supplements and less than 40% of patients discloseo their physicians the usage of herbal remedies, even if they expe-ience severe side effects—because of the fear of censure or rebuke2]. The problem is further compounded by the fact that manyhysicians are themselves not always familiar with the potentialor herb–drug interactions [3]. Hence, it is imperative to promoteredible research on the safety and efficacy of combined herb–drug

reatment for variety of ailments including cardiovascular diseases4].

Diuretics, in particular hydrochlorothiazide (HCTZ), are oftenvoided 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).

009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2009.07.022

© 2009 Elsevier Ireland Ltd. All rights reserved.

patients with ischemic heart diseases. Thiazides affect the renaltubular mechanisms of electrolyte reabsorption, directly increas-ing excretion of sodium and chloride in approximately equivalentamounts. Indirectly, the diuretic action of hydrochlorothiazidereduces plasma volume, with consequent increase in urinary potas-sium loss, plasma renin activity, aldosterone secretion and decreasein serum potassium [5,6]. In patients with cardiac ischemia, heartfailure or left ventricular hypertrophy, even mild-to-moderatehypokalemia increases the likelihood of cardiac arrhythmias [7–9].The combination of HCTZ with ACE-I, aldosterone antagonist orangiotensin II type 1 receptor blocker is found to minimize thepotassium loss characteristically induced by the thiazide compo-nent.

Traditionally, garlic (Allium sativum) and its preparations havebeen widely recognized as agents for the prevention and treat-ment of cardiovascular and other metabolic diseases, such asatherosclerosis, hyperlipidemia, thrombosis, hypertension and dia-betes. Garlic contains biologically active compounds that exertmultiple beneficial 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 withmyocardial infarction. However, there is no scientific report to indi-cate the effect of combined therapy of garlic with HCTZ. The presentstudy was undertaken to evaluate the pharmacokinetic and phar-

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acodynamic interaction of HCTZ with garlic using experimentalodels in rats.

. Materials and methods

.1. Experimental animals

Laboratory bred female Wistar albino rats (200–250 g) wereoused at 25 ± 5 ◦C in a well-ventilated animal house under 12:12 h

ight dark cycle. The rats had free access to standard rat chowAmrut Laboratory Animal feed, Maharashtra, India) containing (%,/w) protein 22.10, oil 4.13, fibre 3.15, ash 5.15, sand (silica) 1.12,

nd water ad libitum. The animals were maintained under stan-ard conditions in an animal house approved by Committee forhe Purpose of Control and Supervision on Experiments on AnimalsCPCSEA).

.2. Chemical

All chemicals used were of analytical grade and purchasedrom standard companies. Pure sample of hydrochlorothiazide wasifted by Bangalore Test House (Bangalore, India). Biochemical kitsere procured from Crest Biosystems (Goa, India).

.3. Preparation of garlic homogenate

Garlic (A. sativum, family: Liliaceae) bulbs were purchased fromhe local market. The cloves were peeled, sliced and ground into aaste and suspended in distilled water. Three different concentra-ions of the garlic homogenate (GH) were prepared, 0.05, 0.1 and.2 g/ml, corresponding to 125, 250 and 500 mg/kg body weight ofnimal [12]. GH was administered within 30 min of preparation.

.4. Determination of diuretic activity

The method of Wiebelhaus et al. [13] was used, with mod-fication, for the determination of diuretic effect. Adult femalelbino rats weighing 200–250 g were divided into followingight groups consisting of eight animals each: group I: vehi-le (1 ml/kg, p.o. for 30 days); groups II–IV: garlic homogenate25, 250 and 500 mg/kg respectively for 30 days orally; group: hydrochlorothiazide 10 mg/kg on the day of experiment, p.o.;roups VI–VIII: garlic homogenate 125, 250 and 500 mg/kg respec-ively for 30 days + HCTZ. Prophylactically treated animals wereasted overnight with water allowed ad libitum. The following

orning rats were given orally 25 ml/kg of normal saline solution,nd immediately after normal saline administration, the rats werelaced individually in a modified funnel having a wire mesh and fit-ed with a graduated test tube. In HCTZ incorporated groups, HCTZas given orally as a fine homogenized suspension in a volume of

5 ml/kg of normal saline solution. Urine excreted for the next 5 has collected and the total volume of urine for each rat was com-ared with the volume of urine produced after the administration oformal saline. The volume of urine excreted during 5 h for each ani-al in the group is expressed as the percent of the liquid (normal

aline) administered. This percentage gives a measure of urinaryxcretion independent of the animal weight. The ratio of urinaryxcretion in the test group to urinary excretion in the control groups used as a measure of the diuretic action for the given dose ofhe drug. As the diuretic action is prone to variability, a param-ter known as diuretic activity was calculated instead. To obtain

he diuretic activity, the diuretic action of the test groups (gar-ic homogenate) was compared with that of the standard (HCTZ)14]. Percentage of saline load excreted = volume of urine/volumef saline load × 100. Urinary excretion = total urinary output/totaliquid administered × 100. Diuretic action = urinary excretion of

ical Interactions 181 (2009) 472–479 473

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 flame photometer,while Cl− content was measured by auto analyzer.

2.5. Isoproterenol (ISO) induced myocardial damage

The animals were divided into seven groups consisting ofeight animals each: group I: vehicle (1 ml/kg, p.o. for 30 days);group II: isoproterenol (175 mg/kg, s.c); group III: hydrochloroth-iazide 10 mg/kg [14] for 7 days, p.o.; group IV: garlic homogenate250 mg/kg for 30 days orally; groups V–VII: garlic homogenate 125,250 and 500 mg/kg respectively for 30 days and HCTZ for last 7days p.o. At the end of treatment period, animals of all groupsexcluding group I were administered ISO (175 mg/kg s.c) for 2 con-secutive days. Blood was withdrawn from retro orbital vein 48 hafter the first dose of ISO under anesthesia and serum was separatedby centrifugation for lactate dehydrogenase (LDH) and creatinephosphokinase-MB (CK-MB) measurement. The blood pressure andECG changes were recorded under appropriate conditions. Theheart was immediately isolated from each animal under ketamine(70 mg/kg, i.p) and xylazine (10 mg/kg, i.p) anesthesia. In eachgroup consisting of eight animals, four excised hearts were homog-enized to prepare heart tissue homogenate (HTH) using sucrose(0.25 M) [15]. The activity of LDH, CK-MB, superoxide dismutase(SOD) [16] and catalase [17] were measured in HTH. Microscopicslides of myocardium were prepared for histopathological studiesfrom the hearts of remaining four animals. The myocardial damagewas determined by giving scores depending on the intensity as fol-lows [18]; no changes – score 00; mild – score 01 (focal myocytesdamage or small multifocal degeneration with slight degree ofinflammatory process); moderate – score 02 (extensive myofibril-lar degeneration and/or diffuse inflammatory process); marked –score 03 (necrosis with diffuse inflammatory process).

2.6. Blood pressure and electrocardiograms measurement

As discussed above, 48 h after first dose ISO administration justprior to collection of blood samples, mean arterial blood pres-sure was measured in awaked animals by the non-invasive bloodpressure module (NIBP pressure meter, LE 5001, V02/0402L, Pan-lab, Hardvard apparatus, Barcelona, Spain) and ECG was recordedin anesthetised animals [ketamine (70 mg/kg, i.p) and xylazine(10 mg/kg, i.p)] by subcutaneous lead II method (Physiograph, EKGcoupler, SO-02, INCO, India), QRS duration, RR interval and QT seg-ment was measured.

2.7. Statistical analysis

Results of pharmacodynamic parameters are expressed asmean ± SEM. The statistical significance was determined using one-way analysis of variance (ANOVA) followed by Bonferroni’s test. Theresults were considered statistically significant when P < 0.05.

2.8. Pharmacokinetic interaction

Both high and moderate doses of garlic homogenate (500 and250 mg/kg, p.o.) were selected for this interaction study. Animalswere divided into three groups consisting of eight animals each:group I: hydrochlorothiazide 10 mg/kg p.o. (single dose), group II:

GH 250 mg/kg for 30 days (p.o.) + HCTZ (single dose) and group III:GH 500 mg/kg for 30 days (p.o.) + HCTZ (single dose). Immediatelyafter HCTZ administration, 0.5 ml of blood samples was withdrawnat each time intervals over 24 h (0, 1, 2, 4, 8, 16 and 24 h) bypuncturing retro orbital vein under partial ether anesthesia and

4 -Biological Interactions 181 (2009) 472–479

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ubjected to analysis. The hypovolaemia is prevented by intraperi-oneal administration of 0.5 ml of normal saline immediately afterach withdrawal of blood.

.9. Extraction procedure [19]

Solid sodium bicarbonate (200 mg) and ethyl acetate (2 ml) wasdded to 1 ml serum. After mixing on a Vortex mixer for 1 minnd centrifuging for 5 min, 1 ml of the organic layer was trans-erred to another tube and 0.5 ml of a 1N sodium hydroxide solutiondded. This solution was mixed and allowed to stand for 1 min atoom temperature, after which the upper layer was discarded. Ether1 ml) was then added to the sodium hydroxide solution, followedy mixing. After standing for 1 min, the ether layer was discardednd traces of ether were removed with a gentle stream of air. Anliquot of the aqueous layer was injected into the liquid chromato-raph. Since initial studies indicated that hydrochlorothiazide wasnstable in strongly alkaline media for prolonged periods, the back-xtraction step into sodium hydroxide solution was performed justrior to the liquid chromatographic analysis.

Liquid chromatography: a Waters 6000 pumping systemWaters Associates) coupled to a Spectroflow SF 770 variable wave-ength detector (Schoeffel Instrument Corp.) was used for thesetudies. A reverse phase system consisting of a 30 cm × 4 mm FBon-apak CIS column (Waters Associates) was utilized with methanol0.01 M sodium dihydrogen phosphate solution (1:4) as the eluent,t a flow rate of 0.6 ml/min. The detector was operated at 271 nm.or the lower levels present in serum, a sensitivity of 0.01 A (fullcale deflection) was required. Peak heights were used for quan-itation. All standard curves were linear and passed through therigin.

.10. Pharmacokinetic determination [20]

For the generated data on hydrochlorothiazide and garlic inter-ction to be analyzed, we assume that the kinetics of HCTZlimination was linear. The data was represented in a plasmaevel-time curve from where the area under time curve (AUC0–24 h)

as calculated using Trapezoid rule. The maximum concentra-ion (Cmax) and maximum time (Tmax) were obtained directlyrom generated data. The elimination constant (Ke) and half-ife (T1/2) were determined from the semi-log plot of the data.he clearance (CL) and apparent volume of distribution (Vd) ofhe drug in the animals were calculated from the equations:L = Vd × Ke, Vd: the administered dose of drug/initial plasma

oncentration of drug obtained at intercept of semi-log plotf plasma drug sample. AUCtotal = AUC0–24 h + C24 h/Ke. The meanlasma concentration–time curve for HCTZ (10 mg/kg) alone andCTZ+ once a day administration of oral garlic homogenate

250 mg/kg) for 30 days was determined. The study was done for

able 1ffect on electrolyte excretion in urine.

Groups Electrolyte concentration (mmol/

Na+

Control 54.35 ± 1.19GH 125 mg/kg, p.o. 65.12 ± 3.67**

GH 250 mg/kg, p.o. 87.73 ± 2.01***

GH 500 mg/kg, p.o. 72.29 ± 2.81***

HCTZ (10 mg/kg, p.o.) 109.20 ± 2.01***

GH 125 mg/kg, p.o. + HCTZ 127.97 ± 4.10***,aa,bb

GH 250 mg/kg, p.o. + HCTZ 138.07 ± 2.07***,aaa,bbb

GH 500 mg/kg, p.o. + HCTZ 123.02 ± 2.91***,aa,bb

ll values are mean ± SEM, n = 8; *P < 0.05, **P < 0.01, ***P < 0.001 when compared to contlone; bP < 0.05, bbP < 0.01, bbbP < 0.001 when compared to HCTZ (comparison between HCn GH groups – 30 days of GH p.o.; in HCTZ group – single dose of HCTZ p.o. and in interac

Fig. 1. Effect of garlic on plasma concentration of HCTZ.

24 h since the half-life of HCTZ is 5.6–8 h [21]. The results wereanalyzed statistically using student’s t-test.

3. Results

3.1. Effect on electrolyte excretion in urine (Table 1)

As shown in Table 1, there was significant increase in excretionof Na+ and Cl− electrolytes in urine of animals previously treatedwith GH 125, GH 250 and GH 500 mg/kg. Further, the extent of Na+,K+ and Cl− excretion is remarkably high in animals treated withHCTZ, indicating its enhanced potency. Moreover, upon addition ofHCTZ in GH treated animals, there was further increase in inhibitionof reabsorption of Na+ and Cl−. However, there was no significantchange in the excretion of K+ in groups pretreated with GH 125,GH 250 and GH 500 mg/kg as compared to control. The GH treatedgroups remained unaltered upon addition of HCTZ in excretion ofK+ when compared to control. The maximum effect was seen withcombination of HCTZ and GH (250 mg/kg, p.o.).

3.2. Effect on percentage of saline load excreted, diuretic actionand diuretic activity (Table 2)

As evident from Table 2, there was significant increase in per-centage of saline load excretion in animals pretreated with GH 250when compared to control group. It is noted that HCTZ significantlyenhances saline load excretion that is further augmented signifi-

cantly in animals pretreated with moderate dose of GH (GH 250).As indicated by Table 2, the diuretic action and diuretic activity wassignificantly elevated when GH and HCTZ were used concurrently.The maximum diuretic activity was seen at moderate doses of GHin presence of HCTZ.

l)

K+ Cl−

45.15 ± 2.23 110.48 ± 1.9641.41 ± 1.49 120.51 ± 1.53*

39.49 ± 1.43 135.98 ± 3.68**

40.21 ± 0.98 113.03 ± 4.34*

59.42 ± 1.71** 137.98 ± 1.58***

45.44 ± 0.82a,b 151.75 ± 3.22***,a,b

40.68 ± 1.31bbb 163.40 ± 0.78***,aa,bb

51.23 ± 0.88*,aa 151.70 ± 1.30***,a,b

rol; aP < 0.05, aaP < 0.01, aaaP< 0.001 when compared to corresponding dose of GHTZ vs HCTZ + GH).tive 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) 472–479 475

Table 2Effect 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 125 mg/kg, p.o. 68.16 ± 2.12* 1.45* 0.53GH 250 mg/kg, p.o. 75.59 ± 13.5*** 1.61* 0.59GH 500 mg/kg, p.o. 64.50 ± 1.32* 1.38* 0.51HCTZ (10 mg/kg, p.o.) 128.74 ± 1.54*** 2.7** –GH 125 mg/kg, p.o. + HCTZ 134.11 ± 1.82***,aaa 2.87**,aaa 1.06GH 250 mg/kg, p.o. + HCTZ 166.45 ± 1.36***,aaa,bb 3.56***,aaa,b 1.31GH 500 mg/kg, p.o. + HCTZ 137.23 ± 2.57***,aaa 2.93**,aaa 1.08

All values are mean ± SEM, n = 8; *P < 0.05, **P < 0.01, ***P < 0.001 when compared to control; aP < 0.05, aaP < 0.01, aaaP < 0.001 when compared to corresponding dose of GHalone; bP < 0.05, bbP < 0.01, bbbP < 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 3Effect on electrocardiographic parameters and hemodynamic findings 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 15.21 ± 1.11 55.11 ± 4.43 14.31 ± 0.87Isoproterenol control 112 ± 4* 386 ± 32* 195 ± 2*** 4.52 ± 0.32* 18.58 ± 2.10* 59.03 ± 5.32 16.21 ± 1.81HCTZ (10 mg/kg, p.o.) 118 ± 3* 357 ± 21• 221 ± 4*,• 4.01 ± 0.22*,• 19.22 ± 2.19** 54.10 ± 4.92 15.21 ± 1.21GH 250 mg/kg, p.o. 113 ± 4* 341 ± 32• 232 ± 6• 3.54 ± 0.31b,• 14.84 ± 1.47* 53.19 ± 2.23• 14.21 ± 1.43•

GH 125 mg/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 ± 1.44GH 250 mg/kg, p.o. + HCTZ 104 ± 4*,b 315 ± 22*,bb,• 242 ± 5b,•• 3.43 ± 0.51b,•• 14.32 ± 2.16*,bb,•• 53.17 ± 1.19• 14.13 ± 1.21•

GH 500 mg/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

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The Cmax, AUC0–24 h and AUCtotal showed significant differ-ence between the HCTZ alone and HCTZ + GH (250 mg/kg) treatedgroups. The time to reach peak (Tmax) in the plasma concentrationof HCTZ occurred at the same time in both groups, whereas, Cmax

ll values are mean ± SEM, n = 8; *P < 0.05, **P < 0.01, ***P < 0.001 when compared to cbP < 0.01, bbbP < 0.001 when compared to HCTZ (comparison between HCTZ vs HCTn GH groups – 30 days of GH p.o.; in HCTZ group – 7 days of HCTZ p.o. and in interall groups except control, were subjected to two dose of ISO 175 mg/kg s.c.

.3. Effect on electrocardiographic parameters and hemodynamicndings (Table 3)

All the treatments showed significant decrease in systolic bloodressure compared to control. The combination of GH 250 mg/kgith HCTZ showed more reduction in systolic blood pressure

ompared to their individual treatments. The heart rate was signif-cantly elevated in isoproterenol (ISO) control compared to normalontrol. However, the chronotropic effect was significantly reducedn all treatment groups compared to ISO control. The combination ofH 250 mg/kg with HCTZ was more effective than individual treat-ent in reducing inclined heart rate as compared to ISO control.The body weight of animal was significantly reduced in iso-

roterenol administered group when compared to normal control.imilarly, fall in body weight was also found in HCTZ, GH 125 + HCTZs well as in GH 500 + HCTZ groups. However, pretreatment of ani-als with GH 250 mg/kg either in presence or absence of HCTZ

ound to revert back the normal weight. Heart weight/body weightatio was augmented in ISO rat which was significantly reducedn all treatment groups. Administration of HCTZ in GH (250 mg/kg,.o.) pretreated animals’ demonstrated maximum fall in elevatedW/BW ratio compared to their individual treatment.

Significant changes in the ECG configuration were observed inhe rats with ISO treatment such as significant prolongation of QRSuration as well as non-significant longer QT segment and RR inter-al. The HCTZ treated animals showed significant prolongation ofRS complex compared to normal control. The combination of GH50 mg/kg with HCTZ was found to significantly decrease the QRSomplex compared to their individual treatment. There was fall inT segment and RR interval in GH 250 mg/kg alone or in presencef HCTZ compared to ISO control.

.4. Effect on biochemical parameters, antioxidants andistological scores (Table 4)

The LDH, CK-MB, SOD and catalase activities were significantlyecreased in heart tissue homogenate (HTH) by ISO treatment

l; P < 0.05, ••P < 0.01, •••P < 0.001 when compared to isoproterenol control; bP < 0.05,), sBP: systolic blood pressure, HR: heart rate, HW/BW: heart weight/body weight.

groups – 30 days of GH treatment p.o. + 7 days of HCTZ p.o. At the end of treatment,

compared to normal control. Prior treatment of rats with HCTZ(P < 0.05) and GH 250 mg/kg (P < 0.001) showed significant increasein these parameters compared to ISO control. Addition of HCTZduring the last 7 days treatment of GH 250 mg/kg provided signif-icant (P < 0.05) rise in LDH and CK-MB activities in HTH comparedto their individual treatment. Similarly, histological examinations(Figs. 2–4) of slides prepared from myocardium of experimen-tal animals treated with HCTZ, GH 250, GH 125 + HCTZ and GH250 + HCTZ indicated a decrease in scores compared to ISO con-trol. However, administration of HCTZ to GH 500 mg/kg treated ratswas unable to show any significant change when compared to ISOcontrol and GH 500 mg/kg alone.

3.5. Effect on pharmacokinetic profile (Table 5)

Fig. 2. H&E (×400) stained microscopic section of isoproterenol (ISO) control. Thereis loss of cellular architecture, nuclear duplication and increased infiltration of leu-cocytes with prominent hyperchromasia.

476 S.M.B. Asdaq, M.N. Inamdar / Chemico-Biological Interactions 181 (2009) 472–479

Fig. 3. H&E (×400) stained microscopic section of heart tissue of animals pretreatedwith GH 250 mg/kg for 30 days orally and subsequently two doses of isoproterenol(f

webpe(rGi(i

Fig. 4. H&E (×400) stained microscopic section of heart tissue of animals pretreatedwith GH 250 mg/kg for 30 days and HCTZ 10 mg/kg for 7 days orally and subsequently

using experimental models in rats. The results observed suggest

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ISO) 175 mg/kg, s.c. Normal architecture restored with focal infiltration as evidentrom the figure.

as remarkably high in presence of moderate dose of GH indicatingnhanced extent of absorption (Fig. 1). This was also demonstratedy a significant difference between AUC0–24 h of HCTZ alone and inresence of GH (250 mg/kg). There was significant prolongation oflimination half-life T1/2 (h) in presence of moderate dose of GH5.6–8 h in human) from 8.8 to 12.7. The HCTZ clearance was alsoeduced significantly from 1.328 to 0.870 (ml/kg h) in presence ofH (250 mg/kg). It is also important to note that there was no signif-

cant difference between the two groups on the rate of absorptionKa). However, the elimination rate of HCTZ is drastically reducedn presence of GH (250 mg/kg).

able 4ffect on biochemical parameters, antioxidants and histological scores in rats.

Groups LDH activity CK-MB activity

Serum (U/l) Heart tissuehomogenate (U/g)

Serum (U/l)

Control 432.38 ± 11.10 716 ± 16.80 25.91 ± 2.73Isoproterenol control 556.86 ± 11.90* 286.25 ± 14.18*** 39.51 ± 1.82**

HCTZ (10 mg/kg, p.o.) 503.21 ± 7.65*,• 333.09 ± 12.43*** 35.28 ± 1.32**

GH 250 mg/kg, p.o. 412.28 ± 13.36*,•• 522.61 ± 14.00***,•• 29.33 ± 1.97*,•

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

452.21 ± 9.21••,b 488.11 ± 4.32***,•••,b 29.29 ± 2.11*,•,b

GH 250 mg/kg,p.o. + HCTZ

429.20 ± 5.79••,bb 569.09 ± 22.43**,•••,bbb 25.87 ± 1.32•••,b

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

588.20 ± 10.01***,b 302.12 ± 9.22*** 34.21 ± 3.77***

ll values are mean ± SEM, n = 8; *P < 0.05, **P < 0.01, ***P < 0.001 when compared to controbP < 0.01, bbbP < 0.001 when compared to HCTZ (comparison between HCTZ vs HCTZ + GHn GH groups – 30 days of GH p.o.; in HCTZ group – 7 days of HCTZ p.o. and in interactive gll groups except control, were subjected to two dose of ISO 175 mg/kg s.c.

able 5harmacokinetic parameters of hydrochlorothiazide (HCTZ).

Parameters HCTZ alone

Cmax (�g/ml) 3.09 ± 0.10Tmax (h) 4.00 ± 0.00AUC0–24 h (�g/h ml) 39.26 ± 0.99AUCtotal (�g/h ml) 46.86 ± 0.32Ke (h−1) 0.079 ± 0.0054CL (ml/kg h) 1.328 ± 0.054T1/2 (h) 8.814 ± 0.607Vd (ml/kg) 16.832 ± 0.623Ka (h−1) 0.563 ± 0.077

alues are mean ± SEM, n = 8; *P < 0.05, **P < 0.01, ***P < 0.001 when compared to HCTZ alon HCTZ group – single dose of HCTZ p.o. and in interactive groups – 30 days of GH treatm

two doses of isoproterenol (ISO) 175 mg/kg, s.c. There is protection from myocardialdamage as revealed by restoration of normal architecture with reduced interstitialspace.

High dose of GH (500 mg/kg) was not able to alter any of thepharmacokinetic parameters measured for HCTZ except slight sig-nificant incline in AUCtotal and decline in CL.

4. Discussion

The present study was undertaken to evaluate the pharma-cokinetic and pharmacodynamic interaction of garlic with HCTZ

that GH (250 mg/kg, p.o.) when combined with HCTZ enhancesthe cardioprotective activity of latter during myocardial damageinduced by ISO in rats. The kaliuretic activity of HCTZ was found to

Heart tissue homogenate Histologicalscores

Heart tissuehomogenate (U/g)

SOD (Units/mgprotein)

Catalase(Units/mg protein)

46.08 ± 2.19 2.51 ± 0.08 3.26 ± 0.05 0.5 ± 0.2221.21 ± 0.36*** 1.53 ± 0.03***,•• 1.66 ± 0.03*** 2.66 ± 0.21***

26.22 ± 2.11***,• 1.91 ± 0.17**,• 1.91 ± 0.27***,• 1.5 ± 0.34**,••

42.22 ± 2.21••• 2.24 ± 0.10••• 3.16 ± 0.04••• 0.5 ± 0.22•••

38.66 ± 0.98*,••,b 2.28 ± 0.05••,b 2.29 ± 0.04*,•••,b 1.5 ± 0.22**,••

b 46.87 ± 0.98•••,bbb 2.55 ± 0.06•••,bb 3.38 ± 0.67•••,bbb 0.5 ± 0.22•••,bb

23.99 ± 2.21*** 1.51 ± 0.13*** 1.65 ± 0.43*** 2.33 ± 0.33***,b

l; P < 0.05, ••P < 0.01, •••P < 0.001 when compared to isoproterenol control; bP < 0.05,).roups – 30 days of GH treatment p.o. + 7 days of HCTZ p.o. At the end of treatment,

HCTZ + GH 250 mg/kg HCTZ + GH 500 mg/kg

7.50 ± 0.29*** 4.10 ± 0.214.00 ± 0.00 4.00 ± 0.00

93.24 ± 6.02*** 53.02 ± 2.32131.94 ± 13.48*** 76.32 ± 2.11*

0.055 ± 0.0058** 0.072 ± 0.00440.870 ± 0.096*** 1.088 ± 0.076*

12.725 ± 1.118*** 9.625 ± 0.51215.622 ± 1.067 15.119 ± 1.072

0.427 ± 0.0528 0.512 ± 0.056

ne; HCTZ: hydrochlorothiazide 10 mg/kg; GH: garlic homogenate.ent p.o. + single dose of HCTZ p.o.

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e substantially reduced in presence of GH (250 mg/kg, p.o.). Theioavailability of HCTZ was significantly increased when given tonimals previously treated with GH (250 mg/kg, p.o.).

Many medicinal herbs and pharmacological drugs are knowno produce therapeutic effect at one dose while being toxic atigher dose. Interactions between herbs and drugs may increaser decrease the pharmacological or toxicological effects of eitheromponent. Herbal medicines are ubiquitous: the dearth of reportsf adverse events and interactions probably reflects a combina-ion of under-reporting and the benign nature of most herbs used.xperimental data in the field of herb–drug interactions are limited;ase reports scarce and case series rare [22]. Nevertheless, recentata indicate that potentially serious interactions exist betweenome common herbal remedies and widely used conventionalrugs [23–25], including those used in the therapy of cardiovas-ular diseases [26–28]. Hence, it is widely accepted that in-depthnd appropriate studies on drug–herb interactions should be car-ied out to confirm the efficacy of combined drug–herb treatments4].

Garlic (A. sativum L., family: Liliaceae) has been considereds a valuable healing agent by people of many different cul-ures for thousands of years. Garlic for the current study wasurchased from the local vegetable market which is the mostidely used form of garlic. The same garlic and its preparations

re commonly employed as spice and condiment as well as phy-otherapeutic agent. Similar quality garlic was used by us andthers for our earlier research purposes [10,11,29–31]. The dosef garlic was selected based on dose-dependent study reported inarlier literature which was further confirmed by our studies. Garlicreparations contain a wide variety of organosulfuric compounds,-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC), whichre mainly derived from alliin. When garlic tissue is disrupted, thenzyme alliinase comes into contact with alliin and catalyzes itsreakdown into allicin [32–35]. Fresh garlic homogenate is knowno possess the highest concentration of active constituent, allicinith half-life upto 2.4 days when compared to normal half-life of

llicin, 2–16 h [36]. The various preparations of garlic have beenescribed as antibacterial, antifungal and anticarcinogenic agentnd have been reported to inhibit platelet aggregation [37]. Allicinallyl 2-propenethiosulfinate) was earlier thought to be the princi-le bioactive compound responsible for the cardioprotective effect.owever, recent studies suggest that allicin is an unstable and

ransient compound with oxidant activity [38] that is virtuallyndetectable in blood circulation after garlic ingestion and decom-oses to form the SAC and SAMC [39] by reacting with an enzymellinase or alliin lyase, which is located only in the vascular bundleheath cells [36]. GH was administered orally for 30 days to availhe bioactivity of SAC and SAMC at highest possible level. Most ofharmacologically active preparations obtained from garlic containctive ingredients and are devoid of the specific smell. Their effi-acy is, however, an arguable issue and depends on the presence ofpecific active ingredients and the manufacturing method.

Hydrochlorothiazide affects the renal tubular mechanisms oflectrolyte reabsorption, directly increasing excretion of sodiumnd chloride in approximately equivalent amounts. Indirectly,he diuretic action of HCTZ reduces plasma volume, with conse-uent increase in urinary potassium loss, plasma renin activity,nd aldosterone secretion, and decrease in serum potassium [6].he clinical manifestations of K+ depletion vary greatly betweenndividual patients, and the severity depends on the degree ofypokalemia. In patients without underlying heart disease, abnor-

alities in cardiac conduction are extremely unusual. In patientsith cardiac ischemia, heart failure or left ventricular hypertro-hy, however, even mild-to-moderate hypokalemia increases the

ikelihood of cardiac arrhythmias [7–9]. Therefore, it is proved thatheir undesirable metabolic consequences have been suspected of

ical Interactions 181 (2009) 472–479 477

contributing to increase in cardiovascular morbidity and mortal-ity. Hence, search for concurrently administered safe therapeuticmedicament continues which can ameliorate the hypokalemia inpatients with ischemic heart diseases.

Previous investigations of diuretic agents have found it advanta-geous to pretreat or prime the test animal with various fluids [40].As diuretics are employed clinically in the treatment of oedema, itwould seem to be most important to demonstrate effectiveness inthe presence of electrolyte and water. Thus, excess water and elec-trolyte was given to stimulate oedema. The result of the currentinvestigations showed increase in diuretic activity of HCTZ in pres-ence of GH which is best at moderate dose of GH (250 mg/kg). Theincreased efficacy could be because of enzyme inducing capacity ofgarlic. Thus it is speculated that the enhanced activity of HCTZ inpresence of GH (especially GH 250) is due to decreased metabolismof HCTZ in liver leading to prolonged natriuretic effect of HCTZ. Thisinformation was later confirmed by pharmacokinetic interactivestudies. As the co administration of garlic and HCTZ reduced theelimination rate constant and the clearance of the drug especiallyin 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 decreased the clearance of the drug, it may be garlic thataltered the metabolism of the HCTZ in the liver. These pharmacoki-netic interactive effects of garlic must be cautiously considered ifpatient consuming garlic must use the HCTZ, as the peak plasmaconcentration of HCTZ in presence of garlic is more than two fold.

One of the important finding of the present study was significantdecrease in kaliuretic activity of HCTZ in presence of GH (250 mg/kg,p.o.). It was also interesting to note the induction of hypochloremiawith hypotension by HCTZ in presence of different doses of GHwithout any prominent signs of hypochloremia such as musclespasm, shallow respiration and tetany [41]. This led to exploringthe effect of combined therapy in animals subjected to myocar-dial damage induced by ISO. The myocardial damage was producedby administration of isoproterenol [1-(3,4-dihydroxyphenyl)-2-isopropylamino-ethanolhydrochloride], which [42] is a syntheticcatecholamine and �-adrenergic agonist that induces severe stressin the cardiac muscle leading to development of myocardial necro-sis. Isoproterenol induced myocardial necrosis showed membranepermeability alterations, which bring about the loss of function andintegrity of myocardial membrane. The administration of two highdoses of ISO was found induce myocardial damage of severe naturewhich is evident from our observation. ISO induces myocardialdamage by various mechanisms such as myocardial hypoperfu-sion [38], glycogen depletion [43,44], electrolyte imbalance [45,46],lipid accumulation [47], lipid peroxidation [48] and free radicaldamage [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 dueto hyperthermia and respiratory failure. The combination of effec-tive diuretic dose of GH (250 mg/kg, p.o.) and HCTZ was found todecrease systolic blood pressure, regulates heart rate and configureelectrocardiographic parameters. The HCTZ induced prolongationof QRS complex was substantially declined in presence of GH, espe-cially with 250 mg/kg, p.o. in rats subjected to myocardial damage.The ST segment elevation was also reduced in animals treated withcombination of HCTZ and GH (250 mg/kg, p.o.).

A number of studies are available that suggest the crucial role offree 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 decreasedendogenous antioxidants such as superoxide dismutase and cata-lase in serum which are structurally and functionally impaired byfree radicals resulting in damage to myocardium. Inclination in

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ndogenous antioxidant activities in HTH is indication for struc-ural integrity and protection to the myocardium that is achievedy prior administration of GH. It is interesting to note the alter-tion in SOD is with concomitant fluctuation in catalase after priorreatment of animals with GH. Elevated activity of catalase in HTH is

ore beneficial than increase in SOD activity alone because withoutsimultaneous increase in catalase activity, increased SOD activityay lead to intracellular accumulation of H2O2 with detrimental

ffects [51]. There was no interference of HCTZ on the exhibi-ion of antioxidant and oxidant properties of moderate doses andigh doses of GH respectively. The membrane of myocardium wasept intact in animals pretreated with GH (250 mg/kg, p.o.) andCTZ as evident from elevated LDH and CK-MB activities in HTHith depleted activities in serum. Damage to cardiac muscula-

ure was also demonstrated and confirmed by histopathologicalcores. An increase in score is indicative of myocardial damage52]. Pretreatment with GH at doses of 250 mg/kg alone or withCTZ substantially decreased the pathological scores and kept theyocardial integrity during ISO damage. This effect might be due to

ugmentation of endogenous antioxidant enzyme synthesis. Theseesults suggest the stabilization of GH mediated protection duringCTZ administration. It was also interesting to note some cardio-rotective effect of HCTZ in histological slides. As HCTZ is a potentialntihypertensive agent. Hypertension increases vascular ROS pro-uction [53]. Agents that can suppress hypertension might also beble to scavenge free radicals. This scavenging of free radicals coulde responsible for cardioprotective effect of HCTZ.

Higher doses of garlic might be containing more amount ofllicin. Normally, upon administration, allicin is metabolically con-erted into safe active substances, SAC and SAMC, which are foundo be antioxidant. However, at high concentration, allicin might notompletely get converted into these safe substances and hence weound marked disturbance in biochemical and histological param-ters at higher doses. Allicin is known to be a transient compoundhich is practically untraceable in blood after ingestion of veryigh doses of garlic [54,55]. Therefore free radical scavengingction of fresh garlic homogenate is attributed to SAC, SAMC andther organosulfur compounds which are readily formed upon itsdministration. However, at high concentration, allicin might notompletely get converted into these safe substances. There was noignificant alteration in blood concentration of HCTZ in presencef GH 500 mg/kg. This is in line with number of studies on garlicuice and garlic homogenate demonstrating injurious effect of highose of garlic on various tissues like intestinal lining and stomach56]. This could be due to enhanced level of allicin instead of protec-ive SAC and SAMC. The potential bioactive constituents might beesponsible for enzyme inhibitory role of garlic, thereby enhancinghe bioavailability of HCTZ in the body.

The results of the present study indicate that combining HCTZith GH (250 mg/kg, p.o.) could provide an opportunity to reduce

he dose of HCTZ, which may help in minimizing the hypokalemias well as achieve enhanced therapeutic effect. At the same time,roper precaution and care should be exercised to avoid hyper-alemia. Therefore, patients on this type of combination should bearefully followed for electrolyte imbalance such as hypokalemia,ypochloremic alkalosis and hyponatremia periodically and whenymptoms (i.e., dry mouth, thirst, lethargy, restlessness, confu-ion, muscle pain, cramps, muscle weakness, oliguria, tachycardia,ausea, vomiting) occur, serum electrolytes should be urgentlyeasured and necessary treatment instituted.

. Conclusion

In conclusion, this study revealed that garlic could causencrease in the bioavailability and half-life along with decrease in

[

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ical Interactions 181 (2009) 472–479

the clearance and elimination rate constant of hydrochlorothiazideper oral. This may pose a negative implication in clinical practice astoxicity of HCTZ may easily be reached especially during multipledosing because of the possibility of drug accumulation. However,careful addition of garlic in moderate doses might result in ben-eficial effect during treatment of hypertension in patients withmyocardial stress as garlic causes substantial fall in excretion ofpotassium when compared to HCTZ alone treatment in rats. Hence,further studies should be carried out to determine the influence ofspecific active constituent of GH when combined with HCTZ in ani-mals subjected to myocardial damage. We hope that this type ofstudy will open new areas of research for interaction and counter-action between herb and conventional drugs when they are takenconcurrently.

Conflict of interest

None.

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