ORIGINAL PAPER

Effect of pomegranate juice on the pharmacokineticsof nitrendipine in rabbits

Swathi Voruganti • Kishore Rapolu •

Santoshkumar Tota • Shravan Kumar Yamsani •

Madhusudan Rao Yamsani

Received: 28 September 2011 / Accepted: 10 November 2011 / Published online: 22 November 2011

� Springer-Verlag France 2011

Abstract Pomegranate juice (PJ) is known to be a potent

inhibitor of human cytochrome enzymes. The purpose of

this study was to investigate the effect of acute and chronic

PJ on the pharmacokinetics of oral nitrendipine (10 mg/kg)

in rabbits. Male New Zealand rabbits were pretreated with

PJ for 1 week and on the last day, a single dose of

nitrendipine was given orally. In another group, both PJ

and nitrendipine were co-administered to evaluate the acute

effect of PJ on nitrendipine pharmacokinetics. The control

group received oral distilled water for 1 week and admin-

istered with nitrendipine on the last day. Blood samples

were collected at different time points and nitrendipine

concentration was estimated by high-performance liquid

chromatography. Relative to control, the area under the

concentration–time curve and peak plasma concentration

of nitrendipine were 2.03- and 2-fold, respectively, greater

in the PJ-pretreated group. However, co-administration of

PJ had no significant effect on these parameters. Further,

there was no significant change in the elimination rate

constant and elimination half-life of nitrendipine in both PJ

co-administered and pretreated groups in comparison with

control. These results suggest that PJ inhibits the intestinal

metabolism of nitrendipine without affecting hepatic

metabolism in rabbits. Although this potential interaction

needs to be explored further, the concomitant use of PJ and

nitrendipine should be avoided.

Keywords Pomegranate juice � Nitrendipine �Pharmacokinetics � Food–drug interaction

1 Introduction

Several fruit juices have been reported to cause food–drug

interactions mainly due to the inhibition of drug-metabo-

lizing enzymes (Lim et al. 2003; Bailey and Dresser 2004;

Hidaka et al. 2005). Pomegranate (Punica granatum)

contains a number of phytochemicals including punicala-

gin, ellagic acid, gallotannins, anthocyanins, and flavo-

noids (Cerda et al. 2003). It has been used in folk medicine

for a wide variety of therapeutic purposes (Langley 2000).

There is growing interest in this fruit because of its

potential health benefits in cases of certain cancers, car-

diovascular, and neurodegenerative disorders (Kim et al.

2002; Aviram et al. 2004). As noted in the United States

patents section, the primary health benefits of PJ have

focused on the antioxidant actions of the juice and its

potential to prevent atherosclerosis as well as slow pro-

gression of atherosclerotic plaques. Five small human

clinical trials testing cardiovascular activities have evalu-

ated PJ for its effects on cholesterol, atherosclerosis,

myocardial perfusion, hypertension, and erectile dysfunc-

tion (Gil et al. 2000; Kaplan et al. 2001; Aviram and

Dornfeld 2001; Aviram et al. 2002, 2004; Noda et al. 2002;

Sumner et al. 2005). Because of these beneficial effects,

pomegranate has been increasingly popularized (Basu and

S. Voruganti � K. Rapolu � S. K. Yamsani � M. R. Yamsani

National Facilities in Engineering and Technology with

Industrial Collaboration (NAFETIC) Centre, University College

of Pharmaceutical Sciences, Kakatiya University,

Warangal 506 009, Andhra Pradesh, India

S. Voruganti (&)

Department of Pharmaceutics, University College

of Pharmaceutical Sciences, Kakatiya University,

Warangal 506 009, Andhra Pradesh, India

e-mail: swathi_1286@yahoo.co.in

S. Tota

Division of Pharmacology, (CSIR) Central Drug Research

Institute, Lucknow 226001, Uttar Pradesh, India

123

Eur J Drug Metab Pharmacokinet (2012) 37:77–81

DOI 10.1007/s13318-011-0075-4

Penugonda 2009). Therefore, with the increased con-

sumption of PJ, clinicians must become aware of its

potential interactions. Some recent in vitro and animal

studies have revealed the effects of PJ on drug-metabo-

lizing enzymes. Hidaka et al. (2005) reported that PJ sig-

nificantly altered the pharmacokinetics of carbamazepine

in rats by inhibiting CYP 3A activity. It was also reported

that components of PJ inhibits human CYP 3A activity in

vitro. Further, it has been found that PJ increases plasma

concentrations of tolbutamide, a substrate of CYP 2C9

(Nagata et al. 2007).

Based on these findings, it can be expected that con-

comitant use of PJ and drugs substrate for CYP 3A4 may

increase the possibility of food–drug interaction. Therefore,

in the present study, we investigated the effect of PJ on the

pharmacokinetics of nitrendipine since it is extensively

metabolized by CYP 3A4 (Bailey and Dresser 2004).

2 Experimental

2.1 Chemicals and reagents

Nitrendipine and felodipine (IS) were generous gifts from

US Vitamins Laboratories Ltd. (Mumbai, India). HPLC

grade acetonitrile, sodium hydroxide, dichloromethane,

and methanol were purchased from Merck Ltd (Mumbai,

India). DMSO AR grade was purchased from S.d. Fine

Chemicals Pvt Limited. Blank plasma was collected from

New Zealand rabbits at Kakatiya University (Warangal,

India). All other chemicals and solvents were of analytical/

reagent grade. Commercially available pomegranate juice

(PJ) was used in this study.

2.2 Animals

Male white New Zealand rabbits (2.15 ± 0.13 kg) were

selected for the study. The rabbits were acclimatized for

7 days to laboratory conditions before initiating the

experiment. They were housed in individual cages and feed

and water were provided ad libitum. Feed was withheld for

at least 10–12 h before and until 4 h after the drug

administration. Necessary approval from the Institutional

Animal Ethics Committee was obtained to carry out the

experiments.

2.3 Pharmacokinetic experiment

Nitrendipine was suspended in 0.25% carboxy methyl

cellulose just before oral administration. Rabbits were

divided into three groups of six each: the control group

(nitrendipine 10 mg/kg, oral), co-administration group

(10 ml/kg of PJ orally co-administered with 10 mg/kg of

nitrendipine), and pretreatment group (10 mg/kg of

nitrendipine was administered orally after 1-week pre-

treatment with PJ 10 ml/kg). Blood samples (1.5 ml) from

marginal ear vein were collected into heparinized micro-

fuge tubes at 0.5, 1, 1.5, 2, 4, 8, 12, and 24 h post-dosing

and plasma was harvested by centrifuging the blood at

13,000 rpm for 10 min and stored frozen at -20�C until

bioanalysis.

2.4 Plasma samples preparation

To 100 ll of serum, 25 ll of methanol, 25 ll of felodipine

(equivalent to 150 ng), and 100 ll of 1 N NaOH were

added and mixed well for 1 min at each step. To this,

750 ll of dichloromethane was added and vortexed for

15 min followed by centrifugation at 15,000 rpm for

15 min. Organic phase was separated and evaporated. The

residue was reconstituted with mobile phase and 20 ll of

this solution was injected on to a HPLC column.

2.5 HPLC conditions for plasma sample analysis

Nitrendipine was estimated in plasma samples using HPLC

method previously validated in our lab (Kumar et al. 2007).

Analysis was carried out using a HPLC system (Shimadzu,

Kyoto, Japan) coupled with Spherisorb ODS2, reversed-

phase C18, 250 mm 9 4.5 mm, 5 lm column (Waters

Spherisorb) maintained at 25�C. The system was run in

isocratic mode with mobile phase consisting of acetonitrile:

water (60:40, v/v) and delivered at a flow rate of 1.5 ml/min.

Mobile phases were duly filtered through 0.22 lm Millipore

filter (Billerica, USA) and degassed ultrasonically for

20 min and then were pumped in isocratic mode. The

detection was performed at 238 nm wavelength.

2.6 Pharmacokinetic analysis

Plasma data were subjected to non-compartmental phar-

macokinetic analysis using WinNonlin (Pharsight Corpo-

ration, Version 5.1). The observed maximum plasma

concentration (Cmax) and the time to reach the maximum

plasma concentration (Tmax) were obtained by visual

inspection of the experimental data. The area under the

plasma concentration time curve from time zero to the last

quantifiable concentration (AUC0–t) was calculated using

linear trapezoidal method. The AUC0–? was calculated

using the AUC0–t plus the quotient of the last measured

concentration divided by Kel. The Kel was estimated by

linear regression of the plasma concentrations in the

log–linear terminal phase. The apparent elimination half-

life (t1/2) was calculated as 0.693/Kel. The apparent oral

clearance of nitrendipine (CL/F) was calculated as

78 Eur J Drug Metab Pharmacokinet (2012) 37:77–81

123

dose/AUC0–?. Additional estimated parameter using non-

compartmental pharmacokinetic analysis was the volume

of distribution (Vd/F).

2.7 Statistical analysis

Experimental values are expressed as mean ± SD. Statis-

tical analysis was performed by one-way ANOVA fol-

lowed by Tukey’s test and Student’s unpaired t test using

Graph Pad prism software. A value of P \ 0.05 was con-

sidered significant.

3 Results

The results are shown in Fig. 1 and the pharmacokinetic

parameters are summarized in Table 1. The statistical

comparison of mean plasma concentration of nitrendipine

in three groups by one-way ANOVA followed by Tukey’s

test revealed significantly higher (P \ 0.01) plasma

nitrendipine level from 2 to 24 h in PJ-pretreated group in

comparison with the control and PJ co-administered groups

[0.5 h: F (2, 15) = 0.41, P [ 0.05; 1.0 h: F (2, 15) = 2.22,

P [ 0.05; 2.0 h: F (2, 15) = 22.26, P \ 0.01; 4.0 h: F (2,

15) = 36.75, P \ 0.01; 8.0 h: F (2, 15) = 45.23, g \ 0.01;

12 h: F (2, 15) = 12.41, P \ 0.05 and 24 h: F (2,

15) = 26.69, P \ 0.01]. However, no significant change

(P [ 0.05) was observed in plasma concentration of

nitrendipine in PJ co-administered group at all the studied

time points in comparison with that of control group.

Further, analysis of pharmacokinetic parameters revealed

that PJ pretreatment caused a significant elevation in Cmax

(twofold) [F (2, 15) = 24.72, P \ 0.05], AUC0–t (2.03-

fold) [F (2, 15) = 62.6, P \ 0.05], and AUCtot (2.18-fold)

[F (2, 15) = 30.81, P \ 0.05] of nitrendipine in compari-

son with the control group and PJ co-administered groups.

On the other hand, the elimination half-life (t1/2) [F (2,

15) = 1.01, P [ 0.05] and elimination rate constant (Kel)

[F (2, 15) = 0.64, P [ 0.05] of nitrendipine did not alter

significantly in PJ-pretreated group as compared with

control and PJ co-administered groups. Further, CL/

F [F (2, 15) = 38.28, P \ 0.05] and Vd/F [F (2,

15) = 6.94, P \ 0.05] of nitrendipine was significantly

altered in PJ-pretreated group in comparison with that of

control and co-administered groups.

When PJ was co-administered, no significant change

(P [ 0.05) in Cmax, AUC0–t, AUCtot, CL/F, Vd/F, t1/2, and

Kel of nitrendipine was observed in comparison with con-

trol. The mean AUC%extrap was less than 20% of AUCtot

in all three groups. Further, Tmax (h) of nitrendipine was not

altered significantly by PJ administration [F (2, 15) = 1.5,

P [ 0.05] (Table 1).

4 Discussion

Nitrendipine is a calcium channel blocker clinically used

for the treatment of hypertension and other cardiovascular

disorders. As it is a P-gp substrate and is extensively

metabolized by CYP 3A enzymes (Bailey and Dresser

2004; Rajnarayana et al. 2008), there is an increased pos-

sibility of pharmacokinetic drug interaction. Pomegranate

has been used extensively because of its potential health

benefits. However, no reports are available till date stating

the effect of PJ on the pharmacokinetics of nitrendipine.

Therefore, in the present study, the effects of PJ on

nitrendipine pharmacokinetics were examined in rabbits.

The plasma nitrendipine concentration–time profiles were

Fig. 1 Plasma concentration–

time profiles of nitrendipine

(10 mg/kg) in rabbits. Data

values are expressed as mean

plasma concentration of

nitrendipine (lg/ml) ± SD.

*Significant difference

(P \ 0.01) in comparison with

control

Eur J Drug Metab Pharmacokinet (2012) 37:77–81 79

123

investigated after oral administration of nitrendipine

(10 mg/kg) to rabbits.

In this study, we demonstrated that pretreatment with PJ

for 1 week enhanced the oral bioavailability of nitrendi-

pine. The area under the concentration–time curve (AUC-

tot) increased by 2.03-fold in PJ-pretreated group when

compared with control group, indicating a potential inter-

action. In comparison with control group, AUCtot of

nitrendipine was not significantly altered when PJ was

administered 1 h before the nitrendipine. We found sig-

nificantly higher concentration of nitrendipine in PJ-pre-

treated group. This may be due to the chronic inhibition of

CYP 3A-mediated metabolism of nitrendipine by PJ. Pre-

vious studies also showed inhibitory effect of PJ on the

drug metabolizing enzymes. PJ contains certain species of

flavonoids and anthocyanins, and has been shown to

improve the plasma concentration of co-administered

drugs. PJ was found to alter the pharmacokinetics of car-

bamazepine, a CYP 3A substrate (Hidaka et al. 2005). PJ

increased the AUC of carbamazepine by 1.5-fold when

administered 1 h prior to drug. Recently, Nagata et al.

(2007) reported that the bioavailability of tolbutamide, a

CYP 2C9 substrate, was significantly increased by PJ due

to inhibition of intestinal CYP 2C9. The improvement in

nitrendipine bioavailability indicates possible inhibition of

CYP 3A activity by chronic PJ though further studies using

purified CYP enzymes are needed to confirm these

findings.

Further, the study showed a significant decrease in the

CL/F and Vd/F along with a significant rise in AUC of

nitrendipine in PJ co-administered group. The study also

showed that the elimination t1/2 and Kel of nitrendipine did

not alter significantly by PJ co-administration and pre-

treatment. Previous studies also demonstrated that PJ

inhibits intestinal metabolism of substrate drugs without

affecting hepatic drug-metabolizing enzymes (Hidaka et al.

2005; Nagata et al. 2007). Since the elimination half-life

and elimination rate constant of nitrendipine were not

altered significantly among different groups and because

previous studies supported these findings, we can speculate

that the observed effect of PJ is mediated via inhibition of

intestinal metabolism. However, further studies are

required to provide a strong conclusion in this regard.

5 Conclusion

The present study suggests that there might be a potential

interaction between PJ and nitrendipine and therefore,

quantitative evaluation of PJ–drug interaction in humans

needs to be verified to avoid food–drug interactions.

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Table 1 Pharmacokinetic

parameters of nitrendipine in

control, pomegranate juice co-

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juice pretreated groups

Data values are expressed as

mean ± SD

* Significant difference

(P \ 0.05) in comparison to

control

Parameters Control PJ (co-administered)

? nitrendipine

PJ (pretreatment)

? nitrendipine

Mean ± SD Mean ± SD Mean ± SD

Cmax (lg/ml) 0.03 ± 0.006 0.04 ± 0.007 0.06 ± 0.007*

Tmax (h) 1.83 ± 0.4 1.83 ± 0.4 2.33 ± 0.8

AUCt (lg h/ml) 0.29 ± 0.04 0.28 ± 0.02 0.59 ± 0.08*

AUCtotal (lg h/ml) 0.32 ± 0.05 0.30 ± 0.02 0.70 ± 0.16*

AUC%Extrap 9.71 ± 4.04 6.84 ± 2.89 14.86 ± 8.29

t1/2 (h) 5.93 ± 1.42 5.26 ± 1.59 7.24 ± 3.68

Kel 0.12 ± 0.02 0.14 ± 0.05 0.12 ± 0.05

CL/F (l/h/kg) 31.67 ± 5.73 33.41 ± 2.93 14.80 ± 2.76*

Vd/F (l/kg) 264.05 ± 42.95 256.12 ± 85.58 143.96 ± 50.22*

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