Grapefruit -Drug Interaction: Isolation, Biological Activities of

Furocoumarins and Their Variation due to Pre- and Post-harvest Factors

Bhimanagouda S. Patil

OO

O

O

O OO

O

OH

O

OOH

O

O

O

OH

OO

O

O

OH

What is Grapefruit Drug Interaction ?

• Concurrent intake of grapefruit juice and certain drugs increases the plasma concentrations of drugs from 1.5 to 15 times.

• GFJ Inhibits the drug metabolizing enzyme system cytochrome P450, P-glycoprotein and OATP.

What is GFJ-Drug Interaction ?

Time

Are

a U

nd

er

Pla

sm

a

Reference

Drugs Influenced by Grapefruit Juice

Drug Increase in Bioavailability

Felodapine ~3 Fold

Cisapride ~1.4 Fold

Cyclosporin ~1.5 Fold

Saquinavir ~2 Fold

Terfenadine ~2.5 Fold

Buspirone ~9 Fold

Lovastatin/Simvastatin ~10 Fold

and....many more Greenblatt et al., 2001

Bioavailability of Drugs

J R Oesterheld : Drug-Drug Interactions

Bioavailability of Drugs

ORAL DOSE

CYP ----------

GRAPFRUITE JUICE

GI TRACT

PORTALCIRCULATION

LIVER(CYP3A)

SYSTEMIC CIRCULATION

Greenblatt et. al. 2001

Flavonoids: Naringin and Naringenin ???Inhibit CYP P450 enzymes in vitro but have

little or no effect in vivo...------------------------------------------------------------------

Bioactive Furocoumarins in Grapefruit 6’, 7’ Dihydroxybergamottin

BergamottinParadisin AParadisin BParadisin CEpoxy-bergamottinBergaptolGeranylcoumarin

Extraction of Grapefruit Juice

• Extraction for isolation and purification.

• 300 L of juice was extracted with Ethyl acetate to get crude mixture, which was reconstituted in methanol.

• 3gms of extract was loaded on to Prep- HPLC column and eluted with aqueous methanol.

GFJ Concentrate Diluted with water Extracted with ethyl acetate Crude extract Loaded to silica gel column chromatography

Eluted with hexane, hexane:ethyl acetate and ethayl actetate

Fraction -1 2 3 4 5 6 7 Preparative HPLC Eluted with methanol and water 1 2 3 (340 mg) (19 mg) (310 mg)

Schematic Representation of Isolation

Girennavar et al 2006

Isolation of Furocoumarins by PREP HPLC

Grapefruit Concentrate Juice(1L)+ Distilled Water (2.4L)

Grapefruit juice + Ethyl Acetate(1:2)

Leave for 2 Hours

Organic layer + Juice Sediment

Twice

Concentrate by Rotary Evaporation

Reconstitute in Methanol

Elute with Methanol

LCMS

NMR

PrepHPLC

Analytical HPLC Profile of Crude mixture

Minutes

0 10 20 30 40 50 60

mAU

0

1000

2000

3000

mAU

0

1000

2000

3000UV6000-240nm

Retention Time

Bergamottin

DHB

Paradisin A

Bergaptol

Preparative HPLC separation

Paradicin AParadicin B

Bergamottin

Minutes

0 10 20 30 40 50 60

0

1000

2000

3000

0

500

1000

2000

0 10 20 30 40 50 60

0

500

1000

0 10 20 30 40 50 60

A

B

C

Ab

so

rba

nc

e (

mA

U)

0 10 20 30 40 50 60

0

500

1000

1500 D

Minutes

0 10 20 30 40 50 60

0

1000

2000

3000

0 10 20 30 40 50 600 10 20 30 40 50 60

0

1000

2000

3000

0

500

1000

2000

0

500

1000

2000

0 10 20 30 40 50 60

0

500

1000

0 10 20 30 40 50 60

0

500

1000

0 10 20 30 40 50 60

A

B

C

Ab

so

rba

nc

e (

mA

U)

0 10 20 30 40 50 60

0

500

1000

1500 D

12.6

12.6

37.7

37.7

43.7

43.7

23.6

OO O

H

H

H

H

O

OH

OH

H

H

O

OH

O

OO O

H

H

H

H

O

O

O

O

H

H

H

H

OH

H

OO O

H

HH

H

O

H

Paradisin A

Dihydroxybergamottin

Bergamottin

Crude Mixture

0

500

250

0

10 20 30

mA

U

0

Run Time (Minutes)

500

250

0

10 20 30

mA

U

Compound2

Compound 1

0

500

250

0

10 20 30

mA

U

0

Run Time (Minutes)

500

250

0

10 20 30

mA

U

OO O

OH

1

2

34

567

8 910

11 OO O

OH

1

2

34

567

8 910

11

89

23

4

o o

1 10

1112

19

13

14

1516

18

17o7

6

5

89

23

4

o o

1 10

1112

19

13

14

1516

18

17o7

6

5

Geranylcoumarin

Bergaptol

NMR Profile of Paradisin AH+ NMR 13C NMR

2D NMR

Identification of furocoumarins by LC/MS

Inhibitory Property of GFJ and Furocumarins on Human Cytochrome

P450 Enzymes

CYP P450 3A4 enzymes

• Involved several reactions such as Phase I.

• Multiple forms Exists

• Isoforms interact with the phytochemicals

• Inhibited reversibly and/or irreversibly by grapefruit juice components.

CYP P450 enzymes system

• 60% of the marketed drugs are metabolized by CYP

3A4, 2C9 and 2D6

Rendic and Di Carlo 1997

Inhibition of CYP3A4 by GFJ and pummelo

0

20

40

60

80

100

120

RIO RUB RAY STA THO MAR DUN PUM

Juices

% I

nhib

itio

n

1% Juice 10% Juice 25% Juice

Inhibition of CYP2C9 by GFJ and pummelo

0

20

40

60

80

100

120

RIO RUB RAY STA THO MAR DUN PUM

Juices

% I

nhib

itio

n

1% Juice 10% Juice 25% Juice

Inhibition of CYP2D6 by GFJ and pummelo

0

20

40

60

80

100

120

RIO RUB RAY STA THO MAR DUN PUM

Juices

% I

nh

ibit

ion

1% Juice 10% Juice 25% Juice

Inhibition of CYP3A4 by furocoumarins

0

25

50

75

100

0.01 0.1 1 10 50 100

Concentration (µM)

% In

hib

itio

n

DHB PARA BERG BTOL GC

IC50

Inhibition of CYP2C9 by furocoumarins

0

25

50

75

100

0.01 0.1 1 10 50 100

Concentration (µM)

% I

nh

ibit

ion

DHB PARA BERG BTOL GC

IC50

Inhibition of CYP2D6 by furocoumarins

0

25

50

75

100

0.01 0.1 1 10 50 100

Concentration (µM)

% In

hib

itio

n

DHB PARA BERG BTOL GC

IC50

IC50 values for different furocoumarins for major drug metabolizing enzymes

Compounds CYP3A4 CYP2C9 CYP2D6

DHB 9.77 1.58 5.63

Paradisin 0.11 0.18 0.30

Bergamottin 22.91 4.508 11.74

Bergaptol 25.82 9.923 37.33

Geranylcoumarin 53.47 21.51 56.21

Levels of DHB in Different Varieties of GFJ and Pummelo

0

0.5

1

1.5

2

2.5

3

RIO RUB RAY STA THO MAR DUN PUM

Varieties

Con

cent

ratio

n of

DH

B

(µg/

ml)

Levels of Paradisin A in Different Varieties of GFJ and Pummelo

0

0.02

0.04

0.06

0.08

0.1

RIO RUB RAY STA THO MAR DUN PUM

Varieties

Con

cent

ratio

n of

par

adis

in A

(µ

g/m

l)

Levels of Bergamottin in Different Varieties of GFJ and Pummelo

0

0.2

0.4

0.6

0.8

1

1.2

RIO RUB RAY STA THO MAR DUN PUM

Varieties

Con

cent

ratio

n of

ber

gam

ottin

(µ

g/m

l)

Influence of Location and Varieties on Levels of DHB

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

RRT RRF RRC MWT MWF

Location and variety

Con

cent

rati

on o

f D

HB

(µ

g/m

l)

Influence of Location and Varieties on Levels of Paradisin A

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

RRT RRF RRC MWT MWF

Location and variety

Con

cen

trat

ion

of

par

adis

in A

(µ

g/m

l)

Influence of Location and Varieties on Levels of Bergamottin

0

0.20.4

0.60.8

11.2

1.41.6

1.8

RRT RRF RRC MWT MWF

Location and variety

Con

cen

trat

ion

of

ber

gam

otti

n

(µg/

ml)

Variation of DHB During Season

0

0.3

0.6

0.9

1.2

1.5

1.8

Nov Dec Jan Feb Mar Apr May

Con

cent

rati

on o

f D

HB

(µg

/ml)

Rio Red Marsh White

Variation of Paradisin A During Season

0

0.03

0.06

0.09

0.12

Nov Dec Jan Feb Mar Apr May

Con

cen

rati

on o

f p

arad

isin

A (

µg/

ml)

Rio Red Marsh White

Variation of Bergamottin During Season

0

0.3

0.6

0.9

1.2

1.5

1.8

Nov Dec Jan Feb Mar Apr May

Rio Red Marsh White

Effect of Storage and Processing on Furocoumarins

• Commercial GFJ production often involves processing at high temperature and mechanical peeling.

• Objective was to quantify the changes if any during processing.

Harvesting Grapefruits at TAMUK- Citrus Center

GFJ Processing Plant at Mission, Texas

Levels of Furocoumarins in Hand Squeezed and Processed GFJ

01

234

56

78

DHB PA BM

Furocoumarins

Co

nce

ntr

atio

n o

f fu

roco

um

arin

s (u

g/m

l)

HS Juice IP Juice

Effect of Different Storage Temperatures on Furocoumarins Level in Rio Red GFJ

Condition Days Levels of Furocoumarins (µg/ml)

    DHB Paradisin A Bergamottin

9 ºC 0 1.201 ± 0.061a 0.09 ± 0.004 1.005 ± 0.049

15 1.137 ± 0.078 0.088 ± 0.002 0.995 ± 0.062

30 1.108 ± 0.069 0.087 ± 0.004 0.978 ± 0.028

45 1.098 ± 0.068 0.076 ± 0.006 0.965 ± 0.37

Room

Temperature 0 1.201 ± 0.061 0.09 ± 0.001 1.005 ± 0.049

15 1.091 ± 0.055 0.077 ± 0.004 0.909 ± 0.055

30 0.998 ± 0.014 0.069 ± 0.007 0.896 ± 0.081aMean ± standard deviation, n=15

Effect of Different Storage Temperatures on Furocoumarins Levels in Marsh White GFJ

Condition Days Levels of Furocoumarins (µg/ml)

    DHB Paradisin A Bergamottin

9 ºC 0 1.704 ± 0.043a 0.092 ± 0.004 1.437 ± 0.038

15 1.689 ± 0.076 0.089 ± 0.006 1.426 ± 0.04

30 1.618 ± 0.091 0.086 ± 0.0052 1.408 ± 0.044

45 1.587 ± 0.068 0.085 ± 0.003 1.397 ± 0.083

Room

Temperature 0 1.704 ± 0.043 0.092 ± 0.005 1.437 ± 0.038

15 1.593 ± 0.099 0.081 ± 0.003 1.406 ± 0.019

  30 1.467 ± 0.065 0.071 ± 0.002 1.396 ± 0.045

aMean ± standard deviation, n=15

Cans

Days Levels of Furocoumarins (µg/ml)

  DHB Paradisin A Bergamottin

0 2.065 ± 0.081a 0.099 ± 0.004 1.255 ± 0.028

15 1.985 ± 0.092 0.088 ± 0.003 1.223 ± 0.046

30 1.885 ± 0.015 0.078 ± 0.003 1.213 ± 0.021

45 1.772 ± 0.064 0.069 ± 0.004 1.193 ± 0.037

60 1.598 ± 0.109 0.065 ± 0.015 1.166 ± 0.045

75 1.499 ± 0.089 0.061± 0.028 1.142 ± 0.013

90 1.411 ± 0.037 0.056 ± 0.007 1.1138 ± 0.049aMean ± standard deviation, n=15

Cardboards

Days Levels of Furocoumarins (µg/ml)

  DHB Paradisin A Bergamottin

0 2.125 ± 0.073a 0.104 ± 0.008 1.487 ± 0.071

15 1.985 ± 0.092 0.097 ± 0.005 1.354 ± 0.087

30 1.818 ± 0.108 0.089 ± 0.001 1.285 ± 0.051

45 1.789 ± 0.089 0.076 ± 0.003 1.116 ± 0.119

60 1.5612 ± 0.089 0.074 ± 0.006 1.099 ± 0.19

75 1.4511 ± 0.061 0.072 ± 0.002 1.037 ± 0.017

90 1.4198 ± 0.183 0.066 ± 0.004 1.004 ± 0.105

aMean ± standard deviation, n=15

Cartoons

aMean ± standard deviation, n=15

Days Levels of Furocoumarins (µg/ml)

  DHB Paradisin A Bergamottin

0 2.3146 ± 0.091 0.1009 ± 0.006 1.8655 ± 0.091

15 2.1485 ± 0.092 0.0981 ± 0.013 1.6813 ± 0.086

30 1.9915 ± 0.105 0.0883 ± 0.008 1.4513 ± 0.107

45 1.8212 ± 0.064 0.0769 ± 0.017 1.3193 ± 0.045

60 1.7598 ± 0.109 0.0685 ± 0.009 1.2168 ± 0.021

Clinical applications of GFJ

• Grapefruit juice may provide a non-toxic and inexpensive alternative to drugs that are used to reduce cyclosporin dose. (Yee GC, Lancet. 1995 Apr 15;345(8955):955-6.)

• Grapefruit juice enhances the bioavailability of the HIV protease inhibitor in man. (Kupferschmidt et. al., Br. J. Clin. Pharmacol 1998: 45: 355-9.)

• Super pill ?

Conclusions

• Various furocoumarins were isolated and characterized from GFJ

• GFJ and furocoumarins are potent inhibitors of CYP3A4, CYP2C9 and CYP2D6 isoenzymes. The order of inhibition poetntial is CYP2D6>CYP3A4>CYP2C9

• Furocoumarins levels are affected by pre- and post-harvest factors.

Girennavar, B., G.K. Jayaprakasha, Jadegouda, G.A. Nagana Gowda, and B.S. Patil. 2007. Radical scavenging and cytochrome P450 3A4 inhibitory activity of bergaptol and geranylcoumarin from grapefruit. Bioorganic and Medicinal Chemistry 15: 3684-3691.

Girennavar, B., G. K. Jayaprakasha, J. L. Jifon and B.S. Patil. 2007. Variation of bioactive furocoumarins and flavonoids in different varieties of grapefruits and pummelo. European Food Research and Technology (in press). DOI:10.1007/s00217.007.0654.4

Girennavar, B., G. K. Jayaprakasha, and B.S. Patil. 2007. Potent Inhibition of Human Cytochrome P450 3A4, 2D6 and 2C9 Isoenzymes by Grapefruit Juice and its Furocoumarins. Journal of Food Science (in Print).

Girennavar, B., S.M. Poulose, G.K. Jayaprakash, N.G. Bhat, B.S. Patil. 2006. Furocoumarins from Grapefruit Juice and their effect on Human CYP 3A4 and CYP 1B1 Isoenzymes. Bioorganic and Medicinal Chemistry 14:2606-2612.

Girennavar, B., G.K. Jayaprakash, B.S. Patil. 2007. Influence of pre- and post- harvest factors and processing on the levels of furocoumarins in grapefruits (Citrus paradisi Macf.). Food Chemistry (under review).

Girennavar, B., Simpkin, S.E., G.K. Jayaprakash, Maxim, J., B.S. Patil. 2007. Influence of Electron Beam Irradiation on Bioactive Compounds in Grapefruits (Citrus paradisi Macf.). Journal of Ag Food Chemistry (under review).

Publications