grapefruit -drug interaction: isolation, biological

Grapefruit -Drug Interaction:

Isolation, Biological Activities of

Furocoumarins and Their Variation

due to Pre- and Post-harvest Factors

Bhimanagouda S. Patil

O O

O

O

O O O

O

OH

O

O OH

O

O

O

OH

O O

O

O

OH

Overview

• What is food-drug interaction?

• Grapefruit-drug interaction

• Media : GFJ & drug interaction

• Isolation of furocoumarins

• Inhibition of CYP enzymes by GFJ

• Benefits of furocoumarins (FC)

• Variation of FC by pre and post harvest

factors

Drug metabolism

CYP

GI TRACT

PORTAL CIRCULATION

LIVER (CYP3A)

SYSTEMIC CIRCULATION

Greenblatt et. al., Clinical Psychopharmacology 21(4):357-359, 2001

ORAL DOSE

VIDEO

http://www.youtube.com/watch?v=sp4tsJ4lbEs

http://www.youtube.com/watch?v=xiuWdJYyIKs&feature=related

Movement of drugs through the

body by

• Absorption

• Distribution

• Metabolism

• Excretion

Food-drug interaction

FOOD

COMPONENTS


• Food-drug interaction:

– Is a broad term that includes interaction of

food-drug and the their effect.

• A food-drug interaction can:

• prevent a medicine from working the way it

should

• cause a side effect from a medicine to get worse

or better

• cause a new side effect

• Give some examples of food-drug

interaction you commonly come

across?


Examples of Food-drug

interaction • Vitamin K rich foods Warfarin

Examples: spinach, turnip greens

• Monoamine oxidase inhibitors (MAOIs)

– metabolism of Tyramine

Examples of MAOI: Resveratrol (wine & grape

skin), Curcumin (Turmeric), Harmine alkaloid

(Coffee), Catechin (Tea)

Sarah. H. Bland., J.Pharmacy Soc. of Wisconsin: 1998, 28-35

• Tea and Coffee Iron supplements

• High fiber diet

– Wheat, bran, oatmeal

• Alcohol Produce additive toxicity

Exp. of Food-drug interaction

Eggs, diary products

Absorption of antidepressents

J.Pharmacy Soc. of Wisconsin: NOV/DEC 1998, 28-35

Grapefruit

Drug Interaction

History

Dr. David Bailey

•FIRST REPORT ON GRAPEFRUIT

JUICE AND DRUG INTERACTION

•Published in the Journal of LANCET

1991

•Results conclude GFJ increase the

bioavailability of tested drugs

YEAR Breakthrough

1989

Serendipitously discovered during exp. designed to

test the effect of ethanol on a calcium-channel

blocker (Felodipine). Clin Invest Med 1989;12:357-62.

1991 FIRST STUDY: Interaction of citrus juices with

felodipine and nifedipine . Lancet 1991;337:268-69

1995

GFJ : pharmacokinetics and pharmacodynamics of

felodipine in healthy subjects.

Eur J Clin Pharmacol 1995;49:61-7

1997 GFJ increases felodipine oral availability in humans

by decreasing intestinal CYP3A protein expression. J Clin Invest 1997;99(10):2545-53.

1997

GFJ: pharmacokinetics and haemodynamics of

intravenously and orally administered felodipine

Eur J Pharmacol 1997;52:139-45.

YEAR Breakthrough

1999

6′,7′-Dihydrobergamottin in GFJ and Seville orange

juice: effects on cyclosporine disposition,

enterocyte CYP3A4, and P-glycoprotein.

Clin Pharmacol Ther 1999;65:237-44.

2000 GFJ –felodipine interaction: Fruit segments and an

extract from unprocessed fruits Clin Pharmacol Ther 2000;67:107.

2001

Seville orange juice–felodipine interaction:

comparison with GFJ and involvement of

furocoumarins. Clin Pharmacol Ther 2001;69(1):14-23

2006

A furanocoumarin-free GFJ proves FC as the

mediators of the GF-Felodipine interaction. Am J Clin Nutr 2006;83:1097–105.

•GFJ interacts with many prescription drugs

which CAN cause potentially serious side

effects

•Interaction listed on the medication labels.

VIDEO



•Woman consumed GFJ along with oral contraceptive

•Developed a blood clot in the limb

•Almost risked loosing the limb.

•Report published in LANCET and speculated the role of

GFJ-drug interaction.

•DRAWBACKS: NO PROOF, Single patient

What is Grapefruit Drug

Interaction ?

• Concurrent intake of GFJ 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

http://dev.newtontalk.net/~dpadilla/images/pill.jpg

Bioavailability of Drugs

J R Oesterheld : Drug-Drug Interactions

Bioavailability of Drugs

CYP ---------- GFJ

GI TRACT

PORTAL CIRCULATION

LIVER (CYP3A)

SYSTEMIC CIRCULATION

ORAL DOSE


Drugs Influenced by GFJ

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


In vivo evidence….. OBJECTIVE: Whether furocoumarins

mediate the GFJ felodipine interaction ?

Design: 18 healthy volunteers ingested

felodipine (10 mg) with 1 of the 3 juices (240

mL).

RESULTS: conc. of felodipine were

significantly greater with consumption of GFJ

than with that of orange or furocoumarin-free

GFJ.

Paine et al., Am J Clin Nutr May 2006 vol. 83 no. 5 1097-1105

In vivo evidence…..

GFJ

ORANGE Juice

Furocoumarin free GFJ

Paine et al., Am J Clin Nutr May 2006 vol. 83 no. 5 1097-1105

In vivo evidence…..

Felodipine+ GFJ

Felodipine+ water

Plasma felodipine concentration time profile

Bailey et al., Br J Clin Pharmacol 1998; 46: 101-110

Bailey et al.,

Br J Clin Pharmacol

1998; 46: 101-110

Drug enters the GI

Drug is not

metabolized in

the stomach

After absorption,

drug enters the

portal vein

In the small

bowel GFJ

inhibits CYP3A4:

90% is

Unmetabolized

In the Liver: the

drug is further

metabolized:

45 % is

unmetabolized

as compared to

15% without GFJ

What are the components in

GFJ that influence drug

interaction….?

GFJ: Components • Initial reports speculated role of naringin

and naringenin in drug interaction

• In vitro tests on human liver models

suggest flavonoids do not inhibit CYP3A4

0

20

40

60

80

Bergapten Quercetin Naringenin Naringin

% I

nh

ibit

ion

of

CY

P3

A4

Ho et al., J Pharma Sci, (2001) 4 : 217-227

• In in vivo test similar results were noticed.

• AUC was measured after administering

felodipine and

– a) Supernatant -- c) GFJ

– b) Particulate -- d) Water

GFJ: Components

Supernatant

Particulate

148 mg of Naringin: 1.85 mg of DHB

7 mg of Naringin: 0.60 mg of DHB

Fuhr et al., Clin Pharmacol Therap 1998: 64, 248-256

GFJ: Components A

UC

(0

-12 h

) (n

mo

l. h

/L

*

*

*

Fuhr et al., Clin Pharmacol Therap 1998: 64, 248-256

Bioactive Furocoumarins in Grapefruit 6’, 7’ Dihydroxybergamottin

Bergamottin

Paradisin A

Paradisin B

Paradisin C

Epoxy-bergamottin

Bergaptol

Geranylcoumarin

GF-1,

GF-2

GFJ: other components

Imperatonin

Pimpenellin

Isopimpenellin

Isolation and Purification of

Furocoumarins from

Grapefruit Juice

Extraction of Grapefruit Juice

• Isolation and purification.

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

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

Schematic of Isolation

Fraction -1 2 3 4 5 6 7

1 2 3

(340 mg) (19 mg) (310 mg)

GFJ Concentrate Diluted with water

Extracted with ethyl acetate

Column chromatography

Crude extract

Prep- HPLC Eluted with MeOH and H2O

Eluted with HEX,

HEX:EtOAc & EtOAc

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

Prep

HPLC

HPLC Profile of Crude mixture

Minutes

0 10 20 30 40 50 60

mA

U

0

1000

2000

3000

mA

U

0

1000

2000

3000UV6000-240nm

Retention Time

Bergamottin

DHB

Paradisin A

Bergaptol

Preparative HPLC separation

Paradisin A Paradisin 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

rban

ce (

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

rban

ce (

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

O O O

H

H

H

H

O

O H

O H

H

H

O

O H

O

O O O

H

H

H

H

O

O

O

O

H

H

H

H

O H

H

O O O

H

H H

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

Compound 2

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

3

4

56

7

89

1011 OO O

OH

1

2

3

4

56

7

89

1011

8

9

2

3

4

o o

110

1112

19

13

14

15

16

18

17o7

6

5

8

9

2

3

4

o o

110

1112

19

13

14

15

16

18

17o7

6

5

Geranylcoumarin

Bergaptol

NMR Profile of Paradisin A H+ NMR 13C NMR

2D NMR

Bergapten

Mass spectrum

of bergapten

1H and 13C NMR spectra of Bergamottin

200 280 360 440 520 600

1.1E+4

0102030405060708090100

200 280 360 440 520 600

1.5E+4

0102030405060708090100

200 280 360 440 520 600

Mass (m/z)

6304

0102030405060708090100 Voyager Spec #1[BP = 433.2, 6304] 433.16

434.15432.15

215.11 361.01

Voyager Spec #1[BP = 339.2, 10766]217.22

321.29

299.29242.39218.22 339.25

Voyager Spec #1[BP = 217.2, 14793]+H

217.23

327.27239.14

+H

+H

% I

nte

nsi

ty

Bergamottin

Bergapten

Heptamethoxyflavone

Mass spectra of

bergamottin

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.

http://rds.yahoo.com/S=96062857/K=enzyme/v=2/TID=I001_0/SID=e/l=II/SS=i/OID=6567d18bdea54816/R=1/*-http:/images.search.yahoo.com/search/images/view?back=http://images.search.yahoo.com/search/images?srch=1&p=enzyme&ei=UTF-8&n=20&fl=0&h=327&w=355&im

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

nh

ibit

ion

1% Juice 10% Juice 25% Juice

Girennavar et al., J FOOD SCI. Vol. 72, Nr. 8, 2007

Inhibition of CYP2C9 by

GFJ and pummelo

0

20

40

60

80

100

120


Juices

% I

nh

ibit

ion



0

20

40

60

80

100

120


Juices

% I

nh

ibit

ion



GFJ and pummelo


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



furocoumarins

0

25

50

75

100

0.01 0.1 1 10 50 100

Concentration (µM)

% I

nh

ibit

ion


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


IC50


IC50 values (µm) of 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


GRAPEFRUIT JUICE &

BREAST CANCER

Controversy

Eating grapefruit every

day could raise the risk of

developing breast cancer

by almost a third, US

scientists say.

• Rationale:

– CYP P450 3A4 (CYP3A4) is involved in the

metabolism of oestrogens.

– GFJ is an inhibitor of CYP3A4, increases

plasma oestrogen concentrations.

– Well established that oestrogen is associated

with breast cancer risk

Does consumption of GF influence

breast cancer in postmenopausal

woman ?

Prospective study: Role of GFJ

in breast cancer

Monroe at al., Br. J. Cancer (2007) 97, 440–445

Prospective study: Role of GFJ

in breast cancer

Monroe at al., Br. J. Cancer (2007) 97, 440–445

Prospective cohort:

•Study size 50,000 postmenopausal women

•1657 breast cancer cases

•GF (1/4th fruit /day) intake increased risk of breast cancer

•RR=1.30, 95% CI 1.06-1.58

•Similar trend noticed in oestrogen + progestin therapy

Are there any beneficial

properties of furocoumarins?

Citrus: Breeding for reducing

furocoumarins

Furocoumarins:

The

GOOD…………………………………… BAD &

• Antiproliferative

• Enhances

bioavailability of

certain low

bioavailable drugs

• Inhibits biofilm

• Used in treatment of

psoriasis

• Inhibits human

intestinal enzyme

cytochrome P450

• Potential for toxicity

• Toxic to fish

• Early clinical trial, study conducted at

University of Chicago Medical Center

• 8 oz of GFJ intake along with the drug

Rapamycin increase the drug levels

allowing lower dose administration.

• Similar combination can be used for treating

various types of cancer.

Furocoumarins: beneficial

properties

1) Increases bioavailability of drugs

2) Antiproliferative

3) Attenuates TNF-α-stimulated

endothelial molecule expression.

4) Inhibit biofilm in bacteria

5) Induce GST

GFJ increases bioavailability

of low bioavailable drugs

• Saquinavir : Is a potent HIV protease

inhibitor drug

• Problem:

– Very low bioavailability

– Expensive

Kupferschimdt et al., Br. J. Cli. Pharmacol. 1998; 355-359

GFJ increases bioavailability of low

bioavailable drugs

Grapefruit Juice (400 mL) Water (400 mL)

Intravenous (12 mg) ORAL (600mg)

SAQUINAVIR

Serial

blood

samples


Pla

sm

a c

on

cen

trati

on

ng

/mL

Water

GFJ

Intravenous (12 mg)


Pla

sm

a c

on

cen

trati

on

ng

/mL

Water

GFJ

ORAL

(600mg)

2 Fold

increase


Furocoumarins: HL-60

Differentiation-Inducing Compounds

Kawaii et al., J. Agric. Food Chem., 1999, 47 (10), pp 4073–4078

Nitro blue tetrazolium

reducing activity

nonspecific

esterase activity

specific

esterase activity

phagocytic activity

cellular proliferation

Nitro blue tetrazolium

reducing activity

nonspecific

esterase activity

specific

esterase activity

phagocytic activity

cellular proliferation

Furocoumarins: HL-60

Differentiation-Inducing Compounds

Kawaii et al., J. Agric. Food Chem., 1999, 47 (10), pp 4073–4078

Bergamottin attenuates TNF-α-stimulated

endothelial molecule expression

Sasaki et al., Am J Physiol Cell Physiol (2004 )286 :931-939

Effects of orally administered imperatorin

and isopimpinellin on GST activity.

Kleiner et al., Carcinogenesis 2001;22:73-82

GFJ & furocoumarins inhibits

autoinducer signaling and biofilm

formation in bacteria

Inhibition of AI-1 activity

Girennevar et al., Int. J Food Microbiol. (2008) 125: 204-208

GFJ & furocoumarins inhibits

autoinducer signaling and biofilm

formation in bacteria

Inhibition of AI-2 activity


Treatment E. coli O157:H7 S. typhimurium P. aeruginosa

Total biofilma %

Inhibition

Total biofilm %

Inhibition

Total biofilm %

Inhibition

(OD590) (OD590) (OD590)

Control 1.32 ± 0.11 1.16 ± 0.03 1.10 ± 0.02

DHB 0.39 ± 0.06 71.9 0.98 ± 0.06 15.5 0.90 ± 0.09 18.1

Bergamottin 0.55 ± 0.08 58.3 0.62 ± 0.03 46.5 0.80 ± 0.10 27.3

Rio red juice 0.49 ± 0.16 64.7 0.79 ± 0.04 31.8 0.84 ± 0.11 23.6

Marsh white

juice

1.18 ± 0.01 10.6 1.13 ± 0.05 2.5 0.88 ± 0.14 20

GFJ & furocoumarins inhibits autoinducer

signaling and biofilm formation in bacteria


http://www.sciencedirect.com/science/article/pii/S0168160508001529

Probable 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 ?

Furocoumarins:

Variation due to Pre- and

Post-harvest Factors

Pre harvest and Post harvest

Variation: commercial products

varieties

location

Season

Storage

Packaging

Degreening

Conditioning

Processing techniques

-Breeding strategies

Fukuda et al., J Chromatogra B, 741 (2000) 195–203

Levels of FC in different commercial products

Levels of DHB in Different Varieties of

GFJ and Pummelo

0

0.5

1

1.5

2

2.5

3


Varieties

Co

nce

ntr

atio

n o

f D

HB

(µg

/ml)

Girennevar et al., Eur Food Res Technol (2008) 226:1269–1275

Levels of Paradisin A in Different

Varieties of GFJ and Pummelo

0

0.02

0.04

0.06

0.08

0.1


Varieties

Co

nce

ntr

atio

n o

f p

ara

dis

in A

(µg

/ml)


Levels of Bergamottin in Different

Varieties of GFJ and Pummelo

0

0.2

0.4

0.6

0.8

1

1.2


Varieties

Concentr

atio

n o

f berg

am

ottin

(µg/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

Co

ncen

tra

tio

n o

f D

HB

(µg

/ml)

B. Girennavar et al., Food Chem. 111 (2008) 387–392


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


Co

nce

ntr

ati

on

of

pa

rad

isin

A

(µg

/ml)

Girennavar et al., Food Chem. 111 (2008) 387–392


Levels of Bergamottin

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

RRT RRF RRC MWT MWF


Con

cen

trati

on

of

ber

gam

ott

in

(µg/m

l)


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

Co

nce

ntr

ati

on

of

DH

B

(µ

g/m

l)

Rio Red Marsh White


Seasonal Variation of Paradisin A

0

0.03

0.06

0.09

0.12

Nov Dec Jan Feb Mar Apr May

Con

cen

rati

on

of

para

dis

in A

(µ

g/m

l)

Rio Red Marsh White

Girennavar et al. Food Chem. 111(2008) 387–392

Seasonal Variation of Bergamottin


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 TAMU-

Citrus Center

GFJ Processing Plant at Mission,

Texas

Levels of Furocoumarins in Hand

Squeezed and Processed GFJ

0

1

2

3

4

5

6

7

8

DHB PA BM

Furocoumarins

Co

ncen

trati

on

of

furo

co

um

ari

ns (

ug

/ml)

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 ± 0.081

aMean ± standard deviation, n=15


Effect of Different Storage Temperatures on

Furocoumarins Levels in Marsh White GFJ

Condition Days Levels of Furocoumarins (µg/ml)


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



Cans

Days Levels of Furocoumarins (µg/ml)


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 ± 0.049



Cardboards



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



Cartons



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



Degreening

• Ethylene gas used

• Early season citrus fruits and

regreened Valencia oranges

• Destruction of chlorophyll and

accumulation of carotenoids.

• Attractive fruit color

Degreening room

0

100

200

300

400

0 7 14 21 28 35

µg/g

dry

wt

Storage (days)

DHB ND DG a a

b

a

b

a

a a

a a

a a

0

10

20

30

40

0 7 14 21 28 35

µg/g

dry

wt

Storage (days)

Bergamottin ND DG

a a

b

a

b

a

a a b

a a

b

0

25

50

75

100

125

150

175

0 7 14 21 28 35

µg/g

dry

wt

Storage (days)

5G7MC ND DG

a a b

a a a

b

a a a

b

a

Effect of degreening on furocoumarins

Temperature Conditioning

• Low temperature used to maintain quality and slow

respiration and metabolic activity after harvest

• Grapefruits are susceptible to chilling injury when stored at

low temperature (< 10°C) for prolonged period

• Chilling injury symptoms – browning of flavedo, pitting,

surface lesions, water soaked tissues

• Temperature conditioning – 7 days at 16C then

stored at low temperature

• Other treatments used – Intermittent warming, waxes

and vegetable oils, modified atmosphere packaging

Furocoumarins

0

30

60

90

120

150

0 4 8 16

DH

B

(µg/

g d

ry w

t)

Storage (weeks)

11ºC 2ºC CD

b

a

b a

a

b

b

c

a

a

a

a

0

30

60

90

120

150

0 4 8 16

Ber

gam

ott

in

(µg/

g d

ry w

t)

Storage (weeks)

11ºC 2ºC CD

a

a

a

c

b

b

a

b a

a

b

ab

0

30

60

90

120

150

0 4 8 16

5G

7M

C

(µg/g

dry

wt)

Storage (weeks)

11ºC 2ºC CD

a

a

a

b

a

a

a

b

b

ab

b

a

House hold processing

techniques

Blending Juicing Squeezing

Furocoumarins

0

20

40

0 2 4 6 8 10 12 14 16 18 20 22 24

0

50

100

2 4 6 8 10 12 14 16 18 20 22 24

0

50

100

2 4 6 8 10 12 14 16 18 20 22 24

0

50

100

2 4 6 8 10 12 14 16 18 20 22 24

DHB

Bergamottin 5-M-7-GC

DHB

DHB

Bergamottin

5-M-7-GC

Bergamottin

Bergamottin

5-M-7-GC

5-M-7-GC

DHB

Furocoumarins

Standard

Blending

Squeezing

JuicingBlending Juicing Squeezing

0.0

0.1

0.2

0.3

0.4

0.5

0.6 DHB

Bergamottin

5-M-7-GC

mg/

100 m

L

a

a

a

b

c c

c

b

b

Uckoo et al., Journal of food science 77 (9), C921-C926

Citrus: Breeding for reducing

furocoumarins

Chen et al., J. AMER. SOC. HORT. SCI. 136(5):358–

363. 2011

Diploid hybrids

identified with low

content of

furocoumarins

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 potential is CYP2D6>CYP3A4>CYP2C9

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

grapefruit -drug interaction: isolation, biological

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