Grapefruit -Drug Interaction: Isolation, Biological Activities of
Furocoumarins and Their Variation due to Pre- and Post-harvest Factors
Bhimanagouda S. Patil
OO
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O OO
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OH
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OH
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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).
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