OH O O OH O O O O O O O OH O O O O O OH O O O Grapefruit -Drug Interaction: Isolation, Biological Activities of Furocoumarins and Their Variation due to Pre- and Post-harvest Factors Bhimanagouda S. Patil
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 ? Area Under Plasma Time Reference
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 Drugs Influenced by Grapefruit Juice Greenblatt et al., 2001
Bioavailability of Drugs J R Oesterheld : Drug-Drug Interactions
Bioavailability of Drugs ORAL DOSE CYP ---------- GRAPFRUITE JUICE GI TRACT PORTAL CIRCULATION 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 Bergamottin Paradisin A Paradisin B Paradisin C Epoxy-bergamottin Bergaptol Geranylcoumarin
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.
Schematic Representation of Isolation Girennavar et al 2006
Analytical HPLC Profile of Crude mixture DHB Bergaptol Bergamottin Paradisin A
Preparative HPLC separation Bergamottin Paradicin B Paradicin A
O H O O H H H H H O O O H Dihydroxybergamottin H O H H O H O O O H O H O O H H H H H O O O H Paradisin A O H H H H O O O H Bergamottin Crude Mixture
500 500 1 Compound 250 250 mAU mAU 0 0 10 10 20 20 0 0 30 30 500 500 2 Compound mAU mAU 250 250 0 0 0 0 20 20 30 30 10 10 Run Time (Minutes) Run Time (Minutes) Bergaptol Geranylcoumarin
NMR Profile of Paradisin A H+ NMR 13C NMR 2D NMR
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
IC50 values for different furocoumarins for major drug metabolizing enzymes
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.
Effect of Different Storage Temperatures on Furocoumarins Level in Rio Red GFJ aMean ± standard deviation, n=15
Effect of Different Storage Temperatures on Furocoumarins Levels in Marsh White GFJ aMean ± standard deviation, n=15
Cans aMean ± standard deviation, n=15
Cardboards aMean ± standard deviation, n=15
Cartoons aMean ± standard deviation, n=15
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.
Publications 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).