1. 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 O O O O O O O

2. 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.

3. What is GFJ-Drug Interaction ? Time Area Under Plasma Reference

4. Drugs Influenced by Grapefruit Juice DrugIncrease 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

5. Bioavailability of Drugs J R Oesterheld : Drug-Drug Interactions

6. Bioavailability of Drugs ORAL DOSE CYP ----------  GRAPFRUITE JUICE GI TRACT PORTAL CIRCULATION LIVER (CYP3A) SYSTEMIC CIRCULATION Greenblatt et. al. 2001

7. 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

8. 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.

9. Schematic Representation of Isolation Girennavar et al 2006

10. Isolation of Furocoumarins by PREP HPLC

11. Analytical HPLC Profile of Crude mixture Bergamottin DHB Paradisin A Bergaptol

12. Preparative HPLC separation Paradicin A Paradicin B Bergamottin

13. Crude Mixture

14. 0 500 250 0 10 20 30 mAU 0 Run Time (Minutes) 500 250 0 10 2030 mAU Compound 2 1 0 500 250 0 10 20 30 mAU 0 Run Time (Minutes) 500 250 0 10 2030 mAU Geranylcoumarin Bergaptol

15. NMR Profile of Paradisin A H + NMR 13 C NMR 2D NMR

16. Identification of furocoumarins by LC/MS

17. Inhibitory Property of GFJ and Furocumarins on Human Cytochrome P450 Enzymes

18. 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.

19. CYP P450 enzymes system 60% of the marketed drugs are metabolized by CYP 3A4, 2C9 and 2D6 Rendic and Di Carlo 1997

20. Inhibition of CYP3A4 by GFJ and pummelo

21. Inhibition of CYP2C9 by GFJ and pummelo

22. Inhibition of CYP2D6 by GFJ and pummelo

23. Inhibition of CYP3A4 by furocoumarins IC 50

24. Inhibition of CYP2C9 by furocoumarins IC 50

25. Inhibition of CYP2D6 by furocoumarins IC 50

26. IC 50 values for different furocoumarins for major drug metabolizing enzymes CompoundsCYP3A4CYP2C9CYP2D6 DHB9.771.585.63 Paradisin0.110.180.30 Bergamottin22.914.50811.74 Bergaptol25.829.92337.33 Geranylcoumarin53.4721.5156.21

27. Levels of DHB in Different Varieties of GFJ and Pummelo

28. Levels of Paradisin A in Different Varieties of GFJ and Pummelo

29. Levels of Bergamottin in Different Varieties of GFJ and Pummelo

30. Influence of Location and Varieties on Levels of DHB

31. Influence of Location and Varieties on Levels of Paradisin A

32. Influence of Location and Varieties on Levels of Bergamottin

33. Variation of DHB During Season

34. Variation of Paradisin A During Season

35. Variation of Bergamottin During Season

36. 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.

37. Harvesting Grapefruits at TAMUK- Citrus Center

38. GFJ Processing Plant at Mission, Texas

39. Levels of Furocoumarins in Hand Squeezed and Processed GFJ

40. Effect of Different Storage Temperatures on Furocoumarins Level in Rio Red GFJ ConditionDaysLevels of Furocoumarins (µg/ml) DHBParadisin ABergamottin 9 ºC01.201 ± 0.061 a 0.09 ± 0.0041.005 ± 0.049 151.137 ± 0.0780.088 ± 0.0020.995 ± 0.062 301.108 ± 0.0690.087 ± 0.0040.978 ± 0.028 451.098 ± 0.0680.076 ± 0.0060.965 ± 0.37 Room Temperature01.201 ± 0.0610.09 ± 0.0011.005 ± 0.049 151.091 ± 0.0550.077 ± 0.0040.909 ± 0.055 300.998 ± 0.0140.069 ± 0.0070.896 ± 0.081 a Mean ± standard deviation, n=15

41. Effect of Different Storage Temperatures on Furocoumarins Levels in Marsh White GFJ ConditionDaysLevels of Furocoumarins (µg/ml) DHBParadisin ABergamottin 9 ºC01.704 ± 0.043 a 0.092 ± 0.0041.437 ± 0.038 151.689 ± 0.0760.089 ± 0.0061.426 ± 0.04 301.618 ± 0.0910.086 ± 0.00521.408 ± 0.044 451.587 ± 0.0680.085 ± 0.0031.397 ± 0.083 Room Temperature01.704 ± 0.0430.092 ± 0.0051.437 ± 0.038 151.593 ± 0.0990.081 ± 0.0031.406 ± 0.019 301.467 ± 0.0650.071 ± 0.0021.396 ± 0.045 a Mean ± standard deviation, n=15

42. Cans DaysLevels of Furocoumarins (µg/ml) DHBParadisin ABergamottin 02.065 ± 0.081a0.099 ± 0.0041.255 ± 0.028 151.985 ± 0.0920.088 ± 0.0031.223 ± 0.046 301.885 ± 0.0150.078 ± 0.0031.213 ± 0.021 451.772 ± 0.0640.069 ± 0.0041.193 ± 0.037 601.598 ± 0.1090.065 ± 0.0151.166 ± 0.045 751.499 ± 0.0890.061± 0.0281.142 ± 0.013 901.411 ± 0.0370.056 ± 0.0071.1138 ± 0.049 a Mean ± standard deviation, n=15

43. Cardboards DaysLevels of Furocoumarins (µg/ml) DHBParadisin ABergamottin 02.125 ± 0.073 a 0.104 ± 0.0081.487 ± 0.071 151.985 ± 0.0920.097 ± 0.0051.354 ± 0.087 301.818 ± 0.1080.089 ± 0.0011.285 ± 0.051 451.789 ± 0.0890.076 ± 0.0031.116 ± 0.119 601.5612 ± 0.0890.074 ± 0.0061.099 ± 0.19 751.4511 ± 0.0610.072 ± 0.0021.037 ± 0.017 901.4198 ± 0.1830.066 ± 0.0041.004 ± 0.105 a Mean ± standard deviation, n=15

44. Cartoons a Mean ± standard deviation, n=15 DaysLevels of Furocoumarins (µg/ml) DHBParadisin ABergamottin 02.3146 ± 0.0910.1009 ± 0.0061.8655 ± 0.091 152.1485 ± 0.0920.0981 ± 0.0131.6813 ± 0.086 301.9915 ± 0.1050.0883 ± 0.0081.4513 ± 0.107 451.8212 ± 0.0640.0769 ± 0.0171.3193 ± 0.045 601.7598 ± 0.1090.0685 ± 0.0091.2168 ± 0.021

45. 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 ?

46. 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.

47. 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