Part 2 Functional Components of Citrus and Their

Part 2

Functional Components of Citrus and Their Relation of Cancer and Chronic Disease Prevention Bhimu Patil Part II (Continued)

Vit. C

Vitamin C deficiency q Scurvy q Cutaneous hemorrhages q Improper bone development q Poor collagen production q Anemia q Susceptibility to infection

Clinical Trials? • Citrus fruits were first suggested for treatment of scurvy in 1593 and again in 1608 - then in 1747 came the clinical trial which was not randomized but was pretty conclusive- two patients got well on citrus fruit and then other ten, on other five treatments go worse. It was 40 years before British navy required lemons and limes and other citrus fruits on board ships at sea. • It took 154 years from innovation to the trial, 40 years from the trial to practice. From Chalmers: A Potpouri of RCT topics. Controlled Clinical Trials 1982: 3: 285

Limonoids Potassium Vit. C Flavonoids Carotenoids Fiber Pectin Coumarins Folic Acid

Carotenoids • • Lycopene-Prostate Cancer Beta-carotene Beta cryptoxanthin Lutein and zeaxanthin-Macular degeneration

Lycopene

Enhancing Carotenoids • Preharvest Factors – Genetics – Season – Location/Climate • Postharvest Factors – Postharvest Storage

Patil 2001, Proc. International Citrus Congress, Orlando, FL.

Enhancing Carotenoids • Preharvest Factors – Genetics – Season – Location/Climate • Postharvest Factors – Postharvest Storage

Enhancing Carotenoids • Preharvest Factors – Genetics – Season – Location/Climate • Postharvest Factors – Postharvest Storage

Postharvest Storage Effect

Enhancing Phytochemicals • Preharvest Factors – Genetics – Season – Location/Climate • Postharvest Factors – Storage • Juice Storage • Fruit storage • Irradiation

Methodology • Storage time § 0 day § 30 day § 60 day • Storage temperature § 9 C § 15 C § 24 C

Changes in Functional Components During Storage of Citrus Juice

Paper board cartons (100% Juice-single strength) processed not from concentrate Patil 2004, ACS Symposium Series 871

Paper board cartons (100% Juice-single strength) processed not from concentrate Patil 2004, ACS Symposium Series 871

Quarantine Treatments Current: • Cold treatment (10 -12 days) • Methyl Bromide Fumigation • Vapor Heat (4 h + 30 min for cooling) • High-temperature forced-air (4 h + 30 m) • Modification of vapor heat Research Under Progress • Refrigerated Ultra-low oxygen storage • Irradiation (15 -20 min)

Refrigerated Ultra Low Filteration • • • Replaces Chemical fumigation Reduces Green Mold HTFA- Fruit quality affected Heat treatment: not fully ripe Extend shelf life Nutritive Value: Very little research on functional components

Storage Conditions • Three commercial shipping cartoons harvested, washed and waxed. • Treatment: 21 d inside marine container under 0. 05 k. Pa Oxygen at 14 C + 14 additional days in air at 21 C. • Control: 21 d inside walk in cooler at 10 C + 14 d in air at 21 C

Fruit Evaluation

* * Patil and Shellie 2004, Acta Hort 628. 321 -327.

* Patil and Shellie 2004, Acta Hort 628. 321 -327.

* Patil and Shellie 2004, Acta Hort 628. 321 -327.

Quarantine Treatments Current: • Cold treatment (10 -12 days) • Methyl Bromide Fumigation • Vapor Heat (4 h + 30 min for cooling) • High-temperature forced-air (4 h + 30 m) • Modification of vapor heat Research Under Progress • Refrigerated Ultra-low oxygen storage • Irradiation (15 -20 min)

Irradiation effect on Cut and Whole fruit. Naringin 8. 22% 13. 15% 123% 26. 36% a a a b Vanamala, …. . Patil 2007. Food Chemistry 1404 -14011 a a (P = 0. 05)

Narirutin in cut and whole fruits 16. 1% 50. 0% 57. 6% a 23. 3% b a a a a (P = 0. 05) Vanamala, …. . Patil 2007. Food Chemistry 1404 -14011

Foods for Health: Opportunities and Challenges

Well balanced diet • Macronutrients-Energy (Carbohydrates, lipids and proteins) 800 million people do not have in their diet • Micronutrients-Good Health • Essential-nutritional disorders – – – 17 minerals and 13 vitamins 250 m Vitamin A Deficiency 2 billion iron deficiency 1. 5 billon iodine deficiency Poor eating habit in Developed countries • Non Essential -promotion of good health – Phytochemicals – 80, 000 -100, 000 Della. Penna 1999, Science

Disease combating 274 million- obesity $1 billion-heart $1 billion-diabetes Farm to Table Health and Human Service Prevention HHS sponsoring Workshops: Agriculture, $$? and Health Table to Farm

Vitamins Chemical names Contribution of F&V A Retinol, beta-carotene 30 -35 % D Colecalciferol, ergocalciferol E Tocopherols K Phylloquinone, Menaquinone B 1 Thiamin B 2 Riboflavin ~ 10 % PP or B 3 Nicotinic acid or nicotinamide ~ 10 % B 5 Panthotenic acid B 6 Pyridoxin, pyridoxal 20 -22 % B 8 Biotin legumes B 9 Folic acid polyglutamates B 12 Cobalamins C Ascorbic acid and dehydroascorbic acid 15 -22 % Vegetables >> 17 -24 % Legumes > 50% 70% FV + potatoes 22%

Vitamin A deficiency and Xerophthalmia Undernutrition - low serum retinol status

Money spent person/year Annual Cost for Cancer alone $ 102 billion

Average Life Span Extended

What do we need to do to change from Myth to Realty? Meta-Analysis Systematic Review Randomized Controlled Trials Cohort studies Case Control studies Case Series/Case Reports Animal research In vitro and In vivo experiments

Consumption of Fruit & Vegetables and Relative Risk of Cancer Vegetable & Fruit Improvement Center · Horticultural Sciences · TAES

Protective against cancers - compilation of epidemiological studies Products Target Protective effect Fruits and vegetables convincing Fish Upper aero-digestive tract, stomach, lung, colon Colon Fibers Breast, Colo-rectum probable Cereals Breast possible Vegetables Prostate possible probable (CNERNA, 1996 ; World Cancer Research Fund, USA, 1997 ; COMA, 1998)

Opportunity • identify bioactive compounds in F & V that promote health and reduce the risk for chronic diseases • develop new agricultural and food processing technology that optimize the bioactive compounds in F &V • Identify agricultural practices that improve the value of crops for growers and help ensure the long term viability of the state, nation and global agricultural enterprise

Isolation, Purification and Characterization of Putative Bioactive Compounds

DRIED SEEDS CITRUS FRUITS SOLVENT EXTRACTION GROUND SEEDS ROTOEVAPORATION SILICA GEL COLUMN SEPARATION HPLC LIMONOID CHARACTERIZATION LIMONIN CRYSTALLIZATION AND PURIFICATION PURIFIED LIMONOID AGLYCONES

Isolated Citrus Bioactive Compounds Limonoids and Flavonoids Limonoid Aglycone Limonoid Glucosides

Confirmation of Limonoids using High Resolution Mass Spectrometry Rapid Communications in Mass Spectrometry 2003: 17: 2517 -2522 Proposed fragmentation of Limonin Proposed fragmentation of Nomilin Proposed fragmentation of Obacunone Proposed fragmentation of Deacetylnomilin

Phase II Enzymes GST and QR Isolation and Characterization Bioactive Compounds Animal Studies Cell Culture Phase I Enzymes CYP 450’s Flavonoids Limonoids

Colon Cancer

Jairam Vanamala Diets Containing GFPP, IGFPP, Naringin, and Limonin significantly reduced AC and HMACF GFPP – Grapefruit Pulp Powder IG – Irradiated Grapefruit Pulp Powder Vanamala et al. , 2006. Carcinogenesis 1257 -1264

Objective To investigate the possibilities of various citrus limonoids and flavonoids to reduce plasma cholesterol

LDL/HDL Cholesterol Ratio d d b a Jun Yu d d c

GST-catalyzed Conjugation of CDNB to GSH GST GSH Cl NO 2 1 -chloro-2, 4 -dinitrobenzene GS NO 2 HCl NO 2 Dinitrophenyl glutathione INSOLUBLE Toxic Less toxic

Jiaxling Li

Limonoids stimulate caspase activation Shibu Poulose

Limonoids and Caspase activity. CAMP OG LG NAG DNAG Control Caspase 3/7 activity 120 80 40 0 0 6 12 Hours 18 24 Induction of caspase activity in human neuroblastoma cells by limonoid glucosides. (A) Time-dependent change in caspase 3/7 activity in cells treated with 10 µmol/L limonoid glucosides or CAMP. Poulose, Harris and Patil. J. Nutr. 2005 135: 870 -877.

Citrus Juice and Osteoporosis Activity (nm) Deyhim et al. , 2006. Nutrition 559 -563 .

In Vitro Effects of Citrus Pectin on the FGF Signaling System

Pectin Content in Different Fruits (% fresh wt) Baker, 1997

Dr. Yan Liu M. D. Graduated in 2000

Factor-Receptor • Cell needs to communicate to each other.

FGFR Stimulation FGF FGFR FGF Inhibition FGFR No Stimulation No Inhibition

Inhibitors (Heparin Mimics) • • • Suramin analogs pentosan polysulfate Carrageenans Dextran derivatives

Specific bound (% of control) Pectin Inhibit FGF-1 Binding to FGFR 1 Pectin Concentration ( g/ml) Liu et al. , 2001. Journal of Agric. and Food Chemistry 49(6): 3051 -3057.

% of Maximum Binding Pectin Inhibit FGF-1 Binding to FGFR 1 Pectin Added Heparin Only Heparin Concentration ( g/ml) Liu et al. , 2001. Journal of Agric. and Food Chemistry 49(6): 3051 -3057.

Variation of Pectin Content and Composition in Different Citrus Species

Percentage of binding Inhibition Activities of Pectin a a a b b c Liu et al. , 2001. Journal of Agric. and Food Chemistry 49(6): 3051 -3057.

Percentage of binding Inhibition activities of pectin a a ab a a a b ab a a bc Liu et al. , 2002. Journal of Science Food and Agriculture 82: 469 -477

Challenges • Develop F & V that contain group of nutrients and bioactive compounds tailored to individual needs • Many bioactive compounds are bitter, acrid, astringent or pungent- consumer preference • Bioavailability of bioactive compounds – Interaction with bioactive compounds – Type – Food Matrix • Carotenoids dissolved in oil or aqueous dispersion (>50%) v/s tomato juice (<3%)

Bioavailability of carotenoids High Low Raw Carrots Tomato Juice Spinach Processed Mild Cooked Carrots Processed tomato Juice with Oil Tomato Paste with Oil Mild Cooked Spinach Formulated carotenoids Oil Solutions Water-dispersible Beadlets Yeum and Russell, 2002

First Study-Bioavailability of Limonoids Manners et al. , 2003

Students/Post Doc/Res Professors Undergraduate Students Ceclie Tixier Marc Villalobos Erika Cantu Julian Ortega Jose Perez Jasmine Williams Sonia Del Rio Etem Chu Jose Garcia Erica Salinas Robert Cooper Denise Santa Ana Michael Gutierrez Charlie Balli Marlene Perez Undergraduate students Ph. D. Yarania Diaz Jairam Vanamala Mike Deleon Jun Yu Katie Sanford Justin Liska Conrad Ojong Rachel Hoerster Eron Reid M. S. Marco Ponce Omar Montemeyor Jiaxing Li Yan Liu, M. D. Kranthi Kiran Mandadi Melissa Etlinger Savitha M. Post Doc / Research Scientist/Faculty Dr. Deepak Dandekar Dr. Jayaprakasha, G. K. Shibu Poulose Basavaraj Girennavar Dr. Girija Raman Dr. Ananthkrishnan, S Amit Vikram Dr. Quingguo Tian Kranthi Chebrolu Dr. Gerson Peltz, M. D. Hajeen Bae Dr. C. R. Sankar Dr. K. N. C. Murthy Jin Hee Kim Dr. Kil Sun Yoo Ram Uchoo Dr. Hae Jeen Bang Dr. Sun Hun Park

Dr. Leonard Pike Breeder Dr. Edward Miller Biomedical Science Dr. Jennifer Brodbelt Chemistry Dr. Narayan Bhat Chemistry Dr. Hassan Ahmad Biochemistry Dr. Edward Harris Biochemistry Dr. Joanne Lupton Dr. Nancy Turner Nutrition-Colon Cancer Dr. Wallace Mckeehan Cancer Biology Dr. Jenna Anding Outreach activities Dr. Richard Mayer Entomologist USDA-ARS

Peer-reviewed Publications on Citrus USDA-IFAFS Grants

Peer-reviewed Publications on Citrus USDA-IFAFS Grants

New Book released on June 6, 2006 Editors Vegetable & Fruit Improvement Center · Horticultural Sciences · TAES

Book chapters on Citrus- USDA-IFAFS grant

Increasing importance Garlic Cabbage Licorice Soybeans Ginger Umbelliferae (carrots, celery, parsnips) Onions Tea Turmeric Citrus (orange, lemon, grapefruit) Whole Wheat Flax Brown Rice Solanacae (tomato, eggplant, peppers) Cruciferous (broccoli, cauliflower, Brussels sprouts) Oats Rosemary Cantaloupe Mints Oregano Sage Potato Basil Tarragon Cucumber Thyme Chives Barley Berries Foods with cancer preventative properties

Connie Sebesta

. . . Learning to eat healthy

Cost of Not consuming F&V • Global mortality attributed to inadequate of F&V is 2, 635 million death per year • Increase 600 g/day could reduce worldwide burden of coronary heart disease by 31% and stroke by 19% (1) • Higher F&V consumption- savings of Medicare charges $2, 000 (2) 1. Lock et al. , Bull World Health Org 2005. 100 -108 2. Daviglus et al. , J. Am Diet Assoc 2005, 1735 -1744.

$$$$$ USDA NIH

Money spent on Clinical Trials- 2006 NIH $3. 0 billion Biotechnology and Pharmaceutical industry $22. 6 billion

Participant Flow in the Dietary Modification Component of the Women's Health Initiative $415 Million Multi-institute Study Prentice, R. L. et al. JAMA 2006; 295: 629 -642. Copyright restrictions may apply.

Kaplan-Meier Estimated Cumulative Hazards for Invasive Colorectal Cancer (N = 48, 835) Beresford, S. A. A. et al. JAMA 2006; 295: 643 -654. Copyright restrictions may apply.

Kaplan-Meier Estimates of the Cumulative Hazard for Invasive Breast Cancer Prentice, R. L. et al. JAMA 2006; 295: 629 -642. Copyright restrictions may apply.

Kaplan-Meier Estimates of Cumulative Hazards for CHD (MI, CHD Death, or Revascularization) and Stroke Howard, B. V. et al. JAMA 2006; 295: 655 -666. Copyright restrictions may apply.

WHI study did not consider phytochemical levels of F&V from harvest to consumption It is time for NIH and USDA to marry and conduct multidisciplinary research

Feedback from all students • Opportunities in foods for health • Challenges for foods for health program • What do you think about conducting research in interdisciplinary research- Foods for Health? • Are you conducting biological activities studies? • What is your major?