Part 2. Functional Components of Citrus and Their Relation of Cancer and Chronic Disease Prevention Bhimu Patil Part II (Continued)

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 Scurvy

Cutaneous hemorrhages

Improper bone development

Poor collagen production

Anemia

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 Vit. C

Folic Acid

Pectin

Carotenoids

Flavonoids

Fiber

Potassium

Coumarins

Carotenoids

• Lycopene-Prostate Cancer

• Beta-carotene

• Beta cryptoxanthin

• Lutein and zeaxanthin-Macular degeneration

http://www.clipartconnection.com/searchsite/download?oid=1233244&fmt=GIF



Lycopene

Clinton, 1998. Nutrition Reviews 56(2):35-51.

Food Lycopene content mg/100g

Tomato (fresh) 0.88-4.20

Grapefruit (raw pink) 3.36

Tomato (cooked) 3.7

Tomato (sauce) 6.2

Tomato Paste 5.40-150.00

Tomato soup, condensed 7.99

Tomato Powder, drum or spray dried

112.63-126.49

Tomato Juice 5.00-11.60

Guava (fresh) 5.4

Watermelon 2.3

Papaya (fresh) 2.00-5.30

Ketchup 9.90-13.44

Enhancing Carotenoids

• Preharvest Factors– Genetics – Season– Location/Climate

• Postharvest Factors– Postharvest Storage


Lycopene-1999

Star Rubya

I-48b

Rio Reda

Ray Rubyc

Hendersond

Ruby Redd

Thompsone

Marshe

Duncane

0

5

10

15

20

25

30ug/g fresh wt

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




Seasonal Changes-Comparision 1998-99 combined

Jun July Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun99

Harvesting time

0

10

20

30

40

50

60Total carotenoids-ppm

Rio Red Star Ruby




Growing Location Effect- Star Ruby-1999

TXa

FLb

TXa

FLa

TXa

FLa

TXa

FLa

TXa

FLb

TXa

FLb

TXa

FLb

Harvesting time

0

10

20

30

40

50Lycopene (ppm)

Ju99 Aug Oct Dec Feb Apr June

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

Postharvest storage time and temperature effect on limonin 17-beta-D glucopyranoside

Storage time (days)

0 10 20 30 40 50 60 70

LG

co

ncn

. (u

g/g

)

140

160

180

200

220

240

260

280

300

9 C 15 C 24 C

Postharvest storage time and temperature effect on vitamin C concentrations.

Storage time (days)

0 10 20 30 40 50 60 70

Vit

am

in C

co

nc

n. (u

g/g

)

150

200

250

300

350

400

9 C 15 C 24 C

Changes in Functional Components During Storage of

Citrus Juice

Storage (months)

0 2 4 6

vit

C /

LG

(u

g/g

)

60

80

100

120

140

160

180

200

Vitamin C Limonin Glucoside

Paper board cartons

(100% Juice-single strength) processed not from concentratePatil 2004, ACS Symposium Series 871

Storage (weeks)

0 2 4 6

Car

ote

no

ids

(ug

/g)

0

2

4

6

8

10

Beta carotene Lycopene Total carotenoids

Paper board cartons

(100% Juice-single strength) processed not from concentratePatil 2004, ACS Symposium Series 871

Quarantine TreatmentsCurrent: • Cold treatment (10-12 days)• Methyl Bromide Fumigation• Vapor Heat (4h + 30 min for cooling)• High-temperature forced-air (4h + 30m) • Modification of vapor heatResearch 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 kPa Oxygen at 14C + 14 additional days in air at 21C.

• Control: 21 d inside walk in cooler at 10C + 14 d in air at 21 C

Fruit Evaluation

Beta Carotene Levels

Storage time21 days 35 days

co

nc

en

tra

tio

ns

(ug

/g)

0

1

2

3

4

5

Control ULO

Lycopene Levels


Co

nc

en

tra

tio

ns

(u

g/g

)

0

5

10

15

20

25

30

ControlULO

*

*

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

Naringin Levels


con

cen

trat

ion

s (u

g/g

)

0

200

400

600

800

1000

1200

1400ControlULO

Narirutin Levels


Co

nce

ntr

atio

ns

(ug

/g)

0

50

100

150

200

250ControlULO

*


Vitamin C Levels


Co

nc

en

tra

tio

ns

(u

g/g

)

0

50

100

150

200

250

300

Control ULO

Limonin 17-beta-D Glucopyranoside Levels


Co

nc

en

tra

tio

ns

(u

g/g

)

0

100

200

300

400Control ULO

*


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

0

100

200

300

400

500

600

700

C-Pulp C-Peel W-Pulp W-Peel

Fruit Part Used

Na

rin

gin

mg

/10

0g F

W

Non-Irradiated

Irradiated

Naringin

a b

a a

a

(P = 0.05)

a a a

13.15%

26.36%

8.22%

123%

Irradiation effect on Cut and Whole fruit.

Vanamala, ….. Patil 2007. Food Chemistry 1404-14011

Narirutin in cut and whole fruits

0102030405060708090

C-Pulp C-Peel W-Pulp W-Peel

Fruit Part Used

Nar

irut

in (m

g/10

0g F

W) Non-Irradiated

Irradiated

57.6%

50.0%

23.3%

16.1%

a b

(P = 0.05)

a b a a a a

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

DellaPenna 1999, Science

Farm to Table Table to Farm

PreventionHHS sponsoring

Workshops: Agriculture,and HealthHealth and

Human Service

$$?

Disease combating274 million- obesity

$1 billion-heart $1 billion-diabetes

Vitamins Chemical names Contribution of F&V

A Retinol, beta-carotene 30-35 %

D Colecalciferol, ergocalciferol -

E Tocopherols 15-22 %

K Phylloquinone, Menaquinone Vegetables >>

B1 Thiamin 17-24 %

B2 Riboflavin ~ 10 %

PP or B3 Nicotinic acid or nicotinamide ~ 10 %

B5 Panthotenic acid Legumes

B6 Pyridoxin, pyridoxal 20-22 %

B8 Biotin legumes

B9 Folic acid polyglutamates > 50%

B12 Cobalamins -

C Ascorbic acid and dehydroascorbic acid

70% FV + potatoes 22%

Vitamin A deficiency and Xerophthalmia

Undernutrition - low serum retinol status

Money spent per person/year

1.21

1390

0200400600800

1000120014001600

Mon

ey s

pen

t ($

)

Annual Cost for Cancer alone $ 102 billion

Average Life Span Extended

25

5

0

5

10

15

20

25

30

Ave

rage

life

sp

an e

xten

ded

Meta-Analysis

Systematic Review

Randomized Controlled Trials

Cohort studies

Case Control studies

Case Series/Case Reports

Animal research

In vitro and In vivo experiments

What do we need to do to change from Myth to Realty?

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

Upper aero-digestive tract,stomach, lung, colon

convincing

Fish Colon probable

Fibers Breast, Colo-rectum probable

Cereals Breast possible

Vegetables Prostate possible

(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

ROTOEVAPORATIONROTOEVAPORATIONSOLVENT SOLVENT EXTRACTIONEXTRACTION

SILICA GEL COLUMN SILICA GEL COLUMN SEPARATIONSEPARATION

HPLC LIMONOID HPLC LIMONOID CHARACTERIZATIONCHARACTERIZATION

PURIFIED LIMONOID PURIFIED LIMONOID AGLYCONESAGLYCONES

LIMONIN LIMONIN CRYSTALLIZATION CRYSTALLIZATION AND PURIFICATIONAND PURIFICATION

GROUND SEEDS GROUND SEEDS DRIED SEEDSDRIED SEEDSCITRUS FRUITSCITRUS FRUITS

Isolated Citrus Bioactive Compounds

Limonoid Aglycone Limonoid Glucosides

Limonoids and Flavonoids

Confirmation of Limonoids using High Resolution Mass Spectrometry

Proposed fragmentation of Limonin

Proposed fragmentation of Nomilin

Proposed fragmentation of Obacunone

Proposed fragmentation of Deacetylnomilin

Rapid Communications in Mass Spectrometry2003:17:2517-2522

Isolation and Characterization

Bioactive Compounds

Flavonoids

Limonoids

Cell Culture

Phase II EnzymesGST and

QR

Phase I EnzymesCYP450’s

Animal Studies

Colon Cancer

Colon Cancer

Basal Diet GFPP IGFPP Naringin Limonin

Tota

l Num

ber

of

AC

0

50

100

150

200

250

a

b b b

P = 0.02

b

Basal Diet GFPP IGFPP Naringin Limonin

Num

ber

of

HM

AC

F

0

2

4

6

8

10

12

14

16

18a

b b

b

P = 0.01

b

Diets Containing GFPP, IGFPP, Naringin, and Limonin significantly reduced AC and HMACF

GFPP – Grapefruit Pulp Powder

IG – Irradiated Grapefruit Pulp Powder

Jairam Vanamala

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

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

C 1 Lim LG Ng Ngn Pulp C 2

Per

cen

tag

e o

f H

amst

er P

lasm

a L

DL

/HD

L

a

bc

d dddJun Yu

NO2

NO2ClGSH GST

NO2

NO2GS HCl

GST-catalyzed Conjugation of CDNB to GSH

1-chloro-2,4-dinitrobenzene Dinitrophenyl glutathione

INSOLUBLE SOLUBLE

Toxic Less toxic

GST in Liver for Aglycone Groups

Treatment

oil AglMix Nomilin Limonin

Sp

Ac

t ( m

ol/m

in/m

g p

rote

in)

0.0

0.2

0.4

0.6

0.8

Jiaxling Li

Shibu Poulose

Limonoids stimulate caspase activation

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.

Limonoids and Caspase activity.

0

40

80

120

0 6 12 18 24 Hours

Cas

pas

e 3/

7 ac

tivi

ty

Control

LGOG

NAGDNAG

CAMP

Antioxidant capacity

0

0.5

1

1.5

2

SHAM ORX OJ GJ

Groups

Activity (nm)

Citrus Juice and Osteoporosis

Deyhim et al., 2006. Nutrition 559-563.

In Vitro Effects of Citrus Pectin on the FGF Signaling System

Apples 0.71-0.84Apricots 0.71-1.32Bananas 0.59-1.28Beans 0.27-1.11Blackberries 0.68-1.19Carrots 1.17-2.92Cherries 0.24-0.54Dewberries 0.51-1.00Grapes 0.09-0.28Grapefruit 3.30-4.50Lemons 2.8-2.99Loganberries 0.59Oranges 2.34-2.38Raspberries 0.97Squash 1.00-2.00

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 FGF

FGFR

FGF FGFR

FGF

FGFR FGF

Stimulation

Inhibition

No StimulationNo Inhibition

Inhibitors (Heparin Mimics)

• Suramin

• Suramin analogs

• pentosan polysulfate

• Carrageenans

• Dextran

• Dextran derivatives

Spe

cifi

c bo

und

(% o

f co

ntro

l)

Pectin Concentration (g/ml)

Pectin Inhibit FGF-1 Binding to FGFR1

-20

0

20

40

60

80

100

0 3 30 300

Liu et al., 2001. Journal of Agric. and Food Chemistry 49(6):3051-3057.

0

20

40

60

80

100

120

0 0.001 0.01 0.1 1 10

Pectin Added Heparin Only

% o

f M

axim

um B

indi

ng

Heparin Concentration (g/ml)

Pectin Inhibit FGF-1 Binding to FGFR1


Variation of Pectin Content and Composition in Different

Citrus Species

-0.2

0

0.2

0.4

0.6

0.8

1

Lemon Grapefruit Tangerine Orange

Citrus Species

Flavedo/Albedo Lamella

Inhibition Activities of PectinP

erce

ntag

e of

bin

ding

c

a a aa abb


0

0.2

0.4

0.6

0.8

1

AUG SEP NOV JAN MAR MAY

F A L

Inhibition activities of pectinP

erce

ntag

e of

bin

ding

a

a a ab

a

a a a a b ab a a b a a bc a a ab

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

Raw Carrots

Tomato JuiceSpinach

Low High

ProcessedMild Cooked Carrots

Processed tomato Juice with Oil

Tomato Paste with OilMild Cooked Spinach

Formulated carotenoidsOil Solutions

Water-dispersible Beadlets

Yeum and Russell, 2002

First Study-Bioavailability of Limonoids

O OH

R

OH

OH

COOH

O

O

O

O

B

C

R=CH2OH

OO

A'

A

OA'

Limonin 17--D-GlucopyranosideO

O

O

O

OO

O

A'

A

B

C D

O

Epilimonin

O

O

O

O

OO

O

A'

A

B

C D

O

Limonin

Manners et al., 2003

Students/Post Doc/Res ProfessorsUndergraduate StudentsCeclie TixierMarc VillalobosErika Cantu Julian Ortega Jose Perez Jasmine Williams Sonia Del RioEtem ChuJose Garcia Erica SalinasRobert Cooper Denise Santa Ana Michael Gutierrez Charlie Balli Marlene Perez

Undergraduate studentsYarania DiazMike DeleonKatie SanfordJustin LiskaConrad OjongRachel HoersterEron Reid

M.S. Marco PonceOmar MontemeyorJiaxing Li Yan Liu, M.D.Kranthi Kiran Mandadi Melissa EtlingerSavitha M.

Ph.D.Jairam Vanamala

Jun Yu

Shibu Poulose

Basavaraj Girennavar

Amit Vikram

Kranthi Chebrolu

Hajeen Bae

Jin Hee Kim

Ram Uchoo

Post Doc / Research Scientist/FacultyDr. Deepak DandekarDr. Jayaprakasha, G.K.Dr. Girija Raman Dr. Ananthkrishnan, SDr. Quingguo Tian Dr. Gerson Peltz, M.D. Dr. C.R. SankarDr. K.N.C. MurthyDr. Kil Sun YooDr. Hae Jeen BangDr. Sun Hun Park

Dr. Leonard PikeBreeder

Dr. Hassan AhmadBiochemistry

Dr. Narayan BhatChemistry

Dr. Edward MillerBiomedical Science

Dr. Wallace MckeehanCancer Biology

Dr. Jennifer BrodbeltChemistry

Dr. Joanne LuptonNutrition-Colon Cancer

Dr. Jenna AndingOutreach activities

Dr. Nancy TurnerNutrition-Colon Cancer

Dr. Richard MayerEntomologistUSDA-ARS

Dr. Edward HarrisBiochemistry

Peer-reviewed Publications on Citrus

USDA-IFAFS Grants

Peer-reviewed Publications on CitrusUSDA-IFAFS Grants

New Book released on June 6, 2006

Vegetable & Fruit Improvement Center · Horticultural Sciences · TAES

Editors

Book chapters on Citrus- USDA-IFAFS grant

Foods with cancer preventative properties

Incr

easi

ng

imp

orta

nce

GarlicCabbageLicorice

Soybeans GingerUmbelliferae

(carrots, celery, parsnips)

Onions Tea TurmericCitrus (orange, lemon, grapefruit)

Whole Wheat Flax Brown RiceSolanacae (tomato, eggplant, peppers)

Cruciferous (broccoli, cauliflower, Brussels sprouts)

Oats Mints Oregano CucumberRosemary Sage Potato Thyme Chives

Cantaloupe Basil Tarragon Barley Berries

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. 100-1082. Daviglus et al., J. Am Diet Assoc 2005, 1735-1744.

$$$$$

Total

Prevention

Total

Prevention

USDA NIH

Money spent on Clinical Trials- 2006

NIH

$3.0 billion

Biotechnology and Pharmaceutical industry

$22.6 billion

Copyright restrictions may apply.

Prentice, R. L. et al. JAMA 2006;295:629-642.

Participant Flow in the Dietary Modification Component of the Women's Health Initiative

$415 Million Multi-institute Study


Beresford, S. A. A. et al. JAMA 2006;295:643-654.

Kaplan-Meier Estimated Cumulative Hazards for Invasive Colorectal Cancer (N = 48, 835)


Prentice, R. L. et al. JAMA 2006;295:629-642.

Kaplan-Meier Estimates of the Cumulative Hazard for Invasive Breast Cancer


Howard, B. V. et al. JAMA 2006;295:655-666.

Kaplan-Meier Estimates of Cumulative Hazards for CHD (MI, CHD Death, or Revascularization) and Stroke

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?

Documents

Part 2. Functional Components of Citrus and Their Relation of Cancer and Chronic Disease Prevention Bhimu Patil Part II (Continued)