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
Potassium
Vit. C
Flavonoids
Carotenoids
Fiber
Pectin
Coumarins
Folic Acid
Carotenoids
•
•
•
•
Lycopene-Prostate Cancer
Beta-carotene
Beta cryptoxanthin
Lutein and zeaxanthin-Macular
degeneration
Lycopene
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)
Watermelon
Papaya (fresh)
Ketchup
5.4
2.3
2.00-5.30
9.90-13.44
Clinton, 1998. Nutrition Rev iews 56(2):35-51.
Enhancing Carotenoids
• Preharvest Factors
– Genetics
– Season
– Location/Climate
• Postharvest Factors
– Postharvest Storage
Lycopene-1999
30
ug/g fresh wt
25
20
15
10
5
0
Star Ruby
a
I-48
b
Rio Red
a
Ray Ruby Henderson Ruby Red Thompson
c
d
d
e
Marsh
e
Duncan
e
Patil 2001, Proc. International Citrus Congress, Orlando, FL.
Enhancing Carotenoids
• Preharvest Factors
– Genetics
– Season
– Location/Climate
• Postharvest Factors
– Postharvest Storage
Seasonal Changes-Comparision 1998-99 combined
60
Total carotenoids-ppm
Rio Red
50
Star Ruby
40
30
20
10
0
Jun
July
Aug
Sep
Oct
Nov
Dec
99
Jan
Harv esting time
Feb
Mar
Apr
May
Jun
Enhancing Carotenoids
• Preharvest Factors
– Genetics
– Season
– Location/Climate
• Postharvest Factors
– Postharvest Storage
Growing Location Effect- Star Ruby-1999
50
Ly copene (ppm)
Ju99
Aug
Oct
Dec
Feb
Apr
June
40
30
20
10
0
TX FL
a b
TX FL
a a
TX FL
a a
TX FL
a a
TX FL
a b
Harv esting time
TX FL
a b
TX FL
a b
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
9C
 15 C
 24 C
300
LG concn. (ug/g)
280
9C
15 C
24 C
260
240
220
200
180
160
140
0
10
20
30
40
50
60
70
Storage time (days)
Postharvest storage time and temperature
effect on limonin 17-beta-D glucopyranoside
Vitamin C concn. (ug/g)
400
9C
15 C
24 C
350
300
250
200
150
0
10
20
30
40
50
60
70
Storage time (days)
Postharvest storage time and temperature
effect on vitamin C concentrations.
Changes in Functional
Components During Storage of
Citrus Juice
Paper board cartons
200
vit C / LG (ug/g)
180
160
140
120
100
80
Vitamin C
Limonin Glucoside
60
0
2
4
6
Storage (months)
(100% Juice-single strength) processed not from concentrate
Patil 2004, ACS Symposium Series 871
Paper board cartons
Carotenoids (ug/g)
10
8
6
Beta carotene
Lycopene
Total carotenoids
4
2
0
0
2
4
6
Storage (weeks)
(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 (4h + 30 min for cooling)
• High-temperature forced-air (4h + 30m)
• 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 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
concentrations (ug/g)
5
*
4
3
2
Control
ULO
1
0
21 days
35 days
Storage time
Lycopene Levels
Concentrations (ug/g)
30
25
*
20
15
Control
ULO
10
5
0
21 days
35 days
Storage time
Patil and Shellie 2004, Acta Hort 628. 321-327.
Naringin Levels
concentrations (ug/g)
1400
Control
ULO
1200
1000
800
600
400
200
0
21 days
35 days
Storage time
Narirutin Levels
Concentrations (ug/g)
250
Control
ULO
200
*
150
100
50
0
21 days
35 days
Storage time
Patil and Shellie 2004, Acta Hort 628. 321-327.
Vitamin C Levels
Concentrations (ug/g)
300
250
*
Control
ULO
200
150
100
50
0
21 days
35 days
Storage time
Limonin 17-beta-D Glucopyranoside Levels
Concentrations (ug/g)
400
Control
ULO
300
200
100
0
21 days
35 days
Storage time
Patil and Shellie 2004, Acta Hort 628. 321-327.
Quarantine Treatments
Current:
• Cold treatment (10-12 days)
• Methyl Bromide Fumigation
• Vapor Heat (4h + 30 min for cooling)
• High-temperature forced-air (4h + 30m)
• Modification of vapor heat
Research Under Progress
• Refrigerated Ultra-low oxygen storage
• Irradiation (15-20 min)
Irradiation effect on Cut and Whole fruit.
Naringin mg/100g FW
Naringin
700
8.22%
13.15%
600
Non-Irradiated
Irradiated
500
400
300
200
123%
100
26.36%
a a
0
C-Pulp
a b
C-Peel
W-Pulp
a a
W-Peel
a a
Fruit Part Used
Vanamala, ….. Patil 2007. Food Chemistry 1404-14011
(P = 0.05)
Narirutin (mg/100g FW)
Narirutin in cut and whole fruits
90
80
70
60
50
40
30
20
10
0
Non-Irradiated
50.0%
Irradiated
57.6%
a
23.3%
b
C-Pulp
a
b
C-Peel
a
a
W-Pulp
Fruit Part Used
Vanamala, ….. Patil 2007. Food Chemistry 1404-14011
16.1%
a
a
W-Peel
(P = 0.05)
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
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
B1
Thiamin
B2
Riboflavin
~ 10 %
PP or B3
Nicotinic acid or nicotinamide
~ 10 %
B5
Panthotenic acid
B6
Pyridoxin, pyridoxal
20-22 %
B8
Biotin
legumes
B9
Folic acid polyglutamates
B12
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
1600
1400
1200
1000
800
600
400
200
0
1390
C
Pr
ev
en
t
ur
e
1.21
io
n
Money spent ($)
Money spent per person/year
Annual Cost for Cancer alone $ 102 billion
30
25
25
20
15
10
5
5
ur
e
C
io
n
0
Pr
ev
en
t
Average life span extended
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
GROUND SEEDS
ROTOEVAPORATION
SOLVENT EXTRACTION
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
CYP450’s
Flavonoids
Limonoids
Colon Cancer
Colon
Cancer
Jairam
Vanamala
Diets Containing GFPP, IGFPP, Naringin, and
Limonin significantly reduced AC and HMACF
250
18
P = 0.02
16
200
150
b
b
b
b
100
50
Number of HMACF
Total Number of AC
a
P = 0.01
a
14
12
10
8
b
b
b
b
6
4
2
0
0
Basal Diet GFPP
IGFPP Naringin Limonin
Basal Diet GFPP
IGFPP Naringin Limonin
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
Percentage of Hamster Plasma LDL/HDL
LDL/HDL Cholesterol Ratio
d
90%
80%
Jun Yu
d
d
70%
c
b
60%
50%
d
a
40%
30%
20%
10%
0%
C1
Lim
LG
Ng
Ngn
Pulp
C2
GST-catalyzed Conjugation of CDNB to
GSH
GST
GSH  Cl
NO2
NO2
1-chloro-2,4-dinitrobenzene
GS
NO2  HCl
NO2
Dinitrophenyl glutathione
INSOLUBLE
SOLUBLE
Toxic
Less toxic
GST in Liver for Aglycone Groups
Sp Act (mol/min/mg protein)
0.8
0.6
Jiaxling Li
0.4
0.2
0.0
oil
AglMix
Nomilin
Treatment
Limonin
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
Antioxidant capacity
2
1.5
1
Activity (nm)
0.5
0
SHAM
ORX
OJ
GJ
Groups
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)
Apples
Apricots
Bananas
Beans
Blackberries
Carrots
Cherries
Dewberries
Grapes
Grapefruit
Lemons
Loganberries
Oranges
Raspberries
Squash
Baker, 1997
0.71-0.84
0.71-1.32
0.59-1.28
0.27-1.11
0.68-1.19
1.17-2.92
0.24-0.54
0.51-1.00
0.09-0.28
3.30-4.50
2.8-2.99
0.59
2.34-2.38
0.97
1.00-2.00
Dr. Yan Liu M.D.
Graduated in 2000
Factor-Receptor
• Cell needs to communicate to each other.
FGFR
Stimulation
FGF
FGFR
FGF
FGFR
FGF
Inhibition
FGFR
FGF
No Stimulation
No Inhibition
Inhibitors (Heparin Mimics)
•
•
•
•
•
•
Suramin
Suramin analogs
pentosan polysulfate
Carrageenans
Dextran
Dextran derivatives
Specific bound (% of control)
Pectin Inhibit FGF-1 Binding to FGFR1
100
80
60
40
20
0
-20
0
3
30
Pectin Concentration (g/ml)
Liu et al., 2001. Journal of Agric. and Food Chemistry 49(6):3051-3057.
300
Pectin Inhibit FGF-1 Binding to FGFR1
% of Maximum Binding
120
Pectin Added
100
Heparin Only
80
60
40
20
0
0
0.001
0.01
0.1
1
Heparin Concentration (g/ml)
Liu et al., 2001. Journal of Agric. and Food Chemistry 49(6):3051-3057.
10
Variation of Pectin Content
and Composition in Different
Citrus Species
Inhibition Activities of Pectin
Flavedo/Albedo
Lamella
Percentage of binding
1
0.8
0.6
0.4
a
a
a
a
b
0.2
0
a
b
c
-0.2
Lemon
Grapefruit
Tangerine
Orange
Citrus Species
Liu et al., 2001. Journal of Agric. and Food Chemistry 49(6):3051-3057.
Inhibition activities of pectin
F
A
L
Percentage of binding
1
0.8
0.6
0.4
0.2
0
a a ab
a a ab
a a
a a a
AUG
SEP
NOV
a b ab
a a b
a a bc
JAN
MAR
MAY
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
O
First Study-Bioavailability of Limonoids
O
O
O
C
A
D
O
O
O
O
B
A'
OH
O
O
O
O
A
OH
C
R
COOH
O
A'
A'
O
B
OH
R=CH2OH
Limonin
O
O
O
Limonin 17--D-Glucopyranoside
O
O
O
C
A
D
O
A'
Manners et al., 2003
O
B
O
O
Epilimonin
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 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
Total
Total
Prevention
Prevention
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?