Citrus
Phytochemicals
Designer Foods
Functional Foods
Hypernutritious Foods
Nutraceuticals
Phytochemicals
• Naturally
occurring
biologically
active components
• Found in plant
Phytonutrient
Plant chemical with known nutrien
properties
• Consumed as part of a usual diet
•
Biologically active or Protect against Disease
Health Canada, 1998
Bioactive
Compounds
“Extranutritional” constituents that
typically are naturally occurring in small
quantities in plant products and lipid rich
foods.
Kitts, 1994 Can J. Physiol Pharmacology 1994
Nutraceutical
• Product
isolated or purified from foods
• Sold in medicinal forms - pill, powder, capsule
Functional Food
• A conventional
food
• Consumed as part of a usual diet
• Provide health benefit beyond basic
nutrition
Biologically active or Protect against Disease
Health Canada, 1998
Chemopreventive agents
• Micronutrients
–vitamins, beta carotene,
molybdenum, calcium
• Phytochemicals
• Synthetics
–vitamin derivatives
–piroxicam
–tamoxifen
Phytochemicals
• Carotenoids
• Indole
• Saponins
•
•
•
•
Coumarins
Dietary Fiber
Isoflavones
Protease
inhibitors
Phytochemicals
•
•
•
•
Organosulfides
Isothiocynates
Indoles
Dithiolthiones
•
•
•
•
Polyphenols
Flavonoids
Tannins
Folic acid
Contd.
Chemopreventive agents
(Based on their mechanisms of action)
• Blocking agents
• Suppressing agents
CANCER CHEMOPREVENTIVE
AGENTS
1. BLOCKING AGENTS -- prevent carcinogens
from reaching or reacting with the DNA, the
genetic information.
2. SUPPRESSIVE AGENTS -- inhibit the
expression of cancer in cells that have
already been exposed to a carcinogen.
Cancer Producing Compounds
Blocking
Agents
Cells Attacked By Cancer Producing Compounds
Suppressing
Agents
Cancer
Wattenburg, 1993
Chemopreventive agents
• Blocking agents
– Flavonoids
– Indoles
– Isothiocynates
– Diallyl sulfides
– D-limonene
MECHANISM OF ACTION
(BLOCKING AGENTS)
1. Inhibit the formation of the active
carcinogen.
2. Increase the rate at which the active
carcinogen is inactivated.
3. Act as scavengers for the active
forms of carcinogens.
Chemopreventive agents
• Suppressing agents
–D-limonene
–Diallyl sulfides
– vitamin D
–vitamin A and retinoids
–monoterpenes
–carotenoids
– polyphenols
Anticarcingenic mechanisms
• Antioxidant effects
• Increased activity of enzymes that
detoxify carcinogens
• Effect on cell differentiation
• Blocked formation of nitrosamines
• Altered estrogen metabolism
• Decreased cell proliferation
• Maintenance of normal DNA repair
THREE-PHASE MECHANISM FOR
CHEMICAL CARCINOGENESIS
1. INITIATION - normal cells to latent
tumor cells.
2. PROMOTION - latent tumor cells to
carcinoma in situ.
3. PROGRESSION - carcinoma in situ
to invasive carcinoma.
Procarcinogens
Phase I enzymes
Phase II enzymes
Glutathione S-transferase
Carcinogens
Electrophiles
Limonoids
Flavonoids
Promotion
DNA Damage
Lycopene, Lutein,
Zeaxanthin and Beta carotene
Initiation
Oxidative damage
Free radicals
Tumor
Health Promoting Compounds
• Carotenoids
– Lycopene -Prostate Cancer
– Beta carotene
– Lutein and zeaxanthin-Blindness
– Beta cryptoxanthin
Carotenoid Concentrations
• Lycopene- Grapefruit
– 3362 ug/100 g wet wt (Mangels et
al.,1993)
– 350 ug/100 g wet wt (Gross et al., 1987)
Carotenoid Concentrations
(ug/100g)
Oranges Pink
Grf
1310
Beta Carotene 39
Carotenoids
Alpha
Carotene
Lutein +
Zeaxanthin
Lycopene
White
Grf
14
Mandarins
Lemons
38
3
20
0
1
20
0
14
0
10
20
12
0
3362
0
0
0
0
0
106
0
149
Beta
Cryptoxanthin
Food
Tomato (fresh)
Lycopene content mg/100g
0.88-4.20
Grapefruit (raw pink)
Tomato (cooked)
Tomato (sauce)
3.36
3.7
6.2
Tomato Paste
Tomato soup, condensed
Tomato Powder, drum or spray
dried
5.40-150.00
7.99
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 Reviews 56(2):35-51.
Lycopene Variation Among Texas Grapefruits
14
ppm
12
10
8
6
4
2
0
a
St
y
b
u
R
r
I-4
8
io
R
ed
R
R
ay
R
y
b
u
n
o
rs
H
e
d
en
y
b
u
ed
R
n
so
R
T
m
o
h
nk
i
P
sh
r
a
M
te
i
h
W
u
D
an
c
n
te
i
h
W
Beta carotene
10
ug/g fresh wt
8
6
4
2
0
Star Ruby
a
I-48
b
Henderson Rio Red
bc
cd
Ray Ruby Ruby Red Thomson
d
e
f
Marsh
g
Duncan
g
16
14
12
10
8
6
4
2
0
Texas
a
Florida
b
Lycopene
Beta carotene
Texas
Florida
LIMONOIDS WITH
ANTICANCER ACTIVITY
• Limonin
• Limonin 17-ß-D-glucopyranoside
• Limonin carboxymethoxime
• Deoxylimonin
INACTIVE LIMONOIDS
• Limonol
• Deoxylimonic acid
• Ichangensin
• 17,19-didehydrolimonoic acid
• Nomilinic acid 17-ß-D-glucopyranoside
LIMONOIDS WITH
PARTIAL ACTIVITY
• Nomilin
• Nomilin 17-ß-D-glucopyranoside
• Obacunone
EPIDEMILOGICAL EVIDENCE
1.
2.
3.
4.
oral cavity.
larynx.
esophagus.
stomach.
5.
6.
7.
8.
pancreas.
lung.
colon.
rectum.
LIMONOID GLUCOSIDES
1. Tasteless.
2. Soluble in water.
3. Human consumption (already present in citrus and citrus products
in relatively high concentrations).
4. Can be prepared from by-products
of juice processing plants (seeds
and citrus molasses).
Limonoid Concentrations
• Limonoid glucosides
– Limonin 17-beta D-glucopyranoside
(54-180 ppm)
– Oranges-320 ppm
– Grapefruit -195 ppm
– Lemon-90 ppm
•
LG 1000
ROLE OF METABOLISM IN CHEMICAL CARCINOGENESIS
P-450
PHASE I
PRO
CARCINOGENS
OXIDATION
PHASE II
REDUCTION
HYDROLYSIS
ULTIMATE CARCINOGEN
DNA DAMAGE
GST
GSH
GSH-CONJUGATION
PRODUCT
EXCRETION
TUMOR
GST-catalyzed Conjugation of CDNB
to GSH
GST
GSH  Cl
NO2
NO2
GS
NO2  HCl
NO2
1-chloro-2,4-dinitrobenzene
Dinitrophenyl glutathione
INSOLUBLE
SOLUBLE
Toxic
Less toxic
GST in Liver for Aglycone Groups
Sp Act (mol/min/mg protein)
0.8
0.6
0.4
0.2
0.0
oil
AglMix
Nomilin Limonin
Treatment
GST in Intestine for Aglycone Groups
Sp Act (mol/min/mg protein)
0.6
0.5
0.4
0.3
0.2
0.1
0.0
oil
AglMix
Nomilin
Treatment
Limonin
GST in Intestine for Glucoside Groups
Sp Act (mol/min/mg protein)
0.5
0.4
0.3
0.2
0.1
0.0
Water
GluMixA
GluMixB
Treatment
Naringin
Isolation, Purification and
Characterization of
Putative Bioactive
Compounds
Isolated Citrus Bioactive
Compounds
Limonoids and Flavonoids
Limonoid Aglycone
Limonoid Glucosides
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
Experimental Diets Significantly
Reduced Total ACF Number
200
a
p<0.05
b
TOTAL ACF
150
100
50
0
Basal Diet
GFPP
IGFPP
Naringin
Limonin
Experimental Diets Significantly
Reduced
High Multiplicity ACF
a
14
p<0.05
12
HMACF
10
b
8
6
4
2
0
Basal Diet
GFPP
IGFPP
Naringin
Limonin
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
Comparison
d
90%
80%
d
70%
Jun Yu
d
c
b
60%
d
a
50%
40%
30%
20%
10%
0%
C1
Lim
LG
Ng
Ngn
Pulp
C2
Total Antioxidant capacity
• ORAC- Oxygen Radical
Absorbance Capacity can be
measured by COBAS FARA II
analyzer
ORAC of commercial orange juice and vitamin C from juice
20
ORAC (micromoles Trolox eq./ml)
Grape
Grapefruit
Tomato
Orange
Apple
15
10
5
0
Juice
Vit. C
Juice
Vit. C
Juice
Vit. C
Wang et al., 1996. J. Agric. Food Chem. 14(3):701-705.
Juice
Vit. C
Juice
Vit. C
Antioxidant Activity of Fruits
ORAC (micromoles of Trolox equvivalents/g of dry matter)
160
140
120
100
80
60
40
20
0
Strawberry
Plum
Orange
Grapefruit Grapefruit
Wang et al., 1996. J. Agric. Food Chem. 14(3):701-705.
Tomato
Grape
Grape
Pectin
• Used traditionally for jelly
preparation
• Modified pectin can prevent prostate
cancer
• Pectin can reduce levels of serum
cholesterol
How much we need?
• To consume about 6g of pectin
only about 170 g of grapefruit
pulp is sufficient (Baker, 1994)
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
Dietary Fiber
Dietary fiber
denotes all plant
cell wall
components that
cannot be
digested by an
animal’s own
enzymes.
•
•
•
•
•
•
Pectin
gums
lignin
Cellulose
Hemicellulose
pentosans
Citrus Pectin Health Benefits
• Modified Citrus Pectin (MCP) prevent
cancer metastasis, inhibiting cancer cell
proliferation
• Hypoglycemic Effect
• Hypocholesterolemic Effect
• Hemostasis
• Modulate human immune function
• Detoxification
Steps in the process of tumor dissemination
Schematic representation of aggregation of tumor
cell to normal cell and pectin function
Raz and LOtan, 1987
Probable mechanism of pectin
hypocholesterol effect
Farnandez, et al., 1990
Pectin Hypocholesterolemic
Action
Pectin Hemostasis
Function
Pectin can shorten the coagulation
time of blood and act as an
antagonist of heparin when injected
intravenously.
Pectin sulfate can behave as strong
anticoagulant.
Parts of the citrus fruit which contain pectin
Components of Pectin
•
•
•
•
•
•
•
Molecular Weight
Polygalacturonic Acid
Galacturonic Acid Content
Methoxyl Esters
Neutral Sugar Content
Ions
Proteins
Structure of Pectin

Rhamnogalacturonan
Side Chain


Linear galacturonan
Structure of Pectin
Fibroblast Growth Factor
Signaling System
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
FPLC Elution Profile
Variation of Pectin Content
and Composition in
Different Citrus Species
Extraction Rate (% of fresh weight)
Pectin Extraction Rate in Different Citrus Species
6.00
F/A
L
5.00
4.00
3.00
2.00
1.00
0.00
d
b
lem
a
a
tan
b
b
org
c
b
gra
Changes of Pectin Content
and Composition Due to
Harvest Season
Extraction Rate (% of fresh weight)
Pectin Extraction Rate
6
F
A
5
4
3
2
1
0
Aug
Sep
Nov
Jan
Mar
May
L
Variation of Pectin Sugar Content
mg/g of Pectin Weight
200
F
180
A
160
140
120
100
80
60
40
20
0
Aug
Sep
Nov
Jan
Mar
May
L
Variation of pectin sugar composition
Percentage (% of total sugar wt.)
70
rhamnose
mannose
arabinose
galactose
xylose
glucose
60
50
40
30
20
10
0
Aug
Sep
Nov
Jan
Harvest Month
Mar
May
In Vitro Effects of
Citrus Pectin on the
FGF Signaling
System
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)
300
Pectin Inhibit FGF-1 Binding to FGFR1
% of Maximum Binding
120
Pectin Added
Heparin Only
100
80
60
40
20
0
0
0.001
0.01
0.1
1
Heparin Concentration (g/ml)
10
Inhibition activities of pectin
Flavedo/Albedo
Lamella
Inhibition Activity
1
0.8
0.6
0.4
0.2
0
-0.2
a
c
Lemon
a
a
Grapefruit
a b
Tangerine
Citrus Species
a b
Orange
Inhibition activities of pectin
F
A
L
Inhibition Activity
1
0.8
0.6
0.4
0.2
0
a a ab
AUG
a a ab
SEP
a a
a a a
NOV
a b ab
JAN
a a b
MAR
a a bc
MAY
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
Case Control and Cohort Studies
of All Types of Cancer
Fruit
Vegetables
No. of studies
Inverse
Positive
55
9
Fruits
29
5
Tomatoes
35
10
Carrots
50
7
Citrus Fruit
26
6
Exploring Potential Drug Interaction
with Texas Rio Red Grapefruit
Variation of Furocoumarins
in Different Varieties
• 9 different varieties are
analyzed for the quantitative
and qualitative differences in
Furocoumarins.
• Bottom line: Optimization of
furocoumarin levels.
Analytical HPLC Profile of Grapefruit Juice
UV6000-240nm
3000
3000
Retention Time
DHB
2000
2000
mAU
mAU
WHITE GFJ
1000
1000
Bergamottin
Paradisin A
0
0
10
20
30
0
40
50
60
Minutes
UV6000-240nm
Retention Time
3000
3000
2000
2000
mAU
mAU
RED GFJ
1000
Bergamottin
DHB
1000
Paradisin A
0
0
0
10
20
30
40
Minutes
50
60
Varieties Used for this Study…
Rio Red
Star Ruby
Ray Red
Henderson
Ruby Red
Thompson Pink
Duncan
Marsh White
Pumello
Variation of DHB in different
Varieties
9
8
7
6
5
g/ml 4
3
2
1
0
n
llo
an
ed
ed
ed Pink
by
o
ite
c
e
s
R
R
R
u
h
r
n
R
y
o
e
T
w
u
um
ay
r
b
d
D
Ri
h
P
a
u
R
n
R
rs
St
a
He
M
Variation of Bergamottin in
different Varieties
4
3.5
3
2.5
g/ml
2
1.5
1
0.5
0
n
llo
an
ed
ed
ed Pink
by
o
ite
c
e
s
R
R
R
u
h
r
n
R
y
o
e
T
w
u
um
ay
r
b
d
D
Ri
h
P
a
u
R
n
R
rs
St
a
He
M
Variation of GF-I-1 in different
Varieties.
0.8
0.7
0.6
0.5
g/ml0.4
0.3
0.2
0.1
0
n
llo
an
ed
ed
ed Pink
by
o
ite
c
e
s
R
R
R
u
h
r
n
R
y
o
e
T
w
u
um
ay
r
b
d
D
Ri
h
P
a
u
R
n
R
rs
St
a
He
M