1 Anthocyanin Pigments: Anthocyanin Pigments: Stability, Availability, and Stability, Availability, and Biotransformation in the Biotransformation in the Science of Foods for Health Science of Foods for Health Gastrointestinal Tract Gastrointestinal Tract M. Mónica Giusti M. Mónica Giusti Department of Food Science & Technology Department of Food Science & Technology Ohio State University, Columbus, OH 43210 USA Ohio State University, Columbus, OH 43210 USA Anthocyanins Anthocyanins O CH2OH O OH HO O + OH 3 Basics about anthocyanins Basics about anthocyanins Natural pigments (blue, purple, red) Potent antioxidants (1-4 times > vitamin E) 637 found in nature (Andersen and Jordheim, 2008) Structure and color change with pH und und O OH HO O + R1 OH R2 O OH OH HO O OH O Backgro Backgro Aglycone Glycosylation Acylation Value of anthocyanins Value of anthocyanins Research – health benefits • Prevention of cardiovascular diseases (Day et al. 1997) • Relief of oxidative stress (Ramirez-Tortosa et al. 2001) • Anti-cancer (Reen et al. 2006; Harris et al. 2001) • Prevention of obesity (Kwon et al. 2007; Tsuda 2008) und und Prevention of obesity (Kwon et al. 2007; Tsuda 2008) Backgro Backgro Food industry • Natural colorant • Added value ingredient • Good image Anthocyanin chemical structure R1 OH Flavonoids C6-C3-C6 skeleton – C – C – C – OH OH HO O + anthocyanin R2 •Each Aglycone has a characteristic color and spectra OH HO O + R1 OH R2 Different aglycones found in nature Aglycon R1 R2 λ max (nm) visible / color Pelargonidin H H 494 nm / orange Cyanidin OH H 506 nm / orange-red Peonidin OMe H 506 nm / orange-red Delphinidin OH OH 508 nm / bluish-red Petunidin OMe OH 508 nm / bluish-red Malvidin OMe OMe 510 nm / bluish-red OH anthocyanin
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Anthocyanin Pigments:Anthocyanin Pigments:Stability, Availability, and Stability, Availability, and Biotransformation in the Biotransformation in the
Science of Foods for HealthScience of Foods for Health
Gastrointestinal TractGastrointestinal Tract
M. Mónica GiustiM. Mónica GiustiDepartment of Food Science & TechnologyDepartment of Food Science & Technology
Ohio State University, Columbus, OH 43210 USAOhio State University, Columbus, OH 43210 USA
AnthocyaninsAnthocyaninsO
CH2OH
O
OH
HO O+
OH
3
Basics about anthocyaninsBasics about anthocyanins
Natural pigments (blue, purple, red)
Potent antioxidants (1-4 times > vitamin E)
637 found in nature (Andersen and Jordheim, 2008)
Structure and color change with pH
ou
nd
ou
nd
O
OH
HO O+
R1
OH
R2
O
OHOH
HO
O
OH
O
g p
Ba
ck
gro
Ba
ck
gro
Aglycone
Glycosylation
Acylation
Value of anthocyaninsValue of anthocyanins
Research – health benefits• Prevention of cardiovascular diseases (Day et al. 1997)
• Relief of oxidative stress (Ramirez-Tortosa et al. 2001)
• Anti-cancer (Reen et al. 2006; Harris et al. 2001)
• Prevention of obesity (Kwon et al. 2007; Tsuda 2008)
ou
nd
ou
nd
Prevention of obesity (Kwon et al. 2007; Tsuda 2008)
Ba
ck
gro
Ba
ck
gro
Food industry• Natural colorant
• Added value ingredient
• Good image
Anthocyanin chemical structure
HO O+
R1
OHFlavonoids
C6-C3-C6 skeleton
– C – C – C –
OH
OH
HO O+
anthocyanin
R2
•Each Aglycone has a characteristic color and spectra
OH
OH
HO O+
R1
OH
R2
Different aglycones found in nature
Aglycon R1 R2 λ max (nm) visible / color
Pelargonidin H H 494 nm / orangeCyanidin OH H 506 nm / orange-redPeonidin OMe H 506 nm / orange-redDelphinidin OH OH 508 nm / bluish-redPetunidin OMe OH 508 nm / bluish-redMalvidin OMe OMe 510 nm / bluish-red
OH
anthocyanin
2
In nature, anthocyanins are always glycosylated and can be acylated
Anthocyanin chemical structure may affect anthocyanins absorption and excretion.
He et al., 2006
Effect of AREs on Aberrant Crypt FociEffect of AREs on Aberrant Crypt Foci
* * *80
100
r
Control Bilberry Chokeberry Grape
Anthocyanin levels in feces correlated with inhibition of early cancer lesions, suggesting unabsorbed anthocyanins may be chemoprotective
* *0
20
40
60
Small (2-3) Middle(4-5) Large(>5) TotalACF Multiplicity
Mea
n AC
F N
umbe
r
Lala et al. 2006. Nutr Cancer 54(1)
7
The GIThe GI5050 of Anthocyaninof Anthocyanin--rich Extracts rich Extracts from Different Natural Sourcesfrom Different Natural Sources
Subset IIISubset I Subset II
L)
130.3
107.7
Bi il bilit
700~2000µg/g in Feces(GI tract)
13.831.2 32.2
68.5 71.2
GI 50
(ug/
m
Jing et al., 2008
Bioavailability
<1.3µg/mL in plasma
Anthocyanin impact on GITAnthocyanin impact on GIT
Importance in the GIT• Improve lumen
condition• Protect epithelial
cells30
40
50
60
controlchokeberrybilberrygrape
Anthocyanin-rich diets vs. rat colon cancer development
ou
nd
ou
nd
• Dose dependant effects
Stability in the GIT• In vivo evidence is
currently scarce 0
10
20
Colonic cellproliferation
index
Large ACFmultiplicity
Fecalanthocyanin(nmol/100g)
Fecal moisturecontent (%)
Fecal bile acids(μmol/g)
ND
Colon cancer development Anthocyanins in colonic lumen(Source: Lala et al. 2006)
Ba
ck
gro
Ba
ck
gro
Experimental DesignExperimental Design
Black raspberry anthocyanin-rich extracts, and lyophilized were used in feeding6 male Fischer 344 rats (11 wks of age) per groupSingle dose of 12 ± 3 mg anthocyanins in 0.1% citric acid solution by stomach tube after 24 hr fasting
180 min
in t
he
GIT
in t
he
GIT
Bladder urine Gastric and small intestinal contents
Cyanidin aglycon was found in the gastric and intestinal contents.• Attributed to acid hydrolysis in
the stomach and
8.8a
6.3b6
8
10
f Cy-
3-gl
u
in t
he
GIT
in t
he
GIT
Abs
orba
nce
(at 5
6
6
Time (min)
1 5
0 5 10 15 20 25
12
34
5
Small intestinal content
Gastric content
12
3
4
5
1 5
0 5 10 15 20 25
12
34
5
12
3
4
5
• β-glucosidase activity in the small intestine – membrane bound (?)
Relative proportion of Cy-3-glu decreased in the small intestine
0
2
4
6
Stomachcontents
Intestinecontents
Peak
Are
a% o
n=23
n=23
An
tho
cya
nin
s i
An
tho
cya
nin
s i
Anthocyanins in Stomach and Anthocyanins in Stomach and Small IntestineSmall Intestine
Anthocyanins in the gastric content linearly decreased with t½ of 120 min.
d (m
g cy
-3-g
lu e
q.)
GIT content
aab6
8
10
12
Stomach
Combined
a
b
abab
Intestine
Anthocyanins in the small intestinal content reached maximum at 120 min before ain
th
e G
ITin
th
e G
IT
Time (min)
Ant
hocy
anin
Rec
over
ed
bcd
ab
0
2
4
6
0 30 60 90 120 150 180 210Anthocyanins recovered from gastric and small intestinal lumen accounted for 75-79% of the administered dose between 30 and 120 min.
maximum at 120 min before decreasing.
b
abab
An
tho
cya
nin
s i
An
tho
cya
nin
s i
Anthocyanins in Stomach and Anthocyanins in Stomach and Small IntestineSmall Intestine
Anthocyanins in the small intestinal tissue also reached maximum at 120 min before decreasing.
The small intestine tissue 0
2
4
6
8
10
12StomachIntestineCombined
red
(mg
cy-3
-glu
eq.
)
A) GIT content
aab
bcd
b
a
abab
a
b
abab
in t
he
GIT
in t
he
GIT
The small intestine tissue took up 7.5% of the administered anthocyanins, very high compared to absorption into the plasma levels.
Urine anthocyanin profile closely reflected that in the lumen of absorption sites
An
tho
cya
nin
s i
An
tho
cya
nin
s i
AnthocyaninAnthocyanin--protein Complex in protein Complex in Stomach TissueStomach Tissue
Stomach tissue extracts exhibited red color that decreased over time. However, free anthocyanins were not detected by HPLCSpectral data obtained by altering the pH of the solution confirmed the presence of anthocyanins, suggesting binding to other compounds, possibly a protein transporter for anthocyanins (Passamonti, S. et al. 2003).
Enzyme activity in the GITEnzyme activity in the GIT
O
OH
HO O+
OH
OH
Gly O
OH
HO O+
OCH3
OH
Gly
OH
OH
OCH3
OH
Cy-3-gly
Dp 3 gly
Pn-3-gly
Pt 3 gly
LPH substrate specificity on the 5 anthocyanin glucosides in blueberry
IT
IT EE
nzy
me
s
nzy
me
s
0
0.1
0.2
0.3
0 30 60 90 120 150
Con
cent
ratio
Time (min)
O
OH
HO O+
Gly O
OH
HO O+
Gly
O
OH
HO O+
OCH3
OH
Gly
OCH3
OH OH
Dp-3-gly
Mv-3-gly
Pt-3-gly
Th
e
Th
e EE
ffe
ct
of
GI
ffe
ct
of
GI
Some concluding remarksSome concluding remarks
Results obtained in in-vitro and animal experiments were in agreement, suggesting a protective effect of anthocyanins against colon cancer.
Anthocyanins were relatively stable in the gastric and small intestinal lumens of fasted rats in contrast to reports from some in vitro studies.
A significant portion of anthocyanins was taken up into the GIT tissues, but neither extensively absorbed or retained. This suggests that anthocyanins may have in-situ protective effects in this tissue.
The chemical structure of the anthocyanin molecule will impact its stability to the conditions of the GIT, and therefore, its chances to interact with the GIT lining tissues.
Anthocyanins could be incorporated into foods as food colorants with added value
Some concluding remarksSome concluding remarks
Anthocyanins are abundant in nature, and could be incorporated into foods as food colorants with added value
Not always we require absorption of compounds in order to exert a protective action
Th ti it f h t h i l i th b d ill b ff t d bThe activity of phytochemicals in the body will be affected by other components present in the food and compounds present in the GIT.
Some compounds can act synergistically, in an additive way or can inhibit each other’s action.
In the news…
August, 2007
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What do you think???What do you think???
Do you think anthocyanins can provide protection against colon cancer?
Could a compound that is not absorbed into the plasma have an impact on health?
How and or why could the chemical structure affect the bioactivity of the compounds?
What are some important questions to ask when working with bioactive components using in-vitro experiments?
How much can we conclude from an animal model test?
What would be some advantages and disadvantages of using an in-vitro test vs an animal test vs a clinical trial?
AcknowledgementsAcknowledgements
Collaborators: • OSU:Joshua Bomser, Mark Failla, Steven Schwartz, Laura Kresty.• MD: Berna Magnuson• UNALM: D. Campos, F. Salas, F. Ludena, V. Noriega, I. Betalleluz, B.
Hatta, R. Chirinos.• China: P. Jing
Students and Research Assistants• Pu Jing, Jian He, Taylor Wallace, Kristin Keatley, Lucy Zhao, Minnie
Malik, Geeta Lala, Qingguo Tian.
Artemis International, Inc / Polyphenolics, Inc. / GlobeNatural International / Agricomseeds
Research supported by USDA-NRI competitive grants and OARDC