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Anthocyanins: For Color and Health
M. Mónica Giusti
Department of Food Science and Technology
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The C6-C3-C6 skeleton of flavonoid compounds
– C – C – C –Isoflavones
� Isoflavones– Phytoestrogens
� Anthocyanins– Natural colorants– Phytonutrients
Research Areas
Compounds of interest: Flavonoids
Areas of work
� Analytical� Horticultural� Processing� Bioavailability� Health benefits
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Introduction
� Epidemiological studies suggest that diets high in fruits and vegetables reduce the risk of a variety of chronic diseases– Cancer
� Strong evidence for GI tract types
– Cardiovascular disease– Obesity
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What compounds in fruits and vegetables are responsible for their protective effects?
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AnthocyaninsO
CH2OH
O
OH
HO O+
OH
3
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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
•Each Aglycone has a characteristic color and spectra
OH
OH
HO O+
R1
OH
anthocyanin
R2
Chemical Structure
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In nature, Anthocyanins are always glycosylated
OCH2OH
O
OH
HO O+
OH
3
… with one sugar on position 3
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In nature, Anthocyanins are always glycosylated and can be acylated
… with acids attached to the sugars
OHOH2C
OO
CH2O- acid
O
HO O+
OH
HO OHO
O
OH
O
HO
Aliphatic acid (malonic acid) Cinnamic acid (p-coumaric acid)
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Anthocyanins as Natural Food Colorants
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Importance of food color
� People judge quality by the appearance of the product
� To keep product standardized
� Many times also correlates with (perceived) flavor or other quality characteristics
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What are we looking for?
� “Right” color– RED is the color with the highest demand in US– Other “attractive” colors are also desirable
� Compatibility with matrix
� Color and pigment stability� No undesirable aromas / flavors
� Added value : potential health benefits
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Evaluate plant materialsNew and exotic / traditional
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Anthocyanin SourcesO
CH2OH
O
OH
HO O+
OH
3
Berries:Simple pigments Other Sources:
Complex pigments
OH2C
OO
CH2
O
HO O+
OH
OO
HOH2C
35
O
O
O
HO OH
O
O
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Berberis boliviana L.
� The Peruvian berry Berberis boliviana L. contains unusually
high concentration of non-acylated anthocyanins
– ~ 7% by dry weight
� Dried berries could be used as colorants without pigment
extraction.
B. boliviana L.
15X – 20X the NORMAL
Anthocyanin Concentration of
Regular Berries!!!
Del Carpio Jimenez, Wallace, and Giusti, 2007
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5.0
1.0
2.0
3.0
4.0
6.0
7.0
250 500 750 1000 m/z0.0
303
250 500 750 1000 m/z0.0
2.5
5.0
7.5465
Peak 1: Delphininidin 3 glucoside
TIM
SIM
Berberis Anthocyanin Characterization
0 5 10 15 20 min
1
3
4
5
67
8
9
102 250 500 nm
5
20
35
mAU
524
342
Peak 1: Delphininidin-3-glucoside
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����
Hue Angle of Acylated Anthocyanins in Model Juices
FD&C Red # 40
Radish Extract
Potato Extract
Black Carrot Extract
MegaNatural Red Grape
Red Cabbage Extract
����
����
����
����
����
330°
30°
60°90°
����
����
����
����
����
����
0°
Model juicespH = 3.5
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� Radish pigments provided excellent red color and stability to maraschino cherries and model juices and could be used as alternative to the use of FD&C Red No. 40.
Giusti and Wrolstad, 1996
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Potato Anthocyanins
CH 2OH
A
B
OH
O
O
HO
OHHO
O
O
O
HO
(+)
CH 2OHCH 2OH
A
B
OH
O
O
HO
OH
HO
O
O
OH
OH
HO
OOH
OH
HO
OO
HO
(+)
CH3
OH
OO
O
OH
O
OCH3
OHOCH3
Pelargonidin-derivatives
Petunidin-derivatives
Malvidin-derivatives
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0 5 10 15 20 25 30 35 40 45
Wayru
Pokko lomocho
Levitts Pink
All Red
5538-13
5538-11
5538-9
5538-7
5538-5
5538-3
5538-1
5589-2
5847-3
5847-1
6236-2
ND5538-2
ND04069-4
Monomeric Anthocyanin content (mg/100g tuber)
Greenhouse
Field
ND5849-1
6236-1
4239
5847-2
5589-1
3261-5R
5538-2
5538-4
5538-6
5538-8
5538-10
5538-12
Huckelberry
Cherry Jubilee
Blossom
Leque Chaqui
0 5 10 15 20 25 30 35 40 45
Wayru
Pokko lomocho
Levitts Pink
All Red
5538-13
5538-11
5538-9
5538-7
5538-5
5538-3
5538-1
5589-2
5847-3
5847-1
6236-2
ND5538-2
ND04069-4
Monomeric Anthocyanin content (mg/100g tuber)
Greenhouse
Field
ND5849-1
6236-1
4239
5847-2
5589-1
3261-5R
5538-2
5538-4
5538-6
5538-8
5538-10
5538-12
Huckelberry
Cherry Jubilee
Blossom
Leque Chaqui
Rodriguez-Soana, Giusti and Wrolstad, 1998
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Pigment Stability
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○
○
����
○
○
○
����
0
5
10
15
0 10 20 30 40 50 60
○
����
���� ����
○
○
���� ���� ��������
����
○
○○
���� ��������
○
○○
����
Room T°(25°C)Mon
omer
ic A
CN
(m
g/10
0mL)
Refrigeration (2°C)
Time (weeks)
Potato
Radish
Potato ACN, t½ = 11 wks
Radish ACN, t ½ = 24 wks
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Challenges in Colorant Production
Goal: Produce a Vegetable Juice concentrate
Challenges:� PPO activity� Starch Content� Presence of Glycoalkaloids
Solution:� Acidity� PH treatments� Processing - concentration� Enzymes?
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Evaluating new Radish Cultivars
in China!
Jiangsu Province, PR China, November 2007
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Chemical Structure of a Pg-derivative From Radish
OH2C
OO
CH2
O
HO O+
OH
OO
HOH2C
35
O
O
O
HO OH
O
O
Arrows indicate hydrogens in high proximity
Giusti, et al., 1998. J. Agric Food Chem.
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Main challenge: radish colorant free of undesirable aromas / flavors
Solution:• Cultivar selection
• Sensory tests• Processing steps
• Water as extraction media• Physical means for
concentration / separation• Enzymes?
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CONCLUSIONS
� Anthocyanins provided excellent color and stability to a variety of food matrices
� Acylation of anthocyanins is responsible for the added stability of the compounds
� Use of anthocyanins may be challenging. Added value would justify the extra effort!
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There is evidence that anthocyanins may contribute to the chemo protective effect of fruits and vegetables
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Supporting evidence
� Purple Sweet Potato (Ipomoea batatas)– Colorectal carcinogenesis in rats (Hagiwara et al.,
2002)– Chemoprotective properties using model systems
(Konczak-Islam, 2003)– Anti-hyperglycemic effect (Matsui et al., 2002)– Direct absorption into rats (Suda et al., 2002)
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Comparing different anthocyanin pigment profiles
Pet-3-G
alC
ya-3-Glu
Del-3-G
al
Del-3-G
lu
Cya-3-G
alD
el-3-Ara
Cya-3-A
ra
Mal -3-gal
Peo-3-glu
Mal-3-A
rabM
al-3-Glu
Pet-3-glu P
et-3-ara
Bilberry
5 10 15 20 25 30 minutes
Del-3-glu
Cya-3-glu
Pet-3-glu
Peo-3-glu
Mal-3-glu
Acylated anthocyanins
Grape
Chokeberry
Cy-3-G
al
Cy-3-G
lu
Cy-3-A
rab
Cy-3-xyl
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Our cell line studies showed that…
� All AREs inhibited growth of HT29 colon cancer cells, with little effect on NCM460 non-tumorogenic cells
� Data suggests that the protective effect of specific anthocyanins or extracts may depend on the chemical structure of anthocyanins present Malik et al., Nutr Cancer. 2003
0
1
2
3
4
Normal HT-29T
otal
Cel
l No.
(x10
6 )
Control
ARE (50ug/ml)
0
20
40
60
80
100
24 hrs 48hrs 72hrsTime of Exposure
Per
cent
age
Inhi
bitio
n
GrapeBilberryChokeberry
Zhao et al., JAFC, 2004
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Animal models : Anthocyanin absorption and chemoprotection
Animal trial: 11 F344 rats / treatment
15 wk ARE treatments: 4g anthocyanin/kg diet
Week 1 Week 15
AOM injection
(20 mg/kg body wt)
Urine, feces Colon, Plasma
Week 14
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Effect of AREs on Aberrant Crypt FociEffect of AREs on Aberrant Crypt Foci
* * *
* *0
20
40
60
80
100
Small (2-3) Middle(4-5) Large(>5) TotalACF Multiplicity
Mea
n A
CF
Num
ber
Control Bilberry Chokeberry Grape
Lala et al. 2006. Nutr Cancer 54(1)
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Pd-3-G
lu
Urine
Cy-3-A
rab
Cy-3-G
al
Pd-3-G
al
Pd-3-A
ra
Chokeberry ARE
Cy-3-G
al Cy-3-G
lu
Cy-3-A
rab
Cy-3-xyl
Plasma
diet
fecesurine
Cy-3-Glu
Pt-3-Glu
Mv-3-Arab
� Presence of intact anthocyanins and metabolites in plasma and urine, demonstrating absorption
� Anthocyanin chemical structure may affect anthocyanins absorption and excretion.
Our Animal Studies Show...
� Anthocyanin levels in feces correlated with inhibition of early cancer lesions, suggesting unabsorbed anthocyanins may be chemoprotective
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Anthocyanin-Rich Extracts (AREs)
Chokeberry (Aronia meloncarpa E.)
Bilberry(Vaccinium myrtillus L.)
Elderberry (Sambucus nigra L.)
Non-acylated Anthocyanins
Grape (Vitis vinifera)
Purple carrot (Daucus carota L.)
Radish(Raphanus sativus)
Purple corn (Zea mays L.)
Acylated Anthocyanins
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Anthocyanin profiles in seven commercial anthocyanin-rich sources
Source Anthocyanidin Glycosylation Acylation Purple corn (Zea may L.) Cy, Pt, Pn C3: mono-glycoside Malonic acid Chokeberry (Aronia meloncarpa E.) Cy C3: mono-glycoside None
Bilberry (Vaccinium myrtillus L.)
Dp, Cy, Pt, Pn, Mv C3:mono-glycoside None
Purple carrot (Daucus carota L.)
Cy C3: di-, tri-glycoside One cinnamic acid
Grape (Vitis inifera)
Cy, Dp, Pt, Pn Mv C3: mono-glucoside One cinnamic acid
Elderberry (Sambucus nigra L.) Cy
C3: mono-;diglucoside C3 and C5: di-, tri-glucoside
None
Radish (Raphanus sativusL.) Pg C3 & C5: tri-glucoside More than one
cinnamic acids
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Experimental design
Purple corn
Chokeberry
Bilberry
Elderberry
Grape
Purple carrot
Red radish
In vitro model :
HT29 cell line
SRB test
GI50
SPE
Semi-purification ARE
OPF
ACN
Fractionation (C18 SPE)
ARE: anthocyanin-rich extract
ACN: anthocyanin fraction
OPF: other phenols fraction
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Fractionation of Anthocyanins
� Different solvents used to separate anthocyanin fraction from other phenolics fraction
� Very high efficiency and low cost
� More than 95% pure
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0
20
40
60
80
100
120
140
0 20 40 60 80 100Dose (ug/mL)
Gro
wth
Inhi
bitio
n (%
con
trol
) ARE : Anthocyanin-rich extract
OPF : Other phenolics fraction
ACN: Anthocyanins fraction
AP: ACN+OPF
Inhibitory effects of fractionations of chokeberry on HT-29 colon cells
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The GI50 of Anthocyanin-rich Extracts from 7 Natural Sources
Bioavailability
700~2000µg/g in Feces(GI tract)
<1.3µg/mL in plasma
Subset IIISubset I Subset II
Purple corn
Chokeberry
Bilberry
Purple carrot
Grape
Elderberry
Radish
13.8
31.2 32.2
68.5 71.2
GI 5
0(u
g/m
L)
130.3
107.7
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Summary
� Anthocyanins offer an attractive alternative to the use of artificial colors for products in a wide variety of food products.
� Studies in our laboratory and others are providing convincing evidence that anthocyanin-rich extracts and foods may be protective against chronic disease
� Anthocyanin-rich commodities may have special appeal due to their potential applications as food colorants and their potential health benefits.
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In the news…
August, 2007
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Functional Foods Research at OSU…
CAFFRE: Center for Functional Foods Research and Entrepreneurship
– Research from Crop to Clinic to Consumer– Chemistry of phytonutrients – Analytical methods development– Processing effects / chemical alterations of phytonutrients – Bioavailability, metabolism, and physiological significance
of phytochemicals.
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THANK YOU !!!
� CREAS - Chile� USDA NRI 71.1 Competitive Grants Program � Collaborators:
– OSU / MD: Berna Magnuson, Joshua Bomser, Steven Schwartz– 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, Lucy Zhao, Minnie Malik, Geeta Lala, Qingguo
Tian
� Artemis International, Inc / Polyphenolics, Inc. / GlobeNatural International / Agricomseeds / RFI Ingredients
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AnthocyaninsO
CH2OH
O
OH
HO O+
OH
3