anthocyanins: for color and health - altavoz
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Anthocyanins: For Color and Health
M. Mónica Giusti
Department of Food Science and Technology
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
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
What compounds in fruits and vegetables are responsible for their protective effects?
AnthocyaninsO
CH2OH
O
OH
HO O+
OH
3
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
In nature, Anthocyanins are always glycosylated
OCH2OH
O
OH
HO O+
OH
3
… with one sugar on position 3
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)
Anthocyanins as Natural Food Colorants
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
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
Evaluate plant materialsNew and exotic / traditional
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
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
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
����
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
����
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330°
30°
60°90°
����
����
����
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0°
Model juicespH = 3.5
� 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
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
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
Pigment Stability
����
○
○
����
○
○
○
����
0
5
10
15
0 10 20 30 40 50 60
○
����
���� ����
○
○
���� ���� ��������
����
○
○○
���� ��������
○
○○
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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
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?
Evaluating new Radish Cultivars
in China!
Jiangsu Province, PR China, November 2007
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.
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?
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!
There is evidence that anthocyanins may contribute to the chemo protective effect of fruits and vegetables
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)
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
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
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
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)
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
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
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
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
Fractionation of Anthocyanins
� Different solvents used to separate anthocyanin fraction from other phenolics fraction
� Very high efficiency and low cost
� More than 95% pure
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
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
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.
In the news…
August, 2007
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.
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
AnthocyaninsO
CH2OH
O
OH
HO O+
OH
3
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