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

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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

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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

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○

○

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○

○

○

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0

5

10

15

0 10 20 30 40 50 60

○

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○

○

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○

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○

○○

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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