basics about anthocyanins value of anthocyanins

1

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)

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nd

ou

nd

Prevention of obesity (Kwon et al. 2007; Tsuda 2008)

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gro

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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

HOH C

OO

CH2O- acidHO O+

OH

… with acids attached to the sugars

OHOH2C O

O

HO OHO

O

OH

O

HO

Aliphatic acid (malonic acid) Cinnamic acid (p-coumaric acid)

Anthocyanin sourcesAnthocyanin sources

OCH2OH

O

OH

HO O+

OH

3

OH2C

OO

CH2

O

HO O+

OH

OO

HOH2C

35

O

O

O

HO OH

O

O

Berries:Simple pigments Other Sources:

Complex acylated pigments

HPLC anthocyanin profiles HPLC anthocyanin profiles

Anthocyanin profiles can vary a lot among plants

• Chokeberry• simple profile• one aglycone (cy)• 4 different sugars

A

8

10

1922

B

Chokeberry

Bilberry

5

19

0 nm

0 nm

4 different sugars

• Bilberry• 5 different aglycones• 3 different sugars

• Grape• One sugar: glucose• 5 different aglycones• acylation

24

5,6

89,10

12

14,15

16,17

18

21

Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

13

4 7 8

11

16

21

2324 25

26

27

28 29

Bilberry

GrapeCAbs

orpt

ion

@ 5

20A

bsor

ptio

n @

520

Comparing different anthocyanin Comparing different anthocyanin pigment profilespigment profiles

Cya-

Del-

Del C

y D C

Peo

Mal

Pet- Bilberry

Chokeberry

Cy-3-G

al

Cy-3-G

lu

Cy-3-A

rab

Cy-3-xyl

Pet-3-Gal

-3-Glu

-3-Gal

-3-Glu

ya-3-Gal

el-3-Ara

Cya-3-A

ra

Mal -3-gal

o-3-glu

Mal-3-A

rabl-3-G

lu

-3-glu Pet-3-ara

5 10 15 20 25 30 minutes

Del-3-glu

Cya-3-glu

Pet-3-glu

Peo-3-gluM

al-3-glu

Acylated anthocyanins

Grape


Anthocyanins can be used as food colorants

… of added value

There is evidence that anthocyanins may contribute to the protective effect y pof fruits and vegetables

3

Anthocyanins are poorly Anthocyanins are poorly absorbedabsorbed

Anthocyanins are widely distributed in nature and in foods

High daily intake (100+ mg/day)

HOWEVER… levels of anthocyanins found in plasma are very low

Less than 1% of the dietary intake is absorbed

So… can compounds that are not even absorbed have any impact on health?

In vitro tests: Anthocyanins In vitro tests: Anthocyanins and colon cancer and colon cancer

t tit ti


protectionprotection

Cells lining the intestine can be affected by compounds entering the blood as well as by compounds that are not absorbed due to direct contact.

What is colorectal cancer?What is colorectal cancer?

Epithelial cell layer of the colon, rectum, and appendixFive stagesSymptoms• Unexplained weight loss• Fatigue• Anemia• Change in bowel habits• Liver metastasis

Anthocyanins in direct contact with the GI tract

American Cancer Society

Who does colorectal cancer Who does colorectal cancer affect?affect?

Third most prevalent cancer in the Western World

7% lifetime risk of developing

Risk factors• AgeAge• Heredity• Diet• Smoking• Alcohol• Physical inactivity

American Cancer Society

Anthocyanin extract preparationAnthocyanin extract preparation

C18 Semi-purification

Plant Material

Acetone/ChloroformExtraction

Acylated Anthocyanin

Fractionated w/ Ethyl Acetate

Rotoevaporated

Lyophilized

Saponified Anthocyanins

Saponified w/ KOH

Rotoevaporated

Lyophilized

HTHT--29 Cell treatment29 Cell treatment

HT-29 cells seeded at 10,000 cells/mL24-hrs

HT-29 cells treated with anthocyanin extracts48-hrs

SRB Assay

Plate Reader @ 490 nm

4

HTHT--29 Cell treatment procedure29 Cell treatment procedure

Blank

AB

XX

XX

100ug 50ug 25ug 12.5ug XB C X100ug 50ug 25ug 12.5ug

ControlAnthocyanin Treatment Concentrations

UnusedBlank

AB

XX

XX



UnusedBlank

AB

XX

XX



UnusedBlank

AB

XX

XX



Unused

AB

XX

XX


ABAB

XXXXXX

XXXXXX

100ug 50ug 25ug 12.5ug XB C X100ug 50ug 25ug 12.5ug100ug 50ug 25ug 12.5ug XB C X100ug 50ug 25ug 12.5ug


Unused

BCDEFGH

X

XXXX

XX

X

XXXX

XX

AcylatedAnthocyanins

SaponifiedAnthocyanins

BCDEFGH

X

XXXX

XX

X

XXXX

XX


BCDEFGH

X

XXXX

XX

X

XXXX

XX

BCDEFGH

X

XXXX

XX

X

XXXX

XX

BCDEFGH

X

XXXX

XX

X

XXXX

XX

BCDEFGH

BCDEFGH

X

XXXX

XX

X

XXXX

XX

X

XXXX

XX

X

XXXX

XX

X

XXXX

XX

X

XXXX

XX


SaponifiedAnthocyanins

Percent inhibition of HTPercent inhibition of HT--29 Cells 29 Cells

% Growth Inhibition = 100 – (Ttrt – T0) x 100/ (Tctr – T0)

Where:

T0 is time zeroTtrt is the absorbance with treatment at 72 hrsTctr is the absorbance with out treatment at 72 hrs

GI50 : amount of extract required to inhibit 50% growth

80

100

120

140

on (%

con

trol

)

ARE : Anthocyanin-rich extract

Inhibitory effects of fractionations of Inhibitory effects of fractionations of chokeberry on HTchokeberry on HT--29 colon cells29 colon cells

orm

atio

nor

mat

ion

0

20

40

60

80

0 20 40 60 80 100Dose (ug/mL)

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wth

Inhi

bitio

Zhao et al., 2005Back

grou

nd In

foBa

ckgr

ound

Info

Fractionation of anthocyaninsFractionation of anthocyanins

Different solvents used to separate anthocyanin fraction f h h lifrom other phenolics fraction

Very high efficiency and low cost

More than 95% pure

Experimental designExperimental design

Purple corn

Chokeberry

Bilberry

ARE: anthocyanin-rich extract

ACN: anthocyanin fraction

OPF: other phenols fraction

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)

The GIThe GI5050 of anthocyaninof anthocyanin--rich extracts rich extracts from different natural sourcesfrom different natural sources

Subset IIISubset I Subset II

/mL)

130.3

107.7

orm

atio

nor

mat

ion

13.831.2 32.2

68.5 71.2

GI 50

(ug/

Jing et al., 2008

Back

grou

nd In

foBa

ckgr

ound

Info

5

80

100

120

140

on (%

con

trol

)

ARE : Anthocyanin-rich extract

Inhibitory effects of fractionations of Inhibitory effects of fractionations of chokeberry on HTchokeberry on HT--29 colon cells29 colon cells

orm

atio

nor

mat

ion

AP: ACN+OPF

0

20

40

60

80

0 20 40 60 80 100Dose (ug/mL)

Gro

wth

Inhi

bitio

OPF : Other phenolics fraction

ACN: Anthocyanins fraction

Jing et al., 2008Back

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nd In

foBa

ckgr

ound

Info

Combination Index: Combination Index: Interaction Interaction Between Anthocyanins and other PhenolsBetween Anthocyanins and other Phenols

It is used to determine if the compounds tested work more efficiently alone or combined.

CIi = [ACN0]

[ACN1]

[OPF0]

[OPF1]+

SourceCombination Index

GI25 GI50 GI100

Anthocyanins + Other Phenols 1.222 1.144 1.221

Combination Effect Additive Additive Additive

Chou and Talalay (1984).

Our cell line studies showed Our cell line studies showed that…that…

All AREs inhibited growth of HT29 colon cancer cells, with little effect on NCM460 non-tumorogenic cells

D t t th t th

3

4

No.

(x10

6 )

ControlARE (50ug/ml)

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. 20030

1

2

Normal HT-29

Tota

l Cel

l N

What questions should we ask…?What questions should we ask…?

Are these compounds actually reaching the tissue?

And if so… are these the actual forms of the compounds that reach the tissue (or have they been transformed?)

Are they present in concentrations that are high enough to make any impact?make any impact?

Are the effects observed really due to the compounds of interest? Or are there other potentially bioactive compounds?

How will the compounds impact cancer cells… and how will they impact normal cells?

Will these results be reproduced in an in-vivo system?

Anthocyanin absorption Anthocyanin absorption and chemoprevention in an and chemoprevention in an animal modelanimal model

FST Fall 2008 Seminar Series FST Fall 2008 Seminar Series –– M. Mónica GiustiM. Mónica Giusti

animal modelanimal model

Animal modelsAnimal models: : Anthocyanin absorption and chemoprotectionAnthocyanin absorption and chemoprotection

Animal trial: 11 F344 rats / treatment

15 k ARE 4 h i /k di15 wk ARE treatments: 4g anthocyanin/kg diet

Week 1 Week 15

AOM injection(20 mg/kg body wt)

Urine, feces Colon, Plasma

Week 14

6

0.0005

0.0010

0.0015

0.0020

4

5

910

1112

Chokeberry

0.0020 2 5 Bilberry

2

Anthocyanins in Plasma Anthocyanins in Plasma (520 nm)(520 nm)

0.1 to 1 μg/mL plasma

Major peaks detected

0.0005

0.0010

0.0015

14

1011

12

0.0000

0.0005

0.0010

0.0015

0.0020

5.00 10.00 15.00 20.00 25.00 30.00 35.00

43 6

78

101112

13

Grape

Acylated anthocyanin detected

Anthocyanin metabolites

Anthocyanin metabolites in urine

Urine

Cy-3-A

rab

Cy-3-G

al ??

5 10 15 20 25 30

Chokeberry ARE

Cy-3-G

al

Cy-3-G

lu

Cy-3-A

rab

Cy-3-xyl

?

O

OH

OHOH

CH 2OHO

OH

OH

OH

OH

O+

Anthocyanin methylation vs Anthocyanin methylation vs glucuronidation glucuronidation

Methylation O

OH

OHOH

CH 2OHO

OH

OH

OCH 3

OH

O+

+14Cy-3-glu M+ 449(449 - 287)

OH OH

Glucuronidation

O

OH

OHOH

COOH

O

OH

OH

OH

OH

O+

Pn-3-glu M+ 463

+14

Cy-3-glucuronide M+ 463

(463 -- 301)

(463 -- 287)

Methylated anthocyanin were found in urine

Urine

Cy-3-A

rab

Cy-3-G

al

Pd-3-Gal Pd-3-G

Pd-3-Ara

~ 35% methylated pigments

5 10 15 20 25 30

Chokeberry ARE

Cy-3-G

al

Cy-3-G

lu

Cy-3-A

rab

Glu

Cy-3-xyl

Chokeberry ARE

Cy-3-G

al Cy-3-G

luC

y-3-Arab

Cy-3-xyldiet

Cy-3-Glu

Pt-3-Glu

Mv-3-Arab

Presence of intact anthocyanins and metabolites in plasma and urine, demonstrating absorption

Our Animal Studies Show...Our Animal Studies Show...

Pd-3-Glu

Urine

Cy-3-A

rab

Cy-3-G

al

Pd-3-Gal

Pd-3-Ara

Plasma

fecesurine

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

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gro

Ba

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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

Gastric and small intestinal tissues

HPLC-PDA-MS

60 min

120 min

30 minControl

(0.1% citric acid)

Treatments

An

tho

cya

nin

s i

An

tho

cya

nin

s i

Anthocyanins Profile ChangeAnthocyanins Profile Change

520

nm)

6Cy aglycone standard

6

2

3

4 Black raspberry extract

2

3

4

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.

00 30 60 90 120 150 180 210

0

0.2

0.4

0.6

0.8

1

1.2

0 30 60 90 120 150 180 210

Intestine

Time (min)

Tota

l Ant

hocy

anin

s R

ecov

er

B) GIT tissue

a

aa

a

An

tho

cya

nin

s i

An

tho

cya

nin

s i

8

Bladder Urine AnthocyaninsBladder Urine Anthocyanins

6.47.4

8.69.3

6

8

10

Are

a%

Cy-3-glu% in urine

8.8a

6.3b6

8

10

Are

a%

Cy-3-glu% in GIT

in t

he

GIT

in t

he

GIT

0

2

4

30 60 120 180

Pea

k A

Time (min)

n=3

n=5

n=5

n=2

0

2

4

Gastriccontent

Intestinalcontent

Pea

k A

n=23

n=23

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).

in t

he

GIT

in t

he

GIT

An

tho

cya

nin

s i

An

tho

cya

nin

s i

Abs

orba

nce

(AU

)

700600500400300

Bound anthocyanins pH 1Bound anthocyanins pH 4.5Free anthocyanins pH1

Wavelength (nm)700600500400300



0.4

0.5

0.6

Dp-3-gluCy-3-gluPt-3-gluPn-3-gluMv-3-glu

n (µ

M)

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

9

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

basics about anthocyanins value of anthocyanins

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