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  • Science of Foods for HealthScience of Foods for Health

    Anthocyanin Pigments:Anthocyanin Pigments: Stability, Availability, and Stability, Availability, and Stability, Availability, and Stability, Availability, and Biotransformation in the Biotransformation in the Gastrointestinal TractGastrointestinal Tract

    M Mó i Gi tiM Mó i Gi tiM. Mónica GiustiM. Mónica Giusti Department of Food Science & TechnologyDepartment of Food Science & Technology

    Ohio State University, Columbus, OH 43210 USAOhio State University, Columbus, OH 43210 USA

  • AnthocyaninsAnthocyanins CH2OH

    HO O+

    OH

    O 2

    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)dd

    R1

    OH

    Structure and color change with pH

    g ro

    u n

    d g

    ro u

    n d

    HO O+

    OH

    R2 OB a

    c k

    g B

    a c k

    g Aglycone

    Acylation

    O

    OH

    O

    HO

    O

    OH

    BB

    OHOHGlycosylation

  • 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 (R t l 2006 H i t l 2001)dd • Anti-cancer (Reen et al. 2006; Harris et al. 2001) • Prevention of obesity (Kwon et al. 2007; Tsuda 2008)

    g ro

    u n

    d g

    ro u

    n d

    B a

    c k

    g B

    a c k

    g

    Food industry • Natural colorantBB • Natural colorant • Added value ingredient • Good image

  • Anthocyanin chemical structure

    R1

    OH Flavonoids

    – C – C – C – HO O+

    R2

    C6-C3-C6 skeleton OH

    OHOH

    anthocyanin

  • Different aglycones found in nature R1

    OH

    •Each Aglycone has a characteristic color and spectra

    HO O+

    R2

    spectra OH

    OH

    anthocyanin

    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-redDelphinidin OH OH 508 nm / bluish-red Petunidin OMe OH 508 nm / bluish-red Malvidin OMe OMe 510 nm / bluish-red

  • In nature, anthocyanins are always glycosylated and can be acylated

    OH

    CH2O- acid HO O+

    O HOH2C

    O O

    O

    … with acids attached to the sugars

    O

    … with acids attached to the sugars

    OO

    HO OHOOHHO

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

  • Anthocyanin sourcesAnthocyanin sources HO O+

    OH

    H2C

    O O

    CH2

    HO O+

    OH

    3 5

    O

    O

    O

    HO OH

    O

    O

    O CH2OH

    O

    OH

    3

    O O

    O O

    HOH2C

    5

    Berries:Berries: Simple pigments Other Sources:Complex acylated pigments

  • HPLC anthocyanin profiles HPLC anthocyanin profiles

    Anthocyanin profiles can vary a lot among plantsA Chokeberry

    5 y g p

    • Chokeberry • simple profile8

    10

    19 22 simple profile

    • one aglycone (cy) • 4 different sugars

    • Bilb

    8 22

    B

    2 4

    8

    12 16,17

    Bilberry19

    @ 5

    20 n

    m @

    5 20

    n m

    • Bilberry • 5 different aglycones • 3 different sugars

    2

    5,6

    8 9,10

    14,15

    18

    21

    CA bs

    or pt

    io n

    @ A

    bs or

    pt io

    n @

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

    11

    21

    27

    GrapeC

    AA

    y

    Minutes 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

    1 3

    4 7 8 16

    23 24 25

    26 28 29

  • Comparing different anthocyanin Comparing different anthocyanin pigment profilespigment profiles

    Chokeberry

    C y-3

    C y-3 C3-G

    al

    C y-3-G

    lu

    3-A rab

    C y-3-xyl

    Pet-3 C

    ya-3-G lu

    D el-3-G

    al

    D el-3-G

    lu

    C ya-3-G

    D el-3-A

    C ya-3-A

    M a

    Peo-3-gl u

    M al-

    M al-3-G

    lu

    Pet-3-glu P e Bilberry

    3-G al

    u G al

    A ra Ara

    l -3-gal

    u -3-A rab

    uet-3-ara

    C

    D el-3-

    C ya-3-glu

    Pet-3-glu

    Peo-3-g M

    al-3-glu

    Acylated anthocyanins

    Grape

    5 10 15 20 25 30 minutes

    -glu

    glu Acylated anthocyanins

  • Science of Foods for HealthScience of Foods for Health

    Anthocyanins can be Anthocyanins can be used as food colorants

    … of added value

  • There is evidence that anthocyanins e e s e de ce t at a t ocya s may contribute to the protective effect of fruits and vegetablesg

  • Anthocyanins are poorly Anthocyanins are poorly absorbedabsorbed

    A th i id l di t ib t d i t dAnthocyanins are widely distributed in nature and in foods

    High daily intake (100+ mg/day)

    HOWEVER… levels of anthocyanins found in y plasma are very low

    Less than 1% of the dietary intake is absorbedLess than 1% of the dietary intake is absorbed

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

  • Science of Foods for HealthScience of Foods for Health

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

    protectionprotection

    Cells lining the intestine can be affectedCells 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 colonEpithelial cell layer of the colon, rectum, and appendix Five stages SymptomsSymptoms • Unexplained weight loss • Fatigue • Anemia• 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 theThird most prevalent cancer in the Western World

    7% lifetime risk of developing

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

    American Cancer Society

  • Anthocyanin extract preparationAnthocyanin extract preparation

    Plant Material

    Acetone/Chloroform Extraction

    Fractionated w/ Ethyl Acetate

    C18 Semi-purification

    Saponified w/ KOHw/ Ethyl Acetate

    Rotoevaporated

    w/ KOH

    Rotoevaporated

    Lyophilized Lyophilized

    Acylated Anthocyanin Saponified Anthocyanins

  • HTHT--29 Cell treatment29 Cell treatment

    HT-29 cells seeded at 10,000 cells/mL 24-hrs

    HT 29 ll t t d ith th i t tHT-29 cells treated with anthocyanin extracts 48-hrs

    SRB Assay

    Plate Reader @ 490 nm

  • HTHT--29 Cell treatment procedure29 Cell treatment procedure

    Anthocyanin Treatment ConcentrationsAnthocyanin Treatment ConcentrationsAnthocyanin Treatment ConcentrationsAnthocyanin Treatment ConcentrationsAnthocyanin Treatment Concentrations Blank

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

    Control Anthocyanin Treatment Concentrations

    UnusedBlank

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

    Control Anthocyanin Treatment Concentrations

    UnusedBlank

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

    Control Anthocyanin Treatment Concentrations

    UnusedBlank

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

    Control Anthocyanin Treatment Concentrations

    Unused

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

    Control Anthocyanin Treatment Concentrations

    Unused

    A B C D

    X X

    X X

    X X

    X X

    g g g g g g g g

    A B C D

    X X

    X X

    X X

    X X

    g g g g g g g g

    A B C D

    X X

    X X

    X X

    X X

    g g g g g g g g

    A B C D

    X X

    X X

    X X

    X X

    g g g g g g g g

    A B C D

    X X

    X X

    X X

    X X

    g g g g g g g g

    A B C D

    A B C D

    X X

    X X

    X X

    X X

    X X

    X X

    X X

    X X

    X X

    X X

    X X

    X X

    g g g g g g g gg g g g g g g g

    D E F G

    X X X

    X X X X

    X

    D E F G

    X X X

    X X X X

    X

    D E F G

    X X X

    X X X X

    X

    D E F G

    X X X

    X X X X

    X

    D E F G

    X X X

    X X X X

    X

    D E F G

    D E F G

    X X X

    X X X X

    X X X X

    X X X X

    X X X X

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