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Bioactive compounds in food plants and their impact on human health

Lars Porskjær ChristensenInstitute for Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, University of Southern Denmark

E-mail: lpc@kbm.sdu.dk

ThymeCarrots

Elderberries

Spinach

Bioactive natural products in plants

> 200.000 secondary metabolites have so far beenisolated from plants. Potential bioactive compounds.

Plants can contain > 1.000 secondary metabolites.

Bioactive secondary metabolites from plants:Fatty acid derivatives (e.g., glycolipids such as galactolipids, fatty acids, polyacetylenes). Origin: Acetate pathway.Terpenoids and steroids. Origin: Build up by isoprene units (C5).Polyphenols (e.g., flavonoids, isoflavones, anthocyanins, stilbenes, phenolic acids, lignans). Origin: shikimic acid and/or acetate pathway. Alkaloids. Origin: Derived from amino acids + other pathwaysGlucosinolates and cyanogenic glycosides. Origin: Derived from amino acids.

Plants produce secondary metabolites mainly for the protection against attack from microorganisms, herbivores etc.

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Characteristics of bioactive natural products – Mode of action

Binding to specific receptors (i) Size and shape resemblance those of endogen metabolites

being able to bind to specific receptors. (ii) Induction/inhibition of specific genes/enzymes/metabolites

resulting in a physiological response.

Antioxidant activity(i) Direct antioxidant activity. Redox active compounds that inactivate reactive oxygen

species (ROS). Particular relevant in the prevention of cardiovascular diseases.(ii) Indirect antioxidant activity. Compounds (not necessarily redox active), which induces

the production of cytoprotective proteins (phase 2 enzymes).

Inhibition of ion-channels and disruption of cell membranes

Covalent binding to biomoleculesApoptosis (programme cell death), inhibition of cell proliferation, inhibition of metabolicprocesses, immune system stimulation.

BioavailabilityAbsorption from the gastrointestinal tract into plasma. Bioactivecompounds has to be relative hydrophobic to be absorbedthrough the lipophilic cell membrane layers.

Bioactive compounds in food plants – Fatty acid derivatives

OH

Oα-Linolenic acid

OH

OLinoleic acid

O

CH2

CH

H2C

OH

OHHO

CH2OH

O

O

O

O

O

Monogalactosyldiacylglycerol (galactolipid)

Bioactivity: Essential fatty acids. Precursor for prostaglandins that are important regulators of metabolic processes. Source: Rapeseed, soya and flaxseed oil and many other plants.

Bioactivity: Anti-inflammatory and anticancer activity in vitro. In vivo activity unknown(bioavailable?). Source: Mainly green vegetables.

Spinach

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Bioactive compounds in food plants – Fatty acid derivatives

HO

HO

Falcarinol (= Panaxynol)

Falcarindiol

HO

Panaxydiol

OH

OH

Bioactivity: Anti-inflammatory, anticancer, anti-platelet-aggregatory, anti-bacterial, anti-fungal and more. Mode of action: Covalent-binding to biomolecules (strong alkylatingagents). Source: Food plants of the Apiaceae (Umbelliferae) family.

Carrots

Celeriac

Celery

Bioactive compounds in food plants – Terpenoids

Limonene (−)-Menthol

OH OH

Thymol

Monoterpenes (C10)

(−)-α-bisabolol

OH

OO

Lactucin

Sesquiterpenes (C15)

OHO

HO

OHHO

HOOC

OHHO

O

O

CO2H

OH

Carnosoic acid

Diterpenes (C20)

Carnosol Steviol

Bioactivity: Anti-inflammatory, anti-bacterial, anti-fungal, and anti-diabetic activity. Mode of action: Disruption of membranes, covalent binding to biomolecules, and binding to specificreceptors. Source: Mainly herbs and spices.

Sweet leaf

Thyme

Lettuce

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Bioactive compounds in food plants – Triterpenoids

OH

OAc

OH

O

HO

O

Cucurbitacin E

Cucurbitacins present in cucumber, melon, and marrow. Bitter tasting, purgative and extremely cytotoxic.

MarrowCucumber

O

OO

O

O

O

OO

O

O

O

OO

O

OAc

O

Limonin Nomilin

Limonoids present in citrus fruits, seeds, and juice. Bitter tasting and antifeedant activity. Anticancer effect and prevent cardiovascular diseases (reduces LDL cholesterol levels).

Bioactive compounds in food plants – Tetraterpenes

β-Carotene (orange)

Lutein (yellow)HO

OH

Lycopene (red)

Tetraterpenes (Carotenoids, C40)

Carotenoids are widespread in plants and have provitamin A activity, antioxidant capacity, and possible anticancer effect (e.g., lycopene)

Carrots

Tomatoes

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HO O

O

OH

OH

OHO-Glc−Rha

GlcO O

O

OH

OH

OH

GlcO O

O

OH

OCH3

OH

HO O

OH

OH

OH

OH

Quercetin 3-O-rutinoside (rutin, flavonol) Luteolin 3-O-glucoside (flavone)

Hesperetin 7-O-rutinoside (flavanone) (+)-Catechin (flavan-3-ol)

Bioactivity: Antioxidants. Anti-inflammatory, anticancer and immunostimulatory effect and prevention of cardiovascular diseases? Glycosides low bioavailability. Source: Widely distributed in plants.

O

C6-C3-C6 flavonoid skeleton

1

45

7 91'

4'

3

Green tea

Lemon

Bioactive compounds in food plants – Flavonoids

HO O

OOH

GlcO O

OOH

Daidzein

Daidzein 7-O-glucoside

OH

HO O

OOH

Genistein

GlcO O

OOH

Genistein 7-O-glucoside

OH

Soybean (Glycine max)

Bioactivity: Regulates oestrogenic response being able to bind to oestrogen receptors. Prevent the incidence of breastcancer and prostate cancer. Effect against type 2 diabetes? Source: Legumes (in particular soybeans). Oestradiol

OH

HO

O

OH

O

HO

Daidzein

Steroidal oestrogen

Phyto-oestrogen

Bioactive compounds in food plants – Isoflavones

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BlackcurrantElderberry

O

OGlcOH

HO

OHOH

+

Cyanidin 3-O-glucoside

O

OGlc-RhaOH

HO

OHOH

+

Delphinidin 3-O-rutinoside

OH

Bioactivity: Antioxidants. Anti-inflammatory and anticancer activity and prevention of cardiovascular diseases? Low bioavailability. Source: Widely distributed in plants and present in high concentrations in berries.

Bioactive compounds in food plants – Anthocyanins

trans-Resveratrol

OH

HO

OH

trans-Resveratrol 3-O-glucoside

OH

GlcO

OH

Bioactivity: Antioxidant, anti-inflammatory and anticancer activity. Prevent the development of cardiovascular diseases(inhibiting LDL-oxidation and platelet aggregation). Source: Grapes, wine, soya and peanut products and in the Itadori plant (roots used to produce tea in Asia).

Peanuts

Itadori plant (Japanese knotweed)

Red grapes

Bioactive compounds in food plants – Stilbenes

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Bioactive compounds in food plants – Gingerols and Curcuminoids

MeO

HO

O OH

MeO

HO

O OHOMe

OH

6-Gingerol

Curcumin

Bioactivity: Anti-inflammatory, antiulcer and anticancer activity. Source: Ginger family(Zingiberaceae)

Ginger (Zingiber officinale)

Turmeric(Curcuma longa)

Curry powder

OHOH

OHHOOCOH O

OH

OH

HOOC

O

OHOH

O

O

OHOHCOOH

OHHO

O

HO O

O

OH

OCH3

OCH3OH

Gallic acid(e.g., red grapes)

Rosmarinic acid(herbs and spices)

3-O-Caffeoylquinic acid(widespread)

1

3

8-O-4' Diferulic acid(cereals)

Bioactivity: Antioxidants. Prevents the development of cardio-vascular diseases. Anticancer and immunostimulatory effect? Bioavailable?

Red grapes

Oregano

Cereals

Bioactive compounds in food plants – Phenolic acids

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Bioactive compounds in food plants – Alkaloids

N

RO

Galactose−Glucose

Galactose−Glucose

α-Solanine, R =Rhamnose

α-Chaconine, R =Rhamnose

Bioactivity: Toxic to humans in high concentrations. Potential anticancer effectin low concentrations. Source: Many plants of the genus Solanum (Solanaceae), including potatoes and tomatoes.

Glycoalkaloids (steroidal alkaloids)

Potato

Tomato

N

S-GlucoseR

OSO3 K+

S C N R CH2

CH2CH2

S

O

Glucosinolates

Myrosinase

Isothiocyanates

Anticancer effect

- −Glucose−Sulphate

Bioactive compounds in food plants – Glucosinolates

Indol-3-carbinol (IC3)

NH

HO

NH

S-Glc

NOSO3 K

+

Glucobrassicin

_

Anticancer effect• Potent inducers of detoxification enzymes resulting in excretion of potential carcinogens

prior to harmful effects. Removal of xenobiotics.• Antiproliferative effects on cancer cells (apoptosis).• Phyto-oestrogenic effect. Prevention of breast/prostate cancer (IC3).

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State of the art within the field of bioactive compounds in food plants– Direct antioxidants (especially glycosides) are not major contributors to the

health effects of plant-based foods due to: (i) low bioavailability, (ii) metabolization and absorption in vivo unclear, (iii) general low bioactivitycompared to other bioactive compounds etc.

– Health effects of plant-based foods are most likely due to the presence of bioactive compounds with bioactivity unrelated to antioxidant activity.

Future work(i) Focus on identification of highly bioactive compounds with effects towards

specific diseases and their possible mode of action.

(ii) Bioavailability and metabolization of bioactive compounds.

HypothesesBioactive secondary metabolites with different bioactivities and mode of actions are major contributors to the health promoting effects of plant-based foods.

Bioactive compounds in plants: Anticancer effect and otherhealth promoting effects of carrots

Carrots are used for foods all over the world and have been used in centuries in traditional medicine in the West.

Carrot is closely related with food plants and medicinal plants of the Apiaceae and Araliaceae plant families that are known to possessimportant pharmacological effects such as:

Anti-inflammatory effect

Anticancer effect

Anti-platelet-aggregatory effects

Anti-diabetic effects

........ and many other pharmacological effects

Carrots

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Epidemiological studies have shown that the intake of vegetables rich in α- and β-carotene, and/or a high level of β-carotene in blood samples, is correlated with a reduced risk of cancer.

Human intervention studies: supplements of β-carotene increased the incidence of cancer!

‘β -Carotene paradox’

Do carrots contain other potential substances with the ability to prevent cancer?

Intake of α- and β-carotene is closely correlated with a high intake of carrots.

Anticancer effect of carrots

Potential bioactive compounds of carrots

Limonenep-Cymene γ-TerpineneTerpinolene

(E)-γ-bisabolene

H

H

β-Caryophyllene

β-Carotene

Monoterpenes

Sesquiterpenes

Tetraterpenes

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Potential bioactive compounds of carrots

OO

H3CO

O

O

O

O

O

OOH

H3CO

OCH3

OCH3

H3CO

CHO

2-Epilaserine

6-Methoxymellein

Gazarin

Cytotoxic

Anti-fungal

Anti-inflammatory

O

OGlcOH

HO

OHOH

+

Cyanidin 3-O-glucoside

Anti-inflammatory?Antioxidant?

+ other cyanidin derivatives

HO

HO

OH

Falcarinol (= Panaxynol)

Falcarindiol

Potential bioactive compounds of carrots

HO

OH

H3COCO

OH

Panaxydiol

Falcarindiol 3-acetate

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Bioassay-guided fractionation for identification of potential anticancer principles of carrots

HO

HO

Falcarinol (= Panaxynol)

FalcarindiolOH

Potential anticancer and anti-inflammatory principles in carrots

+

Fractionation and testing

Methanolextract

Processing/extraction Testing for effects in relevant in vitro bioassays: (i) Anticancer(ii) Anti-inflammatory(iii) Antibiotic(iv) Anti-diabeticEtc.

Effect of falcarinol and falcarindiol on the proliferation of human colon cancer cells (Caco-2)

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

K 0,625%FCS 0 ppb

1 ppb 10 ppb 100 ppb 1 ppm 2,5 ppm 5 ppm 10 ppm 20 ppm

Concentration (μg/ml)

350 x 104

250 x 104

150 x 104

50 x 104

00 1 2.5 5 10 200.10.010.001

Fluo

resc

ense

(via

bilit

y)

Falcarindiol■

● Falcarinol

Larsen, Purup & Christensen (2009). J. Agric. Food Chem. 57, 8290-8296

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Effect of falcarinol and falcarindiol on the proliferation of normal small intestinal cells

Fluo

resc

ense

(via

bilit

y)

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

K 0,625%FCS 0 ppb

1 ppb 10 ppb 100 ppb 1 ppm 2,5 ppm 5 ppm 10 ppm 20 ppm

Concentration (μg/ml)

0 1 2.5 5 10 200.10.010.001

800 x 104

600 x 104

400 x 104

200 x 104

0

Falcarindiol■

● Falcarinol

Larsen, Purup & Christensen (2009). J. Agric. Food Chem. 57, 8290-8296

Hansen et al. (2003). J. Sci. Food Agric. 83, 1010-1017; Young et al. (2007). J. Agric. Food Chem. 55, 618-623

Effe

cton

cell

prol

ifera

tion

ofpr

imar

ym

amm

ary

epith

elia

lcel

ls

0

0,5

1

1,5

0 0.001 0.005 0.01 0.005 0.1 0.5 1 5 10

Falcarinol (μg/ml)

No effect

†

**

***

† P < 0.09** P < 0.01*** P < 0.001

†1.5

0.5

Stimulating effect

Inhibiting effect

Effe

cton

cell

prol

ifera

tion

ofpr

imar

ym

amm

ary

epith

elia

lcel

ls

0

0,5

1

1,5

0 0.001 0.005 0.01 0.005 0.1 0.5 1 5 10

Falcarinol (μg/ml)

No effect

†

**

***

† P < 0.09** P < 0.01*** P < 0.001

†1.5

0.5

Stimulating effect

Inhibiting effect

Effects of falcarinol on cell proliferation of normal cellsand Caco-2 cells

Effe

cton

cell

prol

ifera

tion

14

β-Carotene (ng/ml)

0

0.25

0.5

0.75

1

1.25

1.5

0 1 10 100 1000 10000 100000

Effe

ct o

n ce

ll pr

olife

ratio

n of

prim

ary

mam

mar

y e

pith

elia

l cel

ls

No effect

β-Carotene (ng/ml)

0

0.25

0.5

0.75

1

1.25

1.5

0 1 10 100 1000 10000 100000

Effe

ct o

n ce

ll pr

olife

ratio

n of

prim

ary

mam

mar

y e

pith

elia

l cel

ls

No effect

Hansen et al. (2003). J. Sci. Food Agric. 83, 1010-1017

Effects of β-carotene on cell proliferation of normal and Caco-2 cells

Effe

cton

cell

prol

ifera

tion

Christensen & Brandt (2006). J. Pharm. Biomed. Anal. 41, 683-693

Bioavailability of polyacetylenes determined by multiple reaction monotioring (MRM) LC-MS/MS

Mass transition

plasma+0

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00Time23

100

%

23

100

%

23

100

%

fc190302-72 Sm (Mn, 2x3) MRM of 2 Channels ES+ 227 > 91

5.54e37.20

6.345.112.880.228.43 8.59

fc190302-73 Sm (Mn, 2x3) MRM of 2 Channels ES+ 227 > 91

5.54e3

7.20

2.852.33

0.62

3.44 4.05 5.794.856.10

7.638.11

fc190302-26 Sm (Mn, 2x3) MRM of 2 Channels ES+ 227 > 91

5.54e3

2.912.30

0.22

4.12 7.645.57 6.55 7.12 8.37

Plasma blank

Plasma added 3.3 ng/mL

Standard: 3.3 ng/mL

Plasma blank

Plasma + falcarinol

Falcarinol standard (3.3 ng/ml) Falcarinol

m/z 91m/z 227 [M + H – H2O]+

Proteins removed by precipitation withacetonitrile

Plasma samples

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Bioavailability studies of falcarinol in humans

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 1 2 3 4 5 6 7 8

900 ml juice 600 ml juice 300 ml juice

Falc

arin

ol(n

g/m

l)

Time (hours)

Concentration of falcarinol in plasma as function of time after ingestion of a breakfast meal consisting of carrot juice (13 mg/L). n = 14.

Brandt et al. (2004). Trends Food Sci. Technol. 15, 384-393

Potential anticancer effect of carrots and polyacetylenesdemonstrated in rats induced with colon cancer

ACF (»Aberrant Crypt Foci«)= biomarkers for advancedsteps on the progression towards cancer

ACF = 5

Male BDIX (rat model)

Tumours in rat colon

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Type and size of lesion

% o

f mea

nnu

mbe

rin

cont

rolt

reat

men

t

Carrot

Falcarinol

Large ACF(> 7)

Tumours(≥ 1 mm)

Small ACF(1−3)

Medium ACF(4−6)

0

20

40

60

80

100

120

----

Linear (carrot)

Linear (falcarinol)

Normalised effect of treatments

Increasing steps on the progression towards cancer. The trend for reducing the size of lesion was significant (P = 0.028)

Standard rat feed + 10% maize starch (control)

Standard rat feed + 10% freeze-dried

carrots

Kobæk-Larsen et al. (2005). J. Agric. Food Chem. 53, 1823-1827

Effect of treatments with carrot or falcarinol in physiological relevant concentrations of four types of (pre)cancerous lesions in rat colons

HOH+

HS

R

–H+

S

R

RSH

H2O

–H2OFalcarinol

+

Resonance stabilised carbocation

+

+

Falcarinol coupled to biomolecules (e.g., a protein)

Falcarinol-type polyacetylenes strong alkylating agents

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Effect of falcarinol and falcarinon on the proliferation of human colon cancer cells (Caco-2)

0

20

40

60

80

100

120

0 5 10 15 20

Concentration (μg/ml)

Viab

ility

(rel

ativ

e)

Larsen, Purup & Christensen (2009). J. Agric. Food Chem. 57, 8290-8296

O

Falcarinon

HO

Falcarinol

Immunomodulatory effect: Enhanced production of T-lymphocytes and macrophages through interaction with proteins, e.g., formation of hapten-protein complexes (antigens).

Inactivation of proteins/enzymes:(i) Inhibition of enzymes responsible for the proliferation of cancer cells

such as COX-2 and nuclear factor κB. Anti-inflammatory activity! (ii) Inhibition of COX-1 and COX-2 related to the anti-inflammatory effect of

falcarinol-type polyacetylenes, and hence also their possible anti-plateletaggregatory effects. Regulation of prostaglandin production.

Health promoting effects of falcarinol-type polyacetylenesprobably related to their lipophilic and alkylating properties

Apoptosis (programme cell death).(i) DNA damage.(ii) Induction of cell cycle arrest.(iii) Cell damage caused by increased production of macrophages.

........ and many other possible explanations.

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Thank you for yourattention!

Bioactive compounds in food plants and their impact on human health Bioactive natural products in plantsCharacteristics of bioactive natural products – Mode of actionBioactive compounds in food plants – Fatty acid derivativesBioactive compounds in food plants – Alkaloids