Bioactive Compounds in Food Plants and Their Impact on Human Health

Bioactive compounds in food plants and their impact on human health

Lars Porskjær Christensen

Institute for Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, University of Southern Denmark E-mail: [email protected]

Spinach Carrots Thyme

Elderberries

Bioactive natural products in plants

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

> 200.000 secondary metabolites have so far been isolated 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 pathways Glucosinolates and cyanogenic glycosides. Origin: Derived from amino acids.

1 Characteristics of bioactive natural products – Mode of action

9 Bioavailability Absorption from the gastrointestinal tract into plasma. Bioactive compounds has to be relative hydrophobic to be absorbed through the lipophilic cell membrane layers.

9 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.

9 Inhibition of ion-channels and disruption of cell membranes

9 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).

9 Covalent binding to biomolecules Apoptosis (programme cell death), inhibition of cell proliferation, inhibition of metabolic processes, immune system stimulation.

Bioactive compounds in food plants – Fatty acid derivatives

O α-Linolenic acid

O Linoleic acid

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

OH CH OH Spinach 2 O

O O HO CH OH 2 H C O

H2C O

Monogalactosyldiacylglycerol (galactolipid)

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

2 Bioactive compounds in food plants – Fatty acid derivatives

HO Carrots

Falcarinol (= Panaxynol) Celeriac

Falcarindiol Celery

Panaxydiol

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

Bioactive compounds in food plants – Terpenoids

Thyme

OH OH Monoterpenes (C10)

Limonene (−)-Menthol Thymol

OH O OH

Sesquiterpenes (C15) HO O O Lettuce (−)-α-bisabolol Lactucin

OH OH HO HO OH O HOOC

O Diterpenes (C20)

CO2H Carnosoic acid Carnosol Steviol Sweet leaf

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

3 Bioactive compounds in food plants – Triterpenoids

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

O OAc OH HO

O Cucumber Marrow Cucurbitacin E

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

O O O

O O OAc O

O O O O O O O O O

Limonin Nomilin

Bioactive compounds in food plants – Tetraterpenes

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

Carrots β-Carotene (orange)

HO Lutein (yellow)

Lycopene (red)

Tetraterpenes (Carotenoids, C ) 40 Tomatoes

4 Bioactive compounds in food plants – Flavonoids

OH OH 4' 1 OH OH 7 9 O 1' HO O GlcO O 3 5 4 O-Glc−Rha OH O OH O C6-C3-C6 flavonoid skeleton

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

OH OH

OCH3 OH

GlcO O HO O

OH OH O OH Green tea 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. Lemon

Bioactive compounds in food plants – Isoflavones

HO O HO O

O OH O OH OH Soybean (Glycine max) Daidzein Genistein

GlcO O GlcO O

O OH O OH OH Phyto-oestrogen OH

Daidzein 7-O-glucoside Genistein 7-O-glucoside Daidzein O

O OH

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

5 Bioactive compounds in food plants – Anthocyanins

OH OH OH OH + + HO O HO O OH

OGlc OGlc-Rha OH OH

Cyanidin 3-O-glucoside Delphinidin 3-O-rutinoside

Elderberry Blackcurrant

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 – Stilbenes

trans-Resveratrol Red grapes OH Itadori plant GlcO (Japanese knotweed)

trans-Resveratrol 3-O-glucoside

Bioactivity: Antioxidant, anti-inflammatory and anticancer Peanuts 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).

6 Bioactive compounds in food plants – Gingerols and Curcuminoids

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

OOH MeO

HO 6-Gingerol Ginger (Zingiber officinale)

Curry powder OOH MeO OMe

HO OH Curcumin Turmeric (Curcuma longa)

Bioactive compounds in food plants – Phenolic acids

OH OH OH 1 OH OH OH HOOC 3 OH O HOOC OH O Gallic acid (e.g., red grapes) 3-O-Caffeoylquinic acid Red grapes (widespread) O

OH HO O

OH O COOH OH OCH3 O Oregano HO OCH OH O 3 OH

Rosmarinic acid 8-O-4' Diferulic acid (herbs and spices) (cereals)

Bioactivity: Antioxidants. Prevents the development of cardiovascular diseases. Anticancer and immunostimulatory effect? Bioavailable? Cereals

7 Bioactive compounds in food plants – Alkaloids

Potato N

Glucose α-Solanine, R = Galactose− Rhamnose Tomato

Glucose α-Chaconine, R = Galactose− Rhamnose

Glycoalkaloids (steroidal alkaloids)

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

Bioactive compounds in food plants – Glucosinolates

HO S-Glc

N _ N + OSO3 K H N H Glucobrassicin Indol-3-carbinol (IC3)

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).

CH2CH2

R S-Glucose Myrosinase SCNRCH2 N + OSO- K −Glucose 3 Isothiocyanates −Sulphate O Glucosinolates S Anticancer effect

8 9 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 bioactivity compared 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.

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

9 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.

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

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

9 Carrot is closely related with food plants and medicinal plants of the Apiaceae and Araliaceae plant families that are known to possess important pharmacological effects such as: Anti-inflammatory effect Carrots Anticancer effect Anti-platelet-aggregatory effects Anti-diabetic effects ...... and many other pharmacological effects

9 Anticancer effect of carrots

9 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.

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

9 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?

Potential bioactive compounds of carrots

Monoterpenes

p-Cymene Terpinolene Limonene γ-Terpinene

Sesquiterpenes H

β-Caryophyllene (E)-γ-bisabolene

Tetraterpenes

β-Carotene

10 Potential bioactive compounds of carrots

Anti-fungal

Cytotoxic OH O O O O H CO H3CO 3 Anti-inflammatory

O 6-Methoxymellein CHO OCH3 O O H3CO

OCH3 O Anti-inflammatory? 2-Epilaserine Antioxidant? Gazarin OH OH + HO O + other cyanidin derivatives

OGlc OH

Cyanidin 3-O-glucoside

Potential bioactive compounds of carrots

Falcarinol (= Panaxynol)

OH Falcarindiol H3COCO

Falcarindiol 3-acetate

OH Panaxydiol

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

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

Fractionation and testing HO

Falcarinol (= Panaxynol)

OH + Falcarindiol Potential anticancer and anti- inflammatory principles in carrots

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

350 x 3500000104 ■ Falcarindiol 3000000 ● Falcarinol 250 x 2500000104

2000000

150 x1500000 104

1000000 Fluorescense (viability)

50 x 105000004

0 0 K 0,625% 1 ppb 10 ppb 100 ppb 1 ppm 2,5 ppm 5 ppm 10 ppm 20 ppm FCS0 0 ppb 0.001 0.01 0.1 1 2.5 5 10 20

Concentration (μg/ml)

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

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

800 x 8000000104 ■ Falcarindiol

7000000 ● Falcarinol 600 x6000000 104

5000000

400 x4000000 104

3000000

200 x 2000000104 Fluorescense (viability)

1000000

0 0 K 0,625% 1 ppb 10 ppb 100 ppb 1 ppm 2,5 ppm 5 ppm 10 ppm 20 ppm FCS 0 ppb 0.001 0.01 0.1 1 2.5 5 10 20

Concentration (μg/ml)

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

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

Stimulating effect

1,51.5 † †

Inhibiting effect

1 No effect

† P < 0.09 ** P < 0.01 0.50,5 *** P < 0.001 ** Effect on cell proliferation of Effect on cell proliferation *** primary mammary epithelial cells

0 0 0.001 0.005 0.01 0.005 0.1 0.5 1 5 10

Falcarinol (μg/ml)

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

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

1.5

1.25

1 No effect

0.75

0.5

mammary epithelial cells epithelial mammary 0.25 Effect on cell proliferation Effect on cell proliferation of primary of primary proliferation cell on Effect 0 0 1 10 100 1000 10000 100000

β-Carotene (ng/ml)

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

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

Mass transition + m/z 227 [M + H – H2O] m/z 91

Plasma samples plasma+0 fc190302-72 Sm (Mn, 2x3) MRM of 2 Channels ES+ 7.20 100 227 > 91 Falcarinol 5.54e3 Falcarinol standard (3.3 ng/ml) Standard: 3.3 ng/mL

8.43 8.59 5.11 6.34 0.22 2.88

23 fc190302-73 Sm (Mn, 2x3) MRM of 2 Channels ES+ 100 227 > 91 Plasma + falcarinol 5.54e3 Plasma added 3.3 ng/mL 7.20

Proteins removed by % 2.85 2.33 3.44 4.05 6.10 precipitation with 4.85 5.79 7.63 0.62 8.11

acetonitrile 23 fc190302-26 Sm (Mn, 2x3) MRM of 2 Channels ES+ 100 227 > 91 Plasma blank 5.54e3 Plasma blank

% 2.91 2.30 4.12 5.57 6.55 7.12 7.64 8.37 0.22

23 Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

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

14 Bioavailability studies of falcarinol in humans

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

3.0 900 ml juice 2.5 600 ml juice 300 ml juice 2.0

1.5

1.0 Falcarinol (ng/ml) 0.5

0.0 012345678

Time (hours)

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

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

Male BDIX (rat model)

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

Tumours in rat colon ACF = 5

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

Standard rat feed + Normalised effect of treatments 10% freeze-dried 120 Carrot carrots 100 Falcarinol Linear (carrot) 80 ---- Linear (falcarinol)

40 control treatment

% of mean of % number in 20

0 Small ACF Medium ACF Large ACF Tumours (1−3) (4−6) (> 7) (≥ 1 mm) Type and size of lesion

Standard rat feed + 10% Increasing steps on the progression towards cancer. The maize starch (control) trend for reducing the size of lesion was significant (P = 0.028)

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

Falcarinol-type polyacetylenes strong alkylating agents

+ HO H O H+ 2

Falcarinol –H2O

R HS+ RSH +

Resonance stabilised carbocation –H+

R S

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

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

120 O

100 Falcarinon

40 HO Via bility (re la tiv e ) Falcarinol 20

0 0 5 10 15 20 Concentration (μg/ml)

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

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

¾ Immunomodulatory effect: Enhanced production of T-lymphocytes and macrophages through interaction with proteins, e.g., formation of hapten- protein complexes (antigens).

¾ 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-platelet aggregatory effects. Regulation of prostaglandin production.

¾ 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.

17 Thank you for your attention!