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TRANSCRIPT
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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: [email protected]
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
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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