plant-derived substances and cardiovascular diseases
TRANSCRIPT
Linköping University Medical Dissertations No. 1097
Plant-Derived Substances and Cardiovascular Diseases
-Effects of Flavonoids, Terpenes and Sterols on Angiotensin-Converting Enzyme and Nitric Oxide
Ingrid A-L Persson
Division of Drug Research / Pharmacology
Department of Medical and Health Sciences
Linköping University, Sweden
Linköping 2009
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© Ingrid A-L Persson 2009
Photographs: Ginkgo biloba © Sven Persson, others © Ingrid A-L Persson
Published articles have been reprinted with the permission of the copyright
holder.
Printed in Sweden by Linköpings Tryckeri AB, 2009
ISBN 978-91-7393-706-1
ISSN 0345-0082
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”Blommor är vilsamma att betrakta. De har
varken känslor eller konflikter”
Sigmund Freud
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CONTENTS
ABSTRACT 7
LIST OF PAPERS 8
ABBREVIATIONS 9
INTRODUCTION 12
BACKGROUND 14
ANTIOXIDANTS 14
THE BIOSYNTHESIS OF SECONDARY METABOLITES IN PLANTS 15
Flavonoids 19
Purines 22
Terpenes 22
Sterols 25
HOMEOSTASIS OF THE VASCULAR WALL – REGARDING
ANGIOTENSIN II, NITRIC OXIDE AND REACTIVE OXYGEN SPECIES 26
THE ENDOTHELIUM 27
NITRIC OXIDE 29
THE RENIN-ANGIOTENSIN ALDOSTERONE SYSTEM 30
The Angiotensin-Converting Enzyme 35
AIMS 39
METHODS 41
INFUSIONS AND EXTRACTIONS 41
Tea Infusion, in vitro (Paper I) 41
Tea Infusion, in vivo (Paper V) 41
Coffee Infusion, in vitro 42
Cacao Extraction, in vitro 42
Bilberry 25E Extraction, in vitro (Paper IV) 42
Liquorice Extraction, in vitro 42
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Ginkgo biloba Extraction, in vitro (Paper III) 43
Panax ginseng Extraction, in vitro (Paper II) 43
CULTURED ENDOTHELIAL CELLS FROM HUMAN UMBILICAL VEINS (HUVEC) 43
ANGIOTENSIN-CONVERTING ENZYME ACTIVITY IN HUVEC 44
ANGIOTENSIN-CONVERTING ENZYME RADIOENZYMATIC ASSAY 46
NITRITE/NITRATE ASSAY 46
LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY-MASS MASS SPECTROMETRY (LC-MS-MS) 47
TENSION STUDIES 48
IN VIVO STUDY 49
ANGIOTENSIN-CONVERTING ENZYME GENOTYPE 50
CHEMICALS 52
CALCULATIONS 53
RESULTS AND DISCUSSION 54
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF BEVERAGES CONTAINING FLAVONOIDS AND XANTHINES 55
Green Tea, Black Tea and Rooibos Tea Infusions, in vitro (Paper I) 55
Coffee Infusion, in vitro 56
Cacao Extract, in vitro 56
Xanthines, in vitro (Paper I) 57
Green Tea, Black Tea and Rooibos Tea in Healthy Volunteers (Paper V) 57
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF DIETARY PRODUCTS CONTAINING ANTHOCYANINS OR ISOFLAVONOIDS 60
Bilberry Extract 25E, in vitro (Paper IV) 60
Liqourice Extract, in vitro 62
Genistein, in vitro 63
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EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF HERBAL MEDICINAL PLANT EXTRACTS 64
Panax Ginseng extract, in vitro (Paper II) 64
Ginkgo Biloba extract, in vitro (Paper III) 66
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF THE ANTIOXIDANTS ALPHA-TOCOPHEROL AND BETA-CAROTENE
Alpha-Tocopherol, in vitro 69
Beta-carotene, in vitro 70
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF STEROLS AND BLOOD LIPID-LOWERING DRUGS 72
Sterols, in vitro 72
Blood lipid –Lowering Drugs, in vitro 73
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF PRECURSOR MOLECULES 73
THE MECHANISM OF THE INHIBITORY EFFECT ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF THE FLAVONOIDS 74
EFFECTS ON NITRIC OXIDE 78
Nitric oxide, in vitro (Paper I, II, III) 78
Nitric oxide, in vivo (Paper V) 82
INTERACTIONS BETWEEN DIETARY PRODUCTS AND DRUGS 83
CLINICAL IMPORTANCE 84
SUMMARY 88
TABLE OF RESULTS 90
TACK 102
REFERENCES 105
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ABSTRACT
Diet has for many years been known to play a key role in the development of chronic diseases. There are clear associations between consumption of vegetables, fruits and berries, and risk of cardiovascular diseases, the number one cause of death in the world. To maintain homeostasis of the vascular wall the balance between angiotensin II, nitric oxide and reactive oxygen species is of great importance in order to affect the development of cardiovascular diseases. Angiotensin II, a potent vasoconstrictor causing cell growth and nitric oxide, a signalling molecule influencing the vascular system as a vasodilatator, inhibiting cell proliferation and reactive oxygen species, are linked together in the renin-angiotensin aldosteron system. Angiotensin-converting enzyme will as a key enzyme in the renin-angiotensin aldosteron system convert angiotensin I to form angiotensin II and nitric oxide is known to inhibit angiotensin-converting enzyme and act as a scavenger of reactive oxygen species. Plant-derived substances as flavonoids, tocopherols and carotenoids are shown to have beneficial effects on the cardiovascular system due to their antioxidative effects. The aims of this study were to investigate beverages, dietary products, herbal medicinal plants, α- tocopherol, β-carotene, sterols and lipid-lowering drugs on angiotensin-converting enzyme activity and nitric oxide concentrations. This was done to investigate if the sole mechanism of plant-derived substances is their antioxidative properties and to investigate if there is any connection between effect and biosynthesis/structure of plant substances. The tested infusions and extracts containing high amounts of flavonoids, the flavonoids and β-carotene significantly inhibited angiotensin-converting enzyme activity in vitro. The other substances tested did not affect, or in some cases significantly increased, angiotensin-converting enzyme activity. The infusions and extracts containing high amounts of flavonoids, the flavonoids and β-carotene showed an increase on nitric oxide concentrations in vitro. Oral intake of a single dose of Rooibos tea significantly inhibited angiotensin-converting enzyme activity. A significant inhibition of angiotensin-converting enzyme activity was seen with the green tea for the angiotensin-converting enzyme genotypes II and ID. A significant inhibition of angiotensin-converting enzyme activity was also seen with the Rooibos tea for the angiotensin-converting enzyme genotype II.
Conclusion; flavonoids and β-carotene interact with the cardiovascular system in several ways, by reducing reactive oxygen species (as shown in several studies), increasing nitric oxide concentrations (as shown here and by others) and also by inhibiting angiotensin-converting enzyme activity (as shown here). Infusions and extracts as tea containing high amounts of flavonoids function as angiotensin-converting enzyme inhibitors. Angiotensin-converting enzyme contains two zink-dependent catalytic domains and angiotensin-converting enzyme inhibitors are designed to bind to the Zn2+ at the active site. If the inhibitory mechanism of flavonoids on angiotensin-converting enzyme activity is due to their ability to bind to Zn2+ ions then it would be possible for the flavonoids to also inhibit other zinc metallopeptidases, i.e. endothelin-converting enzyme, matrix metallopeptidases, neutral endopeptidase and maybe insulin-degrading enzyme, thereby exerting several additional positive effects on the cardiovascular system.
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LIST OF PAPERS
This thesis is based on the following papers. Unpublished results are also
included in Results and Discussion. The roman numerals are used when
referring to the papers in the text.
I. Tea flavanols inhibit angiotensin-converting enzyme activity and increase nitric oxide production in human endothelial cells.
Persson, I.A-L., Josefsson, M., Persson, K. & Andersson, R.G.G. Journal of Pharmacy and Pharmacology 58: 1139-1144, 2006.
II. Effects of Panax ginseng extract (G115) on angiotensin-converting enzyme (ACE) activity and nitric oxide (NO) production.
Persson, I.A-L., Dong, L. & Persson, K.
Journal of Ethnopharmacology 105: 321-325, 2006.
III. Effects of Gingko biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity and nitric oxide production in human endothelial cells.
Persson, I.A-L., Lindén, E., Andersson, M. & Persson, K.
Asian Journal of Traditional Medicines 3: 42-51, 2008.
IV. The Effect of Vaccinium myrtillus and its Polyphenols on Angiotensin-Converting Enzyme Activity in Human Endothelial Cells.
Persson, I.A-L., Persson, K. & Andersson, R.G.G. Submitted.
V. Effects of Green Tea, Black Tea and Rooibos Tea on Human Angiotensin-Converting Enzyme and Nitric Oxide in Healthy Volunteers.
Persson, I.A-L., Persson, K., Hägg, S. & Andersson, R.G.G. Submitted.
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ABBREVIATIONS
ACC Acetyl-CoA carboxylase
ACE Angiotensin-converting enzyme
ADP Adenosine diphosphate
AMPA Aminopeptidase A
AMPN Aminopeptidase N
ANOVA One way analysis of variance
ANP Atrial natriuretic peptides
ANS Anthocyanidin synthase
AP Area postrema
APP Aminopeptidase P
ARPE-19 A retinal pigmented epithelium
AT1 Angiotensin receptor 1
AT2 Angiotensin receptor 2
AT3 Angiotensin receptor 3
AT4 Angiotensin receptor 4
ATP Adenosine triphosphate
BBB Blood-brain barrier
CAD Collision activated dissociation
cGMP Cyclic guanosine 3´, 5´monophosphate
CNS Central nervous system
CPM Carboxypeptidase M
CPN Carboxypeptidase N
CVO Circumventricular organs
D Deletion
DMAPP Dimethyl allyl diphosphate
DMSO Dimethylsulfoxide
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ECE Endothelin-converting enzyme
ECGF Endothelial cell growth factor
EDRF Endothelium-derived relaxing factor
eNOS Endothelial nitric oxide synthase
ESI Electrospray ionisation
F3H Flavanone 3β-hydroxylase
FCS Fetal calf serum
FPP Farnesyl pyrophosphate
GGPP Geranylgeranyl pyrophosphate
GPP Geranyl pyrophosphate
HPLC High performance liquid chromatography
HUVEC Culured endothelial cells from human umbilical veins
I Insertion
IDE Insulin-degrading enzyme
iNOS Inducible nitric oxide synthase
IPP Isopentenyl diphosphate
IRAP Insulin-regulated aminopeptidase
LC Liquid chromatography
LDL Low-density lipoprotein
L-NMMA N G-monomethyl-L-arginine
MAP Mitogen-activated protein
MMP Matrix metallopeptidase
MS Mass spectrometry
NADPH Nicotinamide-adenine dinucleotide phosphate
NCEP National Cholesterol Education Program
NEP Neutral endopeptidase
nNOS Neuronal nitric oxide synthase
NO Nitric oxide
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NOS Nitric oxide synthase
OVLT Organum vasculosum of the lamina terminalis
PAI-1 Plasminogen activator inhibitor-1
PBS Phosphate-buffered saline
PCR Polymerase chain reaction
PEP Prolyl endopeptidase
RAAS Renin-angiotensin aldosterone system
RHS Reactive halogen species
RNS Reactive nitrogen species
RSS Reactive sulphur species
ROS Reactive oxygen species
SCB Statistics Sweden
SFO Subfonical organs
WHO World Health Organization
WHOSIS World Health Organization Statistical Information System
1400-W N-3-(aminomethyl) benzylacetamidine
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INTRODUCTION
Diet has for many years been known to play a key role in the development of
chronic diseases. During the latter half of the 2000 century there has been a
great change in diet habits almost all over the world. Food consumption has
changed from a traditionally plant-based diet containing complex carbohydrates
and dietary fibers present in vegetables, fruits and berries to a diet rich in
saturated fats and simple carbohydrates i.e. a diet consisting mainly of meat and
products with high energy content (WHO, 2003). Changes in diet patterns over
time are due to a complex interaction between many factors such as individual
preferences and beliefs, cultural traditions, geographical, environmental, social
and economic factors. As a result of these interactions, associations between
consumption of fibres (Truswell, 2002), unsaturated fats (Russo, 2008),
vegetables, fruits and berries, and risk of cardiovascular diseases have been
shown (Hertog et al., 1993; Keli et al., 1996; Joshipura et al., 1999; Bazzano et
al., 2002; Rissanen et al., 2003; Bruckdorfer, 2008).
Approximately 60% of total reported deaths in the world and approximately
46% of the global burden of diseases (WHO, 2003) are contributed to chronical
diseases. Almost half of the deaths related to chronical diseases are attributed to
cardiovascular events; i.e cardiovascular diseases are the number one cause of
death. As well as in the world, cardiovascular diseases is the number one cause
of death in Sweden; in 1996, 49% of all deaths were due to cardiovascular
diseases according to the Statistics Sweden (SCB) database.
WHO recommends an intake of 400-500 gram of vegetables (apart from
potatoes), fruits, berries and green leaves per day to reduce the risk of coronary
heart disease, stroke and high blood pressure (WHO, 2008). Only a very small
and negligible minority of the world population consumes this recommended
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intake of vegetables, fruits and berries. However, in 2003 only 19 countries in
the WHO European Region had 600 grams of vegetables and fruits available per
capita and day, this to ensure the possible consumption of 400 g intake per day
and Sweden was not one of these 19 countries (WHO, 2008). In Sweden, the
recommended intake of vegetables and fruits is 500g per capita per day
(National Food Administration, Sweden).
Thus, lifestyle, i.e. diet and nutrition, is of great importance to human health
(Joshipura et al., 1999; Bazzano et al., 2002; Truswell, 2002; Rissanen et al.,
2003; Vanharanta et al., 2003; Jansen et al., 2004; Johnsen et al., 2004; Allen et
al., 2008; Chen et al., 2008; López et al., 2008; Patterson et al., 2008).
Vegetables, fruits and berries are well-known for their protection against and
prevention of non-communicable chronical diseases, such as cardiovascular
diseases, obesity, diabetes, cancer and osteoporosis (Steinmetz & Potter, 1991;
Hertog et al., 1995; Ness & Powles, 1997; WHO, 2003). These actions are
attributed to a diversity of effects such as antioxidative (Dragsted, 2003),
antithrombotic and anti-inflammatory properties, activation of endothelial nitric
oxide synthase (eNOS) and inhibition of low-density lipoprotein (LDL)
oxidation (Hwang et al., 2003; Ikizler et al., 2007; Kaliora & Dedoussis, 2007;
Aron et al., 2008; Boots et al., 2008;). Anticarcinogenic, antiatherogenic and
estrogenic effects have also been shown (Morton et al., 2000; Birt et al., 2001;
Nijveldt et al., 2001) as well as prevention of diabetes type 2 (Liese et al.,
2008). These proposed effects of vegetables, fruits and berries are
predominantly attributed to antioxidative mechanisms.
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BACKGROUND
ANTIOXIDANTS
Antioxidants are chemical substances that delay or prevent oxidation and
oxidazible substrates include almost every molecule found in vivo. All living
organisms, except some anaerobic and aerotolerant species, require oxygen in
order to produce energy/ATP by electron transport chains that donate electrons
to dioxygen (O2) and reduce it to water as in the mitochondria of eukaryotic
cells. Under certain circumstances, e.g. certain diets, smoking, ultraviolet-light,
cold and heating, reactive oxygen species (ROS) are generated as side products
of this energy process. Apart from ROS, also called oxygen free radicals (a free
radical means any species capable of independent existence and containing one
or more unpaired electrons), ROS also include reactive nitrogen species (RNS),
reactive halogen species (RHS) and reactive sulphur species (RSS) (Halliwell &
Gutteridge, 2007). Thus, all ROS are not oxygen species, but all reactive species
are usually called ROS (Halliwell & Gutteridge, 2007).
Most of the damaging effects of oxygen are due to oxygen radicals (Muller et
al., 2007). As the oxygen content of the atmosphere increased many primitive
species i.e. anaerobs died out while other organisms began to evolve antioxidant
defence systems for protection against oxygen toxicity in order to survive.
Aerobic organisms survive in the presence of free radicals solely because they
have evolved antioxidant defences. There are two major mechanisms
contributed to antioxidative effects, free radical scavenging and metal chelating.
In healthy aerobs, the production of ROS is approximately in balance with the
antioxidant defence systems. If this balance fails, and the production of ROS
will outweigh the antioxidants defence, then oxidative damage will occur
(Halliwell & Whiteman, 2004). Thus, oxidative damage is the biomolecular
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damage caused by ROS upon living organisms, as a result of diminished
antioxidants or increased production of ROS. This oxidative damage may induce
or become a result of endothelial cell damage, inflammation, atherosclerosis,
proliferation of vascular smooth muscle cells and cardiac remodelling associated
with hypertension and cardiovascular diseases (Grossman, 2008).
Antioxidants can either be syntesised in vivo e.g. glutathione, thiols, bilirubin,
transferin, lactoferin, erythrocytes, albumins or be administered via diet, e.g.
plant-derived substances like flavonoids (see below, section “Flavonoids”),
Vitamin E (see below, section “Terpenes”), carotenoids (see below, section
“Terpenes”) and Vitamin C. Plant-derived antioxidants as flavonoids,
tocopherols and carotenoids (Voutilainen et al., 2006; Kaliora et al., 2007;
Stocker, 2007) are shown to have beneficial effects on the cardiovascular system
(Devaraj et al., 2007; Svarcova et al., 2007; Aron et al., 2008; Choudhary et al.,
2008; Milman et al., 2008), and cancer prevention (Nandakumar et al., 2008).
Flavonoids are also shown to have effects on cognition and behaviour (DeKosky
et al., 2006; Williams et al., 2008). These beneficial properties of plant-derived
substances are proposed to be due to their antioxidative effects. From an
evolutionary point of view, oxygen and thereby antioxidative properties
appeared in significant amounts in the Earth atmosphere almost simultaneously
as the photosynthesis and biosynthesis of the secondary metabolites evolved in
cyanobacteria (Liang et al., 2006).
THE BIOSYNTHESIS OF SECONDARY METABOLITES IN PLANTS
The photosynthesis (Ingenhousz, 1779) can be divided into two major processes;
the energy-transduction reaction and the carbon-fixation reaction. In the energy-
transduction reaction, light energy is used to form adenosine triphosphate (ATP)
from adenosine diphosphate (ADP) and to reduce the oxidized electron-carrier
molecules nicotinamide-adenine dinucleotide phosphate (NADP+) to NADPH.
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The received ATP and NADPH are used by the cells to provide energy for the
further biosynthetic pathways. The energy-transduction reaction is followed by
the second process, the carbon-fixation reaction. In this process the energy of
ATP is used to link carbondioxide, CO2 to an organic molecule and NADPH is
then used to reduce the newly fixed carbon atoms into a simple sugar (the Calvin
cycle after Melvin Calvin, NobelPrize winner in 1961). In the photosynthesis,
carbohydrates (sugars), carboxylic acids, α-amino acids, fats, proteins and
nucleic acids are produced; products involved in and essential for life processes
and suitable for storage as starch and the carbon skeleton from which all other
organic molecules can be built in further processes. Products derived from the
photosynthesis, so called primary metabolites, are also the precursors and
starting materials for the biosynthesis of the so called secondary metabolites. In
contrast to the primary metabolites, the secondary metabolites are not present in
all plants but are found in certain plants, species or families. Furthermore,
primary metabolites are essential to life, while the secondary metabolites are
not, but the secondary metabolites do contribute to the survival of the species
involved in e.g. the defense system and reproduction. Flavonoids, terpenes and
phytosterols are example of plant-derived secondary metabolites synthesised
from monosaccharides (glyceraldehyde-3-phosphate). All secondary metabolites
are linked to primary metabolites and most metabolites originate from a very
limited number of precursor molecules. There are three main biosynthesis
pathways of secondary metabolites: the shikimic, the polyketide and the
mevalonic pathways. As shown in figure 1, plant-sterols are derived from the
mevalonic pathway. The terpenes are an example of secondary metabolites
derived from two pathways, dependent on the type of terpene, from
glyceraldehyd-3-phosphate and pyruvic acid or from the mevalonic pathway
(figure 1). Several groups of metabolites have mixed origin, e.g the flavonoids
where one part of the flavonoid structure derives from the shikimic pathway and
the other part of the structure derives from the mevalonic pathway (figure 1).
Fig
ure
1. T
he
bios
ynth
esis
of f
lavo
noi
ds, t
erpe
nes
, st
erol
s an
d pu
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text
.
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ds, t
erpe
nes
, st
erol
s an
d pu
rine
s/xa
nthin
es. S
econ
dary
met
abo
lites
test
ed in
this
stu
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are
prin
ted
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Sec
onda
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etab
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es te
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in th
is s
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re p
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Figu
re 2. Basic chem
ical structures of flavonoids, stilbenes, aurones and ap
iforol.
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hemical structures of flavonoids, stilbenes, aurone
s and apiforol.
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Flavonoids
All dietary products originating from plants potentially contain flavonoids (Latin
flavus, yellow) and over 4000 compounds have been identified and the list is
still growing (Harborne & Williams, 2000). Flavonoids are water-soluble
pigments present in the cytosol and/or stored in the vacuole of the plant cell and
the flavonoids represent the largest group of phenolic compounds in plants.
Flavonoids are of mixed origin, biosynthesised by the shikimic acid pathway
and the mevalonic acid pathway (figure 1). The biosynthesis pathway of the
flavonoids is part of a larger phenylpropanoid pathway producing a range of
secondary metabolites e.g. phenolic acids, flavonoids, stilbenes, aurones and
apiforols (figure 1). The basic chemical structure of flavonoids is based on two
six-carbon rings linked by a three-carbon unit, the chalcone structure (figure 2).
The main classes/groups of flavonoids according to differences in the C-ring,
OH-substituents and double bondings are chalcones (the basic structure of the
flavonoids and unstable isomers of flavanones), flavanones (e.g.naringenin),
flavones (e.g. luteolin), flavonols (e.g. quercetin), flavanols (catechines),
isoflavones (e.g. genistein) and anthocyanidins (e.g. cyanidin, delphinidin and
malvidin) (figure 2). The difference between the various flavonoids in the
different groups is the number and position of substitution by hydroxylation,
hydrogenation, methylation, glycosylation, malonylation and sulphation
(Andersen & Markham, 2006). The most common forms of flavonoids found in
plants are the glycoside derivatives, except for the catechins which occur
without sugar molecules (Andersen & Markham, 2006). Flavonoid molecules
without sugar molecules are referred to as aglycones.
Flavonoids serve as communicators between the plant and the environment, and
are important for e.g. reproduction, resorption of mineral nutrients and as
antioxidants (Harborne & Williams, 2000); which make the functions of the
flavonoids critical for the survival of the plant. Concerning the antioxidative
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effects of the flavonoids, yellow flavonoids and flavonoids with colours
invisible to the human eye (flavones, flavonols and isoflavonoids) seem to be
primarily involved in the protection against ultraviolet radiation (Stapleton,
1992) and plants often respond to ultraviolet light by activating the flavonoid
biosynthesis (Schmelzer, 1988). In humans, flavonoids are mostly associated
with the ability to donate hydrogen-ions and scavenge of reactive oxygen and
reactive nitrogen species, i.e. antioxidative effects (Sun et al., 2002; Chang et
al., 2007; Jiménez et al., 2007; Tomer et al., 2007).
Some of the flavonoids are more or less specific to particular dietary products
e.g. genistein found in soya-beans (Glycine max (L.) Merr.). Other flavonoids
are found in almost all plant-based dietary products e.g. the flavonol quercetin.
But, dietary products often contain mixtures of different flavonoids. The most
numerous reports on flavonoids and their antioxidative capacity concern the
flavonol quercetin (Rahman, 2006; Ratman et al., 2006) as well as studies on
ingestion of dietary products, mostly everyday beverages, containing large
amount of catechins, found in green tea and black tea, unfermented and
fermented leaves of Camellia sinensis L. (Theaceae) (Persson et al., 2006 (Paper
I); Basu & Lucas, 2007; Lambert et al., 2007). Rooibos or red tea, fermented
leaves and/or bark of Asparalathus linearis Dahlg. (Leguminosae) does not
contain catechins but dihydrochalcones, flavones and flavonols (Bramati et al.,
2002; Bramati et al., 2003). Antioxidative effects have also been reported
concerning procyanidins (polymer chain of catechins), found in beans of the
cacao tree, Theobroma cacao L. (Sterculiaceae) (Keen et al., 2005; Aron et al.,
2008).
In plants, anthocyanins (Greek, antos, flower and kyanos, blue), represent
pink/red/blue/violet to dark blue colours, and it is the anthocyanins that seems to
have the greatest antioxidative effect of all flavonoids and there is a strong
association between anthocyanin biosynthesis and plant stress (Andersen &
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Markham, 2006). The anthocyanins consist of an anthocyanidin (an aglycon),
sugar(s), mostly glucose and galactose and in many cases, acyl group(s).
Anthocyanins are the only flavonoids able to form flavylium cations (figure 2)
and due to this ability the anthocyanins are very reactive towards ROS because
of their electron deficiency. The biosynthesis of the anthocyanins is shown in
figure 1. Anthocyanins may function as general antioxidants and are associated
with a broad range of stressors (Leshem et al., 1996). Anthocyanins are to be
found in dark-coloured foods like bilberries Vaccinium myrtillus L. (Ericaceae).
The anthocyanins are considered responsible for the main pharmacological
effects of Vaccinium myrtillus due to the antioxidative and free radical
scavenging properties (Kähkönen & Heinonen, 2003).
Isoflavonoids e.g. genistein are plant-derived non-steroidal secondary
metabolites also called phytooestrogens due to their structural relationship with
oestrogen and other sex hormones. Isoflavonoids exert both oestrogenic and
antioestrogenic activity by competing for receptor binding with oestrogen
(McCarty, 2006). Apart from acting on oestrogen receptors, genistein is shown
to induce apoptosis of cancer cells, exert antioxidative effect, inhibit cell
proliferation, modulate cell cycling, inhibit angiogenesis and suppress
lymphocyte functions (Polkowski & Mazurek, 2000). The isoflavonoids
originate from flavanons, and consequently the isoflavone genistein is
biosynthesised from the flavanone naringenin (figure 1 and 2). The structural
difference between isoflavonoids and other flavonoids is the linking of the B-
ring to the C-3 rather than to the C-2 position of the C-ring (figure 2).
Glycyrrhiza glabra L. (Fabaceae) contain a numerous of flavonoids, more than
300 species-specific phenolic compounds have been isolated from liquorice.
Glycyrrhiza species contain flavonoids, isoflavonoids, chalcones and bibenzyls
and about 70 of these are present in the root of the plant (Li et al., 2000). The
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major flavonoid in Glycyrrhizin is the isoflavone glabridin (Belinky et al.,
1998).
Purines
Alkaloids (alkali-like) are a group of plant-derived secondary metabolites.
Alkaloids are basic compounds, containing one or more nitrogen atoms and they
are known to have marked physiological effects on humans e.g. caffeine,
morphine and nicotine. Alkaloids are based on amino acids and classified
according to chemical structure and one of the classes is purines. Purines are
heterocyclic aromatic organic compounds consisting of a pyrimidine ring fused
to an imidazole ring. In nature, the purines are widely distributed and their role
is not clear. The name purine (purum uricum) was given by Emil Fischer in
1884 who was the first to synthesise purines in 1899 (Nobel Prize winner in
1902 for his work on sugar and purine syntheses).
The purines are biosynthesised from ribose via pyruvic acid, amino acid and the
purine base xanthine. Purines are derivatives of xanthine, thereby also named
xanthines i.e. caffeine, theobromine and theophylline (figure 1) and present in
beverages as green tea, black tea, coffee and cacao. Pharmacological effects of
purines/ xanthines include bronchodilation, increased cardiac output, alertness
and dependence.
Terpenes
Terpenes (from turpentine, the fluid obtained by distillation of resin
(hydrocarbon secretion), originally from the Greek word terebinthine, Pistacia
terebinthus L., turpentine tree) are secondary metabolites built up from isoprene
units, C5H8 (figure 3), i.e. oligomers of isoprene units (C5)n, according to the
isoprene rule formed by Ruzicka and Wallach (Ruzicka, 1953).
23
Figure 3. Isoprene unit C5H8
Today, about 30000 terpenes have been identified, and most of these compounds
originate from plants. In nature, terpenes mainly exist as hydrocarbons, alcohols
and their glycosides, ethers, aldehydes, ketones, carboxylic acids and esters. The
differentiation of the chemical structure between the terpenes, hemi-(C5), mono-
(C5)2, sesqui-(C5)3, di-(C5)4, sester-(C5)5, tri-(C5)6, tetra-(C5)8 and polyterpenes
(C5)n , is dependent on the number of isoprene units. Isopentenyl diphosphate
(IPP) is the common precursor for all terpenes. Two biosynthetic pathways lead
to the formation of IPP, the formation of IPP in the plastid and the formation of
IPP in the cytoplasm. IPP and its isomer dimethyl allyl diphosphate (DMAPP)
are formed from glyceraldehyd-3-phosphate and pyruvic acid in the chloroplast
for further formation of mono-, di- and tetraterpenes (Lichtenthaler, 2007)
(figure 1). The terpenes derived from IPP formed in the cytoplasm via the
mevalonic pathway are sesqui- and triterpenes (Lichtenthaler, 2007) (figure 1).
Mevalonic acid consists of isoprene units, and the mevalonic acid pathway in
plants and mammals is similar from acetyl CoA to cholesterol except for the
synthesis of terpenes in plants. The biosynthesis of the terpenes is shown in
figure 1.
By combining isoprene units, the pathway produces geranyl pyrophosphate
(GPP) (C5)2, farnesyl pyrophosphate (FPP) (C5)3 and geranylgeranyl
pyrophosphate (GGPP) (C5)4, the precursor of the diterpenes (C5)4.
Tocopherols (Greek tocos, childbirth, phero, to bring forth), a group of
substances consisting of α-, β-, γ- and δ-tocopherols, and α-, β-, γ- and δ-
tocotrienols are cyclic diterpenes, prenylchromanols with a hydroxyl group that
can donate a hydrogen atom to reduce free radicals and a hydrophobic side
24
chain. In plants, tocopherols and tocotrienols serve as antioxidants by protecting
carotenoids (see below) and polyunsaturated fatty acids in the biomembranes
(Matringe et al., 2008). The most active of the tocopherols, α-tocopherol, (2-
prenyl-3,4-dihydro-2H-1-benzopyran-6-ol) is also known as Vitamin E. In
humans, α-tocopherol, as an antioxidant has been suggested to reduce
cardiovascular diseases (Meydani, 2004).
Ginkgo biloba L. (Ginkgoaceae) is considered to have cardioprotective effects
and these effects of Ginkgo biloba are often related to its unique terpene
compounds, the hexacyclic diterpenelactones ginkgolides A, B, C, J and the
tetracyclic sesquiterpene-lactone derivative bilobalide, and flavonol glycosides.
The biosynthesis pathway of the diterpenes and the sequiterpenes is shown in
figure 1.
The precursor of the triterpenes (C5)6 is squalene C30H50. The chemical structure
of the triterpenes is similar to the structure of the steroids (C27, C24, C21, C19,
C18). Triterpenes are also referred to as steroidal phytooestrogens. Effects of
Panax ginseng L. (Araliaceae) on the cardiovascular system are often attributed
to the tetracyclic triterpene saponins, the ginsenosides (Chen, 1996; He at al.,
2007). The biosynthesis pathway of the triterpenes is shown in figure 1.
Tetraterpenes, i.e carotenoids (the first to be isolated was from carrot, Daucus
carota L. in 1831), are based on a (C5)8 isoprene structure. Approximately 600
carotenoids have been identified, but only about 40 are present in ordinary
human diet. The carotenoids are present in all higher plants, in leaves, shoots
and roots. The chloroplast is the site of the photosynthesis containing
chlorophyll (a phytol, an acyclic diterpene alcohol containing a (C5)4 isoprene
side-chain and precursor of Vitamin E) and carotenoid pigments. The
carotenoids are prenol (3-methyl-2-buten-1-ol, an isoprene alcohol) lipids
representing yellow-orange and red colours in relation to the conjugated double
bonds in the carotenoid skeleton. Carotenoids may be acyclic (e.g.lycopene) or
25
having one or both ends modified into rings (e.g. β-carotene). Carotenoids
containing at least one oxygen atom are classified as xanthophylls (e.g.lutein)
and hydrocarbon carotenoids are classified as carotenes. In plants, the
carotenoids contributes to the photosynthetic processes, they have principal
functions as antioxidants by prevention of photo-oxidative damages to the
chlorophyll molecules and serve as communicators between the plant and the
environment. In humans, the effect of β-carotene is related to its antioxidative
activity, preventing cardiovascular diseases (Bjelakovic et al., 2008), cancer
(Bjelakovic et al., 2008)) and protection against light-induced skin damage
(Stahl & Sies, 2002). Beta-carotene is the carotenoid with the highest pro-
vitamin A activity and the most common of the carotenes. The biosynthesis of
the tetraterpenes is shown in figure 1.
Sterols
The terpenes, except the monoterpenes (see above, section “Terpenes”) and the
sterol lipids share a common biosynthetic pathway via IPP and FPP (figure 1),
but they have differences in structure and function. The sterol lipids can be
subdivided according to their biological functions; cholesterol and derivatives
e.g. plantsterols, steroids such as sex hormones and mineralcorticoids,
secosteroids i.e. Vitamin D2 and D3. Plant-derived sterols are also named
phytosterols, e.g. stigmasterol, sitosterol and campesterol. Stigmasterol is an
unsaturated phytosterol, the most common sterol present in plants. Animal-
derived sterols are named zoosterols e.g. cholesterol, the most common sterol of
animal origin also present in plants but not as common as in animals. The
precursor of cholesterol and animal steroids in general is lanosterol, while plant
sterols are formed via squalene, protosterol carbonium I and cycloartenol. The
sterol lipids and the prenol lipids share a common biosynthetic pathway via IPP,
GPP and FPP and are building blocks of cholesterol and thus of all steroids. The
26
biosynthesis of phytosterols and cholesterol is shown in figure 1. In 1976,
stigmasterol was used as starting material for approximately 28% of the world
production of steroids. Studies show that phytosterols have effect on the
cardiovascular system by lowering cholesterol levels (Klingberg et al., 2008;
Poli et al., 2008).
HOMEOSTASIS OF THE VASCULAR WALL
– REGARDING ANGIOTENSIN II, NITRIC OXIDE AND REACTIVE
OXYGEN SPECIES
To maintain homeostasis of the vascular wall, a balance between the
endogenous transmitters angiotensin II (see below, section “The renin-
angiotensin aldosterone system”), nitric oxide; (see below, section “Nitric
Oxide”) and ROS; (see above, section “Antioxidants”) is of great value.
Angiotensin II, nitric oxide and ROS are important participators in the
pathogenetic mechanisms of cardiovascular diseases. It has been shown that
hypertension caused by chronically elevated angiotensin II is mediated in part
by superoxide ions (O2-) and hypertension is a major risk factor for coronary
artery disease, congestive heart failure, cerebrovascular disease, peripheral
vascular disease and renal failure. This suggests that cardiovascular diseases
caused by chronically elevated angiotensin II levels are found to be mediated by
vasoconstriction and furthermore, partially mediated by ROS (Zhang et al.,
2007). Decreased vascular nitric oxide seems to promote angiotensin II
dependent cardiovascular diseases mediated by ROS (deGasparo, 2002).
Angiotensin II acting through angiotensin-1 receptors, AT1, mediates
vasoconstriction and stimulates membrane bound nicotineamide adenine
dinucleotide phosphate (NADPH) oxidase causing accumulation of ROS.
Angiotensin II acting on angiotensin-2 receptors, AT2, results in increased levels
27
of nitric oxide (and bradykinin and prostacyclin). Nitric oxide scavenges ROS
thereby consuming nitric oxide and blocking the beneficial properties of nitric
oxide (Doughan et al., 2008). Accumulation of ROS stimulates mitogen-
activated protein (MAP) kinases which promote cell growth and cell
proliferation (Zhang et al., 2007). The angiotensin receptors AT1 and AT2 are
with their physiologically antagonistic effects maintaining the balance between
nitric oxide and ROS. It is proposed that stimulation of AT1 receptors by
increased circulating or tissue levels of angiotensin II will stimulate cell growth,
cell proliferation, affect homeostasis of the vascular wall and give rise to
inflammation and cardiovascular diseases (deGasparo, 2002). Angiotensin-
converting enzyme (see below, section “The angiotensin-converting enzyme”),
is a key enzyme involved in the formation of the physiological antagonists
angiotensin II and nitric oxide.
THE ENDOTHELIUM
The entire vascular system is covered by endothelial cells, the endothelium, a
single layer of cells between the blood and the vascular smooth muscle cells.
The endothelium responds to neurotransmitters, e.g. acetylcholine, hormones
(e.g. angiotensin II), local mediators (e.g. bradykinin), platelet-derived
substances (e.g. thrombin), and mechanical force (shear stress) (Vanhoutte &
Mombouli, 1996; Lüscher & Barton, 1997). In concequence, endothelium-
derived substances are of a large variety and of importance in the regulation of
vascular tone, smooth muscle cell proliferation, vessel wall inflammation and
platelet function. The endothelium represents a complex interrelationsship
between physiological agonists and antagonists invaluable for homeostasis of
the vascular wall e.g. vasoconstriction versus vasodilatation, fibrinolysis versus
antifibrinolysis, thrombosis versus antithrombosis, growth promotion versus
growth inhibition and oxidation versus antioxidation (Lüscher & Barton, 1997;
de Gasparo, 2002) (figure 4).
Figure 4. Endothelial cell with endothelium
The communication between endothelial cells and smooth muscle cells is for
instance mediated by angiotensin
28
growth inhibition and oxidation versus antioxidation (Lüscher & Barton, 1997;
de Gasparo, 2002) (figure 4).
Figure 4. Endothelial cell with endothelium-derived substances.
The communication between endothelial cells and smooth muscle cells is for
instance mediated by angiotensin-converting enzyme and nitric oxide (Figure 4).
growth inhibition and oxidation versus antioxidation (Lüscher & Barton, 1997;
The communication between endothelial cells and smooth muscle cells is for
converting enzyme and nitric oxide (Figure 4).
29
NITRIC OXIDE
Nitric oxide (NO) was discovered in 1772 by Joseph Priestly as a toxic gas that
is produced by the combustion of air. The first discovered endothelium-derived
relaxing factor (EDRF) (Furchgott & Zawadski, 1980) produced and released by
the endothelium (see above, section “The endothelium”) was later shown to be
identical with NO (Ignarro et al., 1987; Palmer et al., 1987). NO function as a
signalling molecule (a hormone and a neurotransmitter) present throughout the
body. NO is highly reactive and diffuses freely across the membranes, with a
half life of a few seconds.
In humans, NO is biosynthesised from L-arginine (Arg), an α-amino acid
enzymatically transformed by nitric oxide synthase (NOS) forming NO and
citrulline (another α-amino acid, named after Citrullus lanatae, water melon,
from which citrulline was first isolated, and it is proposed that in plants citrulline
may serve as a nitrogen reserve) (Palmer et al., 1988; Sakuma et al., 1988).
Three isoforms of NOS exist: neuronal NOS (nNOS) found in a variety of cells
including endothelial cells, macrophages and neurons (Bredt & Snyder, 1990;
Tsutsui, 2004), inducible NOS (iNOS) found in macrophages and smooth
muscle cells (Hevel et al., 1991) and endothelial NOS (eNOS) found in
endothelial cells (Pollock et al., 1991).
The first known mechanism to produce NO in plants is by the enzyme nitrate
reductase reducing nitrite to NO, but further investigation has shown an
arginine-dependent NO synthesis in plants. This plant-derived NOS behaves like
the human eNOS but is also involved in defence responses as the human iNOS
(Crawford, 2006).
In humans, NO influences the vascular system as a vasodilator by relaxation of
smooth muscle cells (Furchgott & Zawadski, 1980; Rapoport & Murad, 1983)),
inhibition of smooth muscle cell proliferation (Garg & Hassid, 1989), reducing
30
platelet aggregation (Radomski et al., 1987), and platelet and monocyte
adhesion to the endothelium (Roberts et al., 2008). NO activates guanylyl
cyclase producing cyclic guanosine 3´, 5´ monophosphate (cGMP) (Rapoport &
Murad, 1983) which in turn activates proteinkinase G resulting in vasodilatation
and inhibited platelet aggregation. Furthermore, NO inhibits LDL oxidation
(Thomas et al., 2008) and expression of adhesion molecules and endothelin. NO
is also known to inhibit angiotensin-converting enzyme (ACE) (Ackermann et
al., 1998; Persson et al., 2005) (figure 5). NO also acts as a scavenger of ROS
(Doughan et al., 2008). NO together with the noradrenergic nervous system and
endothelin is tonically active in resistance vessels under basal conditions. A
decrease in NO e.g. by scavenging of ROS, thereby increases the risk of
developing atherosclerosis (Puddu et al., 2005) and renin-angiotensin
aldosterone (see below, section “The renin-angiotensin aldosterone system)
dependent diseases as coronary artery disease, diabetes, hypercholesterolemia
hypertension, migraine, peripheral vascular disease, vascular restenosis, stroke
and thrombosis.
THE RENIN-ANGIOTENSIN ALDOSTERONE SYSTEM
The renin-angiotensin aldosterone system (RAAS) (figure 5) is together with the
autonomic nervous system (Lohmeier, 2001), the most important mechanisms in
the body concerning regulation of blood pressure, fluid and electrolyte balance
(Reid, 1985; Ferrario, 1990). RAAS is a kidney-derived mechanism (Tigerstedt
& Bergman, 1898). Prorenin (Hobart et al., 1984), the inactive form of renin, is
released constituitively from the kidney and is found circulating in human
plasma in excess to renin. Prorenin can be activated proteolytically (in the
kidney) and nonproteolytically (in the plasma) (Danser & Deinum, 2005).
Decreased renal blood pressure, low salt content, sympathetic nerve stimulation,
β-adrenoreceptor agonists, circulating catecholamines and prostacyclin stimulate
31
the juxtaglomerular cells in the kidney to release the proteolytic enzyme renin
(Reid, 1985). Renin release is thus controlled by the macula densa cells,
vascular endothelial cells and smooth muscle cells (Davis, 1973). Renin is a
monospecific enzyme persisting in the circulation for 10 minutes to 1 hour.
During this time, renin continuously cleaves the α-2-globulin angiotensinogen
(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser) at the N-terminal
end of the protein, to form the decaamino acid peptide angiotensin I (Asp-Arg-
Val-Tyr-Ile-His-Pro-Phe-His-Leu) (Oparil & Haber, 1974; Ferrario, 1990).
Angiotensinogen is produced constantly and released to the circulation mainly
by the liver and to some extent also by the kidneys (Kobori et al., 2002).
Angiotensin I is a very weak vasoconstrictor with virtually no activity of its
own. Angiotensin I is rapidly converted by angiotensin-converting enzyme
(ACE) (see below, section “The angiotensin-converting enzyme”) by the
removal of two amino acids to form the octaamino acid peptide angiotensin II
(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (Skeggs et al., 1956a). Angiotensin II
causes feedback inhibition of renin release. Other enzymes generating
angiotensin II independently of ACE are tonin, catepsins, carboxypeptidase,
chymotrypsin and chymase (Peach, 1977; Roks et al., 1997). Angiotensin II is
formed both in the circulation and locally/tissue. The circulating form of
angiotensin II regulates systemic blood flow and pressure. The local/tissue
formation of angiotensin II ensures local control of blood flow independently of
blood-borne angiotensin II (Brunner et al., 1972) e.g. in the brain (Ganten et al.,
1971) and in the eye (Danser et al., 1994). An intracellular form of angiotensin
II has recently been discovered (Kumar et al., 2007) and this makes RAAS not
only an endocrine, but also a paracrine and intracrine system. Angiotensin II
functions as a potent vasoconstrictor and also causes cell growth (hypertrophy of
smooth muscle cells) and impairs learning and memory functions. Angiotensin
II regulates blood volume by release of aldosterone. Aldosterone is a
mineralcorticoid/steroid hormone produced by the adrenal cortex. A decrease in
32
sodium ion (Na+) concentration in the blood is sensed by the macula densa cells
in the kidney stimulating release of renin. Renin stimulates the formation of
angiotensin I and subsequently angiotensin II (see above) leading to the
stimulation of synthesis and release of aldosterone (Laragh et al., 1960; Sancho
et al., 1976) by the adrenal cortex (figure 5). Aldosterone increases the
reabsorption of Na+ and water, and decreases the reabsoption of potassium ions
(K+) from the renal collection duct (Brunner & Gavras, 1980). Apart from this
effect, aldosterone is involved in hypertension and cardiovascular diseases by
contributing to vascular inflammation, oxidative stress, fibrosis and vascular
injury (Brown, 2008). Concerning aldosterone and behaviour, amygdala, septum
pellucidum and hippocampus are the regions in the brain with the highest uptake
of aldosterone (Monder & White, 1993).
As the main effector of the RAAS, angiotensin II is involved in the development
of cardiovascular diseases. Angiotensin II binds to the angiotensin receptors
(Chiu et al., 1989) AT1, AT2 (Bumpus et al., 1991), AT3 and AT4 (Gulati, 1996;
Unger et al., 1996) which thereby are mediators of the actions of angiotensin II.
AT1 and AT2 receptors are specific membrane-bound G-protein coupled
receptors (Unger et al., 1996). AT1 receptors are expressed in blood vessels,
heart, kidneys, adrenal glands, liver, brain, lungs (Campbell, 1987; Chai et al.,
1993), in the endometrium (Ahmed et al., 1995) and in adipose tissue (Engeli et
al., 1999). Activation of AT1 receptors is associated with endothelial
dysfunction (Nishimura, 2000), vasoconstriction (Nishimura, 2000), cell
proliferation, cell growth in the heart and arteries (Benson et al., 2008), platelet
aggregation (Fogari & Zoppi, 2006), inhibition of nitric oxide synthase (Rush &
Aultman, 2008), increased aldosterone release from the adrenal glands and
increase in reactive oxygen species (ROS) (see above, section “Antioxidants”)
O2- by NADHoxidas (Reed et al., 2008). Activation of AT1 receptors is also
involved in hypertension, heart failure, salt and water retention (Weiss et al.,
33
2001), vasopressin release, thirst and cognitive and behavioural processes such
as depression, anxiety, decrease in learning and memory (Gard, 2002;
Birkenhäger & Staessen, 2006; McGuinness, 2006) and dementia (McGuiness,
2006). Several polymorphisms in the human AT1 receptor gene have been
discovered of which one (A1166C) is more frequent in hypertensive humans
(Bonnardeaux et al., 1994). It has been shown that AT2 receptors are expressed
in fetal life and in brain (Wright & Harding, 1997) and are supposed to be
involved in growth, differentiation and exploratory behavior (DeGasparo et al.,
2000). Effects of AT2 receptors on cardiovascular diseases e.g. hypertension,
cell proliferation and cell growth, seem to be relatively minor in contrast to the
effects of AT1. AT1 and AT2 receptors may act as physiological antagonists, i.e.
AT2 decrease while AT1 receptors increase blood pressure and cell proliferation
(Ichiki et al., 1995; Nakajima et al., 1995; Stoll et al., 1995). Stimulation of AT1
receptors generates ROS while AT2 stimulation generates the vasodilators
bradykinin and NO (see above, section “Nitric Oxide”). Plasma half life of
angiotensin II in the circulation is less than 2 minutes, then it is inactivated by
blood and tissue enzymes (Reid, 1985), i.e. aminopeptidase A and N, forming
peptide fragments, i.e. angiotensin III (angiotensin 2-8, Arg-Val-Tyr-Ile-His-
Pro-Phe), angiotensin IV (angiotensin 3-8, Val-Tyr-Ile-His-Pro-Phe) and
angiotensin 1-7 (Asp-Arg-Val-Tyr-Ile-His-Pro) (Peach, 1977; Schiavone et al.,
1990; Ferrario et al., 1991; Wright et al., 1995; Ferrario & Iyer, 1998; Ardaillou,
1999;). Effects of angiotensin III are mediated by AT1 and AT2 receptors; it
stimulates secretion of aldosterone and is involved in thirst (Fitzsimons, 1998).
Furthermore, angiotensin III acts as a major effector of RAAS in the brain (Zini
et al., 1996) by regulation of blood-pressure and vasopressin release (Reaux et
al., 2001; Bodineau et al., 2008). Apart from AT1 and AT2 receptors, AT3 and
AT4 receptors have been found (Gulati, 1996; Unger et al., 1996). The AT3
receptor has low affinity for angiotensin III and was initially described in mouse
neuroblastoma cells (Chaki & Inagami, 1992). The effects of the receptor AT3
34
are still unknown (Stanton, 2003). Angiotensin IV affects the endothelium to
release plasminogen activator inhibitor-1 (PAI-1) and this effect is mediated by
the AT4 receptor (DeGasparo et al., 2000; Stanton, 2003) present in human
prostate (DeGasparo et al., 2000) and in the brain e.g. in the amygdala,
hippocampus and thalamus (Wright et al., 2008) affecting learning and memory
(Bodineau et al., 2008). It has also been suggested that brain RAAS is involved
in stress responses (Ruiz-Ortega et al., 2001) and depression (Gard, 2004).
Even if angiotensin II is unable to penetrate the blood-brain barrier easily there
is a strong connection between brain structures outside and inside of the blood-
brain barrier. Furthermore, angiotensin II-sensitive structures inside the blood-
brain barrier may normally be stimulated by angiotensin II generated locally as
components of brain RAAS (Ganten et al., 1971). Many of these angiotensin II-
sensitive neurons in the central nervous system (Bickerton & Buckley, 1961) are
present in highly vascularized nervous structures, the circumventricular organs
(CVO) where the blood-brain barrier is deficient and these neurons are therefore
accessible to circulating molecules such as angiotensin II, although isolated
from the rest of the brain by other barriers. Blood-brain barrier is present in all
brain regions except the CVO including area postrema (AP) in the brainstem (a
part of the brain that controls vomiting), median eminence (connecting the
hypothalamus with the pituitary gland), posterior pituitary gland (neuronal
projections of the hypothalamus that secrete peptide-hormones vasopressin and
oxytocin into the circulation), pineal gland, subfonical organs (SFO) (involved
in vasopressin secretion) and organum vasculosum of the lamina terminalis
(OVLT) (controlling thirst, sodium excretion, blood volume regulation and
vasopressin secretion) (Lind & Johnson, 1982; Fitzsimons, 1998). These brain
structures are implicated in body fluid homeostasis and rich in angiotensin II
receptors and important concerning drinking behaviour, renal function and blood
pressure, and extremely sensitive to the action of angiotensin II. They also have
35
extensive connections with the hypothalamus and other limbic structures
translating information about the inner milieu into behaviour including
emotional-related behaviour, learning and memory (Mosimann et al., 1996). In
addition to thirst, sodium appetite and blood pressure control, angiotensin II in
the central nervous system (CNS) affects cell growth, membrane function,
protein synthesis, prostaglandin release, pituitary hormone synthesis and release
(Mosimann et al., 1996). The RAAS is shown in figure 5.
The Angiotensin-Converting Enzyme
Angiotensin-converting enzyme (ACE; EC 3.4.15.1) (Skeggs et al., 1956b),
initially known as kininase II (Yang & Erdös, 1967) a zink carboxypeptidase is
synthesized by the endothelium and present on the luminal surface of the
membrane of the endothelial cells (Baudin et al., 1997). ACE is bound with part
of the hydrophobic C-terminal in the cell membrane and with the active sites
protruding out into the vessel lumen (Ryan et al., 1975; Hooper et al., 1987; Wei
et al., 1991a) (figure 5). ACE may loose its C-terminal end and become
dissolved in plasma as circulating ACE (Hooper et al, 1987, Wei et al, 1991a,
Baudin et al, 1997). ACE is found mainly in the lungs due to their vast surface
of vascular endothelium (Ng &Vane, 1967). ACE is also present in other
vascular tissues than endothelium; such as smooth muscle cells (in tunica media
and tunica adventitia) (André et al., 1990; Arnal et al., 1994; Battle et al., 1994),
the heart (Lindpaintner et al., 1987), fibroblasts (Arnal et al., 1994; Battle et al.,
1994), the kidney, CNS, placenta and testis (Erdös & Skidgel, 1986; Erdös,
1990). ACE is a polyspecific enzyme metabolising angiotensin I (see above,
section “The renin-angiotensin aldosteron system”), angiotensin 1-7 (see above,
section “The renin-angiotensin aldosterone system”), and furthermore e.g.
enkephalins, substance P, luteinizing hormone-releasing hormone (LH-RH),
desArg9-bradykinin (Erdös & Skidgel, 1986; Berecek & Zhang, 1995,). In
36
insects, ACE is also involved in digestion (as a gastrointestinal hormone),
reproduction and immune defence (Macours & Hens, 2004). Furthermore, ACE
metabolise bradykinin, kallidin (that can be converted to bradykinin) and
kininogen (responsible for bradykinin generation) (Scharfstein et al., 2007).
Bradykinin is a 9-amino acid peptid (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg),
an endothelial cell dependent vasodilator (Regoli & Barabé, 1980; Hall, 1992)
synthesised by the liver and present in plasma and tissues. Bradykinin is formed
as a result of increased vascular permeability (tissue injury) (Sharma et al.,
1996) i.e. inflammation, activated Hageman Factor (factor XII) and the effects
of bradykinin are mediated by the bradykinin receptors, B1, B2, B3 and B4.
Bradykinin activates eNOS and thereby generates NO (figure 5). Bradykinin is
involved in acute and chronic inflammation (Sharma et al., 1996), vasodilatation
(Regoli & Barabé, 1980; Hall, 1992), increases vascular permeability (Sharma et
al., 1996), stimulates and sensities sensory neurons (Whalley et al., 1987) and
increases airway secretion (Hall, 1992). Bradykinin is inactivated by ACE,
kininase I, aminopeptidase P (APP), neutral endopeptidase (NEP) and
carboxypeptidase M and N (CPM, CPN) (Kuoppala et al., 2000) generating des-
Arg-bradykinin a specific agonist to B1 receptor. Thus, ACE is not only
activating the vasoconstrictor angiotensin II but it also inactivates the
vasodilatator bradykinin (figure 5).
ACE contains two homologous sites (Soubrier et al., 1988; Bernstein et al.,
1989), catalytically and independently active (Wei et al., 1991b). Testicular
ACE contains a shorter amino acid sequence and only one active site (the C-
terminal site) (Erdös, 1990), suggesting a gene duplication during the evolution
(Corvol et al., 1995). Both active sites contain Zn2+ ion (Ehlers & Riordan,
1991, Wei et al, 1991b). ACE inhibitors are designed to bind to the Zn2+ ion at
the active sites of ACE (Sturrock et al., 2004).
37
The variation of plasma ACE activity between different individuals is very high
(Alhenc-Gelas et al., 1983), but there seems to be a familiar resemblance of
ACE activity levels (Cambien et al., 1988). The ACE gene is found on
chromosome 17 and the ACE gene polymorphic sites is an insertion/deletion
(I/D) type and explains 47% of the variations between individuals (Rigat et al.,
1990; Tiret et al., 1992). Individuals with the DD genotype have two-three fold
higher levels of ACE than those with the II genotype, and the ID genotype has
an intermediate level of ACE activity (Rigat et al., 1990; Beohar et al., 1995).
The D allele of the ACE gene is suggested to correlate with cardiovascular
diseases (Cambien et al., 1992; Samani et al., 1996). Hence, it seems that the
genotype DD could be a risk factor for cardiovascular diseases (Beohar et al.,
1995).
Figu
re 5. Th
e renin-angiotensin aldosterone syste
mA
minopeptidase A
(AM
PA
), Am
inopeptidase N (A
MP
N), Brad
ykinin (BK
), Brad
ykinin receptors (BK
R), Insulin
(IRA
P), M
as oncogen
e receptor (Mas), N
eutral endopeptidase (N
EP
), Nitric oxide (N
O),
endopeptidase (PE
P), R
enin/prorenin receptors (R
PR
38
angiotensin aldosterone syste
m. A
ngiotensin-converting enzyme (A
CE
), An
giotensin receptors (AT
Am
inopeptidase A (A
MP
A), A
minopeptidase N
(AM
PN
), Brad
ykinin (BK
), Brad
ykinin receptors (BK
R), Insulin
(IRA
P), M
as oncogen
e receptor (Mas), N
eutral endopeptidase (N
EP
), Nitric oxide (N
O), E
ndothelial nitric oxide synthase (eN
OS
),endopeptidase (P
EP
), Re
nin/prorenin receptors (RP
R).
enzyme (A
CE
), An
giotensin receptors (AT
1 , AT
2 , AT
3 , AT
4 ), A
minopeptidase A
(AM
PA
), Am
inopeptidase N (A
MP
N), Brad
ykinin (BK
), Brad
ykinin receptors (BK
R), Insulin-re
gulated aminopeptidase
Endothelial nitric oxide synth
ase (eNO
S),
Prolyl
39
AIMS
The effects of flavonoids are previously associated with their effects as
antioxidants. Epidemiological studies and meta-analyses strongly suggest that
long term consumption of a diet rich in vegetables and fruits will protect against
cardiovascular diseases (Hertog et al., 1993; Hertog et al., 1995; Ness &
Powles, 1997; Carlson et al., 2008) due to the involvement of reactive oxygen
species. Focus has been on the possible role of free radical scavenging and
radical suppression of nutrients in explaining the beneficial effect of diet
compounds. As for the beneficial protection from cardiovascular diseases, the
antioxidative effects of plant-derived substances as flavonoids are but one
explanation of several.
The antioxidative properties of flavonoids, tocopherols and carotenoids are of
importance, but to maintain homeostasis of the vascular wall in order to affect
the development of cardiovascular diseases the balance between angiotensin II,
nitric oxide (NO) and reactive oxygen species (ROS) is of great importance.
The aims of this study were to …..
…… investigate if the sole mechanism according to the benificial properties of
vegetables, fruits and berries on cardiovascular diseases, is their antioxidative
properties
…… investigate plant extracts with alleged effect on the cardiovascular system
on angiotensin-converting enzyme activity and nitric oxide concentration, in
vitro, and in vivo (after administration of green, black tea and Rooibos). Plant
extracts used as everyday beverage, dietary products, food supplements, herbal
medicinal products and traditional antioxidants
40
…… map the active substances with effect on angiotensin-converting enzyme
activity and nitric oxide concentration
…… investigate if there is any connection between effect and
biosynthesis/structure
41
METHODS
The studies on cultured endothelial cells from human umbilical veins (HUVEC)
(Paper I-IV) were approved by the regional ethics committee at the Faculty of
Health Sciences, Linköping, Sweden (Dnr 03-602).
The in vivo study (Paper V) was approved by the regional ethics committee at
the Faculty of Health Sciences, Linköping, Sweden (Dnr M56-07).
INFUSIONS AND EXTRACTIONS
Tea Infusion, in vitro (Paper I)
Tea infusions were prepared from green tea (Camellia sinensis, L. Theaceae;
Japanese Sencha), black tea (Camellia sinensis, L. Theaceae; Indian Assam
B.O.P.) and Rooibos (Aspalathus linearis, Dahlg. Leguminosae). Infusions were
made with 1 g tea in 20 ml sterile phosphate-buffered saline (PBS) for 5 min
(the green tea and the black tea) or 10 min (the Rooibos tea). The infusions were
filtered twice, first through a standard filter 0.45µm (Munktell, Grycksbo,
Sweden) and then through a sterile filter 0.2µm (Millipore). The obtained
filtrates were considered as 1:20 and were frozen at -20ºC in aliquots.
Tea Infusion, in vivo (Paper V)
The tea was prepared from green tea Japanese Sencha imported by Charabang,
Stockholm, Sweden, black tea Indian Assam B.O.P. imported by Norrköping
Kolonial, Sweden, and Rooibos tea imported by Norrköping Kolonial, Sweden.
The teas were bought at Tebladet, Linköping, Sweden. Infusions were made
with 10 g tea in 400 ml fresh-boiled water for 5 min with the green tea and the
black tea, and 10 min with the Rooibos tea, using a tea filter (Agatha´s Bester,
Germany).
42
Coffee Infusion, in vitro
Coffee infusion (Coffea arabica, L. Rubiaceae) was prepared from Santos Brazil
coffee. Infusion was made with 2 g coffee in 20 ml sterile PBS for 3 min. The
infusion was filtered twice, first through a standard filter 0.45µm (Munktell,
Grycksbo, Sweden) and then through a sterile filter 0.2µm (Millipore). The
obtained filtrate was considered 1:10 and was frozen at -20ºC in aliquots.
Cacao Extraction, in vitro
Cacao extract (Theobroma cacao, L. Sterculiaceae), containing approximately
10% cacao fat was used for preparation of solution. Solution was made of 1 g
cacao extract in 20 ml sterile PBS for 15 minutes in boiling waterbath. The
solution was filtered twice through a standard filter 0.45 µm (Munktell,
Grycksbo, Sweden). The obtained filtrate was considered as 50 mg/ml and was
frozen at -20ºC in aliquots.
Bilberry 25E Extraction, in vitro (Paper IV)
Bilberry extract 25E (Vaccinium myrtillus, L. Ericaceae) standardized for 25%
anthocyanidins (cyanidin, delphinidin and malvidin) was used for preparation of
solution. Solution was made of 1 g Bilberry 25E extract in 20 ml sterile PBS for
15 minutes in boiling waterbath. The solution was filtered twice through a
standard filter 0.45 µm (Munktell, Grycksbo, Sweden). The obtained filtrate was
considered as 50 mg/ml and was frozen at –20°C in aliquots.
Liquorice Extraction, in vitro
Liquorice (Glycyrrhiza glabra L. Fabaceae) dried powder was used for
preparation of solution. 2 g powder was dissolved in 20 ml sterile PBS and
submitted to boiling waterbath for 1 hour. The Glycyrrhiza suspension was then
43
filtered twice through a standard filter 0.45 µm (Munktell, Grycksbo, Sweden).
The obtained filtrate was considered as 100 mg/ml and was frozen at -20ºC in
aliquots (Persson et al., 2008).
Ginkgo biloba Extraction, in vitro (Paper III)
Ginkgo biloba, L. (Ginkgoaceae) standardized extract EGb 761, was used for
preparation of solution. One g EGb 761 was dissolved in 10 ml sterile PBS and
placed in 60ºC waterbath for 1 hour. The suspension was then filtered twice
through a standard filter 0.45µm (Munktell, Grycksbo, Sweden). The obtained
filtrate was considered as 100 mg/ml and was frozen at -20ºC in aliquots.
Panax ginseng Extraction, in vitro (Paper II)
Panax ginseng L. (Araliaceae) standardized extract G115, was used for
preparing the solution. 1 g G115 was dissolved in 10 ml sterile PBS and placed
in 60ºC waterbath for 1 hour as described previously (Friedl et al., 2001). The
suspension was filtered twice, first through a standard filter, then through a
sterile filter 0.2µm (Millipore). The obtained filtrate was considered as 100
mg/ml and frozen at -20ºC in aliqouts.
CULTURED ENDOTHELIAL CELLS FROM HUMAN UMBILICAL VEINS
(HUVEC) (PAPER I-IV)
Human umbilical cords were obtained after normal vaginal delivery (after
informed consent from the mothers), and kept in sterile bottles containing PBS
and antibiotics. Endothelial cells were isolated according to the method of
Nyhlén et al., (2000). In short, the veins were cannulated at each end, washed
with PBS, and then treated with collagenase in 37ºC for 25 minutes. The
collagenase+cell perfusate was washed twice, and then resuspended in cell
culture medium (Dulbecco´s modified Eagle´s medium, DMEM) supplemented
with nonessential amino acids (1:100), oxalacetic acid (1.2 mM), insulin (0.24
44
IE/ml), penicillin (5 U/ml), streptomycin (0.5 µg/ml), hepes (10 mM),
endothelial cell growth factor (ECGF, 30 µg/ml), heparin (20 U/ml) and 17 %
inactivated fetal calf serum (FCS). Resuspended HUVEC were seeded in 25
cm2 tissue culture flasks coated with 0.2 % gelatin, kept in an incubation
chamber, and medium was replaced every 48-72 hour. At confluence, cells were
harvested with trypsin-EDTA for 5-10 minutes, and then reseeded 1:2. Second
passage was seeded in a 96-well microtiter plate, and allowed to reach
confluence.
ANGIOTENSIN-CONVERTING ENZYME ACTIVITY IN HUVEC (PAPER
I-IV)
HUVEC cultured in 96-well microtiter plates (as described above; 2nd passage)
was used. Immediately prior to treating the cells with drugs or extract, the
medium was removed and replaced with serum free medium. This was done to
avoid discrepancies in results due to ACE present in the fetal calf serum
(Bramucci et al., 1999). Cells were treated with flavonoids, terpenes, sterols,
purines, precursor molecules, plant infusions and extracts, human steroids and
lipid-lowering drugs for 10 minutes. The flavonoids tested were the isoflavone
genistein (0.1, 0.5 and 1 mg/ml), the flavonol quercetin (0.1, 0.5 and 1 mg/ml),
the epi-flavan-3-ols (catechins) epicatechin (0.1, 0.5 and 1 mg/ml),
epicatechingallate (0.1, 0.5 and 1 mg/ml), epigallocatechin (0.1, 1 and 2 mg/ml)
and epigallocatechingallate (0.05, 0.1, 1 and 2 mg/ml), procyanidin (0.1, 0.5 and
1 mg/ml), the anthocyanidins cyanidin, delphinidin and malvidin (0.01, 0.025
and 0.05 mg/ml), myrthillin chloride (0.01, 0.025 and 0.05 mg/ml) containing
cyanidin, delphinidin and malvidin, and the biflavan sciadopitysin (0.1, 0.5 and
1 mg/ml). The terpenes tested were the diterpenes α-tocopherol (0.1, 0.5 and 1
mg/ml) and ginkgolides A, B, C, (0.1, 0.5 and 1 mg/ml), the sesquiterpene
bilobalide (0.1, 0.5 and 1 mg/ml), the triterpenes ginsenoside Rb1, Rb2, Rc, Rd,
Re, Rf, Rg1 (0.1, 0.5 and 1 mg/ml) and the tetraterpene β-carotene (0.1, 0.5 and
45
1 mg/ml). The sterols tested were stigmasterol (0.1, 0.5 and 1 mg/ml), lanosterol
(10-7, 10-6, 10-5 and 10-3 M) and cholesterol (10-5, 10-4 and 10-3 M). The purines
caffeine (0.1, 0,5 and 1 mg/ml), theobromine (0.01, 0.05 and 0.1 mg/ml) and
theophylline (0.1, 0.5 and 1 mg/ml) were also tested. The biosynthesis precursor
molecules tested were mevalonic acid, malonic acid, shikimic acid, chorismic
acid and the progenitor squalene (10-7, 10-6, 10-5 and 10-3 M). The phenol salicin
(0.1, 0.5 and 1 mg/ml) were tested. Bilberry extract 25E (0.00625, 0.0125,
0.025, 0.05 and 0.1 mg/ml) containing 25% anthocyanidins, cacao extract
(0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/ml) containing procyanidin oligomers
derived from epicatechin were tested. Green tea, black tea and Rooibos tea
infusions, coffee infusion (1:3200, 1:1600, 1:800, 1:400 and 1:200), Ginkgo
biloba Egb761 extract (0.1, 0.5, 1, 5 and 10 mg/ml), Panax ginseng G115
extract (0.1, 0.5, 1, 5 and 10 mg/ml), liquorice extract (0.00625, 0.0125, 0.025,
0.05 and 0.1 mg/ml) were tested. The human-derived steroids aldosterone,
estradiol and testosterone (0.1, 0.5 and 1 mg/ml) were also tested. Furthermore,
the blood lipid-lowering drugs simvastatin and pravastatin were tested (10-8, 10-7
and 10-6 M). The bilberry extract, the cacao extract, the green, black and
Rooibos tea infusions, the coffee infusion, the Ginkgo biloba extract, the Panax
ginseng extract, the liquorice extract, salicin and cholesterol were dissolved in
PBS; all other drugs in dimethylsulfoxide (DMSO). Corresponding volumes of
PBS or DMSO were used as controls. Blank and standard serum, from the
commercial kit was added to wells with corresponding volumes of medium
without FCS. After 10 minutes incubation with drugs, ACE activity was
analysed as is described below.
46
ANGIOTENSIN-CONVERTING ENZYME RADIOENZYMATIC ASSAY
(PAPER I-V)
After incubating cells with drugs, extracts, infusions (Paper I-IV), or serum
(Paper V) ACE activity was analysed with a commercial radioenzymatic assay
(ACE-direct REA, Bühlmann Laboratories, Allschwil, Switzerland). In short,
the synthetic substrate 3H-hippuryl-glycyl-glycine was added to the samples and
cleaved by ACE to 3H-hippuric acid. After 2 hours for the in vitro experiments
and 1 hour for the in vivo study (Paper V) of incubation in 37ºC, the reaction
was stopped with 1 M HCl, scintillation liquid was added, and each sample was
counted in a scintillation counter.
NITRITE/NITRATE ASSAY (PAPER I-III, V)
After incubating the cells with drugs, extracts or infusions, medium was
removed and stored at -20ºC until nitrite/nitrate analysis, or for the in vivo study
(Paper V), serum was stored at -70ºC until analysis. Nitrite was analysed as a
marker of NO concentration as proposed by Lauer et al. (2001). Nitrite and
nitrate concentration in the tea infusions, cacao, Ginkgo biloba and Panax
ginseng extracts were also analysed. Nitrite/nitrate concentration was analysed
with a commercial nitrite/nitrate assay (Nitric oxide (NO2-/NO3
-) assay, R&D
Systems, UK). Nitrate was first reduced to nitrite which was then analysed. The
principle for the colorimetric detection of nitrite is the Griess reaction;
diazonium ions are produced when acidified nitrite reacts with sulfanilic acid.
Diazonium ions form chromophore agents when reacting with N-(1-
naphthyl)ethylenediamine. Optical density was determined with a Spectramax
reader at 540 nm.
47
LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY-MASS
SPECTROMETRY (LC-MS-MS) (PAPER I)
For quantifications of epicatechin, epigallocatechin, epicatechingallate and
epigallocatechingallate, an ESI-LC-MS-MS system for gradient chromatography
was used (Paper I). The instrumentation consisted of a Perkin Elmer 200
chromatographic system equipped with two micro pumps, a solvent degasser
and an autosampler (Norwalk, CT, USA). Mass detection was performed on a
Sciex API 2000 triple quadrupole instrument equipped with a turbo ion-spray
interface (PE Sciex, Ontario, Canada) operating in positive ion mode. The
interface probe was set at 350ºC and the ion-spray needle was operated at -4500
V. Nitrogen was used as nebulizer-, auxiliary-, curtain- and CAD-gas and was
set at 25, 50, 30 psi and a value of 5 respectively. The high performance liquid
chromatography (HPLC) was carried out on a Hypersil Polar-RP 150 x 3.0 mm
(Phenomenex, Torrance, CA, USA) equipped with an Opti-Solv 2 µm column
inlet filter (Optimize, Portland, Oregon, USA). The mobile phases consisted of
acetonitrile, methanol and 20 mM ammonium formiate buffer, pH 3, in mixtures
of 5:5:90 (v/v/v) for phase A and 40:40:20 (v/v/v) for phase B. A linear gradient
chromatography from 0-100 % B-phase over seven minutes was run. Total
runtime including wash and reconditioning was 10 minutes. A flow-rate of 0.25
ml/min at ambient temperature was used. The detection and quantification of the
selected compounds were performed in the Multiple Reaction Monitoring mode
(MRM). MS-MS product ion spectra were produced by collision activated
dissociation (CAD) of the de-protonated molecule ion (M+1)-. The most intense
transitions; 289/245 (epicatechin), 305/179 (epigallocatechin) 441/169
(epicatechingallate), 457/169 (epigallocatechingallate) were used for
quantification. Linear calibration curves were performed ranging from 0.5 µg/ml
to 20 mg/ml. Tea extracts were diluted in methanol and buffer 1:10 and 1:100
48
respectively before analysis. Instrument control, integration and calculation were
performed with the PC based PE Sciex software, Analyst 1.4.
TENSION STUDIES (PAPER II)
Bovine mesenteric arteries were obtained at the local slaughterhouse and then
transported to the laboratory in Krebs´ buffer. The Krebs´ buffer had the
following composition (in mM): 137 Na+, 5.89 K+, 2.50 Ca2+, 1.20 Mg2+, 134 Cl-
, 18.0 HCO3-, 1.20 H2PO4
- and 5.60 glucose. The arteries were carefully cleaned
of adipose tissue, cut into rings of 3-5 mm and then cut open, forming arterial
strips. The strips were mounted between a fixed point in organ chambers
containing Krebs´ buffer, and a Grass FT 03 strain gauge transducer connected
to a Grass Polygraph (Grass Instrument, Quincy, MA, USA), allowing
continuous measurement of auxometric tension. The strips were stretched to 2-
2.5 g and allowed to equilibrate for 45 min, resulting in a passive tension of 0.5-
1.0 g (the optimal passive tension for these vessels). After wash, arteries were
incubated with or without ginseng extract for 10 min and then angiotensin I-
induced contractions were examined by submitting the strips to 10-5 M
angiotensin I. Then, the preparations were submitted to several washes until
tension returned to basal value. Maximal contraction of each strip was
determined by using a Krebs´ buffer with a high K+ concentration (128 mM K+,
15.4 mM Na+ and other ions as described above).
When endothelium-dependent relaxation was studied, two strips were used from
each cow. In one strip the endothelium was left intact, while on the other strip
the endothelium was removed by gently rubbing the luminal side of the strip
with a wooden stick as previously described (Persson and Andersson, 1998).
Then, the strips were precontracted with 10-5 M phenylephrine, and when a
stable contraction was obtained ginseng extract was added in cumulative doses.
49
IN VIVO STUDY (PAPER V)
Twenty healthy volunteers were randomised in a single-blind cross-over study to
receive either 400 ml green tea (Japanese Sencha), black tea (Indian Assam
B.O.P.) or Rooibos tea. The effect of single dose of the different teas was
compared with a 1-4 weeks washout period between the tea regimens. All
experiments started at 8 am and the subjects were not fasting. Three subjects
were excluded; two because of difficulties when collecting blood and one used
oral tobacco. The participants did not use any kind of drugs (medical or herbal)
2 weeks prior to and during the study. The volunteers did not use nicotine in any
form. Intake of beverages or food containing high amount of catechins, the
flavonol quercetin and anthocyanins, i.e. chokeberries, aubergine, blackberries,
bilberries, elderberries, raspberries, strawberries, coffee, cacao/chocolate,
cherries, onions, plums, pears, radishes, red and black currants, red cabbage,
black beans, cranberries, tea, wine, grapes and apples were not allowed 48 h
before the experiment. A list of food not allowed was distributed to the
participants when they registered to the study. The participants did also answer a
questionnaire concerning everyday intake of certain food i.e. vegetables, fruits
and berries containing the flavonoids catechins, quercetin and anthocyanins.
Data collected at each treatment were blood pressure, heart rate and venous
blood samples for analyses of ACE activity, NO concentration and ACE
genotype. The tea was newly made and cooled so the participants could drink it
within approximately 2 minutes. Blood pressure, heart rate and blood samples
for analyses of ACE activity and NO concentration were taken before drinking
tea and after 30 min, 60 minutes and 3 hours. Vaccutainer tubes without
anticoagulantia were used to collect blood for serum-ACE and serum-nitrite
analyses. After 2 hours the tubes were centrifugated 1000g for 20 minutes, at
4ºC. Serum was transferred to plastic tubes and ACE activity were analysed the
same day as described above. Remaining serum was frozen -70ºC until nitrite
50
analysis. Vaccutainer tubes with EDTA were used to collect blood for
determination of polymorphism concerning the ACE genotype. The blood was
transferred to sterile test tubes and frozen -70ºC until analysis (see above).
ANGIOTENSIN-CONVERTING ENZYME GENOTYPE (PAPER V)
Genomic DNA was isolated from venous blood using QIAmp DNA Mini Kit
(QIAGEN, Hilden, Germany).
The ACE gene contains a polymorphism based on the absence and/or presence
of a 287 base pair nonsense DNA domain in intron 16, which results in three
different genotypes: deletion/deletion (DD) homozygote, insertion/insertion (II)
homozygote and insertion/deletion (ID) heterozygote. Three primers 5´-CTG
CAG GTG TCT GCA GCA TGT GC-3´, 5´-GAT TAC AGG CGT GAT ACA
GTC ACT TTT-3´ and 5´-GCC ATC ACA TTC GTC AGA TCT GGT AG-3´
(Invitrogen Ltd., Paisley, UK) were used according to the method of Cheon et
al. (Cheon et al., 2000).
For the polymerase chain reaction (PCR) reaction a “ready-to-use” reaction
mixture (REDExtract-N-Amp Blood PCR ReadyMix, Sigma-Aldrich, St. Louis,
MO, USA) was used in a total volume of 20 µl. Each primer was used at a
concentration of 0.4 µM and for each reaction 2 µl DNA solution was added to
the reaction mixture.
The PCR was performed in a Perkin Elmer Cetus DNA Thermal Cycler and
started with 5 minutes of initial denaturation at 94ºC. Then the samples
underwent 30 cycles of amplification: 45 seconds of denaturation (94ºC), 45
seconds of annealing (65ºC) and 45 seconds of extension (72ºC). The reaction
was finished with 7 minutes of final extension (72ºC) and hold at 4ºC. The PCR
products were separated by electrophoresis on a 2% agarose gel, then stained
51
with ethidium bromide and visualized with ultraviolet light. The PCR products
detected with the primers are one 237 base pair fragment for the deleted allele,
and one 525 and one 155 base pair fragment for the inserted allele.
52
CHEMICALS
All chemicals for culturing cells were obtained from Life Technologies,
Scotland, UK, except Endothelial Cell Growth Factor that was bought at
Boehringer-Mannheim, Germany and heparin (Heparin LEO) from LEO
Pharma AB, Malmö, Sweden. Ginkgolides A, B, C, bilobalide, ginsenosides
Rb1, Rb2, Rc, Rd, Re, Rf and Rg1, cyanidin, delphinidin, malvidin and myrtillin
chloride were purchased from Extrasynthese Genay, France. Genistein,
quercetin, epicatechin, epicatechingallate, epigallocatechin,
epigallocatechingallate, procyanidin, sciadopitysin, α-tocopherol, β-carotene,
stigmasterol, lanosterol, cholesterol, mevalonic acid, malonic acid, shikimic
acid, chorismic acid, squalene, aldosterone, estradiol, testosterone, caffeine,
theobromine, theophylline, simvastatin, pravastatin, angiotensin I and
phenylephrine were bought from Sigma-Aldrich Chemical Co, MO, USA.
Enalaprilat (Renitec® 1 mg/ml) was bought from Merck Sharp and Dohme,
Haarlem, The Netherlands. NG-monomethyl-L-arginine (L-NMMA) and N-3-
(aminomethyl) benzylacetamidine (1400W) were bought from Alexis
Biochemicals, San Diego, USA. Salicin was a gift from Naturapoteket,
Norrköping, Sweden. The Bilberry extract 25E was a gift from Ferrosan,
Copenhagen, Denmark; the cacao extract and the liquorice powder was a gift
from Cloetta-Fazer, Ljungsbro, Sweden; the green tea, the black tea, the
Rooibos and the coffee was a gift from Tebladet, Linköping, Sweden; Ginkgo
biloba EGb 761 was a gift from Cederroth International AB, Upplands Väsby,
Sweden, and Dr Willmar Schwabe, Karlsruhe, Germany; Panax ginseng G115
was a gift from Pharmaton S.A. Switzerland.
53
CALCULATIONS
Results are presented as mean ±s.e.m. (standard error of the mean).
One unit (U) of ACE activity is defined as the amount of the enzyme required to
release 1 µmole of hippuric acid per minute and liter. Statistical calculations
were done with Graph Pad PrismTM 3.0 (Paper I and IV) Graph Pad PrismTM 4.0
(Paper II) and Graph Pad PrismTM 5.0 (Paper III and V). One way analysis of
variance (ANOVA) for repeated measures was performed followed by
Dunnett´s post test. Statistical significance is denoted as * p<0.05, **p<0.01 and
*** p<0.001.
54
RESULTS AND DISCUSSION
Results and discussion starts with effects seen on ACE activity followed by
effects on NO concentration, food-drug interactions and the results applied on
clinical importance. The results concerning ACE activity are divided in
beverages, other dietary products, herbal medicinal products, traditional
antioxidants, sterols, blood-lipid lowering drugs, precursor molecules and
proposed mechanism of effects of flavonoids on ACE activity.
Results on ACE activity and NO concentration in HUVEC and chemical
structure of the substances tested are summarised in Table 1, at the end of this
section.
NO concentrations was not measured with Bilberry extract 25E or its
anthocyanidins cyanidin, delphinidin and malvidin due to light absorption at 540
nm which is the same wavelength used in the nitrite/nitrate analysis. Neither was
NO concentration measured with genistein, ginsenosides, β-carotene, α-
tocopherol, stigmasterol, pravastatin and simvastatin.
55
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF
BEVERAGES CONTAINING FLAVONOIDS AND XANTHINES
Green Tea, Black Tea and Rooibos Tea, in vitro (Paper I)
Due to the climate, the tea tree/bush is cultivated all over the World, but mostly
in China, Japan, India and Sri Lanka, and tea is one of the most ordinary
beverages in the World. Green tea and black tea is produced from Camellia
sinensis L. (Theaceae); green tea from heated leaves to preserve the green
colour, and black tea from fermented leaves. Pharmacological effects on the
cardiovascular system of green and black tea have been attributed to
antioxidative effects of the catechin substances. Rooibos tea is a caffeine-free
alternative produced from fermented leaves and bark of a tree growing in South
Africa, Aspalathus linearis Dahlg. (Leguminosae). Rooibos tea do not contain
catechins but dihydrochalcones, flavones and flavonols.
In this study, 10 minutes incubation with green tea, black tea and Rooibos tea
showed a significant and dose-dependent inhibition of ACE activity in HUVEC
with the green tea 1:1600 *p<0.05, 1:800, 1:400 and 1:200 **p<0.01, with the
black tea 1:800 *p<0.05, 1:400 and 1:200 **p<0.01, while no significant effect
was seen with the Rooibos tea compared to the PBS control.
After ten minutes incubation a significant inhibition of ACE activity was seen
with the flavanols epicatechin 1 mg/ml *p<0.05, epicatechingallate 0.1mg/ml
*p<0.05, 0.5 mg/ml and 1 mg/ml **p<0.01, epigallocatechin 0.1 mg/ml
*p<0.05, 1 and 2 mg/ml **p<0.01 and epigallocatechingallate 0.1, 1 and 2
mg/ml ***p<0.001 compared to the DMSO control.
The green tea showed a stronger ACE inhibitory effect compared to the black
tea, probably because of the higher amount of catechins in green tea, especially
epigallocatechingallate. The tendency towards inhibition seen with the Rooibos
tea could be due to the content of flavonoids other than catechins. In contrast to
green and black tea, Rooibos tea do not contain any catechins, but the flavonol
56
quercetin is present in Rooibos tea (Bramati et al., 2003). The slight inhibition
of ACE activity in vitro by Rooibos tea might depend on its quercetin content,
since quercetin exhibits an ACE inhibitory action in vitro (Paper III).
The degree of ACE inhibition varied between the different flavanols/catechins
tested; lowest effect was seen with epicatechin, then epigallocatechin,
epicatechingallate, and the greatest effect was seen with epigallocatechingallate,
and consequently between extracts containing different amounts of catechins;
green tea and black tea. An increased number of hydroxyl groups and addition
of double-bound oxygen seemed to increase the inhibitory effect of the different
catechins on ACE activity (Table 1).
Coffee Infusion, in vitro
Coffee is produced by roasting the beans from the coffee bush, Coffea arabica
L. (Rubiaceae). Coffee is an everyday beverage mostly consumed due to its
amount of the psychostimulant caffeine. Studies investigating coffee in relation
to cardiovascular diseases are mostly focusing on its content of caffeine (George
et al., 2008). Besides caffeine, coffee contains catechin and epicatechin (Chen et
al., 2006).
After 10 minutes incubation with the coffee extract a significant inhibition on
ACE activity in HUVEC was seen with 5.0 mg/ml *p<0.05 compared to the
PBS control. This inhibition is proposed to be due to its content of catechins.
Cacao Extract, in vitro
Cacao is produced from the beans of Theobroma cacao L. (Sterculiaceae), a tree
cultivated in South-America, Africa and Asia. Cacao and chocolate is produced
from fermented, dried, roasted and crushed cacao-beans. Cacao contains
57
procyanidins which are oligomeric catechins linked together; dimers and trimers
are most common, e.g. epicatechin-epicatechin.
After 10 minutes incubation with cacao extract a significant and dose-dependent
inhibition of ACE activity in HUVEC was seen with 0.00625, 0.0125, 0.025,
0.05 and 0.1 mg/ml **p<0.01 compared to the PBS control. Compared to the tea
infusions, the cacao extract seems to have a stronger inhibitory effect on ACE
activity but to be noted; pure cacao extract was used not cacao-powder as we
usually drink or eat.
Xanthines, in vitro
The xanthines/purines caffeine, theobromine and theophylline are components
in coffee, tea and cacao and are known to have various pharmacological effects
on the cardiovascular system. Neither, caffeine, theobromine or theophylline did
show any significant effect on ACE activity in HUVEC after ten minutes
incubation compared to DMSO control, thus they are not responsible for the
effect on ACE activity of tea, coffee nor cacao.
Green Tea, Black Tea and Rooibos Tea in Healthy Volunteers (Paper V)
After oral intake of 400 ml green tea, black tea or Rooibos tea, a significant
**p<0.01 inhibition of serum ACE activity was seen with the Rooibos tea after
30 minutes and *p<0.05 after 60 minutes.
As previously mentioned, studies show that tea reduces mortality in
cardiovascular diseases (Mukamal et al., 2002; Hakim et al., 2003; Babu & Liu,
2008). The effects of tea on cardiovascular diseases are suggested to be due to
the high content of catechins i.e. epicatechin, epigallocatechin,
epicatechingallate and epigallocatechingallate (Paper I).
58
In HUVEC, green tea and black tea significantly inhibited ACE activity, in
contrast to Rooibos tea where no significant inhibition of ACE activity was seen
(Paper I). In the in vivo study, with healthy volunteers drinking tea, a significant
inhibition of serum ACE activity after intake of Rooibos tea was seen. This
discrepancy between the in vitro and in vivo studies may either be due to the
content of flavonoids, their absorption and/or metabolism or to a possible
different mechanism of action of the Rooibos tea compared to the green tea and
the black tea. The Rooibos tea did not show any significant effect on ACE
activity in vitro, but the flavonol quercetin found in Rooibos tea showed
significant inhibition of ACE activity in vitro.
To understand the mechanisms of action, and to be able to validate the results
from the in vitro experiments and to confirm the inhibiting effect on ACE
activity in vivo, it is necessary to gain information about the pharmacokinetic of
the flavonoids. During metabolism of flavonoids different metabolites are
formed (Kühnau, 1976; Williamson & Manach, 2005) which might be
responsible for the effects on ACE activity in vivo. An in vitro model of
degradation of quercetin by microorganisms formed the metabolites
dihydroquercetin (taxifolin), the auronol alphitonin and chalcones (Braune et al.,
2001) and, consequently, quercetin and its metabolites may affect ACE activity
in vivo. Tea catechins undergo phase II metabolism, methylation,
glucuronidation and sulphation. Epigallocatechingallate, the catechin with the
strongest effect on ACE activity in HUVEC, is hence degraded into methylated,
glucuronided and sulphated forms of epigallocatechin (Lambert et al., 2007)
which may explain the discrepancy of the results in vitro compared to in vivo of
green tea and black tea. Data available on flavonoids and their pharmacokinetics
consider unmetabolised flavonoids i.e. the ingested flavonoid or its conjugates.
Flavonoids are absorbed in the small intestine as glycosides or hydrolyzed to
their aglycones by microorganisms and then absorbed (Walle, 2004). The half-
59
life of catechins seems to be rather short, 1-3 hours, compared to genistein 6-10
hours and quercetin 11-28 hours (Manach et al., 2005). This discrepance in half-
life of the different flavonoids may also affect the results in vitro and in vivo
concerning tea.
When sub-grouping the participants, according to ACE genotype a significant
inhibition of serum ACE activity was seen with the green tea for the genotype II
*p<0.05, 30 minutes after intake, and for the genotype ID *p<0.05, 60 minutes
after intake. A significant inhibition of serum ACE activity was also seen with
the Rooibos tea for the genotype II *p<0.05, 60 minutes after intake. In
accordance with earlier studies (Rigat et al., 1990) the basal levels of serum
ACE activity was significantly higher ***p<0.001 for the participants with ID
and DD genotype compared to those with II genotype. The DD genotype is
proposed to be a risk factor for cardiovascular diseases, and in this study no
effect was seen on ACE activity for the DD genotype.
The difference on serum ACE activity between genotypes when drinking tea
may be a question of dosage or long term intake. ID and even more DD
genotype might need higher amount or long term intake of flavonoids to affect
serum ACE activity compared to II genotype. It is previously shown that the
effect of the ACE inhibitor drug enalaprilat is significantly greater among
individuals of II genotype (Ueda et al., 1998). Even so, in this study one cup of
tea was enough to significantly inhibit serum ACE activity in individuals with II
and ID genotypes. It is therefore tempting to suggest that “chronic” tea drinkers
have lower ACE activity no matter which genotype. The ACE gene
polymorphism explains 47% of the variations of serum ACE levels between
individuals (Rigat et al., 1990; Tiret et al., 1992). Although plasma levels of
ACE concentrations in one individual seems to be stable over time (Alhenc-
Gelas et al., 1991) environmental influence on gene expression can not be
excluded (Sing et al., 2003). Cardiovascular diseases have a complex
60
multifactorial etiology and can not be explained by nor genes neither
environmental factors alone. Over time environmental factors as e.g. culture and
diet may influence gene expression and consequently change gene expression
(Sing et al., 2003). The “evolution” of culture according to diet; from a
traditionally plant-based diet containing complex carbohydrates and dietary
fibers, vegetables, fruits and berries to a diet rich in saturated fats, and simple
carbohydrates i.e. a diet consisting mainly of meat and products with high
energy content (WHO, 2003), may have changed the expression of the ACE
genotypes. Changes of gene expressions due to long term intake of one-sided
food e.g. unsaturated fat and lack of vegetables, fruits and berries may be
underestimated.
In conclusion, the beneficial properties of tea besides antioxidative properties,
on cardiovascular diseases may be due to its inhibitory effect on ACE activity.
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME
ACTIVITY OF DIETARY PRODUCTS CONTAINING
ANTHOCYANINS OR ISOFLAVONOIDS
Bilberry Extract 25E, in vitro (Paper IV)
Bilberry, Vaccinium myrtillus L. (Ericaceae), grows in the north of Europe and
is one of the most common plants in Sweden and bilberry is known to contain
high amounts of anthocyanins.
The bilberry extract 25E, according to the manufacturer standardized for
containing 25% anthocyanins (the glucosides), showed a significantly inhibitory
effect on ACE activity in HUVEC. After incubation with Bilberry extract 25E
for 10 minutes, a significant and dose-dependent inhibition was seen with
61
0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/ml ***p<0.001 compared to the PBS
control.
The anthocyanidins (the aglycones) cyanidin, delphinidin and malvidin did not
show any significant inhibition of ACE activity, while the combination of these
three anthocyanidins in myrtillin chloride 0.05 mg/ml *p<0.05 showed
significant inhibition of ACE activity compared to the DMSO control.
Previous studies show effect of anthocyanidins on blood circulation, especially
in vision disorders such as retinopathy caused by diabetes or hypertension and
venous insufficiency (Detre et al., 1986; Favot et al., 2003; Matsumoto et al.,
2005). Its mechanism of action is proposed to be due to its antioxidative and free
radical scavenging properties (Kähkönen & Heinonen, 2003).
The effect of bilberry and its anthocyanidins on ACE activity has not previously
been studied. The effect on ACE activity showed a more pronounced effect
using the bilberry extract compared to the respective anthocyanidins (combined
in myrtillin chloride or separately). The difference in inhibition of ACE activity
between the anthocyanidins and/or the bilberry extract may be due to other
compounds than anthocyanins present in the bilberry extract, e.g. the stilbene
resveratrol and the carotenoid lutein. The anthocyanidin substances are not
water soluble in contrast to anthocyanins (anthocyanidin with sugar) in the
Bilberry extract and this makes the anthocyanidins difficult to study as isolated
substances. In this study, the Bilberry extract 25E was dissolved in PBS while
myrtillin chloride and the anthocyanidins were dissolved in DMSO, and this
organic solvent is most likely of importance for the outcome of the results on
ACE activity. The ability of the anthocyanidins to form the natural electron
deficient flavylium cation makes them very sensitive to changes in pH, and
sensitive also to temperature, oxygen and light (Kim et al., 2003).
Supplementation based on bilberry extract is used for improvement of vision
(Canter & Ernst, 2004), macular degeneration and diabetic retinopathy (Zafra-
62
Stone et al., 2007). As vascular factors contribute to the development of various
ocular diseases such as diabetic retinophaty, macular degeneration and
glaucoma, the inhibition of ACE activity by Bilberry extract in HUVEC might
indicate that bilberry has positive effects on the eye. Supplementation based on
bilberry often also contain other substances as lutein (a carotenoid) and Vitamin
A, which also might be/probably is involved in the proposed effect on vision by
bilberry supplement (see below, section “Beta-carotene, in vitro”).
Liqourice Extract, in vitro (Persson et al., 2008)
Liqourice is produced from the roots of Glycyrrhiza glabra L. (Fabaceae) and
most frequently used as a flavouring/sweetening agent in drugs, herbal teas,
tobacco and as sweets. Glycyrrhiza species contain two types of main chemical
substances; the triterpene saponins glycyrrhizic acid and glycyrrhizinic acid, and
flavonoids. More than 300 different flavonoids have been isolated from
Glycyrrhiza species, numerous isoflavones and other flavonoids (Li et al.,
2000).
After 10 minutes incubation of HUVEC with Glycyrrhizin extract, a significant
and dose-dependent inhibition of ACE activity was seen with 0.00625, 0.0125,
0.025, 0.05 and 0.1 mg/ml ***p<0.001 compared to the PBS control (Persson et
al., 2008). This implies that liquorice can have positive effects on cardiovascular
diseases. However, liquorice is contraindicated in patients with hypertension due
to its content of the triterpene saponins glycyrrhizin, although it has been
reported that liquorice is valid against cardiovascular diseases due to its
antiatherosclerotic and antioxidative effects (Aviram & Fuhrman, 1998;
Fuhrman et al., 2002). Liquorice can induce hypertension (Russo et al., 2000)
by interaction with the hormonal system, i.e. aldosterone and cortisol, involved
in blood pressure homeostasis (Schambelan, 1994). Liquorice intake may
decrease the release of aldosterone and markedly increase cortisol levels thereby
63
inducing pseudohyperaldosteronism resulting in severe hypertension and
hypokalemia. Glycyrrhizin is believed to be responsible for the effects of
liqourice on the adrenal glands (Hammer & Stewart, 2006). The chemical
structure of glycyrrhizin is related to the structure of steroidal hormones in
humans and like these hormones, liquorice i.e. glycyrrhizin affects RAAS by
inhibition of renin release (Sigurjonsdottir et al., 2006). Fuhrman et al. have
shown that liquorice extract free of glycyrrhizinic acid reduces systolic blood
pressure (Fuhrman et al., 2002), and Quaschning et al. have shown that
glycyrrhizic acid increases systolic blood pressure (Quaschning et al., 2001).
Taken together, these two previous studies and our results imply that the
flavonoid/isoflavonoid fraction of the plant may act as an ACE inhibitor. It is
well known that angiotensin II increases the release of aldosterone from the
adrenal glands. The mechanism by which liquorice inhibits aldosterone release
is unknown, but may be explained by the inhibition of ACE activity in HUVEC
seen in this study. In this study aldosterone did not show any significant effect
on ACE activity in HUVEC (see below, section “Sterols, in vitro” and Table 1),
while other studies have shown significant increase of ACE activity by
aldosterone (Sugiyama et al., 2005).
In conclusion, even if liqourice is contraindicated in hypertension, this study
demonstrates that liquorice extract inhibits ACE activity in HUVEC. These are
benifical indicators on the cardiovascular system and on the blood pressure.
Substances in liquorice responsible for these effects are not clarified, but most
likely it is the flavonoids/isoflavonoids.
Genistein, in vitro
Genistein is an isoflavone found in soya-beans Glycine max (L.) Merr.
(Fabaceae). Isoflavonoids are so called phytooestrogens, according to their
structural resemblance with oestrogen and oestrogen-like effects in humans.
64
After ten minutes incubation of HUVEC, a significant and dose-dependent
inhibition of ACE activity was seen with genistein 0.1 mg/ml *p<0.05, 0.5
mg/ml ***p<0.001 and 1 mg/ml **p<0.01 compared to the DMSO control.
Prevention of cardiovascular diseases mediated by antioxidative effects and by
lowering of cholesterol and LDL has been reported by soy protein containing
high amounts of isoflavonoids as genistein (Sirtori & Lovati, 2001, Clarkson,
2002).
The inhibition of ACE activity by genistein seen in this study has also been seen
in rats by others (Xu et al., 2006). In contrast to genistein, oestrogen showed no
inhibition of ACE activity in HUVEC.
This study indicates that isoflavonoids as well as flavonoids inhibits ACE
activity in vitro.
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF
HERBAL MEDICINAL PLANTS EXTRACTS
Panax Ginseng extract, in vitro (Paper II)
Panax ginseng L. (Araliaceae) is a traditional herbal plant from Asia. Panax
ginseng extract is made from root and/or rhizome and used as a so called
“adaptogen”. Adaptogens are referred to as natural plant products that are
proposed to balance the homoestasis of the body i.e. by increase of mental and
physical performance (Keifer & Pantuso, 2003).
After 10 minutes incubation with Panax ginseng G115 extract, a significant and
dose-dependent inhibition of ACE activity in HUVEC was seen with 5 and 10
mg/ml **p<0.01 compared to the PBS control.
The effects of Panax ginseng are traditionally(?) suggested to be due to the
triterpene saponins, the ginsenosides.
65
After ten minutes incubation with the triterpenes ginsenosides Rb1, Rf and Rg1,
no significant inhibition of ACE activity was seen compared to the DMSO
control. The ginsenosides Rb2, Rc and Rd significantly increased ACE activity
(Rb2 0.5 mg/ml **p<0.01 and 1 mg/ml *p<0.05, Rc 0.05 mg/ml **p<0.01 and 1
mg/ml ***p<0.001, Rd 0.1, 0.5 mg/ml *p<0.05 and 1 mg/ml **p<0.01), while
the ginsenoside Re 1 mg/ml *p<0.05 significantly inhibited ACE activity in
HUVEC compared to the DMSO control (Table 1, unpublished data).
Beside the rather non-specific aphrodisiac and adaptogenic effects (Nocerino et
al., 2000), Panax ginseng is also believed to reduce blood pressure (Kim et al.,
1994; Han et al., 1998; Jeon et al., 2000; Sung et al., 2000), act as an
antioxidant (Kim et al., 1992) and increase nitric oxide concentration (Gillis,
1997). The cardiovascular effects of Panax ginseng, the antioxidative
properties, are proposed to be linked to NO (Gillis, 1997; Nocerino et al., 2000)
(see below, section “Nitric Oxide, in vitro” and Table 2).
The ginsenoside Re was the only ginsenoside to inhibit ACE activity, while
Rb2, Rc and Rd significantly increased ACE activity in HUVEC. This increase
of ACE activity seen in HUVEC could either be due to increased biosynthesis of
ACE in the endothelial cells or a direct increase of ACE activity. The
differentiation in effect of the ginsenosides on ACE activity in HUVEC might
be due to the location of the different glycosides attached to the aglycone
ginsenoside structures. There are divergent opinions about however ginseng is
appropriate as treatment of cardiovascular diseases (Coon & Ernst, 2002;
Messina, 2006) and a possible side effect reported for Panax ginseng is
hypertension (Coon & Ernst, 2002). The ginsenosides are triterpene saponins
and the chemical structure of the triterpenes is comparable to a steroid structure
and triterpenes are known to interact with the hormonal system (see above,
section “Liqourice Extract”). Hypertension due to Panax ginseng may be due to
increased ACE activity by the ginsenoside Rb2, Rc and Rd (seen in this study)
66
or the ginsenosides affecting the adrenal glands; a proposed possible mechanism
of the adaptogenic action of Panax ginseng is the augmentation of adrenal
steroidogenesis (Nocerino et al., 2000). Studies show that Panax ginseng may
slightly decrease blood pressure (Siegel, 1979) while other studies show
increase in blood pressure (Coon & Ernst, 2002). This discrepancy might
depend on the amount of ginsenosides and flavonoids in the extract tested.
Although, there is a standardized extract of Panax ginseng the amount of
respective compound may vary. Panax ginseng G115 extract is standardized
after the amount of ginsenosides (4%); however, the amount of and the
characterization of the flavonoids are usually not documented. The inhibitory
effect of Panax ginseng G115 on ACE activity in HUVEC may be associated
with the amount of flavonoids present in the extract. This conceivable inhibition
of ACE activity by the flavonoids in the Panax ginseng extract may interact
with the increase of ACE activity by the ginsenosides resulting in opposite
effects on ACE activity. Whether the inhibition of ACE activity by Panax
ginseng extract or the increase of ACE activity by the ginsenosides Rb2, Rc and
Rd seen in vitro also occur in vivo is dependent on the bioavailability of the
respective substances.
Ginkgo Biloba extract, in vitro (Paper III)
The standardized extract EGb 761 of Ginkgo biloba L. (Ginkgoaceae) is
prepared from leaves of the Ginkgo biloba tree growing in Asia. Ginkgo biloba
is the only living representative of the Ginkgoales.
After 10 minutes incubation with Ginkgo biloba EGb 761 extract, a significant
and dose-dependent inhibition of ACE activity in HUVEC was seen with 5 and
10 mg/ml ***p<0.001 compared to the PBS control.
67
The extract, EGb 761 is standardized concerning the content of the presumed or
proposed most active substances; 6% terpene lactones (ginkgolides A, B, C, J
and bilobalide), 24% flavonol glycosides (principally derivatives of quercetin,
kaempferol and isorhamnetin), 7% proanthocyanidins (e.g. procyanidin), 13%
carboxylic acids, 2% catechins and <0.005% ginkgotoxin (Van Beck, 2002;
Mesbath et al., 2005; Xie et al., 2006).
No significant effect on ACE activity in HUVEC was seen with the diterpenes
ginkgolides A, B, C or the sequiterpene bilobalide in contrast to the Gingko
biloba extract EGb 761.
The flavonol quercetin present in Gingko biloba extract was shown to affect
ACE activity. After ten minutes incubation with the flavonol quercetin a
significant inhibition of ACE activity in HUVEC was seen with 0.1 mg/ml
*p<0.05, 0.5 mg/ml ***p<0.01 and 1 mg/ml ***p<0.001 compared with the
DMSO control. This inhibition of ACE with quercetin is also described in other
studies (Häckl et al., 2002; Loizzo et al., 2007).
Procyanidin did not significantly affect ACE activity in HUVEC compared to
the DMSO control. However, there was a significant difference **p<0.01
between the concentrations 0.1 and 1 mg/ml of procyanidin.
Ginkgo leaves, but not the standardised extract EGb 761, also contain small
amounts of the biflavone sciadopitysin (Van Beck, 2002). Sciadopitysin did not
show any significant effect on ACE activity in HUVEC compared to the DMSO
control.
Ginkgo biloba is considered to have cardioprotective effects (Liebgott et al.,
2000; Pietri et al., 1997), antioxidative effects (Pietri et al., 1997), increase nitric
oxide concentration (Koltermann et al., 2007) and effects on cerebral blood flow
(Ahlemeyer & Krieglstein, 2003), dementia (DeKosky, 2006) and cognition
(Ward et al., 2002).
68
The inhibitory effect of Ginko biloba extract EGb 761 on ACE activity in
HUVEC is suggested to be associated with its content of flavonoids. This
inhibition of ACE activity may be one explanation of the pharmacological
effects of Ginkgo biloba on the cardiovascular system. Mechanisms related to
the effects of Ginkgo biloba on dementia and Alzheimer´s desease are related to
the increase of NO and to NO as a scavenger (Bastianetto & Quirion, 2002;
DeKosky et al., 2006) and antioxidant resulting in reduction of age-related loss
of cognitive functions (Ward et al., 2002). Studies have been performed with the
ginkgolides and the ginsenosides on cognitive functions like learning and
memory, showing that the ginkgolides may have effects in vitro and in animal
models (Birks & Grimley Evans, 2008) and that the ginsenosides are possibly
interacting with the steroid hormone receptors (Leung et al., 2007). However,
inhibition of ACE activity will increase local blood flow (in the CNS).
Furthermore, as ACE activity i.e. angiotensin II, III and IV, is involved in
cognitive functions (Chaki & Inagami, 1992; Ruiz-Ortega et al., 2001; Gard,
2004; Bodineau et al., 2008), the inhibition of ACE activity might also, at least
partly explain the proposed effects of Ginkgo biloba and Panax ginseng on
cognitive functions. Studies have been performed on flavonoids concerning
effect on behaviour and cognition showing a possible restoring beta-amyloid-
induced effect by epigallocatechingallate (found in tea) (Rasoolijazi et al.,
2007), and antioxidative effects altering stress signalling and neuronal
communication by vegetables and fruits containing polyphenols (Joseph et al.,
2007; Shukitt-Hale et al., 2008; Williams et al,. 2008). It has also been shown
that liqourice extract may improve learning and memory in mice (Dhingra et al.,
2004). There is shown to be a relationship between cardiovascular diseases and
emotional expressions like anger, hostility and depression (Sirois & Burg, 2003;
Davidson, 2008). A diversity of emotional expressions derives as a result of
cardiovascular disease, as part of a coping strategy. There is also a physiological
interaction between emotions like depression and cardiovascular diseases due to
69
e.g. the impact on RAAS, the importance of serotonin and the immune system
and dysfunction in the hypothalamic-pituitary-adrenocortal and
sympathoadrenal axis in response to stress (Bondy, 2007). Like cardiovascular
diseases, depression is a common disorder in developed countries and there is a
psychological as well as physiological connection between the brain, the
endrocrine system (including RAAS) and the immune system. Is there a
connection between cardiovascular diseases and mental disorders e.g. depression
and diet? An improvement of quality of life has been reported in patients using
ACE inhibitors and angiotensin receptor blockers (Maggioni, 2006) as well as
with a vegan diet (Link et al., 2008).
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF
THE ANTIOXIDANTS ALPHA-TOCOPHEROL AND BETA-CAROTENE
Alpha-Tocopherol, in vitro
The diterpene α-tocopherol showed no significant effect on ACE activity in
HUVEC.
Alfa-tocopherol is associated with antioxidative effects (Matringe et al., 2008).
However, α-tocopherol does not seem to lower cardiovascular mortality (Buijsse
et al., 2008). The fact that a substance is an antioxidant may not prove that the
same substance will prevent cardiovascular diseases. It could be speculated that
to have positive effects on cardiovascular diseases, an antioxidative effect is not
sufficient. Some additional mechanism of action may be required, perhaps ACE
inhibition.
70
Beta-Carotene, in vitro
The carotenoids are tetraterpenes and have some characteristics in common with
the flavonoids; they are plant-pigments present in the plastid. The carotenoids
derive from a different biosynthesis pathway compared to the flavonoids and in
contrast to the flavonoids, the carotenoids are lipid-soluble substances.
After ten minutes incubation with β-carotene, a significant and dose-dependent
inhibition of ACE activity in HUVEC was seen with 0.5 mg/ml **p<0.01 and 1
mg/ml ***p<0.001 compared to the DMSO control.
Carotenoids have principal functions as antioxidants in plants, and β-carotene is
the most common of the carotenes. In humans, the effect of β-carotene is related
to its antioxidant activity in prevention of a variety of diseases e.g.
cardiovascular diseases (Bjelakovic et al., 2008). The use of β-carotene as
antioxidative supplementation is debated as it has been shown that treatment
with β-carotene as well as α-tocopherol may increase mortality in various
diseases (Voutilainen et al., 2006; Bjelakovic et al., 2008).
Beta-carotene was the only non-flavonoid substance tested with a strong
inhibitory effect on ACE activity. Approximately 60 carotenoids (carotenes and
xanthophylls) have been found in human diet and few of these are present in
blood in measurable amounts e.g α-, β-carotene, lycopene, β-cryptoxanthin,
lutein and zeaxanthin. Do all carotenoids inhibit ACE activity or is it just β-
carotene? The difference in chemical structure between the carotenoids is;
acyclic or having one or both ends modified into rings, containing at least one
oxygen atom, or hydrocarbon atom (chemical structure of β-carotene is shown in
Table 1). The chemical similarities with flavonoids are the OH-groups and the
double bondings. From a speculative point of view probably all carotenoids
inhibit ACE activity, at least in vitro. To confirm this speculation further in
vitro/in vivo studies on ACE activity have to be performed.
71
The bioavailability of carotenoids is highly variable, dependent on e.g. species,
amount consumed in a meal, genetic factors and interactions. Food processing,
as cooking (β-carotene is absorbed 65% from cooked carrots and 41% from raw
carrots), and intake of fat improve the bioavailability of the carotenoids. The
bioavailability of carotenoids as supplements is usually higher than of
carotenoids in fruits and vegetables (Yonekura & Nagao, 2007). Beta-carotene is
the carotene best known for its function as a vitamin A precursor and vitamin A
is mainly studied for its role in eye function and vision.
After ten minutes incubation with β-carotene a significant inhibition of ACE
activity in vitro in the eye retinal pigmented epithelium cell line ARPE-19 was
seen with 0.5 mg/ml *p<0.05 and 1 mg/ml **p<0.01 compared with DMSO
control (figure 6; unpublished data).
Figure 6. ACE activity in ARPE-19 after incubation with β-carotene for 10 minutes. Statistics were calculated with one way ANOVA repeated measures and denoted as *p<0.05 and **p<0.01 compared with DMSO, n=3.
Beta-carotene
DMSO
0.1
mg/m
l
0.5
mg/m
l
1 m
g/ml
0.0
0.5
1.0
1.5
*
**
AC
E a
ctiv
ity (U
)
72
Carotenoids have been studied concerning effects on macular degeneration,
indicating a protective effect of carotenoid-rich food especially lutein and
zeaxanthin (Seddon, 2007). Decrease in ocular blood flow is one risk factor for
macular degeneration (Pemp & Schmetterer, 2008). Macular degeneration is a
common condition in the elderly and exists in two forms; as a cause of cellular
debris and angiogenesis of blood vessels from the choroid. Diabetic retinophaty
is described as changes in the retinal microvessels that can lead to macular
edema and vision loss. The major components of RAAS have been identified in
eye tissue and hypertension is a risk factor for diabetic retinopathy and local
formation of angiotensin II ensure local control of blood flow in e.g. the eye
(Ganten et al., 1971).
Activation of the angiotensin II receptors AT1 is proposed to be involved in
diabetic retinopathy (Clermont et al., 2006). ACE inhibiting drugs are shown to
inhibit capillary degeneration and to block the progress of diabetic retinopathy
(Clermont et al., 2006). The inhibition by β-carotene of ACE activity on ARPE-
19 in vitro is an indication of a possible mechanism of carotenoids on vision
disorders.
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF
STEROLS AND BLOOD LIPID-LOWERING DRUGS
Sterols, in vitro
So far, β-carotene, except for the ginsenoside Re, is the only lipid tested that
significantly inhibited ACE activity in HUVEC. Due to this we also tested the
sterol lipid, the phytosterol stigmasterol, cholesterol, lanosterol, the human
steroids oestradiol, testosterone and the mineralcorticoid aldosterone.
73
After ten minutes incubation with the sterols, no significant inhibition of ACE
activity in HUVEC was seen with stigmasterol, oestradiol, testosterone or
aldosterone compared to the DMSO control, nor with cholesterol compared to
the PBS control. Lanosterol significantly increased ACE activity in HUVEC 10-3
M **p<0.01 compared to the DMSO control. In this study, aldosterone did not
show any significant effect on ACE activity, while another study has shown a
significant increase of ACE activity by aldosterone (Sugiyama et al., 2005).
Blood lipid –Lowering Drugs, in vitro
In relation to the results on ACE activity in HUVEC in this study by the sterols
and their previously shown effects on the cardiovascular system by lowering
cholesterol levels (Klingberg et al., 2008; Poli et al., 2008), the blood lipid-
lowering drugs pravastatin and simvastatin were tested on ACE activity in
HUVEC.
After ten minutes incubation with the bloodlipid-lowering drugs pravastatin and
simvastatin, a significant increase of ACE activity was seen in HUVEC, 10-7 and
10-6 M *p<0.05, 10-7 M, **p<0.01 and 10-6 M *p<0.05, respectively.
These drugs are used as treatment of cardiovascular diseases by lowering
bloodlipid levels but might, as this study shows, increase ACE activity. This
might be a negative side effect of blood lipid-lowering drugs on the
cardiovascular system.
EFFECTS ON ANGIOTENSIN-CONVERTING ENZYME ACTIVITY OF
PRECURSOR MOLECULES
To investigate if the effect on ACE activity were associated to the biosynthesis
of plant substances (a biosynthesis-activity connection) the precursor molecules
involved in the biosynthesis of flavonoids, and the phenol salicin derived solely
from the shikimic pathway (figure 1) were studied.
74
After ten minutes incubation with the precursors, mevalonic acid, malonic acid,
shikimic acid, chorismic acid and the progenitor squalene, no significant effect
on ACE activity was seen in HUVEC compared to the DMSO control. Neither
did salicin show any significant effect on ACE activity after ten minutes
incubation of HUVEC compared to the PBS control. Studies show an inhibiting
effect on platelet aggregation/adhesion by flavonoids (Nardini et al., 2007; Babu
& Liu, 2008). This effect on platelets probably derives from the shikimic acid
pathway and coumaric acid, which is the connection between salicin (the
ancestor of acetylsalicylic acid) and the flavonoids (see figure 1).
THE MECHANISM OF THE INHIBITORY EFFECT ON ANGIOTENSIN-
CONVERTING ENZYME ACTIVITY OF THE FLAVONOIDS
Epidemiological studies have shown reduced cardiovascular morbidity among
tea drinkers (Mukamal et al., 2002; Hakim et al., 2003; Babu & Liu, 2008). It
has been proposed that this positive effect is due to the antioxidative effect of tea
catechins. Previously shown effects of green tea and black tea include
antioxidative (Locher et al., 2002; Kurihara et al., 2004), antiproliferative and
anti-angiogenic activity (Locher et al., 2002; Oak et al.,2005) and nitric oxide
synthase activation (Nakagawa & Yokozawa, 2002; Anter et al., 2004; Lorenz et
al., 2004). Pharmacological effects attributed to Rooibos tea are antioxidative
activity (Standley et al., 2001; Lee & Lang, 2004) and immunostimulatory
effects (Lambert & Yang, 2003).
Cacao contains catechin-related flavonoids as procyanidins and catechins as in
tea and coffee. Cacao is proposed to play a beneficial role on the cardiovascular
system as a anti-inflammatory modulator (Selmi et al., 2008) and procyanidins
found in cacao are shown to have antioxidative effects (Keen et al., 2005; Aron
et al., 2008).
75
The inhibition on ACE activity in HUVEC shown in this study may explain the
positive effects of green tea and black tea seen in habitual tea drinkers. The
results seen in this study on ACE activity in HUVEC using tea as well as cacao
and coffee add an additional and novel mechanism to the antioxidative effect;
flavonoids i.e. catechins as ACE inhibitors.
Do all flavonoids inhibit ACE activity and which is the possible molecular
mechanism of the ACE inhibitory action of the flavonoids and β-carotene?
Flavonoids are known to bind to enzymes and proteins (Andersen & Markham,
2006). Some proteins need additional components for their functions, So called
co-factors. In plants, as well as in humans, co-factors may be metal ions, e.g.
Fe2+ or Mg2+. Flavonoids as a group are known to have metal chelating capacity
to ions like Fe, Cu and Al (Raven, Evert and Eichhorn, 2003; Kostyuk et al.,
2001). The major flavonoid in liquorice, the isoflavone glabridin is known to
have metal chelating capacity (Belinky et al., 1998). ACE have two active sites
containing Zn2+ ion and the pharmacological mechanism of medicinal drugs
inhibiting ACE may be their ability to bind to the Zn2+ at the active site of ACE
and thereby decreasing its activity (Berecek & Zhang, 1995). Metal ions like
Mg2+ and Fe2+ are also involved in the biosynthesis of flavonoids. The
biosynthesis of the flavonoid precursor chalchone, the flavanols, the isoflavanols
and the anthocyanins are dependent on enzymes with metal chelating activity
(Andersen & Markham, 2006). A common feature of the biosynthesis of
flavanols (catechins), flavonols, isoflavanols and anthocyanins is that the
enzymes involved, flavanone 3β-hydroxylase (F3H), and anthocyanidin
synthase (ANS), bind and are dependent on Fe2+. Also Mg2+ functions as a
cofactor in the biosynthesis, for acetyl-CoA carboxylase (ACC) to form
malonyl-Co-A. It is possible that the flavonoid substrates bind to these enzymes
via the metal ions. The mechanism of this enzyme- and protein binding,
including the mechanism of the ACE inhibition, is so far speculative. However,
76
it seems that the flavonoids i.e. flavanols (catechins), flavonols (e.g.quercetin)
and anthocyanins do have the potential to bind Zn2+ at the active site of the
ACE. The flavonoids are relatively small chemical structures, with the ability to
form covalent bondings (Andersen & Markham, 2006). Furthermore, the
phenolic nucleus is a structural unit suitable for molecular, non-covalent
interactions with proteins related to the double bondings and OH-group(s) of the
flavonoids (Andersen &Markham 2006). This might be one explanation of the
ACE inhibitory effect of the flavonoids seen in this study.
In fact, flavonoids are known to be metal chelaters (Kostyuk & Potapovich,
1998; Kostyuk et al., 2001; Havsteen, 2002), to bind metal ions like Fe2+ and
Cu2+. It seems that flavonoids are involved in the resistance and protection of
plants against exposure to high levels of metals in soils (Havsteen, 2002;
Moreno et al., 2003). Furthermore, plants exposed to high levels of e.g.
aluminium have been found to synthesize high levels of flavonoids (Riesenberg
& Soltis, 1987). For instance, bilberries grow in soil contaminated with metal
ions like Zn2+ without being affected by this (Vallino et al., 2005). The metal-
chelating effect of the flavonoids might explain the ACE inhibiting effect of
these substances. Many enzymes, as well as ACE, contain Zn2+ due to the ability
of Zn2+ to easily react with other compounds. These enzymes are said to belong
to the zinc metallopeptidase family. Other zinc metallopeptidases except ACE
are e.g. aminopeptidase A (AMPA), aminopeptidase N (AMPN), insulin-
regulated aminopeptidase (IRAP), endothelin-converting enzyme (ECE), matrix
metallopeptidase (MMP), neutral endopeptidase (NEP) and insulin-degrading
enzyme (IDE). Peptidases are classified according to catalytic activity and
converged properties (Rawlings & Barrett, 1993). AMPA converts angiotensin
II to angiotensin III, AMPN converts angiotensin III to angiotensin IV, and
angiotensin IV activates IRAP which action may play a role in the development
of complications in type 2 diabetes (Keller, 2004). ECE catalyses the synthesis
77
of endothelin whith function as a vasoconstrictor, bronchoconstrictor and
stimulator of aldosterone secretion (Barton & Yanagisawa, 2008). MMPs are
involved in cell growth and tissue destruction in various diseases e.g myocardial
restenosis and macular degeneration (Marder & Greenwald, 2003; Dormán et
al., 2007). NEP catalyses the degradation of a variety of renal and CNS-active
peptides including substance P, bradykinin, enkephalins, atrial natriuretic
peptides (ANP), endothelin and angiotensin II (Aulakh et al., 2007). IDE,
degrading a range of substrates e.g. insulin, is a zinc metalloendopeptidase
(Authier et al., 1996), an evolutionary conserved neutral thiol-
metalloendopeptidase with structural similarities to NEP. If the inhibitory
mechanism of flavonoids on ACE activity is due to their ability to bind to Zn2+
ions then it would be possible for the flavonoids to also inhibit ECE, MMP, NEP
and maybe IDE, thereby exerting several additional positive effects on the
cardiovascular system. Kiss et al. have shown that Ligustrum vulgare L.
(Oleaceae) extract inhibit ACE and NEP in vitro (Kiss et al., 2008). Ligustrum
vulgare contain the flavonols kaempferol and quercetin (Romani et al., 2000).
No in vivo studies have been performed investigating the effects of flavonoids
on NEP, MMP, ECE or IDE.
In this study, β-carotene was the only non-flavonoid substance, except from the
ginsenoside Re, with a strong inhibitory effect on ACE activity.
Common features of the flavonoids and β-carotene are; they are plant pigments,
present in the plastid and antioxidants. It is unknown whether β-carotene has
metal-chelating properties and thereby exhibits ACE inhibition. The enzyme β-
carotene hydroxylase, like the enzymes involved in the biosynthesis of
catechins, flavonols, isoflavanols and anthocyanins, involved in the biosynthesis
of β-carotene is a Fe dependent enzyme. If β-carotene is a metal-chelator, the
metal-chelating mechanism could be the common feature of the plant-derived
78
substances affecting ACE activity, and thereby even all of the zinc
metallopeptidases.
Part of the aim of this study was to investigate if the sole mechanism according
to flavonoids and their protection against cardiovascular diseases is their
antioxidative effect. The results presented in this study show that ACE inhibition
is an additional mechanism.
Seen from an evolutionary perspective the flavonoids developed simultaneously
as vascular plants, approximately 440-430 million years ago (Raven et al.,
2003). Anthocyanins have been found in various orders of bryophytes (mosses),
the most primitive group of land plants, estimated 470 million years of evolution
(Gould, 2002). At this time the formation of soils was established. Between
approximately 470-430 million years ago plants went through a time of major
innovations: the colonization of land, development of a specialized system to
distribute water, solutes, photosynthetic products and the development of a
sexual reproductive system (Willis & McElwain, 2002). Seen from the
perspective of this study; it might be of importance that flavonoids developed
simultaneously as vascular plants, and are interacting with and affecting the
cardiovascular system as ACE inhibitors.
EFFECTS ON NITRIC OXIDE
Nitric oxide, in vitro (Paper I, II, III)
As the results on ACE activity showed, in some cases, a strong inhibition by the
flavonoids in vitro, it was interesting to investigate if there was a connection
between the effects on ACE activity, the antioxidative effects and the effects on
NO concentration.
79
After 24 hours incubation with the flavonoids and flavonoid-containing extracts,
a significant increase in NO concentration was seen in HUVEC with two of the
catechins, epigallocatechin 1 and 2 mg/ml **p<0.01 and epigallocatechingallate
0.5, 1 and 2 mg/ml ***p<0.001. Further, a significant increase in NO
concentration was seen with procyanidin 0.5 and 1 mg/ml **p<0.01, quercetin
0.1 mg/ml *p<0.05, 0.5 and 1 mg/ml ***p<0.001 and the biflavane
sciadopitysin 0.5 and 1 mg/ml ***p<0.001. Results were compared to DMSO
control.
After 24 hours incubation with plant-extracts containing high amounts of
flavonoids, a significant increase of NO concentration in HUVEC was seen with
the green tea 1:1600, 1:800, 1:400 and 1:200 ***p<0.001, black tea 1:800, 1:400
and 1:200 ***p<0.001, Rooibos tea 1:400 *p<0.05 and 1:200 ***p<0.001,
coffee extract 1:200 *p<0.05, cacao extract 100µg/ml ***p<0.001, liquorice
extract 50 and 100µg/ml ***p<0.001, Ginkgo biloba extract 5 and 10 mg/ml
***p<0.001 and Panax ginseng extract 0.5, 1, 5 and 10 mg/ml *p<0.05. Results
were compared to PBS control.
Other compounds significantly affecting NO concentration in HUVEC, apart
from the flavonoids, were caffeine increasing NO concentration, 1 mg/ml
*p<0.05, and the sesquiterpene bilobalide decreasing NO levels 0.5 mg/ml
*p<0.05 and 1 mg/ml ***p<0.001 compared to DMSO control.
Plants and plant-derived substances like green tea, black tea and various
catechins, cacao and procyanidins, bilberry and other anthocyanin-containing
berries, quercetin and genistein and their effect on NO concentration is a rather
well documented field of research (Squadrito et al., 2002; Hodgson, 2006;
Taubert et al., 2007; DalBó et al., 2008; Erlund et al., 2008; Faridi et al., 2008;
Grassi et al., 2008a; Hall et al., 2008; Loke et al., 2008; Nantz et al., 2008). NO
is known to influence the vascular system, and the increase in NO concentration
by flavonoids (Grassi et al., 2008b) will result in vasodilatation by relaxation of
80
smooth muscle cells (Squadrito et al., 2002; Hodgson, 2006; DalBó et al., 2008;
Grassi et al., 2008a; Hall et al., 2008), inhibition of smooth muscle cell
proliferation, reduced platelet aggregation (Erlund et al., 2008), reduced platelet
and monocyte adhesion to the endothelium, inhibition of LDL oxidation (Erlund
et al., 2008; Nantz et al., 2008,) and blood pressure reduction (Erlund et al.,
2008; Faridi et al., 2008; Grassi et al 2008; Nantz et al., 2008). An increase of
NO concentration by the flavonoids would thereby decrease the risk of
developing cardiovascular diseases (Napoli et al., 2006, Yetic-Anacak &
Catravas, 2006).
The results in this study showing an increase in NO concentration with the
flavonoids and flavonoid-containing extracts are in accordance with these
previous reports.
Is the increase in NO levels from HUVEC seen in this study related to an
increase of the cellular synthesis or to the nitrate present in the extracts
themselves? Studies have shown that inorganic nitrate found in vegetables and
fruits have the ability to convert to nitrite and NO in humans (Lundberg &
Govoni, 2004; Lundberg & Weitzberg, 2005; Lundberg et al., 2006). As shown
in Table 2, green tea, black tea, Rooibos, Ginkgo biloba and Panax ginseng
extracts themselves contain both nitrite and nitrate, and this could explain part of
the effect seen on NO concentration. However, the cacao extract did not contain
any nitrate but showed a significant increase in NO concentration from HUVEC
(Table 1). Furthermore, the Panax ginseng extract contains high amounts of
nitrate but did not show any significant increase on NO concentration after 10
minutes incubation (Paper I) and a significant but small increase, p<0.05 of NO
concentration after 24 hours incubation (Table 1). This indicates that the
increase in NO concentration seen with the flavonoids and extracts containing
high amounts of flavonoids is due to increases in cell-mediated NO
concentration and not nitrite/nitrate concentration of the extracts.
81
Table 2. The quantity of nitrite, nitrate and nitrite/nitrate in green tea, black tea, Rooibos, cacao extract, Ginkgo biloba extract and Panax ginseng extract compared to PBS.
Extract Nitrite µM Nitrate µM
Nitrite/Nitrate µM
PBS 0.32 0.53 0.85 Green tea 1:200 0.87 2.96 3.83 Green tea 1:800 0.74 2.73 3.47 Green tea 1:3200 0.42 2.57 2.99 Black tea 1:200 1.70 2.72 4.42 Black tea 1:800 0.42 3.29 3.71 Black tea 1:3200 0.32 3.15 3.47 Rooibos tea 1:200 1.93 2.37 4.30 Rooibos tea 1:800 0.64 1.76 2.40 Rooibos tea 1:3200 0.32 1.72 2.04 Cacao 0.1 mg/ml 0.32 0.53 0.85 Cacao 0.05 mg/ml 0.32 1.36 1.68 Cacao 0.025 mg/ml 0.32 1.36 1.68 Ginkgo biloba 10mg/ml 5.57 12.43 18.00 Ginkgo biloba 5mg/ml 3.21 13.36 16.57 Ginkgo biloba 1mg/ml 0.87 8.20 9.07 Gingko biloba 0.5mg/ml 0.32 4.93 5.25 Panax ginseng 10mg/ml 0.64 139.17 139.81 Panax ginseng 5mgml 0.42 66.04 66.46 Panax ginseng 1mg/ml 0.32 12.32 12.64
82
Nitric oxide, in vivo (Paper V)
After oral intake of 400 ml green tea, black tea or Rooibos tea, no significant
change in serum NO concentration was seen neither in the entire group of
volunteers nor in any of the genotype sub-groups.
Increased NO concentration may be the result of either an increased
synthesis/production due to increased NOS activity, or a prolonged half life of
NO. Increased NOS activity would result in a slow effect; the increase in NO
concentration in HUVEC was shown after incubation for 24 hours compared to
incubation for 10 minutes (Paper I, II and Table 1). Nicholson et al. has shown
that the stilbene resveratrol (figure 2) and quercetin affect the expression of the
gene encoding eNOS (Nicholson et al., 2008). Prolonged half life of NO may be
due to decreased metabolism of NO. NO is metabolised by reaction with O2-
forming peroxynitrit (Doughan et al., 2008). Flavonoids scavenging O2-, as
proposed in various studies (Sun et al., 2002; Chang et al., 2007; Jiménez et al.,
2007; Tomer et al., 2007), thereby inhibit the metabolism of NO and this
mechanism would result in rapid effect on NO concentration. Whether
flavonoids excert their antioxidative effects by scavenging free radicals or by
metal-chelating properties the effects will be the same. There are several known
connections between ACE and NO. NO inhibits ACE (Ackermann et al., 1998;
Persson et al., 2005) and downregulates AT1 receptors (Ichiki et al., 1998). ACE
degrades bradykinin, thereby inhibiting its stimulation of NO synthesis. Also,
angiotensin II activates NADH oxidase thereby increasing the synthesis of the
NO-scavenging radical O2-.
83
INTERACTIONS BETWEEN DIETARY PRODUCTS AND DRUGS
So far, interactions between herbal medicines and medicinal drugs are a rather
unexplored field. Results of this study showed a synergistic effect on ACE
activity in HUVEC by ginseng extract and the ACE inhibiting drug enalaprilat,
(Paper II) and by ginkgo biloba extract and enalaprilat (figure 7; unpublished
data). This indicates a possible interaction between ACE inhibiting drugs and
herbal medicines containing high amount of flavonoids with effect on ACE
activity.
Figure 7. ACE activity in HUVEC after incubation for 10 minutes with enalaprilat (E) alone
or in combination with Ginkgo biloba (GB) extract (ACE activity in PBS control 12.8±3.1 U),
n=3. Statistics calculated with one way ANOVA repeated measures and denoted as **p<0.01,
n=4 from different individuals.
-10 M
Enalap
rilat
10
M+G
B 0.1 m
g/ml
-10
E 10M
+GB 0.
5 mg/m
l
-10
E 10
GB 1.0 m
g/ml
M+
-10
E 10
M+G
B 5.0
mg/m
l
-10
E 10
M+G
B 10.0
mg/m
l
-10
E 10
0
5
10
15
****
AC
E a
ctiv
ity (U
)
84
As reported by Harris et al. (2003), a number of food-drug interactions in liver
metabolism have been reported (Harris et al., 2003). Food containing complex
mixtures of phytochemicals, e.g. vegetables, fruits and tea is more likely to
induce or inhibit the activity of drug-metabolising enzymes, and particularly
large interactions may occur due to intake of herbal dietary supplements (Harris
et al., 2003). Cytochrome P450 appears to be especially sensitive to dietary
effects (Cermak, 2008) and food-drug interactions may occur when food
substances use the same metabolizing enzymes as medicinal drugs. There are
indications on changes in gene expression of CYP 450 according to drug
metabolism (Ek et al., 2007) and this might also correspond to food intake.
Flavonoid-induced effects on drug bioavailability have also been reported
(Cermak, 2008).
CLINICAL IMPORTANCE
According to the WHO Statistical Information System (WHOSIS),
cardiovascular diseases is the number one cause of death and an estimated 20
million people will die from cardiovascular diseases in 2015; this will represent
30% of all global deaths (WHOSIS). The results of this investigation on plant-
derived substances inhibiting ACE activity and increasing NO concentration
could lead to new to ACE inhibiting drugs. Examining and mapping which
foods, i.e. vegetables, fruits and berries that contain these substances should lead
to specific diet recommendations concerning prevention and treatment of
cardiovascular diseases. ACE inhibiting drugs are used for treatment of
hypertension (Lim, 2007), congestive heart failure (Lim, 2007), ventricular
dysfunction (Lakhdar et al., 2008) and nephropathy in diabetes mellitus (Koitka
& Tikellis, 2008). Dependent on flavonoid(s) and/or carotenoid content,
vegetables, fruits and berries, containing plant-derived ACE inhibitors could be
an addition to medical drugs; food as medicine. Diet in the perspective of
85
lifestyle and cardiovascular diseases as a major cause of morbidity and mortality
is a combination of great interest, psychologically and physiologically. The
concomitant change in diet behavioural pattern and worldwide treatment of
cardiovascular diseases with medicinal drugs is probably not a mere accident.
Why do we eat; essentially for the same reason as we take medicinal drugs, to
survive and secondary, to stay healthy. Today, it seems that we eat to satisfy the
emotions and take medicinal drugs to survive and stay healthy.
Furthermore, flavonoids and β-carotene, due to inhibiting ACE activity, might
also increase blood flow and thereby positively affect cardiovascular diseases
and cognitive abilities.
Seen from a nutritional perspective, the role of the flavonoids in health and
disease is not clarified. As opposed to other well-known/recognized plant-
derived micronutrients e.g. Vitamins, lack of flavonoids does not induce obvious
deficiency syndroms. The initial classification of flavonoids as Vitamin P,
according to the action on vascular permeability and fragility (Rusznyak &
Szent-Györgyi, 1936) was withdrawn (Kühnau, 1976), and today flavonoids are
not classified as Vitamins, due to their lack of obvious deficiency syndroms.
Early analysis and identification of flavonoids in vegetables and fruits lead to an
estimated recommended dosage of a total of 1 g/day (Kühnau, 1976), distributed
between flavanones, flanonols and flavones (160-175 mg/day), anthocyanins
(180-215 mg/day), catechins (220 mg/day) and biflavans (460 mg/day)
(Kühnau, 1976). This estimated intake of flavonoids was applied until the 1990s,
when new results indicated this to be a highly overestimated dietary flavonoid
intake due to the accually flavonoid intake. An estimated daily intake of
flavonoids in Western population is about 100-200 mg (Manach et al., 2004).
However, considering the great number of flavonoids present in vegetables,
fruits and berries, an intake of 1 g per day could easily be acheived.
86
The Swedish National Food Administration recommends 500 g intake of
vegetables and fruits per capita and day, distributed over 5 times a day. This
recommendation is based on what is considered necessary for maintaining or
improving health and general well being, even if secondary metabolites from
plants i.e. flavonoids is not considered as essential nutritients. Essential (Latin,
unknown) nutrients are often described as necessary to maintain normal life
or/and normal health and must be administered to the body via food (Swedish
National Food Administration; WHO). So far, vitamins and minerals are the
only minor food components derived from plant biosynthesis classified as
necessary/essential for maintaining and/or improving health and general well
being. However, do flavonoids have to be administered to the human body via
food and necessary for health? When it comes to essential nutritients as vitamins
and minerals, there is a recommended average intake to minimize the risk of
inadequate intake and a lowest recommended intake to avoid the risk of
developing deficiency symptom. According to the result of the questionnaire
(Paper V), average intake of vegetables and fruits was 3.16 times a day for the
participants of this study. A population based questionnaire in Sweden, showed
an average intake of vegetables and fruits of 3.0 times a day; less than 10% ate
vegetables and fruits 5 times a day, and 25% ate vegetables and fruits 3-4 times
a day (Sepp et al., 2004). According to this, there is an inadequate intake of
vegetables and fruits. Long term inadequate intake increases the risk of
developing defiency symptom e.g. inadequate intake of flavonoids will increase
the risk of cardiovascular diseases. Appropriate recommended average intake
values to regulate the intake of flavonoids might give substance to their
importance and involvement in maintaining and/or improving health and general
well being. It might also prevent intake of too high concentrations of flavonoids
in e.g. supplements, as high intake may give rise to side effects as kidney and/or
liver toxicity.
87
Statins are used as lipid-lowering drugs and according to guidelines of the
National Cholesterol Education Program (NCEP) statins play an important role
in prevention of coronary heart disease, myocardial infarction, stroke and
peripheral artery disease. Studies also show that statins appear to have effect on
inflammation (Jones & Farmer, 2008), dementia (Orr, 2008), cancer (Xie &
Itkowitz, 2008), nuclear cataracts (Bariciak & Nichols, 1995) and hypertension
(Cernes et al., 2008). The fact that the statins i.e. pravastatin and simvastatin
increase ACE activity in vitro, as shown in this study is to be considered as a
negative effect of these drugs. After ten minutes incubation with simvastatin and
pravastatin, a significantly increased ACE activity was seen in HUVEC.
Simvastatin 10-8, 10-6 M *p<0.05 and 10-7 M **p<0.01, pravastatin 10-7 and 10-6
M *p<0.05 compared to the DMSO control (Table 1). The pharmacological
mechanism of the effect of statins on ACE activity is unknown. Intake of
flavonoid-rich food might also as prevention or complement lipid-lowering
drugs since studies show that flavonoids lower lipids (Hwang et al., 2003;
Ikizler et al., 2007; Kaliora et al., 2007; Aron et al., 2008; Boots et al., 2008) as
well as is shown in this study inhibit ACE activity, while lipid-lowering drugs as
this study shows increase ACE activity in human endothelial cells. Is it possible
to replace lipid-lowering drugs with vegetables, fruits and berries containing
high enough doses of flavonoids, at least in patients with low to modest elevated
blood lipids?
88
SUMMARY
Flavonoids interact with the cardiovascular system in several ways, by reducing
ROS (as shown in several studies), increasing NO concentration (as shown here
and by others) and also by inhibiting ACE activity (as shown here).
In plants, flavonoids have antioxidative effects and metal chelating properties
and the antioxidative effects affect NO. There are two major mechanisms
contributed to antioxidative effects; free radical scavenging and metal chelating.
The two active sites of ACE contain Zn2+ and ACE inhibitors are designed to
bind to the Zn2+ at the active sites. The effects seen in this study on ACE activity
could be due to the metal chelating properties of the flavonoids. Substances
affecting ACE activity, NO concentration and thereby the cardiovascular system
will affect the individual as a whole; ACE as a part of the endocrine system and
NO as a part of different systems in the human body. Physiologically as well as
psychologically the endocrine system, the immune system and the
cardiovascular system are linked together.
Green tea and black tea infusions, cacao extract, Bilberry extract 25E, liquorice
extract, Ginkgo biloba extract EGb 761 and Panax ginseng extract G115 showed
significant inhibition on ACE activity in vitro.
The most potent ACE inhibitors in vitro of the plant-derived substances tested
were the flavonoids, i.e. epicatechingallate, epigallocatechin.
epigallocatechingallate, quercetin, genistein and the tetraterpene β-carotene.
In vivo, Rooibos tea significantly inhibited ACE activity.
In vivo, Green tea significantly inhibited ACE activity for the genotype II and
ID.
In vivo, Rooibos tea significantly inhibited ACE activity for the genotype II.
89
Among the terpenes tested, the tetraterpene β-carotene was the only one
showing a strong ACE inhibiting effect, i.e β-carotene was the only non-
flavonoid with a strong inhibitory effect on ACE activity.
Bilobalide, ginsenoside Rb2, Rc, Rd and the statins, i.e. pravastatin and
simvastatin increased ACE activity, in vitro.
Green tea, black tea and Rooibos tea infusion, cacao extract, liqourice extract,
Ginkgo biloba extract EGb 761 and Panax ginseng extract G115 significantly
increased NO concentration in vitro. The most potent plant-derived substances
increasing NO concentration in vitro were epigallocatechingallate, procyanidin,
quercetin and sciadopitysin. Bilobalide significantly decreased NO
concentration in vitro.
It has been proposed that the positive effects of flavonoids are due to the
antioxidative effects and NO effect. However, a substance with antioxidative
effects, do not automatically have to prevent cardiovascular diseases, e.g. α-
tocopherol impliying an additional mechanism. Our results on ACE activity
show an additional important mechanism; extracts containing high amounts of
flavonoids and flavonoids as ACE inhibitors.
90
Table 1. A
CE
activity and N
O concentration in H
UV
EC
after incubation
with different con
centration of flavonoids, purine
s, terpenes, sterols,
salicin, biosynthesis precursor m
olecules, human steroids, blood lipid-low
ering drugs and plant extrac
t for ten minutes resp
ective 24 hours
compared to control (P
BS
or DM
SO
as stated). Statis
tics were calculated w
ith one wa
y AN
OV
A repeated m
easures, *p<
0.05, **p<0.01 and
***p<0.001. n=
5-7 from different individuals. a
ap<0.01 betw
een 0.1 and 1 m
g/ml. A
CE
activity is exp
ressed in Units (U
) and N
O concentration
in µM
.
Flavonoids
Epicatechin
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 11.9±1.6
11.2±2.2 9.4±1.9
7.9±2.1*
N
O concentration
4.3±1.8
5.2±1.7 6.2±1.9
5.9±2.2
Epicatechingallate
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 11.9±1.6
7.7±1.9* 3.8±1.7**
1.2±0.6**
N
O concentration
4.3±1.8
5.7±2.0 11.1±4.1
17.0±6.0
OO
H
OH
OH
OH
OH
O
O
OH
OH
OH
OH
OO
H
OH
OH
91
Epi
gallo
cate
chin
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
1mg/
ml
2mg/
ml
AC
E a
ctiv
ity
11.9
±1.6
7.
5±2.
0*
3.1±
0.8*
* 0*
*
N
O c
once
ntra
tion
4.
3±1.
8 6.
7±2.
3 21
.0±7
.8*
23.6
±6.5
*
Epi
gallo
cate
chin
galla
te
Con
cent
ratio
n
DM
SO
50
µg/
ml
0.1m
g/m
l 0.
5mg/
ml
1mg/
ml
2m
g/m
l A
CE
act
ivity
9.
1±1.
5 5.
5±1.
7 5.
3±2.
0 1.
1±0.
8***
0.
1±0
.1**
* 0*
**
NO
con
cent
ratio
n
4.3±
1.8
25.7
±3.7
***
31.8
±2.9
***
40.4
±3.0
***
Gre
en te
a ex
trac
t Jap
anes
e S
ench
a
Con
cent
ratio
n
PB
S
1:32
00
1:16
00
1:80
0 1:
400
1:20
0 A
CE
act
ivity
14
.9±3
.9
10.2
±3.2
7.
7±2.
0*
5.7±
1.8*
* 2
.2±0
.9**
0.
8±0.
6**
NO
con
cent
ratio
n
2.2±
0.9
2.5±
0.9
3.3±
1.0*
**
4.0±
1.1
***
5.0±
1.1*
**
7.1±
1.2*
**
Bla
ck te
a ex
trac
t Ind
ian
Ass
am B
.O.P
.
Con
cent
ratio
n
PB
S
1:32
00
1:16
00
1:80
0 1:
400
1:20
0 A
CE
act
ivity
14
.9±3
.9
11.8
±2.8
9.
4±2.
5 6.
4±1.
4*
4.7
±0.5
**
2.3±
0.6*
* N
O c
once
ntra
tion
2.
2±0.
9 2.
4±0.
9 2.
6±1.
0 3.
6±1.
0***
4.
8±1.
1***
7.
0±1.
1***
R
ooib
os te
a ex
trac
t
Con
cent
ratio
n
PB
S
1:32
00
1:16
00
1:80
0 1:
400
1:20
0 A
CE
act
ivity
14
.9±3
.9
15.4
±5.1
13
.3±3
.8
13.4
±3.7
11
.9±3
.2
8.5±
3.9
NO
con
cent
ratio
n
2.2±
0.9
2.3±
1.0
2.3±
1.0
2.5±
1.1
2.8±
1.0*
4.
5±1.
3***
O
OH
OH
OH
OH
OH
OH
O
O
OH
OH
OH
OH
OH
OO
H
OH
OH
92
Coffee extract S
antos Brasil
Concentration
P
BS
1:3200
1:1600 1:800
1:400 1:200
AC
E activity
19.2±3.7 18.4±4.2
17.0±3.8 15.0±3.2
9.5±2.6
4.0±1.7* N
O concentration
3.7±0.8
4.9±0.9 5.5±1.0
4.9±0.9 6.
1±0.9 7.0±0.6*
Procyanidin E
picatechin-epicatechin
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 4.8±0.5
7.8±0.9 6.8±0.5
4.7±0.7a
a
N
O concentration
2.0±0.5
2.0±0.5 3.7±1.0**
8.2±0.6**
Cacao extract
Concentration
P
BS
6.25µ
g/ml
12.5µg/m
l 25µ
g/ml
50µg/
ml
100µg/m
l A
CE
activity 30.8±4.1
18.1±2.9** 11.7±2.9**
5.9±2.2** 1.4±1.2**
0** N
O concentration
3.6±1.4
3.4±1.4 3.5±1.4
3.8±1.2 4.
3±1.2 5.9±1.2***
Genistein
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 7.7±1.3
4.7±1.2* 2.7±0.6***
3.6±1.1**
Quercetin
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 4.8±0.5
3.0±0.9* 1.7±0.4***
1.5±0.4***
N
O concentration
2.0±0.5
6.1±1.3* 9.1±0.6***
9.8±0.7***
OO
OH
OH
OH
OO
OH
OH
OH
OH
93
Sci
adop
itysi
n
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
4.
8±0.
5 5.
4±0.
7 5.
3±0.
6 6.
1±0.
6
N
O c
once
ntra
tion
2.
0±0.
5 5.
8±0.
6 19
.2±1
.6**
* 46
.1±2.5**
*
Cya
nidi
n
R1=
H
R2=
OH
Con
cent
ratio
n
DM
SO
0.
01m
g/m
l 0.
025m
g/m
l 0.
05m
g/m
l
A
CE
act
ivity
8.
4±2.
0 13
.5±3
.2
4.1±
1.4
2.6±
1.1
Del
phin
idin
R
1=O
H
R2=
OH
Con
cent
ratio
n
DM
SO
0.
01m
g/m
l 0.
025m
g/m
l 0.
05m
g/m
l
A
CE
act
ivity
8.
4±2.
0 8.
4±2.
4 5.
7±1.
7 2.
9±1.
3
M
alvi
din
R1=
OC
H 3 R
2=O
CH 3
Con
cent
ratio
n
DM
SO
0.
01m
g/m
l 0.
025m
g/m
l 0.
05m
g/m
l
A
CE
act
ivity
8.
4±2.
0 5.
0±1.
8 5.
3±2.
1 3.
3±1.
3
M
yrtil
lin c
hlor
id
Con
cent
ratio
n
DM
SO
0.
01m
g/m
l 0.
025m
g/m
l 0.
05m
g/m
l
A
CE
act
ivity
5.
5±1.
9 2.
5±0.
9 0.
9±0.
5*
0.9±
0.6*
B
ilber
ry e
xtra
ct 2
5E
Con
cent
ratio
n
PB
S
6.25
µg/
ml
12.5
µg/
ml
25µ
g/m
l 50
µg/
ml
100µ
g/m
l A
CE
act
ivity
25
.0±5
.4
5.7±
3.2*
**
2.7±
1.8*
**
1.0±
0.7**
* 0.
2±0.
2***
0*
**
O
O OH
OH
O O
OC
H3
OC
H3
OH
O
CH
3
O+
OH
OH
OH
R1
OH
R2
94
Liquorice extract
Concentration
P
BS
6.25µ
g/ml
12.5µg/m
l 25µ
g/ml
50µg/
ml
100µg/m
l A
CE
activity 29.4±4.0
13.4±4.5*** 11.3±3.5***
5.8±2.3*** 2.4±1.4***
0.7±0.5*** N
O concentration
2.8±1.5
3.4±1.6 4.2±1.6
5.9±1.7 9.
1±2.1*** 22.8±3.2***
Purines
Caffeine
R1=C
H3 R
2=C
H3 R
3=C
H3
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 11.9±1.6
15.8±3.3 18.5±3.1
18.8±3.4
N
O concentration
4.3±1.8
4.5±1.5 4.2±1.5
5.2±1.7*
T
heobromine R
1=H
R2=
CH3 R
3=C
H3
Concentration
D
MS
O
0.01mg/m
l 0.05m
g/ml
0.1mg/m
l
A
CE
activity 11.9±1.6
13.7±2.4 14.2±2.2
11.1±2.1
N
O concentration
4.3±1.8
4.4±1.6 4.5±1.6
8.6±3.7
T
heophylline R1=
CH
3 R2=
CH
3 R3=
H
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 11.9±1.6
11.3±1.6 12.6±1.7
9.7±2.2
N
O concentration
4.3±1.8
4.5±1.8 5.7±1.9
4.7±1.7
N
NN N
O
O
R1
R3
R2
95
Ter
pene
s
Gin
kgol
ide
A
R1=
OH
R
2=H
R
3=H
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
15
.4±1
.7
12.9
±2.5
13
.7±3
.6
12.8
±2.6
N
O c
once
ntra
tion
3.
2±0.
6 2.
9±0.
4 2.
8±0.
4 2.
9±0.
4
G
inkg
olid
e B
R
1=O
H
R2=
OH
R
3=
H
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
15
.4±1
.7
15.1
±3.4
15
.5±4
.4
14.4
±2.7
N
O c
once
ntra
tion
3.
2±0.
6 2.
8±0.
4 2.
7±0.
4 2.
7±0.
4
G
inkg
olid
e C
R
1=O
H
R2=
OH
R
3=
OH
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
15
.4±1
.7
17.0
±5.1
18
.1±5
.8
23.3
±6.9
N
O c
once
ntra
tion
3.
2±0.
6 3.
2±0.
5 3.
1±0.
5 3.
0±0.
5
Bilo
balid
e
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
15
.4±1
.7
13.4
±2.5
12
.3±2
.0
13.5
±2.3
N
O c
once
ntra
tion
3.
2±0.
6 2.
8±0.
4 2.
6±0.
4*
2.4±
0.4*
**
O O
OH
OO
H
R3
OH
R2
O
R1
OO
OH
OH
OO
OH
OO
OH
96
Ginkgo biloba extract E
Gb 761
Concentration
P
BS
0.1m
g/ml
0.5mg/m
l 1m
g/ml
5mg/m
l 1
0mg/m
l A
CE
activity 33.0±1.9
29.8±4.8 23.4±4.6
15.6±4.5***
2.7±1.2*** 0.6±0.3***
NO
concentration
3.5±0.6 3.1±0.4
3.4±0.4 4.2±0.5
11.0±1.3***
19.2±2.4***
Ginsenoside R
b1 R1=
Glc- 2G
lc R2=
Glc- 2G
lc- R3=
Glc- 6G
lc
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 24.7±6.3
25.0±6.6 25.9±5.8
27.4±5.1
G
insenoside Rb2 R1=
Glc- 2G
lc R2=
Glc- 2G
lc- R3=
Ara(p)- 6G
lc
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 24.7±6.3
29.8±6.2 33.3±8.5**
32.4±7.7*
Ginsenoside R
c R1=G
lc- 2Glc R
2=G
lc- 2Glc- R
3=A
ra(f)- 6G
lc-
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 24.7±6.3
26.4±6.3 32.4±8.0**
34.7±9.0***
Ginsenoside R
d R1=G
lc- 2Glc R
2=H
R3=
Glc-
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 24.7±6.3
33.2±8.6* 33.2±7.8*
35.4±10.2**
Ginsenoside R
e R1=H
R2=
Rha- 2G
lc-O- R
3=G
lc-
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 24.7±6.3
26.3±7.1 27.0±6.8
19.3±4.4*
R2O
R3
R1O
OH
97
Gin
seno
side
Rf
R1=
H
R2=
Glc
-2 Glc
-O
R3=
H
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
24
.7±6
.3
18.2
±3.4
18
.4±2
.8
20.3
±3.1
G
inse
nosi
de R
g1
R
1=H
R
2=G
lc-O
- R
3=G
lc-
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
24
.7±6
.3
23.3
±5.2
20
.9±6
.6
21.2
±5.6
P
anax
gin
seng
ext
ract
G11
5
Con
cent
ratio
n
PB
S
0.1m
g/m
l 0.
5mg/
ml
1mg/
ml
5mg/
ml
10m
g/m
l A
CE
act
ivity
16
.5±3
.6
16.8
±3.0
15
.9±3
.7
13.7
±2.7
8.
1±2.
8**
5.4±
2.0*
* N
O c
once
ntra
tion
6.
5±1.
4 6.
1±1.
3 5.
1±1.
1*
5.1±
0.8*
5.
0±1.
0*
4.8±
0.9*
β-c
arot
ene
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
7.
7±1.
2 4.
7±1.
3 3.
5±1.
4**
1.9±
0.8*
**
α-t
ocop
hero
l
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
7.
7±1.
3 7.
3±0.
9 7.
4±0.
8 8.
6±1.
3
H
H
O
CH
3
CH
3
OH
CH
3
98
Sterols
Cholesterol
Concentration
P
BS
10 -5M
10
-4M
10-3M
A
CE
activity 20.7±8.0
19.5±6.8 20.5±7.4
20.1±7.2
Lanosterol
Concentration
D
MS
O
10 -7M
10-6M
10
-5M
10-3M
AC
E activity
5.1±1.9 6.8±0.9
7.1±0.9 7.2±0.7
8.0±2.1**
Stigm
asterol
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 7.7±1.3
6.1±0.9 7.2±1.5
6.4±0.9
OH
HH
H
H
HO
H
OH
H
H
H
99
Phe
nol
Sal
icin
Con
cent
ratio
n
PB
S
0.1m
g/m
l 0.
5mg/
ml
1mg/
ml
AC
E a
ctiv
ity
28.1
±2.9
29
.4±2
.9
26.8
±3.0
27
.4±3
.0
NO
con
cent
ratio
n
4.5±
0.3
4.4±
0.3
4.7±
0.1
4.5±
0.3
Pre
curs
or m
olec
ules
Cho
rism
ic a
cid
Con
cent
ratio
n
PB
S
10-6M
10
-5M
10
-4M
A
CE
act
ivity
26
.2±5
.9
16.7
±4.6
17
.3±4
.5
18.0
±4.6
Mal
onic
aci
d
Con
cent
ratio
n
PB
S
10-6M
10
-5M
10
-4M
A
CE
act
ivity
24
.1±6
.8
19.0
±5.7
22
.2±7
.0
20.6
±7.1
O
OHO
OH
OH
OH
O
OH
CO
OH
CO
OH
CH
2
OH
OH
OO
10
0
Mevalonic acid
Concentration
P
BS
10 -6M
10
-5M
10-4M
A
CE
activity 29.6±5.3
29.0±6.2 27.2±6.7
28.1±6.9
N
O concentration
4.5±0.3
4.9±0.2 4.8±0.2
5.2±0.2
Shikim
ic acid
Concentration
P
BS
10 -6M
10
-5M
10-4M
A
CE
activity 26.3±5.8
23.4±6.1 23.8±6.2
22.6±5.3
N
O concentration
4.4±0.3
4.4±0.2 5.0±0.3
4.9±0.5
Squalene
Concentration
D
MS
O
10 -7M
10-6M
10
-5M
10-3M
AC
E activity
5.1±1.9 7.5±3.4
7.2±2.4 6.4±1.9
7.4±1.7
H
uman steroids
Aldosterone
Concentration
D
MS
O
0.1mg/m
l 0.5m
g/ml
1mg/m
l
A
CE
activity 7.7±1.3
7.1±1.3 9.0±1.9
9.9±2.3
OH
OH
OO
H
OH
O
OH
OH
OH
O
H
H
OO
OH
10
1
Oes
trad
iol
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
7.
7±1.
3 6.
3±1.
0 6.
1±1.
2 6.
5±1.
6
Tes
tost
eron
e
Con
cent
ratio
n
DM
SO
0.
1mg/
ml
0.5m
g/m
l 1m
g/m
l
A
CE
act
ivity
7.
7±1.
3 8.
5±2.
9 8.
1±2.
0 6.
7±2.
2
B
lood
lipid
-low
erin
g dr
ugs
Pra
vast
atin
Con
cent
ratio
n
PB
S
10-8M
10
-7M
10
-6M
A
CE
act
ivity
32
.1±9
.0
34.6
±9.8
35
.7±9
.6*
35.6
±9.3
*
Sim
vast
atin
Con
cent
ratio
n
DM
SO
10-8
M
10-7
M
10-6
M
AC
E a
ctiv
ity
1.6±
1.0
7.9±
2.7*
9.
5±3.
4**
7.2±
2.0*
OH
H
OH
HH
O
H
OH
HH
CH
3
O
CO
OH
CH
3
O
HH
3C
HO
OH
CH
3
CH
3
O
CH
3
CH
3C
H3
OO
O
HHO
102
TACK……..
Forska…Forska???....Tänk om jag skall forska..????
Det var många funderingar och känslor som dök upp i mitt huvud.
Jo, jag vill, men??…kan jag???? Typiskt mig, jag vill (jag vill det mesta), men
vad är det jag skall göra och kan jag ????? Jag förstod att det måste handla om
att tänka, systematik och vetenskap. För mig är det jobbigt att tänka, men jag är
bra på att känna, och systematik… vad är det??
Jag började med att slå upp ordet forskning, och läste: forskning är en aktiv (jag
är aktiv, så det klarar jag) och systematisk process (här får jag problem), jag
läser vidare i ordboken, forskningen drivs av forskarens nyfikenhet (nyfikenhet
har jag inbyggt i generna, det ligger i släkten, så det klarar jag) och intresse ???
(i ordboken står det: intresse t.ex. fågelskådning. Fågelskådning är roligt och jag
har fågelskådat i många år, så då klarar jag intressedelen också).
Mitt liv som forskare kunde börja!
Det behövs visst en handledare också…….
För mig var det självklart, det måste bli mannen som erbjudit mig att bli
forskare, Rolf Andersson. Jag fick, fast det var nog snarare så att vi kom överens
om att han skulle bli min handledare, Rolf Andersson alltså………..
Professor Rolf G.G. Andersson….. undrar vad G.G. betyder? Great Guru
kanske, (Guru är ”den som skingrar mörkret”). Det är en verkligt bra sak när
handledaren tror att det skall gå bra, för det händer att självförtroendet sviktar
ibland, det hände i alla fall mig. Handledare är en person som tar dig i handen
och leder dig längs vägen fram till målet. Fast nu är det ju så att jag har alltid
gått mina egna vägar, och lycka på jorden, du tillät mig att bana min egen väg i
103
forskningssnåret. Jag tror, om jag får bli lite djup, att det är detta som jag är mest
tacksam över, att jag fick bana min egen väg, att det har varit min forskning. Jag
är också tacksam för din entusiasm och din alltid så positiva inställning till min
forskning.
Anita……när jag arbetade med min ”dricka-te studie” kom jag till jobbet tidigt
på morgonen, men hur tidigt jag än kom så fanns Anita där. Det är bra när någon
alltid finns där, det är tryggt. Anita har många bra egenskaper och en är hennes
goda kakor, onsdags-kakorna har varit en mitt-i-veckan ljuspunkt i den mörka
forskningstunneln. ”De som vandrar i mörker skall se ett stort ljus”, jag såg
Anita.
Tack till co-forskare, co-kollegor och alla som har visat intresse för min
forskning och bidragit med en hjälpande hand när jag har behövt och bett om
hjäälp!
Familjen Ranidae, Temporaria, Arvalis och Dalmatina. Era alltid glada hopp och
vackra sång, ni är mina prinsar.
Fresse…… nu undrar ni förståss vad det finns för anledning att tacka en katt?
Men alla ni som efter en lång dag och en ännu längre vecka fullproppad av
forskning och för att inte tala om allt tänkande, har fått vila ert trötta huvud på
en katt ni förstår precis varför jag tackar Fresse. Med en katt som huvudkudde
kan Tempur och allt vad ni nu heter slänga er i väggen. Nu sitter Fresse i
katthimlen och jag saknar min mjuka, sköna och trogne huvudkudde.
Karin, min lillasyster. I ordboken, under ordet lillasyster står det: ”Lillasyster är
ett svenskt rockband som bildades 2006, bandets debutalbum heter Hjärndöd
musik för en Hjärndöd generation”. Det är något som inte stämmer; när Karin
spelar Julkväll på Hawaii på orgeln låter det visserligen som hjärndöd musik för
en hjärndöd generation, men Lillasyster, rockbandet alltså bildades 2006 och jag
är säker på att lillasyster, Karin alltså är äldre än 3 år. Det är bra att du har lagt
104
orgeln på hyllan. Karin har också intresse, fågelskådning alltså och vi utövar
intresse tillsammans. Det har varit många roliga och vilsamma dagar med fika,
fika och fågelskådning för att inte tala om alla steg vi har gått (ca 100 miljoner).
Vi har nött ut många par vandringskängor och trampat ner i många hål i marken
(Karin i alla fall). Under våra vandringar har havsörn, skäggmes, majvivor,
naturguider, ACE och kväveoxid blandats i en salig röra.
Det har varit roligt!
I.P.
105
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