Plant-Derived Substances and Cardiovascular Diseases

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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

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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

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”Blommor är vilsamma att betrakta. De har varken känslor eller konflikter”

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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 renin-angiotensin I to form renin-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.

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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

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

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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 NG-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

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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

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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

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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

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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 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).

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F ig u re 1 . T h e b io sy n th es is o f fl av o n o id s, t er p en es , st er o ls a n d p u ri n e te x t. 1 7 d s, t er p en es , st er o ls a n d p u ri n es /x an th in es . S ec o n d ar y m et ab o li te s te st ed i n t h is s tu d y a re p ri n te d i n b o ld n es . S ec o n d ar y m et ab o li te s te st ed i n t h is s tu d y a re p ri n te d i n b o ld

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F ig u re 2 . B as ic c h em ic al s tr u ct u re s o f f la v o n o id s, st ilb en es , a u ro n es a n d a p if o ro l. 1 8 h em ic al s tr u ct u re s o f f la v o n o id s, st ilb en es , a u ro n es a n d a p if o ro l.

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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

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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-B-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

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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).

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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 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

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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

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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

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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.

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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-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

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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

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).

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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

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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).

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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

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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.,

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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

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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 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

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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

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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).

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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).

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F ig u re 5 . T h e re n in -a n g io te n si n a ld o st er o n e sy st e m A m in o p ep tid as e A ( A M P A ), A m in o p ep tid as e N ( A M P N ), B ra d y k in in ( B K ), B ra d y k in in r ec ep to rs ( B K R ), In su lin (I R A P ), M as o n co g en e re ce p to r ( M as ), N eu tr al e n d o p ep tid as e (N E P ), N itr ic o x id e (N O ), en d o p ep tid as e (P E P ), R en in /p ro re n in r ec ep to rs ( R P R 3 8 an g io te n si n a ld o st er o n e sy st e m . A n g io te n si n -c o n v er tin g e n z y m e (A C E ), A n g io te n si n r ec ep to rs ( A T A m in o p ep tid as e A ( A M P A ), A m in o p ep tid as e N ( A M P N ), B ra d y k in in ( B K ), B ra d y k in in r ec ep to rs ( B K R ), In su lin (I R A P ), M as o n co g en e re ce p to r ( M as ), N eu tr al e n d o p ep tid as e (N E P ), N itr ic o x id e (N O ), E n d o th el ia l n itr ic o x id e sy n th as e (e N O S ), en d o p ep tid as e (P E P ), R en in /p ro re n in r ec ep to rs ( R P R ). en z y m e (A C E ), A n g io te n si n r ec ep to rs ( A T 1 , A T 2 , A T 3 , A T 4 ), A m in o p ep tid as e A ( A M P A ), A m in o p ep tid as e N ( A M P N ), B ra d y k in in ( B K ), B ra d y k in in r ec ep to rs ( B K R ), In su lin -r e g u la te d a m in o p ep tid as e E n d o th el ia l n itr ic o x id e sy n th as e (e N O S ), P ro ly l

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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

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…… 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

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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).

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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

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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

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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,

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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.

Plant-Derived Substances and Cardiovascular Diseases

Linköping University Medical Dissertations No. 1097