Introduction
1. INTRODUCTION
Natural products constitute the best possible library with maximum diversity
of chemical structural types. With modernization and deforestation, the marine
environment have become a rich source of biologically active natural products many
of which have not been found in terrestrial sources. The first living organisms have
appeared in the sea more than 3,500 million years ago (Argulis et al, 1982). The
oceans are full of living organisms and contain more flora and fauna compared to the
land. Marine organisms encompass a complex and diverse assemblage of life forms,
which occur throughout the oceans. These organisms live in complex communities
and in close association with other organisms both macro (e.g. algae, sponges,
ascidians etc.,) and micro (e.g. bacteria, fungi, actinomycetes etc.,) organisms
(Maudougall, 1996) . In the course of evolution, marine organisms have adapted
excellently to the marine environment such as high salinity, low temperature, high
pressure and low nutrient availability.
Marine organisms have developed unique metabolic and physiological
capability that not only ensure survival in a great variety of extreme habitats but also
offer the production of metabolites, which could not be observed in terrestrial
organisms leading to the development of new natural products. With marine species
comprising approximately a half of the total biodiversity of the earth, the sea provides
enormous novel compounds and it is classified as the natural bioactive reservoir that
can be explored for drug activity.
Research on modern bioactive natural products began approximately 40 years
ago with the study of organisms from intertidal and shallow sub tidal environments.
So far, about 7000 marine natural products have been isolated. Out of which 33 % is
Page 1
Introduction
from sponges, 25 % from algae 18 % is from coelenterates (sea whips, sea fans, soft
corals) and 25 % of other invertebrate fila such as ascidians, opistho branch molluscus,
echinoderms and bryozoans. (Radhika G et al, 2008).
Tyrian purple was the first marine metabolite whose structure was correctly
deduced and proven in 1909. Every year, an increasing number of metabolites from
marine are reported. Approximately 2,500 new metabolites were reported from marine
organisms and nearly 2070 papers were published during the period 1977-1987
(Ireland et al, 1993). In the next decade 1988-1997, there was an increase of the total
number of publications (4,791) and in the number of isolated metabolites to 7099.
From humble beginnings the number of compounds isolated from various marine
organisms has virtually soared and now exceeds even 10,000 (MarinLit VS, 2003).
Seaweeds
Nearly 5,000,000 species available in the sea are virtually untapped source of
secondary metabolites. Seaweeds are the extraordinary sustainable resources of
marine ecosystem and man has been using the seaweeds as food, feed and medicine. It
was estimated that about 90 % of the plant species of marine are algae and about 50 %
of the global photosynthesis is contributed from algae (Dhargalkar, 2005). About 271
genera and 1153 species of marine algae have been reported from Indian coast
(Kaliaperumal, 2002). These algae form an important renewable resource in the
marine environment and have been a part of
human cultivation from time
immemorial.
Seaweeds have been used as food stuff in Asian diet as it contains carotenoids,
proteins, vitamins, dietary fibres, essential fatty acids and minerals. Marine algae are
exploited mainly for the industrial production of phycocolloids. Bio-stimulant and the
antimicrobial properties of seaweeds are used in agriculture and in the development of
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Introduction
novel antibiotics respectively. Seaweeds have been reported to have valuable
medicinal principles such as antibiotics, laxatives, anticoagulants, anti-ulcer products,
neurotoxins and suspending agents in radiological preparations (Scheuer, 1990).
The metabolic active compounds already isolated from seaweeds have helped
in the development of new drugs against cancer, microbial infections and
inflammation (Francisco, M et al, 2001). Preventing disease outbreaks or treating the
disease with drugs or chemicals tackles these problems. Nowadays, there is an
increase in use of antibiotics due to heavy infections and the pathogenic bacteria
becoming resistant because of indiscriminate usage of antibiotics. It becomes a greater
problem of giving treatment against resistant pathogenic bacteria (Sieradski et al,
1999). Moreover, the cost of the drugs is high and they cause adverse effect like
hypersensitivity reactions and depletion of beneficial microbes in the gut (Idose et al,
1968). Hence it became necessary for the development of new alternatives (Smith et
al, 1994). The increasing demand for antibiotics in the screening programs seeking
therapeutic drugs from marine natural source has led to interest, especially seaweed
(Kellam et al, 1998).
The study of algae is called phycology and its history is quite old. Phykos is a
Greek word which means seaweed. Seaweeds, the marine macro-algae supposedly
primitive, photosynthetic organisms live in sea or in brackish water. Nowadays,
macroalgae are divided into three main groups according to the distribution and
occurrence of several photosynthetic pigments, which are the main features for algae
classification. They are classified on the basis of their pigment constituents in to 4500
species of red algae (Rhodophyta) with r-phycoerythrin and c-phycocyanin pigments,
3000 species of brown algae (Phaeophyta) having pigments like xanthophylls, and
fucoxanthins and some 7000 species of green algae (Chlorophyta) with chlorophylla
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Introduction
and β-carotenes and various xanthophylls and the references to algae are available in
the early Chinese, Roman and Greek literatures.
Early Chinese pharmacopoeia and Materia Medica (Sher-Nung Ben Cao
Jingo 200 BC and Ben Cao Gang Muc 1518-1593 AD) recommended seaweeds for
diverse maladies as anti-goitre, anti-oedema, antipyretic, antitumor and diuretic agents.
The first medicinal use of brown algae in the literature appeared in Li
Shihchen’s sixteenth century herbal book Pen Ts’ao Kang Mu where brown algae
were listed as a remedy for goiter by virtue of the iodine they contained.
Types of Seaweeds
Algae are considered a big group of autotrophic organisms, ranging from
unicellular to multicellular forms. These organisms can be found in both marine and
fresh waters, and are divided according to their size into microalgae and macroalgae
or seaweeds. Microalgae constitute a polyphyletic group of prokaryotic and
eukaryotic microscopic organisms with a simple cellular structure. This group of
unicellular microorganisms live individually or in groups and is known as
phytoplankton. Among the most common microalgae, are the Cyanophyceae (blue
green algae), Bacillariophyceae (diatoms) and Chrysophyceae (golden algae). Among
these, diatoms are the dominant life form in phytoplankton and represent the largest
group of biomass producers on earth.
Macroalgae can be defined as photosynthetic multicellular eukaryotic
organisms, with a wide variety of cell morphologies and life cycles. These organisms
are found in a multiple variety of habitats and their morphological diversity results
from their polyphyletic origin within the eukaryotic tree of life. In their natural
environment, macroalgae grow on rocky substrates and form stable, multi-layered,
perennial vegetation. A wide range of specific requirements, such as nutrients, salinity,
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Introduction
temperature, light, depth and currents, are determinant factors that affect macroalgae
growth and nutrients production. Nevertheless, the fact of being fixed on a substrate
enhances macroalgae growth and productivity, contrary to what happens with
phytoplankton. These organisms are of extreme importance as primary producers,
they play a key role in the productivity of oceans and constitute the basis of the
marine food chain. Additionally to the organic material they produce through sun light,
carbon dioxide (CO2) and water (H2O), macroalgae are one of the major responsible
for oxygen production on earth. Several classifications have been proposed over the
years. Both terminologies “macroalgae” and “seaweed” have been used when
referring to these organisms, although the second respects to macroalgae living in
marine waters. Along this dissertation, the term “macroalgae” will be used in a
generalized context and the term “seaweed” will be confined to macroalgae species
exclusive or collected in marine waters (Kang et al, 2012).
Chlorophyta
Chlorophyta are considered to be the ancient of terrestrial plants and, due to
their similarities with them, they are grouped in the kingdom Plantae. This phylum is
better represented in fresh water (90 %) than in the sea (10 %) and counts more than
7000 known species. Contrary to fresh water species, which have a cosmopolitan
distribution, Chlorophyta species belonging to the marine environment from northern
and southern hemispheres present clear differences. In this phylum, the chloroplasts
contain chlorophyll a and b, β-carotene and xanthophylls in a proportion resembling
terrestrial plants. Lutein is the main carotenoid. Their major photosynthetic pigment,
responsible for their green color, is chlorophyll b, although, some tropical species are
pigmented by siphonoxanthin and siphonein. Concerning storage products obtained
from photosynthesis, Chlorophyta differs from other eukaryotic macroalgae since they
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Introduction
reserve starch in organelles called pyrenoids, within chloroplasts, instead of
cytoplasm. The cell walls of these organisms are composed by cellulose as the major
polysaccharide, although xylans and manans can replace cellulose in some species.
Rhodophyta
Rhodophyta, one of the oldest eukaryotic algae, are thought to be descendent
from a cyanome in the Glaucophyta. More than 6000 species have been identified to
date, of which only a few are found in fresh water. Rhodophyta can occur at all
latitudes, but predominantly from equator to colder seas. Few Rhodophyta are found
in polar and sub-polar regions, where green and brown algae are predominant,
contrary to what happens in temperate and tropical regions where this phylum prevails.
Members from this phylum share a combination of characters that do not occur
together in any other eukaryote, such as the complete lack of flagellated stages and
the absence of centrioles.
Pigments like chlorophyll, phycobiliproteins, red phycoerythrin, blue
phycocyanin, carotenes, lutein and zeaxanthin are present in these species.
Phycoerythrin, provides the algae's red pigmentation by absorbing blue light and
reflecting red light. Red algae have the ability to live below 200 m depth in the ocean.
The photosynthetic reserves are stored in plastids as floridean starch grains.
Several species from this phylum are important food crops, particularly in
Asian countries, where members of the genus Porphyria are highly consumed. In
Portugal, algae are consumed in trace quantities, the consumption of red algae being
almost limited to the species Osmundea pinnatifida (Hudson) Stackhouse, also known
as pepper grass, particularly in Azores Islands.
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Introduction
Phaeophyta
Brown algae belong to the phylum Heterokontophyta or Phaeophyta,
according to the taxonomic classifications. As a matter of standardization, brown
algae will be referred as Phaeophyta.
Phaeophyta mainly consists of macroscopic organisms inhabiting marine
waters. Of the approximately 2000 known species, less than 1 % colonizes fresh water
habitats (Wilce, 1966). These species have adapted to a great variety of marine
ecological niches, including the tidal zone, rock pools, all intertidal zone and
relatively deep waters near the coast. A large number of Phaeophyta are found in the
intertidal or upper littoral area, where the waters are predominantly cool and the
currents of nutrient-filled cold waters inflow the land. Species belonging to this
phylum present a wide range of sizes and forms, varying from groups of threadlike
cells with few centimeters to giant kelps with dozens of meters. These species grow
so profusely that they form offshore kelp forests, very efficient at capturing sunlight
and serving as home to a diverse group of marine animals. In a general way, brown
algae species are composed by a holdfast (root like structure) thallus (twig), stipe,
lamina and pneumatocyst (also known as gas bladder, a floating structure containing
gas). Pneumatocysts are found only in brown algae and exert an upward force that
allows the organism to receive more sunlight for photosynthesis .
Members of Phaeophyta are distinguished by their brown or yellow brown
color, resulting from their main carotenoid fucoxanthin. Along with fucoxanthin, the
main photosynthetic pigments of brown algae are chlorophylls a, c1 and c2,
β-carotene, violaxanthin and diatoxanthin. Their brown color is also influenced by the
presence of phlorotannins stored in vesicles called physodes. Contrary to Chlorophyta
and Rhodophyta, their main photosynthetic storage product is not starch but laminarin,
Page 7
Introduction
a water soluble β-(1→3)-glucan. Their cell walls are composed of cellulose, alginic
acid and sulfated polysaccharides, produced in high quantities in some marine species
and harvested for commercial purposes. In fact, some Phaeophyta species are of
sufficient commercial importance and have become subjects of extensive research.
The alginates of some brown algae are harvested for commercial use as thickening
agents in the textile, cosmetic and food industries. For many years macroalgae were
also the source of iodine, a trace nutrient required in thyroid disorders. Despite the
interest of the pharmaceutical industry regarding the search for new bioactive
compounds, brown seaweeds are particularly attractive for cosmetics, where their
bioactive ingredients show benefits. (Schurch et al, 2007).
Chemical composition of the seaweeds
In the classical Indian Ayurvedic system a little has been reported regarding
the medicinal use of seaweeds. The word Shaiwal has been employed for it in the
Ayurveda. However, there exist records (shavaprakas 16th century AD) that Laminaria
(brown seaweed) and Porphyra (red seaweed) species were used for treating various
ailments. In this Ayurveda, Susurutha, a famous physician in ancient times (16th
century), used seaweeds in the form of decoction, paste and fresh algal extracts for the
ailments of ulcer, blood clotting, unnatural semen discharge, piles etc.
Since ancient times, seaweeds play an important role in Asian population diet,
where they are also widely used by food industries because of their rich chemical
composition and high content of fiber, minerals, vitamins and different antioxidants
(Figure 1.1). H2O makes up 70 to 90 % of the weight of fresh seaweeds. The
composition of the dry seaweed varies with the species, but the approximate
proportions are 45-75 % of carbohydrates and fiber, 7-35 % proteins and less than 5 %
fats. The main sugars are mannitol in brown seaweeds, sorbitol in red and saccharose
Page 8
Introduction
in green. The primary mineral components are iodine, calcium (Ca2+), phosphorous,
magnesium, iron, sodium, potassium and chlorine. Concerning vitamins, seaweeds do
not have vitamin D, but are rich in vitamin A, vitamins from B complex and vitamin
C. Taurin, an important amino acid present in seaweeds, is essential for the formation
of bile salts (Mouritsen, 2013).
Figure 1.1 Main classes of compounds found in seaweeds
Many active bioactive molecules have been isolated from various species of
seaweeds. Chemically the bioactive metabolites of marine flora include brominated
phenols,
heterocyclics
of
oxygen,
nitrogen,
sulphur,
sterols,
terpenoids,
polysaccharides, proteins and peptides. A review have been made on the chemistry
and biological activities of the isolated compounds (Bhakuni D et al, 1974, Blunt J et
al, 2005).
Some of the secondary metabolites of seaweeds arose further attention, not
only for their commercial application, but also for their biological activities and
interesting health effects (Figure 1.1). Among them, polysaccharides like agar,
carrageenans and alginate can be highlighted, as they are, by far, the seaweeds most
economically important compounds.
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Introduction
The extensive use of sulfated polysaccharides obtained from seaweeds in food
and medicine made them economically most important products. Of the four
major seaweed classes, the Rhodophyceae, Phaeophyceae, Cyanophyceae and
Chlorophyceae, polysaccharides of interest are produced by the first two classes.
The red algae produce carrageenan, agar, agarose, furcellaran or Danish agar.
Alginic acid, fucoidan are obtained from brown algae. The use of seaweed extracts in
food and medicine have been reviewed. (Upham SD, 1968). Among which Sulphated
Polysaccharide from brown algae has been proven to have various medicinal values.
Especially fucose rich sulphated polysaccharides from sargassum species was
reported to possess wide pharmacological actions, as potent free radical scavenging
(Park PJ et al, 2005) and antioxidant (Xue CH et al, 2001), hepatoprotective.
(Raghavendran et al, 2004, Wong et al, 2000) immunomodulatory, analgesic, antiinflammatory, antipyretic and anticancer property.
These polysaccharides of high molecular weight are the main structural
components of seaweed cell walls and, despite some biological activities have been
reported, they are important because of their extensive use in food and cosmetic
industries, thanks to their gelling, viscosifying and emulsifying properties.
Of great importance are also carotenoids. These 40-carbon atoms structures
are essential constituents of the photosynthetic apparatus, being responsible for light
harvesting during photosynthesis. They also act as protein stabilizers and antioxidants.
Carotenoids consist of a carbon skeleton derived from the polymerization of isoprene
units that may undergo cyclization in one or both ends. The formed ring can be
additionally substituted by oxo, hydroxyl (OH) or epoxy groups, originating different
xanthophylls (Pinto et al, 2000).
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Introduction
Fatty acids are essential for cell function and have entered the biomedical and
nutraceutical areas as result of their biological roles in clinical conditions, such as
obesity and cardiovascular diseases. These compounds consist of a carboxyl group
linked to a saturated or unsaturated aliphatic chain and play key roles in cellular and
tissue metabolism, membrane fluidity, electron and oxygen transport and thermal
adaptation. Seaweeds are rich in health benefit long chain polyunsaturated fatty acids.
Halogenated compounds mainly produced by red and brown seaweeds,
comprise a vast and heterogeneous group of molecules dispersed in several different
classes of primary and secondary metabolites, including indoles, terpenes, acetogenins,
phenols and volatile hydrocarbons. The halogens present in these compounds are
mainly bromine and chlorine and the antibacterial and antitumor activities of those are
of pharmacological interest.
Polyketides and lectins are also interesting secondary metabolites present in
seaweeds. Polyketides, like macrolides, for their antibiotic application and lectins for
their carbohydrate binding specificity, are useful in immunological and histochemical
studies .
Mostly because of their rich and diverse chemical composition, seaweeds
importance increased in such a way that in the past few decades the emphasis has
moved from wild harvest to farming and controlled cultivation, in order to produce
valuable and profitable new products in a large scale (Wijesinghe et al, 2012).
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Introduction
Courtesy: Lei Liu et al., Journal of Ethnopharmacology,2012 ,142;591-619
Figure 1.2 Medicinal uses of Sargassum species in Traditional Chinese medicine
Sargassum species occupy a very important place in traditional chinese
medicine (Figure1.2). Around 386 articles on Sargassum species was cited in PubMed,
Web of Knowledge and Sci Finder Scholar. Chinese publication database suggests
that there are about 136 peer viewed reports on the medicinal property of Sargassum
in china since 1979 ( Lei Liu et al, 2012).
Sea weed resources have attracted the attention of scientists all over the world.
While other countries are already ahead in the research from marine source, India is
lacking behind. In India seaweeds are abundant in the regions of Palk Bay, Gulf of
Mannar, and Laccadive archipelago of India.
Algae appear to be the panacea of bioresource especially in tropical countries.
From considering the algae as organisms of nuisance value and scum, to treating them
as “treasure trove” and “gold mine” in Biotechnology, they have metamorphosed form
being organisms of sheer academic curiosity to the status of preferred candidates. Be
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Introduction
it fuel or fine chemical, drugs or waste treatment algae are being identified as the best
bioresources.
Oxidative stress
Oxidative stress can be defined as a biological condition occurring from the
imbalance between the production of reactive species and their detoxification through
biological systems (enzymatic and non-enzymatic) that remove or repair the damage
they cause. All living organisms have biological systems responsible for removing
and repairing the damage caused by free radicals, existing a balance between them
and the molecules responsible for their neutralization. Every time this redox balance
is disturbed, and there is an increment in the production of reactive species, that will
cause damage to all components of cell, including proteins, lipids and DNA, resulting
in a state of oxidative stress. Thus, the maintenance of the equilibrium between the
production of reactive species and antioxidant defenses is an essential condition for
normal organism functioning. Nevertheless, despite their association with several
diseases and pathological conditions, such as neurological diseases and inflammatory
states, and with the aging process, reactive species can be beneficial to the organism
when used by the immune system to destroy the pathogens, or when acting as
messenger molecules in cell signaling pathways (Romano et al, 2010).
Oxidative stress may result from natural causes, like situations of extreme
exercise or inflammation, and from non-natural causes, such as the presence of
xenobiotics in the body. There is a continuous need for the inactivation of oxidants
formed as a normal product of aerobic metabolism. The intake of dietary rich
antioxidants helps to maintain or balance the levels of endogenous antioxidants.
Moreover, it is recognized that antioxidants other than vitamins can constitute a major
factor responsible for such protective effects (Romano et al., 2010).
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Introduction
Reactive species
There are several reactive species directly involved in the establishment of
oxidative stress. Nevertheless, the most common are the reactive oxygen species
(ROS) and the reactive nitrogen species (RNS) (Bast et al, 1997).
Oxygen molecule has low reactivity, however, due to its electronic
configuration, it can be activated or reduced, leading to the formation of toxic
molecules. ROS include radical and non-radical species derived from living systems.
Among the radical species, hydroxyl (•OH), peroxyl (ROO•), alkoxyl (RO•) and
superoxide anion (O2•-) are the most representative. In turn, hydrogen peroxide
(H2O2), hypochlorous acid (HOCl), singlet oxygen (1O2) are the non-radical ones.
O2•- is formed by reduction of triplet molecular oxygen (3O2), in a process mediated
by enzymes, such as NADPH oxidases and xanthine oxidase (XO) or, nonenzymatically, by redox-reactive compounds like the semi-ubiquinone compound of
the mitochondrial electron transport chain (Figure 1.3). Superoxide dismutase (SOD)
is responsible for the conversion of superoxide into hydrogen peroxide. In biological
tissues, O2•- can also be converted non-enzymatically into the non-radical species
H2O2 and O2. In the presence of reduced transition metals, such as ferrous or cuprous
ions, H2O2 can be converted into the highly reactive •OH, or into H2O, by the
enzymes catalase (CAT) or glutathione peroxidase (GPx) (Figure 1.3). O2•- is the
primary ROS produced in cells. Due to the various conversions that O2•- might be
targeted, most of the effects observed in cellular and non-cellular systems are indeed
not directly mediated by O2•-, but rather by its derived ROS. Of those, the reactions
with cellular lipids, proteins and DNA may be highlighted, since they can lead to cell
damage and death (Wei et al, 2012).
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Introduction
Figure 1.3 Schematic representation of the generation of several ROS and RNS
NADPH - nicotinamide adenine dinucleotide phosphate reduced form;
O2•- - superoxide radical; SOD – superoxide dismutase; H2O2 – hydrogen peroxide;
GPx – glutathione peroxidase; •OH – hydroxyl radical; PUFAs – polyunsaturated
fatty acids; ROO• - peroxyl radicals; RO• - alkoxyl radicals; NO – nitric oxide;
ONOO- - peroxynitrite; GABA –γ aminobutyric acid.
Among RNS, the nitric oxide radical (•NO) and the non-radical reactive
species peroxynitrous acid (ONOOH) and peroxynitrite (ONOO-) can be highlighted,
the last ones being extremely harmful for the central nervous system (CNS). •NO is
produced in higher organisms by the oxidation of L-arginine, in a process catalyzed
by the enzyme nitric oxide synthase (NOS) (Palmer et al, 1988). (Figure 1.4)
The role of NO in biological systems and its relation with pathological states
will be further explored in the section concerning inflammation (Chiueh, 1999).
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Introduction
NO and Inflammation
Inflammation can be defined as a mechanism of innate immunity by which the
organism responds to harmful stimuli. As a complex biological response of vascular
tissues, inflammation is mainly characterized by vasodilatation, which results, in a
large extent, from NO-dependent processes.
NO is highly permeable and diffuses rapidly across the membranes. Because it
is responsible for promoting blood vessels relaxation, it was also termed as
endothelium relaxing factor. In inflammatory responses this effector molecule can
modulate the release of a wide range of inflammatory modulators, enzymes activity,
adhesion of leucocytes to the vascular endothelium. Under normal physiological
conditions NO produces an anti-inflammatory effect. Once over-produced, it acts as a
pro-inflammatory mediator and induces inflammation and is involved in immune
responses by cytokine-activated macrophages. As a neurotransmitter, it acts at neuron
synapses and regulates apoptosis. Face to this, molecules with the capacity to suppress
the overproduction of NO and avoid tissue destruction, have been seen as potential
anti-inflammatory agents. Both inhibitors of NO biosynthesis and synthetic L-arginine
analogous demonstrated a successful effect on this field (Sharma et al, 2007).
NO is produced through its precursor, L-arginine, by the action of the specific
enzyme NOS. From the activity of NOS, by oxidation, also results the amino acid
L-citrulline (Figure 1.4). There are three isoforms of NOS. These enzymes are
constitutively expressed in cells and responsible for synthesizing NO in response to an
increase in Ca2+ or stress stimuli (Figure 1.4). The third isoform, the inducible NOS
(iNOS ), is constitutively expressed in some tissues and is typically synthesized in
response to inflammatory or pro-inflammatory mediators, such as cytokines or
endotoxins (like bacteria products) (Fujiwara, 2005).
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Introduction
Figure 1.4 Schematic representation of NO production by iNOS in macrophages.
LPS – lipopolysaccharide; TLR4 – toll like receptor 4; NF-kB –nuclear factor kappa;
iNOS – inducible nitric oxide synthase; mRNA – messenger ribonucleic acid;
O2•- - superoxide anion; OONO- - peroxynitrite anion; •OH - hydroxyl radical.
Macrophages are a major component of the mononuclear phagocyte system
and play a critical role in initiation, maintenance and resolution of inflammation.
These cells start the production of NO, for example, after exposure to bacterial
lipopolysaccharide (LPS), and continue its production for several hours. The NO
produced can combine with O2•- in the phagosomes, forming OONO- and
consequently •OH, which is used by these cells to kill ingested bacteria (Figure 1.4)
(Fujiwara, 2005). The over-production of NO by iNOS can lead to septic shock,
which is characterized by hypotension, inadequate tissue perfusion and organ failure,
often resulting in death. Sepsis can be managed by drugs with the capacity to inhibit
NO production or action (Kolb-Bachofen et al, 2006).
Antioxidants
Antioxidants can be defined as substances that delay or prevent the oxidation
of an oxidizable substrate. Defense against free radicals-induced oxidative stress
involves preventive and repair mechanisms and physical antioxidant defenses. The
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Introduction
antioxidant defenses can be exogenous or endogenous and, enzymatic (SOD, GPx and
CAT) and non-enzymatic ascorbic acid (Vitamin C), α-tocopherol (Vitamin E),
glutathione (GSH), carotenoids, flavonoids, among other antioxidants. In normal
conditions, there is always a balance between both the activities and the intracellular
levels of these antioxidants, which is essential for organism’s homeostasis.
Antioxidants can act in vivo or in vitro by inhibiting the generation of reactive species,
or by direct sequestration of these species. In vivo, an antioxidant can also act
indirectly and increase the endogenous antioxidant defenses, by increasing the
expression of genes encoding for SOD, CAT or GPx, which prevent the formation of
reactive species. The various defenses complement each other and act on different
oxidizing agents or cellular compartments (Halliwell, 1995).
Macroalgae have been deeply explored in the last decades and, apart from the
isolation of compounds from different chemical groups, several kinds of extracts have
been evaluated for their capacity to scavenge free radicals. Because macroalgae are
photosynthetic organisms, they are exposed to sun’s radiation and to high
concentrations of oxygen, which promotes the formation of free radicals. The absence
of structural damage in these organisms indicates a good protection against oxidation
and, for this reason, these organisms are a good source of secondary metabolites with
antioxidant potential (Le Tutour, 1990).
Inflammation
Inflammation, frequently occurs in living tissues, and is the major cause for
numerous death and precursor of deadly diseases. A major cause of morbidity of the
working force throughout world is inflammatory diseases including different types of
rheumatic diseases. Rheumatism is one of the oldest known diseases and affects a
large population of the world and no substantial progress has been made in achieving
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Introduction
a permanent cure. The search involving screening and development of drugs for antiinflammatory activity is an unending problem.
One of the most important autoimmune disease is arthritis. Elderly being most
affected. Arthritis is a painful swelling of joints. About 1 % of the world's population
is afflicted by rheumatoid arthritis, women three times more often than men.
Osteoarthritis, rheumatoid arthritis (Figure 1.5) and gout are some of the common
types prevalent worldwide, each occurring at different stages and involved with
different mechanism of action.
Figure 1.5 Normal and Arthritic joints
Rheumatoid arthritis is a chronic inflammatory disease of joints. Progression
of the disease (Figure 1.6) results in joint destruction, deformity and significant
disability. Osteoarthritis is characterized by loss of articular cartilage, bone
remodeling, subchrondal bone sclerosis and bone cysts. (VanItallie, 2010). Gouty
arthritis, uric acid crystals gets deposited in the joints causing inflammation. The
clinical manifestation include acute inflammatory arthritis, uric acid kidney stones,
gouty nephropathy and periarticular inflammation. (Ali, 2009)
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Introduction
Figure 1.6 Stages of Rheumatoid Arthritis
Figure 1.7 Drugs used in the management of Arthritis
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Introduction
Anti-inflammatory drugs from Seaweeds
In recent years, it has been paid growing attention to the anti-inflammatory
activity of both extracts and isolated phlorotannins from seaweeds (Vo et al, 2012).
Jung and co-workers isolated five phlorotannins from an Eisenia species (eckol,
dieckol, 7-phloroeckol, phlorofucofuroeckol A and dioxinodehydroeckol) and found
that they dose-dependently inhibited LPS-induced NO production at non-toxic
concentrations. In another work, Sugiura and co-workers went further and studied the
same seaweed species in an in vivo model for the first time. These authors tested four
isolated phlorotannins and the results were as expected, with a good antiinflammatory
activity
for
the
tested
compounds
(eckol,
8,8’-bieckol,
phlorofucofuroeckol A and B). Kim and colleagues evaluated the antioxidant and antiinflammatory activity of phlorotannins isolated from a species of Ecklonia and
concluded that phlorofucofuroeckol significantly inhibited the LPS-induced
production of NO and PGE2 through the down-regulation of iNOS and
cyclooxygenase 2 protein expression (Vo et al, 2012). Other works performed with
seaweeds extracts attribute their anti-inflammatory activity to the polyphenols
(Mhadhebi et al, 2012).
Along with phlorotannins, the anti-inflammatory activity of glycerolipids has
been studied in different cell lines. These compounds have the ability to downregulate iNOS expression in cells exposed to LPS, thus exerting an anti-inflammatory
action by the reduction of NO production.It is known that glycerolipids with higher
levels of unsaturation are more effective in inhibiting iNOS. (Banskota et al, 2012).
Fucoidan, a type of complex carbohydrate available from kelp such as kombu
do have anti-inflammatory, anti-tumor and free radical scavenging activity. A few
reports on this complex carbohydrate in recent years have found that the use of brown
Page 21
Introduction
algae extract had shown a promising results in controlling liver and lung cancer and
promotes collagen synthesis. The high fiber content of kelp also slows down fat
absorption and promote weight loss. (http://theconsciouslife.com/ top-10-antiinflammatory-foods.htm ).
The revival of herbal and other complementary therapies in the management
of chronic diseases such as RA and other inflammatory disorders is well documented.
Since many compounds were introduced as a result of laboratory search for drugs
with anti-inflammatory activity, though many of them produced a dramatic
symptomatic improvement in rheumatic processes, did not arrest the progress of the
disease and they shared the common side effect i.e. gastro-intestinal irritations.
Seaweed derived anti-inflammatory compounds, have attracted interest and
promising replacer of current anti-inflammatory drugs. Macro algae derived bioactive
principles have both pro- and anti-inflammatory molecules, the later include sulphated
polysaccharides (fucoidans) from brown seaweeds, alkaloids (Caulerpin I, II and III)
from red and green seaweeds, polyunsaturated fatty acids (Docosahexaenoic acidEDA, Eicosapentaenoic acid- EPA, Stearidonic acid- SA and Eicosatrienoic acidETA), carotenoids (fucoxanthin and astaxanthin), Pheophytin A and Vidalols A and B.
(Irwandi Jaswir et al, 2011)
In India many Ayurvedic practitioners are using various indigenous plants for
the treatment of different types of arthritic and inflammatory conditions which has
showed improvement in the progression of disease. Natural bioactive compounds
include a broad difference in structures and functions that provide an excellent pool of
molecules for the production of nutraceuticals, functional foods, and food additives.
Unique compounds from marine have shown pharmacological activities for many
deadly diseases like Cancer, AIDS, Diabetes, Arthritis etc (De et al, 2006).
Page 22
Introduction
Approximately 10-15 different marine natural products are currently in clinical trials
in the areas of cancer, pain and inflammatory diseases including arthritis. (Thakur, NL
and Muller, WE, 2004). Seaweeds are the eukaryotic organisms that live in salty
water and recognized as a potential source of bioactive natural products but less
explored.
Hence the present study was an attempt to explore a bioactive principle from
marine seaweed for the treatment of arthritis.
Page 23
Introduction
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