CHAPTER - I

General Introduction

Discoveries are often made by not following instructions; by going off the

main road; by trying the untried.

-Frank Tyger

Chapter - I General Introduction

1

1.1. Eicosanoids and their biological role:-

Discovery of eicosanoids dates back to beginning of 19th Century. Burr

and Burr observed a deficiency state can be induced in the rats on fat free diet

(Burr and Burr, 1929; Burr and Burr, 1930). At the same time Swedish

physiologist and Nobel laureate, Ulf von Euler and other investigators found

that extracts of seminal vesicles or human semen lowered blood pressure and

caused contraction of human uterine smooth muscle. Von Euler coined the

term prostaglandin (PG) because he assumed that the active material came

exclusively from the prostate gland. Now it is well known that arachidonic acid

(AA) is the substrate for eicosanoids which plays an important role in

inflammation which is an immunological response of our body towards foreign

antigens. It comprises of both innate and acquired immune system. But, when

this response becomes severe and lasts for long time, it will cause the damage.

There are many biomolecules which play vital role in these processes.

Eicosanoids (eicosa-Greek for twenty) are twenty carbons oxidized

signaling molecules derived from polyunsaturated fatty acids (PUFAs). They are

oxygenated essential fatty acids not stored within the cells, but are generated

as and when required. These eicosanoids are called as local hormones because

of their short life span and due to their autocrine and paracrine effects. Unlike

hormones, they have various activities in different kinds of cells. They are not

tissue specific and are not stored or concentrated in specific cells. It is well

known that releases of these biomolecules are synonymous with their

synthesis. The above features have made the study of eicosanoid as difficult.

Eicosanoids have numerous and diverse effects. Diversity could be appreciated

and at the same time perplexing. They have broad spectrum of actions and

show different activities both quantitative and qualitative. They play a vital role

in inflammation and as messengers in central nervous system (CNS) and

maintenance of many organs.

There are mainly two families of eicosanoids – prostanoids and

leukotrienes (LTs). Prostanoids further includes PGs, thromboxanes (TXs),

prostacyclins (PGIs). Once released from the immune cells these eicosanoids

are immediately inactivated by β-oxidation and/or modification brought by

Chapter-I General Introduction

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different mechanisms in the body (Funk, 2001). Although fatty acids are

symmetric, their oxidized product eicosanoids are chiral and oxidation

proceeds with high stereo-specificity. There are many other bioactive molecules

included in eicosanoids family. In cells, synthesis of eicosanoids is

compartmentalized. Based on the enzymes involved in the biosynthesis of these

eicosanoids suggests that their synthesis is evolved from detoxification of

reactive oxygen species (ROS). These groups of molecules are classified as

classical eicosanoids and non-classical eicosanoids, each having its own

biological functions.

Figure 1.1. Different eicosanoids and enzymes involved in their biosynthesis. Adapted and modified from Wikipedia.

Because of the vital role of eicosanoids in the human body, three Nobel

Prize have been awarded for the research in this field. Von Euler identified PGs

received Nobel Prize in the field of medicine in 1970 and the same was shared

by Samuelsson, Vane and Bergstrom in 1982. John Vane received Nobel Prize

for the identification of aspirin action and others received it for their elucidation

of the mechanism of biosynthetic pathway. E.J. Corey received Nobel Prize in

the field of chemistry (1990) for synthesis of PGs (Bergsrom et al., 1964; Vane, 1971).

Chapter-I General Introduction

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Eicosanoids as mentioned are biologically active lipid mediators of C20

fatty acids and their derivatives. Key precursor fatty acids as shown in the

figure 1.2 involved in biosynthesis of eicosanoids are eicosatrienoic (dihomo-γ-

linolenic DGLA) acids 20:3(n-6), AA 20:4(n-6) and eicosapentaenoic acids (EPA)

20:5(n-3). There are other molecules with similar functions like docosanoids

(resolvins and protectins) derived from docosahexaenoicacid (DHA) 22:6 (n-3)

and plant products such as jasmonates and oxylipins derived from octatrienoic

(α-linolenic acid ALA/ALNA) acid 18:3(n-3). Precursor fatty acids belong to the

ω-6 or ω-3 families. All eicosanoids are active at nanomolar (nM) concentrations

and are important molecules in the maintenance of homeostasis of many

tissues.

“The arachidonic acid content of active tissues is high… and it is

natural to assume some important role for this highly unsaturated, long

chain fatty acid” (Burr and Burr, 1930)

The term n-6/ ω-6 and n-3/ ω-3 signify that the first double bond exists

as the six and third carbon-carbon bond from the terminal methyl end (ω) of

the hydrocarbon chain respectively.

Figure 1.2. Structure of fatty acids and its numbering. Picture is adapted

and modified from European Food Information Council (EUFIC).

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1.1.1. Inflammation: “to set on fire” and Eicosanoids:-

Eicosanoids plays an important role in all the types and stages of

inflammation and are important molecules responsible for the signs of

inflammation as shown in the figure1.3. – Heat (calor), Redness (rubor),

Swelling (tumor), Pain (dolor) and Loss of function (functiolaesa).

Figure 1.3. Historical signs of inflammation. This whimsical picture was drawn for a 2002 review article in Nature by the Department of Medical

Illustration at St. Bartholomew’s College. Nature Reviews

Immunology 2, 787-795.

Inflammation is an immunological response comprising of both innate

and acquired immune system. It is of three stages:

1) Vasoconstriction- smooth muscles surrounding the vessels will

constrict and lead to the decreased blood flow which will enhance the

interaction between the pathogens and blood cells. This process gives

opportunity for leukocytes to adhere to vessel wall.

2) Vasodilatation- endothelial cells contract and increase space between

the cells. This results in increased capillary permeability. During this process,

there will be increase in blood vessel diameter and increase the blood flow and

causes erythema (flare).

3) Diapedesis / Extravasation- adhesion molecules are activated and

lead to release of blood cells into the surrounding media and causes edema

(wheal).

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Among the blood cells white blood cells (WBCs) plays the major role in

the defensive system. There are different types of WBCs. They all have many

things in common, but are all distinct in form and function. A major

distinguishing feature of some leukocytes is the presence of granules; and

based on these features WBCs can be classified as presented in the figure 1.4

and 1.5 as granulocytes and agranulocytes. Table 1.1 indicates the percentage

of occurrence of these cells and their physiological functions.

Granulocytes (polymorphonuclear leukocytes PMNLs): leukocytes

characterized by the huge amount of granules in the cytoplasm which can be

stained and viewed under light microscopy. These granules are enriched with

enzymes and many other bioactive molecules which will be released or utilized

within the cell to kill the pathogens. There are three types of granulocytes

which were classified and named based on their staining properties:

neutrophils (60% of WBC), basophils (1% of WBC) and eosinophils (4% of

WBC). Eicosanoids are also one of the mediators released by these granules.

Even these cells count will vary based on the conditions. During parasitic

infection eosinophils will increase and if there is threat from bacteria,

neutrophils will increase. Therefore differential count of WBC is important for

diagnosis.

Agranulocytes (mononuclear leukocytes): These are group of WBCs that

does not have any granules except non-specific lysosomes. Monocytes,

lymphocytes and macrophages belong to these classes of cells.

All these cells derived from hematopoietic stem cells (figure 1.4) in the

bone marrow which further gives pro-erythroblast, monoblast, myeloblast,

lymphoblast and megakaryoblast. Majorly the cells derived from myeloid

lineage are involved in the synthesis of eicosanoids.

Chapter-I General Introduction

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Figure 1.4. Hematopoietic system of blood cells. Picture adapted from Tom Hank’s a level notes.

Figure 1.5. Classes of WBCs and their percentage of occurrence. Picture

adapted from lymphomation.org.

Chapter-I General Introduction

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Table 1.1. Granulocytes of WBC and their percentage of occurrence and functions.

Inflammation is a defensive mechanism of an organ to avoid the invasion

by pathogens or wound repair. But, some microorganisms are able to evade the

clearance by immune system for example, by possessing cell wall that enable

them to resist phagocytosis. This will leads to the damage of surrounding

regions due to mediators released by immune cells. Inflammation can be

classified as acute and chronic based on their persistence in the system. Unlike

acute inflammation, chronic inflammation is the one which exists for longer

duration and causes damage to the tissue. Acute and chronic inflammation

differs by the kind of cells involved. In case of acute inflammation, majority of

the cells involved are the cells which plays the role in innate immune system

like PMNLs and in case of chronic inflammation, it is lymphocytes which plays

the vital role in acquired immune system. Such an inappropriate and damaging

immune response can be called as hypersensitive reactions / allergic reactions.

These responses can be even some times against non-harmful antigens.

Hypersensitive/allergic reactions are classified by Gell and Coombs as in the

Table. 1.2:

Type I: Anaphylactic / Ig E mediated reactions

Type II: Cytotoxic / cytolytic reactions

Type III: Immune complex mediated reactions

Type IV: Delayed type hypersensitive (DTH) reactions / T-cell mediated

/ tuberculin reactions.

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Table 1.2. Different types of hypersensitive reactions and their chatacteristics. Adapted and modified from Microbiology and

Immunology, University of South Carolina School of Medicine.

These hypersensitive reactions can be classified into different phases like

initialization, activation and effectors phases. Eicosaniods as mentioned in the

previous paragraph are involved in all the types of hypersensitive / allergic

reactions and plays an important role especially in the effectors stage. Immune

cells like mast cells which play the vital role during allergic reactions release

two types of mediators (as in the Table.1.3) which are preformed or newly

synthesized mediators. Preformed mediators are histamines, serotonin,

eosinophilic chemotactic factors (ECF), neutrophil chemotactic factors (NCF)

and heparin. Eicosanoids are the major lipid mediators formed along with

platelet activating factors (PAF) which were formed from membrane

phospholipids during inflammation and therefore eicosanoids are important

newly synthesized mediators. They play an important role in late phase

Chapter-I General Introduction

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reactions by acting as a chemotactic agents and helps in vasodilation and

extravasation of immune cells. The Figure 1.6 explains the role of these

mediators in the extravasation which will leads to edema and if it is systemic,

then leads to shock and death.

Table 1.3. Different types of mediators of hypersensitive reactions and their effects. Adapted and modified from Microbiology and Immunology,

University of South Carolina School of Medicine.

Figure 1.6. Inflammation: - Entry of immune cells in the tissue. Picture is adapted and modified from microbiologybytes.com.

Chapter-I General Introduction

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Table 1.4. Shows the source of eicosanoids and their biological role.

Eicosanoid Major site(s) of synthesis Major biological activities

LXA4

Lipoxins

platelets, endothelial cells, mucosal epithelial cells and

other leukocytes via inteactions with PMNs

reduce PMN and eosinophil infiltration to sites of

inflammation, stimulate nonphlogistic (non-inflammatory-inducing) monocyte recruitment, stimulate macrophage

phagocytosis of apoptotic PMNs, block IL-8 (chemokine) expression, block TNF-α release and actions, stimulate TGF-β action

LXB4

platelets, endothelial cells, mucosal epithelial cells and other leukocytes via inteactions with PMNs

same as for LXA4

PGD2 mast cells inhibits platelet and leukocyte aggregation, decreases T-cell

proliferation and lymphocyte migration and secretion of IL-1α

and IL-2; induces vasodilation and production of cAMP

PGE1 from DGLA of the membrane in kidney, spleen, heart

induces vasodilation and inhibits platelet aggregation

PGE2 from AA of the membrane in kidney, spleen, heart

increases vasodilation and cAMP production, enhancement of

the effects of bradykinin and histamine, induction of uterine contractions and of platelet aggregation, maintaining the open passageway of the fetal ductus arteriosus; decreases T-cell

proliferation and lymphocyte migration and secretion of IL-1α and IL-2

PGF2α kidney, spleen, heart increases vasoconstriction, bronchoconstriction and smooth muscle contraction

PGH2 precursor of all prostaoids precursor to thromboxanes A2 and B2, induction of platelet aggregation and vasoconstriction

PGI2 heart, vascular endothelial cells

inhibits platelet and leukocyte aggregation, decreases T-cell proliferation and lymphocyte migration and secretion of IL-1α and IL-2; induces vasodilation and production of cAMP

TXA1 from DGLA of the membrane in platelets

induces vasodilation and inhibits platelet aggregation

TXA2 platelets induces platelet aggregation, vasoconstriction, lymphocyte

proliferation and bronchoconstriction

TXB2 platelets induces vasoconstriction

LTB4 monocytes, basophils, neutrophils, eosinophils, mast cells, epithelial cells

powerful inducer of leukocyte chemotaxis and aggregation, vascular permeability, T-cell proliferation and secretion of INF-γ, IL-1 and IL-2

LTC4

monocytes and alveolar macrophages, basophils, eosinophils, mast cells,

epithelial cells

component of SRS-A, microvascular vasoconstrictor, vascular permeability and bronchoconstriction and secretion of INF-γ, recruitment of leukocytes to sites of inflammation, enhance

mucus secretions in gut and airway

LTD4 monocytes and alveolar

macrophages, eosinophils, mast cells, epithelial cells

same as LTC4

LTE4 mast cells and basophils same as LTC4

1.1.2. Biosynthesis of eicosanoids:-

Chapter-I General Introduction

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Biosynthetic pathway of eicosanoids begins with the activation of

phospholipase A2 (PLA2) which will act on membrane phospholipids and

releases fatty acid. These free fatty acids (FFA) will be acted by cytosolic

lipoxygenase (LOX) enzyme and releases LTs. This pathway is called as linear

pathway in an eicosanoid metabolism. Released FFA will also act by

cyclooxygenase (COX) and releases PGs, TXs and PGIs. This pathway is called

as cyclic pathway in eicosanoid biosynthesis.

Figure 1.7. Biosynthetic pathways for eicosaniods. Adapted from Journals.prous.com.

1.1.3. Leukotrienes:-

LTs derive their name from their discovery in leukocytes and their three

conjugated double bonds (Stella, 1999). The discovery of LTs is dependent on

Schultz-Dale reaction with guinea pig smooth muscle. These smooth muscles

with histamine showed reversible constriction. When mast cell supernatant

was added to the reaction mixture, slow prolonged contraction resulted which

cannot be easily reversed by washing and therefore they are named as slow

reacting substance of anaphylaxis (SRS-A) (Murphy et al., 1979). They are

metabolite of AA called LTs mainly produced by the reaction of LOX enzyme. LT

pathway also called as linear pathway mainly involves cytosolic LOX, LTA4

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hydrolase and two integral nuclear envelope proteins such as 5- LOX activating

protein (FLAP) and LTC4 synthase (Geotzl et al., 1995). LTs play a major role in

immediate hypersensitive reactions and inflammation which are the key

players during many disease conditions like asthma, arthritis and many other

allergic conditions (Samuelsson, 1983; Nicosia et al., 2001; James, 1997). They

are considered to be the main cause of anti-histamine resistance in asthmatics.

In the biosynthetic pathway of LTs, formation of epoxide LTA4 is the committed

step. LTs are mainly classified as LTB4 and Cysteinyl LTs (CysLTs). CysLTs are

distinguished by the presence of cysteine in their chemical structure. The term

CysLTs distinguishes itself from the non-cysteine-containing dihydroxy LT-

LTB4. LTB4 is an inflammatory mediator, a potent chemotactic agent and

attracts many pro-inflammatory cells like neutrophils and eosinophils to the

site of inflammation and helps in extravasations or diapedesis (Ford-Hutchinson

et al., 1980).

CysLTs are a family of potent inflammatory lipid mediators synthesized

from AA majorly by mast cells, eosinophils, basophils and macrophages.

CysLTs includes LTC4, LTD4 and LTE4, which are potent biological mediators in

the pathophysiology of inflammatory diseases. They trigger contractile effects

during inflammatory processes through the interaction with specific cell

surface receptors, belonging to the super family of G protein-coupled receptors

(GPCR).

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Figure 1.8. Structure of LTs.

Pharmacological characterizations have suggested the existence of at

least 2 types of CysLT receptors based on potency of agonist and antagonist,

designated as CysLT1 and CysLT2. The CysLT1 receptors are mostly expressed

in lung smooth muscle cells, interstitial lung macrophages and spleen. On the

other hand, CysLT2 receptors are present in heart, brain and adrenal glands.

CysLTs contract air-way and vascular smooth muscles stimulate mucus

secretion, increases micro-vascular permeability and they are very potent

bronchoconstrictors (Stella, 1999; Nicosia et al., 2001). Different types of

CysLTs such as LTC4, D4 and E4 are equipotent in causing bronchospasm and

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are potent stimulators of mucus secretion from airway tissues. As little as

nanomole (nmoles nM) concentration of CysLTs elicits erythema and wheal

formations like histamine. Human and animal studies have revealed that, LTs

are 1000-fold more potent than that of histamine (Dahlen et al., 1980). The

bronchoconstrictor activity of LTC4 was studied in artificially ventilated

monkeys (Smedegard et al., 1982). When both histamine and CysLTs were

administered intravenously, they are equipotent in their effects, but when given

as an aerosol of LTC4 (20 nmoles) were 100 fold more potent compared to that

of histamine (1000 to 5000 nmoles). LTs are involved in all the stages of

inflammation from constricting smooth muscles around large blood vessels and

vasodilation to diapedesis. It is well known fact that, prolonged use of aspirin,

a potent inhibitor of COX will increase the LOX activity (Kuna et al., 1997).

These classes of eicosanoids play an important role in systemic as well as

localized hypersensitive reactions (atopy). Tendency to manifest localized

hypersensitive reactions is inherited and called atopy. Atopic allergies, which

afflict at least 20% of the population in developed countries, include wide range

of IgE mediated disorders, including allergic rhinitis (Hay fever), asthma, atopic

dermatitis (eczema) and food allergies. LTs contribute to

the pathophysiology of allergic conditions like asthma as mentioned below:

airflow obstruction

increased secretion of mucus

mucosal accumulation

bronchoconstriction

infiltration of inflammatory cells in the airway wall

Allergic reactions of the respiratory system are becoming very common

because of the growing population and due to air pollution. Especially, India

with growing population and vast industrial development has drastic increase

in number of population suffering from asthma and other allergic reactions.

According to the report published in Times of India (TOI) on 4th of February,

2011- Dayanand Medical College and Hospital Vice-Principal Jagdeep Whig

states to Times Of India, ''Nearly 80 patients visit outpatient department (OPD)

with chest blockage and 30% of them are those suffering from asthma. There

aren't any exact reasons for its spread among city residents, though high air

pollution is one of its major causes. Air pollution in Ludhiana includes

Chapter-I General Introduction

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industrial and vehicular pollution and its chief components are sulphur dioxide

(SO2) and nitrous oxide (N2O). As the city is extremely polluted, its residents are

more prone to the disease''. In a study from Bangalore, they have observed the

increase in allergic respiratory disorders by 30% in the children below 18 years

(Paramesh, 2002).

Figure 1.9. Effect of LTs in asthmatic patients. Adapted from

singulair.ae.

In humans, LTB4 is mainly synthesized in monocytes, alveolar

macrophages and neutrophils, whereas CysLTs are synthesised by eosinophils,

basophils, mast cells and alveolar macrophages. They are synthesized by trans-

cellular metabolism from neutrophil-derived LTA4 by platelets and vascular

endothelial cells. This trans-cellular biosynthesis is considered very important

because it could generate remarkably high concentrations of CysLTs in a local

environment, ultimately affecting organ function (Dahinden et al., 1985;

Feinmark and Cannon, 1986; Feinmark, 1990; Maclouf and Murphy, 1988;

Maclouf et al., 1989; Maclouf et al., 1996) Such trans-cellular biosynthesis of

LTs has been reported from mast cells (Bigby and Meslier,1989) peripheral

blood monocytes (Bigby et al., 1989), human airway epithelial cells, alveolar

macrophages, kidney-derived endothelial cells (Brady and Serhan, 1992),

keratinocytes (Iversen et al., 1994) and chondrocytes (Amat et al., 1994). As

mentioned earlier, CysLTs have very short life span and are inactivated by 3

Chapter-I General Introduction

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major mechanisms viz.,N-acetyl derivatization, reaction with hypochlorous acid

to form sulfoxide and hydroxylation followed by carboxylation which can be

further metabolized by β-oxidation to form shortened metabolites (Keppler,

1992; Lewis and Austen 1984; Nicosia et al., 2001; Sala, 1990).

Figure 1.10. AA cascade for formation of LTs and their effects. Adapted and modified from Canadian Medical Association Journal, 1999.

LTB4 exerts its effects mainly by two G-protein coupled seven trans-

membrane receptors BLT1 and BLT2 (as in the Figure 1.10). BLT1 acts by

phspholipase C (PLC) pathway and mobilizes intracellular Ca2+ level in the

target cell. BLT1 is also involved in the activation of peroxisome proliferator

activated receptorα (PPARα) and involved in the feedback inhibition. BLT2 is

involved in the chemotaxis function of the LTB4. There are many antagonists

for these receptors, among which CP-105, U-75302 are effective against BLT1

(Figure 1.11) (Nicosia et al., 2001). They are also known to enhance the activity

of PLA2 activating protein (PLAP) and increase the release of AA from the

membrane. LTs exert their effects by GPCR which mainly activate PLC resulting

in increase of Ca2+ concentration and inhibition of adenylate cyclase pathway

(Nicosia et al., 2001; Funk, 2001).

Chapter-I General Introduction

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Effects of LTs can be nullified as mentioned by either inhibiting LOX or

by blockage of LTs receptors (Figure 1.11). There are many inhibitors available

in the market like zileuton which exert its effect by competitive inhibition of

LOX and zafirlukast that binds to the LTE4 and D4 receptors ultimately

blocking inflammatory signals. ZD2138 can directly inhibit LOX and MK-886,

BAYx1005 acts by inhibiting 5-LOX activating protein (FLAP) a protein which

enhances the interaction between AA and LOX. There are many antagonists for

CysLT receptors. Cys LT are classified based on their sensitivity for classical

antagonists like SK & F 104353 (pobilukast), ICI 204, 219 (zafirlukast), MK-

571, MK-476 (montelukast), ONO-1078 (pranlukast), CGP 45715A (iralukast)

and Ro 24-5913 (cinalukast). Receptors sensitive for these classical antagonists

are called CysLT1. A new drug BAY u9773 is available with dual action which

acts on both the receptors CysLT1 and Cys LT2. CysLT1 is predominantly

activated by LTD4/LTE4 and CysLT2 by LTC4 (Nicosia et al., 2001). Ketotifen,

azelastine and oxatomide are new generation H1 receptor antagonists which

are used for treating patients suffering from allergic diseases. Ketotifen and

oxatomide inhibits (PLA2) activity whereas, azelastine inhibits LTC4 formation

by inhibiting PLA2 and LTC4 synthase (Hamasaki et al., 1996).

LTA4H-LTA4 hydratase, LTC4S- LTC4 synthase

Figure 1.11. Target of different drugs designed for LT biosynthesis

pathways. Adapted from Leukotriene Signaling in Atherosclerosis and Ischemia, Cardiovascular Drugs and Therapy 23 (1) (2008).

Chapter-I General Introduction

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1.1.4. Prostanoids:-

As mentioned prostanoids are eicosanoids involving PGs, TXs and PGIs.

PGs were named because it was thought that it is a product of prostate gland.

Later it was found that it is synthesized in the seminal vesicles and is rich in

semen. It is a family of closely related derivatives of hypothetical C20 molecule

prostanoic acid. About 1 mg of PG produced in humans every day. Seminal

vesicles, lung and renal tissues have greatest capacity to synthesize PGs

whereas aorta and spleen can produce small amounts. PGs along with LTs play

an important role in maintaining the normal homeostasis of different tissue

and also play a crucial role in inflammation. The systematic nomenclature of

PGs and its metabolites is based on prostanoic acid (Figure 1.12), a

monocarboxylic acid with 20 carbon atoms, arranged as two side chains with 7

and 8 carbons, respectively linked to central cyclopentane ring. PGs are

classified by the functional groups of the cyclopentane ring. PGs differ from

each other in two ways: (1) the substituents of the pentane ring (indicated by

the last letter, eg., E and F in PGE and PGF) and (2) the number of double

bonds in the side chains (indicated by the subscript, eg., PGE1 and PGE2). PGH2

is metabolized by prostacyclin (PGI), thromboxane (TX) and PGF synthases (S)

to PGI2, TXA2 and PGF2 respectively.

Figure 1.12. Structure of prostanoic acid and prostanoid ring structure. Adapted from lipidlibrary.aocs.org.

Chapter-I General Introduction

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Prostanoids, thromboxanes (TX) and prostacyclins (PGI) are synthesized

mainly by COX-1 / PGH Synthase-1 (PGHS1) and COX-2 / PGH Synthase-2

(PGHS2) (Figure 1.13) which is a multi-enzyme complex called as PG

endoperoxide synthase having two subunits- COX and peroxidase. COX-1

mainly has housekeeping role and is located in the endoplasmic reticulum

(ER). They are involved in the secretions of PGs into the surrounding medium

which helps in maintenance of normal homeostasis in kidney, stomach,

platelets and endothelial cells. COX-2 is located in the nuclear envelope where

it is activated only during the inflammation and other pathogenic conditions.

COX-2 genes are immediate early genes and are not constitutively expressed

(Goetzl et al., 1995).

Figure 1.13. Effects of PGs on human tissues.

PGs have wide range of functions in human body. PGE and PGA are

potent vasodilators and have anti-hypertensive action by lowering the blood

pressure. They stimulate renin secretion from JG cells which leads to increase

in levels of angiotensin II. PGE1 is a potent inhibitor of platelet aggregation and

are proved to be useful in storage of blood platelets in blood banks. PGE1, PGE2

and PGA1 inhibit gastric acid secretion and have been used for preventing

gastric ulcers, but at the same time they increase pancreatic secretion of

bicarbonate and also enhance the mucus secretion in the intestine. PGE and F

contracts longitudinal muscles of stomach to colon and show purgative action.

PGE1 and E2 are potent vasodilators and this property has been used for

treatment of asthma. PGE and F induce uterine contraction and therefore these

synthetic PGs are used to induce child birth- parturition / abortion. PGE2

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(0.5µg/mL) is used for the induction of labor and at higher concentration

(5µg/mL) termination of pregnancy in first and second trimesters

(abotrification). PGE2 decreases water absorption in distal tubules, increases

urine volume and output of Na+ and K+. PGE2 and PGD2 play the vital role in

inflammation by increasing capillary permeability, vasodilation which leads to

wheal and flare reaction. PGD2 is an important mediator of anaphylaxis. TXB2

and PGI2 as in the figure 1.14, play an important role in maintaining the proper

tone of blood vessels. Imbalance in their level leads to ischemia (Dubois et al.,

1998; Stack et al., 2001).

Figure 1.14. Role of TX and PGI in regulation of vascular tone.

The diverse effects of PGs have forced scientists to artificially synthesize

the PGs. Presently these synthetic PGs are used to induce child birth

(parturition) or abortion, to treat peptic ulcer, as a vasodilator in severe

Raynaud’s phenomenon, in pulmonary hypertension, treatment of glaucoma

(an eye disorder in which the optic nerve suffers damage, permanently

damaging vision in the affected eye and/or eyes), treatment of erectile

dysfunction and to prevent closure of patent ductus arteriosus (is a congenital

disorder in the heart wherein a neonate's ductus arteriosus fails to close after

birth) in newborns with particular cyanotic heart defects. Alprostadil (PGE1)

may be used for its smooth muscle relaxing effects to maintain the

ductusarteriosus patient in some neonates awaiting cardiac surgery and in the

treatment of impotence. Misoprostol, a PGE1 derivative, is a cytoprotective PG

used in preventing peptic ulcer and in combination with mifepristone (RU-486)

for terminating early pregnancies. PGE2 and PGF2 are used in obstetrics to

Chapter-I General Introduction

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induce labor. Latanoprost and several similar compounds are topically active

PGF2 derivatives used in ophthalmology to treat open angle glaucoma. PGI

(epoprostenol) is synthesized mainly by the vascular endothelium and is a

powerful vasodilator and inhibitor of platelet aggregation. It is used clinically to

treat pulmonary hypertension and portopulmonary hypertension. In contrast,

TXA has undesirable properties (aggregation of platelets, vasoconstriction).

Figure 1.15. Biosynthesis of prostanoids and their receptor distribution. Adapted from Current Opinion in Pharmacology 5 (2005) 204-210.

Aspirin a potent inhibitor of COX causes decrease in platelet aggregation

will also decreases PGI2, which will be regenerated by endothelial cells unlike

TXB2 by platelets which are enucleated cells. Long term intake of aspirin will

cause increase in 5-LOX activity and will also damage the gastrointestinal (GI)

tract. There are many chemical classes (Table 1.4) of anti-inflammatory drugs

with different effects. These drugs can be classified as non-steroidal anti-

inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs

(glucocorticoids) (Figure 1.15). NSAIDs act by inhibiting COX and causes

benefits like analgesia, anti-inflammatory action and anti-pyretic (fever

reducing) action.

Chapter-I General Introduction

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Chemical Class

Examples

Physiological Effects

Salicylic acids

Propionic acids

Acetic acids

Para-aminophenols

Oxicams

Pyrazolones

Fenemates

Aspirin

Ibuprofen

Indomethacin

Paracetamol

Piroxicam

Phenylbutazone

Mefenamic acid

Analgesic, anti-pyretic, anti-inflammatory.

Analgesic, anti-pyretic, anti-

inflammatory.

Analgesic, anti-pyretic, anti-

inflammatory.

Analgesic, anti-pyretic.

Analgesic, anti-pyretic, anti-

inflammatory.

Anti-pyretic, anti-inflammatory.

Analgesic, anti-pyretic.

Table 1.5. Different classes of NSAIDs and their physiological effects.

Aspirin acts by irreversible inhibition of COX, ibuprofen acts as a

competitive substrate and paracetamol acts by its free radical scavenging

action which will interfere with hydroperoxide production.

Figure 1.16. NSAIDs commonly used and its chemical class and their targets. Adapted from doctorsgates.blogspot.com.

Chapter-I General Introduction

23

But all these NSAIDs have side effects such as GI upset, nephrotoxicity,

nausea and vomiting. It is a well-known fact that paracetamol in excess causes

hepatotoxicity by formation of metabolite N-acetyl-p-benzoquinone. Many COX-

2 specific inhibitors are available to reduce the side effects and among them

commonly used are rofecoxib and celecoxib. Therapeutic doses of

glucocorticoids have anti-inflammatory effects by stabilizing the lysosomal

membrane, by preventing kinin formation and decreasing the permeability of

capillary walls. Glucocorticoids exert their effects by altering the corticosteroid

responsive genes. They reduce the release of AA from membrane through

inhibition of PLA2 by inducing formation of polypeptide lipocortin. They also

reduce the activity of macrophages and fibroblasts. Reduces the release of IL-1

from granulocytes and histamine from mast cells. Prolonged treatment with

glucocorticoids results in osteoporosis and gastrointestinal ulcers. Due to the

side effects of these NSAIDs, there is a sentence in medical science-

“Cardiologist wants his patient to take aspirin daily and the same will upset the

gastroenterologist”.

This is very pertinent to restrict pro-inflammatory eicosanoids from its

action under certain inflammatory conditions. There are many NSAIDs

available in drug stores. Both steroid and NSAIDs have side effects; hence

nutraceuticals are gaining the importance in treating these inflammatory

diseases and reducing the levels of pro-inflammatory eicosanoids. In this study,

dietary spices and their active principles and n-3 rich garden cress oil (GCO)

were used to study their modulatory effect on eicosanoids.

1.2. Spices:-

A spice is a dried seed, fruit, root, bark or vegetative substance used in

nutritionally insignificant quantities as a food additive for flavor, color or as a

preservative that kills harmful bacteria or prevents their growth. Since ages

spices and its active principles are known for their beneficial effects on human

health. They are the natural stimulators of appetite. Along with their usage in

the food flavorings they have also immensely used in the perfumery, cosmetics

and toiletries. Spices and their derivatives like essential oils are being widely

used for food flavoring, food preservation, personal hygiene products, aroma

Chapter-I General Introduction

24

therapy, pharmaceuticals and beverage industries for flavoring and fragrances.

Spices impart aroma, colour and taste to food preparations and sometimes

mask undesirable odors. Volatile oils give the aroma and oleoresins impart the

taste. Aroma compounds play a significant role in the production of

flavourants, which are used in the food industry to flavour, improve and

increase the appeal of their products.

The Economic Times (April 4, 2011) has reported “India to become global

spice hub soon” on press trust of India (PTI). India is the largest producer,

consumer and exporter of spices in the world today, contributing about 48 % of

the world's requirement of spices. The current production of spices in the

country (2010-2011) was at 5.5 million tons. Export of spices play a significant

role in earning foreign revenue for the country. Recent reports show that total

export of spices from India during the current financial year up to November

2011 is 3,51,900 tons valued at Rs.6,209.08 crores, which is US dollars

1332.25 Million. Among the major spices exported from India, Chili contributes

132,500 tons occupies the first place. Other major spices that are exported

from India include turmeric (58,000 tons), Cumin (26,500 tons), Coriander

(18,200 tons), Pepper (17,000 tons), Fenugreek (14,700 tons), Ginger (11,250

tons), Fennel (5,100 tons), Nutmeg & mace (2,550 tons), Celery (2,450 tons),

Cardamom small (3,100 tons) and Cardamom Large (475 tons), Garlic (1075

tons). Mint products, curry power and pastes, spice oils, oleoresins and other

spices like Tamarind, Asafoetida, Cassia and Saffron etc also contribute to the

Indian exports.

India 1 600 000 86 %

China 99 000 5 %

Bangladesh 48 000 3 %

Pakistan 45 300 2 %

Nepal 15 500 1 %

Other countries 60 900 3 %

Total 1 868 700 100 %

Table 1.6. Total global spice production.

Chapter-I General Introduction

25

Table 1.7. Spice producing areas in India.

Spices States of India

Pepper Kerala, Karnataka, Tamil Nadu.

Cardamom (Small) Kerala, Karnataka, Tamil Nadu.

Cardamom (Large) Sikkim, West Bengal.

Ginger Andhra Pradesh, Karnataka, Kerala, Madhya Pradesh,

Meghalaya, Orissa, Arunachal Pradesh, West Bengal,

Mizoram, Sikkim, Himachal Pradesh, Tamil Nadu,

Uttaranchal, Chattisgarh, Jharkhand.

Turmeric Andhra Pradesh, Karnataka, Orissa, Tamil Nadu,

West Bengal, Maharashtra, Kerala, Assam, Bihar,

Meghalaya, Tripura, Uttar Pradesh, Arunachal

Pradesh

Chilli Andhra Pradesh, Gujarat, Karnataka, Maharashtra,

Orissa, Rajasthan, Tamil Nadu, Uttar Pradesh, West

Bengal, Madhya Pradesh, Uttaranchal.

Coriander Rajasthan, Uttar Pradesh, Uttaranchal.

Cumin Rajasthan, Gujarat, Uttar Pradesh

Fennel Gujarat, Rajasthan, Uttar Pradesh

Fenugreek Rajasthan, Uttar Pradesh, Gujarat

Clove Kerala, Tamil Nadu, Karnataka

Nutmeg & Mace Kerala, Tamil Nadu, Karnataka

Cinnamon &

Cassia

Kerala, Tamil Nadu

Vanilla Kerala, Karnataka, Tamil Nadu

Garlic Haryana, Madhya Pradesh, Maharashtra, Orissa,

Uttar Pradesh, Gujarat, Karnataka, Rajasthan,

Chattisgarh, Bihar

Ajowan Bihar, Jammu & Kashmir

Kokam Karnataka

Mustard Uttar Pradesh, Bihar, Andhra Pradesh

Chapter-I General Introduction

26

It is well established that the spices have many medicinal values such as

anti-microbial, anti-oxidant, carminative and immunomodulatory properties

(Shobana and Akhilender, 2000; Srinivasan, 2005; Gruenwald, 2010). Medicinal

spices have been used since centuries in traditional medicinal systems like

Ayurveda and Unani medicine. Spices and herbs can be the flowers, fruits,

seeds, roots, leaves, bark of the plant. Medicinal properties of spices can have

preventive / prophylactic action and are being investigated to understand their

nutraceutical properties. Spices have been tested for its anti-microbial

activities. In a study, anti-bacterial potentials of six crude plant extracts (Allium

sativum, Zingiber officinale, Allium cepa, Coriandrum sativum, Piper nigrum and

Citrus aurantifolia) were tested against five Escherichia coli isolated from potable

water sources at kushtia, Bangladesh. Spices might have anti-bacterial activity

against enteric pathogens and could be used for prevention of diarrheal

diseases (Shahedur et al., 2011). Cinnamon oil has proven more effective than

ampicillin in inhibiting the growth of Staphylococcal infections and unlike

conventional antibiotic drugs, essential oils tend to leave beneficial bacteria

intact while killing virulent bacteria (pathogens). Essential oils from clove and

eugenol show various degrees of inhibition against Aspergillus niger,

Sacchromyces cerevisiae, Mycoderma sp., Lactobacillus acidophilus, Bacillus

cereus, Fusarium verticilloides and Listeria monocytogenes (Meena and Sethi,

1994; Veluti et al., 2004). Clove oil / eugenol show antifungal activity against

Eurotium, Cladosporium herbarum, Penicillium glabrum, Penicillium expansum

and Aspergillus niger (Martini et al., 1996). Additionally, bacteria do not acquire

resistance to the oils as they do with antibiotics. Today when so many illnesses

and bacteria are becoming resistant to antibiotics, the therapeutic effects of

essential oils and their immune-boosting abilities may be just what we need. In

animal experiments, effect of garlic and onion on regression of established

gallstones and formation of gallstones have been reported (Vidyashankar et al.,

2009; Vidyashankar et al., 2010). Nutritional points of view, some spices are

enriched with dietary fibers and proteins. A wide variety of phenolic compounds

and flavonoids present in spices possess potent anti-oxidant, anti-mutagenic

and anti-carcinogenic activities.

Chapter-I General Introduction

27

Curcuminoids and other constituents of turmeric are well known for

their anti-inflammatory activity. Turmeric extract, volatile oils from turmeric

and curcuminoids were reported to possess this property in different

experimental models of inflammation viz., mice, rats, rabbits and pigeons

(Arora et al., 1971; Chandra and Gupta, 1972; Ghatak and Basu, 1972).

Curcumin the active principle of turmeric is shown to possess

immunomodulatory activity by suppressing lymphocyte proliferation and pro-

inflammatory cytokine production in vitro (Xiaohua et al., 2004). Eugenol and

its derivatives have shown to possess anti-inflammatory properties and used in

dental treatment (Reddy and Lokesh 1994; Yukio et al., 2005). Oral

administration of curcumin at a dose of 3 mg/kg was also found to be effective

in reducing inflammation associated with various forms of arthritis (Chandra

and Gupta, 1972; Srimal and Dhawan, 1973).

Polyphenols are found in many dietary plant products, including fruits,

vegetables, beverages, herbs and spices. Several of these compounds have been

found to inhibit the inflammation process as well as tumorigenesis in

experimental animals. They can also exhibit potent biological properties. In

addition, epidemiological studies have indicated that populations who consume

foods rich in specific polyphenols have lower incidences of inflammatory

disease. Anti-inflammatory activities of most of the polyphenols suggested to

include, but not limited to, the inhibition of enzymes related to inflammation,

such as COX and LOX and many others including peroxisomal prolifrerated

activated receptor (PPAR), nitric oxide synthase (NOS), nuclear factor-κB (NF-

κB) and NSAID activated gene-1(NAG-1). COX, LOX, and PLA2 are considered

as AA-dependent pathway proteins by polyphenols, whereas NOS, NF-κB, PPAR

and NAG-1 are considered as AA-independent pathway proteins (Joo and

Seung, 2005; Marion-Letellier et al., 2009). The o-Methoxyphenols such as

eugenol and isoeugenol components of clover oil inhibits lipopolysaccharide

(LPS) induced NF-κB activation and COX-2 expression in macrophages (Yukio

et al., 2005). But, due to low levels of spice consumption, their impact on

nutrient make-up may not be as dramatic as that of other food ingredients.

Extensive animal studies have shown that many varieties of spice consumption

even at higher levels (100 fold the normal intake) have no effect on growth,

Chapter-I General Introduction

28

organ weights, feed efficiency ratio, nitrogen balance and blood constituents

(Smith et al., 2002; Srinivasan, 2005).

The components of spices responsible for the quality attributes have

been designated as active principles which are well studied for their beneficial

physiological effects. Most of these active principles belong to the class of

flavanoids or alkaloids or to an umbrella term polyphenolic molecules.

Observational studies have suggested that life style risk factors such as

tobacco, alcohol, high-fat diet, radiation and infections can cause cancer and

that a diet consisting of fruits and vegetables can prevent cancer. Evidence

from many studies, suggests that agents either causing or preventing cancer

are linked through the regulation of inflammatory pathways. Genes regulated

by the transcription factor NF-κB have been shown to mediate inflammation,

cellular transformation, tumor cell survival, proliferation, invasion,

angiogenesis and metastasis. Whereas various life style risk factors have been

found to activate NF- κB and NF- κB -regulated gene products. Flavonoids

derived from fruits and vegetables have been found to suppress this pathway

(Prasad et al., 2010). During recent years, spices and their active principles

have been studied as possible ameliorative or preventive agents. Spices have

been proved for their hypolipidemic and hypocholesterolemic effects in animal

models. Cinnamom has been found to reduce triglycerides, bad cholesterol and

sugar in the blood, thus helping those with high cholesterol, diabetes and heart

disease. Experiments using curcumin and capsaicin have proved their role in

lowering blood cholesterol and also increase the conversion of cholesterol to

bile acids and these spice principles are also proved for their anti-lithogenic

effects (Srinivasan, 2005; Shubha et al., 2011). Curcumin and capsaicin have

also shown modulatory effect on AA metabolism and lysosomal enzymes such

as collagenase, elastase and hyaluronidase secretion by rat peritoneal

macrophages which play vital role in inflammation. These studies

demonstrated that curcumin and capsaicin can control the release of

inflammatory mediators such as eicosanoids and hydrolytic enzymes secreted

by macrophages and thereby may exhibit anti-inflammatory properties (Bina

and Lokesh, 1997). Curcumin has been shown in the last two decades to be a

potent immunomodulatory agent that can modulate the activation of T cells, B

cells, macrophages, neutrophils, natural killer (NK) cells and dendritic cells.

Chapter-I General Introduction

29

Curcumin can also down regulate the expression of various pro-inflammatory

cytokines including tumor necrosis factor (TNF), interleukin (IL)-1, IL-2, IL-6,

IL-8, IL-12 and chemokines most likely through inactivation of the

transcription factor NF-κB (Jagetia and Aggarwal, 2007). Inhibitory effects of

these spices and its active principles are reversible. The substance that

gives turmeric its color shows promise in fighting Alzheimer's disease, Cystic

Fibrosis and even certain types of cancer. Both an anti-flammatory and an

antibacterial, turmeric has long been used for treatment of enhancing digestion

and preventing a cold and flu. Among other findings, researchers discovered

that turmeric (especially the curcumin component) has rich stores of

biomolecules which plays the vital role in reducing oxidative stress in the body

and in turn reduces free radicals that can otherwise damage cells. Studies

indicate that curcumin is as powerful an anti-oxidant as vitamins C and E and

even beta-carotene. Anti-oxidants are also powerful preservatives, which helps

explain why turmeric has long been sprinkled on food to help retain its

freshness. In an animal study, curcumin has shown anti-arthritic activity

(Ramadan et al., 2011). The inhibitory effects of spices and its active principles

will be evident only when they are intact in the cells (Vijayalakshmi and

Chandrasekhra, 1981; Bina and Lokesh, 1997). It has been shown that

phenolic anti-oxidants eugenol inhibit NF-κB activation induced by TNF-α and

block COX-2 expression in LPS-stimulated macrophages. TNF-α, is also known

to mediate inflammation and carcinogenesis in various pathophysiological

processes, acts in part through activation of NF-κB, an important

transcriptional factor that regulates inflammatory response and expression of

inflammatory cytokines. Additionally, TNF-α stimulates the secretary activity of

airway smooth muscle cells, having a main role in orchestrating and

perpetuating the inflammatory process. Eugenol effectively improved functional

and structural pulmonary changes induced by LPS, modulating lung

inflammation and remodeling in an in vivo model of acute lung injury (ALI),

through a mechanism involving inhibition of TNF-α release and NF-κB

activation (Magalhaes et al., 2010). Spices have also shown anti-platelet

activities and modulation of platelet prostanoid metabolism.

Chapter-I General Introduction

30

Eugenol (2-methoxy-4-(2-propenyl) phenol) (Figure 1.16) is the active

principle of clove (Eugenia aromaticum) is the major component in clove oil and

it is used in dental care, as an antiseptic, analgesic and antibacterial agent

against oral bacteria associated with dental caries and periodontal disease. In

addition to its anti-microbial activity, eugenol possesses anti-inflammatory,

cytotoxic, insect repellent, insecticidal, anticancer and anesthetic properties

(Markowitz et al., 1992; Cai and Wu, 1996; Banerjee et al., 2006; Chaieb et al.,

2007). The expression analysis of genes involved in apoptosis

and inflammation revealed significant down-regulation of Bcl-2, COX-2, and IL-

1β on treatment with eugenol. Thus, Studies proves that eugenol exerts its

anti-cancer activities via apoptosis induction and anti-inflammatory properties

(Hussain et al., 2011). Anti-platelet and calcium inhibitory properties of eugenol

have been proved by a scientific study conducted using human blood (Chen et

al., 1996). Inhibitory effect of curcumin, a food spice from turmeric, on platelet-

activating factor (PAF) and AA-mediated platelet aggregation through inhibition

of thromboxane formation and Ca2+ signaling has been reported (Shah et al.,

1999). Another group has proved the inhibitory effect of curcumin on platelet

aggregation induced by collagen, AA and adrenaline in the human blood (Saeed

and Gilani, 1994; Srivastava et al., 1995). Cinnamomum cassia is a well-known

traditional medicine for improvement of blood circulation. An extract of this

plant showed both platelet anti-aggregation and blood anti-coagulation effects

in preliminary testing. Composition of oil was enriched with eugenol,

cinnamaldehyde, cinnamic acid and coumarin (Kim et al., 2010). Garlic oil has

been tested for its platelet aggregation properties. Acute effects of garlic have

been tested on platelet aggregation in 14 healthy volunteers using a

randomized, double-blind, placebo-controlled, crossover research method.

Results have shown that, oil was successfully inhibiting adrenaline induced

platelet aggregation (Wojcikowski et al., 2007). In a study, scientists have

examined the effect of phenolic and non-phenolic active principles of common

spices on copper ion-induced lipid peroxidation of human low density

lipoprotein (LDL) by measuring the formation of thiobarbituric acid reactive

substance (TBARS) and relative electrophoretic mobility (REM) of LDL on

agarose gel. Curcumin, capsaicin, quercetin, piperine, eugenol and allyl sulfide

inhibited the formation of TBARS effectively and decreased the REM of LDL

(Naidu and Thippeswamy, 2002).

Chapter-I General Introduction

31

Essential oil of Syzygium aromaticum, as well as its main component

(eugenol), possesses anti-ulcer activity. The data suggest that the effectiveness

of the essential oil and eugenol is based on its ability to stimulate the synthesis

of mucus, an important gastroprotective factor (Santin et al., 2011). Spices and

their active principles have beneficial effects for indigestion, excess gas

(flatulence), bloating and other mild stomach upset. Reinforcing an ancient use

for turmeric, German health authorities have declared turmeric tea a valuable

remedy for stomach upset. The curcumin as described an active principle of

turmeric fights bacteria commonly responsible for infectious diarrhea.

Curcumin has been proved for its ameliorative effect on chronic experimental

colitis by modulating the action of mitogen activated protein kinase (MAPK) p38

which in turn alter the activity of COX-2 and inducible nitric oxide synthase

(iNOS) expression (Laura et al., 2007). Curcumin has also shown inhibitory

effects on trinitrobenzenesulphonic acid (TNBS) induced colitis in the

experimental animals by activating PPARγ (Figure 1.17) (Ming et al., 2006).

PPARγ was initially identified for its role in adipocyte differentiation and

regulation of genes involved in lipid and glucose metabolism. However,

activation of PPARγ also can antagonize NF-κB action in macrophages resulting

in down-regulation of pro-inflammatory cytokines. Implicated in these anti-

inflammatory properties, PPARγ is not only expressed in adipocytes but also in

a number of other cell types, such as macrophages, lymphocytes, hepatocytes

and skeletal muscle. Very high expression levels are found in the colonic

epithelium (Rogler, 2006).

TLR-Toll Like Receptor

Figure 1.17. Anti-inflammatory role of PPAR-γ in intestinal epithelial cells. Adapted and modified from The Lancet 360 (2002) 1410–1418.

Chapter-I General Introduction

32

Since spices and its active principles have been proved for its anti-

inflammation, anti-oxidant and anti-platelet aggregation properties, it is very

much pertinent to further explore their effects in this direction.

In this study following seven different spices and its active principles

were used.

Figure 1.18. Pictures of spices used for the study.

Chapter-I General Introduction

33

Figure 1.19. Structure of spice active principles used for the study.

Molecular structures were adapted from Wikipedia.

Chapter-I General Introduction

34

1.3. Omega 3-fatty acids :-

Studies on Paleolithic nutrition and hunter gathering populations, it

appears that human beings evolved consuming diet that was low in saturated

fatty acids and equal amounts of n-6 and n-3 fatty acids (Eaton and Konner,

1985; Simopoulos, 1991). Intake of n-3 fatty acids is much lower today because

of decrease in fish consumption and industrial production of animal feeds rich

in grains containing n-6 fatty acids leading to production of meat rich in n-6

fatty acids and eggs (Crawford, 1968). Even cultivated vegetables contain fewer

n-3 fatty acids than do plants in the wild (Simopoulos and Salem, 1986;

Simopoulos et al., 1995). In summary, modern agriculture, with its emphasis on

increase in crop production and yields, has decreased the n-3 fatty acid

content in many foods including green leafy vegetables, animal meats, eggs and

even fish. Fatty acids present in our daily diet plays a vital role in synthesis of

eicosanoids. Earlier, it was known that by replacing saturated fatty acids of the

diet by PUFA one can reduce the risk of heart diseases. But, the modern diet

has forced the researcher to look for new type of oils enriched with n-3 fatty

acids to overcome the deficiency of n-3 fatty acids in vegetable oils. An article

published in American Journal of Nutrition pointed out that ratio of n-6 to n-3 of

1:1 from pre-historic times has shifted to 10:1 because of unhealthy dietary

practices.

Important n-3 fatty acids in human nutrition are ALA/ALNA, EPA and

DHA. Human body cannot synthesize n-3 fatty acids, but can synthesize long

chain n-3 fatty acids from ALA. EPA and DHA is found mainly in oily cold water

fish such as tuna, salmon, trout, herring, sardines, bass, swordfish and

mackerel (Figure 1.20). With the exception of sea weed, most plants do not

contain EPA and DHA. Many marine plants, especially algae can synthesize

very long chain fatty acids (VLCFAs) like EPA and DHA. Fish surviving on this

phytoplankton therefore will be the good source of these fatty acids (Calder,

2001). Plants such as green leafy vegetables contain small amounts of ALA but

flaxseed oil, soybean oil, canola oil, walnuts and fish oil (Figure 1.20) are rich

sources of ALA. Human milk has small but nutritionally significant amount of

EPA and DHA. Enzymes present in the human body can convert ALA to EPA

and DHA. Mammals cannot interconvert n-6 and n-3 fatty acids as there is a

Chapter-I General Introduction

35

deficiency of enzyme required for this conversion (Calder, 2001). In humans,

major product of fatty acid synthase is palmitate and further elongation and

unsaturation will be carried out by enzymes present in the ER. Humans lack

the enzyme which can bring unsaturation beyond carbon-9, for this reason LA

and ALA are essential fatty acids and rest of the PUFA can be synthesized from

these two fatty acids.

Figure 1.20. Oil-sources of different fatty acids.

Chapter-I General Introduction

36

Table 1.8. Shows important n-3 fatty acids with their double bonding

positions.

Common name Lipid

name Chemical name

Hexadecatrienoic acid 16:3 (n−3) all-cis-7,10,13-hexadecatrienoic acid

α-Linolenic acid 18:3 (n−3) all-cis-9,12,15-octadecatrienoic acid

Stearidonic acid 18:4 (n−3) all-cis-6,9,12,15-octadecatetraenoic acid

Eicosatrienoic acid 20:3 (n−3) all-cis-11,14,17-eicosatrienoic acid

Eicosatetraenoic acid 20:4 (n−3) all-cis-8,11,14,17-eicosatetraenoic acid

Eicosapentaenoic acid 20:5 (n−3) all-cis-5,8,11,14,17-eicosapentaenoic acid

Heneicosapentaenoic acid 21:5 (n−3) all-cis-6,9,12,15,18-heneicosapentaenoic acid

Docosapentaenoic acid,

Clupanodonic acid 22:5 (n−3) all-cis-7,10,13,16,19-docosapentaenoic acid

Docosahexaenoic acid 22:6 (n−3) all-cis-4,7,10,13,16,19-docosahexaenoic acid

Tetracosapentaenoic acid 24:5 (n−3) all-cis-9,12,15,18,21-tetracosapentaenoic acid

Tetracosahexaenoic acid (Nisinic

acid) 24:6 (n−3) all-cis-6,9,12,15,18,21-tetracosahexaenoic acid

Chapter-I General Introduction

37

Common name Lipid

name Chemical name

Linoleic acid 18:2 (n−6) all-cis-9,12-octadecadienoic acid

Gamma-linolenic acid 18:3 (n−6) all-cis-6,9,12-octadecatrienoic acid

Eicosadienoic acid 20:2 (n−6) all-cis-11,14-eicosadienoic acid

Dihomo-gamma-linolenic acid 20:3 (n−6) all-cis-8,11,14-eicosatrienoic acid

Arachidonic acid 20:4 (n−6) all-cis-5,8,11,14-eicosatetraenoic acid

Docosadienoic acid 22:2 (n−6) all-cis-13,16-docosadienoic acid

Adrenic acid 22:4 (n−6) all-cis-7,10,13,16-docosatetraenoic acid

Docosapentaenoic acid 22:5 (n−6) all-cis-4,7,10,13,16-docosapentaenoic acid

Tetracosatetraenoic acid 24:4 (n−6) all-cis-9,12,15,18-tetracosatetraenoic acid

Tetracosapentaenoic acid 24:5 (n−6) all-cis-6,9,12,15,18-tetracosapentaenoic acid

Calendic acid 18:3 (n−6) 8E,10E,12Z-octadecatrienoic acid

Table 1.9. Shows important n-6 fatty acids with their double bonding

positions.

Fatty acid composition of membrane phospholipids of inflammatory cells

like monocytes / macrophages plays a vital role during inflammation. These

phospholipids not only alter the fluidity of the membrane which will further

Chapter-I General Introduction

38

alter the position of receptors involved in cellular signaling like

phosphoinositide cascade. Fatty acid composition will also alter the

phospholipase activity involved in the release of substrate for these signaling

processes (Miles and Calder, 1998; Calder, 2003). Inflammatory cells will be

typically enriched with AA in the membrane phospholipids because of intake of

diet rich in n-6 fatty acids. The fatty acid compositions can be altered by intake

of diet rich in n-3 fatty acids like EPA and DHA (fish oil) (Gibney and Hunter,

1993; Sperling et al, 1993; Calder, 2003). Increasing in the consumption of n-3

rich oil will partially replace n-6 fatty acids in the cell membranes of platelets,

erythrocytes, neutrophils, monocytes and liver cells. As a result, intake of EPA

and DHA from fish or fish oil leads to (Figure 1.20 and 1.21) (i) decreased

production of PGE2 metabolites; ii) Decreased production of TXA2, a potent

platelet aggregator and vasoconstrictor; iii) Decreased formation of LTB4, an

inducer of inflammation and leukocyte chemotaxis and adherence; iv)

Increased TXA3, LTB5 which are weak platelet aggregators, vasoconstrictors and

weak inducer of leukocyte chemotaxis respectively (Lewis et al., 1986; Weber et

al., 1986; Kang and Weylandt, 2008; Sabater et al., 2011; Tartibian et al., 2011).

Figure 1.21. Interaction of n-3 PUFA with AA in the synthesis of eicosanoids.

Chapter-I General Introduction

39

1. ∆12- Desaturase (only in plants) 2. ∆15- Desaturase (only in plants)

Figure 1.22. Metabolism of n-6 and n-3 PUFA. Adapted and modified from

Annual Review of Nutrition 30 (2010) 237-255.

With respect to eicosanoid biosynthesis, COX activity renders PGI, PGs

and TXs of 1, 2 and 3 series from ETA, AA and EPA respectively. The 5-LOX is

responsible for synthesis of 3, 4 and 5 series of LTs from the same PUFA

mentioned above. These eicosanoids derived from ETA and EPA has less

potency in exerting their role as that derived from AA. The 5-series of LTs

derived from EPA are weak chemotactic and vasoconstrictors. TXA3 is a weak

platelet aggregator and PGI3 has lower vasodilatation and anti-platelet

aggregation property. PGD3, PGE3 and PGF3 are weak vasodilators and therefore

cause less edema (Horrobin and Morse, 1995; Caldera, 2001; Gil, 2002; Caldera,

2003; Sabater et al., 2011). Infusion of lipids enriched with omega-3 fatty acids

produces significant short-term changes in eicosanoid values, which may be

accompanied by an immunomodulatory effect. Beneficial effect of diet rich in

EPA and DGLA (20:3 n-6) for patient with inflammatory skin disease is well

established (Gil, 2002). Modulation of PG synthesis has been shown in rat

Chapter-I General Introduction

40

peritoneal macrophages with n-3 fatty acids (Linda and Patricia, 1983). Long

term administration of highly purified EPA ethyl esters (EPA-E) prevents

diabetes and abnormalities of blood coagulation in the experimental animals

(Hidefumi et al., 2000). Highly purified EPA-E has diverse pharmacological

activities that include a lipid (especially triglycerides) lowering effect,

antithrombotic effect and anti-inflammatory properties in an animal model

(Terano et al., 1986; Mizuchi et al., 1993 and Sato et al., 1993). Studies also

have shown the effects of moderate dietary supplementations with n-3 fatty

acids on macrophage and lymphocyte phospholipids and macrophage

eicosanoid synthesis in the rat (Christophe and Marc, 1990). They have shown

EPA, provided by fish oil or synthesized from dietary ALA may be incorporated

like AA into the second position of phospholipids. It is converted via LOX

pathway to LTB5 and to CysLTs of 5 series. EPA is slightly better substrate than

AA for 5- LOX (Soberman et al., 1985). Although EPA is poor substrate for COX

leading to prostanoids of 3-series, it has been reported that it is an inhibitor of

AA conversion by COX and LOX (Lokesh et al., 1988). These studies prove that

n-3 fatty acids can modify the activity of macrophages which play important

role in inflammation conditions. Macrophages play important role among blood

cells in the production of eicosanoids, since they release both COX and LOX

products in contrast to PMNLs which release only LOX products (Rouzer et al.,

1980; Parker, 1984; Bina and Lokesh, 1994). Clinical interventions provide

further support for the beneficial effects of n-3 fatty acids in the prevention and

management of cardiovascular disease, hyperinsulinemia and possibly type 2

diabetes.

Indu and Ghafoorunissa, 1992 indicated that increasing dietary intake

of ALA increases EPA concentration in plasma phospholipids after both 3 and 6

weeks of dietary intervention (Indu and Ghafoorunissa, 1992). Dihomo-γ-

linolenic acid (20:3n-6) (DGLA) concentrations were reduced but AA

concentrations were not altered. The reduction in the ratio of n-6 long-chain

PUFAs (LCPUFAs) to n-3 LCPUFAs was greater after 6 weeks than after 3

weeks. They were able to show anti-thrombotic effects by reducing the ratio of

n-6 to n-3 fatty acids with ALA-rich vegetable oil. After ALA supplementation,

there was an increase in n-3 LCPUFA in plasma and platelet phospholipids and

a decrease in platelet aggregation. ALA supplementation did not alter

triacylglycerol concentrations. As shown by others, only LCPUFAs have

Chapter-I General Introduction

41

triacylglycerol-lowering effects (Emken, 1994). Recent studies have proved the

effect of omega-3 fatty acids in attenuation of immune mediated diseases

(Simopoulos, 2001). Studies have shown the effect of n-3 enriched diet in

reducing the ROS level of peritoneal macrophages (Bina and Lokesh, 1994).

Same authors have also shown the effect of n-3 fatty acids in attenuating

adjuvant induced arthritis in rats (Bina and Lokesh, 1997). The diets of

Western countries have large amounts of LA, which has been promoted for its

cholesterol-lowering effect. It is now recognized that dietary LA favors oxidative

modification of LDL cholesterol (Reaven et al., 1991; Abbey et al., 1993),

increases platelet aggregation (Renaud, 1990) and suppresses the immune

system (Endres et al., 1989). In contrast, ALA intake is associated with

inhibitory effects on the clotting activity of platelets (Renaud et al., 1986;

Renauda et al., 1986) and on the regulation of AA metabolism (Budowski and

Crawford, 1985). In clinical studies, ALA contributed to lowering of the blood

pressure (Berry and Hirsch, 1986) and in a prospective study, (Ascherio et al.,

1996) ALA is inversely related to the risk of coronary heart disease in humans.

ALA is not equivalent in its biological effects compared to the LCPUFA

found in marine oils. EPA and DHA are rapidly incorporated into plasma and

membrane lipids and produce rapid effects than does ALA. Relatively large

reserves of LA is found in body fat of vegans or in the diet of omnivores in

Western societies, would tend to slow down the formation of LCPUFAs from

ALA. Therefore, the role of ALA in human nutrition becomes important in terms

of long-term dietary intake. One advantages of consumption of ALA over n-3

fatty acids from fish is that the problem of insufficient vitamin E intake which

does not exist with high intake of ALA from plant sources.

The body response to insults such as infection, surgery and injury

includes an activation of some components of the immune system. The result is

the local release of chemical mediators and the appearance of increased

concentrations of some of these mediators in the bloodstream. The mediators

released include eicosanoids, cytokines, reactive oxygen (superoxide anions,

hydrogen peroxide H2O2), nitrogen (nitric oxide NO) species and PAF.

Collectively, these mediators are known as inflammatory mediators and the

process that produces them is termed the inflammatory response. Some of the

inflammatory mediators are involved in direct destruction of pathogens, while

Chapter-I General Introduction

42

others play a regulatory role within the immune or whole-body responses to

insult. The overall aim of the inflammatory response appears to be the creation

of an environment characterised by oxidative stress and inflammation that is

hostile to pathogens and the initiation of cellular immune responses involved in

pathogen elimination. Cell-culture-based studies with human endothelial cells

have suggested that LA may play a role in inflammation through activation of

NF-κB and increased production of TNF-α, IL-6 and other inflammatory

mediators (Calder, 2006). Most epidemiological studies and clinical trials using

n-3 fatty acids in the form of fish or fish oil have been carried out in patients

with coronary heart disease. However, studies have also been carried out on

the effects of ALA in normal subjects and in patients with myocardial

infarction. It is well known that, cardiovascular disease is the leading cause of

death worldwide and preventive approaches, particularly achievable dietary

changes, have major public health implications. An increased dietary intake of

n–3 PUFA is one such dietary approach. Most studies have shown an inverse

association between fish consumption and the risk of coronary heart

disease. Furthermore, both consumption of fish and higher blood

concentrations of omega 3 fatty acids are associated with a reduced risk of

sudden death.

Possible mechanisms of action of omega 3 fatty acids:

Anti-arrhythmic

Anti-thrombotic

Anti-atherosclerotic

Anti-inflammatory

Improves endothelial function

Lowers blood pressure

Lowers triglyceride concentrations

The hypo-lipidemic effects of n-3 fatty acids are similar to those of n-6

fatty acids, provided that they replace saturated fats in the diet. The n-3 Fatty

acids have the added benefit of consistently lowering serum triacylglycerol

concentrations; whereas the n-6 fatty acids do not decrease and may even

Chapter-I General Introduction

43

increase them. Furthermore, consumption of high amounts of fish oil blunted

the expected rise in plasma cholesterol concentrations in humans. These

findings are consistent with the low rate of coronary artery disease found in

fish-eating populations. Studies in humans have shown that fish oils reduce

the rate of hepatic secretion of very low density lipoproteins (VLDL)

triacylglycerol. In normolipidemic subjects, n-3 fatty acids prevent and rapidly

reverse carbohydrate-induced hyper-triglyceridemia. There is also evidence

from kinetic studies that fish oil increases the fractional catabolic rate of VLDL

(Phillipson et al., 1985; Weber and Leaf 1991; Jehangir, 2004; Carlos et al.,

2011; Gajos et al., 2011; Moyers et al., 2011; Umberto et al., 2011). A case-

control study in 94 men has shown a relation between serum fatty acids and

incident coronary heart disease who were enrolled in the usual-care group of

the Multiple Risk Factor Intervention Trial between December 1973 and

February 1976 (Simon et al., 1995). The results are consistent with other

evidence indicating that saturated fatty acids are directly correlated, and n-3

PUFAs are inversely correlated with coronary heart disease. Because these

associations were present after adjustment for blood lipid concentrations, other

mechanisms, such as a direct effect on blood clotting, may be involved (Kumar

et al., 2011).

The n-3 Fatty acids, affect coronary heart disease beneficially not by

changing serum lipid concentrations, although EPA and DHA do lower

triacylglycerol concentrations, but by reducing blood clotting in vessel walls

and ventricular arrhythmias (Sellmayer et al., 1995; Simon et al., 1995; Hong et

al., 2011). Composition of the oil in our diet plays a vital role in the

cardiovascular disease and platelet aggregation (Fukushima et al., 1996 and

Ramaprasad et al., 2005). The n-3 fatty acids have many beneficial effects on

cardiovascular diseases by altering the lipoprotein metabolism and by

decreasing the serum triglyceride levels (William et al., 1996). Earlier study has

shown the effect of dietary unsaturated fatty acids if fed along with vitamin E,

curcumin and eugenol will alter the serum and liver lipid peroxidation in

experimental animals (Reddy and Lokesh 1994). The n-3 PUFA consumption

may protect against both the pathological processes leading to the

cardiovascular disease (i.e. atherosclerosis) and the processes that ultimately

cause death (e.g. myocardial infarction MI and stroke) (Calder, 2004). The n-3

LCPUFA favorably affect a number of factors involved in the development of

Chapter-I General Introduction

44

atherosclerosis, indicating that they most likely slow the progression of the

disease. For example, elevated fasting and post-prandial plasma triacylglycerol

concentrations are now recognized to increase the risk of cardiovascular

disease and n-3 LCPUFAs lower both. Typically, a 25–30% lowering of fasting

triacylglycerol concentrations could be expected from an intake of more than 2

g of EPA+DHA/day. The n-3 PUFA also decrease chemoattractant, growth

factor and adhesion molecule production and so could down-regulate processes

leading to leukocyte and smooth muscle migration into the vessel wall intima.

The n-3 LCPUFA are also anti-inflammatory and so could decrease

inflammatory processes within the vessel wall, which are now recognized to be

a major contributory factor in the atherosclerosis. Enrichment of n-3 LCPUFAs

into the diet also has a small, but significant, hypotensive effect in both

normotensive and hypertensive individuals, as confirmed in a recent meta-

analysis. Finally, these fatty acids cause endothelial relaxation and promote

arterial compliance, which might be related to alter NO production. Indeed,

including n-3 LCPUFAs in the diet has been demonstrated to decrease

atherosclerosis in a variety of animal models. Thus the dietary intervention by

n-3 LCPUFAs might act to stabilize atherosclerotic plaques by decreasing

infiltration of inflammatory and immune cells (e.g. monocyte / macrophages

and lymphocytes) into the plaques and/or by decreasing the activity of those

cells once in the plaque. Thus n-3 LCPUFAs exert effects at many steps

involved in the process of atherosclerosis and so they might be expected to

decrease or slow this disease.

The observations made by a study (Thies et al., 2003) suggest that the

primary effect of n-3 PUFAs might be on macrophages. Macrophage numbers

within the plaque might be decreased due to fewer monocyte / macrophages

entering the plaque as a result of decreased adhesion molecule expression on

endothelial cells and/or the monocyte / macrophage itself, which would act to

limit movement of monocyte / macrophages into the plaque. Cell culture

studies have shown that n-3 PUFAs can decrease the expression of intercellular

cell-adhesion molecule-1(ICAM-1) and VCAM-1 on the surface of endothelial

cells and monocytes. Furthermore, feeding fish oil decreased the expression of

several adhesion molecules, including ICAM-1, on the surface of rat

lymphocytes, mouse macrophages and human monocytes. A second

mechanism by which monocyte/macrophage entry into the plaque might be

Chapter-I General Introduction

45

decreased is through decreased generation of chemo attractants. There is

evidence that dietary fish oil decreases the production of a range of

chemoattractants including LTB4, platelet-derived growth factor (PDGF), PAF

and monocyte chemoattractant protein-1 (MCP-1). An alternative means by

which macrophage numbers within the plaque could be decreased is by an

increased rate of cell death by either apoptosis or necrosis. Although feeding

fish oil to mice was shown to increase the level of Fas expression on

lymphocytes and to increase lymphocyte apoptosis, there is little published

information about dietary n-3 PUFAs and monocyte / macrophage apoptosis.

However, both EPA and DHA have been shown to increase apoptosis of human

monocytes and monocytic cell lines in culture.

There is much evidence for the beneficial role of n-3 fatty acids in

chronic inflammatory diseases. Clinical studies have shown that n-3 PUFA

supplementation has beneficial effects in atherosclerosis, inflammatory bowel

disease (IBD), psoriasis, rheumatoid arthritis and asthma which support the

idea that n-3 PUFA are anti-inflammatory and immunomodulatory (Figure

1.22)(Caldera, 2001; Gil, 2002). The supplementation of n-3 PUFA to

rheumatoid arthritis patient has resulted in significant reduction in morning

stiffness, reduced swollen joints, reduced joint pain (Caldera, 2001; Gil, 2002

and Kremer, 2000). The n-3 PUFA supplemented to experimental animals has

shown reduction in formation of pro-inflammatory eicosanoids in lung

diseases. Studies have also shown that intake of EPA and DHA supplements in

children with asthma reduce their symptomology (Nagakura et al., 2000).

Chapter-I General Introduction

46

Figure 1.23. Anti-inflammatory effect of GLA and EPA in epidermis.

Eicosanoids plays a very important role in the skin inflammation. The n-

3 PUFA from fish oil has also shown to inhibit the enzymes involved in the

biosynthesis of these eicosanoids (Anna, 2012) and also alter the production of

inflammatory cytokines. EPA does not activate NF-κB in a monocytic cell line,

while both EPA and DHA inhibit endotoxin-stimulated production of IL-6 and

IL-8 by cultured human endothelial cells. Recent studies have shown that EPA

does not induce TNF-α, IL-1α or IL-1β or IL-6 in osteoblasts and even counters

the up regulating effect of AA. EPA and DHA can totally abolish cytokine-

induced up-regulation of TNF-α, IL-1α and IL-1β in cultured bovine

chondrocytes and in human osteoarthritic cartilage explants. EPA is also less

potent than AA in inducing IL-6 expression by macrophages. EPA prevents NF-

κB activation by TNF-α in cultured pancreatic cell, an effect that involves

decreased degradation of the inhibitory subunit of NF-κB, perhaps through

decreased phosphorylation. Similarly, EPA or fish oil decrease endotoxin-

induced activation of NF-κB in human monocytes, which is associated with

decreased phosphorylation of inhibitory subunit of NF-κB, perhaps because of

decreased activation of MAPK. These observations suggest direct effects of n-3

LCPUFA on inflammatory gene expression via inhibition of activation of

transcription factor NF-κB. Recent studies as in the figure 1.24 suggest that

one aspect of anti-inflammatory action of fish oil resulted in lower level of NF-

Chapter-I General Introduction

47

κB in the nucleus in LPS stimulated murine spleen lymphocytes (Xi et al.,

2001; Calder, 2006).

Figure 1.24. The n-3 PUFA and its effect on inflammatory genes.

Second group of transcription factors activated by n-3 PUFA are PPAR.

PPAR act through dimerization with retinoid X receptor and subsequent

regulation of gene expression. Absence of PPAR-α have prolonged response to

inflammation (Devchand et al., 1996). PPARα and γ shown to inhibit the

induction of inflammatory genes involving TNF-α, IL1β, IL-6, IL-8, COX -2,

VCAM-1, NOS, matrix metalloprotease (MMP) and acute phase proteins.

Activation of PPARγ has been demonstrated to result in monocyte /

macrophage apoptosis, and studies suggest that n-3 PUFAs can induce PPARγ

activation. PPARγ is found in atherosclerotic plaques and it is tempting to

speculate that dietary fish oil might result in activation of PPARγ in plaque

monocyte / macrophages, driving them towards apoptosis. Cell culture studies

indicate that activation of PPARγ in human monocytes also results in inhibition

of production and activity of MMP-9 (Marx et al., 1998). Since MMP are a major

contributor to plaque instability, this might provide a mechanism by which n-3

PUFAs improve plaque stability.

Chapter-I General Introduction

48

In addition to their role in inflammation and other diseases, dietary fatty

acids have multiple effects on human metabolism. Fatty acids serve as an

important source of energy; they specifically influence numerous metabolic

pathways in a variety of organs. Many of these effects are achieved by altering

mRNA expression. In the past decade important new information has emerged

about the mechanisms by which fatty acids influence DNA transcription and

regulate mRNA expression. It has been known for some time that dietary PUFA

inhibit the expression of several genes involved in lipogenesis, such as fatty

acid synthase and stearoyl-CoA desaturase. However, only recently-detailed

insights into the molecular mechanisms behind this regulation have started to

emerge.

The transcription factor PPAR, was ruled out as the factor mediating the

effects of PUFA on lipogenic gene expression. In contrast, it appears that a

pivotal role in this regulation is played by sterol regulatory element-binding

protein-1c (SREBP-1c), a helix-turn-helix transcription factor that is present in

both liver and adipose tissue. In liver SREBP-1c induces the expression of a

whole set of genes involved in fatty acid and triacylglycerol synthesis. It was

found that dietary PUFA potently lower SREBP-1c mRNA levels in mouse liver

as well as in hepatoma and other cell lines (Hannah et al., 2001). Liver X

receptor-α (LXRα) is a potent activator of SREBP-1 gene transcription and

stimulates the expression of several lipogenic genes. Indeed, a response

element for LXRα so-called LXRE has been identified in the promoter of the

SREBP-1 gene. Interestingly, activation of the SREBP-1 promoter activity by

LXRα is suppressed by EPA by inhibiting binding of the LXR – retinoid X

receptor complex, which is the actual binding unit, to the LXRE. It was

observed that PUFA compete with oxysterols for binding to LXR, suggesting

that PUFA behave as LXR antagonists (Yoshikawa et al., 2002). The most

potent fatty acid was AA, followed by EPA and DHA. Saturated fatty acids

showed little or no effect. Thus, it appears that PUFA down regulate lipogenic

genes by serving as antagonists of the nuclear receptor LXRα.

In the past few years it has become clear that induction of gene

transcription by fatty acids is often mediated by a subclass of nuclear hormone

receptors, peroxisomal proliferated activated receptor (PPAR). The PPAR are

ligand-activated transcription factors that stimulate gene transcription by

Chapter-I General Introduction

49

binding to a small sequence element in the promoter of certain genes. As

explained in the earlier paragraph their activity is induced by binding of small

fatty acid-like molecules. Three PPAR subtypes can be distinguished: α, α’ and

γ. While PPARα is mostly expressed in brown adipose tissue and liver, PPARα’

is present at high concentrations in numerous tissues but is especially

abundant in the intestine. PPARγ, in turn, is most abundant in white adipose

tissue and to a lesser extent in colon and macrophages. Fatty acids have an

important regulatory function in hepatic energy metabolism by two different

mechanisms, one involving the transcription factors LXRα and SREBP-1 and

another involving the transcription factor PPARα, they up or down regulate the

expression of a whole set of genes involved in fatty acid synthesis and fatty acid

oxidation and/or ketogenesis, thereby controlling their own metabolic fate

(Sander, 2002).

Ulcerative colitis (UC) and Crohn disease (CD) are chronic inflammatory

diseases of the alimentary tract. In UC, the mucosa of the colon is mainly

affected, whereas in CD, any part of the alimentary tract from mouth to anus

can be affected, although it is usually the ileum and colon. In both the

diseases, intestinal mucosa contains elevated concentrations of inflammatory

cytokines and eicosanoids such as LTB4 (Sharon and Stenson, 1984). The

established role of AA–derived eicosanoids in the pathophysiology of IBD

suggests that a high dietary intake of n–6 PUFAs may play a part in

establishing or perpetuating the disease. Several randomized, placebo-

controlled, double-blind studies of fish oil in IBD have been reported. The dose

of n–3 LCPUFAs used in these trials was between 2.7 and 5.6 g/day and

averaged ≈4.5 g/day. Some of these trials indicate the benefits of fish oil,

including improved clinical score, gut mucosal histology, sigmoidoscopic score,

lower rate of relapse, and decreased use of corticosteroids. At sufficiently high

intakes, LCPUFAs, as found in oily fish and fish oils, decrease the production of

inflammatory eicosanoids, cytokines and ROS and the expression of adhesion

molecules. LCPUFAs act both directly (eg. by replacing AA as an eicosanoid

substrate and inhibiting AA metabolism) and indirectly (eg, by altering the

expression of inflammatory genes through effects on transcription factor

activation). LCPUFAs also give rise to a family of anti-inflammatory mediators

such as resolvins, protectants etc. Thus, n–3 PUFAs are potent anti-

inflammatory agents. As such, they may be of therapeutic use in a variety of

Chapter-I General Introduction

50

acute and chronic inflammatory settings. Evidence of their clinical efficacy is

reasonably strong in some settings (eg. in rheumatoid arthritis) but is weak in

others (eg. in IBD and asthma) (Caldera, 2006; Calder, 2010).

Figure 1.25. Garden cress seeds.

The garden cress grows in regions of Madhya Pradesh, Chhattisgarh,

Uttar Pradesh, Maharashtra and Northern Karnataka. It is domesticated by

population in the above said regions mainly for seeds to be used in diet or

medicinal purposes. The morphology of garden cress seeds resembles that of an

oil seed with 80–85% of the seed matter is dicotyledonous endosperm

(Sumangala et al., 2004). There are reports about the garden cress seeds to

possess varied medicinal properties like galactogogue (improve lactation),

diuretic, alterative (blood purification), tonic, aphrodisiac (increase sexual

desire), carminative and emmenagogue (increasing blood flow in the region of

pelvic and uterus) (Umang et al., 2009). Seeds are also useful in hiccup,

dysentery, diarrhea and skin diseases caused by impurities and toxins in blood

and chronic enlargements of spleen (Nadkarni and Nadkarni, 1954).

Pharmacological studies on seeds of L. sativum have suggested the presence of

cardioactive substance and also are effective in alleviating the symptoms of

asthma in patients (Vohora and Khan, 1977; Paranjape and Mehta, 2006).

Plants such as green leafy vegetables, flaxseed oil, camelina oil, canola

oil, soybean oil as mentioned are rich sources of ALA. Since vegetarians avoid

fish and fish oil, there is a need for n-3 fatty acids supplementation from plant

Chapter-I General Introduction

51

sources. Garden cress oil (GCO) is one of the vegetable oils rich in ALA of 34%

(Mathews et al., 1993). It is relatively stable oil than flax seed oil (Moustafa et

al., 1957). The chemoprotective effect of garden cress (Lepidium sativum) and

its constituents, glucotropaeolin (GT) and benzylisothiocyanate (BITC), a

breakdown product of GT, towards 2-amino-3-methyl-imidazo [4,5-f] quinoline

(IQ)-induced genotoxic effects and colonic pre-neoplastic lesions was

investigated in single cell gel electrophoresis (SCGE) assays and in aberrant

crypt foci (ACF) experiments, respectively. The modulation of activities of

cytochrome P4501A2, glutathione-S-transferase (GST) and UDP glucuronosyl

transferase (UDPGT) by garden cress juice, GT and BITC was studied. Whereas

GT and BITC did not affect the activity of any of the enzymes significantly,

garden cress juice caused a significant increase in the activity of hepatic

UDPGT-2 (Fekadu et al., 2002). Studies have shown that GCO is rich in

phenolics (Aranda et al., 2007). Bioavailability studies with GCO showed

significant increase in ALA levels in serum and conversion to EPA and DHA in

tissues in experimental rats (Diwakar et al., 2008). Diet rich in GCO have

modulatory effect on inflammatory cytokines and differential

immunomodulation with LCPUFA in health and chronic disease have been

proved in the animal studies (Sijben and Calder, 2007; Diwakar et al., 2011).

Classic mechanism of the anti-inflammatory action of LCPUFA, EPA and DHA

is by decreasing the amounts of AA available as a substrate for eicosanoid

synthesis and also inhibit the metabolism of AA. Recent studies have identified

garden cress as unexplored, alternative low input oil owing to nutritionally and

industrially desirable fatty acid composition in the garden cress oil (Angelini et

al., 1997). There are reports with regard to the use of oil obtained from garden

cress seeds (Uphof, 1959; Lotfy et al. 1957). The purpose of the study was to

determine the modulatory effect of n-3 rich GCO which is a good source of ALA,

on eicosanoids metabolism in in vivo systems.

This research work aims to give a comprehensive insight into the

understanding of effect of spices and its active principles on the eicosanoids

biosynthesis. Further, modulatory effect of spice active principles and GCO on

platelet aggregation and UC were studied in an experimental model.

Chapter-I General Introduction

52

The major objectives of the thesis are:

1. To study the effect of spice active principles on phospholipase A2, key

enzyme involved in release of arachidonic acid from cell membranes.

2. To study the effect of spice active principles on 5-LOX and COX-1, key

enzymes involved in eicosanoids metabolism in suitable model systems.

3. To assess the modulatory effect of spice active principles and omega-3

fatty acids from garden cress seeds on leukotrienes, prostaglandins and

platelet aggregation.

4. To assess the modulatory effect of combination of spice active principles

and omega-3 fatty acids on above eicosanoids in inflammatory animal

model system.

Work Plan and Hypothesis of the Current Study

53

Figure 1.26. Work plan and hypothesis of the study.