IN VIVO AND IN VITRO ANTI-INFLAMMATORY EFFECT OF THE CHLOROFORM EXTRACT OF ANNONA MURICATA (SOURSOP) LEAVES

ABSTRACT

This  study  ascertained  the  anti-inflammatory effect  and  the  mechanisms  of  action  of  the chloroform extract of Annona muricata leaves. The plant material was extracted using a mixture of chloroform and methanol in a ratio of 2:1. The chloroform extract was used to further investigate the in vivo effect on agar-induced rat paw oedema, and the in vitro activities of phospholipase A2  and prostaglandin synthase, membrane stabilization and platelet aggregatory response. The percentage yields of the chloroform and methanol extracts were 6.80 and 0.29% respectively. Phytochemical analyses of the extracts revealed the presence of phenols (26.290 ±

0.004 mg/g), flavonoids (24.123 ± 0.003 mg/g), terpenoids (19.040 ± 0.005 mg/g), alkaloids (15.691 ± 0.004 mg/g), tannins (5.446 ± 0.004 mg/g), reducing sugars (4.783 ± 0.004 mg/g), saponins (1.007 ± 0.005 mg/g), glycosides (0.576 ± 0.004 mg/g), steroids (0.236 ± 0.003 mg/g), hydrogen cyanides (0.147 ± 0.002 mg/g) and soluble carbohydrates (0.105 ± 0.003 mg/g) for the chloroform  extract  and  phenols  (18.790  ±  0.006  mg/g),  alkaloids  (10.382  ±  0.005  mg/g), reducing sugars (6.848 ± 0.003 mg/g), terpenoids (5.857 ± 0.004 mg/g), tannins (4.947 ± 0.003 mg/g), flavonoids (4.163 ± 0.003 mg/g), saponins (0.736 ± 0.004 mg/g), hydrogen cyanides (0.503 ± 0.002 mg/g), steroids (0.464 ± 0.003 mg/g), glycosides (0.448 ± 0.007 mg/g) and soluble carbohydrates (0.153 ± 0.017 mg/g) for the methanol extract respectively. Acute toxicity study on the chloroform extract showed it was toxic at 1000 mg/kg body weight. The LD50 was calculated as 316.23 mg/kg body weight. Agar-induced rat paw oedema test showed that at 50,

100 and 150 mg/kg body weight, the extract exhibited a significant (p < 0.05) reduction in paw volume from 1.5 to 5.5 hr compared to the control. The extract also significantly (p < 0.05) inhibited phospholipase A2  activity in a concentration-related manner compared to the control, with a range of 0.2 to 0.6 mg/ml inhibiting the enzyme activity by 23.91 to 43.48%. The effect of the extract on prostaglandin synthase activity showed a significant (p < 0.05) inhibition of enzyme activity at the concentrations 0.1, 0.5 and 1.0 mg/ml compared to the control. The highest percentage inhibition (87.46) attained at 0.5 mg/ml was comparable to that of 1.0 mg/ml indomethacin. At various concentrations (0.1 – 0.8 mg/ml), the chloroform extract also significantly (p < 0.05) inhibited heat and hypotonicity-induced haemolysis of human red blood cells  (HRBCs) compared to  the  control. The highest percentage inhibition of heat-induced haemolysis (53.03) was obtained at  0.4 mg/ml of the extract while the highest percentage inhibition (77.91) of hypotonicity-induced haemolysis was obtained at 0.8 mg/ml. The extract also  significantly  (p  <  0.05)  and  concentration-dependently inhibited  platelet  aggregatory response compared to  the  control. The anti-inflammatory effect  exerted by the chloroform extract was found to be higher than that of the standard anti-inflammatory drugs, indomethacin and prednisolone. These results thus demonstrated that the chloroform extract of A. muricata leaves has potentials in curtailing inflammatory disorders through the inhibition of phospholipase A2  and  prostaglandin  synthase  activities,  membrane  stabilization  and  inhibition  of  platelet aggregation.

CHAPTER ONE

INTRODUCTION

Natural products, especially those derived from plants, have been the basis of many traditional medicine systems throughout the world (Moghadamtousi et al., 2015). Plants remain the major source of structurally important chemical substances that lead to the development of innovative drugs (Jachak and Saklani, 2007; Yadav and Agarwala, 2011). Plant-based drugs are considered to be the major point of focus because they are easily available, less expensive and have little side effects (Fabricant and Farnsworth, 2001). Unlike modern drugs which are single active components that target one specific pathway, herbal medicines work in an orchestral manner, as the chemical compounds they contain act synergistically on targeted elements of a complex cellular pathway (Kumar et al., 2013). Plants are used in traditional medicine to treat several diseases including inflammatory-related ones (Deepa and Renuka, 2014).

Inflammation is  a  protective response to  tissue  injury caused  by physical trauma,  noxious chemicals or microbiological agents (Kumar et al., 2013). It can be acute or chronic (Ferrero- Miliani et al., 2007). Chronic inflammation is associated with many diseases of advanced age such as heart attacks, Alzheimer’s disease and cancer (Coussens and Werb, 2002; Libby et al.,

2002). Non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids are classically used to  alleviate inflammation. Long term uses of NSAIDs causes side effects  including gastric ulceration and renal toxicity (Payne, 2000; Ezekwesili et al., 2011) since they concurrently inhibit  both  isoforms  of  cyclooxygenase  (COX).  The  development  of  NSAIDs  which  are selective COX inhibitors still have side effects as reports have connected these drugs with an increased risk of heart  attack and stroke (Salmon, 2006; Nelson and Cox, 2008). There  is therefore, a need for potent anti-inflammatory drugs with fewer side effects. This has prompted the research into plants used in folk medicine to ameliorate inflammation. An example of such plant, which is a rich source of active phytoconstituents that provide medicinal benefits against various ailments and diseases, is Annona muricata Linn (Soursop).

Annona muricata is a shrub belonging to the Annonaceae family, and widely distributed throughout tropical and subtropical parts of the world, including Nigeria (Adewole and Caxton- Martins, 2006). It abounds in the southern part of Nigeria and cultivated mainly in home gardens. All parts of this plant are extensively used as traditional medicine against an array of human

ailments and diseases. Baskar et al. (2007) observed that the plant has anti-oxidant effect. It also possesses anti-proliferative and anti-trypanosomal effects (Pieme et  al., 2014; Onyeyili and Aliyoo, 2015). The antinociceptive and anti-inflammatory effects of the ethanol extract of the plant in animal models have also been reported (De Sousa et al., 2010; Roslida et al., 2010; Chan and Roslida, 2012). However, despite the studies on the anti-inflammatory activity of Annona muricata leaves, there is little or no empirical evidence on its anti-inflammatory mechanisms of action, hence, this study.

1.1 Description of Annona muricata

Annona muricata is a member of the Annonaceae family of flowering plants. Annonaceae is the largest family of the Magnoliales order and comprises of approximately 130 genera and 2,300 species (Mishra et al., 2013). Among the 130 genera of Annonaceae family, 7 genera are very commonly  available  and  widely  distributed.  They  include:  Annona,  Anonidium,  Rolliania, Uvaria, Melodorum, Asimina and Stelechocarpus.

Annona muricata is a species of the genus Annona, derived from the Latin word ‘anon’ which means ‘yearly produce’ or ‘annual harvest’. As the name suggests, various species in this genus generally have annual fruit production and about 119 species have  been identified (SCUC,

2006). Other common ones include; Annona squamosa (sweetsop), Annona senegalensis (wild soursop), Annona reticulata (custard apple), Annona cherimoya (cherimoya) and Annona atemoya, which is a hybrid between cherimoya and sweetsop. Annona muricata is the most tropical of the group and bears the largest fruit.

Annona muricata is a shrub and low-branching tree growing 5-6 m in height (Vijayameena et al., 2013). It  is an underutilized (ICUC, 2002) tree with alternate, large, glossy, leaves oval to lanceolate in shape. The leaves are dark green on the upper surface and lighter green beneath. They have short petioles, 14 to 16 cm in length and 5 to 7 cm in width (Pinto et al., 2005). Annona muricata has an ovate or heart shaped, though sometimes irregular fruits, weighing from

0.5 to 2 kg, 10 to 30 cm long and 15 to 20 cm in diameter. It bears its fruits indiscriminately on twigs, branches and trunk. The fruits are covered with leathery-appearing but tender, inedible, bitter skin which are dark-green when unripe and slightly lighter green when ripe (Lima and Alves, 2011). The skin contains well-spaced, short, slightly curved spines, whose tips break off

easily when the fruit is fully ripe. The edible part of the fruit is the pulp or flesh, which is white, cottony-fibrous and juicy, with slightly acidic flavour. The pulp is divided into segments with closely-packed segments seedless and other segments having a single oval, smooth, hard seed. One piece of large fruit can contain a dozen to 200 or more seeds with a black colour soon after harvest, but becoming dark-brown later (Lima and Alves, 2011). The seed is about 4% of the whole fruit, with size varying from 1 to 2 cm in length and 0.33 to 0.59 kg in weight. The stems are rounded, rough and not pubescent, with a dark-brown colour. The flowers are greenish yellow, 3.2 to 3.8 cm in length and may emerge anywhere on the twigs, branches and trunk like the fruit. They are short-stalked, with petals plump and conical, 4-5 cm long. Anthesis begins early morning and complete anthesis takes approximately 6 hours depending on the climate (Pinto  et  al.,  2005).  Annona  muricata  experiences  inefficient  natural pollination,  which  is normally done by beetles, and frequently poor fruit set. Hand pollination is thus, an important orchard management practice.

Figure 1: Diagrammatic representation of Annona muricata leaves and fruit

1.1.1 Taxonomic Classification of Annona muricata

Kingdom:                   Plantae Subkingdom:              Tracheobionta Super-division:           Spermatophyta Division:                     Magnoliophyta Class:                          Magnoliopsida Subclass:                    Magnoliidae Order:                         Magnoliales Family:                       Annonaceae Genus:                        Annona Species:                      muricata

Source: (Alqasim, 2013)

1.1.2   Common Names of Annona muricata

Annona muricata is commonly known in English as Soursop due to its slightly acidic taste when ripe. Other common names include; Ebo  (Yoruba), Tuwon Biri (Hausa), Sawonsop (Igbo), Guanábana (Spanish), Corossol and Sappadille (French), Zuurzak (German), Munolla and Mamphal (India), Graviola (Portuguese) and Mamon (Spanish, Philippines).

1.1.3 Geographical Distribution, Climate and Soil Type

Plants of the Annonaceae family can be  found in tropical and subtropical regions. Annona muricata is indigenous to the warmest tropical areas in South and North America, but now widely distributed  throughout  tropical  and  subtropical parts  of  the  world,  including  India, Malaysia and Nigeria (Adewole and Caxton-Martins, 2006). Only few are found in temperate regions. Annona muricata is adapted and related to areas of high humidity and relatively warm winters.  Temperature  below  50C  will  cause  damage  to  its  leaves  and  small  branches  and

temperature below 30C can be fatal. In the tropics, A. muricata is grown from sea level to 1000

m.  It cannot tolerate standing water, and with its shallow roots, it does not require a very deep soil base (ICUC, 2002). Its best growth is thus achieved in rich, well-drained, semi-dry soil. It can also be grown in acidic and sandy soil.

1.2 Phytochemical Study of Annona muricata

Phytochemical evaluations on different parts of A. muricata have shown the presence of various phytoconstituents and compounds. Annonaceous acetogenins are powerful phytochemicals found only in Annonaceae family. The biological activities of acetogenins are primarily characterized with toxicity against cancer cells (Chang et al., 2003). They induce cytotoxicity by inhibiting the mitochondrial   complex   I   (NADH:   ubiquinone   oxidoreductase)   involved   in   adenosine triphosphate (ATP) synthesis (McLaughlin, 2008) and ubiquinone-linked nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in cancer cells cytoplasmic membranes (Konno et al.,

2008). These chemicals also have antiparasitic, pesticidal and anti-microbial activities (Luna et al., 2006). A. muricata produces these natural compounds in its leaves, stem, bark, and seeds. The annonaceous acetogenins isolated from the leaves include; Annomuricin A, B, C and E, annomutacin,  (2,4-cis)-10R-annonacin-A-one, (2,4-trans)-10R-annonacin-A-one, annohexocin, muricapentocin, (2,4-cis)-isoannonacin, (2,4-trans)-isoannonacin, muricatocin A, B and C, gigantetronenin, solamin, annonacin A, annopentocins A, B and C, cis- and trans- annomuricin- D-ones, murihexocins A, B and C, muricoreacin, goniothalamicin, gigantetrocin A, cis- corossolone, annocatalin and annocatacin B, muricatetrocin A and B, epomuricenin A and B, annonacinone and solamin (Moghadamtousi et al., 2015; Patel and Patel, 2016).

Studies on the methanol, ethanol and aqueous extracts of the leaves showed that the three extracts contain flavonoids, carbohydrates, glycosides, proteins, saponins and tannins with the presence of terpenoids and quinones only in the aqueous extract and alkaloids in the methanol and ethanol extracts (Vijayameena et al., 2013). Phytochemical screening of A. muricata leaves aqueous and  methanol extract  by Solomon-Wisdom et  al.  (2014) revealed  the  presence of flavonoids,  saponins,  tannins,  alkaloids,  cardiac  glycosides  and  steroids.  Using  the  same solvents, Chauhan and Mittu (2015) revealed carbohydrate, protein, fat, alkaloid, terpenoids, steroids, flavonoids, tannins, phenolic compounds, saponin and glycoside with the absence of alkaloid in the aqueous extract. Analysis of the dried and powdered leaves indicate the presence of saponins, alkaloids, flavonoids, tannins, β-carotene, ascorbic acid and reducing sugars (Usunobun et  al.,  2015).  Screening  of  the  fruit  extract  indicates  the  presence  of  tannins, saponins, cardiac glycosides, flavonoids and coumarins (Boakye et al., 2015). Analysis on the leaf oil of A. muricata revealed the presence of sesquiterpenes, with the major compound present being β-caryophyllene (Alitonou et al., 2013).

1.3 Benefits of Annona muricata

All portions of the A. muricata tree, including the twigs, leaf, root, fruit and seeds are extensively used as traditional medicine against several diseases. Generally, the fruit and fruit juice are taken to eliminate worms and parasites, cool fever, increase lactation after child birth, and as an astringent for diarrhoea and dysentery. The fruit can also be used as an oral rehydration therapy (Enweani et al., 2004) to correct the deficits associated with acute diarrhoea. The crushed seeds are used as a vermifuge and antihelmintic against internal and external parasites, head lice and worms. In South America and tropical Africa, including Nigeria, leaves of A. muricata are deployed as an ethnomedicine against tumours and cancer (Adewole and Ojewole, 2009). The leaves can be crushed along with raw fruit from the plant and mixed with olive oil which can be used to treat various skin disorders like rashes, boils and sores. The boiled water infusion of the leaves have anti-plasmodic, astringent and gastric properties.

In addition to ethnomedicinal uses, the fruits and leaves are widely employed for the preparation of beverages, candy, ice creams, shakes and syrups (Jaramillo-Flores and Hernandez-Sanchez,

2000; Yenrina et al., 2015). In Indonesia, sweet cake is made by boiling the pulp in water and adding sugar until the mixture hardens. The fruit pulp can also be consumed fresh for dessert when fully ripe or mixed with ice cream or milk to make a delicious drink, as is done in Java, Cuba  and  other  parts of  America.  Incorporation of  A.  muricata  pulp  into  diets  has  great nutritional benefits as it contains appreciable amount of nutrients (Onyechi et al., 2012; Degnon et al., 2013). Annona muricata seed has potentials as an animal feed supplement (Fasakin et al.,

2008). Its seed oil extracts could also be useful for industrial applications (Kimbonguila et al.,

2010).

1.4 Pharmacological Effects of Annona muricata

Studies over the years have validated some of A. muricata uses in herbal medicine, some which include;

1.4.1 Antioxidant Effect

Baskar et al. (2007) investigated the in vitro antioxidant effect of the leaves of Annona species. It was observed that  the  ethanol extract  of A.  muricata  leaves  at  500  µg/ml  showed higher scavenging   activity  of   2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate)  (ABTS)   radical

cation, hydroxyl radical and nitric oxide (NO) compared to the other species. Boakye et al. (2015) also investigated the antioxidant activity of four underutilized tropical fruits in which A. muricata fruit pulp was observed to have free radical scavenging activity of 75.39%. These studies thus, demonstrated the potential of A. muricata leaves and pulp in the scavenging of free radicals.

1.4.2 Anti-proliferative Effect

The anti-proliferative effect of the ethyl acetate extract of A. muricata leaves against lung cancer A549 cells was investigated by Moghadamtousi et al. (2014) while the anti-proliferative effect of the ethanol extract of its leaves, twigs and roots on human promyelocytic leukemia (HL-60 cells) was investigated by Pieme et al. (2014). This effect was found to be mediated through the disruption of membrane mitochondrial potential (MMP) and the arrest of the cell cycle.

1.4.3 Laxative Effect

The study undertaken by Jeevanandham (2013) showed that the ethanol extract of A. muricata leaves  exhibited  laxative  effect.  An  oral administration of the  extract  produced significant increase in faecal output of rats and the stimulation of gastrointestinal motility. The study thus demonstrated the potential of A. muricata leaves in reducing constipation.

1.4.4 Anti-bacterial Effect

Several studies have demonstrated the anti-bacterial effect of A. muricata. Vieira et al. (2010) demonstrated that the aqueous extract of A. muricata peels showed an antibacterial effect against Staphylococcus aureus and Vibro cholerae, with greatest halos of bacterial growth inhibition (16 and 23 mm) observed at 200 µL of the peels homogenate. The methanol extract of A. muricata leaves also show inhibition of Escherichia coli strains isolated from livestock as demonstrated by Chukwuka et al. (2011). The anti-bacterial activity of the aqueous and methanol extracts of the leaves against gram positive and gram negative bacteria was also undertaken by Solomon- Wisdom et al. (2014). This study was substantiated by the anti-listerial activity of the aqueous extract of the leaves conducted by Chauhan and Mittu (2015).

1.4.5 Antitrypanosomal Activity

Onyeyili and Aliyoo (2015) investigated the trypanocidal activity of the chloroform extract of

A.muricata stem bark against Trypanosoma brucei brucei. It was observed that the extract was

effective against the parasite in both the in vivo and in vitro analysis, thus, demonstrating the ability of the extract to control trypanosomosis.

1.4.6 Anti-hepatotoxic Activity

Several studies have demonstrated the hepatoprotective effect of A. muricata. Oyedepo (2014) investigated the effect of the ethanol extract of A. muricata leaves on paracetamol-induced liver damage in rats. It was observed that A. muricata leaves reduced the elevated levels of aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), total bilirubin and total protein. The methanol extract of the seed also had effect on ALT, AST and ALP as investigated by Agbai and Nwanegwo (2013). The ability of the ethanol extract of the leaves to  inhibit  ALT was  confirmed by Usunobun (2014) and  Tanaya  et  al. (2015). The bilirubin lowering potential of the aqueous extract of the leaves was also investigated by Arthur et al. (2012). These studies thus confirmed the efficacy of A. muricata in hepatoprotection.

1.4.7 Wound Healing Activity

A study on the wound healing activity of the alcohol extract of A. muricata stem bark was conducted by Padmaa et al. (2009). Topical application of the extract to albino rats led to contraction of open wounds from the 4th day, with the highest percentage reduction in the wound area (88.85) attained at  the 12th  day. This study thus, demonstrated the possible use of A. muricata in the healing of wounds.

1.5 Overview of Inflammation

Inflammation is a pervasive phenomenon that operates during severe perturbations of homeostasis, such as infection, injury and exposure to contaminants (Ashley et al., 2012). It is triggered by infectious agents such as, viruses, fungi, bacteria and protozoa. It is also due to trauma, physical and chemical agents, tissue necrosis and immune reactions.

The mechanisms involved in the inflammatory process are common to all, regardless of the triggering factor (Ferrero-Miliani et al., 2007). It  involves a cascade of biochemical events comprising of the local vascular system and the immune system (Da Silveira e Sá et al., 2013). It also involves the production of factors that could cause damage to tissues when not properly regulated. As a consequence, genes that play key roles in effector functions of inflammatory responses are actively repressed under normal conditions and are only induced when cells sense

evidence of a triggering factor (Nathan, 2002). The primary functions of inflammation are to eliminate the initial cause of cell injury, remove necrotic cells and tissue, initiate the process of repair and then restore tissue homeostasis (Medzhitov, 2008; Soehnlein and Lindbom, 2010).

1.5.1 Inflammatory Mediators

Inflammatory mediators are substances triggered by inflammatory stimuli. They are derived from inflammatory cells, or released as plasma proteins (Vishal et al., 2014). Most mediators bind to specific target receptors on the cells to elicit their effects. Exceptions are lysosomal enzymes that have a direct enzymatic effect and reactive oxygen species (ROS) that have a direct toxic effect. Mediators can stimulate target cells to release secondary mediators.

1.5.1.1 Cell Derived Mediators

These are mediators derived from stimulated inflammatory cells. They may be pre-formed and stored in the granules of these cells or may be synthesized de novo and secreted as needed.

1.5.1.1.1 Vasoactive Amines

Vasoactive amines include histamine and serotonin. The latter is also known as 5- hydroxytryptamine (5-HT). Like lysosomal enzymes, they are preformed mediators in secretory granules. Histamine is released from mast cells during antigen reaction with mast cell bound antibody molecules and during the inflammatory response to skin injury (Trautmann et al.,

2000). It is also released from basophils and platelets. It causes the contraction of endothelial cells of venules leading to increased vascular permeability. Its effect is rapidly inactivated by histaminase. Serotonin is released from the platelets along with histamine and acts similarly to histamine (Stone et al., 2010).

1.5.1.1.2 Cytokines

These are soluble immune signalling proteins of low molecular weight that modulate the differentiation,  proliferation  and  function  of  immune  cells,  and  coordinate  inflammatory responses (Kidd and Urban, 2001). They are secreted primarily by activated tissue macrophages, lymphocytes and endothelial cells. Their main effects are to induce the acute-phase reaction and to activate vascular endothelium, leukocytes, platelets and fibroblasts, thus, initiating the cascade of vascular, cellular and humoural events which together comprise the inflammatory response (Tan et  al., 1999). Several cytokines play essential roles  in orchestrating the  inflammatory

process, especially interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-α) which are monokines, produced by monocytes and macrophages (Simopoulos, 2002). Lymphokines such as interferon‐gamma (IFN-γ) are produced by lymphocytes and they play an important role in the first line of defence against viral infections.

1.5.1.1.3 Chemokines

These are a family of pro-inflammatory peptides with potent leukocyte chemoattractant and activating properties (Kolaczkowska and Kubes, 2013). Chemokines are inducible molecules. They include, C-X-C (or α) chemokines and C-C (or β) chemokines. C-X-C chemokines are so- called because they have an intervening amino acid between the first two of the four conserved cysteine residues at their amino terminal. C-C chemokines on the other hand, do not have an intervening amino acid between their first two amino-terminal cysteine residues (Tan et al.,

1999). C-X-C chemokines are primarily neutrophils chemoattractants while C-C chemokines are primarily chemoattractants for monocytes and T-cells. Example of C-X-C chemokine is interleukin-8 (IL-8) while an example of C-C chemokine is monocyte chemoattractant protein-1 (MCP-1).  Interleukin-8  and  MCP-1  also  induce  degranulation  and  respiratory  burst  in neutrophils and monocytes respectively.

1.5.1.1.4 Nitric Oxide

Nitric oxide is generated through the action of nitric oxide synthase (NOS), as it oxidizes the terminal guanidine nitrogen atom of L-arginine (Kumar et al., 2013). Three major isoforms of NOS include two constitutively expressed forms, which are calcium/calmodulin dependent and are collectively known as constitutive nitric oxide synthase (cNOS). The third which is calcium/calmodulin independent, induced by cytokines and regulated in the gene by a variety of inflammatory mediators is  inducible  nitric  oxide synthase (iNOS). Nitric  oxide  is  toxic  to bacteria and directly inhibits viral replication. It also combines with ROS to yield peroxynitrate radicals which have potent antimicrobial activity (Chukwuka et al., 2011). Additionally, it causes vascular dilation, increases vascular permeability, stimulates relaxation of smooth muscle cells within the vessel walls and inhibits platelet aggregation. It is produced by macrophages and endothelial cells.

1.5.1.1.5 Prostaglandins

Phospholipase A2  (PLA2), in response to any disturbance of the cell membrane activates the hydrolysis of phospholipids from the lipid bilayer into free fatty acids such as, arachidonic acid. Arachidonic acid plays an important role  in  many metabolic pathways and  is useful when produced in moderation (George et al., 2014). When produced in excess, it acts as a substrate for COX (or prostaglandin H2 synthase) to release prostanoids, comprising of prostaglandins (PGs), thromboxanes (TXs) and prostacyclins (Ricciotti and FitzGerald, 2011). Examples of PGs are prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2). Prostaglandin D2 is a major prostaglandin produced  by  mast  cells.  It  is  a  bronchoconstrictor and  also  acts  as  a  chemoattractant  for leukocytes (Stone et al., 2010). Prostaglandin E2  which is more widely distributed causes pain, vasodilatation and increases vascular permeability. All cells are capable of synthesizing PGs, apart from non-nucleated erythrocytes.

1.5.1.1.6 Thromboxane A2 and Prostacyclin

Thromboxanes and Prostacyclins are also produced through the action of COX on arachidonic acid. Thromboxane A2 (TxA2) is a potent platelet-aggregating agent and a vasoconstrictor. It is unstable and is rapidly converted to inactive Thromboxane B2 (TXB2). Prostacyclin or prostaglandin I2  (PGI2) on the other hand is a vasodilator and a potent inhibitor of platelet aggregation (Pilotto et al., 2010). It increases blood flow as well as blood vessel permeability by assisting in the release of NO from the endothelium (Vishal et al., 2014).

1.5.1.1.7 Leukotrienes

Leukotrienes (LTs) like prostanoids, are released from excess arachidonic acid due to the activity of  5-lipoxygenase  (5-LOX).  The  lipoxygenase  (LOX)  pathway  is  a  parallel  inflammatory pathway to the COX pathway (George et al., 2014). Leukotrienes are released from leukocytes and  mast  cells  and  are  generally  pro-inflammatory.  Leukotriene  B4   (LTB4)  is  a  potent chemotactic agent for neutrophils. It also promotes their adhesion to vascular endothelial cells, their trans-endothelial migration and stimulates the synthesis of pro-inflammatory cytokines from macrophages and lymphocytes (Dalgleish and O’Byrne, 2002; Medzhitov, 2008). Leukotriene B4 also promotes degranulation and the generation of ROS. Cysteinyl-containing leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4) cause intense vasoconstriction, bronchospasm and increased vascular permeability in venules.

1.5.1.1.8 Platelet-activating Factor

Platelet-activating factor (PAF)  is  an ether phospholipid which is  released  from most  pro- inflammatory cells and platelets by the action of PLA2 (Sato et al., 2009). PAF- like molecules are also generated by activated vascular endothelium in a membrane-bound form. They are chemotactic for neutrophils, enhances the adhesion of platelets and leukocytes to endothelium, involved in vasoconstriction and bronchoconstriction and cause increased vascular permeability.

Figure 2: Generation of arachidonic acid metabolites and their roles in inflammation

1.5.1.2 Plasma Derived Mediators

These are proteins that circulate in the plasma as inactive precursors. They undergo proteolytic cleavage  to  become  active.  Mediator-producing  systems  in  plasma  include:  complement, clotting, fibrinolytic and kinin systems.

1.5.1.2.1 Complement System

The complement system is an important part of the innate immune system conferring protection against invading infectious agents, such as, bacteria, viruses and protozoa. Its role is to generate biologically  active  products  from  the  pathways  of  complement  activation  which  include: classical, lectin, alternative, properdin and thrombin pathways (Neher et al., 2011). Peptides generated by complement activation play a critical role in the elimination of invading pathogens. They include, C3b and C4b which opsonise pathogens for phagocytosis, the anaphylatoxins C3a and C5a which acts as chemoattractants for leukocytes and the membrane attack complex (MAC or C5b-9) involved in the direct lyses of pathogens (Alexander et al., 2008; Griffiths et al.,

2009). The generation of anaphylatoxins C3a and C5a further induces degranulation of mast cells,  basophils and eosinophils. They also induce the expression of adhesion molecules on endothelial cells and cause smooth-muscle contraction (Klos et al., 2009).

1.5.1.2.2 Clotting or Coagulation System

The mechanism of coagulation involves activation, adhesion and aggregation of platelets, along with the  deposition and  maturation of fibrin.  Fibrin  activation  involves two  pathways, the intrinsic and the extrinsic cascades. Following vascular injury, the extrinsic clotting cascade is triggered as activated endothelium expresses tissue factor (TF) on their luminal surfaces and expresses increased levels of plasminogen activator inhibitor-1 which inhibits fibrinolysis (Tan et al., 1999). In the presence of clot activating factors, prothrombin is converted to thrombin by prothrombinase. Thrombin in turn converts fibrinogen to fibrin, which is the major product involved in clot formation. The intrinsic cascade occurs with the engagement of Hageman factor (factor XII) which interacts with factors Va and VIIIa and becomes converted to its active form, factor XIIa. Factor XIIa in turn activates factor Xa which directly converts prothrombin to thrombin  and  the  subsequent  generation of  fibrin  from  fibrinogen (Ward,  2010).  Fibrin  is deposited on aggregated platelets to bring about thrombus formation.

1.5.2.2.3 Fibrinolytic System

The fibrinolytic system acts in opposition to the coagulation system, to counterbalance clotting. It is activated by urinary plasminogen activator (uPA) and tissue plasminogen activator (tPA) which converts plasminogen to plasmin. Plasmin directly interacts with fibrin to bring about the breakdown of fibrin clots as they are formed within the intravascular compartment (Ward, 2010).

1.5.2.2.4 Kinin System

Kinins are vasoactive peptides generated through the kinin-generating cascade. The most important product of this cascade is the plasma protein, bradykinin. Prekallikrein is converted to the protease, kallikrein by factor XIIa from the clotting cascade. Kallikrein interacts with high- molecular-weight kininogen (HMWK) to bring about the hydrolysis and release of bradykinin (Stankov, 2012). Bradykinin is a powerful vasopermeability agent. It also causes pain, vasodilatation and oedema, all contributing to inflammation.

Figure 3: Intercommunication between inflammatory cascades and the coagulation cascades

1.5.2 Cellular Components of Inflammation

The  principal cellular  components of the  inflammatory response are  the  white  blood cells (WBCs) or leukocytes. They originate from pluripotent haemopoietic stem cells and are found in tissues during acute inflammatory process and in the superficial aspects of a lesion during sub acute or chronic inflammation (Anosike et al., 2012b). Leukocytes include the granulocytes and agranulocytes. Other cells involved in inflammation are platelets, vascular endothelial cells, fibroblasts and plasma cells.

₦5,000.00 – DOWNLOAD FULL PROJECT DOWNLOAD FULL PROJECT Added to cart

---

---

---

---

Related Project Topics :