ABSTRACT
The aim of this study was to investigate the antimicrobial effect of Cissusaralioides extracts and their anti-inflammatory activity using experimental rat models. Phytochemical analyses of Cissusaralioides showed the presence of flavonoids, saponins, tannins, alkaloids, terpenoids, steroids and glycosides. Antimicrobial activity of C. aralioides was investigated against some gram negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Klebsiellaaerogenes and gram positive bacteria such as Staphyllococcus aureus and Streptococcus pneumonia using agar well diffusion method. Anti-inflammatory activities were tested on egg albumin-induced rat paw oedema, acetic acid-induced vascular permeability in rats, agar-induced leukocyte migration in rats and heat and hypotonic solution-induced haemolysis of human red blood cell membrane.Cissusaralioides extract administered orally up to the dose of 5000 mg/kg caused no deaths after 24 hours indicating that the lethal dose of Cissusaralioides is > 5000 mg/kg. Acetone, methanol and aqueous extracts displayed various degrees of antibacterial activity but the methanol extract showed higher activity against the bacteria examined. The extract significantly (p< 0.05) inhibited egg albumin-induced oedema in rats treated with the extract (100-400 mg/kg) and also significantly (p< 0.05) reduced exudate volume and vascular permeability induced by acetic acid in rats. The extract significantly (p< 0.05) stabilized human erythrocyte membrane subjected to heat and hypotonic-induced lysis in the treated groups (100-800 µg/ml). This study has shown that the acetone, aqueous and methanol extracts of Cissusaralioides possessed antimicrobial properties, and the methanol extract had anti-inflammatory activity.
CHAPTER ONE
INTRODUCTION
Inflammation is the response of tissue to injury (infection, trauma and hypersensitivity), characterized in the acute phase (microscopically) by increased blood flow (vasodilation) and vascular permeability along with the accumulation of fluid, leukocytes, and inflammatory mediators such as cytokines. Macroscopically, it is characterized by redness, swelling, heat, pain and loss of function and these cause considerable pain and discomfort and most often require treatment (Anosikeet al., 2012).At the onset of an inflammation, the cells undergo activation and release inflammatory mediators. These mediators include histamine, serotonin, slow reacting substances of anaphylaxis (SRS-A), prostaglandins and some plasma enzyme systems such as the complement system, the clotting system, the fibrinolytic system and the kinin system(Perianayagamet al., 2006). These mediator molecules work collectively to cause increased vasodilation and permeability of blood vessels. Thus, leading to increased blood flow, exudation of plasma proteins and fluids, and migration of leukocytes, mainly neutrophils, outside the blood vessels into the injured tissues. However, several medicinal plants are used in the treatment of disorders arising from inappropriate deployment of the inflammatory mediators, among them isCissusaralioides.This is even more compelling with the realization that currently available anti-inflammatory agents such as steroids and non-steroidal anti-inflammatory drugs are fraught with several drawbacks or limitations to their use. Increased incidence of stroke, atherosclerosis, cancer, gastric disorder and coronary heart related diseases has been attributed to prolonged use of synthetic inhibitors of cyclooxygenase, the non-steroidal anti-inflammatory drugs such as indomethacin, largely due to the implication of prostanoids in these pathological conditions (Grosser et al., 2006). Specific inhibitors of the cyclooxygenase isoenzymes- cyclooxygenase-1 and cyclooxygenase-2 are associated with gastrointestinal and vascular systems toxicities respectively (Joseph et al., 2005).Recently, these traditional medicines are receiving more scientific support which helps in not only authenticating the use of these medicines for treatment but also understanding the mechanism of action of these drugs. Cissusaralioides found in Asia and Africa shows antidiabetic, antimicrobial as well as anti- inflammatory properties (Maxwellet al., 2015). Cissusaralioides as an anti-inflammatory agent may reduce the formation of pro-inflammatory cytokines that stimulate bone resorption, thereby reducing bone loss and also act as an estrogen receptor agonist. Available experimental evidence showed that the extracts of the leaf caused moderate anti-diabetic activity (Igoliet al., 2012).
Flavonoids and some other compounds isolated from Cissusaralioides inhibit the release of β- hexosaminidase in rat basophilic leukemia cells (Xu et al., 2009). Flavonoids have anti- inflammatory (Yamamoto and Gaynor, 2011), antimicrobial (Cushnine and Lamb, 2005; 2011), anticancer and anti-diarrheal properties (Schuieret al., 2005).Terpenoids have been well studied for their pharmacological activities and are known to have anti-inflammatory properties and are used as anti-cancer drugs and they target the phospholipase, cyclooxygenase and lipoxygenase (Bracaet al., 2010).
1.1 Cissusaralioides:
Fig. 1: Cissusaralioides
Cissusaralioides is a natural climber only found from the countries of Arabia through Eastern and Western Africa.
1.1.1 Description of Cissusaralioides
Cissusaralioides is a lofty climber, woody at the base with stout green succulent stems constricted at the nodes and sometimes sub-succulent leaves. Flowers are greenish or whitish, comparatively large and horizontal. The fruit is 2½ cm long, mostly red in colour. The whole plant is covered with irritating hairs and leaves contain an acid and slightly acrid red sap. They are commonly found in deciduous forests and fringing jungle across the region from Senegal to Northern and Southern Nigeria. Cissusaralioides is found commonly in Tropical Africa especially Cameroon (common name- kindamine) and Nigeria (Igbo name- eririagwo) (Burkill,
2000).
1.1.2 Taxonomic Classification of Cissusaralioides
Taxonomy classification of Cissusaralioides
Kingdom: Plantae
Division: Magnoliophyta (Flowering plants) Class: Magnoliopsida (Dicotyledons) Order: Vitales
Family: Vitaceae (grape fruit family) Genus: Cissus
Species: aralioides (Burkill, 2000). Common name: Guinea Bissau
Nsukka name: Eriri-agwo
1.1.3 Origin and Geographic Distributionof Cissusaralioides
Cissusaralioidesoriginated from Congo extending from Arabia through Eastern Africa
Southwards to Mozambique and the Transvaal (Wild et al., 1963).
1.1.4 Edible Uses of Cissusaralioides
Cissusaralioides is a climbing or prostate shrub found throughout Africa, Egypt and the Arabian Peninsula and is used as a vegetable. It has minor economic importance as a medicinal plant (Balogun and Fetuga, 1986). The stem is sold as a food condiment in local markets in the Eastern and Northern parts of Nigeria.
1.1.5Medicinal Uses of Cissusaralioides
In Nigeria folkloric medicine, the leaves are used for treatment of cuts, wounds, internal and external microbial infections and swellings. It is also used for the treatment of arthritis, rheumatism, dropsy, gout swelling, oedema, analgesic, pulmonary troubles. (Burkill, 2000). In Cameroon traditional medicine, Cissusaralioides leaves and roots are used as antimicrobial agents against microorganisms of the gastrointestinal and urogenital tracts (Assobet al., 2011). In Gabon, the grinded leaves of Cissusaralioides mixed with sugar cane juice are used to combat gonorrhea. The liane (freed from the leaves) is used in Congo for its analgesic and antiseptic attributes to relieve cough, abdominal and kidney problems (Burkill, 2000). Phytochemical analyses of Cissusaralioides showed that it contains alkaloids, tannins, saponins, terpenes, flavonoids and cardiac glycosides (Borokini and Omotayo, 2012).
1.2 Medicinal Plants
1.2.1 History of Medicinal Plant
Medicinal plants have been used as native treatment for numerous human diseases for thousands of years and in many parts of the world and can be alternatives since their reputed efficacies have been experienced and passed on from one generation to another (Akinyemiet al., 2005; Jachak and Saklani, 2007).
In rural areas of developing countries, they continue to be used as the primary source of medicine (Chitmeet al., 2003). About 80% of the people in developing countries use traditional medicines for their health care (Kim, 2005). It is estimated that there are 250,000 to 500,000 species of plants on earth (Borris, 1996). Relatively small percentages (1 to 10%) of these are used as foods by both humans and other animals. It is possible that even more are used for medicinal purposes (Moerman, 1996). Hippocrates (in the late fifth century B.C.) mentioned
300 to 400 medicinal plants (Schultes, 1978). In the first century A.D., Dioscorides wrote De MateriaMedica, a medicinal plant catalogue which became the prototype for modern pharmacopoeias.
The Bible offers descriptions of approximately 30 healing plants (Cowan, 1999). Indeed, frankincense and myrrh probably enjoyed their status of great worth due to their medicinal properties. They were reported to have antiseptic properties and were even employed as mouthwash. Thus the mainstream medicine is increasingly receptive to the use of antimicrobial and other drugs derived from plants, as traditional antibiotics (products of microorganisms or their synthesized derivatives) become ineffective and as new, particularly viral diseases remain intractable to this type of drug. Another driving factor for the renewed interest in plant antimicrobials in the past 20 years has been the rapid rate of plant species extinction (Lewis and Elvin-Lewis, 1995).
1.2.2 The Phytochemicals in Medicinal Plants
Plants have almost a limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen-substituted derivatives (Lahlou, 2004). Most of the derivatives are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total (Schultes, 1978). In many cases, these substances serve as plant defence mechanisms against predation by microorganisms, insects, and herbivores. Some of them, such as terpenoids, give plants their odours; others (quinones and tannins) are responsible for plant pigments. Many compounds areresponsible for plant flavour (the terpenoid capsaicin from chili peppers), and some of the same herbs and spices used by humans to season food yield useful medicinal compounds. Such secondary metabolites like tannins, terpenoids, flavonoids, alkaloids, saponins, reducing sugars and sterols have been found to have anti-diarrhoeal or antimicrobial activity (Yu et al., 2000; Al-Rehailyet al., 2001).
1.3 Antimicrobial Agents
Man is in constant contact with a large number of different bacteria which temporarily or permanently inhabit his body creating temporary or permanent community. Relations which are thus established are dangerous and very complex, from those positive to those whose consequences for man are extremely negative. Very often, both on and in man’s body, bacteria which have the ability to cause an infection are present. This ability of pathogenic bacteria is reflected in possession of certain pathogenicity factors. A set of factors which enable successful invasion and damage of the host are: toxins, surface structures and enzymes. Between the host and the pathogen, very complex relations are established whose outcome depends on host’s characteristics as well as on pathogen’s characteristics (Bartizilaet al., 1992).
Infections caused by bacteria can be prevented, managed and treated through anti- bacterial group of compounds known as antibiotics. Antibiotics are natural, semi-synthetic or synthetic compounds that kill or inhibit the growth of bacteria. When bacteria are exposed to an antibiotic, they respond doubly:
(i) They are sensitive to what caused the inhibition of their growth, division and death or
(ii) ii) They can remain unaffected or resistant (Okorie, 2005).
The resistance of bacteria to antibiotics can be natural (intrinsic) or acquired. Natural resistance is achieved by spontaneous gene mutation. The acquired resistance occurs after the contact of bacteria with an antibiotic and as a result of adaptation of a species to adverse
environmental conditions. In such population, an antibiotic as a selective agent, acts on sensitive organism, while resistant organism survive and become dominant. Bacteria gain antibiotic resistance due to three reasons which are:
(i) Modification of active site of the target site resulting in reduction in the efficiency of binding of the drug
(ii) Direct destruction or modification of the antibiotic by enzymes produced by the organism (iii) Efflux of antibiotic from the cell (Sheldon, 2005).
The evolution of antibacterial resistance in human pathogenic and commensal microorganisms is the result of the interaction between antibiotic exposure and the transmission of resistance within and between individuals. It is especially interesting in the phenomenon of horizontally gene transfer, extra-chromosomal DNA material, so-called plasmids, often carry genes of resistance and can transfer information within and between the individuals of the same or related bacterial species, thus also spreading the resistance.
Having in mind the current progress of resistance spreading and resilience of larger and larger number of bacteria to traditional antibiotics as well as a way of transmitting the gene of resistance, above all via plasmids, one can conclude that the ability of obtaining bacterium resistance to antibiotics represents a very dynamic and unpredictable phenomenon. For that reason, bacterial resistance to antibiotics represents a major health problem. Solving this problem and search for new sources of antimicrobial agents is a worldwide challenge and the aim of many scientific researches, academic institutions and pharmaceutical companies is testing biologically active compounds of plant origin (Cowan, 1999).
1.3.1 History of Antimicrobial Agents
The first anti-microbial agent was Salvarsan, a remedy for syphilis that was synthesized by Ehrlich in 1910. In 1935, sulfonamides were developed by Domagk and other researchers. These drugs were synthetic compounds and had limitations in terms of safety and efficacy. In 1928, Fleming discovered penicillin. He found that the growth of Staphyllococcus aureus was inhibited in a zone surrounding a contaminated blue mold (a fungus fromPenicilliumgenus) in culture dishes, leading to the finding that a microorganism would produce substances that would inhibit the growth of other microorganisms (Cushnie and Lamb, 2011). Penicillin was originally effective against gram-positive organisms such as S. aureus which produces the penicillin hydrolyzing enzyme penicillinase and later methicillin was developed. The drugs have been
developed to achieve better pharmacodynamics including the absorption of oral drugs, concentration in the blood and distribution to the inflammatory focus.
Antimicrobial agents that are associated with serious side effects have been replaced by other safer drugs. Quinolone antimicrobials represent an example of drugs with improved pharmacodynamics and safety. Nalidixic acid, the first drug of this class, was active only against gram-negative bacteria. S. aureus is the resistant bacterium that rapidly acquired resistance to sulfonamides when they were in use. However, in 1961, methicillin-resistant S. aureus (MRSA) was isolated in the UK. MRSA acquires resistance to most β-lactam antibiotics, through its acquisition of the penicillin-binding protein (PBP)2 gene. PBP2 is an enzyme involved in cell wall synthesis that has low binding affinity for β-lactam antibiotics. Although P. aeruginosa are intrinsically resistant to many antimicrobial agents, the emergence of P. aeruginosa strains resistant to all the classes of antimicrobials, i.e., carbapenems, quinolones and amino glycosides is a recent concern(Chambers, 1997). This has spurred our interest to undertake antimicrobial screening especially against P. aeruginosa.
1.4 Biochemistry of Inflammation
Inflammation is a complex reaction of the body in response to cellular injury that is marked by tissue swelling, capillary dilation, anti-histamine activity, redness, heat and pain. It serves as a mechanism initiating the elimination of noxious agents of damaged tissue by intercepting and destroying invading microorganism (Anosikeet al., 2012b).At the onset of inflammation, the cells undergo activation and release inflammatory mediators. These mediators include histamine, serotonin, slow reacting substances of anaphylaxis (SRS-A), prostaglandins and some plasma enzymes such as the complement system, the clotting system, the fibrinolytic system and the kinnin system (Perianayagamet al., 2006). These mediator molecules work collectively to cause
increased vasodilation and permeability of blood vessels. The damaged cell signals inflammatory response releasing NF Kappa B, the key regulator to our inflammatory response system. This results in the expression of several pro-inflammatory proteins such as COX-2 and iNOS that cause pain, fever, swelling and heat in an affected area. Simultaneously, a series of pro-inflammatory cytokines such as IL-2, TNF-α and interferon-γ are released (Cushnine and Lamb, 2005).Following inflammation, injured tissue is usually replaced by new cells and extracellular materials, with undamaging surrounding cells proliferating and migrating to fill the void, although some tissues especially surface epithelium can grow back efficiently (Ferrero et al., 2007).The basic components of inflammatory response are:
1.4.1 Increased Vascular Permeability
The endothelial lining of capillaries becomes leakier, allowing more fluid (blood plasma) to exude into the connective tissue spaces. There is normally a balance between fluid leaving vascular spaces and fluid re-entering the system. Inflammation shifts this balance, causing accumulation of interstitial fluid. The fluid build-up, which follows this permeability change, is called oedema and is visible as puffiness or swelling (Albertset al., 2002).
1.4.2 Emigration of Leukocytes
Vasodilation and increased vascular perfusion are designed to prepare the way for the inflammatory infiltrate to enter the inflamed tissue.A combination of vasodilation with thickening of the blood (due to fluid leaking out of the vessels) causes a slowing of flow rate, which encourages leukocytes to stick to the sides of the vessels. This is called “margination” or “pavementing” (the white blood cells gather along the endothelium, like bricks paving a road).From here the leukocytes crawl between the endothelial cells and enter the inflamed connective tissue. Increased metabolic activity associated with leukocyte activity also generates heat, contributing to local warmth (Pancer and Cooper, 2006).
1.4.3 Inflammatory Infiltrate
The inflammatory or leukocytic infiltrate consists of white blood cells which leave the blood and enter (infiltrate) the inflamed connective tissue. Cells of the inflammatory infiltrate include neutrophils, lymphocytes and monocytes. Immigration of these cells into peripheral tissues is one of the principal purposes for inflammation, bringing to a site of injury the immune cells which combat infection and clean up damaged tissue (Moreau et al., 2001).
1.4.3.1 Neutrophils
Neutrophilic leukocytes are the first white blood cells to enter the tissue during acute inflammation. Neutrophils are anti-bacterial cells which lyse (break down) bacterial cells by releasing lysosomal enzymes (Langermanset al., 1994). Neutrophils are fairly uniform in size with a diameter between 12 and 15 micrometers and are polymorphonuclear. The nucleus consists of two to five lobes joined together by hair like filaments. Neutrophils move with amoeboid motion. They extend their long projection called pseudopodium into which their granules flow; this action is followed by contraction of filaments based in the cytoplasm, which draws the nucleus and rear of the cell forward. In this way neutrophils rapidly advance along a surface. The bone marrow of a normal adult produces about 100 billion neutrophils daily. It takes about one week to form a mature neutrophil from a precursor cell in the marrow; yet, once in the blood, the mature cells live only a few hours or perhaps a little longer after migrating to the tissues (James and Michael, 1996). To guard against rapid depletion of the short-lived neutrophils (for example, during infection), the bone marrow holds a large number of them in reserve to be mobilized in response to inflammation or infection.
Within the body, the neutrophils migrate to areas of infection or tissue injury. The force of attraction that determines the direction in which neutrophils will move is known as chemotaxis and is attributed to substances liberated at sites of tissue damage. Of the 100 billion neutrophils circulating outside the bone marrow, half are in the tissues and half in the blood vessels; of those in the blood vessels, half are within the mainstreams of rapidly circulating blood and the other half move slowly along the inner walls of the blood vessels (marginal pool), ready to enter tissues on receiving a chemotactic signal from them (Agerberth and Gudmunsson, 2000).
Neutrophils are actively phagocytic; they engulf bacteria and other microorganisms and microscopic particles. The granules of the neutrophils are microscopic packets of potent enzymes capable of digesting many types of cellular materials. When bacterium is engulfed by a neutrophil, it is encased in a vacuole lined by the invaginated membrane. The granules discharge their contents into the vacuole containing the organism. As this occurs, the granules of the neutrophil are depleted (degranulation). A metabolic process within the granules produces hydrogen peroxide and a highly active form of oxygen (superoxide), which destroy the ingested bacteria. Final digestion of the invading organism is accomplished by enzymes.
Neutrophils constitute 40 to 70% of total WBCs; they are the first line of defense against infection. Mature neutrophils have a half-life of about 2 to 3 days (Albertset al., 2002). During acute inflammatory response (e.g., infection), neutrophils are drawn by chemotactic factors and alerted by the expression of adhesion molecules on blood vessel endothelium, leave the circulation and enter tissues. Their purpose is to phagocytose and digest pathogens. Microorganisms are killed when phagocytosis generates lytic enzymes and reactive O2 compounds (e.g., superoxide, hypochlorous acid) and triggers release of granule contents (e.g., defensins, proteases, bactericidal permeability-increasing protein, lactoferrin, and lysozymes). DNA and histones are also released, and they, with granule contents such as elastase, generate fibers in the surrounding tissues; the fibers may facilitate killing by trapping bacteria and
focusing enzyme activity. Severe inflammation may increase the numbers of neutrophils in blood, resulting in neutrophilia (Langermanset al., 1994).
1.4.3.2 Lymphocytes
Lymphocytes are the cells responsible for the body’s ability to distinguish and react to an almost infinite number of different foreign substances, including those of which microbes are composed (Moreau et al., 2001). Lymphocytes are mainly a dormant population, awaiting the appropriate signals to be stirred to action. Lymphocytesaccumulate somewhat later during the inflammatory process. Their presence in large numbers indicates the continuing presence of antigen and thus may suggest an established infection (Boyton and Openshaw, 2000).
Lymphocytes produce the multitude of diverse antibody molecules (one specific type of antibody per lymphocyte) which provide the mechanism for chemical recognition of foreign materials (distinguishing between self and non-self) and also for mediating and regulating immune responses. Lymphocytes travel in the blood, but they routinely leave capillaries and wander through connective tissue. Therefore, lymphocytes may be normally encountered at any time in any location. They even enter epithelial tissue, crawling between the epithelial cells. They re-enter circulation via lymphatic system channels (hence their name) (Yenuguet al., 2003). Lymph channels drain into lymph nodes, where dense aggregations of lymphocytes form lymph nodules. Each lymph nodule has a “germinal center”, where activated lymphocytes proliferate. Lymph nodules with proliferating lymphocytes also characterize the tonsils and the appendix and may be encountered in other sites as well (James and Michael, 1996).
Recent research suggests that some types of lymphocytes are compartmentalized to particular tissues or body regions. Lymphocytes are small cells, 7-9 micrometer in diameter in blood smears, and are the second most common white blood cell type (about 30% of the WBCs). They have a round heterochromatic (deeply staining) nucleus surrounded by a relatively thin rim of cytoplasm. Lymphocytes are most easily recognized in histological sections as small “naked” nuclei (the cytoplasm is usually inconspicuous) which occur here and there in most tissues and especially commonly near mucous membranes. Lymphocytes are found densely packed in lymphoid tissue-spleen and lymph nodes. Plasma cells are lymphocytes which are specialized for mass production and secretion of circulating antibodies. Plasma cells have more extensive cytoplasm filled with rough endoplasmic reticulum (for synthesizing protein, specifically antibody molecules). This cytoplasm is distinctly basophilic, a consequence of the large numbers of ribosomes associated with the rough ER, and typically forms a lopsided bulge on one side of the nucleus. The heterochromatin of plasma cells is typically clumped in a characteristic “spoke-wheel” arrangement which also aids plasma cell recognition (Agerberth and Gudmunsson, 2000).
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ANTIMICROBIAL SCREENING AND ANTIINFLAMMATORY ACTIVITIES OF METHANOL EXTRACT OF CISSUSARALIOIDES
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