University of Ghana http://ugspace.ug.edu.gh QP752.A7 Ain7 bite C.l G365701 University of Ghana http://ugspace.ug.edu.gh ANTI-INFLAMMATORY MEDICINAL PLANTS AS ANTI-OXIDANTS AND INHIBITORS OF PRO- INFLAMMATORY EICOSANOID BIOSYNTHESIS A THESIS SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF GHANA BY KWABENA AMPONSAH-MANAGER IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF A MASTER OF PHILOSOPHY DEGREE IN BIOCHEMISTRY MAY, 2001 University of Ghana http://ugspace.ug.edu.gh Grace is inexplicable You understand it only by experiencing it Those who have found it need no explanation They already know what it is Those who have not found it, or at least not yet Will never understand it, no matter how often you explain Kwabena Amponsah-Manager University of Ghana http://ugspace.ug.edu.gh DECLARATION I carried out the experimental work contained in this thesis in the laboratories of the Department of Biochemistry and the Noguchi Memorial Institute for Medical Research, both of the University of Ghana, under the supervision of Prof. Marian Ewurama Addy and Dr. Alexander Kwadwo Nyarko. Kwacena Amponsah-Manager (Student) Prof. M. E. Addy Dr. A.K. Nyarko (Supervisor) (Co-supervisor) University of Ghana http://ugspace.ug.edu.gh DEDICATION TO MY MOTHER, FOR OBVIOUS REASONS University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I wish to put on record my deep appreciation of the support and encouragement that were given to me by my supervisors, Prof. Marian Ewurama Addy and Dr. Alexander Kwadwo Nyarko. I cannot thank them enough for their sacrifices throughout this research. I am highly indebted to Dr. Phyllis Addo, the Head of the Experimental Animals Unit of the Noguchi Memorial Inst, for Medical Research (NMIMR), for offering her laboratory and making some timely suggestions and criticisms. I also thank Messrs Mark Ofosuhene and Kwesi Teye of the Clinical Pathology Unit and the Technical Staff of the Department of Biochemistry, University of Ghana for technical assistance. I acknowledge the role various friends and colleagues played in this work. I am grateful to them all, especially my best friend Mr. Kojo Aikins who gave so much to get this work done, Ps and Mrs. Dokosi for all the materials they provided and Mr. John Dadzie-Mensah who made some fine suggestions during the initial stages of this work. Finally, I am grateful, as always, to my family for their prayers and support throughout my studies. University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS CONTENTS Page CITATION i DECLARATION ii DEDICATION iii ACKNOWLEDGEMENT iv TABLE OF CONTENTS v LIST OF FIGURE viii LIST OF TABLES ix ABSTRACT x CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW General introduction 1 Research objectives 7 Significance of the study 8 Literature review 11 Inflammation 11 Pro- and anti-inflammatory eicosanoids 13 Leukotrienes 14 Thromboxanes 17 Estimation of eicosanoids 21 Anti-oxidants 22 Asthma as inflammatory disorder 24 Anti-inflammatory medicinal plants 28 v University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO: MATERIALS AND METHODS Material 33 Methods 3 4 Preparation of plant extracts 34 Pre-treatment of animals 35 Preparation of isolated organ 36 Sampling of lung effluent 37 Preparation of microsomes 38 Protein determination 39 In vitro synthesis of pro-inflammatory eicosanoids 39 Quantitative estimation of eicosanoids 40 Anti-oxidant properties 42 Effect on hydroxyl radical generation 42 Quantitative determination of Total phenol 43 Water soluble phenols 44 Statistical analysis 44 CHAPTER THREE: RESULTS Release of Eicosanoids 45 Effects of 'Tina A' 45 Effect of T. sanguineaon the release of thromboxane B2 52 Synthesis of eicosanoids 52 Thromboxane synthesis 52 vi University of Ghana http://ugspace.ug.edu.gh Cysteinyl leukotrienes 58 Effect on hydroxyl radical generation 59 Total and soluble phenols 61 CHAPTER FOUR: DISCUSSION AND CONCLUSION 63 Reference: 77 Appendix 93 vii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure Title Page Fig 1.1: The lipooxygenase pathway 15 Fig 1.2: The cycloxygenase pathway 20 Fig 3.1: Effect of 'Tina A' on the release of TXB2 46 Fig 3.2: Effect on the release of cysteinyl leukotrienes 49 Fig 3.3: Effects of extracts on synthesis of thromboxanes 54 Fig 3.4: Effect of 'Tina A' on TX synthesis 56 Fig 3.5: Effect on hydroxyl radical generation 60 University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table Title Page Table 1.1: Effect of 'Tina A' on the release of TXB2 and Cysteinyl LT under different conditions of treatment. 51 Table 3.2: Effects of T. sanguinea and L. multiflora on TXB2 synthesis 57 Table 3.3:Effect of 'Tina A' and T. sanguinea on LT synthesis 58 Table 3.4: Phenolic content of extracts 61 Table 3.5:Total phenolic content and inhibition hydroxyl radical generation 62 University of Ghana http://ugspace.ug.edu.gh ABSTRACT Arachidonic acid (AA) metabolism that leads to the production of both anti- and pro-inflammatory eicosanoids is a standard assay used to investigate the basis for the therapeutic action of anti-inflammatory medicinal plants. Earlier investigations have established the efficacy of some herbal preparations in terms of their ability to increase the amounts of anti-inflammatory eicosanoids. Desm.od.ium adscendens and Parkitina sp. ('Tina A' ) increased PGE2 and PGI2 synthesis and inhibited phospholipase A2 activity while Thonningia sanguinea decreased the release of histamine and slow reacting substances of anaphylaxis (SRA-S). So far none of these extracts has shown any significant effect on thromboxane (TX) synthesis. The present study was conducted to evaluate the efficacy of four plant extracts, D. adscendens, Tina A, T. sanguinea and L. multi flora in decreasing the amounts of the pro-inflammatory eicosanoids, TXB2 and cysteinyl leukotrienes (cyst.LT), and their antioxidant properties to act as anti-oxidants. Isolated guinea pig lungs perfused via the trachea were used as a model to study the release and inhibition of the release of the TXB2 and cyst.LTs by the extracts. The organs were perfused with Kreb's solution with and without the plant extracts and TXB2 and cyst.LT released were estimated by an x University of Ghana http://ugspace.ug.edu.gh ELISA. There were significant decreases in the amounts of TX B2 and cyst.LT released from lungs perfused with the extract of 'Tina A' or T. sanguinea and lungs from animals treated with 'Tina A'. Generally, the treatment of the animals with the extract decreased the release of TX more than LTs, while perfusion of the isolated lungs (i.e. short term treatment) had more effect on cyst.LT than TX. The effects of D. adscendens, 'Tina A' , L. multiflora and T. sanguinea on the in vitro synthesis of TXB2 using blood platelet microsomes were investigated. Microsomes for this study were prepared from blood platelets. Except L. multiflora, for which higher concentration showed minimum effects on TXB2 synthesis, there was a concentration-dependent inhibition of TXB2 by all the extracts. The effect of T. sanguinea became significant at a relatively higher concentration compared to that of D. adscendens or 'Tina A'. At the highest concentration of 100ug/ml, D. adscendens, 'Tina A’ and T. sanguinea caused 81%, 81.6% and 87.5% decreases respectively in the amounts of TXB2 synthesized. The effects of the extracts on hydroxyl radical generation and total and water- soluble phenolic content were determined. There was a concentration-dependent inhibition of hydroxyl generation by all the extracts. Large amounts of phenolic compounds were identified in all the extracts. For 'Tina A', 74% of the total phenols was water soluble while T. sanguinea which University of Ghana http://ugspace.ug.edu.gh gave the highest amount of total phenols had 31% being water soluble. There was a positive correlation between total phenolic content and inhibition of hydroxyl radical generation. T. sanguinea and D. adscendens which had the highest and lowest amounts of total phenols respectively, showed the highest and lowest inhibition of hydroxyl radical generations respectively at all concentrations studied. These findings suggest that the inhibition of both synthesis/release of pro-inflammatory eicosanoids and generation of reactive oxygen species by the plant extracts studied validates their use in folk medicine in the management of asthma and other inflammatory disorders. University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW GENERAL INTRODUCTION Medicinal plants are a major source of healthcare management for a large number of the world's population (Farnsworth et al., 1985) . It is estimated that about eighty per cent of the world's population depend solely on local plants as medicine (Lewington, 1990). Today, due to science and technology and improvement in synthetic and phyto-chemistry, the use of non-plant drugs is gradually increasing, thereby changing this percentage. However, due to increasing costs of orthodox drugs, the majority of the poor in the world, especially those in developing countries, continue to use plant medicine for their healthcare management. In Ghana medicinal plants play a vital role in the Health Delivery System (Anokbonggo, 1992). In many local communities, plant-based drugs are used for the treatment or management of cancer, ulcer, reproductive problems and hypertension. Others are used as contraceptives, laxatives, and for managing a host of inflammatory disorders. For example, Desmodium adscendens, Indigofera errecta, Trema occidentalis, Capparis erythrocarpus and Thonningia sanguinea are currently used for the treatment of asthma, diabetes 1 University of Ghana http://ugspace.ug.edu.gh mellitus, hepatitis, arthritis and asthma respectively at the Center for Scientific Research into Plant Medicine (CSRPM) at Mampong in Ghana (Gyamfi et al., 1999; Nyarko and Addy, 1994) . In most communities, it is believed that the knowledge of the curative value of herbs are given to individuals by God, through dreams, psychic powers or some extra sensory perception. As a result such preparations are expected to be administered in a certain form or should follow a certain procedure even when such procedures are unacceptable to a large number of the people. At the moment western medicine is preferred to these local plant extracts by the majority of the people even though they are more expensive. The reasons for this include proper quality control, standardized dosage, documented pharmacological effects and how to handle those effects, safety, and attractive forms of presentation. It is important that the African medicinal herbal preparations are improved upon to make them more acceptable to the majority of the people including those from other continents. Putting science into the practice of herbal medicine will go a long way to address the above concerns. Currently, a number of plant extracts, the majority in their crude forms, are being used to manage inflammatory diseases. 2 University of Ghana http://ugspace.ug.edu.gh In spite of their usefulness, detailed scientific studies have not been carried out on these preparations to validate their use in therapy. Such studies are necessary considering the fact that most plant preparations may elicit both therapeutic and toxic effects by interacting with enzymes and/or metabolites of the pathways that those enzymes are involved in. An example is the extract of fever few plant, Tanacetum parthenium which has been found to inhibit normal aggregatory and secretory response of blood platelets (Heptinstall, 1987) . The fever few plant has been in use since ancient times to treat fever but its effect on platelets became known only recently. Phytochemical studies of some of these plant preparations have shown that they contain alkaloids, some of which can be toxic (Mclean, 1970). Herbal preparations can be misused, especially by the rural and urban folk who cannot afford orthodox medicine. Lately, on buses and at the market places diverse medicinal plant products are advertised and sold. Some of these materials originate from charlatans and may not have any therapeutic action against the ailment they are claimed to heal. The use of most of these plant preparations is not supported by any scientific data with respect to their efficacy, dosage, shelf life, etc. In order to improve the quality of herbal medicine, the Center for Scientific Research into Plant 3 University of Ghana http://ugspace.ug.edu.gh Medicine (CSRPM) at Mampong-Akwapim in Ghana was set up to carry out scientific investigation on herbal preparation with the view to addressing these concerns indicated. The Center stocks medicinal plants, some of which are useful in the management of several diseases including asthma. Some of the plants used to manage asthmatic attacks are Thonningia sanguinea and Desmodium adscendes (Addy and Nyarko, 1985). The anti-asthmatic effects of these plants have been studied in the Department of Biochemistry and at the Noguchi Memorial Institute for Medical Research, both of the University of Ghana. The early investigations used anaphylaxis in the guinea pig as a model to study the anti-asthmatic effects of the plant preparations (Addy and Dzandu, 1986; Addy and Nyarko, 1985; Addy and Awumey, 1984; Addy and Gbewonyo, 1980) . As a result, the effects of the extracts on the biosynthesis of prostanoids/eicosanoids are now known (Addy and Schwartzman, 1995, 1992; Addy and Burka, 1987). A good anti-inflammatory plant product will modulate the metabolism of AA and hence synthesis of eicosanoids such that more anti- and less pro-inflammatory products will be synthesized in response to the administration of the plant material. Inhibition of the cyclooxygenase enzyme protects against inflammation. The non-steroidal anti-inflammatory 4 University of Ghana http://ugspace.ug.edu.gh drugs (NSAIDs) which have aromatic carboxylic acid moiety, such acetylsalicylic acid (aspirin) and aspirin-like drugs such as indomethacin, inhibit the cyclooxygenase enzyme. These drugs consequently reduce the amounts of pro- inflammatory eicosanoids such as thromboxane A2 and the prostaglandin of the F series. The disadvantage in the mode of inhibition described above is that it leads to a reduction of anti-inflammatory eicosanoids, which protect against inflammations. Therefore, a mechanism-based assay that involves the inhibition of the key enzymes, and the consequent reduction in the amounts of pro-inflammatory eicosanoids can be established for use to evaluate potential anti-inflammatory drugs. In this regard, the selective inhibition of AA metabolism will be a precise assay to use in order to evaluate the mode of action of putative anti­ inflammatory materials. Previous studies have established the efficacy of some anti-asthmatic herbal preparations. One of these, extracts of D. adscendens has been shown to increase the anti-inflammatory prostanoid PGE2 (Addy and Schwartzman, 1995) . This extract however did not decrease the amounts of pro-inflammatory prostanoid PGF2a. In another study the extracts of D. adscendens greatly increased the synthesis of prostacyclin, PGI2, another anti-inflammatory prostanoid, but did not affect the synthesis of thromboxane A2 (Dadzie-Mensah, 2000), a pro-inflammatory eicosanoid whose 5 University of Ghana http://ugspace.ug.edu.gh biosynthesis is related to that of PGI2. The reason for this lack of effect on pro-inflammatory eicosanoid is not apparent but it is likely that the conditions of the assays used were not optimal for their synthesis. For example, for the in vitro studies in some of the previous investigations, microsomes used as the source of the cyclooxygenase enzyme were prepared from either the lung or the kidney. These organs, even though rich in the cyclooxygenase enzymes, may not be good sources of the enzyme system for TX biosynthesis. The major product of AA metabolism in platelets is thromboxanes (Ali and Mohammed, 1986) . In view of the fact that thrombocytes are a rich source of the enzyme system for TX synthesis (Moore et al, 1991), it was important to use microsomes from blood platelets in order to evaluate the effect of these plant extracts on production of pro- inflammatory eicosanoids such as TXs and cysteinyl- leukotrienes. In vivo measurement of TX has so far not been significantly successful over the years. Measurement of TXB2, the dominating compound in circulation after injection of AA indicates that, there is great artifactual formation of the compound during sampling (Samuelsson, 1978). Consequently "peripheral plasma levels" of TXB2 is no doubt very high and certainly does not reflect the true endogenous circulating 6 University of Ghana http://ugspace.ug.edu.gh levels of the compound. Another problem has to do with the post release metabolism of the compound in the whole animal. This possibility can be minimized by employing an isolated whole organ, which, unlike tissue homogenates, adequately retains vital physiological characteristics, but at the same time has the advantage of excluding some of the complexities associated with whole animal experimentation. Research Objectives The overall objective of the research was to investigate the potential of certain plant extracts that are used by herbalist to treat various forms of inflammatory diseases by assaying for their ability to decrease the synthesis of pro- inflammatory eicosanoids, such as TXA2 and the cysteinyl- leukotriene, and to act as anti-oxidants. The specific objectives included: • Establishing of an assay for the biosynthesis of the pro- inflammatory eicosanoids, TX and cysteinyl-leukotrienes, using isolated guinea pig lung. • Establishing an assay for the synthesis of these eicosanoids in vitro using microsomes from platelets as a source of enzyme. 7 University of Ghana http://ugspace.ug.edu.gh • Using the established assays to evaluate the effects of these plant extracts purported to be used to manage inflammatory diseases. • Evaluating the anti-oxidant effects of the plant extracts by measurement of hydroxyl radical levels as well as total and water soluble phenols. It is reported that mediators that contribute to immediate and late asthmatic responses include reactive oxygen species (ROS) produced by epithelial cells and inflammatory cells recruited to the sites of antigen challenge (Comhair et al., 2000). The effects of the ROS produced depend on local anti­ oxidant defenses available within the airway epithelial lining fluid. This study therefore proceeded to investigate the anti-oxidant properties of the herbal plant extracts. The results include the effects of the extracts on the hydroxyl radical generation, total and water-soluble phenols. Significance of the Study Aspirin, a non-steroidal anti-inflammatory drug (NSAID), has remained the primary drug of choice for the treatment of inflammatory diseases and the majority of articular and musculoskeletal disorders for the past century (Payan and Shearn, 1992) . It is the standard against which all anti­ inflammatory agents are evaluated. This may be due to its 8 University of Ghana http://ugspace.ug.edu.gh low cost and minimum side effects. Other NSAIDs, such as indomethacin and phenylbutazone, like aspirin, block prostaglandin production by irreversible inhibition of the cyclooxygenase enzyme (Payan et al., 1992). The steroidal anti-inflammatory drugs (SAIDs) act by inhibiting phospholipase A2 and consequently interfere with the mobilization of AA and hence the production of all eicosanoids. These two groups of drugs have significant undesirable side effects. For example, phenylbutazone therapy may cause plastic anemia (Glew, 1992) . Some of the NSAIDs and SAIDs are very expensive and beyond the means of the majority poor. Our forests however have in abundance trees and herbaceous plants that are used to manage various inflammatory diseases. Screening putative plants for their anti-inflammatory effects will provide data that could * be employed as a protocol to standardize herbal preparations that are being used to treat inflammatory diseases * lead to a more discriminate use of anti- inflammatory herbal products and also increase their acceptance and patronage 9 University of Ghana http://ugspace.ug.edu.gh stimulate young and emerging scientists to enter into scientific research into plant medicine thereby providing another avenue for employment • contribute to our growing economy by cutting down government expenditure on drug importation • lead to the production of drugs for local as well as foreign markets and hence earn some foreign exchange for Ghana. 10 University of Ghana http://ugspace.ug.edu.gh LITERATURE REVIEW Inflammation Inflammation is the basic cause of a host of diseases and signifies cell injury. It is the body's reaction to invasion by an infectious agent, an antigen or even just a physical damage (Payan and Shearn, 1992) . Inflammation occurs when immunologically competent cells are activated in response to foreign organisms or antigenic proteins thereby directing elements of the immune system to sites of infection or injury. The outcome of this response is generally beneficial, as when it causes invading organisms to be neutralized. It could however be deleterious as in the case of arthritis, when it leads to the destruction of bone and cartilage resulting in the limitation of joint function (Roitt et al., 1993) . The manifestations of inflammation are heat, pain, redness and swelling. Redness or rubor is usually the first thing to be noted when inflammation sets in. This is due to the dilation of arteries supplying blood to the area with the result that capillaries, which were previously empty or only partially distended, become rapidly filled with blood. The body controls this condition called hyperemia both neurogenically and chemically via the release of mediator 11 University of Ghana http://ugspace.ug.edu.gh substances such as histamine. The pain, or dolor, may be the result of change in local pH or in the concentration of certain ions or release of certain chemicals that stimulate nerve endings. Heat, or calor, is the result of more blood being conducted from the inside of the body to the surface, which is normally cooler than 37°C. For inflamed areas deep within the body, local warmth is not a problem since such tissues will already be at the core temperature, and local hyperemia would make no difference. Swelling is the most striking aspect of acute inflammation. It results from the transfer of fluid and cells from the bloodstream to the interstitial tissues. These manifestations are mediated by pro-inflammatory compounds. Some of these pro-inflammatory compounds are eicosanoids that are produced from the metabolism of arachidonic acid (AA) catalyzed by cyclooxygenase, lipoxygenase and monooxygenase enzymes. The release of AA from membrane phospholipids is the rate- limiting step. Once AA is released, it is subsequently metabolized to various eicosanoids (Bhagavan, 1992; Smith, 1992). The excessive production of the pro-inflammatory metabolites is the result of a derangement in the signal transduction mechanism that involves the metabolism of AA. 12 University of Ghana http://ugspace.ug.edu.gh Pro-and anti-inflammatory Eicosanoids AA metabolism via different pathways gives rise to various C- 20 fatty acid (FA) oxygenation products, the eicosanoids. Three of the major classes of eicosanoids namely prostaglandin, prostacyclin and thromboxane, are produced from the cyclooxygenase-mediated metabolism of AA. The lipoxygenase reaction leads to the production of leukotrienes and lipoxins (Fig. 1.1). A third pathway of AA metabolism is mediated by an NADPH-dependent monooxygenase enzyme, a flavoprotein reductase present in the endoplasmic reticulum. Even though the eicosanoids are produced from the metabolism of a common substrate, some of them are anti-inflammatory while others are pro-inflammatory. Generally, prostaglandin E2 and prostaglandin I2 also known as prostacyclin, are anti­ inflammatory while thromboxane A2, prostaglandin F2a and leukotrienes are pro-inflammatory (Whaley and MacSween, 1992) . Many anti-inflammatory drugs act by modulating the formation of these eicosanoids or by blocking their action on the target cells. In this study cysteinyl-leukotrienes and thromboxanes are the pro-inflammatory eicosanoids that were measured in response to treatment with the plant extracts. 13 University of Ghana http://ugspace.ug.edu.gh Leukotrien&s The LTs, which mediate acute inflammatory reactions are formed from AA in leukocytes (Hardie, 1991; Sammuelsson et al., 1978). Their biosynthesis has subsequently been demonstrated in other bone marrow-derived cells that express the 5-lipoxygenase including eosinophils, mast cells, and macrophages. Leukotrienes are commonly found in vascular tissues of the lung, heart and platelets. They are synthesized via a lipoxygenase, which catalyze direct oxidation at the 5-carbon of AA to form a hydroxyl peroxy fatty acid intermediate (Fig. 1.1) . In the synthesis of LTs, the first product formed is 5-hydroperoxyeicosatetraenoic acid (5-HPETE). 5-HPETE is converted to a monohydroxyeicosatetraenoic acid (5-HETE) by a peroxidase or to the 5, 6-epoxide also known as leukotriene A4 (LTA4) by a dehydratase. Aspirin and other NSAIDs inhibit the formation of 5-HETE. LTA4 is transformed into 5,12- dihydroxyeicosatetraenoic acid (LTB4) by a hydrolase or into a glutathione adduct with the formation of a thioether linkage at carbon-6 by a glutathione-S-transferase to give LTC4 as the product. LTD4 results from the removal of the glutamyl residue from LTC4, while LTE4 results from the removal of glycyl residue from LTD4. The addition of a glutamyl residue to LTE4 results in the final product LTF4 (Bhagavan 1992). 14 University of Ghana http://ugspace.ug.edu.gh Fig. 1.1: Lipoxygenase pathway of arachidonic acid metabolism for leukotriene synthesis COOH Aracliidonic Acid Lypoxygenase I OOII c c 5-1tPETE Ueltydia-f ^ \ ^ J X ^ COOM ,CsHii Lp iiko lrio n n A ,| (I T A 4 ) IIjO COOH LeukoU iene " ^ (! T B ^) Y-GIu Leukolnene C 4 (L., ■„ Glutamyl trans peptidase HO „H , •• COOH LTD 4 11 V Cys-Gly Di peptidase I s and HE! Es other than the 5 derivatives aie also produced (not shown) 1 University of Ghana http://ugspace.ug.edu.gh The cysteinyl-leukotrienes ( LT C4 , LTD4 and LTE4) are potent bronchoconstrictors in several species, including humans, with specific effects on the peripheral airways (Piper and Tippins, 1982). They are involved in several immune-mediated inflammatory reactions of anaphylaxis and are constituents of substances originally referred to as "slow-reacting substance of anaphylaxis" (SRS-A). L T C 4 and LTD4 have been shown to mimic the effects of SRS-A when injected intradermally into guinea pigs (Samuelsson, 1983). They contract smooth muscle and affect vascular permeability. LTs, which are important mediators of the inflammatory processes, are more potent than histamine in constricting airways and promoting formation of tissue edema (Bogie et al., 1998; Bhagavan 1992). Inhibition of the 5-lipoxygenase reaction that leads to the formation of LTs, can therefore be used in the therapy of inflammatory diseases (Higgs et al., 1984). The biological activity of LTE4 is much lower in most systems, but its presence reflects the prior existence of LTC4 and LTD4, which are inactivated by conversion to more polar metabolites. LTC4 and LTD4 can also be subjected to co-oxidation (Higgs et al., 1984; Samuelsson et al, 1978). Cysteinyl-leukotrienes can accumulate to relatively high concentration in the effusion fluids such as synovial fluids, pleural effusions, and pericardial or intraventicular 16 University of Ghana http://ugspace.ug.edu.gh aspirates that are associated with inflammation. Since LT metabolism is incomplete in these circumstances, substantial amounts of LTC4, LTD4, and LTE4 may be present. For example, bronchoalveolar lavage fluid from asthmatic subjects may contain 700-1000pg/mol cysteinyl-leukotrienes comprising mainly LTC4 and LTD4 (Westcott et al., 1990). Consequently, analysis of such fluids could be a useful assay for the evaluation of putative anti-inflammatory plant materials. Thromboxanes The cyclooxygenase enzyme complex catalyses oxidative reactions leading to the formation of prostaglandin H2 (PG H2) , a cyclic endoperoxide with the characteristic 5-membered ring structure of the PGs (Hardie, 1991). Thrombaxanes (TXs) are synthesized by microsomal thromboxane synthases from prostaglandin H2 via isomerization reaction (Smith 1992; Hardie, 1991). Thromboxanes were first isolated from human and equine thrombocytes (platelets), which have been shown to be the major source of TX (Moore et al. , 1991). Thromboxane A2 has a very short half life (Ti/2 =30s at 37°C and pH 7.5). It undergoes rapid non-enzymatic hydrolysis to produce the inactive TXB2 (Bhagavan, 1992). The cyclooxygenase pathway for eicosanoid biosynthesis is shown in Fig. 1.2. 17 University of Ghana http://ugspace.ug.edu.gh Thromboxane A2 has an oxane:oxetane structure. It derives its name from both its structure and origin, thrombocytes. It is a potent inducer of platelet aggregation, platelet release reaction (Ali and Mohammed, 1986; Hamberg et al., 1974) and smooth muscle contraction (Samuelsson et al., 1978) . In fact, rabbit aorta contracting substance (RCS), which was originally believed to be a PG endoperoxide has been shown to consist of mainly TXA2 and rather low levels of PGH2, the intermediate (Heptinstall et al., 1987; Hamberg et al., 1974) . Thromboxane A synthase, present in the endoplasmic reticulum is abundant in the lung and platelets (Glew, 1992; Moore et al., 1991). TX production has been demonstrated in platelets, lungs, spleen, polymorphonuclear leukocytes, brain and inflammatory granuloma (Sameulsson et al., 1978). Cultured vascular endothelial cells (Ingerman-Wojenski et al., 1981) as well as intact blood vessels (Salzma et al., 1980) are also capable of synthesizing TX. Vascular synthesis of TXs may thus contribute to platelet aggregation and vasoconstriction. In an experiment involving the formation of rabbit aorta contracting substance and platelet aggregation factors, Needleman et al. (1977) reported that although PGHi, PGH2 and PGH3 were formed by sheep vesicular glands, only PGH2 and PGH3 were converted to TX by platelet microsomes. 18 University of Ghana http://ugspace.ug.edu.gh Thromboxanes have been implicated in the alteration of microvascular permeability from acute lung injury (Sprangue et al., 1992). Recently, Sprangue et al. (1992) reported that the TX synthase inhibitor, OKY-04 6 prevents pulmonary edema induced by phorbol myristate acetate (PMA). TXs are important in alcoholic liver injury and inhibition of their production is accompanied by amelioration of liver injury. Nanji (1993) has demonstrated that compared to feeding with saturated fat and ethanol, liver nonparenchymal cell production of TXB2 and leukotriene B4 was higher in rats fed corn oil and ethanol. Feeding rats with corn oil and ethanol caused liver injury and the pathologic changes correlated with plasma levels of TXB2. Thromboxanes may also be important in synovial inflammation and certain destructive events in arthritis (Hayes et al., 1994). This stems from the observation that the synthesis of the key metabolites in these conditions, 1,25- dihydroxyvitamin D3 is increased by thromboxanes, leukotrienes and interferon gamma - a macrophage activating factor secreted by activated T-lymphocytes (Hayes et al., 1994) . 19 University of Ghana http://ugspace.ug.edu.gh Fig. 1.2: Cyclooxygenase pathway for prostanoid biosynthesis / S coot: V - / AA /' " K, c:» o oonr errors (y1 r/ \ / COO I, o OOM PUG* Oil \IV 0 PFROXCVVSP 6-oxo-P G c^ ♦ \ ’' 'v—/ X ] \ CV?hydrogennsi» a U-6K)M0OXAN£A SYNFmSE . , . FRTYJNOFSUTACYC.UN/ flSG / s/^vA OH TXAr U N / hy o (iIfi oelyn Sz'vSm OH k g i2 / CXX) (Prostacyclin) on j Nun-enzym_c / hydrolysis TXFJj / ROS7AGLANON P / SYNTH ASE^X/S^COO / 2NAIJPH/ / ^TTO SsTYANGitAmMseO N D l rrtosmGLANaNr - Oh / 6-oxo-PGF-j, 2NADP Oil \ K IK) Oil OM r>GE, PGF*, pen* , rTCOST/\GLANl»l e, * NADPH 9-REDUCrASE NA!»' 20 University of Ghana http://ugspace.ug.edu.gh It has long been established that the PG endoperoxides are converted to only a minor extent to stable PG, PGE2, and PGF2a in certain tissues and cells such as platelets and lung tissues (Hamberg and Samuelsson, 1974) . Instead the major part of the endoperoxide is converted into the highly unstable TXA2, which is subsequently converted into the stable but inactive TXB2 (Samuelsson et al., 1978). Thus, in order to get a reliable picture of biochemical events in these cells, it is necessary to monitor TXs instead of PGs. Methods for the Estimation of Eicosaniods Enzyme immunoassay (EIA) and radio-immuno-assay (RIA) have been developed for eicosanoids and their analogues including thromboxanes and TX derivatives. Biological fluids that have been employed for analyses include blood plasma/serum and urine. Tissue and isolated cells have also been analyzed. The sensitivity of EIA and RIA has increased greatly during recent years. Recently produced antibodies often have higher avidity and specificity, which may be explained by the use of more suitable carrier molecules for preparation of the conjugates, the use of better coupling methods, as well as the use of appropriate species for antibody production (Hubbard and Gould, 1988; Blake and Gould, 1984). 21 University of Ghana http://ugspace.ug.edu.gh RIA and EIA assays have been developed for the determination of TXB2 in various minute samples and is thus suitable for detailed kinetic studies such as the events occurring during platelet aggregation. TX measurements are conveniently carried out in vitro, either in platelet experiments or in perfusion studies of various organs (Samuelsson et al., 1978) . The difficulty of measuring TXB2 in vivo stem from artifactual formation of the compound of interest during sampling, which does not allow 'peripheral plasma levels' of TXA2 to reflect the true endogenous circulating amounts of the compound. In the case of PGs, this problem is solved by monitoring the 15-keto-13, 14-dihydro metabolites instead. These are not formed artifactually during sampling and reliably reflect endogenous levels. In the case of in vivo measurements of TXs, no major circulating metabolites of the compound seem to exist (Samuelsson et al., 1978). Anti-oxidants Reactive oxygen species (ROS) are involved in the pathogenesis of a number of diseases including rheumatoid arthritis, arteriosclerosis, skin aging, nephritis, diabetes mellitus and asthma (Florence, 1995; Cerutti, 1994; Stadman and Oliver, 1991; Stadman, 1990;Steingberg et al., 1989). 22 University of Ghana http://ugspace.ug.edu.gh ROS include superoxide ion, singlet oxygen, and hydroxyl radical, which are generated from exogenous factor or as byproducts of biological reactions (Cerutti, 1991). These free radicals characteristically possess one or more unpaired electrons. Superoxide radical is formed in small amounts as oxyhemoglobin is converted to methemoglobin, and a small percentage of the electrons passing down the mitochondrial electron transport chain leak directly into oxygen (02) , thereby producing O2'-. Also, during the inflammatory process, inflammatory cells such as mast cells, macrophages, eosinophils and neotrophils become activated by a number of stimuli and produce superoxide radicals during the respiratory burst. This potentially dangerous free radical is acted on by superoxide dismutase and converted to hydrogen peroxide, another ROS, which is removed by catalase and glutathione peroxidase. Hydrogen peroxide can also react with free iron or copper to from hydroxyl radicals. Iron is normally tightly bound to ferritin and transferrin and copper is usually bound to ceruloplasmin. Under conditions of chronic inflammation and tissue damage on the airways, iron is liberated and can catalyze the production of highly reactive hydroxyl radicals, further exacerbating the inflammation and airways tissue damage (Chabot et al., 1998; Barnes, 1990; Heffner and Repine, 1989). Aerobic bacteria produce ROS, and those that are catalase negative can be 23 University of Ghana http://ugspace.ug.edu.gh another source of chronic exogenous hydrogen peroxide in infected airways (Gabridge et al., 1985) There are systems, both enzymatic and non-enzymatic, which are available within the airway epithelial lining fluid as local anti-oxidant defenses. These include Cu Zn, superoxide dismutase and reduced glutathione (Maynard et al., 1992). The effect of the ROS on the airways depends on the availability of these local anti-oxidant defenses. It is known that the respiratory tract anti-oxidant capacity is altered in mild stable asthma (Kelly at al., 1999). Therefore a search for natural anti-oxidants and other preparation of plant origin is highly commendable. Asthma as an Inflammatory Disorder. Asthma is a clinically defined condition marked by recurrent, discrete episodes of reversible bronchial narrowing, separated by periods in which ventilation approaches normality (Green, 1999). It is an inflammatory disease that involves mast cells, airway epithelium, eosinophils, and neutrophils. These cells produce the broad array of mediators and cytokines that cause bronchoconstriction, mucosal edema, mucus secretion and bronchial hyper­ responsiveness that characterize asthma (Lazarus, 1998). 24 University of Ghana http://ugspace.ug.edu.gh Current guidelines for asthma therapy recommend that all patients whose asthma is more severe than mild intermittent receive chronic treatment with drugs that interrupt the inflammatory cascade mentioned above. There is established and growing evidence that asthmatic attacks are also inflammatory responses. Most pharmacological agents used in the management of asthma, for a example, theophylline (a bronchodilator in acute and chronic asthma) and corticosteriods, have anti-inflammatory properties (Minoguchi et al., 1998). Again, asthmatic airways are infiltrated with inflammatory cells that release mediators and cytokines into the microenvironment. It is believed that this inflammatory cell density in peripheral airways in severe asthma may relate to the peripheral airway obstruction that is characteristic of this condition (Maclean et al., 1999; Haley et al., 1998). In fact Takami and Tsukada, (1998) reported that a marked and sustained bronchoconstriction occurs after antigen challenge in actively sensitized guinea pigs, and the bronchoconstriction observed correlated with increased TXA2 levels in both the plasma and bronchoalveolar lavage fluid (Takami and Tsukada, 1998) . , An inhibitor of TX synthase 25 University of Ghana http://ugspace.ug.edu.gh (DP-1904) was shown to attenuate TXA2 levels and the bronchoconstriction in a dose-dependent manner. In platelet-depleted animals, antigen-induced broncho constriction and TXA2 release in the plasma were significantly reduced compared to those of non-platelet - depleted animals, a further indication that platelets are a major source of TXA2 production. Leukotrienes, which are lipoxygenase products of AA metabolism, play an important role in inflammatory reactions (Bogie et al., 1998) and this has been well studied in bronchial asthma. Sulfidopeptide leukotrienes ( LTC4, LTD4 and LTE4) are potent bronhoconstrictors that produce mucous secretions and alter vascular permeability (Bogie et al., 1998; Drazen, 1998). They also participate in the inflammation process as well as in early and late asthmatic responses and the administration of specific leukotriene receptor antagonist or leukotriene synthesis inhibitors ameliorates the symptoms and signs of bronchial asthma (Bogie et al., 1998). The foregoing indicates that asthmatic attacks have similar pathogenesis as other inflammatory disorders. It is in this light that inflammation is emphasized in the management of persistent asthma (Szefler and Nelson, 1998), with anti­ 26 University of Ghana http://ugspace.ug.edu.gh inflammatory agents recommended as medications for primary long-term control of the disease (Szefler and Nelson, 1998). One such long-term preferred medication is inhaled corticosteroids (Jatakanon et al., 1998). However recent report indicates that asthmatic patients who rely on long­ term, high-dose corticosteroid therapy are at increased risk for osteoporosis (Niewoehner and Niewoehner, 1999). Again, available data provides evidence to show that the cost involved in the therapy of asthma is substantial all over the world. In Switzerland, for instance, this amounts to approximately 1,200 million Swiss France (CHF) per year (Szucs et al., 1999). In Ghana, the cost of drugs for the management of asthma is beyond the means of many asthmatic patients who therefore turn to self-medication usually with plant medicines. At the Center for Scientific Research into Plant Medicine (CSRPM) in Ghana, the extract of various plants, e.g. Desmodium adscendens, Thonningia sanguinea and Parquetina sp., also known as Tina 'A' are used to treat asthmatic patients who visit the out-patient clinic of the Center. In the case of Tina A, it is reported that in some instances this plant extract provides immediate relief from the symptoms of asthma (Robertson, Personal communication). Scientific investigation into this and other local tl University of Ghana http://ugspace.ug.edu.gh preparations that are used in the management of asthma and other inflammatory disorders could lead to the discovery of locally affordable, efficacious and safe plant medicines that can substitute the imported and expensive orthodox drugs. In view of the fact that eicosanoids such as TXs and LTs mediate both asthmatic and other inflammatory disorders, and the fact that anti-inflammatory and anti-asthmatic agents modulate eicosanoid metabolism, assays involving the release and/or synthesis of these eicosanoids can be used to evaluate the efficacy of herbal preparations that are used to manage asthma and other inflammatory disorders. Medicinal Plant with Anti-inflammatory Effects Thonningia sanguinea vahl (Balanophoraceae) : This is one of the plants in a herbal preparations used prophylactically against bronchial asthma at the Center for Scientific Research into Plant Medicine. The powdered dried roots of T. sanguinea are used in combination with the stem or leaves of Desmodium adscendens for the management of asthma. The extracts of the plant were found to be anti-anaphylactic when given to guinea pigs orally. These experiments showed the extracts of T. sanguinea caused a decrease in the release of histamine and mepyramine-resistant spasmogens, inhibited 28 University of Ghana http://ugspace.ug.edu.gh anaphylactic contractions in isolated ileal pieces, and reduced the sensitivity of ileal pieces to exogenous histamine (Nyarko and Addy, 1994; Addy and Nyarko, 1985). The extracts of this plant are reported to have a scavenging action for the stable free radical 1,l-diphenyl-2- picrylhydrazyl (Gyamfi et al., 1999). Desmodiun adscendens Sw. DC var. adscendens (Papilionaceae) : The extract of Desmodium adscendens has been used by herbalist and studied to some extent by some scientists. According to Ampofo (1977), the plant material is useful against poor lactation and dysmernorrhoea. There are reports that the extract of the plant is used to treat ringworm, constipation, convulsion (Ayensu, 1978), urinary problems, abdominal tumors and catarrh (Ayensu 1978) . At the CSRPM in Ghana, aqueous decoction from the stem and leaves of the plant is used to manage asthma. Preparations from the roots are used as cough mixtures and also applied on wounds and abscesses. Extracts of this plant have been found to reduce anaphylactic contractions and interfere with histamine- and antigen- induced contractions of smooth muscle. It also reduces the amounts of histamine released from lung tissue in a dose- dependent manner (Addy and Dzandu, 1986; Addy and Awumey, 29 University of Ghana http://ugspace.ug.edu.gh 1984). it was found to activate the cyclooxygenase enzyme and increase the synthesis of the anti-inflammatory prostanoid, PGE2 (Addy and Schwartzman, 1995) and also inhibited NADPH-dependent oxygenation of arachidonic acid and AA-induced contractions of smooth muscles (Addy and Schwartzman, 1992). Several chemical constituents have been isolated from aqueous extract of different species of D. adscendens. Some of these are N, N-dimethyltyramine, N, N, dimethyltryptamine, salsoline (Asante-Poku et al., 1988), triterpenoid glycosides dehydrosoyasaponin I (DHS-I), soyasaponin I, soyasaponin II and soyasapogenol B and E (McManus et al., 1993). Tenacetum parthenium: The extract of this plant has been used in the treatment of migraine, an inflammatory disease resulting from vasospasm and dilation of intra-cranial arteries and their branches resulting from the intermittent release of hydroxytryptamine and prostaglandins (Their and Smith, 1981). Dioscores sp. (the yam family): Steroidal compounds with anti-inflammatory properties have been identified in many plants of this species. These include cortisone and hydrocortisone, which inhibit phospholipase A2 and hence block AA mobilization in eicosanoid biosynthesis. 30 University of Ghana http://ugspace.ug.edu.gh Lippia multiflora Moldenke (Family Verbanaceae) ; Syn. Lippia adoensis Hochst.; Lippia gxandifolia Hochst. ExWalp: L. multiflora, also known as Power Tea (Noamesi, 1977) is a herbaceous savannah plant widely distributed in West Africa (Chanh et al., 19881; Noamesi et al., 1985) . It is used on a large scale in the African folk medicine. In Ivory Coast, infusion of L. multiflora is used as for the treatment of hypertension (Chanh et al., 19882; Noamesi et al., 1985). In Ghana some people in villages hang the dried leaves of the plant in bedrooms and the characteristic aromatic scent that emanates from the leaves is said to repel mosquitoes (Noamesi et al., 1985). Leaves of the plant have also been reported to have anti-malarial, anti-viral and anti-fungal effects (Taboubi et al. , 1997; Benoit et al., 1996; Valentin, 1995). The total phenolic extract from L . multiflora was found to have a dose-related inhibition of the biosynthesis of TXA2 (Chanh et al., 19882) , suggesting an anti-inflammatory action of the plant. This anti-thromboxane synthase activity may be the major contributor to the hypotensive effect of the plant. Euphorbia hirta Linn. (Syn Euphorbia pilulifera Linn) also known as Australian asthma herb, Queensland asthma weed or Cat's hair. It is referred to as 'Nimakoa' (in the Twi language) in Ghana. It is a common weed in towns and 31 University of Ghana http://ugspace.ug.edu.gh villages near drains, roadsides and waste places. Chemical compounds that have been isolated from E. hirta include diterpenes (phorbol esters), triterpenes, flavonoids, hydrolysable tannins, aromatic acids, alkaloids, coumarins and anthocyanidins. It is widely used as anti-asthmatic and anti-spasmodic and in some communities it is used against bronchitis, dysentry and amoebiosis (Ayiku, 1992). 32 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO MATERIAL AND METHODS MATERIALS Guinea pigs Male guinea pigs weighing between 150 and 350g initially were initially purchased from local breeders and quarantined in large cages for at least 14 days prior to their being used for the study. The animals were maintained on a standard laboratory diet, obtained from the Tema Food Complex Ltd, Tema, Ghana, and daily rations of fresh grass (Panicum maximum). Drinking water, which was occasionally supplemented with vitamin C, was freely provided. The animals were kept in two main groups, A and B. Plant Materials The crude powdered product of Parguetina sp. (Tina A) was obtained from the Center for Scientific Research into Plant Medicine (CSRPM), Mampong-Akuapem, Ghana. Roots of Cryptolepsis sanguinolenta and leaves of Lippia multi flora were supplied as dried powdered material by Phyto-Riker (GIHOC) Pharmaceuticals, Ghana. Freeze-dried products of Thonningia sanguinea and D. adscendens were obtained from the NMIMR, Legon. 33 University of Ghana http://ugspace.ug.edu.gh Chemicals and Reagents Biorad reagent was obtained from BIORAD Life Sciences Group, CA, U.S.A. Cysteinyl-leukotriene (Cat # 520501) and thromboxane B2 (cat # 519031) immunoassay kits were obtained from Cayman Chemical Company, Ann Arbor, U.S.A. Reduced glutathione (GSH), Bovine Serum Albumin (BSA) and arachidonic acid (AA) were obtained from Sigma Chemical Company, St. Louis, MO. USA. Other chemicals used were of analytical grade and obtained from Fluka Chemie, Switzerland. METHODS Preparation of Plant Extract a) For pretreatment of guinea pigs Amounts of plant materials used and volumes administered to the guinea pigs were based on the dosage administered to humans by CSRPM. Approximately 9.4 g of the powdered product of Parquetina sp. (Tina A) was boiled in 150ml of tap water for 2 minutes. The mixture was strained and the solution stored in a refrigerator until needed. The extract which was prepared and stored this way was warmed to room temperature prior to its administration to the animals and used within five days after which the remainder was discarded. 34 University of Ghana http://ugspace.ug.edu.gh b) For perfusion studies Four grams of the powdered product of 'Tina A' and T. sanguinea were boiled in 20 ml of water for 2 minutes and strained. The extract was always freshly prepared. c) For the in vitro effect on arachidonic acid metabolism Pre-determined amounts of the plant material (Tina A' , L. multiflora and C. sanguinolenta) were boiled in distilled water such that concentration of the extract was 0.3g/ml. This was kept as the stock solution. For D. adscendens and C. sanguinea a solution of 100ijg/ml was prepared with the freeze-dried material. All extracts were kept at 4°C for not more than three days. Appropriate dilutions were made with potassium phosphate buffer (pH 7.4) at the time of the experiment. Pretreatment of Animals Group A) : Animals in this group were pre-treated with the extract of 'Tina A' as prepared above. The extract was administered orally using graduated syringes at a dose of 8ml/kg body weight daily. After fifteen days the animals were sensitized by injection with 2ml (1ml intra-peritoneal and 1ml sub-cutaneous) of egg albumin (lOOmg/ml) as antigen in normal physiological saline. This group of guinea pigs was sub-divided into two. Extract administration was continued in 35 University of Ghana http://ugspace.ug.edu.gh one group (TT) while it was terminated in the other (TS) . Eight days following the first sensitization, the animals were injected with a booster dose of the same antigen (5mg/ml) as described above. Group B The guinea pigs (18 for A and B) in this group without pre­ treatment with the extract were sensitized with egg albumin, (lOOmg/ml, 1ml i.p. and 1ml s.c.) at the same time that those in group A were sensitized. Each animal received a booster dose of 5mg/ml egg albumin 8 days following the initial sensitization as described for group A. Preparation of Isolated Organ Fourteen days following the second injection with the antigen, each of the guinea pigs (now 300 to 500g-body weight) was anaesthetized with chloroform. The trachea was canulated following mid-thoracotomy after which the lungs were removed and suspended in a vessel. The lungs were washed of blood and equilibrated by perfusion via the trachea with warm (37°C) Kreb's solution, gassed with 95% 02, 5% C02, at a rate of 2.0 to 2 .2ml/min for 10-12 min. 36 University of Ghana http://ugspace.ug.edu.gh Sampling of Lung Perfusate After the equilibration period (about 10-12 min.), lungs removed from pre-treated and non-pretreated guinea pigs were infused with 0.6ml egg albumin (lOOyg/ml) through the Kreb's stream for 3min.at approximately 0.2ml/min. The lung effluent for the first three minutes was discarded. The perfusate collected between the 4th and 5th minutes was pooled for the estimation of pro-inflammatory eicosanoids by enzyme immunoassay. This was based on previous observation by Serios et al, (1987) and de Nucci et al. (1986) that maximum concentration of eicosanoids under these conditions were reached in the perfusate 4 to 5 minutes following anaphylactic reaction. Isolated lungs used to investigate the ex vivo effects of the extract were treated as follows: 1ml extract of 'Tina A' was infused (~0 .2ml/min) into the isolated organs 10 minutes before antigen challenge. During the antigenic challenge, as described above, 0.5ml of the extract was continuously given as infusion into the lung. The lung perfusate collected between the 4th and 5th minutes were pooled for the estimation of pro-inflammatory eicosanoids, TXB2 and cysteinyl leukotrienes. After sampling for the 'Tina A' , the no pretreatment (NPT) lungs were washed for ten minutes to get rid of excess 'Tina 37 University of Ghana http://ugspace.ug.edu.gh A' in the organs. The effect of T. sanguinea on the release of TX B2 was evaluated. One set of lungs was again challenged with 0.6ml of the antigen (0.2ml/min for 3 minutes) and the lung perfusate was collected between the 4th and 5th minutes. Another set of lungs was perfused with 1.0 ml of the solution of T. sanguinea ten minutes before the antigenic challenge. During the antigen challenge, 0.5ml of the extract was continuously given as infusion into the lungs. The lung perfusate was sampled again at the 4th and 5th minutes for the estimation of TXB2. Preparation of Microsomes The method of Needleman et al. (1976) was used with the following modification. Two liters of whole goat blood was collected in 3.8%w/v trisodium citrate (TSC) to achieve a final concentration of 10% TSC. The mixture was spun at 200g for 10 min. at 4°C to remove red blood cells. The platelet rich plasma (top 2/3 fraction) was separated and centrifuged at 2000g for 15 min. at 4°C. The pellet of platelets was re­ suspended in 20ml of Tris-Cl buffer (lOOmM, pH 7.5). The platelet suspension was subjected to three rounds of freezing and thawing to disrupt the membranes. The disrupted platelets were centrifuged at 5000g for 15 min. at 4°C using a high speed refrigerated centrifuge (Model 20PR-52D, Hitachi Koki Co., Japan) to remove cell debris. The supernatant was 38 University of Ghana http://ugspace.ug.edu.gh centrifuged at 100,000g using an automatic preparative ultracentrifuge (Model 80P-7, Hitachi Koki Co. Ltd., Japan). The resultant pellet was suspended in 2ml of lOOmM Tris-Cl, pH 7.5 and homogenized. The pellet was re-suspended in 6ml Tris buffer, and divided into aliquots and stored at -80°C. Protein Determination: The Bradford method for protein determination was used (Bradford, 1976): Five serial dilutions of a protein standard BSA were prepared with concentration range between 0 and lmg/ml. A volume of lOOjil of each standard solution and sample to be determined (dilutedl in 50 dilution) was pipetted into clean dry test tubes. Five milliliters of diluted (1:4) dye reagent (Coomasie Brilliant Blue) was added to each tube and vortexed. All samples were incubated at room temperature for eight minutes and the absorbances were read at 595nm. The protein concentration of the sample was estimated from a standard curve generated with the protein standard. Effect of Plant Extracts on the in vitro Synthesis of Pro- inflammatory Eicosanoids: The total of 1ml reaction mixture used consisted of 0.86mg/ml microsomal protein, 0. 5mM GSH (freshly prepared), and 15]_iM AA (freshly prepared) in 0. 1M potassium phosphate buffer, pH 39 University of Ghana http://ugspace.ug.edu.gh 7.4. The mixture was kept in a refrigerator prior to use. It was then placed in a water bath maintained at 37°C for at least 2 minutes and the reaction was initiated by the addition of lOOuL AA (15uM in 0.1M phosphate buffer, pH 7.4). For the test reaction, the mixture was pre-incubated with the plant extract (0.05 to 100 yg/ml) for lOmin before initiating the reaction. The reaction was terminated after 5 minutes by the addition of lOOyl of 1M citric acid to bring the pH below 3. The terminated reaction mixture was stored in a refrigerator and the amounts of eicosanoids present were determined within four hours. The pH of the mixture was adjusted to slightly alkaline range by the addition of lOOpl concentrated NaOH solution just before use. The extracts evaluated were 'Tina A', L. multi flora, D. adscendens, C. sanguinolenta, and T. sanguinea. Quantitative Estimation of Eicosanoids An ELISA was used for the estimation of the eicosanoids. The reaction mixture was composed of the eicosanoid released or synthesized (X) and X-acetylcholinesterase complex (known as the tracer) for a limited number of X-specific rabbit antiserum binding sites. The concentration of the tracer was held constant while the concentration of free X was varied. The complex between the X or tracer and the specific rabbit 40 University of Ghana http://ugspace.ug.edu.gh antiserum then bind to mouse monoclonal anti-rabbit antibody that had been previously attached to the well. After washing the wells once with wash buffer, 50ul of tracer and 50pl of antibody were put into each well. Fifty micro­ liters of sample (lung perfusate or in vitro reaction mixture) were added to each well. All samples were assayed in duplicates. The plates were covered with a plastic film and incubated for 20 hours at room temperature. The wells were washed five times with wash buffer to remove any unbound reagent. Exactly 200ial of Ellman's reagent containing acetylcholine, the substrate for the acetylcholinesterase, were put into each well and incubated for 90 - 120 minutes at room temperature in the dark. The product of the reaction gave a yellow color, the intensity of which was measured at 405 - 450nm. The intensity determined this way is inversely proportional to the amount of free X contained in the samples analyzed. Concentrations of eicosanoids were calculated using software provided by Cayman Chemical Company, USA. The eicosanoids estimated were TXB2 for all plant extracts evaluated and cysteinyl leukotrienes for only 'Tina A' and T. sanguinea. 41 University of Ghana http://ugspace.ug.edu.gh Anti-oxidant Properties of the Herbal Plant Extracts Effect on hydroxyl radical generation Hydroxyl radicals were generated according to the method of Suarez et al. (1998). Ascorbic acid is oxidized with Fe3+ and hydroxyl radicals generated combine with methyl groups donated by DMSO to form formaldehyde. Approximately 0.375g of the freeze-dried extract of Desmodium adscendens and Thonningia sanguinea were dissolved in 10ml of lOOmM potassium phosphate (PB) buffer (pH 7.4). Appropriate pre-determined amounts of powdered product of Parquetina sp.and Lippia multiflora were boiled in the buffer for two minutes such that the concentration of the extracts would be approximately 0.38g/ml. The solutions were strained using filter paper. All solutions of extracts were stored at 4°C until used. For concentration dependent assays, appropriate volumes of the extract prepared as described above were added to the buffer such that the total volume was 300)il. Mannitol was used as a standard inhibitor at a concentration of 3.0 mg/ml reaction mixture. The control was made up of just 300]jl of PB buffer. Blanks were prepared for each of the assays, that is, controls, mannitol and each of the concentration of the extracts. Exactly 250pl of Fe3+-EDTA mixture and 50)jl DMSO (1.771ml of DMSO diluted to 50ml with PB buffer, pH 7.4) were added to each tube. 250pl of trichloroacetic acid (TCA, 42 University of Ghana http://ugspace.ug.edu.gh 1.75g dissolved in 10ml of distilled water) were added to each of the blanks and vortexed before initiating the reaction. The reaction was started by the addition of 150vil of freshly prepared ascorbic acid (1.7 61mg/ml in PB, pH 7.4). Each tube was incubated at 37°C for 3.5 hours. The reaction was stopped by the addition of 250pl of TCA. One ml of freshly prepared Nash (1953) reagent (made up of 15g ammonium acetate, 0.2ml acetylacetone and 0.3ml acetic acid made up to 100ml with distilled water) was added to each of the reaction mixtures and each tube incubated for 45 minutes at 37°C. Absorbance was immediately read at 412nm. Results were expressed as percentage inhibition of formaldehyde formation in the presence of the extract: i.e. (ABScontroi - ABStest) /^BScontrol ̂ 100. Quantitative Determination of Total Phenols Total phenolic content was determined by the method of Lowman and Box (1983) with the following modifications. Concentrations of 0.3g/ml plant extract solutions were made with distilled water. A volume of 83.6iil of this solution was made up to 25ml with 50% v/v methanol giving a final concentration of 0.lmg plant extract solution. The solutions were boiled for 3 minutes and made up to 25ml with 50% v/v methanol. Five millilitres of each solution was diluted to 25ml with de-ionized water. Successive addition of 1.50ml of 43 University of Ghana http://ugspace.ug.edu.gh Na2C03 (200g/l) and 0.50ml 2N Folin-Ciocalteu reagent was made to each mixture and absorbance at 750nm read after one hour. Catechin was used as the standard and the amounts of phenols expressed as mg catechin equivalents per gram plant extract. Quantitative Determination of Water Soluble Phenols Plant extracts and catechol (0.4mg/ml) were prepared in 50% v/v methanol and allowed to stand at room temperature for 24 hours. The solutions were centrifuged at 3500rpm for 5 minutes and filtered with a filter paper. A volume of 1.50ml Na2C03 and 0.5ml of 2N Folin-Ciocalteu's reagent were added to each solution and left at room temperature for one hour. Absorbance was read at 750nm. The content of water-soluble phenols were calculated as mg catechin equivalents per gram plant extract. Statistical Analysis The student t-test or analysis of variance were used for the statistical analysis in the report. Statistical significance was calculated at p ^ 0.05. 44 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE RESULTS A: Release of Eicosanoids Effects of 'Tina A' The effects of Tina A on the release of thromboxane B2 (TX B2) and cysteinyl leukotrienes (cyst.LT) during anaphylaxis in guinea pig lungs are presented in Figs 3.1 and 3.2. Fig. 3.1 shows the effects of Tina A on the release of TXB2. In these figures NT denotes the group of animals that were not treated with the extract. TS refers to the group of guinea pigs that were pre-treated with the extract before sensitization, while TT applies to the group that were given the extract before sensitization and throughout the experiment. The figure shows that the highest amount of TXB2 was released in the NT group. Within this group the amounts of TXB2 released from lungs with and without extract infusion were significantly different (p = 0.05). Infusion of the extract into the isolated lungs caused 25.4% decrease in the amount of TXB2 released. Amounts of thromboxanes released from the TS set of guinea pigs were lower than the NT group. Among these animals, infusion of the extract caused a 45 University of Ghana http://ugspace.ug.edu.gh 600 500 1 N2 400 (D p ■S 300 1 | §o 200