QP752. A7 D12 blthr C.1 G364642 3 0692 1078 5896 9 University of Ghana http://ugspace.ug.edu.gh MODULATION OF EICOSANOEDS BIOSYNTHESES IN VIVO AS A MECHANISM-BASED EVALUATION OF PUTATIVE ANTI-INFLAMMATORY PLANT EXTRACTS BY JOHN D ADZIE-MEN S AH JUNE, 2000 University of Ghana http://ugspace.ug.edu.gh I achieve nothing if I live for myself but I achieve ALL if I live for the truth and social justice, And love, and serve my God and humanity in all sincerity. JOHN DADZie-MCNSAH University of Ghana http://ugspace.ug.edu.gh MODULATION OF EICOSANOEDS BIOSYNTHESES IN VIVO AS A MECHANISM-BASED EVALUATION OF PUTATIVE ANTI-INFLAMMATORY PLANT EXTRACTS A THESIS SUBMITTED BY JOHN DADZIE-MENSAH IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A MASTER OF PHILOSOPHY DEGREE DEPARTMENT OF BIOCHEMISTRY FACULTY OF SCIENCE UNIVERSITY OF GHANA LEGON JUNE, 2000 University of Ghana http://ugspace.ug.edu.gh DECLARATION The experimental work contained in this thesis was carried out by me in the laboratories of the Department of Biochemistry and the Noguchi Memorial Institute for Medical Research both of the University of Ghana, and the findings therefrom, my exclusive contribution to the science of plant medicine. JOHN DADZIE-MENSAH (Student) i i .2 - p . A i ........ DR. ALEXANDER KWADWO NY ARK O PROF. MARIAN EWURASt^ADDY (Co-supervisor) (Supervisor) University of Ghana http://ugspace.ug.edu.gh DEDICATION TO M Y PARENTS University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT To the glory of the living God! I wish to express my heartfelt appreciation to all who helped in diverse ways to making this piece of work a success. I am most grateful to my supervisors; Prof. Marian Ewurama Addy and Dr. Alexander Kwadwo Nyarko, whose guidance, patience and respect encouraged me to go through this exercise successfully. I could not have enjoyed working in the laboratory better at the near point of exhaustion without the company of Maame Aba Coleman and Kisha Green from the Duke University, U.S.A., who, on an exchange programme, worked in the same laboratory. I am so grateful to my family for the encouragement, love, prayers and financial support. I must also express my profound gratitude to the Association of African Universities for the award of a grant that contributed, in no small way, to the successful completion of this thesis. To the management and staff of the Green Earth Organization I say, God richly bless you for allowing me the full usage of your electronic facilities, without which this work University of Ghana http://ugspace.ug.edu.gh would have been completed with an unprecedented difficulty. I also thank the organization greatly for its financial support. To the technical staff of the Department of Biochemistry and of the Chemical Pathology, Electron Microscopy and the Research Animal Breeding Units of the Noguchi Memorial Institute for Medical Research (NM3MR), University of Ghana, and to Mrs. Sarah Dwamenah Abassah and Sarah Acheampong of the Green Earth Organization, I say a big thank you for your assistance. My special appreciation goes to Dr. Addo , the head of the Research Animal Breeding Unit, for allowing me the use of the facilities at her Unit. May God richly bless you all. vii University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS COVER PAGE--------------------------------------------------------------------------------- i RECITATION---------------------------------------------------------------------------------- ii COVER PAGE--------------------------------------------------------------------------------- iii DECLARATION------------------------------------------------------------------------------ iv DEDICATION----------------------------------------------------------------------------------v ACKNOWLEDGEMENT ---------------------------------------------------------------- vi TABLE OF CONTENTS ------------------------------------------------------------- viii LIST OF TABLES---------------------------------------------------------------------------- xi LIST OF FIGURES -------------------------------------------------------------------------- xii ABSTRACT---------------------------------------------------------------------------- xiii CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW General introduction--------------------------------- ---------------------------------- 1 Inflammation and its mediators--------------------------------------------------------- 5 Biosyntheses of eicosanoids ---------------------------------------------------------- 7 - Phospholipase A2 activation----------------------------- *------------------- 7 - The cyclooxygenase pathway------------------------------------------------ 12 - The lipoxygenase pathway---------------------------------------------------- 1 5 Content Page University of Ghana http://ugspace.ug.edu.gh - The monooxygenase pathway--------------------------------------------- 17 Eicosanoid function in inflammation---------------------------------------------------- 19 Anti-inflammatory drugs---------------------------------------------------------------- 21 Anti-inflammatory medicinal plants---------------------------------------------------- 23 CHAPTER TWO: MATERIALS AND METHODS MATERIALS------------------------------------------ 31 Guinea pigs............................................................................................................ 31 Medicinal plant parts/preparations------------------------------------------------------ 31 Chemicals and reagents....................................................................................... 31 METHODS Animals and pretreatment-------------------------------------- 32 Preparation of plant extracts------------------------------------------------------------ 32 Administration of plant extracts---------------------------------------------------------- 33 Blood sampling and treatment------------------------------------------------------------ 34 Preparation of microsomes-------------------------------------------------------------- 35 Protein determinations------------------------------------------------------------------- 36 - (i) Folin-Lowry method---------------------------------------------------- 36 - (ii) Biuret method----------------------------------------------------------- 37 Eicosanoid biosynthesis----------------------------------------------------------------- 3 7 Quantitative estimation of the eicosanoids---------------------------------------------- 38 Assay for sPLA2 activity---------------------------------------------------------------- 3 9 ix University of Ghana http://ugspace.ug.edu.gh Assay for plasma SPLA2 protein amounts---------------------------------------------- 41 Statistical analysis-------------------------------------------------------------------------- 43 CHAPTER THREE : RESULTS EICOSANOID BIOSYNTHESIS-------------------------------------------------- 44 Effect of Desmodium adscendens extract -------------------------------------------- 44 Effect of other medicinal plant extracts------------------------------------------------- 56 PHOSPHOLIPASE A2 ACTIVATION -------------------------------------------- 62 Enzyme activity ------------------------------------------------------------------------ 62 SPLA2 protein ------------------------------------------------------------------------------- 62 Total serum protein--------------------------------- 63 CHAPTER FOUR: DISCUSSION AND CONCLUSION------------------------ 6 6 REFERENCES -------------------------------------------------------------------------- 84 APPENDICES ......................................... 96 x University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table Table A1 Table A2 Table A3 Table A4 Table A5 Table A6 Table A7 Table A8 Table B1 Table B2 Eicosanoid production in the presence of GSH as a coenzyme 45 Prostanoid production in the presence of NADPH as a coenzyme— 49 Prostanoid production without the addition of a coenzyme---------- 50 Ratio of anti- to pro-inflammatory prostanoids (PGE2/PGF20) compared to PGE2 only----------------------------------------------- 51 Ratio of anti- to pro-inflammatory prostanoids (6 -keto-PGFia/TXB2) Compared to 6 -keto-PGFia only-------------------------------------- 52 Title Page Effect of Desmodium adscendens on peptido-leukotriene level in the presence and absence of cofactors----------------------------- 54 Effect of different plant extracts on types and amounts of prostanoids produced-------------------------------------------------- 59 Ratio of anti- to pro-inflammatory prostanoids for different plant extract treatments compared to anti-inflammatory prostanoids alone— 60 Effect of different plant extracts on serum secretory phospholipase A2 activity---------------------------------------------------------------------- 64 Effect of different plant extracts on serum secretory phospholipase A2 protein---------------------------------------- 65 xi University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES F ig l Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8 Fig 9 Figure Arachidonic acid release and oxygenative metabolism-------------- 11 Cyclooxygenase pathway for prostanoid biosynthesis---------------- 14 Lipoxygenase pathway of arachidonic acid metabolism for leukotriene synthesis-------------------------------------------------- 16 Monooxygenase pathway of arachidonic acid metabolism----------- 18 Some isolated chemical constituents of Desmodium adscendens— 27 Effect of Desmodium adscendens extract on prostanoid production with GSH as cofactor (at lower dose) expressed as percentage change over control--------------------------------------------------------------------- 46 Effect of Desmodium adscendens extract on prostanoid production with GSH as cofactor (at higher dose) expressed as percentage change over Control---------------------------------------------------------------------- 47 Effect of Desmodium adscendens on peptido-leukotriene production expressed as percentage change over control on addition of different cofactors--------------------------------------------------------------------- 55 Effect of Desmodium adscendens, Tina A and Kenken extracts on anti-inflammatory prostanoid production expressed as percentage change over control------------------------------------------------------- 61 Title Page University of Ghana http://ugspace.ug.edu.gh ABSTRACT The eicosanoids are a group of oxygenated unsaturated 20-carbon chemical compounds produced from arachidonic acid (AA), that mediate almost every step in the inflammatory process naturally. Some of the eicosanoids are anti-inflammatory while others are pro- inflammatory. Desmodium adscendens, a medicinal plant used by local herbalists to manage asthma, had been shown to be anti-anaphylactic in vivo, and to modulate AA metabolism in vitro. In this work, the anti-inflammatory properties of D. adscendens in vivo were examined by measuring the effect of its aqueous extract on eicosanoid production. Two other putative anti-inflammatory medicinal plants, Parquetina sp {‘Tina A 0 and Cassia sieberiana (‘Kenkeri’) were also examined. The effects of the extracts on phospholipase A2 (PLA2), the enzyme responsible for the mobilization of arachidonic acid (AA) from membrane glycerophospholipid stores to initiate the de novo biosyntheses of the eicosanoids were also examined. Microsomal enzymes prepared from the lungs of both extract-treated (test) and untreated (control) male guinea pigs were used to catalyze the metabolism of arachidonic acid (AA), the natural substrate for eicosanoid synthesis, via the cyclo-. mono- and the lipoxygenase pathways to produce various eicosanoids. Reduced glutathione, GSH, was added to the reaction mixture for the cyclooxygenase pathway while NADPH was added xiii University of Ghana http://ugspace.ug.edu.gh for the monoxygenase pathway. For the lipoxygenase pathway no cofactor was added. The monooxygenase metabolites were not quantified due to limited resources. However, the influence of added NADPH on the production of the metabolites of the other two pathways was evaluated. All synthesized eicosanoids were assayed by ELISA. As sources of secretory phospholipase A2 (sPLA2), blood samples were taken from the experimental animals and used to assess the effects of the plant extracts on PLA2 activity. The cyclooxygenase and lipoxygenase pathways were identified as good bioassay systems for assessing the anti-inflammatory properties of D. adscendens. The plant’s extract inhibited the pro-inflammatory lipoxygenase pathway in a dose-dependent manner (6 8 % and 98% reductions in peptidoleukotrienes syntheses for the lower and higher doses respectively). In the cyclooxygenase system, the extract enhanced the syntheses of the anti-inflammatory prostanoids; PGI2 (6437% increase) and PGE2 (581% increase). The effect on the synthesis of the pro-inflammatory prostanoid PGF2a was insignificant (0.5% increase) and that on TXA2 was 49% increase, both at the higher dose of the extract. Using D. adscendens as a model, the anti-inflammatory effects of ‘ Tina A ’ and ‘Kenken’ were assessed using the cyclooxygenase bioassay system only. Like D. adscendens, the two medicinal plants also increased PGI2 and PGE2 production and hardly showed any xiv University of Ghana http://ugspace.ug.edu.gh effect on PGF2„ and TXA2 syntheses. The increases in PGI2 production ranged between 290% and 1417% ; those for PGE2 were 57% and 78%, all at the higher doses of the extracts. The effects at the lower doses were not significant except for ‘Tina A \ In all, D. adscendens proved to be the best anti-inflammatory plant with respect to enhancing anti­ inflammatory eicosanoids syntheses, followed by ‘Tina A \ All three extracts inhibited phospholipase A2 activity with ‘7/no A ’ showing a dose- dependent inhibitory effect even with a small dose difference of 1: 2.5. ‘Tina A’ was the best PLA2 inhibitor followed by ‘Kenken The results indicate that the medicinal plants evaluated provide therapeutic relief to inflammatory disorders by the following mechanisms: (a) directly reducing PLA2 activity and thus release of AA which the rate determining step in eicosanoid production, and/or by (b) enhancing PGI2 and PGE2 production when arachidonic acid has been released. Inhibition of peptido-leukotriene synthesis could also be said to be a good determinant of the anti-inflammatory status of D. adscendens in particular. Increased synthesis of the anti-inflammatory prostanoids PGE2 and PGI2, and/or inhibition of phospholipase A2 activity appear to be good bioassays for evaluating medicinal plants claimed to have anti-inflammatory properties. It was therefore concluded that good bioassay systems have been developed for in vivo evaluation of putative anti-inflammatory drugs with respect to eicosanoid biosynthesis. xv University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW General Introduction Eighty percent of the world’s population relies entirely on local medicines made almost exclusively from plants (Lewington, 1990). People belonging to the most advanced civilizations and the simplest cultures the world over have relied on plants to keep them healthy. Only recently, with the advances in synthetic chemistry have developed countries broken their dependence on cures that came almost exclusively from plants (Lewington, 1990). Though this practice of dependence on non-plant based drugs is gradually creeping into simple cultures, it may perhaps, never gain roots fully in these cultures which utilize such drugs because of the ever-increasing cost of Western medicine. Interestingly, the complex nature of many plant compounds essential to modem medicine has made their synthesis either too expensive for chemical companies to venture into, or practically impossible. For these reasons, plants will continue to be the source of medical care for most patients of the world, particularly those in the tropics where two-thirds of all the plant species of the world exist. The tropics also have the largest resource of traditional knowledge and experience in the use of plants as medicines, possessing the greatest diversity of as yet unknown active compounds. l University of Ghana http://ugspace.ug.edu.gh The mystery surrounding the time-proven curative properties and the effectiveness of different plant extracts in the management of several diseases, including those declared incurable by Western medicine, coupled with the low treatment cost are the reasons why many countries the world over have held on to folk medicine. These countries, including Ghana, have done so inspite of the rather contemptuous attitudes in some areas of Western medicine that exist towards the use of crude plant extracts in disease management (Abbiw, 1990; Lewington, 1990). Other countries like the United States of America have increased their interest in plants as sources of raw materials for discovering and developing new pharmaceutical products (Komen, 1991). It is estimated that between 35,000 and 70,000 different species of plants have been used as medicines by various peoples of the world with the commercial value of plant-derived drugs by Western medicine standing at about 40 billion U.S. dollars every year (Lewington, 1990). At least some 7000 different plant-derived medicinal compounds have been introduced into Western pharmacopoeia. Out of these, only 120 or so plant-based drugs, coming from just 95 plant species, are prescribed for use worldwide. Out of the 250,000 species of flowering plants, approximately 5000 only have had their pharmaceutical potential tested in laboratories and very few have been acknowledged in the West to have any real therapeutic value (Krogsgaard-Larsen et al, 1984). 2 University of Ghana http://ugspace.ug.edu.gh The plant-based drugs prescribed for worldwide use include analgesics, anesthetics, antibiotics, anti-cancer, anti-parasitic compounds and drugs for diseases of the heart. Others are anti-inflammatory drugs, oral contraceptives, hormones, laxatives, diuretics and for ulcer treatment. These indicate that plants possess rich medicinal properties that could be exploited to treat almost all known diseases of man and animals. The contribution of plant medicine to health care has great economic potential, with a large and rapidly growing global market for affordable and effective plant-based remedies. Ideally therefore, both Western and traditional medicine (which is plant- based) should complement each other. It is in recognition of this feet that the World Health Organization (WHO) is officially encouraging the integration of traditional medicine into the health care systems of developing countries where the majority of the population cannot afford the high cost of Western medicine. Several plant species have been documented to be effective in the management of human diseases. These include, Hemamelis virginiana (Wych hazel), Eucalyptus globulus, Cephaelis impecacuanha, Papaver somniferum, Ephedra spp, Catharanthus roseus (rosy periwinkle), Rauwolfia serpentina, Erythroxylum coca and Cinchona spp. Crude extracts from these plants are used to treat a number of diseases including colds and fevers, coughs, bilharziasis, guinea worm infestations, bronchitis, pain and anxiety, cancers, hypotension and hypertension, menstrual tension, menopausal problems, psychiatric conditions and malaria. With respect to 3 University of Ghana http://ugspace.ug.edu.gh inflammation, a pathological condition which is the subject of study of this thesis, crude extracts from H, virginiana (Wych hazel), Glycyrrhiza spp, Salix alba (White willow), Filipendula ulmaria, Tenacetum parthenium, Oenothera spp and pineapple have been effectively used over the years to manage a broad spectrum of inflammatory diseases including rheumatoid arthritis, thrombosis, migraine and asthma (Lewington, 1990). Scientific investigations into Desmodium adscendens, a medicinal plant, whose extracts are used among Ghanaians to remedy asthmatic attacks, have confirmed its efficacy. It has been shown that D. adscendens extracts are anti-anaphylactic both in vivo and in vitro (Addy and Awumey, 1984; Addy and Burka, 1988; Addy and Dzandu, 1986). The extracts also reduced the production of some spasmogens from lungs (Addy and Dzandu, 1986). Following the anti-anaphylactic effects in vivo, the effect of the extract on some eicosanoids biosyntheses in vitro were analyzed using radiochromatographic methods. The results indicated an increase in prostaglandin E2 synthesis (Addy and Schwartzman, 1995) and a decrease in NADPH-dependent oxygenation of arachidonic acid (Addy and Schwartzman, 1992). The research work reported here seeks to investigate the effect of aqueous extracts from D. adscendens on eicosanoids biosyntheses in vivo. Since eicosanoids are typical spasmogens that mediate inflammation, changes in their production could be used to assay for the anti-inflammatory effects of putative medicinal plants. Using the results from D. adscendens studies as a model, the anti­ 4 University of Ghana http://ugspace.ug.edu.gh inflammatory effects of extracts from Cassia sieberiana and Parquetina sp on eicosanoids biosyntheses will be evaluated. Inflammation and its mediators Inflammation is the local response to cell injury, involving small blood vessels, the cells circulating within these vessels, and nearby connective tissues. The response characteristically begins with hyperemia, edema, and adherence of the circulating white blood cells to the endothelial cells. The white cells then migrate between the endothelial cells of the blood vessel into the tissue to effect the necessary immunological response to the injury (Lagunoflf, 1994). Symptoms of inflammation include redness, swelling, heat and pain. Increased chemotactic movement of blood to the site of injury through the small blood vessels causes heat and redness. The loss of water from circulation to the extracellular edematous connective tissue causes edema/swellings through the engorged and distended capillaries. The collective effects of these events may lead to reduced blood flow rate in the immediate vicinity of the injury, with subsequent cessation of flow (stasis) and clotting of the concentrated blood cells in the constricted venules. This is the cause of thrombosis. Stimulation of nerve endings by agents released during the inflammatory process causes pain. Inflammation is fundamentally a protective response whose ultimate goal is to rid the organism of both the initial cause of cell injury (e.g., microbes, toxins) and the consequences of such injury (the necrotic cells and tissues). However, 5 University of Ghana http://ugspace.ug.edu.gh inflammation and repair may be potentially harmful. Inflammatory reactions, for example, underly life-threatening hypersensitivity reactions to insect bites, drugs, toxins and antigens in asthmatics as well as some chronic diseases of modem times. These include rheumatoid arthritis, atherosclerosis, tuberculosis, lung fibrosis, common cold, influenza, gastroenteritis in salmonellae “food poisoning”, bacillary dysentery (acute colitis), diphtheria, pseudomembranous colitis, fibrinous inflammation, abscesses e.g. boils, ulcers and migraine (Robbins et al., 1994; Thier and Smith, 1981; Whaley and MacSween, 1992). The process of inflammation, both vascular and cellular, is orchestrated by an array of molecules produced locally. Many anti-inflammatory drugs function by preventing the formation of these mediators or by blocking their actions on the target cells whose behaviour is modified by the mediators (Lagunoff, 1994). These mediators include histamine, various peptides, complement components, kinins, antibodies, interleukins , and various eicosanoids. The eicosanoids are very important in inflammatory disorders because of their various and sometimes conflicting pharmacological effects that enable them to mediate virtually every step in inflammation (Devlin, 1997; Robbins et al., 1994). They include leukotrienes, prostaglandins, thromboxanes, prostacyclins, lipoxins and a number of hydroperoxy and hydroxy fatty acids (Smith, 1989). These myriad pharmacological effects are often species-, sex- and tissue-dependent (Taylor and Ritter, 1986). Being natural mediators of inflammation, the eicosanoids are produced by virtually all mammalian tissues (Smith, 1987). They are local 6 University of Ghana http://ugspace.ug.edu.gh hormones synthesized de novo on induction by an appropriate stimulus and rapidly get metabolized (Smith et al., 1991). Though produced as products from the metabolism of a common substrate, arachidonic acid, some of the eicosanoids exhibit pro-inflammatory effects while others are anti-inflammatory. For example, prostaglandin F20, thromboxane A2 and the leukotrienes are pro-inflammatory while prostaglandin E2 and prostaglandin I2 (prostacyclin) are generally anti-inflammatory (Whaley and MacSween, 1992). Therefore, a putative anti-inflammatory drug acting on the metabolism of arachidonic acid, may reasonably do so by either blocking a common pathway in the metabolic process, or modulate the process to yield more of the anti­ inflammatory eicosanoids, and either maintain or reduce the levels of the pro- inflammatory eicosanoids. Biosyntheses of Eicosanoids Phospholipase A 2 Activation Eicosanoids are synthesized from arachidonic acid (AA), a 20-carbon poly­ unsaturated fatty acid commonly found in the sn-2 position of membrane phospholipids. The common glycerophospholipids with AA are phosphatidyl- serine, -inositol, -choline and phosphatidyl-ethanolamine (Smith et ah, 1991). Since unesterified free AA is the substrate for eicosanoid biosynthesis, the 7 University of Ghana http://ugspace.ug.edu.gh reactions involved are initiated by the hydrolytic release of AA from the sn-2 position by a stimulus-activated phospholipase A2 (Murayama et al., 1990; Silk et al., 1989). Alternatively, phosphatidylinositol may be degraded by phosphatidylinositol-specific phospholipase C to yield diacylglycerol, from which AA may be obtained (Smith et al., 1991; Zubay, 1983). The phospholipases A2 (PLA2) have been found to be either free and soluble (sPLA2) (Channon and Leslie, 1990; Leslie et al., 1988) or membrane-associated (Dennis, 1987; Lister et al., 1988, 1989; Ross et al., 1985; Ulevitch et al., 1988). The activated PLA2 appears to be a soluble enzyme, which becomes reversibly associated with membrane in the presence of higher Ca2+ concentrations (Channon and Leslie, 1990). The movement of SPLA2 from solution into the membrane is essential to bring it to close proximity to the glycerophospholipid whose AA is to be hydrolyzed. Such phospholipids are normally in the vicinity of the membrane associated AA metabolizing enzymes for the rapid metabolism of the AA when released (DeWitt et al., 1981; Rollins and Smith, 1980; Smith, 1986, 1987). Though some calcium- independent phospholipases have been characterized (MacDonald and Maxey, 1998; Smith et al., 1991), it has been observed that almost all phospholipases operate optimally at higher Ca2+ concentrations (Smith et al., 1991). For example, the activity of phospholipase Ai does not necessarily require Ca2+ but its hydrolytic effect is enhanced by Ca2+ and charged amphipaths because of their role in altering 8 University of Ghana http://ugspace.ug.edu.gh the surface charge on the substrate micelle or membrane (Gurr and Harwood, 1991). Most of the external stimuli for PLA2 activation therefore, operate by elevating intracellular calcium ion concentration (Smith et al, 1991). PLA2 activities are present in both the soluble and membrane-associated fractions of cellular preparations (Kramer et al., 1986). The bulk of AA metabolizing enzyme activity, in general, is associated with endoplasmic reticulum (DeWitt et al., 1981 ;Rollins and Smith, 1980; Smith, 1986). The free AA is rapidly metabolized into specific eicosaniods via any of three principal enzyme catalyzed pathways, namely, the cyclooxygenase, lipoxygenase, and monooxygenase (epoxygenase) pathways (Smith, 1989). All the membrane- associated enzymes for the metabolisms of AA are already present and active (Gurr and Harwood, 1991). The release of AA is thus said to be the rate-limiting step. The biosynthesis of the eicosanoids is thus thought to be regulated, at least acutely, at the level of arachidonic acid release (Bettazoli et al., 1990; Dennis, 1987). Therefore, an anti-inflammatory drug acting against eicosanoid biosynthesis may do so at the level of AA release through the inhibition of the activity of PLA2. Although the substrate is the same (AA) the concentration and availability of enzymes and coenzymes for the three pathways differ in different tissues, giving rise to various metabolites in the different tissues. These metabolites exhibit 9 University of Ghana http://ugspace.ug.edu.gh different physiological and pharmacological effects on various organs and tissues, and therefore, defects in AA metabolism could lead to different diseases associated with different organs and tissues (Dunn, 1976; Samuelsson, 1983;Schibouta et al., 1981). The cyclooxygenase pathway yields the prostanoids; prostaglandins, prostacyclins and thromboxanes, while leukotrienes and lipoxins are formed via one or more lipoxygenase reactions (Samuelsson et al., 1987; Smith, 1989), and lipid epoxides and diols are formed through cytochrome P-450-dependent reactions of the epoxygenase pathway (Capdevila et al., 1990; Fitzpatrick and Murphy, 1989; Laniado-Schwartzman et al, 1988). All enzymes of the arachidonate cascade are inactivated during catalysis. A summary of the oxygenative metabolism of AA is shown in figure 1 . 10 University of Ghana http://ugspace.ug.edu.gh Figure 1. Arachidonic acid release and oxygenative metabolism (Huber et al., 1993) r\ / o ii c h 2- o - c - r o c h 2- o - p - o - x * i O" Glycerophospholipid H2 0 PHOSPHOLIPASE A 2 0 II CH2- 0 - C - R I H O -C H o I II c h 2- o - p - o - x * 1 O' Lyso-glycerophospholipid Arachidonate EPOXIDES HETEs DIOLS LEUKOTRIENES HETEs PROSTANOIDS X* = choline, ethanolamine, inositol or serine R = Alkyl group 11 University of Ghana http://ugspace.ug.edu.gh The Cyclooxygenase Pathway This pathway involves the incorporation of two moles of molecular oxygen (2 O2) into a mole of arachidonic acid by the cyclooxygenase activity of the bifunctional enzyme, prostaglandin endoperoxide synthase (PES). The unstable initial product formed, prostaglandin G2 (PGG2), is reduced by the peroxidase activity of PES to the stable prostaglandin H2 (PGH2). The conversion of PGG2 to PGH2 requires two moles of reduced glutathione (GSH) as cofactor. The resulting PGH2 undergoes further reduction or rearrangement to yield what are considered to be the biologically active prostanoids: prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin I2 (PGI2), prostaglandin F2o (PGF20) and thromboxane A2 (TXA2) (Smith etal., 1991) The mechanism of conversion of PGH2 to the various prostanoid products varies from one product to the other. While PGE2, PGD2, PGI2 and TXA2 are synthesized from PGH2 by non-oxidative rearrangements, PGF2a is formed from PGH2 by a net two-electron reduction by the endoperoxide reductase (PGF synthase), which uses NAPDH as a reducing agent (Bergstrom et al., 1963; Dunn et al., 1978; Watanabe et al., 1985). In contrast, PGE2 is reported to be formed non-oxidatively from PGH2 by the catalytic action of PGH-PGE isomerizing enzyme, PGE synthase, which does not consume the GSH coenzyme in contrast to the NADPH consuming function of PGF synthase (Moonen et al., 1982; Ogino et al., 1977; Tanaka et al., 1987). However, nonenzymic formation of PGE from PGHz at relatively fester rates has also been reported (Hamberg and Samuelsson, 1973; Nugteren and 12 University of Ghana http://ugspace.ug.edu.gh Hazelhof, 1973). Conversion of PGH2 to PGD2 is by catalysis of PGD synthase which may either be GSH-dependent (Christ-Hazelhof and Nugteren, 1982; Ujihara et al., 1988; Urade et al., 1987) or GSH-independent (Urade et al., 1985). The conversion of PGH2 to PGI2 involves PGI synthase activity, which is high in vascular endothelial cells and in both vascular and non-vascular smooth muscle cells (DeWitt et al., 1983; Smith et al., 1983). PGI2 is hydrolyzed to the stable and physiologically inert product, 6 -keto-PGF)a with an approximate half-life of 2 minutes (Salmon and Flower, 1982; Whaley and MacSween, 1992). The amount of PGI2 produced from the metabolism of AA can therefore be quantified indirectly by measuring the amount of 6 -keto-PGFi0 present in the medium The conversion of PGH2 to TXA2 is catalysed by TXA synthase. TXA is very unstable with a half-life of 30 seconds. It is hydrolysed to the stable product TXB2 which lacks appreciable biological activity (Haurand and Ullrich, 1985; Nusing et al., 1990; Zubay, 1984). The amount of the biologically active TXA2 resulting from the metabolism of AA can, therefore, be estimated by measuring the amount of the inactive stable product, TXB2 present. Details of the cyclooxygenase pathway are shown in figure 2 . 13 University of Ghana http://ugspace.ug.edu.gh Figure 2. Cyclooxygenase pathway for prostanoid biosynthesis (Huber et al., 1993; Taylor and Ritter, 1986) ,COOH pgi2 (Pro*taeycfcn) m O S ’ AGLAhCM E SrN TH *SE PftjSfA G LA PG f* O S Y N T t*S £ raO S IA O L A fO X F S V M T w J f 2NADP NADPM 14 University of Ghana http://ugspace.ug.edu.gh The Lipoxygenase Pathway The conversion of unbound arachidonic acid (AA) to the leukotrienes involves an initial enzyme-catalyzed incorporation of one mole o f molecular oxygen (O2) into a molecule of AA. The hydroperoxy substitution of AA by lipoxygenases may occur at position 5, 12 or 15 to produce the hydroperoxyeicosatetraenoic acids (HPETEs). The 5-HPETE is the major lipoxygenase product in basophils, polymorphonuclear (PMN) leukocytes, macrophages, mast cells and any organ undergoing an inflammatory response (Devlin, 1997). The HPETEs undergo dehydration among many other reactions, to produce the epoxy fatty acids. The epoxy eicosatrienoic acids and their metabolic products are the leukotrienes. The 5-HPETE is responsible for leukotriene production and is important in neutrophils, eosinophils, monocytes, mast cells and keratinocytes as well as lung, spleen, brain and heart (Gurr and Harwood, 1991). In the formation of leukotrienes, the epoxide intermediate, leukotriene A4, is converted enzymatically by hydration to leukotriene B4 and to leukotriene C4 (LTC4) by addition of reduced glutathione (GSH). LTC4 is further metabolized to leukotrienes D4 and E4 by the successive elimination of glutamyl residue by glutamyl transferase catalysis to yield LTD4, and further elimination of glycine from LTD4 by a dipeptidase to yield LTE4 (Samuelsson, 1983). Details of the lipoxygenase pathway are shown in figure 3. 15 University of Ghana http://ugspace.ug.edu.gh Figure 3: Lipoxygenase pathway of arachidonic acid metabolism for leukotriene synthesis (Samuelsson, B., 1983) XX COOHV A rach idon ic A c id Lypoxygenase H OOH / —"N ^ C00i_ \ — — yX/ Xy ' 5-H PETE COOH Leukotriene B 4 (LTB 4 ) r -G iu Leukotriene C 4 (L T C 4 ) Glutam yl transpeptidase HO. H COOH Cys-Gty Dipeptidase HO ,H k ^ C 5H„ " S Cys DHTs and HETEs other than the 5 derivatives are also produced (not shown) COOH i6 University of Ghana http://ugspace.ug.edu.gh The Monooxygenase/Epoxygenase Pathway There are three known reactions in this third pathway, all of which involve oxygenation of the AA using the NADPH-dependent monooxygenase enzyme. One atom of oxygen is incorporated into a molecule of AA while NADPH reduces the other atom to water. The enzyme complex consists of a flavoprotein reductase and cytochrome P-450 already present in the membranous subcellular structures of the cell as electron acceptors in microsomal electron transfer from NADPH to oxygen (White and Coon, 1980). The different actions in this pathway lead to the formation of various products, including different regiospecific isomers of epoxy-eicosatrienoic acid (EET): 5,6-, 8,9-, 11,12- and 14,15-EET. Other products are the 19- and 20- hydroxyeicosatetraenoic acids (19- and 20-HETE) and various regioisomeric monohydroxyeicosatetraenoic acids (HETEs). The EETs produced are rapidly hydrolyzed to their corresponding dihydroxyeicosatrienoic acids (DHTs) by epoxide hydrolases (Chacos et al., 1983). Details of the monooxygenase pathway are shown in figure 4. 17 University of Ghana http://ugspace.ug.edu.gh Figure 4: Monooxygenase pathway of Arachidonic Acid Metabolism (Huber et al., 1993) 5-6-epoxy-eicosatrienoate (5-6-EETs) f h 2o hydrolase HO OH / = 0 “ 0 \ ^ CO°" 5-6-DHT 5-hydroxyeicosatrienoic acid (5-HETEs) 8-HETEs 9-HETEs 11-HETEs 12-HETEs 15-HETEs 8-9-EETs 11-12-EETs 14-15-EETs And their corresponding DHTs are also produced 18 University of Ghana http://ugspace.ug.edu.gh Eicosanoid function in inflammation Unlike most chemical messengers, the eicosanoids are not stored in cells but instead, are synthesized and released immediately in response to a stimulus (Devlin, 1997). They exert a range of profound biological activities including effects on smooth muscle contraction/relaxation, inhibition or stimulation of platelet aggregation, bronchoconstriction/dilation and vasoconstriction/dilation (Gurr and Harwood, (1991). Typical responses of a cell to eicosanoids include changes in intracellular concentration of cAMP (Sonnenburg and Smith, 1988), cGMP (Zubay, 1984) and Ca2+ (Negishi et al., 1989) upon binding of the eicosanoid to appropriate cell membrane surface receptors (Smith, 1989). However, thromboxane A2 (TXA2) is reported to cause an increase in cytosolic Ca2+ by acting as Ca2+ ionophore (Zubay, 1984). The modulation of cell function by the eicosanoids during inflammation has been reported to be influenced by the relative levels of these secondary messengers (Whaley and MacSween, 1992). Agents like prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2), which increase cAMP levels with resultant activation of cAMP dependent protein kinases, reduce inflammation (anti-inflammatory). In contrast, agents like prostaglandin F2a (PGF2«) and thromboxane A2 (TXA2), which increase cytosolic Ca2+ ion levels through the elevation of cGMP concentrations, 19 University of Ghana http://ugspace.ug.edu.gh enhance inflammatory cell function (pro-inflammatory) (Whaley and MacSween, 1992). PGE2 and PGI2 are described as being strongly anti-inflammatory (pro- inflammatory in edema) with PGI2 proving much more potent than PGE2 in most systems (Samuelsson, 1983). They act as vasodilators, inhibitors of platelet aggregation, smooth muscle relaxants, relaxers of coronary arteries and preventors of platelet binding to arterial walls (Robbins et al., 1994; Samuelsson, 1983; Zubay, 1984). They are thus opposite in biological function to TXA2 which is an unstable platelet aggregating, smooth muscle contracting, serotonin releasing and vasoconstrictor substance (Gurr and Harwood, 1991; Samuelsson, 1983; Zubay, 1984). Similarly, PGE2 and PGI2 are antagonistic in function to PGFja which is a pro-inflammatory substance that causes smooth muscles to contract (Devlin, 1997). The monohydroxyeicosatetraenoic acids (HETES) particularly 5-HETE and leukotriene B4 (LTB4), work synergistically to cause adhesion and chemotactic movement of leukocytes, and stimulate aggregation, lysosomal hydrolytic enzyme release, adenylate cyclase, polymorphonuclear neutrophils (PMNs) degradation and generation of superoxide in neutrophils. They thus function as pro-inflammatory substances (Devlin, 1997; Samuelsson, 1983). Leukotrienes LTC4, LTD4 and LTE4, collectively referred to as the peptido- leukotrienes, are humoral agents usually released from the lung tissue of asthmatic 20 University of Ghana http://ugspace.ug.edu.gh subjects exposed to specific allergens. They cause slowly evolving, but protracted contraction of smooth muscles in the airways and gastrointestinal tract, and enhance capillary permeability. They are thus referred to as slow-reacting substances of anaphylaxis (SRS-A). By these effects, they play a pathological role in immediate hypersensitivity reactions (Devlin, 1997; Samuelsson, 1983). They are thus pro-inflammatory in function. Generally therefore, the anti-inflammatory effects of PGE2 and PGI2 antagonize the pro-inflammatory functions of the peptido-LTs, PGF2a and TXA2 that cause bronchial asthma and other hypersensivity related diseases. Products from the monooxygenase pathway have a wide range of biological activities (Capdevila et al., 1983; Schlondorff et al., 1986; Snyder et al., 1983). The 5,6-epoxyeicosatrienoic acid (5,6-EET) is reported to be a potent vasodilator and a relaxer of arterial rings (Schwartzman et al., 1987). Anti-inflammatory drugs The effectiveness of a therapeutic agent in the management of inflammatory disorders might, therefore, be based on its ability to modulate arachidonic acid metabolism in favour of the anti-inflammatory substances. It may also be based on its ability to inhibit the PLA2 enzyme responsible for the mobilization of AA, or its inhibition of the prostaglandin endoperoxide synthetase enzyme that possesses the cyclooxygenase activity. In this connection it is known that the non-steroidal anti­ inflammatory drugs (NSAIDs) such as aspirin (acetylsalicylic acid), ibuprofen, 21 University of Ghana http://ugspace.ug.edu.gh indomethacin and phenylbutazone, block prostaglandin production by inhibiting the cyclooxygenase enzyme. (Flower, 1974; Mizuno et al 1982; Van der Ouderaa et al., 1980; Vane and Botting, 1987). Aspirin irreversibly inhibits the enzyme by acetylating the side-chain hydroxyl group of a seryl residue (Ser 530) on the enzyme (DeWitt and Smith, 1988), while the others inhibit cyclooxygenase by binding non-covalently to it (Devlin, 1997). The steroidal anti-inflammatory drugs like hydrocortisone, prednisone, and betamethasone, block prostaglandin synthesis by inhibiting phopholipase A2 activity so as to interfere with the mobilization of AA (Delvin, 1997). Glucocorticoids induce the synthesis of a protein, lipocortin (macrocortin, lipomodulin), which inhibits phospholipase A2 activity (Robbins et ah, 1994; Whaley and MacSween, 1992). In studying the effect of a putative anti-inflammatory drug therefore, the absolute amounts of the eicosanoids produced, or the relative amounts of anti-and pro- inflammatory prostanoids, or the activity of PLA2 are some of the parameters to be measured. Drugs that are effective against inflammatory disorders are expected not to decrease the amounts of anti-inflammatory eicosanoids, nor increase the levels of pro-inflammatory eicosanoids. The PLA2 activity is expected to significantly decrease in response to such drugs. Ratios of the absolute amounts of anti- and pro- inflammatory eicosanoids could also be used to assess anti-inflammatory properties of drugs. PGI2 is produced in significantly high amounts in the endothelial lining of blood vessels and it antagonizes the platelet aggregating effect of TXA2, which is 22 University of Ghana http://ugspace.ug.edu.gh produced in high amounts by the platelets themselves during inflammation (Whaley and MacSween, 1992; Zubay, 1983). The ratio PGI2/TXA2 would therefore be a useful parameter for assessing anti-inflammatory activity. The ratio PGE2/PGF2a is also a useful parameter because o f the direct antagonistic effects of the two prostanoids involved, which are also commonly produced together in all tissue where they are found. A ratio of PGE2+PGI2/PGF201+TXA2 could also be useful. Anti-inflammatory Medicinal Plants A number of plants have been recorded to have anti-inflammatory properties (Lewington, 1990). These include the following: Hemamelis virginiana (Wych hazel): Alcohol extracts from the leaves and bark of this plant help prevent inflammation and control bleeding. Tea prepared from the leaves and bark of the plant is drunk to alleviate colds, fevers and sore throats, and also used to wash sores and wounds. Over one million gallons of Wych hazel are sold in the United States alone each year. Glycyrrhiza sp: Crude extracts from the dried roots and rhizomes of Glycyrrhiza glabra (liquorice) is used as an expectorant and anti-inflammatory drug, and is common in cough syrups, sweets and pastilles. The main ingredient is saponin-like glycosides of which glycyrrizin is the most important. Salix alba and Filipendula ulmaria: The parent chemical compound of Aspirin (salicylic acid), the celebrated anti-inflammatory drug that irreversibly inhibits the 23 University of Ghana http://ugspace.ug.edu.gh cyclooxygenase enzyme by acetylation, was extracted from the leaves and bark of the white willow, Salix alba, and the perennial herb meadowsweet, Filipendula ulmaria Oenothera sp: Oil extracted from the evening primrose, a species of Oenothera, is reported for its possible remedy for arthritis, migraine, asthma, eczema, high blood pressure and premenstrual tension. The medical profession now officially recognizes primrose oil as a treatment for atopic eczema. Tenacetum parthenium: Extracts from the plant have been found to be specific in the treatment of migraine, an inflammatory disease resulting from vasospasm and dilation of intracranial arteries and their branches, due to intermittent release of 5- hydroxytryptamine (serotonin) and prostaglandins (Their and Smith, 1981). Dioscorea sp. (the yam family): Many plants in the species are the source of several steroids. Products of steroidal sapogenins include cortisone and hydrocortisone, very important steroidal anti-inflammatory agents that block AA mobilization in eicosanoid biosynthesis by inhibiting phospholiphase A2 activity. They are thus used to treat rheumatoid arthritis, rheumatoid fever and sciatica, which are common inflammatory disorders. Ananas comosus (Pineapple): Extracts from the stems, fruits and leaves of pineapple are reported to contain bromelain, an enzyme that breaks down proteins including fibrin which is responsible for blood clots in thrombosis, an inflammatory disorder enhanced by TXA2 release. Inspite of the successes achieved in the isolation, purification and identification of active chemical constituents of plants as well as the successful chemical synthesis of quite a reasonable number of these chemicals and their derivatives, the crude extracts from most of these plants have been found to be more potent, with less or 24 University of Ghana http://ugspace.ug.edu.gh no quantifiable side effects, with broader spectra of performance, and less expensive (Lewington, 1990). It has often been found that while the effect of a plant may be marked when all its compounds are used together, isolation of those thought to be responsible (active principles) do not always produce cures (Lewington, 1990). It has therefore become increasingly important that the use and dosage of medicinal plant extracts are examined and refined to incorporate them into modem treatments as well as conduct thorough studies that will scientifically verify the claims made on them to facilitate their acceptance into future medicine. It is for this reason that the Department of Biochemistry of the University of Ghana has, over the years, been investigating the scientific basis or the mechanism of action and the toxicology of several putative medicinal plants including those used in the treatment of malaria, diabetes mellitus, asthma and other inflammatory diseases. One such anti-inflammatory plant that has been investigated scientifically is Desmodium adscendens (Sw) DC. var adscendens locally known as Ananse nkateeor Akwanfamu or Nkatenkate. D. adscendens is a leguminous plant belonging to the family, Papilionaceae. Both herbalists and medical practitioners at the Centre for Scientific Research into Plant Medicine (CSRPM) in Ghana use aqueous decoction from the dried stem leaves of the plant to manage asthma. It is also used for treating abdominal colic, diarrhoea, and dysmenorrhoea (Ampofo, 25 University of Ghana http://ugspace.ug.edu.gh 1977). According to Ayensu, the plant is also used to treat constipation, ringworm, convulsions, veneral sores and for dressing wounds (Ayensu, 1978). Other species of Desmodium are also known for their medicinal properties. Preparations from different parts of Desmodium gangeticum (Linn). DC are used to treat urinary problems, diarrhoea, chronic fever, asthma, abdominal tumors, nasal polyps, febrifuge, and catarrh (Ayensu, 1978). Preparations from the roots are also used as astringents, tonics and diuretics (Ayensu, 1978). The leaves of Desmodium triflorum (L.) DC. are used to induce lactation, as a remedy for diarrhoea, dysentery and convulsion. The roots are used in coughs, asthma, and also applied to wounds and abscesses. These properties are usually ascribed to the alkloidal content of the various parts of the plant. (Ghosal et al., 1971; Ghosal and Bhattacharya, 1972). Several chemical constituents have been isolated from the stem-leaves of aqueous extracts of D. adscendens. These include tyramine, N, N-dimethyltyramine, 3,4- dimethoxy-P-phenethylamine, salsoline, N, N-dimethyltryptamine, in addition to several unidentified indole and other minor basic components (Asante-Poku et al., 1988). Also purified and identified from the extracts are the triterpenoid glycosides dehydrosoyasaponin I (DHS-I), soyasaponin I, soyasaponin III, soyasapogenol B and E (McManus et al, 1993). Chemical structures of some of the listed isolated chemical constituents of D. adscendens are shown in figure 5. 26 University of Ghana http://ugspace.ug.edu.gh Figure 5: Some isolated chemical constituents of Desmodium adscendens (Asante-Poku etal., 1988; McManus etal., 1993) c h 2c h 2n h 2 CH2CH2N(CH3)2 c x ? OH Tyram ine N ,N -d im ethy lty ram ine N ,N -d im e thy ltryp tam ine C H , S a lso line 3 ,4 -d im e thoxy- p -phene thy lam ine ■ T O HO OH Compound Ri r 2 Rs Soyasaponin I H, OH I II Dehydrosoyasaponin I 0 I II Soyasaponin III H, OH I H Soyasapogenol B H, OH H 27 University of Ghana http://ugspace.ug.edu.gh Extracts from D. adscendens have been found to reduce anaphylactic contractions, interfere with histamine-induced and antigen-induced contractions of smooth muscles and to reduce the histamine content, as well as the amount of the amine released from lung tissue in a dose dependent fashion (Addy and Awumey, 1984; Addy and Dzandu, 1986). The extract also inhibited AA- and peptidoleukotriene- induced contractions of guinea pig tracheal spirals and lung parenchymal strips (Addy and Burka, 1988; 1989). The extracts have also been found to inhibit NADPH-dependent oxygenation of arachidonic acid (Addy and Schwartzman, 1992). As noted earlier, this third pathway of arachidonic acid metabolism produces some pro-inflammatory eicosannoids. Desmodium adscendens extracts were also found to activate cyclooxygenase and increase the synthesis of the anti­ inflammatory prostanoid PGE2 (Addy and Schwartzman, 1995). In these experiments PGF20 also varied depending on the concentration of cyclooxygenase enzyme, presence or absence of GSH, and concentration of the D. adscendens extract. Some of the experiments involving antigen-induced contractions of smooth muscle were carried out in vivo that is, the extracts were administered to sensitized laboratory animals from which organs or tissues containing smooth muscles were removed and challenged with antigen. The experiments on eicosanoid production, AA- and peptidoleukotriene-induced contractions were carried out in vitro. In vivo conditions may alter significantly the effective concentrations as well as the overall effect of the extracts on eicosanoid synthesis due to possible barriers to absorption and the stability of the active compounds in the gastro intestinal tract. One cannot, 28 University of Ghana http://ugspace.ug.edu.gh therefore easily extrapolate in vitro results to apply to in vivo conditions. However, by administering plant extracts to experimental animals and using tissues from such animals as a source of enzymes for metabolizing AA, the in vivo effect of the extract on the enzymes involved in eicosanoid synthesis could be evaluated. The overall aim of this thesis therefore, was to find out if the effects of D. adscendens on the biosynthesis o f eicosanoids from AA, which have been shown to occur in vitro, would occur in vivo. For the in vitro studies, the substrate, AA, was freely supplied. However, in vivo mobilization of AA via the activity of phospholipase A2, is the rate-limiting step in the de novo biosynthesis of the eicosanoids. Therefore, the in vivo studies also included an investigation into the effect of the plant extract on PLA2 activity. After establishing effects of D. adscendens, it was worth finding out if the effects could be used as an assay to evaluate other medicinal plants purported to have anti-inflammatory properties. At the Center for Scientific Research into Plant Medicine, an aqueous suspension of powdered root bark of Cassia sieberiana (‘Kenken') is used to treat abdominal colic and pains associated with the joints. Likewise, hot aqueous decoction from grated whole shoot of Parquetina sp (‘Tina A ’) is used to treat asthma, and it is claimed to effect relief to patients under severe asthmatic attacks within a period of about 15 minutes (Mills-Robertson, personal communication). These plants could also be anti-inflammatory and may exert their effect by modulating eicosanoid production. 29 University of Ghana http://ugspace.ug.edu.gh Another aim of the work reported here was to use extracts from these two other plants, Kenken and Tina A, to verify the in vivo anti inflammatory assay developed using D. adscendens. This thesis, therefore, contains results of experiments carried out specifically i. to study the effect of extracts from D. adscendens on the in vivo production of eicosanoids with special reference to the relative production of pro-and anti-inflammatory eicosanoids ii. to investigate the effect of the plant on the activity of phospholipase A2 iii. to use these parameters for D. adscendens to assay for anti-inflammatory effects of extracts from Cassia sieberiana ( ‘Kenken’) and Parkitina sp V Tina A ’) 30 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO MATERIALS AND METHODS MATERIALS Guinea pigs Male guinea pigs were obtained from the Noguchi Memorial Institute for Medical research (NMIMR), the Korle-Bu Teaching Hospital, the Achimota School and from the open market. Medicinal Plant parts/preparations Powdered root bark of Cassia sieberiana (Kenken), grated whole shoot of Parquetina sp (Tina A), and a preserved aqueous extract of Desmodium adscendens, as dispensed to patients were obtained from the Center for Scientific Research into Plant Medicine (CSRPM), Mampong-Akuapem, Ghana. Chemicals and reagents The following enzyme immunoassay kits; prostaglandin E2 (PGE2) (Cat # 514010), 6 -keto-prostaglandin Fia (6 -keto-PGFia) (Cat # 515211), prostaglandin F2o(PGF2a (Cat # 516011), peptido-leukotriene (peptido-LT) (Cat #520501), thromboxane B2 (TXB2) (Cat #519031) and secretory phospholipase A2 (sPLA?) (human synovial) (Cat # 585000), as well as secretory phospholipase A2 (SPLA2) activity (Cat # 765001), were obtained from Cayman Chemical Company, Ann Arbor, U.S.A. 31 University of Ghana http://ugspace.ug.edu.gh Arachidonic acid (5,8,11,14-eicosatetraenoic acid), reduced nicotinamide adenosine dinucleotide triphosphate (NADPH), reduced glutathione (GSH) and Bovine Serum Albumin (BSA) were obtained from Sigma Chemical Company, St. Louis, U.S.A. Folin-Ciocalteau reagent and all other chemicals of analytical or higher grade which were used, were obtained from Fluka Chemie, Switzerland. METHODS Animals and Pretreatment Male guinea pigs weighing between 250 g and 350 g were selected. They were quarantined for, at least, two weeks in the Research Animals Unit of the NMIMR for clinical examination/observation before used. They were fed mainly on the leaves of the elephant grass (Panicum maximum) supplemented with pelleted animal feed. Drinking water was freely provided. Daily weight measurements were taken and plotted against time as a means of monitoring intake of food supply and detecting any possible defects resulting from handling. The guinea pigs were put in groups of five for the various treatments. Preparation of plant extracts The preserved aqueous extract of D. adscendens prepared for the treatment of patients attending the out-patient clinic at the Centre for Scientific Research into Plant Medicine (CSRPM), was kept at 4°C and used as such without any further modification A sample of 277.2 g of powdered Cassia sieberiana (Kenken) was 32 University of Ghana http://ugspace.ug.edu.gh mixed with 200ml of distilled water and stored at 4°C as stock solution. For Parquetina sp {Tina A), a sample of 9.4 g of the grated plant shoot was boiled in 150mls of distilled water for 2 minutes and strained using a muslin cloth. It was allowed to cool to room temperature and stored at 4°C. With the exception of the D. adscendens extract that was preserved, fresh stocks were prepared for “Kenken” and “Tim A” every five days. The quantities of plant materials used to prepare the stock solutions were estimated based on the proportions prescribed by the CSRPM for outpatients. Administration of plant extracts All three extracts were administered orally to separate groups of animals using graduated sterile syringes. Two different volumes of each of the extracts were administered to two groups of guinea pigs during the experimental period o f 28 consecutive days to obtain a high and a low dose for each extract. Control groups received water in place of the extracts. In one set of experiments three groups of five guinea pigs each, weighing between 250 g and 350 g, were used to evaluate the in vivo effect of D. adscendens extract. Animals receiving the low dose of extract were given 2 ml of the preserved extract of D. adscendens per day, and those receiving the high dose were given 5 ml of the same extract per day. The corresponding approximate dosages estimated for the animals are, 32.6 mg of freeze-dried extract/ kg body weight of guinea pig/day for the lower dose of 2 ml (designated as 2D), and 81.6 mg body weight of guinea 33 University of Ghana http://ugspace.ug.edu.gh pig/day for the higher dose of 5 ml (designated as 5D) (see Appendix II). The control group (designated as C) received 2 or 5 ml of water in place of the extract. In a different set of experiments, thirty-five male guinea pigs weighing between 250 g and 350 g were separated into seven groups of five animals per group. Extracts from D. adscendens and two other plants, C. sieberiana (Kenken) and Parquetina sp. (Tina A ) claimed to have anti-inflammatory properties, were orally administered over the experimental period of 28 consecutive days. Two groups of animals were given two different doses of each extract; a lower dose o f 2 ml and a higher dose of 5 ml per day. These were respectively estimated as 32.6 mg of freeze-dried extract/kg body wt/day (2D) and 81.6 mg of freeze dried extract/kg body wt/day (5D) for D. adscendens; and as 111.4 mg of freeze-dried extract/kg body wt/day (designated as 2T) and 278.4 mg/kg body wt/day (designated as 5T) for ‘Tina A ’ extract. For ‘Kenken’ however, the estimated dosages were for the fine-powdered plant material and not the freeze-dried extract: 285.3 mg of plant material/kg body wt/day (designated as 2K) and 713.3 mg of plant material/kg body wt/day (designated as 5K) respectively for the lower and higher doses. The control group, again, received water in place of the extract. Blood sampling and treatment On the 29* day, a maximum of 5 mis of blood was obtained from each animal by cardiac puncture. A sample of 100 ul of blood from each animal was quickly transferred into an eppendorf tube containing 900 nl of pre-chilled 25 mM Tris-HCl 34 University of Ghana http://ugspace.ug.edu.gh buffer (pH 7.5) to obtain 1:10 dilution This sample was used for determination of concentration of secretory phospholipase A2 (SPLA2). The remaining blood samples in the test tubes were allowed to clot at 4°C until a separated clear serum was observed. They were then centrifuged at 1500 r.p.m for 5 minutes using a bench centrifuge (Kubota KN-70, Japan). The serum for each sample was divided into small aliquots and kept in dry sterile clean eppendorf tubes. A tube of serum for each sample was kept on ice for use on the same day for estimating SPLA2 activity. The remaining tubes were kept frozen at -20°C and used within two weeks for the estimation of total protein content. Preparation of Microsomes Animals were sacrificed on day 29 by cervical dislocation. The lungs were removed and rinsed in pre-chilled 0.15 M KC1 solution on ice to get rid of blood. Each tissue was transferred into, and kept in a fresh solution of 0.15 M KC1 on ice before being used for preparation of microsomes. Each tissue was finely chopped with a clean pair of scissors in a prechilled beaker kept on ice. Approximately 40 mis of pre-chilled homogenizing buffer (0.25 M sucrose-10 mM Tris-HCl, pH 7.5) were added to approximately 6 g of the chopped lung tissue, which was then homogenized using a teflon-glass homogenizer (Glas- Col® Terre Haute, U.S.A.). The homogenate was transferred into a centrifuge tube and centrifuged at 10,000 g for 20 minutes at 4°C using a high-speed refrigerated centrifuge (Model 20PR-52D, Hitachi Koki Co., Ltd., Japan). The supernatant fraction was centrifuged at 105,000 g for 60 minutes at 4°C using a preparative 35 University of Ghana http://ugspace.ug.edu.gh ultracentrifuge (Model 80P-7, Hitachi Koki Co., Ltd., Japan). The microsomal pellet was homogenized in 0.1 M potassium phosphate buffer (pH 7.6) using a volume of buffer, not exceeding 3 mis, for a yield from a lung tissue weighing approximately 6 g. The resuspended microsomal preparation from each lung tissue was divided into small aliquots, dispensed into 1.5 ml capacity eppendorf tubes and stored at -80°C until ready for use. Microsomes were used within a maximum of three months. Protein determinations Two different methods for protein determination were employed: L Folin-Lowry method A volume of 5mls of alkaline copper sulphate solution was added to 1ml of test sample diluted to 1:10 with 0.1 M potassium phosphate buffer (pH 7.4). The mixture was allowed to incubate at 40°C on a water bath for 15 minutes. A volume of 0.5 ml of commercial Folin-Ciocalteau reagent (1:2 dilution) was then added to the mixture with rapid mixing. The final mixture was allowed to stand for 30 minutes at room temperature. Absorbance at 750 nm was read against a blank containing 1 ml of the buffer instead of the 1 ml 1:10 buffer-diluted sample. The protein concentrations were estimated from a standard curve of absorbances against corresponding concentrations of five serial dilutions from 1 mg/ml stock solution of Bovine Serum Albumin (BSA). Dissolution and serial dilutions of BSA were made with 0.1 M potassium phosphate buffer (pH 7.4). 36 University of Ghana http://ugspace.ug.edu.gh L Biuret method A volume o f 3 mis of Biuret reagent was added to 2 mis of test sample diluted 1:10 (for microsomal samples) and 1:100 (for serum samples) with 0.2 M NaOH solution and thoroughly mixed. The mixture was incubated at 37°C for 10 minutes, cooled to room temperature and the absorbance at 540 nm read against a blank containing 2 mis of 0.2 M NaOH in place of the diluted sample. The protein concentrations were estimated from a standard curve prepared with five serial dilutions from a stock solution of 5 mg/ml Bovine Serum Albumin (BSA). Dissolution and dilutions of BSA were made with 0.2 M NaOH solution. Eicosanoid Biosynthesis A total volume of 1ml reaction mixture consisted o f nricrosomes (0.92 mg/ml protein), 0.5 mM of either GSH or NADPH freshly prepared, and freshly prepared 15 oM arachidonic acid (AA) in 0.1 M potassium phosphate buffer (pH 7.4). All solutions/suspensions of the reaction mixture were maintained on ice before use. The mixture, without AA, was placed in a water bath with a shaker, maintained at 37°C, allowed to incubate for two minutes, before the addition of the substrate, 100 ql of freshly prepared 150 oM AA, to start the reaction. The reaction was terminated after 5 minutes for the cyclooxygenase reaction, in which GSH was the coenzyme, and after 15 minutes for the monooxygenase reaction with NADPH as the coenzyme. This was done by the addition of 100 ul of 1 M citric acid to bring the pH to < 3. The reaction mixture was quickly removed, kept on ice or stored in 37 University of Ghana http://ugspace.ug.edu.gh the refrigerator, and used within 8 hours for the determination of the type and amount of eicosanoids produced. Just before use, the pH of the mixture was adjusted upwards to pH 7.4 - 7.6 by adding 100 ul of 5M NaOH solution and mixing well by shaking. Reaction mixtures with no coenzyme were similarly treated. Quantitative Estimation of the Eicosanoids , The assay is based on competition between the specific eicosanoid (X) and an X- acetylcholinesterase conjugate (X-tracer) for a limited amount of X monoclonal antibody. Because the concentration of the tracer is held constant while concentration of X varied, the amount of the tracer that was able to bind to the monoclonal antibody would be inversely proportional to the concentration of the specific eicosanoid (X) in the well. This antibody-X complex got bound to a goat anti-mouse polyclonal antibody already coated on the inner walls of the wells. The wells of the microtitre plates were rinsed once with wash buffer. Fifty microlitres (50 ul) of the reaction mixture was pipetted into a well to which 50 ul of the tracer and 50 ul of the antibody were added. This was done in duplicates for each sample. The plates were covered with plastic film and incubated for 18 hours at 4°C or room temperature as prescribed for a particular eicosanoid kit. After the incubation period, the wells were washed five times with wash buffer to remove any unbound reagent. The buffer was removed from the wells by inverting the plate and shaking to remove the last drops. Two hundred microlitres (200 ul) of 38 University of Ghana http://ugspace.ug.edu.gh Ellman’s reagent which contains the substrate for acetylcholinesterase (acetylcholine) were added to each well and the plate covered with plastic film. The plates were allowed to develop in the dark for 60-120 minutes depending on the type of eicosanoid kit, and the extinction read at 405-450 nm. The intensity of the yellow colour resulting from the acetylcholinesterase activity, determined spectrophotometricaUy, is proportional to the amount of tracer bound to the well and inversely proportional to the amount of free eicosanoid (X) in the reaction mixture. Appropriate standards were included in the determination of the various eicosanoids. Also included were various incomplete reaction mixtures containing all but cofactor, or AA or microsomes. All resuspended reagents from the ELISA kits were kept at 4°C until used. A special CAYMAN ELISA microplate reader software was used to convert the absorbance values to the corresponding concentrations in pg/ml. The various eicosanoids evaluated were prostaglandins E2, F2o, 6 -keto Fia (for prostacyclin), thromboxane B2 (for thromboxane A2) and the peptidoleukotrienes. Assay for SPLA2 Activity This assay is based on the following principles: upon hydrolysis of the thioester bond at the sn-2 position by PLA2, free thiols react with DTNB to give a coloured product. Thus, the higher the rate of colour development, the higher the rate of release of free thiols, and the more active the PLA2 catalysing the hydrolysis. This assay was used for free or secretory PLA2 in serum 39 University of Ghana http://ugspace.ug.edu.gh Ten microlitres (10 ul) of undiluted serum sample, 10 ul of 5,5'- dithiobis (2- nitrobenzoic acid) (DTNB), and 5 ul of assay buffer (25mM Tris-HCl, pH 7.5 containing 10 mM CaCl2, 100 mMKCl, 0.3 mM Triton X-100, and 1 mg/ml BSA) were pipetted into wells and mixed by shaking the plate. Each sample was run in duplicate. Blank wells contained assay buffer in place of sample. Bee venom PLA2 replaced sample in the positive control wells. The reactions were initiated by the addition of 2 0 0 ul of reconstituted substrate (diheptanoylthio- phosphatidylcholine) solution. The contents of the wells were mixed by careful shaking. The absorbances at 405 nm were read at 10 different time intervals starting from the 2nd to the 50th minute after the introduction of substrate. Straight-line curves of absorbances against time were plotted for each specific sample well. The slope AA405, that is the rate of substrate breakdown, was determined from the line of best fit for each plot including plots obtained from the blank wells. Duplicate values for the slopes determined for each sample, control and the blank were averaged and the value for the blank, subtracted from those of the samples and positive control. The SPLA2 activity for each sample was then calculated from the following formula: sPLA2 Activity = Slope x *VI x sample dilution * e *v 40 University of Ghana http://ugspace.ug.edu.gh *£ = extinction coefficient for DTNB at 405nm (adjusted for solution path length in the well)=10.0mM‘l *V, = total volume (m l) o f m ixture in well *v = volume (m l) o f sample in well sPLA2 Activity = A A ^ /min x 0.225m l x 1 10.0 m M '1 0.01ml = mnol/min/ml Assay for Plasma SPLA2 Amounts This immunometric assay is based on a double-antibody “sandwich” technique. Each of the wells of the microtitre plate is coated with a monoclonal antibody specific for secretory phospholipase A2 (SPLA2 capture antibody). Any sPLA2 introduced into the well gets bound to the antibody. An acetylcholinesterase: Fab1 conjugate (AChE: Fab1), which binds selectively to a different epitope on the SPLA2 molecule, is introduced and it binds to the SPLA2 molecule forming a “sandwich” immobilized inside the wells. Excess reagent is washed away and the concentration of the analyte determined by measuring the enzymic activity of the AChE on addition of Ellman’s reagent containing the substrate for AChE. The product is coloured and can be measured spectrophotometrically. The intensity of 41 University of Ghana http://ugspace.ug.edu.gh the colour is directly proportional to the amount of bound conjugate, which, in turn, is proportional to the concentration of the SPLA2. Secretory phospholipase A2 Enzyme Immunoassay kit was used to assay for the amount of plasma SPLA2 as follows: the wells of the microtitre plates were rinsed once with reconstituted wash buffer. Hundred microlitres (100 ul) each of serially diluted sPLA standard solutions and blood samples diluted 1:10 with 25 mM Tris- HC1 buffer (pH 7.5) were pipetted into the wells. One hundred microlitres (100 nl) o f acetycholinesterase: SPLA2 Fab1 conjugate was added to each of the wells except the ones labelled blank. The plate was then covered with plastic film and incubated overnight (18 hrs) at 4°C. The wells contents were discarded and rinsed five times with reconstituted wash buffer. A volume of 200 ml of reconstituted F'lm an’ s reagent was added to each well, the plate was covered with a plastic film and allowed to develop in the dark for at least 60 minutes. The absorbances of the yellow colour developed were read at 405-450 nm on a microplate spectrophotometer (WF043 Denley “We” Scan, England) Secretory Phospholipase A2 (sPLA2) concentrations in pg/ml were estimated from a standard curve obtained by plotting absorbances against concentration of serially diluted SPLA2 standards. Average absorbances for duplicate readings for each sample and standards were used. 42 University of Ghana http://ugspace.ug.edu.gh Statistical Analysis Analysis of variance (ANOVA) was used for all statistical analyses because three or more treatment conditions were compared for each set of experiments. Statistical significance was calculated at p < 0.05. 43 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE RESULTS A. EICOSANOID BIOSYNTHESIS I: Effect of Desmodium adscendens extract Eicosanoids produced using microsomes prepared from lungs of guinea pigs treated with or without aqueous extracts from Desmodium adscendens are presented in this section. The group of guinea pigs which did not receive any extract, the control group, is designated C, the group which received a lower dose of the extract is designated 2D and that which received a higher dose of the extract is designated 5D. When GSH was used as a cofactor, there was an overall increase of prostanoids compared to the controls except for PGF2a. Prostaglandin E2 production in the 2D group was not significantly different from that for the control; its production in the 5D group was comparatively higher than in both the C and 2D animals. The production of 6 -keto-PGFia in the 2D was significantly higher than that in the control group. Its production in the 5D group was very high compared to both the C and 2D groups. Extract administration at both doses, did not affect PGFm production levels. Although production of TXB2 in both 2D and 5D animals were 44 University of Ghana http://ugspace.ug.edu.gh significantly higher than in the control group. There was no statistically significant difference between the two values (Table Al). TABLE Al Eicosanoid production in the presence of GSH as a coenzyme Values are mean + SEM, n=5 Type and amount of prostanoid (ng/ml) Sample p g e 2 6-Keto-PGFja PGF2a TXB2 C 2.5±0.7 0.03±0.004 1 .0 2 ±0 . 2 2 3.20±0.48 2D 2.7±0.4 *0.06±0.010 1.30±0.39 *6.30±2.10 5D *+17.0±5.0 *+1.9±0.020 1.02±0.35 *4.80±1.10 * _ significantly different from control + _ significantly different from corresponding 2D value C = co n tro l: no extract administered 2D = lower extract dosage: ~32.6mg/kg body wt/day 5D = higher extract dosage: ~ 8 1.6mg/kg body wt/day see appendix II The data expressed as percentage increases over the controls are shown in figures 6 and 7. As indicated in figure 6 , higher percentage increases were recorded for 6 - keto-PGFia and TXB2 compared to the increases in PGE2 and PGF20 for the 2D 45 University of Ghana http://ugspace.ug.edu.gh treatments. On administration of a higher dose of extract (5D) however, very high percentage increases over the controls were recorded for both PGE2 and 6 -keto- PGFla (Figure 7). Fig. 6: Effect of D. adscendens extract on prostanoid production with GSH as cofactor expressed as % change over control; dose = 2ml of preserved extract/animal/day (2D). 2D = lower extract dosage: ~32.6mg/kg body wt/day see appendix II 46 University of Ghana http://ugspace.ug.edu.gh TYPE OF PROSTANOID Fig. 7: Effect of D. adscendens extract on prostanoid production with GSH as cofactor expressed as % change over control; dose = 5ml of preserved extract/animal/day (5D). 5D = higher extract dosage:- 81.6mg/kg body wt/day see appendix II 47 University of Ghana http://ugspace.ug.edu.gh With NADPH as the coenzyme for eicosanoid production, the monooxygenase pathway was to be favoured. However, the unavailability of appropriate ELISA kit did not allow for the evaluation of products via this pathway. Instead, the effect of adding NADPH on the cyclooxygenase pathway was examined by quantifying the prostanoids produced. The amount of PGE2 produced in response to the lower dose of the extract (2D) indicated significant increase over the control, but that in response to the higher dose (5D) did not indicate a significant difference from the control. The amounts of 6 -keto-PGFia produced with both doses were the same, and significantly higher than the control value. Significant increase in PGF2a production over the control was recorded for the lower dose but not for the higher dose. Significant increase in TXB2 over the control was recorded for the lower dose. The 5D value was not statistically different from the control value, but significantly lower than the value obtained at the lower dose (Table A2). 48 University of Ghana http://ugspace.ug.edu.gh Prostanoid Production in the Presence of NADPH as a Coenzyme Values are + SEM, n**5 TABLE A2 Type and amount of prostanoid (ng/ml) Sample p g e 2 6-Keto-PGFia PGF2a t x b 2 C 4.6±0.9 0.044±0.006 0.60±0.09 2.70±0.66 2D * 7.6±1.3 *0.058±0.009 *0.80±0.10 *4.90±0.86 5D +4.8±0.7 *0.058±0.014 0.66±0.14 +2.80±0.61 * _ significantly different from control + _ significantly different from corresponding 2D value C = control: no extract administered 2D = lower extract dosage: ~32.6m g/kg body wt/day 5D = higher extract dosage:- 81.6mg/kg body wt/day see appendix n Results from reaction m ixtures with no cofactor indicated trem endous variations in prostanoids amounts for the 2D treatm ents compared to their control levels. There was no significant difference in the amount o f PGF2o produced; the amount o f TXB2 declined significantly, while PGE2 and 6- keto-PGFia showed significant increases at 2D. All prostanoid amounts recorded for 5D anim als were very low compared to their corresponding values in both the 2D and C groups (Table A3). 49 University of Ghana http://ugspace.ug.edu.gh Prostanoid production without the addition of a coenzyme Values are mean + SEM, n=S TABLE A3 Type and amount of prostanoid (ug/ml) Sample PGE2 6 -Keto-PGFia p g f 2o TXB2 C 9.2±1.2 32.0±3.4 0.36±0.05 400±70 2D * 14.0±1.4 *61.0±6.5 0.36±0.03 *200±32 5D *+5.4xl0‘4 ±5.3x1 O' 5 *+5.8xl0"4 ±6 . lx l O' 5 *+3.6xlOJt ± 4.7x1 O' 5 *+1.5xl0' 2 ±2.6x1 O' 3 * _ significantly different from control + _ significantly different from corresponding 2D value C = control: no extract administered 2D = lower extract dosage: ~32.6mg/kg body wt/day 5D = higher extract dosage:- 81.6mg/kg body wt/day see appendix II The ratios of anti- to pro-inflammatoiy prostanoids were expressed as PGE2/PGF2a and 6 -keto-PGFio/TXB2 to assess if the effect of the plant extract would be better expressed in this manner. The lower dose of D. adscendens did not affect the PGE2/PGF20 ratio in order of magnitude for all three different reaction mixtures, that is, when GSH and NADPH were used as cofactors and when no cofactor was used. For the higher dose of 50 University of Ghana http://ugspace.ug.edu.gh extract, when GSH was employed as the cofactor, there was an increase in this ratio: an increase one order of magnitude greater than the control value (TableA4). A similar trend was observed for the PGE2 production alone. TABLE A4 Ratio of anti- to pro-inflammatory prostanoids (PGE2/PGF2«) compared to PGE2 only in the presence of GSH Sample p g e 2/p g f 2« PGE2 (ng/ml) C 2.45 2.5 2D 2.07 2.7 5D 16.62 17 C = control: no extract administered 2D = lower extract dosage: ~32.6m g/kg body wt/day 5D = higher extract dosage: ~81.6m g/kg body wt/day see appendix II When GSH was used as a cofactor, the ratio 6 -ketoPGFi„/TXB2 was not changed in response to the lower dose of the extract. However, with a higher dose, there was an increase of two orders of magnitude over the control (Table A5). The trend was the same when production of only 6 -keto-PGFiawas considered. 51 University of Ghana http://ugspace.ug.edu.gh Ratio of anti- to pro-inflammatory prostanoids (6-keto- PGFia/TXB2) compared to 6-keto PGF|„ only in the presence of GSH TABLE A5 Sample 6 -keto PGF1o/TXB2 6 -keto PGFia (ng/ml) C 8.90x10'j 3x1 O’2 2D 9.39x1 O' 3 6 x 1 0 1 5D 3.91x1 O' 1 1.9 C = control: no extract administered 2D = lower extract dosage: ~32.6mg/kg body wt/day 5D= higher extract dosage:- 81.6mg/kg body wt/day see appendix II The effect of D. adscendens on the synthesis of peptido-leukotrienes with different cofactor is shown in table A6 . The data, presented as percentage changes over the control values are shown in figure 8 . When GSH was used as a cofactor, the amount of peptido-leukotrienes synthesized for both the lower (2D) and higher (5D) doses of the extract were not significantly different from each other (i.e. no dose effect), but both were significantly higher than the control value. A higher percentage increase was observed for 2D than for 5D. 52 University of Ghana http://ugspace.ug.edu.gh When NADPH was used as the cofactor, the amount of leukotrienes synthesized in response to extract administration was significantly higher than the control value only at the higher dose of the extract (5D). The values, expressed as percentage changes over the control, is presented in figure 8 , showing virtually no change in 2D but a high percentage increase in 5D. When no cofactor was added, the amount of peptido-leukotrienes produced was much greater than when cofactor was added. In contrast to the reaction mixtures with cofactors, the amount o f peptido-leukotrienes were reduced in response to administration of the extract when no cofactor was added. The reductions in the amount of peptido-leukotrienes produced, which were statistically significant, occurred in a dose dependent manner. 53 University of Ghana http://ugspace.ug.edu.gh TABLE A6 Effect of D. adscendens on peptido-leukotriene level in the presence and absence of cofactors Values are mean ± S. E. M., n=5 Cofactor used values quantified in pg/ml) Sample GSH NADPH NO COFACTOR C 2.53xl02±24.35 77±8.49 2.85xl05±3.74xl04 2D *5.92x102±77.85 82±6.13 *9.19x104±7.25xl 03 5D *4.87x102 ±87.87 *+140±24.71 *+5.08xl03 ±4.90x102 * - significantly different from control + _ significantly different from lower dose C = control: no extract administered 2D = lower extract dosage: 2m 1 o f preserved extract/anim al/day ~32.6mg/kg body wt/day 5D = higher extract dosage: 5ml o f preserved extract/animal/day ~ 81,6mg/kg body wt/day see appendix II 54 University of Ghana http://ugspace.ug.edu.gh TYPE OF COFACTOR Fig. 8 : Effect o fj). adscendens on peptido-leukotriene production expressed as % change over control on addition of different cofactors; doses of extract represented by 2D and 5D. 2D = lower extract dosage: 2ml o f preserved extract/animal/day ~32.6mg/kg body wt/day 5D = higher extract dosage: 5ml o f preserved extract/animal/day ~ 81,6mg/kg body wt/day see appendix II 55 University of Ghana http://ugspace.ug.edu.gh II: Effect of other medicinal plants D. adscendens was used as positive control to which Cassia sabriena (kenken) and Parquetina sp (Tina A) were compared. Extracts from the three plant materials were administered in two doses to the experimental animals daily for 28 consecutive days before the animals were sacrificed, and microsomes, as source of enzymes, prepared from their lungs. The reaction mixtures contained GSH as coenzyme. For convenience, the different aqueous extracts from D.adscendens, Tina A and kenken have been respectively represented by D, T and K. The numbers 2 and 5 preceding any of the letters, represent the lower and higher dosages of the extracts respectively. The results are presented in Table A7. At a higher dose (5D), D. adscendens extract caused significant increases in the amounts of PGE2 and 6 -keto-PGFia synthesized over their corresponding lower dose (2D) and control (C) values. The 2D values in turn, were higher than the control values for both eicosanoids, the increase being significant only for 6 -keto- PGFia. Similar trends with respect to the effect of D.adscendens on the synthesis of PGE2 and 6 -keto-PGFi0 were observed for kenken but not for Tina A. In the case o f Tina A, significant increases in PGE2 and 6 -keto-PGFia productions at both dose levels 56 University of Ghana http://ugspace.ug.edu.gh over their control values were observed. The amount of 6 -keto-PGFia produced in 2T and 5T were not significantly different from each other. With respect to the synthesis of PGF20 and TXB2 , none of the two dose levels for all three extracts caused any significant change from the control value except for a significant reduction in PGF201 value at the higher dose of D. adscendens (5D), and a significant increase in TXB2 produced at the lower dose. Effect of the extracts on PGE2 and 6 -keto-PGFia production as percentage changes over the controls are shown in figure 9. Generally, percentage increases for 6 -keto- PGFiaare higher compared to corresponding PGE2 increases. For 6 -keto-PGFi0 the % increase for the higher dose of D. adscendens (5D) is the most pronounced; there is no significant difference between 2T and 5T (for Tina A) whereas there is a significant difference for kenken (2K and 5K). Ratios of anti- to pro-inflammatory prostanoids expressed as PGE2/PGF2a and 6 - keto-PGFia /TXB2 for all three extracts are shown in Table A8 . Extracts effect on 6 -keto-PGFia /TXB2 were more pronounced than for PGE2/PGF2a. The lower dose of D. adscendens did not seem to affect the control value of the ratio 6 -keto-PGFia/TXB2. However,the lower doses of ‘Tina A ’and 1 Kenken’ caused appreciably high increases. All three extracts effected very large increases in the ratio at their higher doses with D. adscendens indicating the highest effect 57 University of Ghana http://ugspace.ug.edu.gh followed by ‘Tina A'. A similar trend was observed when only the absolute values o f the anti-inflammatory 6 -keto-PGFia were considered, except that the lower dose of D. adscendens effected some appreciable increase in this case (Table A8 ). For the ratio PGE2/PGF2a, the higher doses of all three extracts caused increases in the range of about twice as much the control value, but the lower dose values were not any different from the control value (Table A8 ). The trend was the same when production of PGE2 only was considered (Table A7). 58 University of Ghana http://ugspace.ug.edu.gh TABLE A7 Effect of different plant extracts on types and amounts of prostanoids produced in the presence of GSH as cofactor Values are means ±S . E. M, n=S Type and amount of prostanoid (pg/ml) sample p g e 2 6 -keto-PGFia PGF2n t x b 2 C 0.17±0.01 2.08±0.38 2.89±0.51 9.09x10J±0.00 2D 0.18±0.03 *3.01±0.51 3.50±0.55 *1.77xl04±7.76xl0j 5D *+0.28±0.05 *+31.49±4.24 +2.53±0.40 +9.09xl0J±0.00 2T *0.22±0.04 *8.67±0.49 2.98±0.59 9.09xl03±0.00 5T *+0.30±0.03 *9.29±0.53 2.63±0.41 9.09x10J±0.00 2K 0.18±0.02 *3.21±0.47 2.49±0.56 9.09x10j±0.00 5K *+0.27±0.04 *+8.08±0.64 2.75±0.67 9.09x10^0.00 * _ significantly different from control value + _ significantly different from lower dosage value C = control: no extract administered 2D= lower extract dose for D.adscendens: ~32.6mg/kg body wt/day 5D= higher extract dose for D.adscendens - 81.6mg/kg body wt/day 2T= lower extract dose for Tina A : —111.4 m g/kg body wt/day 5T= higher extract dose for Tina A: -2 7 8 .4 m g/kg body wt/day 2K= lower extract dose for Kenken : -285 .3 mg/kg body wt/day 5K= higher extract dose for Kenken :~ 713.3 mg/kg body wt/day see appendix n 59 University of Ghana http://ugspace.ug.edu.gh Ratio of anti-to pro-inflammatory prostanoids for different plant extract compared to anti-inflammatory prostanoids alone TABLE A8 sample p g e 2/ p g f 2„ 6 -keto-PGF i a/TXB2 C 5.90xl0"2 2.28x1 O' 4 (0.17) (2.08) 2D 5.22xl0 ' 2 1.70x1 O' 4 (0.18) (3.01) 5D 1.09x1 O'* 3.47x1 O' 3 (0.28) (31.49) 2T 7.43x10’2 9.55xl0’4 (0 .2 2 ) (8.67) 5T 1.16x10‘‘ 1 .0 2 x 1 O’" (0.30) (9.29) 2K 7.35xl0"2 3.54x10"* (0.18) (3.21) 5K 9.75xl0"2 8.90X10"4 (0.27) (8.08) Figures in parentheses represent only P G E j or 6-K eto-PG Fia m easured in pg/ml C = control: no extract administered 2D= lower extract dose for D. adscendens: ~32.6mg/kg body wt/day 5D= h igher extract dose for D. adscendens:~& 1,6mg/kg body wt/day 2T= lower extract dose for Tina A : ~ 1 11.4 mg/kg body wt/day 5T= higher extract dose for Tina A: -2 7 8 .4 m g/kg body wt/day 2K= lower extract dose for Kenken : -285 .3 mg/kg body wt/day 5K= higher extract dose for Kenken : -713 .3 m g/kg body wt/day see appendix II 60 University of Ghana http://ugspace.ug.edu.gh oaI- 2Q(J 2o LL a: £ Fig. 9: Effect of D. adscendens (D), Tina A (T) and Kenken (K) extracts on anti­ inflammatory prostanoid production expressed as % change over control;doses represented by 2D, 5D, 2T, 5T, 2K, SK 2D= lower extract dose for D.adscendens: ~32.6m g/kg body wt/day 5D= higher extract dose for D. adscendens:-^ 1,6mg/kg body wt/day 2T= lower extract dose for Tina A : —111.4 m g/kg body wt/day 5T= higher extract dose for Tim A: -278 .4 m g/kg body wt/day 2K= lower extract dose for Kenken : -285 .3 m g/kg body wt/day 5K= higher extract dose for Kenken : -713 .3 m g/kg body wt/day see appendix II 61 University of Ghana http://ugspace.ug.edu.gh B: PHOSPHOLIPASE A2 ACTIVATION Groups of guinea pigs receiving aqueous extracts of either Desmodium adscendens, Parquetina sp. (Tina A) or Cassia sieberiana (Kenken) are designated D, T and K respectively. The numbers 2 and 5 preceding any of the letters represent the lower and higher doses of the extracts administered. C represents the control group that received no extracts. Enzyme activity Oral administration of the different plant extracts in two doses resulted in significant decreases in SPLA2 activity with respect to the basal (control) level. The extent of inhibition was about the same for both the lower (2D) and higher (5D) doses of D. adscendens extracts. The same was true for Kenken which had a greater inhibitory effect. Tina A extracts exerted similar inhibitory effect which was dose-dependent, that is, a significant difference was observed between enzyme activity for the two doses. The extent of inhibition of SPLA2 activity was greater for Tina A at the higher dose than for D. adscendens and Kenken (Table Bl). sPLA2 protein The higher dose of D. adscendens effected a significant increase in