STRUCTURAL ELUCIDATION AND ANTIPLASMODIAL ACTIVITIES OF SOME COMPOUNDS ISOLATED FROM THE RHIZOME OF COCHLOSPERMUM TINCTORIUM A THESIS submitted to the UNIVERSITY OF GHANA In partial fulfilment o f the requirement for the degree o f MASTER OF PHILOSOPHY IN CHEMISTRY B Y PAUL OSEI-FOSU, BSc. (Hons) CHEMISTRY OCTOBER 2001 DEPARTMENT OF CHEMISTRY UNIVERSITY OF GHANA LEGON. University of Ghana http://ugspace.ug.edu.gh 3 6 8 2 3 5 $£ \bS>C6 Osl \ L j k ^ • $ - r v University of Ghana http://ugspace.ug.edu.gh DEDICATION To the omnipotent, the omniscient and to my parents for theirLove and care University of Ghana http://ugspace.ug.edu.gh DECLARATION I declare that the experimental work described in this thesis was preformed by me in the Department of Chemistry, University of Ghana, Legon and the Immunology Unit of Noguchi Memorial Institute for Medical Research, University of Ghana, Legon under supervision and has not been presented for a degree in this University or any other University elsewhere for another degree. SUL OSEI-FOSU (CANDIDATE) DR. I. \{/ OPPONG (SUPERVISOR) (SUPERVISOR) PROF. W. A. ASOMANING (SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I acknowledge my deep appreciation to my supervisors, Prof. W. A. Asomaning. Dr. I. V. Oppong and Dr. R. K, Akuamoah of Chemistry Department, University of Ghana, Legon, Dr. B. D. Akanmori of the Immunology Unit, Noguchi Memorial Institute for Medical Research, (NMIMR) Legon and Dr. A. Sittie of the Centre for Scientific Research into Plant Medicine. (C.S.R.P.M.) Mampong, Akwapim who despite their tight schedules made time to give their constructive criticism and contribution, which made this work a success. I am also indebted to Prof. W. L. Phillips, Dr. L. Duamekpor, Dr. Dorcas Osei-Sarfo and Mr. Asunka for their advice, encouragement and guidance in the course of this project. 1 also wish to express special thanks to Dr. S. B. Christensen, Royal Danish School of Pharmacy, Copenhagen for running the Nuclear Magnetic Resonance (NMR) and Mass spectra of the isolated compounds and for assistance in interpreting the spectra. My sincere thanks and appreciation to Mike Ofori and the staff of the Immunology Unit, Noguchi Memorial Institute for Medical Research, Legon for assistance with the antiplasmodial studies. Special mention must be made of the Danish International Development Assistance (DANIDA) as part of the Accra-Copenhagen Research Link for funding this work. I am indeed very grateful to them. University of Ghana http://ugspace.ug.edu.gh I extend my gratitude to my senior colleagues, G. Duker-Eshun, E. Oppong, A. Aning, A. A. Appiah, Nana Oppong-Wusu, A. Quarcoo, A. Tawiah and S. Afful for their encouragement and assistance in the course of this work. My final appreciation goes to the entire staff of the Departments of Chemistry and Botany, University of Ghana, Legon and to the Centre for Scientific Research into Plant Medicine (C.S.R.P.M.), Mampong Akwapim, for all the assistance they gave me while undertaking this project. University of Ghana http://ugspace.ug.edu.gh ABSTRACT Cochlospernum tinctorium A. Rich was investigated for its chemical constituents and its antiplasmodial activities. The chemical constituents of petroleum ether, dichloromethane and ethyl acetate extracts of the rhizome of the plant were successively studied. The in vitro antiplasmodial activities for petroleum ether, dichloromethane, ethyl acetate, ethanol and water extracts as well as the isolated compounds were determined. Three compounds isolated from the dichloromethane extract have been identified by spectroscopic methods to be triacontanol, triacontanyl p-coumarate and triacontanyl ferulate. This is the first report of the isolation of the two esters and the long chain saturated alcohol from the plant. Another compound was obtained from the dichloromethane extract and coded D9. The petroleum ether extract of the rhizome yielded stigmasterol and two compounds which were coded PE6 and PE11. The ethyl acetate extract of the rhizome yielded two compounds which were coded E13 and E166 Using an in vitro technique, the inhibition of growth of Plasmodium falciparum (chloroquine sensitive, 3D7 and chloroquine resistance DD2, strains,) were investigated based on the principle of uptake of 3H-hypoxanthine by viable parasites on various extracts of Cochlospermum tinctorium and also on the isolated compounds with chloroquine as standard. University of Ghana http://ugspace.ug.edu.gh The percentage inhibition for all the extracts and isolated compounds were found to be dose-dependant, with the aqueous and dichloromethane extracts being more active with IC50 values 32.27 (ig/ml and 24.34 |J.g/ml for the chloroqiune sensitive and 30.90 jig/ml and 23.45 |j.g/m! for the chloroquine resistant strains respectively. University of Ghana http://ugspace.ug.edu.gh CONTENTS Dedication ... ... ........................................................ Declaration ... ... ... .............................. Acknowledgements ... ... ................ ................ Abstract ... ... ................ ... ................. Contents ................................................................................... CHAPTER ONE Introduction ... ... ........................................................ 1.1.Ethnobotanical uses of Cochlospermum tinctorium A.Rich 1.2.Ethnobotanical uses of some species of Cochlospermacene 1.2.1. Cochlospermum vitifolium [.2.2.Cochlospermum religiosum 1.2 3 .Cochlospermum planchonii 1.3. Aim of the project ................ ................ CHAPTER TWO Literature Review ... ........................................... 2.1. C. tinctorium 2.2. C. angolense 2.3. C. regium 2.4. C. gossypium. vii PAGE 1 11 iii v vii 1 2 4 4 4 5 5 7 7 12 13 14 University of Ghana http://ugspace.ug.edu.gh 2.5. C. planchonii ... ... .............................. 2.6 . C. vitifolium 2.7. The Disease Malaria ........................................... 2.7.1. Life cycle of the malaria parasite ................ 2.7.2. Clinical manifestations........................................... 2.7.3.Laboratory Diagnosis .............................. 2.8. Antimalarials ... ................ ................. 2.8.1. Biological classification of antimalarials 2.8.2. Pharmacological classification of antimalarial... 2.8.3. Drug resistance in malaria...................................... 2.8.3.1. Resistance to the common antimalarial drugs 2.9. Plants as sources of antimalarial ... ................ 2.10. In vitro cultivation of Plasmodium CHAPTER THREE Result and Discussion ................ ................ 3.1. Fractionation of Petroleum ether ex trac t... 3.1.1. Characterization of PE6 ................ ................ 3.1.2. Characterization of PE8 (Stigmasterol)................ 3.1.3. Characterization of PEI 1 ... ................ 3.2. Fractionation of Dichloromethane extracts 3.2.1. Characterization of D5 (Triacontanol)................ 3.2.2. Characterization of D65 (triacontanyl ferulate) 15 16 24 25 26 26 27 27 28 30 30 31 33 37 38 39 41 42 45 46 48 viii University of Ghana http://ugspace.ug.edu.gh 3.2.3. Characterization of D6& (triacontanyl p-coumarate) 3.2.4. Characterization of D9 ... 3.3. Fractionation of Ethyl acetate extract ........................................... 3.3.1. Characterization of E13 ................ 3.3.2. Characterization of E166 ...................................................................... 3.4. Antiplasmodial activities of crude extracts and compounds isolated from the rhizome of C. tinctorium (using P. falciparium in vitro culture) CHAPTER FOUR Experimental ... ... ... ... .............................. 4.1. General Methods 4.2. Collection and Treatment of plant material .............................. 4.3. Reagents... ... ................ ................ ................. 4.4. Extraction ........................................................ .............................. 4.5. Phytochemical screening ... ........................................... 4.6. Investigation of extracts........................................................ ................. 4.7. Antiplasmodial assay .............................. .............................. 4.8. M ethods... ........................................................ ................. 4.9. Extraction of the rhizome of C. tinctorium ................ 4.10. Preparation of various concentrations of crude extracts from the rhizome of C. tinctorium 4.11. Preparation of various concentrations of isolated compounds from the rhizome of C. tinctorium ix 53 57 59 60 60 62 77 77 79 79 81 82 84 97 99 100 100 102 University of Ghana http://ugspace.ug.edu.gh 4.12. In vitro cultivation of malaria parasites... ... ... 1"3 4.13. Fixation and Staining ... ••• ••• ••• 1®^ 4.14. Cryopreservation of parasites ... ... ... ••• 105 4.15. Subculture. ... ... ••• 106 4.16. In vitro inhibition assay for extracts and isolated compounds from the rhizome of C. tinctorium ... ... 106 4.17. Dimethylsulfoxide (DMSO) trials ................................... ... ... 107 Reference ... ... 108 Appendix I. Infrared (IR) spectra ... ........................................... ... 116 Appendix 1!. Proton ( 1H) Nuclear Magnetic Resonance (NMR) spectra ... 126 Appendix III. Carbon-13 ( l3C) Nuclear Magnetic Resonance (NMR) spectra 146 Appendix IV. Mass spectra ( M S ) ........................................................................ 154 Appendix V. Ultra Violet (UV) spectra .......................................................... 158 Appendix VI Homonuclear proton-proton shift correlation spectra (COSY) 164 Appendix VII. The life cycle of the malaria parasite... ... 166 Appendix VIII. Buffers and Solutions .............................. 168 x University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION Cochlospermum tinctorium is bushy, attains about 50 cm height with annual shoots arising from a perennial woody stock, and handsome golden yellow flowers which render it worthy of horticultural cultivation. Its time of flowering is during the rainy season. It is of widespread occurrence in savanna and scrubland throughout the drier parts of the West African Region, and, indeed seemingly in the devastated, rocky and annually burnt areas. It occurs also in Cameroun, Sudan and Uganda. In Ghana it is found in the savanna region.5 The Centre for Scientific Research into Plant Medicine at Akwapim-Mampong, Ghana has been vigorously investigating the therapeutic values of various plants used in traditional medicine against common diseases including malaria. In northern Nigeria, the rhizomes of Cochlospermum tinctorium A. Rich is used locally to treat febrile episodes including that due to malaria2. Malaria is an important public health problem, killing over one million people every year and infecting about half a billion in total. It remains endemic in 102 countries and more than half the world’s population is at risk. There are probably more than 100 million cases of the disease throughout the world each year, of which perhaps a million are fatal. Inspite of control programmes in many countries, the malaria situation has shown little improvement within the past 15 years and this is partly 1 University of Ghana http://ugspace.ug.edu.gh due to world economic and/or political problems, notably in India and China. Malaria is the most common infectious disease reported at the outpatient departments of health institutions in Ghana and accounted for 40-42 % of all outpatients attendance from 1985-1987. It also accounted for 7-8% of all certified deaths in the 0-4 year age group4. This situation is aggravated by the development of resistance by various strains of Plasmodium falciparum to some commonly used antimalarials. Nonetheless in the absence of an effective vaccine against the disease prompt diagnosis and appropriate treatment using an effective antimalarial drug remains the bedrock of malaria control throughout the world. In order to avoid the large financial effort to develop new drugs for prophylaxis and therapy, special attention has been paid in recent years to the use of traditional medicine.5, 6' 7’ 8 The great success in this field was the isolation of compounds from the plant Artemisia annua. This plant is used in traditional Chinese medicine.9,10 The scientific basis for the use of Cochlospermum tinctorium against malaria has not been completely validated. The principal aim of the present study is to address this gap in knowledge. 1.1 ETHNOBOTANICAL USES OF COCHLOSPERMUM TINCTORIUM A. RICH The pulped leaves of Cochlospermum tinctorium are used in Cote d’Ivoire to treat abscesses and furuncles. The oil from the seeds is used in treating leprosy. The unripe 2 University of Ghana http://ugspace.ug.edu.gh fruit capsules are however eaten by hunters to allay thirst. The fruit contains floss, which is used to stuff cushions. In Togo the plant is regarded as the father of cotton and it is spun into necklace cords. The Chambas of northern Nigeria use a drink from a mixture of the fruit and that of tamarindas as an antidote for snakebite.1 The powdered root is applied topically in Cote d’Ivoire and Burkina Faso to treat snakebite. The root along with other drug plant is used in Cote d ’Ivoire and Burkina Faso for treating leprosy. The roots are also known to be used against ascites, beriberi and various oedemas in Burkina Faso. In Cote d ’Ivoire and Burkina Faso the roots are used for oedematous conditions, for orchites, schistosomiasis, jaundice, fevers, epilepsy, pneumonia, intercostal pains and bronchial infections, in eye-instillations for conjunctivitis and for indigestion and stomach pains. 1 In Gambia, the aqueous extract of the roots is given to women at childbirth. A root- infusion is used by the Fula cattlemen in Guinea to arrest diarrhoea in calves. The root is chewed in Nigeria as a tonic. In Senegal and Cote d ’Ivoire the root has a reputation as an efficient decongestant, and as a venal vaso constrictor it is said to be effective in reducing haemorrhoids where surgery would normally be indicated. 1 The root is used in Nigerian folk medicine to treat skin infections and water extracts of the roots have shown activity against Gram-positive organisms. The root may be crushed up with potassium salts obtained from vegetable ash and boiled, and with the addition of indigo a wider range of colour is achieved. A yellow or brownish — 3 University of Ghana http://ugspace.ug.edu.gh yellow dye is obtained from the root which is used to colour shea butter and cooking oil and may perhaps also impart some flavour. 1 The root is taken in baths for urino-gential disorders and kidney and intercostal pain. The young stem-bark yields a useful fibre. In Cameroun, cattle will not graze on related Cochlospermum species even in time of shortage of grass, thus suggesting that there may be some toxic substances in the other Cochlospermum species.1 1.2.ETHNOBQTANICAL USES OF SOME SPECIES OF COCHLOSPERMACEAE 1.2.1 Cochlospermum vitifolium (Willdenow) Sprengel The plant is a small tree with attractive yellow flowers. It is found in Mexico, the Caribbean and savanna regions in West Africa. In Ghana it is under cultivation at the Cocoa Research Institute. In Brazil, the wood is used to make fishnet floats and also in Mexico the bark yields fibre, which is used to make rope. Its concoction is taken for jaundice.1 1.2.2. Cochlospermum religiosum (Linn.) Alston The plant is a sparsely branched shrub, it is found in the drier parts of India, but now cultivated in several areas in West Africa. The dried leaves and flowers are said to be a stimulant. The floss surrounding the seeds is an inferior substitute for ‘kapok’. The seeds contain non-drying oil reported in Indian material to amount to 14-15% and are used in soap-manufacture. The residual seed cake is used as manure. The bark contains a cordage fibre.1 4 University of Ghana http://ugspace.ug.edu.gh The tree yields a gum, katira gum, which is insoluble in water. When mixed with gum arable, it gives a water-borne adhesive paste. The gum is sweetish, cooling and sedative and it is used in cough medicines. The gum is used in cigar and ice cream manufacture and can be used as a substitute for gum tragacanth in various industrial processes.1 1.2.3 Cochlospermum planchonii Hook. f. The plant is a shrub, which attains about 2 2.5m height. It is widespread in the region from Senegal to Western Camerouns and into Eastern Cameroun. It borne yellow flowers during its time of flowering in the rainy season.1 In Sierra Leone and Northern Nigeria the stem-bark is used in making string and rope. The seeds are also used as beads, while the root is a source of a yellow dye in Sudan and Northern Nigeria. In Lagos, Nigeria the root is used in cooking soup when oil is not available. A root decoction is drunk in northern Sierra Leone for the treatment of gonorrhoea. An extract of the root is said to control menstruation. The Fula of Northern Nigeria claim that a leaf-infusion bestows magical protection. 1 1.3. Aim of the Project The major aim of this project is two fold (i) To evaluate the in vitro activity of the crude extracts from the rhizome of Cochlospermum tinctorium (Petroleum ether, Dichloromethane, Ethyl Acetate, Ethanol and Water) on strains of Plasmodium falciparum. 5 University of Ghana http://ugspace.ug.edu.gh (ii). To isolate, purify and elucidate the structures of some natural products from the rhizome of Cochlospermum tinctorium and to evaluate their in vitro activity on the strains of Plasmodium falciparum. University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW Cochlospermum tinctorium A. Rich belongs to the family Cochlospermaceae. A review of the literature suggests that inspite of the fact that C. tinctorium has been used in folk medicine for the past century for treating malaria and other diseases, not much has been done to ascertain which compounds in the plant are active. It is interesting to find in the literature that even though most parts of the plant such as fruit, leaves, stem and rhizomes are used extensively in folk medicine the bulk of scientific investigation has been done on the leaves and fruits with few reported studies on the rhizomes. A number of carotenoids and flavonoids have been isolated and characterized from the leaves and flowers. Some polyphenol compounds, tannins, tri acyl benzenes triterpene and quercetin have also been isolated from the rhizome. In this chapter, a brief description of the work done on the Cochlospermaceae species up to the year 2000 is discussed. The report includes chemical and biological studies. 2.1. Cochlospermum tinctorium A. Rich Earliest work on this plant was by Benoit et al 11 in 1995 who analysed the antimalarial activity in vitro of Cochlospermum tinctorium tubercle extracts. The tubercle extracts were obtained by infusion and decoction and were tested in vitro on 2 strains of Plasmodium falciparium, (FcBl-Columbia) chloroquine resistant and (F32-Tanzania) chloroquine sensitive. 7 University of Ghana http://ugspace.ug.edu.gh The 50% inhibitory concentration (IC50) was determined by measuring [3H]- hypoxanthine incorporation into parasite DNA and also by microscopical examination. The IC50 values obtained were of the order l-2ug/ml, about one-tenth of those reported for the extract of neem leaves (Azadirachta indica) and about half the values reported for Artemisia annua extracts. Similar results were obtained with fresh extracts, frozen extracts and lyophilized extracts of C. tinctorium. The results also suggested that the traditional use of C. tinctorium extracts to treat malaria is based on a real anti-parasitic activity. The apparent stability of the anti malarial activity after either freezing or iyophilization is potentially valuable. Fran^oise Benoit-Vical ei al 12 analysed the in vitro antimalarial activity and cytotoxicity of Cochlospermum tinctorium and C. planchonii leaf extracts and essential oil. The leaf extracts were obtained by decoction. The oil components were extracted by hydrodistillation. The crude extracts and oils were tested for in vitro antimalarial activity on Plasmodium falciparium strain (FcBI-Columbia) chloroquine-resistant. The method used was the radioactive micromethod of Desjardins et al.13. The IC50 were evaluated after 24 and 72hr contacts between the oils and the parasite culture, and ranged from 22 to 500 ug/ml. C. planchonii leaf oil yielded the best antimalarial effect (IC50: 22-35 jxg/ml), while the most potent effect from crude leaf extracts was induced by C.tinctorium (1CS0 3 .8 -7 .5 ja.g/ml) The cytotoxicity of the leaf crude extracts and oils was assessed on the K562 cell line and showed IC50 values ranging between 33 and 2000p.g/ml. For C. tinctorium, the essential University of Ghana http://ugspace.ug.edu.gh oil characterized has a high proportion (40%) o f oxygenated aliphatic components such as alcohols, aldehydes, ketones and esters and for C. planchonii the major component was sesquiterpenes (80%) with predominance of hydrocarbons: p-caryophyllene and famesenes. The total yield obtained for the essential oil was 0.05% for C. planchonii and 0.2% for C. tinctorium. From the result it was suggested that since decoction is traditionally used C. tinctorium could be preferable to C. planchonii because C. tinctorium extract was more active than C. planchonii. Diallo et al 14 also reported that C. tinctorium is used in African traditional medicine for the treatment o f liver diseases. The hepatoprotective activity of the rhizome o f C. tinctorium was investigated using carbon-tetrachloride toxicity on mouse and tert-butyl hydroperoxide in vitro induction of lipid peroxidation and hepatocyte lysis. Aqueous hydro-ethanolic and ethanolic extracts showed significant dose-dependent hepatoprotective actions. The ethanolic extract showed a hepatoprotective activity of lower doses. The ethanolic and hydro-ethanolic extracts established remarkable effects against the induction o f lipid peroxidation and hepatocyte lysis; the aqueous extract showed comparatively weaker effects. These differences were related to the chemical composition o f the extracts. Carotenoids such as cochloxanthin (6-hydroxy-8'-apo-6-caroten-3-one-8'-oic acid) and dihydrocochloxanthin (4,5-dihydro-6-hydroxy-8'-apo-e-caroten-3-one-8'-oic acid), flavonoids such as 5,4'-dimethlyquercetm and 7j3 I*dimethyldihydraquercefin) were found in both ethanolic and hydro-ethanolic extracts and only in minute amounts in the 9 University of Ghana http://ugspace.ug.edu.gh aqueous extract and this could be responsible for the protection against the t-BH intoxication. These compounds are antioxidant. Quercetin derivatives (5,4’-dimethylquercetin and 7,3’-dimethylquercetin), arjunolic acid and major triterpene (aromadendrene, 5-cadinene, a-pinene a-selinene and a-humuiene) were also isolated from the ethanolic extract o f C. tinctorium , which showed antihepatotoxic and anti-inflammatory activities. Gallic acid, ellagic acid as well as ellagitannins were isolated from the three extracts studied. These take a prominent part in the antihepatotoxic activity of the traditional preparations from C. tinctorium rhizome. Rabate1' who investigated in 1938 reported the first critical examination o f C. tinctorium that the rhizome contains a large quantity o f starch, which resembles manioc (50-60 % of the dried drug). The friable powdered rhizomes readily yield starch on treatment with water. Diallo and Vanhaelen 16 isolated cochloxanthin and dihydrocochloxanthin from the rhizome o f C. tinctorium using high-speed counter current chromatography on a horizontal flow through coil-planet centrifuge. This method was used because carotenoids are well known for their instability. Analytical method such as HPLC and preparative TLC were also used to separate the constituents. Diallo and Vanhaelen ! 7 did some chemical investigation on extract from the rhizome of C. tinctorium. Powdered dry rhizomes (500g) were extracted exhaustively with cold 10 University of Ghana http://ugspace.ug.edu.gh MeOH by percolation. The extract was evaporated to yield a syrup, which was mixed with cellulose (lOOg). The mixture was percolated successively with 1. I Litre petroleum ether 2. 2.5 Litres ethyl acetate 3. 0.5 Litres CHCI3 4 . 350 ml CHCl3-M e2CO (1:1) mixture 5. 200 ml CHCb-MeOH (9:1) mixture 6. 200 ml CHCl3-MeOH (8:2) mixture 7. 450 ml. CHC13-MeOH (1:1) mixture The CHCI3 MeOH (1:1) fraction was evaporated and partitioned with CHGb-MeOH- H:0 (5:3.5:1.5.600ml). The residue of the chloroform extract was subjected to column chromatography on Silica gel (lOOg). The column elution was achieved with CHCI3 containing increased amounts of MeOH. Arjunolic acid was detected in the CHCh-MeOH (9:1) fractions. It was further purified by preparative TLC on Silica gel in CHCb-EtOAc-HOAc (8:0.5:1.5) and finally on a C|g Silica gel column eluted with MeOH-HzO (6:4, 8:2) to remove pigments. It was crystallized from EtOH / Me2CO / H2O. The Rf value of arjunolic acid on Silica gel in CHCh-MeOH (9.5:0,5) was 0.51. The arjunolic acid, its triacetate derivative and its methyl ester were tested using the short-term in vitro assay on EBV-EA activation in Raji cells induced by 12-0- tetradeconylpho-bol- 13-acetate (TPA). Their inhibitory effects on skin tumor promoters were found to be greater than previously studied natural products as 7-0-acetylcfromosin, retinoic and glycyrrhetinic acids.18 11 University of Ghana http://ugspace.ug.edu.gh In 1987, Diallo et al 19 used a combination of column and preparative thin layer chromatography to isolate polyphenol compounds (gallic and ellagic acids) and carotenoids from the methanol extract of the rhizome of C. tinctorium. The methanol and ethanol extracts were investigated for antihepatotoxic activity using carbon tetrachloride and galactosamine - induced cytotoxicity in primary culture rat hepatocytes. It was detected that the extracts and the isolated compounds exhibited antihepatotoxic effect. In 1991,Diallo et a l20 isolated some triacylbenzenes and long-chain volatile ketones from C. tinctorium rhizome. Eleven compounds were isolated from the volatile fraction through the use of steam distillation under N2 from the air-dried and powdered rhizome. GC, NMR and GC-MS were used to investigate the compositions of the volatile fraction, among the 11 constituents detected, 8 were identified as straight chain ketonic compounds. The triacylbenzenes were isolated from a petroleum ether extract, separated by HPLC and identified by the NMR and MS, 2.2. Cochlospermum angolense (Welw) An aqueous extracts of the roots of C. angolense is used for the treatment of icterus and in combination with other materials for the prophylasis of malaria.21 Presber et a l22 tested a red coloured crystalline product isolated from chloroform extract, on the growth of P. falciparum (M-25 / Zaire) and P. bergbei (Gdansk). The multiplication of P. falciparum was decreased to 50% of the control in the presence of lOjig/ml extracted material and there was a total inhibition at a concentration of 50fJ.g/ml. 12 University of Ghana http://ugspace.ug.edu.gh Mice erythrocytes infected by P. bergbei were incubated for 6 hours with 25ug/ml of the extract. The result showed that the DNA synthesis was depressed to nearly background level. Nogueira Prista and Correia de Silva 23 found flavones and carotenoids in the bark of C. angolense. Also isolated from the plant are the following compounds, Oleanolic acid (mpt 308-309°), Quinone, C |2H20 , (mpt 79-81°), a fatty substance (mpt 63-70°), Carotenoid and a Fiavone.24 2.3. Cochlospermum regium In 1995, Lima et al 25 isolated a dihydrokaempferol 3-0-glucopyranoside, C21H22O 11, melting point 177-179°C (0.4%) from the rhizome of C. regium. The following are the spectral peaks: 'H-NMR (200mHz, MeOH) 7.37 (2H, d, J = 8.6Hz), 6.81 (2H, d, J = 8.6Hz), 5.92 (1H, d, J = 2.1Hz) 5.89 (1H, d, J = 2.1Hz) 5.27 (1H, d, J =10.2Hz), 4.98 (1H, d, J= 10.2Hz), 3.79 (1H, d, J -7.52Hz), 3.76 (1H, dd, J =12.3, 2.2Hz), 3.60 (1H, dd, J =12.3, 5.7Hz), 3.07- 3.35 (3H, m), 2.92-3.05 (1H, m); l3C-NMR (50 MHz, MeOH-cU): 8 83.55 (C-2), 71.52 (C-3), 196.11 (C-4), 103.06 (C-4a), 164.20 (C-5), 97.40 (C-6), 169.03 (C-7), 96.33 (C-8), 165.50(C-8a), 128.52 (C -l’), 130.40 (C-2’), 115.72 (C-3’), 159.30 (C-4’), 116.25 (C-5’), 130.47 (C-6’), 102.60 (C -l”), 74.55 (C-2”), 78.22 (C-3”), 71.24 (C-4”), 77.58 (C-5”), 62.60 (C-6”); 13 University of Ghana http://ugspace.ug.edu.gh DC1-MS (NH3) m/z 468 [(M + 18), 64%], 451 (24), 342 (22), 306 (72), 298 (35), 271 (22), 256 (6), 198 (6), 180 (100), 162 (11), 127 (23), Other compounds that have also been isolated from this plant include Quercetin derivatives, arjunolic acid, 14 apocarotenoids 17 and triacylbenzenes together with long chain volatile Ketones.20 2.4. Cochlospermum gossypium DL, syn C. religiosum Ramachandraiah et a l 26 in their investigation isolated (3-sitosteryl-glucoside (0.3%) from the flowers of C. gossypium. The gum of C. gossypium is sweetish, cooling, sedative, useful in coughs, hoarse throat and scalding in the urine. It is also used for treating diarrhoea and dysentery,27 the young leaves are used for cooling the hair, 27 while the dried leaves and flowers are used as stimulant.28 Purification of the gum was done by precipitation by alcohol from acidified aqueous solutions. The ash-free gum is a white, amorphous, hygroscopic powder.29 Tannins have been identified in the 50% alcoholic extract of the stem bark.30 Naringenin has also been isolated from the flower o f C. gossypium.3I 14 University of Ghana http://ugspace.ug.edu.gh 2.5. Cochlospermum planchonii Hook, f Aliyu et al 32 in their investigation isolated zinc formate from the rhizome of C. planchonii by using inhibition of two rat cytochrome P-450 enzymes, aminopyrine-N- demethylase and aniline hydroxylase, as bioassays to guide fractionation by solvent partitioning, polyamide column chromatography, preparative thin layer chromatography and fractional crystallization. The zinc formate was shown to be a hepatoprotective cytochrome P-450 enzyme inhibitor. The inhibitor did not melt at temperatures up to 300 °C and the ’H-NMR spectrum in dimethylsulfoxide-dg contained broad signals (consistent with poor solubility) at 1.6-2.0 ppm (assigned to H-C=0) and at 3.0-4.0 ppm (water). The 13C-NMR spectrum contained a single signal at 165ppm (carbonyl). The infrared spectrum contained strong absorptions at 1370cm'1 and 1600cm'1 (carboxylate anion stretching). The Ultraviolet absorption spectrum lacked significant absorption at wavelength longer than 250mu. The FAB mass spectrum contained no significant peaks over lOOm/e. Ashing in a gas flame left a copious white powder completely soluble in dilute hydrochloric acid. It was also analyzed by inductively coupled plasma atomic emission spectroscopy. All these results indicated that the crystalline inhibition was zinc formate. Addae-Mensah et al 33 in their investigation isolated four novel long-chain triacylbenzenes from the apolar fraction of C. planchonii rhizomes. The triacylbenzenes were separated by HPLC into 2 major compounds and 2 minor compounds, except for the MS all the compounds showed very similar spectroscopic behavior. 15 University of Ghana http://ugspace.ug.edu.gh 2.6. Cochlospermum vitifolium, Willdenow (Sprengel) The root of C. vitifolium is used as a remedy against jaundice in middle American folk medicine and as a dye. Achenbach et al 34 isolated two new 7'-apocarotenoic acid (vitixanthin and dihydrovitixanthin) from the methanolic root extracts using repeated column chromatography and HPLC. The electronic spectra show maxima (MeOH) at 422, 398, 377 and 359 (sh) nm, which indicates a carotenoid heptaene chromophor. The Homo- and heteronuclear COSY experiments established structural details, which resemble cochloxanthin and dihydrocochloxanthin as far as part of the carotenoid polyene chain, are concerned. 16 University of Ghana http://ugspace.ug.edu.gh Table 1: COMPOUNDS ISOLATED FROM COCHLOSPERMUM SPECIES Name and Molecular formula Structure Source Gallic acid (3.4,5-trihydroxybenzoic acid) c 7h6o 5 c 0 O H ' H 0 0 HO H Rhizome 19 Apigenin 5,7-Dihydroxy-2- (4- hydroxyphenl)-4H-1 - benzopyran-4-oneC 15H 10O5 OH 0 HO/ ^ ^ OH Flower36 l,3-di(dodecanyl)-5-tetradecanoylbenzeneC44H76O3 co(CH2)lda% AH3C(CFfc)12OC COCCH^ idCH, Rhizome 20 and Rootbark33 1,3,5-tridodecanoyl-benzeneC42H72O3 CO(CH2),oCH3 AH3C(CH2)ioOC CO(C5^ );rjCH3 Rhizome 20 and Rootbark33 1,3,5-tri(tetradecanoyi- benzeneC4SH84O3 COCCH2)i2CH3 AH3C(CH2)i20C CO(CH2)i2CH3 Rhizome20 and Rootbark33 .......... 17 University of Ghana http://ugspace.ug.edu.gh 3,5-di(tetradecanoyl)-l-dodecanoylbenzeneC46H80O3 CO(CH2)i0CH3 XXH3C(CH2)12OC CO(CH2),2CH3 Rhizome 20 and Rootbark33 DihydrovitixanthinC33H44O6 / k A , 10 Flower34 1,3-di(tetradecanoy l)-5- hexadecanoylbenzene C5oHg8C>3 c c x c h 2)12c h 3 rSH3C(CH2)i40( r ^ !!S^ C O (C H 2)i2CH3 Rhizome 20 VitixanthinC33H42O6 0 Flower34 Capsanthin(3,3’-Dihydroxy-p,k-caroten-6 ’-one)C40H56O3 „ £ C A ' ' J — r ^ r 4 p - Flower36 Zeaxanthin (P, (3-carotene-3,3’-diol) C40H56Q2 . OH Flower36 18 University of Ghana http://ugspace.ug.edu.gh Kaempferol (3,4’, 5,7-tetrahydroxy- flavone) C 15H 10O6 OH 0 ho^ ^ o- ^ Y ^ I ^ ^ ^ O H L eaf37 Naringeninc 15h 12o 5 OH 0 H O ^ ^ O - N p i ^ ^ O H Entire plant31 Taxifolin (3’ ,4' ,5 ’ ,7-tetrahydroxy- dihydroflavonol)C 15H 12O7 OH O l ^ J l o H 1 1 / L .OHHO O ^ V V ^ ^ O H Entire Plant38 Myricetin(3,3’,4’,5’,7-Hexahydroxyl-flavone)C 15H 10O8 OH 0j ^ ^ l l ^ O H ^-OHHO 0 ^ Y y 5#’^ OH OH Leaf37 Quercetin (3 ’ ,4’ ,5,7-tetrahydroxy- flavonol)C 15H 10O7 OH O JL 1 OH ho^ ^ - oJ y Y ° H Leaf37 19 University of Ghana http://ugspace.ug.edu.gh S-Cadinene C15H24 Rhizome 35 a-Pinene(2,6,6-Trimethylbicyclo-[3,l,l]hept-2-ene) C10H16 Rhizome 35 {3-Bisabolene (1 -methyl-4-(5-methyl-1 - methyJene-4-hexenyl)cyclo- hexene) C15H24 Rhizome 20 a-SelineneC 15H24 Rhizome35 Gentisic acid (2,5-Dihydroxybenzenoic acid)C7H6O4 COOH OH HO Flower 36 AromadendreneC 15H24 Rhizome 35 20 University of Ghana http://ugspace.ug.edu.gh Ellagic acid (23,7,8- Tetrahydroxy[l]benzo- pyrano[5,4,3-cde][ 1 ]benzo- pyran-5,10-dione) CuFUOg 0y ~ ° \ o h HO 0—4 O Leaf 19 Lycopene\j/,\ji-caroteneC40H56 — ™ Flower36 a-Humulene 2,6.6.9-Tetramethyl-1,4,8- cycloundecatriene C 15H24 £ Rhizome35 Trans caryophyllene Ci5H24 Hy Rhizome 35 Arjunolic acid ^ '0 f ] H p c°OH Rhizome 14 Cochloxanthin (6-hydroxy-8'-apo-e- caroten-3-one-8'-oic acid) CioHjgCXt Rhizome 14 21 University of Ghana http://ugspace.ug.edu.gh Dihydrocochloxanthin(4,5-dihydro-6-hydroxy-8'-apo-e-caroten-3-one-8'-oicacid) C30H40O4 Rhizome 14 5,4’-dimethylquercetin OH I . O C H 3 0 H OCH3 0 Rhizome 14 7,3’-dimethylquercetin OCH3 CH3W ° v U I I I 0H OH O Rhizome 14 2-Tridecanone (C ,3H260 ) CH3-(C H 2),o-CO-CH3 Rhizome 20 i -Dodecanol (C I2H260) CH 3-(CH 2) io-CH 2OH Rhizome 20 1-Tetradecanol (C14H30O) CH3-(C H 2) 12-CH2OH Rhizome20 3-Octadecanone(CisH^feO) CH3-(C H 2) ,4-CO— c h 2- c h 3 Rhizome20 22 University of Ghana http://ugspace.ug.edu.gh 1 -Hydroxy-3-octadecanone (C 18H36O2) CH3-(C H 2) 14-CO— CH2-CH2OH Rhizome 20 3-Hexadecanone (C I6H320) CH3- (C H 2) i2-CO-CH2~CH3 Rhizome 20 1 -Hydroxy-3-hexadecanone (C ,6H3202) CH3-(C H 2) 12-CO— c h 2- c h 2o h Rhizome 20 1 -O-Acetyl-3-hexadecanone (C ,8H3602) CH3-(C H 2) 12-CO— CH2-C H 2OAc Rhizome20 2-Pentadecanone (C15H30O) CH3- (C H 2)12-CO-CH3 Rhizome 20 1-Nonadecanol (C19H40O) CH3- (C H 2)i7-CH2OH Rhizome 20 Zinc formate HCOOZn 0I H - C — 0— Zn Rhizome32 23 University of Ghana http://ugspace.ug.edu.gh 2.7. THE DISEASE MALARIA Malaria, a major public health problem worldwide, is caused by a protozoan parasite of the genus Plasmodium, The major species of this genus are Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae. The deadly P. falciparum is becoming increasingly resistant to available drugs and the number of drugs under development is dwindling. Malaria is transmitted from one vertebrate host to another by the female mosquito vector the most efficient of this species being Anopheles gambiae. Parasites of the genus Plasmodium undergo the sexual portion of their life cycle in a mosquito. The sequence begins when a mosquito, after biting a vertebrate ingests a gametocyte-infected blood meal and ends with a second blood meal during which sporozoites are passed with the mosquito’s saliva into a new vertebrate host(39,40). The parasite must overcome a number of barriers to develop in a mosquito. Immediately after ingestion, gametocytes emerge from red blood cells and differentiate into male and female gametes. These gametes are fertilized and the resulting zygotes develop into ookinetes within the gut. The ookinetes enter the body cavity (hemocoel) by crossing the gut lining (peritrophic matrix) and the midgut epithelium, where they subsequently lodge on the hemocoel side of the gut epithelium, beneath the basal lamina. The ookinetes then develop into oocysts. Oocysts undergo many rounds of nuclear division and produce thousands of sporozoites that are released into the hemocoel. Sporozoites invade the 24 University of Ghana http://ugspace.ug.edu.gh salivary glands to be injected into a vertebrate host about 2 weeks after the mosquito 41ingested the first infected blood meal. 2.7.1. Life cycle of the m alaria parasite The development of each of the four species of human plasmodia starts with the phase in which the direct progeny of sporozoites injected into the circulation by the bite of an infected female anopheles mosquito enter the liver where they grow and multiply in the parenchymatous cells. This pre-erythrocytic tissue schizogony is completed towards the end of the incubation period of the infection, which ranges from 7-37 days, when large numbers of tissue merozoites from ruptured tissue schizonts are released into the blood stream. In this form the parasite invades the erythrocytes, grows and multiplies asexually from trophozoites to mature blood schizonts, which release merozoites, and produces all the clinical symptoms of the disease. Some erythrocytic forms develop into two types of sexual parasites (gametocytes), which unite when taken up by a suitable female anopheles mosquito that has ingested the blood of the infected individual. Eventually, after the gradual stages of ookinete and oocyst, large numbers of sporozoites are produced and stored in the salivary glands of the female anopheles mosquito. These ensure the transmission of the disease when injected into a human host. The life cycle of the human malaria parasite is shown in Appendix VII. In some species {P. vivax), the merozoites originating from pre-erythrocytic tissue schizogony re-enter liver cells and continue their development as secondary exoerythroctic forms which are responsible for producing relapses of malaria with 25 University of Ghana http://ugspace.ug.edu.gh clinical symptoms.42' There is evidence to suggest that the relapses may be due to the presence of two or more populations of parasites in the liver cells, one developing as the typical liver schizonts and the other (termed hypnozoites) persisting for some time as small, non-developing uninucleate parasites. The latter may initiate cycles of development weeks, months or years after the initial infection, giving rise to the so-called relapses.43 2.7.2. Clinical manifestations After an incubation period ranging between 7 and 37 days for most cases of naturally transmitted malaria, the first signs of clinical malaria appear suddenly. These consist of headaches, malaise, anorexia, nausea, fatigue and dizziness.44 A typical paroxysm starts with a feeling of cold accompanied by shivering, pallor and cyanosis. In children, it may present as a convulsive seizure. Other symptoms include dry cough, abdominal pain and vomiting. Irregular fever with the usual symptoms is not the only clinical picture of falciparum malaria. Because of the rapid multiplication of the Plasmodia o f this species and their tendency to invade the internal organs, severe complications may appear suddenly. Drowsiness, coma, delirium, bloody diarrhoea, severe haemolytic anaemia, pulmonary oedema hyperpyrexia and renal failure indicate the involvement of various organs. Death may occur unless there is rapid diagnosis and adequate treatment. 2.7.3. Laboratory Diagnosis Diagnosis of malaria is confirmed when a Giemsa-stained blood film obtained from the patient’s thumb shows the presence of malaria parasites under a microscope. Most 26 University of Ghana http://ugspace.ug.edu.gh diagnosis can be made with a well-prepared and stained thick film but in the case of doubtful species diagnosis, or mixed infections, the thin film can often be the final arbiter.45 2.8. Antimalarials 2.8.1. Biological classification of antimalarials Since various stages in the life cycle of malaria parasites show different susceptibility to antimalarial drugs, these drugs have been classified into the following groups: 1. Tissue schizontocides (used for causal prophylaxis): These act on the pre-erythrocytic stages of the parasites (primary tissue forms or primary exo-erythrocytic forms) and thus completely preventing invasion o f the blood cells. 2. Tissue schizontocides (used as antirelapse drugs): These act on the exoerythrocytic stages or tissue forms o f P. vivax and P ovale and thus able to achieve radical cure of these infections. An example is Proguanil 3. Schizontocides (blood schizontocides or schizontocidal drugs): These act on the erythrocytic stages o f parasites commonly associated with acute disease, though these stages may be present in some infections accompanied by few clinical symptoms. They also act on the sexual erythrocytic forms o f P. vivax, P. ovale and P malariae but not directly on the mature gametocytes of P,falciparum. Examples are quinine, chloroquine and amodiaquine. 4. Gametocytocides (gametocytocidal drugs): These act on all sexual forms including those of P..falciparum, they also act on the developmental stages o f malaria parasites in the anophelines. Examples quinine, primaquine. 27 University of Ghana http://ugspace.ug.edu.gh 5. Sporontocides (sporontocidal drugs): These prevent or inhibit the formation of oocyts and sporozoites in anophelines that have fed on carriers of gametocytes. Proguanil and primaquine are examples.46 2.8.2. Pharmacological classification of Antimalarials Antimalarial drugs may be broadly classified in two groups: lysosomotropic quinoline - containing drugs and antimetabolites. The lysosomotropic quinoline - containing drugs include quinine and quinidine, the 4-aminoquinolines, Chloroquine and amodiaquine, amopyroquine and the quinoline-methanol, mefloquine. The antimetabolities drugs include Lincomycin, actinomycin, cycloieucins, mitomycin and many others. Chloroquine Quinine Amopyroquine c f 3Mefloquine 28 University of Ghana http://ugspace.ug.edu.gh Amopyroquine Lincomycin OH Quinoline-containing drugs have been the mainstay of antimalarial treatment for decades and are still widely used. Although the clinical use of quinolines is hampered by various degrees of parasite resistance 47- 48, there is still interest in drugs that share the same biochemical target. The main target for quinoline antimalarial is haempolymerization, process whereby intraerythrocytic - stage malaria parasites detoxify haem in the digestive vacuole. Haem is a by-product of haemoglobin digestion and is used by the parasite as source of most of its essential amino acids 49. It is potentially toxic to biological membrances and parasite enzymes 50 51 and is thus sequestered in the form of an insoluble crystalline polymer haemozoin (or malaria pigment)49. Quinoline antimalarials have been shown to inhibit both synthetic and native polymerization 52 53 54, Chloroquine appears to act by forming quinoline - haem complexes which terminate haemozin chain extension 55' 56. The ability of drugs to inhibit haem polymerization is directly related to their antimalarial potency 53. University of Ghana http://ugspace.ug.edu.gh The classification of antimetabolites is complex and controversial. Their modes of action are very specific, three main mechanisms being in operation: inhibition of synthesis of the bacterial cell well, increased permeability of cytoplasmic membranes and interference with intracellular protein or nucleic acid synthesis. There are some correlations between the mode of action and the general spectrum of activity of antibiotics.57 2.8.3. Drug resistance in malaria Drug resistance in malaria has been defined as the “ ability of a parasite strain to survive and/ or to multiply despite the administration and absorption of a drug given in doses equal to or higher than those usually recommended but within the limit of the subject” . Although the resistance of the drug embraces all species of malaria parasites and all acceptable dosages of blood or tissue schizontocides, gametocytocides and sporontocides, in practice it is most commonly applied to the effect of blood schizontocides on falciparum malaria.57 2.8.3.1 Resistance to the common antimalarial drugs Human malaria parasite, P. falciparum has developed resistance to chloroquine and other drugs including amodiaquine and quinine. The other parasites have also developed resistance to proguanil, pyrimethamine and related compounds. In Ghana resistance of P. falciparum to chloroquine and other antimalarials has been reported.58 Resistance by P.falciparum to chloroquine, as well as all species to proguanil and pyrimethamine, is attributable to selection under drug pressure of resistant mutants 30 University of Ghana http://ugspace.ug.edu.gh which survive by utilizing alternative metabolic pathways to those blocked by the particular drug. In respect of chloroquine, resistance is characterized by a decrease in high-affinity binding sites for the drug. Once selected, and provided that they escape the destructive action of the host immunity, the resistant parasites may be transmitted by local mosquitoes to other people in the immediate area, or may be carried by a migrant host to other places where mosquitoes may or may not be present to establish transmission.57 2.9. Plants as sources of antimalarials The use of medicinal plants as a source of relief from malaria, which is one of the oldest infections mentioned in early writings in Egypt, India and China could be traced back to over 2000 years. Attempts were made to treat it by the use of roots, leaves and flowers of many plants, example was the use of powdered roots of Ch’ang shan (Dichroa febrijuga) in China for a least 2000 years. Today, plants are almost the exclusive source of drugs for the majority of the world’s population.57 In industrialized countries, medicinal plants research has had its ups and downs during the last decades. However, substances derived from higher plants constitute about 25% of prescribed medicines. Extracts of medicinal plants have mainly been superseded by the pharmaceutical preparations of the developed countries. However, natural products from higher plants, fungi and bacteria continue to be used in pharmaceutical preparations either as pure compounds or as extracts.59 31 University of Ghana http://ugspace.ug.edu.gh The structure-activity relationships o f naturally derived antimalarials are studied with a view to improving the therapeutic effects of such drugs and reducing their toxic effects. The importance to medicine of natural product molecules lies not in their pharmaceutical or chemotherapeutic effects but also in their role as template molecules for the production of new drug substances. Quinine, which was isolated from cinchona bark was found to have undesirable toxic side effect such as impaired hearing on prolonged use. Attempts to modify the structure of the drug showed that the quinoline ring was essential for activity. Synthetic antimalarials such as chloroquine were developed in which the activity of quinine was retained but the toxicity reduced.?9 Many tropical countries have a list of useful plants for treating malaria. Examples are Africa (Ghana, Nigeria), North America (Mexico) and South East Asian (Malaysia, Indian) countries. There have been numerous attempts to test plant extracts for antimalarial activity and in the most extensive programme reported in 1947, over 600 plants from 126 families were screened for in vitro activity against P. gallinacewn in chicks and against P.lophurae in ducklings. Species from some 33 genera gave positive results and the most significant levels o f activity were found in extracts o f species from the Amaryllidaceae and from the Simaroubaceae. The latter family contains a number of genera, which are used in indigenous medicine for a range of activities in many different countries. The active ingredients are degraded triterpenes known collectivity as quassinoids or simaroubolides. The biological activites of these compounds include anticancer, antiviral, insecticidal and anti-inflammatory activities. Several quassinoids are potent inhibitors o f protein synthesis in P falciparum in vitro. 59 32 University of Ghana http://ugspace.ug.edu.gh The plants used medicinally for the treatment of malaria include Brucca javanica in Thailand, Castela nichosoni, which was used in Brownsville, Texas for treatment when quinine had failed and Picrasma antidesma in British Honduras. In another study, the antimalarial effect of the stem bark of three Khaya species was investigated. It showed that the order of activity of the three species was K. Ivorensis > K. grandifoila > K. senegalensis while only K. grandifolia at 50 mg/kg/day was active against established infection.59 The antimalarial activities of several African plants used in treating malaria have been investigated. These plants include Cassia siamea, Dialium guineense, Dichapetalum guineense, Gomphrena celosioides, Jatropha gossypiifolia, Nauclea latifolia, Paullinia pinnata and P. crassipes. The aqueous extracts of C. siamea, J. gossypiifolia and P. crassipes were most effective against P. falciparum on a dose-dependent basis. 59 An in vitro antimalarial test, utilising the inhibition of uptake of 3H-hypoxanthine into Plasmodium falciparum cultured in human blood was used to assess the activity of four solvent extracts of Nauclea latifolia-, aqueous, methanol, ethanol and chloroform. The percentage inhibition for all the extracts were found to be dose-dependent, with the methanol extract showing the strongest inhibition of 99% at a concentration of 500 M-m/ml. All the extracts were found to possess moderate activity against P. falciparum . 59 2.10. In vitro cultivation of Plasmodium. For the estimation of the antimalarial effects of plant extracts, either in vivo or in vitro methods are used. In in vivo work, various dilutions of the extract based on LD 50 values 33 University of Ghana http://ugspace.ug.edu.gh are administered to rodents infected with the rodent malaria parasite, P. berghei and the following effects determined: (1) Blood schizontocidal activity on early infection (4-day test). (2) Repository effect, i.e. the ability of the extract to prevent infection or its prophylactic effect. (3) Effect on established infection. A number of studies have been published in which in vivo methods are reported. ^ 61 When in vivo methods are used, one is not certain whether an active ingredient in the extracts or a metabolite of such an active principle is responsible for any observed activity. Briefly, In vitro work for the assessment of plant-derived antimalarials, includes incubation of the extracts or fractions with P. falciparum and using standard methods as discussed by Jensen and Trager 62 to measure the inhibition of growth and multiplication of the parasite. Microscopic examination of Giemsa-stained thin blood films can be used to determine the parasitaemia. Alternatively, the degree of inhibition of uptake of a radiolabelled nucleic acid precursor like 3H-hypoxanthine by the parasites can be used to measure the extent of inhibition of growth of the parasites.63 Labeled hypoxanthine is used because a functional de novo purine biosynthetic pathway has not been demonstrated for the malaria parasite; consequently, they require an exogenous supply of preformed purines. The preferred purine for both P. lophurae and P. falciparum appears to be hypoxanthine, which is derived intraerythrocytically through the 34 University of Ghana http://ugspace.ug.edu.gh monophosphate —» inosine monophosphate —> inosine —» hypoxanthine. In contrast to the malaria parasite, the human host has the ability to synthesize purines de novo. The purine nucleotides are assembled from a variety of precursors. Glycine provides C-4, C-5 and N-7. The N-l atom comes from aspartate. The other two nitrogen atoms, N-3 and N-9, come from the amide group of the side chain of glutamine. Activated derivatives of tetrahydrofolate furnish C-2 and C-8, whereas CO2 is the source ofC-6. The use of the 96- well microtiter plate for such inhibition assays, together with the use of 3H-hypoxanthine as an index of parasite growth provides a simple and convenient method which utilizes a minimum of scarce resources, as microlitre volumes are used. It also yields results rapidly (within 48 hours), large enough for statistical analysis and is easily reproducible. It is also more reliable, because it is based on the metabolism of purine in the parasite and hence more accurate. This method is usually preferred over microscopic methods because of the reasons mentioned. Scintillation counting is used in this method and it gives more reliable results and is less laborious. The microscopic method however is still of importance in studies in which the effect of drugs or extracts on the various growth stages is being studied. However, it is time consuming, strenuous and has a higher degree of subjectivity. A new method that utilizes radioactive ethanolamine instead of hypoxanthine has been reported . The major advantage of this method is that this precursor is incorporated into 35 University of Ghana http://ugspace.ug.edu.gh phospholipid, and the phospholipid incorporated can be used to evaluate P. falciparum in vitro. It is an ideal tool when compounds, which interfere with DNA and / or RNA metabolism, are to be investigated for their effect on Plasmodium growth. The work of O ’Neil et al, 63 shows that in vitro antimalarial testing can be carried out on crude plant extracts using P. falciparum. Initial information on activity may be obtained by using ten-fold dilutions of crude-extracts and solvent fractions. For accurate determination of IC50 values, further results are needed from two-fold dilutions. Unlike the in vivo methods, the in vitro approach indicates whether the plant extract as opposed to its metabolite has or does not have active antimalarial principles. 36 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE Results and Discussion In the present investigation 1.0 kg of shade dried pulverised sample of the whole rhizome of Cochlospermum tinctorium was sequentially extracted with petroleum ether (40-60 °C), dichloromethane, ethyl acetate, ethanol and water for 48 hours each using cold percolation. Although some chemical investigations have been carried out on this plant, the aim of this project is to look at the antiplasmodial activities of the various crude extracts and the compounds isolated from the rhizome extracts. The crude extracts from the petroleum ether, dichloromethane, ethyl acetate, ethanol and water were subjected to a series of phytochemical screening tests71 in the hope of establishing the presence or absence of the various classes of organic compounds. The results are summarised in Table 2 below. Crude extracts and compounds isolated were tested for antiplasmodial activity using a modification of the method of Jensen and Trager.62 Table 2: Summary of results of phytochemical screening tests on petroleum ether, dichloromethane, ethyl acetate, ethanol and water extracts of the rhizome of Cochlospermum tinctorium. Class of compounds Petroleumetherextract Dichloro-methaneextract EthylAcetateextract Ethanolextract Waterextract Alkaloids - - - - - Terpenoids + - - - - Flavonoids - - - + - 37 University of Ghana http://ugspace.ug.edu.gh Leuco-anthocyanins - - - + - Tannins - - + + - Saponins - - - + + Cardiac glycosides + + + + + Anthraquinones and Anthracene derivatives - - — + — Symbols + = present/detected = absent/ not detected The results showed the presence of cardiac glycosides in all the extracts, but no alkaloid was present in any of the extracts. Other compounds detected were anthraquinones and anthracene derivatives (ethanol extract), saponins (water and ethanol extracts), leuco- anthocyanins (ethanol extract), flavonoids (ethanol extract) and terpenoids (petroleum ether extract). 3.1. Fractionation of Petroleum ether extract The petroleum ether extract was analysed as outlined in Scheme 3.1 PEI no resolution Scheme 3.1Dried pulverised rhizome 1. cold percolation(Petroleum ether) 40-60°C 2. concentration of extract Crude Extract column PE2 PE 3 PE4 PE5 PE6 PE7 PE8 no resolution PE9 PE10 PE11 PE12 PEI3 recryst no resolution PE6 white solid (300mg) recryst PE8 white solid (90mg) no resolution recryst. PEI 1 white solid (80mg) 38 University of Ghana http://ugspace.ug.edu.gh Three solid components: PE6, PE8 and PE11 were isolated. The percentage yields of the compounds based on the crude extract and also on the dry plant material are shown in Table 3 below. Table 3. Percentage yields of compounds isolated from the rhizome of C, tinctorium Compound Mass/mg % yield based on crude extract % yield based on plant material PE6 300 17,241 0.030 PE8 90 5,844 0.009 PEI 1 80 7.843 0.008 3.1.1. Characterization of PE6 Fraction PE6 was evaporated to dryness and dissolved in a limited amount of petroleum ether. It was kept in a freezer at a temperature of -20°C for 18 hours. A white solid precipitated from the solution. This was filtered and washed with cold petroleum ether. The sample coded as PE6 had a melting point of 70-72 °C. The 1R spectrum of PE6 (Appendix la) is shown in Table 4 below Table 4: Prominent peaks on IR spectrum of PE6 Frequency of signal (cm 1) Assignments 3400 - 3200(b) O-H stretching vibrations 2960 - 2850 (s) C-H stretching vibrations 1700-1680 (s) C=0 stretching vibrations 1670- 1630 (s) C=C bending vibrations 1470-1360 (m) C-H bending vibrations 1350-1000 (s) C -0 bending vibrations 970 -960 (v) C-H bending vibration (trans alkene) 730 -675 (s) C-H bending vibration (cis alkene) 39 University of Ghana http://ugspace.ug.edu.gh The ‘H-NMR spectra (Appendix Ila, lib and He) were assigned to PE6 as shown in Table 5 below Table 5: 'H-NMR spectrum summary of PE6 Signal (5, ppm) Assignment 6.8 (1H, s ) -C=CH_-CO 5.0 ( 1H, s ) -R -OH 2.9 (1H, d, J =2.1) -CO-CH-CH= 2.65 (2H, t, J = 7.3) C=CH-CH=CH- (cis) 2.58 (1H, d, J =2.1) -CO-CH -CH= 2.42 ( lH ,t, t, J =13.3, 13.3) =CH-CH=CH-CH= (trans) 1.96 ( lH ,t, t, J =13.3, 13.3) =CH-CH=CH-CH= (trans) 1.6 (3H, s) CHb-CO- 1.3 (42H, bs) CH3-CH2-(CI32)2i-C 0.9 (6H, t, J =6.6) CH3-CH2-C The signals in the 13C-NMR spectrum (Appendix Ilia) were assigned as shown in Table 6 below Table 6 : Summary of i;iC-NMR spectrum of PE6 Chemical shifts (8 , ppm) Carbon Assignments 211.53 C=0 (carbonyl) 147.29 CH2 (alkene) 139.10 CH2 (alkene) 77.40-62.6 CH2 (alkane) 40 University of Ghana http://ugspace.ug.edu.gh 51.34-22.56 CH2 (alkane) 13.98 CH3 The l3C-NMR spectrum gave 21 carbon atoms/peaks. At this point it was not possible to deduce the exact structure o f the sample PE6 because the mass spectrum and the expanded ‘H-NMR spectral were not obtained, but from the information available it suggests that the sample composes of a carbonyl group, an alcohol group, a saturated hydrocarbon group and a long chain unsaturated hydrocarbon group. 3.1.2. Characterization of PE8 Fraction PE8 was concentrated into a small volume and kept in a freezer at -20 °C for 8 hours. A white solid precipitated from the solution. The solid was filtered and washed with cold petroleum ether and coded as PE8. The melting point was found to be 123-125 °C. The sample and an authentic sample o f stigmasterol were dissolved in petroleum ether and spotted on the same TLC plate in various solvent systems and they gave the same Rt- values. The IR spectrum (Appendix lb), indicated the presence of hydrogen bonded O-H (3510cm'1), an alkene C-H stretching vibration (2960-2850 cm '1), an alkene C=C stretching vibration (1680-1620 cm’1), a saturated hydrocarbon (C-H) stretching vibration (1600-1500 cm '1), an alkane C-H bending vibration (1485-1350 cm '1) and phenol C -0 bending vibration (1350- 1000 cm '1). 41 University of Ghana http://ugspace.ug.edu.gh Table 7: Comparison of IR spectrum of PE8 and stigmasterol PE8 IR peaks (cm 1) Stigmasterol IR peaks (cm"1) Interpretation 3500-3600 3500-3600 O-H stretch o f alcohols 2960-2850 2950-2850 C-H stretch of alkanes 1680-1620 1670-1620 C=C stretch vibration o f alkenes 1600-1500 1600-1500 C-H stretch o f alkanes 1485-1350 1490-1360 C-H bending vibration o f alkanes 1245 1245 O-H stretch o f secondary alcohols 1015-1010 1020-1010 C -0 stretch o f alcohols Comparison o f the IR spectrum for PE8 and authentic sample of stigmasterol as shown in Table 7 suggests that the sample PE8 was stigmasterol. The mixed melting point (123- 124 °C) and the co-TLC of sample PE8 and the authentic sample o f stigmasterol confirmed that the sample PE8 was stigmasterol. 3.1.3. Characterization of PEI 1 Fraction PEI 1 was kept in the freezer at -20°C for 12 hours. A white solid was formed in the flask. This was filtered and washed several times with cold petroleum ether. The solid was labelled as PEI 1 and the melting point was 45-47 °C. The IR spectrum (Appendix Ic) is shown in Table 8 . 42 University of Ghana http://ugspace.ug.edu.gh Table 8: Summary of IR functional groups of PEI 1 Frequency of signal (cm 1) Assignments 3100 -3000 (m) N-H stretching vibrations 2960 - 2850 (s) C-H stretching vibrations 1700-1630 (s) C=0 stretching vibrations 1600- 1500 (s) C=C bending vibrations 1485-1445 (m) C-H bending vibrations 1350-1000 (s) C -0 bending vibrations 770 -730 (s) Ar -H bending vibration The 'H-NMR spectra (Appendixes lid, He and Ilf) were assigned as shown in Table 9 below Table 9: Summary of 'H-NMR spectrum of PEI 1 Signal (8 , ppm) Assignment 7.3 (2H, d, J =5.1) Aromatic protons 7.05 (IH, m) Aromatic protons 4.16 (1H, t ) -CH -CH-NH-CO-R 2.55 (4H, t, t, J = 15.0,7.6) -CO-CH2-CH 2-N - 2.00 (lH ,m ) - CH -CH-CH2-O - 1.60 (6H, t, J = 6 .6) CH3-CH 2-O- 1.30 (64H, bs) - CH2-(CH 2)32-CH 2- 0.91 (9H, t) c h 3- c h 2 - 43 University of Ghana http://ugspace.ug.edu.gh The COSY spectra (Appendix Via) indicated the coupling of 7.30ppm to 7.05ppm, 2.55ppm to 1.60ppm, 2.00ppm to L,30ppm, i .60ppm lo 1.30ppm and 1.30ppm to 0.91 ppm. The signals in the L’C-NMR spectrum (Appendix 11 lb and 1 lie) were assigned as shown in Table 10 below Table 10: Summary of L’C-NMR spectrum o f PEI 1. Chemical shifts (8, ppm) Carbon Assignments 199.25 C O (carbonyl) 138.51 CH: (alkene) 118.12 CH2 (alkene) 77.59-76.74 CHt (alkane) 37.20-22.75 CHi (alkane) 14.16 CHj The l’C-NMR spectrum gave 20 carbon atoms/peaks. The UV spectra (Appendix Vc) showed two absorption maxima at 272nm, and 375nm. The data obtained was not sufficient to enable full characterisation of sample PEI 1. but the information available suggests that the sample has an aromatic part, an amide part, carbonyl part and a long chain unsaturated hydrocarbon part. Fractions PEI to PE5. PE7. PE9, PE10, PE12 and PE13 were not worked on because of the minute quantities and also the TLC was very complex. 44 University of Ghana http://ugspace.ug.edu.gh 3.2. Fractionation of D ichloromethane extract Isolation and purification work done on the dichloromethane extract is outlined in Scheme 3.2. Scheme 3.2 Crude Extract Column Chromatograph D1 D2 D3 D4 D5 D6 recryst. D7 D5 (W h ile Solid. 80mgj Column Chromatograph D8 D9 DIO D ll recryst. D9 (Yellowish solid) 29mg D12 1 2 3 4 5 6 7 8 9j recrysl. j recrysl. D65 D66(White solid) (While solid)I9mg 23mg Four solid D5, D65, D66 and D9 were isolated. The percentage yields of these compounds based on the masses of the crude extract and also on the dry plant material are given in Table 11 below. Table 11: Percentage yields of compounds isolated from the rhizome of C. tinctorium Compound Mass/ing % yield based on crude extract % yield based on plant materialD5 80 19.51 0.0080 D65 19 3.33 0.0019 D6fi 23 4.04 0.0023 D9 29 11.60 0.0029 45 University of Ghana http://ugspace.ug.edu.gh 3.2.1. Characterization of D5 Fraction D5 on recrystallization from petroleum ether gave white crystals and was coded D5. This compound was found to be triacontanol with melting point 83-85°C, literature melting point, 85-86°C The white solid was filtered and washed with cold petroleum ether. The TLC gave a single spot when an ansialdehyde reagent was used. The white solid was coded D65 (mpt 81-83 °C). The Rf values of sample D65 in a mixture of petroleum ether/ethyl acetate are shown in Table 28. It was however UV inactive. Table 28 R| values of D65 Solvent System Rf Petroleum ether/ethyl acetate (10:1) 0.35 Petroleum ether/ethyl acetate (9:1) 0.46 Petroleum ether/ethyl acetate (8:1) 0.50 The IR spectrum (Appendix Ie) of D65 gave peaks at 3488, 3445, 2958, 2924, 2853, 1696. 1674, 1466, 1378 and 1063 cm '1. The 'H-NMR spectrum (Appendices Ilj, Ilk, III and Urn) contained signals at 8(ppm) = 7.62(d), 7.06(d, d), 6.92(d), 6.30(d), 5.85(s), 4.20(t), 3.93(s), 1.68(q), 1.58(s), 1.38(m), 1.25(s) and 0.88(t). The two dimensional (2D)- NMR, COSY (Appendix VIb) gave a coupling of 1.68ppm to 4.20ppm, 0.88ppm to 1.25ppm, 6.92ppm to 7.06ppm and 6.30ppm to 7.62ppm. 90 University of Ghana http://ugspace.ug.edu.gh In the 1 C-NMR spectrum (Appendix Hid) signals appeared at 8(ppm) 13.97, 22.55, 25.86. 28.63, 29.18, 29.23, 29.57, 31.80, 55.84, 64.55, 70.04, 76.54, 76.96, 77.39, 109.25, 11469, 115.70, 123.08 and 144.69. The Mass spectrum (Appendix IVb) showed a molecular ion peak and a base peak at m/z 614 (100%). Other prominent peaks occurred at m/z 586 (27%), 558 (4%), 194 (34%), 177 (38%), 150 (12%), 137 (18%), 97 (5%), 83 (6%), 57 (9%) and 43 (7%). 4.6.2.3. Isolation of D6 * Fraction D6h was found to have deposited a white solid. This was filtered and washed several times with cold petroleum ether. The solid (23 mg) on TLC in different solvent systems gave a single spot and melted at 79-81 °C. This was coded D6g. The Rf values of the sample D66 in a mixture of petroleum ether/ethyl acetate are shown in Table 29. It was however UV inactive. Table 29: R| values of D66 Solvent system Rr Petroleum ether/ethyl acetate (10:1) 0.28 Petroleum ether/ethyl acetate (9:1) 0.40 Petroleum ether/ethyl acetate (8:1) 0.45 IR spectrum of D6ft (Appendix 10 was determined and gave peaks at 3488, 3445, 2958, 2924, 2853, 1696, 1674, 1466, 1378 and 1063 cm '1. The *H-NMR spectrum (Appendix 11n and 11m) produced signals at 5(ppm)= 7.62(d), 7 .44(d), 6.84(d), 6.30(d), 4 .2(t), 3.93(s), 1.68(q), l.58(s), 1.38(m), 1.25(s)and 0.88(t). 91 University of Ghana http://ugspace.ug.edu.gh The mass spectrum (Appendix IVc) showed prominent peaks at m/z 584 (63%), 556 (237,). 528 (3$). 194 (8%), 147 (63%), 120 (32%), 107 (28%), 83 (13%), 57 (19%) and 43 (15%). The base peak appeared at m/z 164 (100%). Other prominent peaks occurring al m/z greater than 584 are probably due to contamination by sample D65. These are 614 (28f/r ) and 616(10%). 4.6.2.4. Isolation of D9 The orange solid particles in fraction D9 was filtered arid washed several times with cold petroleum ether. The solid (29 mg) showed a single spot on a TLC plate sprayed with anisaldehyde reagent and heated in an oven at a temperature of 110 °C. This was labeled D9 and gave a melting point of 200-202 °C. The solid was UV active. The R, values of the sample D9 in a mixture of petroleum ether/ethyl acetate are shown in Table 30 below. Table30: R, values of D9 Solvent system Rf Petroleum ether/ethyl acetate (3:1) 0.35 Petroleum ether/ethyl acetate (5:2) 0.40 IR spectrum of D9 (Appendix Ig) was determined and gave peak at 2917, 2847, 1712, 1463. 1266, 1171, 1037, 974 and 721 cm '1. The UV spectrum of D9 (Appendix Va) showed maxima (CHCI3) at 290, 315, 322, 422, 442 and 472 nm. 92 University of Ghana http://ugspace.ug.edu.gh The ‘H-NMR spectrum (Appendices Up, Ilq, Hr and IIs) produced signals at 8(ppm) 1,3(s). I.6(m), 2.9(t, t), 3.25(t, t), 4.01(d), 4.05(d), 4.55(d, d), 4.65(m), 4.9(q), 6.58(d) and 6.68(d). The two dimensional (2D)-NMR spectrum (Appendix Vic), COSY gave a coupling of 2.9 to 3.25, 4.05 to 4.55, 2.9 to 4.55, 4.9 to 3.25, 4.55 to 2.9, 4.65 to 6.68 and 4.9 to 6.58 ppm. In the 1 ’C-NMR spectrum (Appendix Hie, Illf and Illg) signals appeared at 8(ppm) 37.22, 37.49, 50.38, 54.64, 62.66, 76.76, 77.40, 78.05,' 127.74, 127.99, 128.05, 128.33, 129.38. 129.64, 129.84, 130.13, 130.26, 132.37, 133.01, 134.35, 135.22, 136.79, 138.20, 168.56, 168.78 and 173.33. 4.6.3. Ethyl Acetate extract of the rhizome of C. tinctorium. Ten grams of the crude extract was dissolved in a minimum volume of ethyl acetate and absorbed onto silica gel (25 g) and introduced onto a column packed with silica gel (500 g). The column was eluted using 100% petroleum ether / ethyl acetate with increasing proportions of the ethyl acetate until 100% of the ethyl acetate was used. This was followed by 100% ethanol. Twenty millilitre of the eluate was collected and monitored bv TLC results the elutes were combined into 19 main fraction labeled E l, E2, E3, E l9. The fractional yields are shown in Table 31 below. Table 31 Fractional Yields of Ethyl acetate extracts. Fraction State Colour Yield / g El i- Oil Colourless 0.15 E2 1 Oil Yellowish 0.13 93 University of Ghana http://ugspace.ug.edu.gh E3 Oil Yellowish 0.13 E4 Oil Yellowish 0.38 E5 Syrup Yellowish-green 0.21 E6 Syrup Yellowish-green 0.14 E7 Oil and Solid Yellowish-green 0.12 E8 Oil and Solid Yellowish-green 0.11 E9 Oil and Solid Yellowish-green 0.15 EIO Oil and Solid Yellowish-green 0.18 El 1 Oil Yellowish 0.19 E12 Oil and Solid Yellowish-green 0.14 E13 Oil and Solid Yellowish-green 0.22 E14 Oil and Solid Yellowish-green 0.17 E15 Oil Yellow 0.10 E16 Oil and Solid Yellowish-brown 0.41 E17 Oil and Solid Yellowish-brown 1.43 E18 Oil and Solid Dark-Brown 1.78 E19 Oil and Solid Dark-Brown 2.97 4.6.3.1. Isolation of E13 The traction E l3 was kept in a freezer for 24hours. It precipitated a yellowish solid. The solid (28 mg) was filtered and washed with cold petroleum ether. TLC of the solid showed as a single spot when sprayed with anisaldehyde spray reagent. This was labeled 94 University of Ghana http://ugspace.ug.edu.gh E l3 and gave a melting point of 69-71 °C. The solid was UV active. The Rf values of the sample EI3 in a mixture of petroleum ether / ethyl acetate are shown in Table 32 below. Table 32. Rr values of E13 Solvent System Rr Petroleum ether / ethyl acetate (3:1) 0.95 Petroleum ether / ethyl acetate (4:1) 0.59 Petroleum ether/ethy l acetate (8:1) 0.40 Petroleum ether/ethy l acetate (9:1) 0.31 The IR spectrum of E13 (Appendix Ih) was determined and gave peak at 2955, 2917, 2849, 1711, 1630, 1464, 1300, 1170 and 721 cm '1. The UV spectrum of E13 (Appendix Vb) was determined. 4.6.3.2. Isolation of E166 Fraction E l4, E l5 and E l6 were combined because they showed similar TLC profile. This was concentrated, relabeled E16. This solid (0.67 g) was put on a column (13.5 g silica gel) and eluted with 100% petroleum ether and mixture of petroleum ether/ethyl acetate with increasing proportion of ethyl acetate until 100% of the ethyl acetate was used. About 10 ml of the eluate were collected and monitored by TLC. Nine (9) main tractions were obtained and labeled E161, E I62, .......... E I69. All the fractions were put into a freezer for 24 hours. 95 University of Ghana http://ugspace.ug.edu.gh Fraction E16h precipitated a dark yellowish solid. The solid (33 mg) was filtered and washed with cold petroleum ether. The TLC gave a single spot when an anisaldehyde reagent was used. The solid was coded as E16& (mpt > 250 °C). The Rf value of the sample E I6& in a mixture of petroleum ether/ethyl acetate are shown in Table 33 below. Table 33:Rr value of E16(, Solvent system Rf Petroleum ether/ethyl acetate (5:2) 0.35 Petroleum ether/ethyl acetate (2:1) 0.54 The IR spectrum of E 16h (Appendix li) was determined and gave peaks at 3408, 3073, 2942. 2870, 1689, 1642, 1451, 1377,1243, 1190 and 1049 cm ' 1 96 University of Ghana http://ugspace.ug.edu.gh 4.7. ANTIMALARIAL ASSAY 4.7.1. Chemicals and Reagents RPMI-1640 with Hepes modification and NaHC03 without glutamine (SIGMA, USA) L-Glutamine (SIGMA, USA) Gentarnycin (stock 50mg/ml, GIBCO, Scotland) Glycerol (BDH, England) Sodium Chloride (SIGMA, USA) Sorbitol (BDH, England) Chloroquine diphosphate salt (SIGMA, USA) 'H-hypoxanthine (NEN BOSTON, USA) Ethanol (SIGMA, USA) Methanol (SIGMA, USA) Dimethylsulfoxide (DMSO) (SIGMA, USA) Geimsa stain (BDH, England) Immersion Oil (BDH, England) 4.7.2. Red Blood Cells and Serum Blood group O Rh+ (Volunteers) Normal Human Serum (NHS) from blood group AB Rh+ (SIGMA, USA) 97 University of Ghana http://ugspace.ug.edu.gh 4.7.3 Malaria Parasite Strains Chloroquine sensitive Plasmodium falciparum 3D7 strain Chloroquine resistant Plasmodium falciparum DD2 strain (both strains were from the Centre for Medical Parasitology, Copenhagen, Denmark) 4.7.4. Materials Candle jar Pure candles Flat bottom 96 well microtitre culturing plates Glass Filter fibers Glass microscope slides Millipore filters 25 cm' culture flasks 5 ml and 10 ml sterile disposable pipettes 5 ml and 50 ml sterile syringes 1.8 ml screw capped cryotubes 4.7.5. PLANT MATERIAL The rhizomes of Cochlospermum tinctorium were collected from an area 9km from Drobo towards Sampa with the assistance of botanists from the Ghana Herbarium, Botany Department, University of Ghana, Legon. The plant material was air-dried for two weeks and ground by machine into coarse powder 98 University of Ghana http://ugspace.ug.edu.gh 4.8.1. Preparation of Citrate-Phosphate Dextrose Buffer (CPD) This was prepared by weighing 0.5 g citric acid, 2.647 g sodium citrate, 0.221 g sodium phosphate, 0.027 g Adenine (6-Aminopurine) and 3.154 g glucose in petri dishes transferring them into a 200 ml beaker and dissolving with 100 ml distilled water with stirring. The resulting solution was sterilized in an autoclave at 120 °C for 30 minutes after which it was stored at 4 °C. For every 20 ml of whole blood, 3 ml of CPD was added as an anticoagulant. 4.8.2. Preparation of Washing medium /Incomplete RPMI medium This was prepared by adding 0.5 ml of gentamycin (50 mg/ml) and 4.0 ml of L-glutamine to 500 ml of RPMI solution. This was stored at 4 °C. 4.8.3. Preparation of Complete RPMI medium. (10% NHS) This was prepared by adding 5 ml filtered (through 0.8 /xm pore filter) NHS (0+) and 50 mg of D-glucose to 45 ml washing medium. This was stored at 4 °C and used for parasite cultivation. 4.8.4. Uninfected Red blood Cells Human blood group O Rh+ was obtained from Korle-Bu Hospital and donors in CPD and stared at 4 "C. For cultivation, a volume of blood was centrifuged for 5 minutes at 2000 rpm. The plasma and buffy coat were removed using a sterile pipette. The packed cells remaining were washed several times (4x) with equal volumes of incomplete RPMI (that 4.8. METHODS 99 University of Ghana http://ugspace.ug.edu.gh is, medium with no serum added) and the buffy coat on top of the cells removed. To the remaining packed cells was added an equal volume of incomplete RPMI medium to produce 50°/( PCV. This was stored at 4 °C and used for cultivation. 4.9. Extraction of the rhizome of Cochlospermum tinctorium One hundred grams (100 g) of the dried and ground Cochlospermum tinctorium were sequentially extracted with 600 ml of Petroleum ether,-dichloromethane, ethyl acetate, ethanol and water using cold maceration. Maceration was carried out in one-liter beaker for a period of 48 hours at room temperature with daily stirring of the plant material for each organic solvent. At the end of the 48 hours period, the extracts were obtained by filtration under pressure with a Bucher funnel and again using a funnel to filter it. Solvents were recovered from the extracts by using a rotary evaporator. These were stored at 4 °C in sealed round bottom flask. The aqueous extract was freeze-dried and stored in a dessicator in a sealed container. The TLC of the extractsrextracted for both the column chromatography and the antimalarial assay gave similar spots. 4.10 Preparation of various concentrations of crude extracts from the rhizome of Cochlospermum tinctorium Each of the plant extracts (5 mg) was weighed into a cryotube. The aqueous extract was dissolved in 50 /xl of water and the other extracts dissolved by shaking with 50 /zl dimethylsulphoxide (DMSO). This gave a stock solution of 105^ g/ml, which was filter- sterilized with a millipore filter (0.20 jul). Ten microlitre (10 /zl) of this was added to 990 Ml of complete RPMI in a lymphocyte tube to give a solution containing 1 fig /l/il. This 100 University of Ghana http://ugspace.ug.edu.gh was regarded as 1:1 dilution. Fifty microlitres (50 /xl) of this solution was then added to 50 /x! of parasitised red blood cells in the well to give a final concentration of 500 /xg/ml. This diluted solution (400 /xl) was added to 400 ji\ of complete RPMI to give a 1:2 dilution. Fifty microlitres (50/nl) of which was added to 50 /xl of parasitised red cells in the well to give a concentration of 250 /xg/ml. Similarly, Four hundred microlitres (400 jU 1) of the 1:2 dilution was added to 400 /xl of complete RPMI to give a 1:4 dilution of which 50 /xl was added to 50 /tI of parasitised red blood cells in the well to give a final concentration of 125 /xl/ml. Again 400 /x 1 of the 1:4 dilution was added to 400 /xl of complete RPMI to give a 1:8 dilution from which 50 .^1 was added to 50 /xl of parasitised , ed blood cell in the well to give a concentration of 62.5 /xg/ml. Four hundred microlitres (400 /il) of the 1:8 dilution was added to 400 /x 1 of complete RPM.! to give a 1:16 from which 50 /t 1 was added to 50 /il of parasitized red blood cells in ihe well to give a concentration of 31.25 /xg/ml. Four hundred microlitres (400 /il) of the 1:16 dilution was added to 400 /xl of complete RPMI to give a 1:32 dilution from which 50/xl was added to 50/xl of parasitised red blood cells in the well to give a concentration of 15.63 /xl/ml. Four hundred microlitres (400 /xl) of the 1:32 dilution was added to 400 /xl of complete RPMI to give a 1:64 dilution from which 50 /x 1 was added to 50/xl ot parasitised red blood cells in the well to give a concentration of 7.81 /xg/ml. Four hundred microlitres (400 /xl) of the 1:64 dilution was added to 400 /xl of complete RPMI to give a 1:128 dilution from which 50 /xl was added to 50 /xl of parasitised red blood cells in the well to give a concentration of 3.91 /xg/ml. 101 University of Ghana http://ugspace.ug.edu.gh The DMSO content of the 500 /xg/ml, 250 /xg/ml, 125 /tg/ml, 62.5 iig/m l, 31.25 /xg/ml, 15.63 /xg/ml, 7.81 fig/ml and 3.91 Mg/ml dilutions of test were respectively 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.0156%, 0.0078% and 0.0039%. 4.11 Preparation of various concentrations of isolated compounds from the rhizome of C. tnctorium Each of the isolated compounds (1 mg) was weighed into cryotube. Fifty microlitres (50 /ri) dimethylsulphoxide (DMSO) were used to dissolve the compounds by shaking. This gave a stock solution of 2x l04 jug/ml, which was filter-sterilized with a millipore filter (0.20 ixI). Ten microlitre (10 /xl) of this was added to 990 /il of complete RPMI in a lymphocyte tube to give a solution containing 200 /xg/ml. This was regarded as 1:1 dilution. Fifty microlitres (50/xl) of this solution was then added to 50/tl of parasitised red blood cells in the well to give a final concentration of 100 /tg/ml. This diluted solution (400/1.1) was added to 400/tl of complete RPMI to give a 1:2 dilution, from which 50/xl was added to 50/xl of parasitised red cells in the well to give a concentration of 50/tg/ml. Similarly, 400/tl of the 1:2 dilution was added to 400/tl of complete RPMI to give a 1:4 dilution of which 50/tl was added to 50/xl of parasitised red blood cells in the well to give a final concentration of 25/zl/ml. Again 400/il of the 1:4 dilution was added to 400/tl of complete RPMI to give a 1:8 dilution from which 50/xl was added to 50/xl of parasitised red blood cell in the well to give a concentration of 12.5/i g/ml. 102 University of Ghana http://ugspace.ug.edu.gh Four hundred microlitres (400/xl) of the 1:8 dilution was added to 400/xl of complete RPMI to give a 1:16 from which 50/xl was added to 50/xl of parasitised red blood cells in the well to give a concentration of 6.25/xg/ml. Four hundred microlitres (400/xl) of the 1:16 dilution was added to 400/xl of complete RPMI to give a 1:32 dilution from which 50(Li 1 was added to 50/xl of parasitised red blood cells in the well to give a concentration of 3.13/xl/ml. Four hundred microlitres (400/4.1) of the 1:32 dilution was added to 400/xl of complete RPMI to give a 1:64 dilution from which 50/xl was added to 50/xl of parasitised red blood cells in the well to give a concentration of 1.56/xg/ml. Four hundred microlitres (400/xl) of the 1:64 dilution was added to 400/xl of complete RPMI to give a 1:128 dilution from which 50/xl was added to 50/xl of parasitised red blood cells in the well to give a concentration of 0.78/xg/ml. The DMSO content of the 100/xg/ml, 50/xg/ml, 25/ig/ml, 1.25/xg/ml, 6.25/xg/ml, 3.13/xg/ml, 1.56/xg/ml and 0.78/xg/ml dilutions of test were respectively 0.1%, 0.05%, 0.025%, 0.0125%, 0.00625%, 0.00313%, 0.00156% and 0.00078%. 4.12. In Vitro cultivation of malaria parasites Parasitised red blood cells containing chloroquine sensitive (3D7) and resistant (DD2) P. falciparium strains were obtained from the Immunology Unit, NMIMR, Legon and kept in continuous culture by the candle jar method of Trager and Jensen. 77 These parasites were chosen because they are sensitive to chloroquine (3D7) and resistant to chloroquine (DD2), which is the standard drug, for treatment of malaria in Ghana. A fair comparison 103 University of Ghana http://ugspace.ug.edu.gh could therefore be made for their response to chloroquine and the plant extracts and isolated compounds. To imitate cultivation cryopreserved parasites were removed from liquid nitrogen (-196 (1C) and quickly disengaged and thawed by placing the tube in water bath set at 37 °C containing sterile water. It was immediately centrifuged at 1500rpm for 10 minutes and the supernant removed. An equal volume of the thawing medium (3.5% Sodium chloride in sterile water) was then added. It was again centrifuged at 1500rpm for 10 minutes and the supernatant removed. The cells were resuspended in 1.0 ml of RPMI 1640 medium 75supplemented with 10% norma! human serum (AB+), L-glutamine and gentamycin. This was washed once again as described and the cells added to a culturing flask containing 5ml of complete culture medium and 200/xI of 0+ packed red blood cells. This was then gassed with a special gas mixture (2.0% oxygen, 5.5% carbon dioxide and 92.5% nitrogen) for 30 seconds and incubated in an incubator set at 37°C. Later, a candle jar was used. The flasks were flamed and placed in the candle jar containing a lighted candle. The jar was closed, the tap shut when the candle went off, and jar transferred into an incubator, kept at 37 °C. The spent medium was changed everyday and slides were prepared and stained with Giemsa to monitor the growth of the parasites expressed as percentage parasitaemia. Five millilitres (5ml) of complete medium was added if the percentage parasitaemia is less than 3%. The cultures were maintained until the schizont stage with a parasitaemia of approximately 3.0% and it was used for the assay. 104 University of Ghana http://ugspace.ug.edu.gh 4.13. Fixation and Staining Parasitaemia was determined to ascertain when the parasites in the culture flasks were ready for subculturing. Glass slides were soaked in 70% ethanol, cleaned well with cotton before the film was prepared. The dried blood films were fixed with methanol and air- dried. The films were stained using a 1:10 dilution of Giemsa stain for 10 minutes. The stained films were washed with water, dried and viewed under the microscope (Olympus BH2 Microscope) at 100X magnification. The parasitaemia was determined as a ratio of the number of parasitised erythrocytes, to the total number of erythrocytes in a microscope field. 4.14. Cryopreservation of Parasites Cryopreservation was used to preserve some malaria parasites for future work. The ring stage of a culture of malaria parasites with parasitaemia between 3 and 5% was centrifuged at 1200rpm for 5 minutes. The supernatant fraction was then removed to obtain a pellet of parasites. An equal volume of freezing solution (28g glycerol, 5g sorbitol and 0.63g NaCl in 100ml distilled water and filter sterilised) was added to the pellet of parasites, gently mixed and transferred by sterile serological pipette into sterile cryotubes. The transfer was rapidly performed to avoid haemolysis during thawing. The cryotubes were rapidly closed and transferred into liquid nitrogen as described by Merryman and Hornblow. 76. They were stored in liquid nitrogen until brought into in vitro culture. 105 University of Ghana http://ugspace.ug.edu.gh 4.15. Subculture This was done when the parasitaemia in culture flasks reached between 3 and 5% from a starting parasitaemia of 0.25%. To 200/il of non-parasitised blood (O Rh+) was added 20jx\ of parasitsed blood in a 25ml culture flask to give a ratio of 10:1. To this was added 5ml of RPMI supplemented with 10% normal human serum. The subcultures were done for about three times to attain a stable growth before it was used for any assay. 4.16. In Vitro inhibition assay for extracts and isolated compounds from the rhizome of C. tinctorium This was preformed using 96-well flat-bottomed microculture plates. The test procedure was based on the method of O’Neil et al.63 Fifty microlitre aliquots of the various concentration of the extracts and isolated compounds of C. tinctorium (that is for extracts, 500/ig/ml, 250/ig/ml, 125/ig/ml, 62.5/tg/ml, 31.25/ig/ml, 15.63/ig/ml, 7.81/tg/ml and 3.91/tg/ml and isolated compound 100/xg/ml, 50/tg/ml, 25/ig/ml, 12.5/ig/ml, 6.25/ig/ml, 3.13/ig/ml, 1.56/tg/ml and 0.78/ig/ml) were introduced in triplicate into the wells. To each of these was added 50/il of parasitised blood of parasitaemia between 1 and 2% and cell suspension of 5x l08/ml. Positive control wells were free of any extract, while chloroquine (CQ) diluted with sterile complete RPMI in various concentrations were introduced into negative control wells. This was prepared from a stock chloroquine diphosphate salt (SIGMA, USA). The microculture plates were placed in a candle jar containing a lighted candle. The jar was closed and the tap shut when the candle went off. The jar was kept at 37°C in an 106 University of Ghana http://ugspace.ug.edu.gh incubator. The plates were brought out after 24 hours and 20/il of 3H-hypoxanthine (40/w.Ci/ml) added to each well. They were incubated again at 37 °C for another 24 hours after which the cells were harvested on a glass-fibre filter using a Packard filter Mate 96 cell Harvester. Radioactivity of !H-hypoxanthine incorporated in the DNA of the parasites were counted by Direct Beta Counter, Matrix 96 (Packard, USA) that uses gas mixture of helium gas (97.5%) and butane (2.5%) for scintillation. The glass-fibre filter was dried at 37 °C and later read using the counter. 4.17. Dimethylsulfoxide (DMSO) Trials DMSO is normally used at a concentration range of 0.1%-0.5% for this type of inhibitor assay in the laboratory where this was performed. Two trial experiments were however performed to test for any possible effect it might have on the 3D7 parasites. This was performed using the method described under section 4.16. Fifty microlitre aliquots of RPMI-1640 medium containing either 0.5% DMSO or 0.5% aqueous extract of C. tinctorium were introduced in triplicate into the wells. The 0.5% DMSO was the highest concentration of DMSO used in the inhibition assay and it was present in the 500^1/ml dilutions. Any inhibition at this level would add to the possible inhibitory effect of the plant extract. 107 University of Ghana http://ugspace.ug.edu.gh REFERENCES 1. H. ML Burkill. (1985), The useful plants o f West Tropical Africa.Edition 2. Vol. 1 p 386-389. 2. Trap. Doctor. (1997) 27. 1: p 12-16 (Dept of Anthropology Unit Hawaii Honolulu. U.S.A.). 3. WHO (1989), Tropical Diseases, Progress in International Research 1987-88. 4. Ahmed. K. (1989) Epidemiology of malaria in Ghana, Ghana Med. J. 23, p. 190-195. 5. Bruhn, J.G. (1989) The use of natural products in modem medicine. Acta Pharm. Nord. 1(3), p. 117-130. 6. Farnsworth, N. R., Akerele, O,, Bingel, A.S., Soejarto, D.D. and Guo, Z. (1985) Medical plants in therapy. Bull. WHO 63 (6), p 965-981. 7. Samuelsson, G. (1989) Nature as source of new drugs. Acta Pharm. Nord. 1 (3), p 111- 116. 8. Tyler, V.E. (1988) Medical plant research. Planta Med. 2, p 95-100. 9. China Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials. (1982), The chemistry and synthesis of qinghaosu derivatives, J. Trad. Chin. Med. 2, p 9-16. 10. Klayman, D.L., Lin, A.J., Acton, N., Scovill, J.P., Hoch, J.M., Milhous, W.K and Theoharides, A.D. (1984), Isolation of artemisinin (Qinghaosu) from Artemisia annua growing in the United States. J. Nat. Prod. 47, p 715-717. 11. Benoit, F; Valentin, A; P61issier, Y; Marion, C; Dakuyo, Z; Mallie, M and Bastide, 1-M. (1995), Antimalarial activity in vitro of Cochlospermum tinctorium tubercle extracts, Trans. Roy. Soc. Trop. Med. Hyg. 89, p 217-218. 108 University of Ghana http://ugspace.ug.edu.gh 12. Benoit-Vical Fran^oise; Valentin, A; Mallie, M; Bastide, J-M and Bessiere J-M (1999). Planta Medica. 65, p 378-381. 13. Desjardins, R. E„ Canfield, C. J., Haynes, J. D. aand Chulay, J. D. (1979). Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy. 16, p. 710-718. 14. Diallo, B; Vanhaelen-fastre R; Vanhaelen, M; Fiegel, C; Joyeux, M; Rolnd A and Fleurentin. J. (1992). Further studies on the hepatoprotective effects of Cochlospermum tinctorium rhizomes. Journal o f ethnopharmacolgy, 36 p 137-142. 15. Rabate. J. (1939). The rhizomes of C. tinctorium. J. pharm. Chim. 29. p 582-3 16. Diallo, B. & Vanhaelen, M. (1988). Large scale purification of apocarotenoids from Cochlospermum tinctorium by counter-current chromatography. J. Liq. Chromatogr. 11(1). p 227-31. 17. Diallo, B and Vanhaelen. M. (1987). Apocarotenoids from Cochlospermum tinctorium. Phytochemistry, Vol. 26. No. 5, p .1491- 1492. 18. Diallo, B; Vanhaelen-fastre R; Vanhaelen, M. (1989). Studies on inhibitors of skin- tumor promotion. Inhibitory effects of triterpenes from Cochlospermum tinctorium on Epstein-barr virus activation. Journal o f Natural products.Vol. 52, No.4. p 879-881. 19. Diallo, B; Vanhaelen, M, Kiso Y and Hikino H. (1987). Antihepatotoxic actions of C. tinctorium rhizomes. Journal o f Elhnopharmacology, Vol. 20, p 239-243. 20. Diallo. B: Vanhaelen-fastre R & Vanhaelen, M. (1991). Triacylbenzenes and long- chain volatile ketones from Cochlospermum tinctorium rhizome. Phytochemistry, Vol. 30, No. 12, p. 4153-4156 109 University of Ghana http://ugspace.ug.edu.gh 21. Batalha Van Dunen, M. M. (1980). Report about the ‘ Encontro National de Medicina traditional Angolana’. Luanda (Angola). 22. Presber. W: Birbel Hegenschaid; Harnaandez-Aluarez.. H; Herrmann. D and Brendel. C. (1992) Inhibition of the growth of P.falciparum and P.berghei in vitro by an extract of Cochlospermum angolense Acta. Tropica, 50 p 331-338. 23. Nogueira Prista L. and Correia da Silva A. C. (1955). Chemical composition of C. angolense. Anais fac. Farm. Porto 15., p, 113-119. 24. Nogueira Prista L. (1956). Chemical study of Cochlospermum angolense. Anais fac. Farm. Porto 16. p 105-120. 25. Lima de D.P; Castro Abreu de M.S; Mello de J.C.P; Kassab N.M. and Siqueira J.M (1995). A flavanone glycoside from Cochlospermum regium. Fitoterapia Vol. LXVI, No 6, p 545-546 26. Ramachandriah R., Adinarayana D. and Syamasundar K.V. (1991) Chemical investigation of some Indian medicinal plants. Fitoterapia. Vol. LXII, No. 6, p 544- 545. 27. Nadkarni A. K. (1957), “Indian Materia Medica”, Popular Book Depot, Bombay, Vol 1, p 362. 28. ‘‘The Wealth of India” Series Raw materials, Vol. II, CSIR, Delhi, 1950, p 261. 29. Hirst E. L. and Dunstan Sonia. (1953) The structure of Karaya gum. J. Chem. Soc. p 2332-7. 30. Aial, C. K.. Srivastava, J. B„ Wali, B. K„ Chakravarty, R. B. and Dhawan, B.N. (1978) Screening of Indian plants for biological activity. Part V III . Indian J. Exp. Biol. 16, p 330- 349. 110 University of Ghana http://ugspace.ug.edu.gh 31 . Adinarayana D„ Ramachandriah Chetty P. (1985) Chemical investigation of some medicinal plants occurring in south India. Indian J. Chem. 24B, p 453. 32. Aliyu R; Okoye Z. S. C and Shier W. T (1995). The hepatoprotective cytochrome P- 450 enzyme inhibitor isolated from the Nigerian medicinal plant Cochlospermum planchonii is a zinc salt. Journal o f ethnopharmacology 48. p 89-97. 33. Addae-Mensah, I., Waibel, R. and Achenbach, H. (1985). Novel long-chain triacylbenzenes from Cochlospermum planchonii). Liebigs Ann. Chem. p 1284- 1287. 34. Achenbach. H., Blumn, E. and Waibel, R. (1989). Vitixanthin and Dihydrovitixanthin - New unusual T - apocarotenoic acid from Cochlospermum vitifolium. Tetrahedron Lett Vol. 30. No. 23, p.3059-3060. 35. Brum, R.L„ Honda, N.K,. Hess, S.C,. Cruz, A.B. and Moretto, E. (1997). Antibacterial activity of Cochlospermum regium essential oil. Fitoterapia 68, 1, p 79-. 36. Dixit, B.S and Srivastava, S.N. (1992). Flavonoids and Carotenoids of Cochlospermum vitifoleum flower. Fitoterapia 63, 3, p 270-. 37. Harborne, J.B. (1975). Flavonoid bisulphates and their co-occurences with ellagic acid in the Bixaceae, Frankeniaceae and related families. Phytochemistry. 14, p 1331-1337. 38. Lopez, J.A. (1981). Flavonoids in Cochlospermum vitifolium willd) Ing. Cienc. Quim. 5, 3, p 101-102. 39. Richman, A. M and Kafatos, F. C. (1998). Nature Med. 4. p 552. 40. Shahabuddin. M and Kaslow, D.C. (1994). Bull, Inst. Pasteur 92, p 119. 41. Shahabuddin M and Schneider D. (2000). Malaria Parasite development in a Drosophila model. Science Vol. 288, p 2376-2379. I l l University of Ghana http://ugspace.ug.edu.gh 42. Bruce-Chwatt, L.J., Ed (1986). Chemotherapy of malaria. Revised second edition. WHO, Geneva, p 22. 43. Duerden, B.I., Reid, T.M.S., Jewsbury, J.M., Turk, D.C. (1987). A New Short Textbook of Microbial and Parasitic Infection. ELBS/Hodder & Stoughton, London, p. 149. 44. Bruce-Chwatt, L.J., Ed (1986). Chemotherapy of malaria. Revised second edition. WHO, Geneva , p. 119-121. 45. Technical Report Series, WHO, 1984, p. 181. 46. Bruce-Chwatt, L.J. Ed (1986). Chemotherapy of malaria. Revised second edition. WHO, Geneva, p 30-32. 47. Wemsdorfer, W. H. (1991). The development and spread of drug-resistant malaria. Parasitology Today 7, p 297-303. 48. White, N. J. (1992). Antimalarial drug resistance the pace quickens. Journal o f Antimicrobial Chemotherapy 30, p. 571-585. 49. Goldberg, D. E., Slater, A. F. G., Cerami, A. H. & Henderson. G. B. (1990). Heamoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle. Proceedings o f the National Academy o f Science o f the USA, 87. p. 2931-5. 50. Fitch, C. D„ Chevil, R„ Banyal, H. S., Pfaller, M. A., Phillips, G. & Krogstad, D. J. (1982). Lysis of Plasmodium falciparum by ferriprotoporphyrin IX and a chloroquine- ferriprotoporphyin IX complex. Antimicrobial Agents and Chemotherapy 21, p. 819- 822 . 112 University of Ghana http://ugspace.ug.edu.gh 51. Gluzman, I. Y., Francis, S. E., Oksman, A., Smith, C. E., Duffin, K. L. & Goldberg, D. E. (1994). Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway. Journal o f Clinical Investigation. 93, p. 1602-1608. 52. Egan, T., Ross, D. & Adams, P. (1994). Quinoline anti-malarial drugs inhibit spontaneous formation of (3-haematin (malaria pigment). FEBS Letters, 352, p. 54-57. 53. Raynes, K., Foley, M„ Tilley, L & Deady, L. W. (1996). Novel bisquinoline antimalarials. Biochemical Pharmacology 52, p. 551-559. 54. Blauer, G.& Akkawi, M. (1997). Investigation of B- and (3-haematin. Journal o f Inorganic Biochemistry, 66, p. 145-152. 55. Sullivan, D. J., Gluzman, I. Y., Russell, D. G. & Goldberg, D. E. (1996). On the molecular mechanism of chloroquine’s antimalarial action. Proceedings o f the National Academy o f Sciences o f the USA, 93, p. 11865-70. 56. Dorn, A., Stoffel, R., Matile, H„ Bubendorf, A. & Ridley, R. (1995). Malarial haemozoin/P-haematin supports haem polymerization in the absence of protein. Nature, 374. p. 269-71. 57. Bruce-Chwatt, L.J. Ed (1986) Chemotherapy of malaria. Revised second edition. WHO, Geneva, p. 9, 48, 102-112. 58. Ofori-Adjei, D„ and Mate-Kole, M. O. (1991). Spurious resistance of P. falciparum to quinine. Ghana Med. Jour. 25. p. 331-333. 59. Quarcoo, A. (1998). Antimalarial properties of Nauclea latifolia using Plasmodium falciparum in vitro culture. M. Phil. (Biochemistry) thesis, p. 29-32, 87-92.. University of Ghana, Legon. 113 University of Ghana http://ugspace.ug.edu.gh 60. Awe. S. O. & Opeke, O. O. (1990). Effect of Alstonia congensis on P. berghei in mice. Fitoterapia LXI, p. 225-229. 61. Awe, S. O. & Makinde, J. M. (1991). Antimalarial effects of the stem bark aqueous extracts of three Kaya Species. Fitoterapia LXII, p. 467-473. 62. Jensen, J. B & Trager, W. (1977). P- falciparum in culture, use of outdated erythrocytes and description of the candle jar method. J. Parasitol, 63, p. 883-886. 63. O’Neil, M. J., Bray, D. H., Boardman, P., Phillipson, J. D. & Warhurst, D.C. (1985). Plants as sources of antimalarial drugs. Part I — In vitro test for evaluation of crude extracts from plants. Planta Medica. p. 394-498. 64. Elabbadi, N., Ancelin, M. L. & Vial, H. J. (1992). Use of radioactive ethanolamine incorporation into phospholipids to assess in vitro antimalarial activity using the semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy, 36, p. 50-55. 65. Body, D. R. (1975). The occurrence of dihydrophytyl wax esters in bovine rumen liquor, Biochim-Biophys-Acta. 380(1), p. 45-51. 66. Kazuya, Kosuge,, Katsuyoshi, Mitsunsga., Kazuo, Koike and Taichi Ohmoto. (1994). Studies on the constituents of Ailanthus integrifolia. Chem, Pharm. Bull. 42(8) p. 1669- 1671. 67. Cong-Jun Li; Ahmed, A. Ahmed; Alicia Del Camen Arias and Tom J.Mabry. (1997). Clerodane diterpenoids, Long chain ester of coumaric acid and other compounds from Buccharis myrsinites. Phytochemistry, Vol. 45, No. 3, p. 571-574. 68. Munandhha, M.S.P, and Van Dyke, K, (1795). Detailed purine salvage metabolism in and outside the free malaria parasite. Exp.Parasitol. 37, p. 138-146. 114 University of Ghana http://ugspace.ug.edu.gh 69. Badam, L. (1987). In vitro antimalarial activity of neem leaf and seed extracts. Indian Journal o f Malariology, 24, p. 111-117. 70. Weenen, H. (1990). Antimalarial activity of Tanzanian medicinal plants. Planta Medica, 56 , p. 368-370. 71. Peach. K. & Tracy, M. V. (1956). Modern Methods o f Plant Analysis. Springer-Verleg, Berlin, p. 40,44. 72. Kirchner, G. J. (1975). Technique o f Organic Chemistry. Vol. XII. Thin Layer Chromatography Interscience Publisher, p. 153. 73. Stahl, E. (1965). Thin Layer Chromatography. A Laboratory Handbook, Academic Press Inc. p. 486. 74. Mann, F. G. & Saunders, B. C. (1972). Practical Organic Chemistry. 4th Ed. Longman, Louse and Bryone Ltd. London, p. 523. 75. Crammer, S. L., Magowan, C., Laing, J., Coppel, R. L. & Cooke, B.M. (1997). An alternative to serum for cultivation of Plasmodium falciparum in vitro. Trans. R. Soc. Trap. Med. Hyg. 91, p. 363-365. 76. Merryman, H. T. and Homblower, M. (1972). A method for freezing and washing red blood cells using a high glycerol concentration. Transmission 12, p. 145-156. 77. Truger. W. and Jensen, J. (1976). Human malaria parasites in continuous culture. Science, 193, p. 673-675. 115 University of Ghana http://ugspace.ug.edu.gh Page 1 1 7 1 1 8 1 1 9 120 121 122 1 2 3 1 2 4 1 2 5 APPENDIX I INFRARED SPECTRUM IR spectrum of PE6 IR spectrum of PE8 IR spectrum of PEI 1 ... ................ IR spectrum of D5 (triacontanol) IR spectrum of D65 (triacontanyl ferulate) IR spectrum of D66 (triacontanyl p-coumarate) IR spectrum of D9 IR spectrum of E13 IR spectrum of El6f, ... 116 University of Ghana http://ugspace.ug.edu.gh Wave n u m b e r[cm-1 j Accumulation Zero Filling Gain Date/Time Update Operator File Name Sample Name Comment 71 ON 4 3/14/01 1:10PM 3/14/01 1:14PM W R P 03 14_2001-006. jw s P E 6 Nujol Mull Resolution Apodization Scanning Speed 4 cm-1 Cosine 2 mm/sec 1:3425 ,47 .86 5: 1699, 11.47 9:1266, 66.14 2: 2956, 2.81 6: 1666, 1.27 10: 1133, 83.94 3: 2915, -0.15 7 :1467 ,15 .27 11:966, 83.34 4 :2851 ,0 .07 8: 1377, 60.80 12: 722. 57.62 University of Ghana http://ugspace.ug.edu.gh Wavenumber[cm-1] Accumulation Zero Filling Gain Date/Time Update Operator File Name Sample Name Comment 64 ON 4 3/14/01 1:23PM 3/14/01 1:27PM W R P 03 14 2001 -007.jws P E 8 Nujol Mull Resolution Apodization Scanning Speed 4 cm-1 Cosine 2 mm/sec 1:3510, 95.82 5: 1725, 86.07 9: 1463, 52.55 2: 2955, 15.84 6: 1640, 99.27 10: 1376, 80.64 3: 2920, 0.75 7: 1604, 101.68 11: 1176, 62.42 4: 2850, 6.78 8: 1519, 89.16 University of Ghana http://ugspace.ug.edu.gh ■10 1 J 1 1 ‘ 1 —------------3800 3000 2000 1000 500 Wavenumber[cm-1 ] Accumulation 111 Resolution 4 cm-1 Zero Filling ON Apodization Cosine Gain 8 Scanning Speed 2 mm/sec Date/Tima 3/27/01 2:34PM Update 3/27/01 2 :44PM Operator W R P File Name 03 27 2001-006.jws Sample Name P E 11 Comment KBr 1:3255, 12.20 5: 2920, 0.07 9: 1468,11.30 2: 3184, 19.13 6: 2852, 0.94 10: 1176, 7.27 3: 3061, 22.10 7: 1609, 2.06 11: 1084, 38.10 4: 2957, 3.26 8: 1509, 3.13 12: 721 ,32 .80 University of Ghana http://ugspace.ug.edu.gh 3800 3000 2000 1000 500 Wavenumber[cm-1 ] Accumulation 74 Resolution 4 cm-1 Zero Filling O N Apodization Cosine Gain 4 Scanning Speed 2 mm/sec Date/Time 3/14/01 2 :05PM Update 3/14/01 2 :07PM Operator W R P File Name 03 14 2001 -010.jws Sample Name D5 Comment KB r disk 1:3350, 95.74 5: 1061, 68.55 2: 2917, 1.54 6: 721, 64.83 3: 2849, 3.83 4: 1463, 44.51 University of Ghana http://ugspace.ug.edu.gh Wavenumber[cm-1] Accumulation Zero Filling Gain Date/Time Update Operator File Name Sample Name Comment 68 ON 4 3/14/01 1:57PM 3/14/01 2 :01PM W R P 03_14_2001 -009. jws D 6 (5) Nujol Mull Resolution Apodization Scanning Speed 4 cm-1 Cosine 2 mm/sec 1:3488, 88.96 5: 2853, 13.62 9: 1378, 58.28 2: 3445, 96.01 6: 1698, 58.76 10: 1063, 76.56 3: 2958, 10.60 7: 1674, 68.07 4: 2924, 1.94 8: 1466, 38.72 University of Ghana http://ugspace.ug.edu.gh If: IR spectrum of D66 (triacontanyl p-coumarate) i i University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh 3800 3000 2000 1000 500 Wavenumber[cm-1 ] Accumulation 83 Resolution 4 cm-1 Zero Filling O N Apodization Cosine Gain 8 Scanning Speed 2 mm/sec Date/Time 3/27/01 1:56PM Update 3/27/01 2 :01PM Operator W R P File Name 03 27 2001 -005.jws Sample Name D 9 Comment KBr 1:2917, 9.25 2: 2847, 14.86 3: 1712, 44.95 4: 1463, 56.61 5:1266, 50.14 6: 1171, 45.19 7: 1037, 69.12 8: 974, 67.40 9 :7 21 ,6 4 .29 University of Ghana http://ugspace.ug.edu.gh Wavenumber[cm-1] Accumulation Zero Filling Gain Date/Time Update Operator File Name Sample Name Comment 74 O N 4 3/27/01 0 :03PM 3/27/01 0 :07PM W R P 03_27_2001 -003. jws E13 KBr Resolution Apodization Scanning Speed 4 cm-1 Cosine 2 mm/sec (N 1:2965, 34.22 5: 1630, 87.51 2: 2917. 3.23 6: 1464, 54.36 3: 2849, 6.66 7: 1300, 76.29 4: 1711. 40.03 8: 1170, 69.91 University of Ghana http://ugspace.ug.edu.gh Wavenumber[cm -1 j Accumulation Zero Filling Gain Date/Time Update Operator File Name Sample Name Comment 73 ON 4 3/27/01 11:27AM 3/27/01 11:32AM W R P 03_27_2001 -002. jws E16 -6 KBr Resolution Apodization Scanning Speed 4 cm-1 Cosine 2 mm/sec cm^ 1 2371.9 ® r2172a |- 1.0OTJ I He: ^-NMR spectrum of PEI 1 (expanded) 7 . 6 7 . 4 7 . 2 7 . 0 1 1 I 1 16 .8 6 . 4 6 . 2 6 . 0 5 . 8 5 . 6 5 . 4 S . 2 ppm University of Ghana http://ugspace.ug.edu.gh expl stdlh SAMPLE DEC. & VT date Mar 2 01 df rq 300 . 061 solvent CDC13 dn HI file exp dpwr 12 ACQUISITION dof 0 sf rq 300.061 dm nnn tn HI dmm c at 3.751 dmf 200 np 20224 PROCESSING sw 2695.7 wtflie fb 1600 proc ft bs 16 f n not used tpwr 50 pw 5.5 werr dl 0 wexp tof -351.9 wbs nt 16 wnt ct 16 alock n ga 1 n not used FLAGS i 1 n 1 n y dp y DISPLAY sp 466 .8 wp 805 .6 vs 1000 sc 0 wc 250 hzmm 3.22 1 s 500.00 rf 1 2371 .9 rfp 2172.4 th 20 1 ns 1.000 nm cdc ph Ilf: XH-NMR spectrum of PEI 1 (expanded) 4 . 2 4 ,0 3 . 8 3 . 6 3 . 4 3 . 2 3 . 0 2 . 8 2 . 6 2 . 4 2 . 2 2 . 0 1 . 8 ppm .5 94 University of Ghana http://ugspace.ug.edu.gh 1— ■— ' 1 ' 1— 1 1 1 1 1 '— ' 1— ' ' ' ' 1 ' 1 ' • 1— ■ 1 ' 1 I ' ' 1 1 I ' ' ' ■ I 8 7 6 5 4 3 2 1 -0 ppm University of Ghana http://ugspace.ug.edu.gh expl stdlh SAMPLE DEC. & VT date Aug 29 00 df rq 300.061 solvent CDC13 dn HI file exp dpwr 12 ACQUISITION dof 0 sf rq 300.061 dm linn tn HI dmm c at 3. 744 dmf 200 np 20736 PROC ESSIM3 sw 2769.5 v/tf i 1 e fb 1600 proc ft bs 16 fn not used tpwr 50 pw 5.5 werr dl 0 wexp tof -371.3 wbs nt 16 wnt ct 16 alock n ga i n not used FLAGS 11 n i n y dp y DISPLAY sp 669 .1 vp 534.2 vs 1000 sc 0 wc 250 hzmm 2.14 i s 600.90 rf 1 2426.7 rfp 2172.4 th 12 i ns 1.000 nm cdc ph Ilh: ’H-NMR spectrum of D5 (triacontanol) expanded CO 3 . S 3 . 5 3 . 4 3 . 2 3 . 0 Z . 8 2 . 6 2 . 4 ppm University of Ghana http://ugspace.ug.edu.gh D5 expl stdlh SAMPLE DEC. & VT date Aug 29 00 dfrq 300.061 solvent CDC13 dn HI file exp dpwr 12 ACQUISITION dof 0 sf rq 300.061 dm nnn tn HI dmm c at 3.744 dmf 200 np 20736 PROCESSING sw 2769 .5 w t f 1le fb 1600 proc ft bs 16 fn not used tpwr SO pw 5.5 werr dl 0 wexp tof -371.9 wbs nt 16 wnt ct 16 alock n gain not used FLAGS i 1 n 1 n y dp y DISPLAY Sp 72.0 wp 534 .2 vs 1000 sc 0 wc 250 hzmm 2.14 i s 600. '!0 rfl 2423.7 rfp 2172.4 th 8 ins 1.000 nm cdc ph Hi: *K m !H-NMR spectrum of D5 (triacontanol) expanded University of Ghana http://ugspace.ug.edu.gh 1--- 1 ' ’ '— I— '---- '— '----'---1— ■— '— T----1----1----'---- 1----1— 1--1— '— '— 1— 1----1— '— 1---- '— --- 1----'----'----1----'----1----1— T ’— 1 I ' ' 8 7 6 5 4 3 2 1 - O p pm University of Ghana http://ugspace.ug.edu.gh DH5 expl stdlh SANPLE DEC. & VT date Aug 29 00 dfrq 300 .061 soIvent CDC 13 dn HI file exp dpwr 12 ACQUISITION dof 0 sf rq 300. 061 dm nnn tn HI dmm c at 3.744 dmf 200 np 20736 PROCESSING sw 2769 .5 w t f 11e fb 1600 proc ft bs 16 f n not used tpwr 50 pw 5 .5 werr dl 0 wexp tof -303 .7 wbs nt 16 wnt ct 16 alock n ga 1 n not used FLAGS 11 n 1 n y dp y DISPLAY sp 1646 . 0 wp 729 • 8 eg vs 5000 co sc 0 ^ wc £5 0 ^ hzmm 2co92 1 s 500“ 00 rf 1 18*.5 rfp 0 th 20 Ins 1 . 00 nm cdc ph JJ cn Ilk: !H-NMR spectrum of D65 (triacontanyl ferulate) expanded «> ^ *W>»W 1 1 I 1 ' 7 . 27 . 8 7 . 6 7 . 4 7 . 0 6 . Q 6 . 6 6 . 4 6 . 2 6 . 0 ppm University of Ghana http://ugspace.ug.edu.gh SAMPLE date Aug 29 00 solvent CD C13 file exp ACQUISITION sfrq tn at np sw fb bs tpwr pw dl tof nt ct alock gain il i n dp [ sp wp vs sc wc hzmm 1 s rfl rfp th i ns DH5 expl stdlh 300.061 Hi 3.744 20736 2769 .5 1600 16 50 5.5 0 -303.7 16 16 n not used FLAGS n y y ISPLAY 706 .4 723 .8 5000 0 250 2.32 500.00 187.50201.000 cdc ph DEC. & VT | df rq 300.061 dn Ha dpwr dof dm dmm dmf PROCESSING wtf i1e proc 2 0 ) fn werr wexp wbs wnt not usei III: H-NMR spectrum of D65 (triacontanyl ferulate) expanded I I I I | I I—I I | 1 University of Ghana http://ugspace.ug.edu.gh DH5 expl stdlh SAMPLE DEC. & VT date Aug 29 00 df rq 300 .061 solvent CDC 13 dn HI file exp dpwr 12 ACQUISITION dof 0 sf rq 300.061 dm nnn tn HI dmm c at 3.744 dmf 200 np 20736 PROCESSING sw 2769 .5 w t f 1 le fb 1600 proc ft bs 16 fn not used tpwr 50 pw 5.5 werr dl 0 wexp tof -303 .7 wbs nt 16 wnt ct 16 alock n gain not used FLAGS 11 n 1 n y I dp y DISPLAY sp -30 .4 wp 726 .4 vs 5000 sc 0 wc 250 hzmm 2.91 1 s 500.00 rfl 187 .5 rfp 0 th 20 1 ns 1.000 nm cdc Ph 1 Ilm: H-NMR spectrum of D65 (triacontanyl ferulate) expanded ppm Onrn University of Ghana http://ugspace.ug.edu.gh DG6 SAMPLE date Aug 29 00 solvent CDC13 file exp ACQUISITION expl stdlh sf rq tn at np sw fb bs tpwr pw dl tof nt Ct alock gal n11 1 n dp C sp wp vs sc wc hzmm t s rf 1r f p th Ins 300.061 HI 3.752 21888 2917.0 1600 16 50 5.50 -388 .9 16 16 not FLAGS cdc ph y yY -347.1 2917.0 50000 250 11.67 711.23 2519.5 2172.4 20 1.000 DEC. & VT dfrq 300 dn dpwr dof dm dmm dmf PROCESSING wtflie proc 061 HI 120 nnn c2 0 0 f n werr wexp wbs wnt not used Iln: 1H-NMR spectrum of D66 (triacontanyl WuL p-coumarate) o ppm University of Ghana http://ugspace.ug.edu.gh SAMPLE date Aug 29 00 solvent CDC13 file exp ACQUISITION DEC. & VT dfrq 300.061 sf rq tn at np sw fb bs tpwr pw dl tof nt ct alock gai n i 1 i n dp 300.061 HI 3.752 21333 23 17.0 1600 1*5 50 5.5 0 -338.9 16 16 n not used FLAGS n y y dn dpwr dof dm dmm dmf PROCESSING Wtfile HI 120nnn c2 0 0 proc f n werr wexpwbs wnt ft not used Ho: ^-NM R spectrum of D66 (triacontanyl p-coumarate) expanded 7.8 7 . 6 7 . 4 7 . 2 7 . 0 6 . 8 6 . 6 6 . 4 6 . 2 6 . 0 ppm University of Ghana http://ugspace.ug.edu.gh SAMPLE DEC. & VT exp3 stdlh date Mar 2 01 df rq 300 .06 1 solvent CDC13 dn HI file exp dpwr 12 ACQUISITION dof 0 sf rq 300.061 dm nnn tn HI dmm c at 3.748 dmf 200 np 21824 PROCESSING sw 2911.4 wtf H e fb 1600 proc ft bs 16 f n not used tpwr 50 pw 5.5 werr dl 0 wexp tof -312.2 wbs nt 16 wntC t 1 6 a l o c k n ~!--- '--- 1--- 1--- *--- 1--- ’--- 1--- 1 1 I 1 1 1 1 1--- 1--- 1--- 1--- 1--- T 8 7 6 5 4 University o Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh SAMPLE DEC. & VT I date Mar 2 01 dfrq 300. oil solvent CDC13 dn [HI file exp dpwr 12 ACQUISITION dof 0 sf rq 300. 061 dm inn tn HI dmm c at 3,748 dmf 200 np 21824 PROCESSING sw 2311.4 v t f 1le fb 1600 proc ft bs 16 fn not u 3 ed tpwr 50 pv 5.5 verr dl 0 wexp tof -312 .2 vbs nt 16 wnt ct 16 a lock n ga i n not used FLAGS i 1 n 1 n dp sp wp vs sc vc hzmm 1 s rf 1 rf p th 1 ns cdc ph 1832.4 602.1 4000 250 2.41 500.00 2440.0 2172.4 8 1 . 0 0 0 Ilq: XH-NMR spectrum of D9 (expanded) cn University of Ghana http://ugspace.ug.edu.gh date Mar 2 01 df rq 300.061 solvent CDC13 dn HI file exp dpwr 12 ACQUISITION dof 0 sf rq 300.061 dm nnn tn HI dmm c at 3.748 dmf 2 00 np 21824 PROCESSING sw 2911.4 w t f 11e fb 1600 proc ft bs 16 fn not used tpwr 50 pw 5.5 werr dl 0 wexp tof -312.2 wbs nt 16 wnt ct 16 a lock n qa 1 n not used FLAGS 11 n 1 n y dp y DISPLAY sp 940 . 1 wp 598 .3 vs 800 sc 0 wc 250 hzmm 2.39 1 S 500.00 rf 1 2440.0 rfp 2172.4 th 8 i ns 1.000 nm cdc ph Hr: XH-NMR spectrum of D9 (expanded) cr> University of Ghana http://ugspace.ug.edu.gh DEC. & VT date Mar 2 01 df r q 300.061 solvent CDC 13 dn HI file exp dpwr 12 ACQUISITION dof 0 Sf rq 300.061 dm nnn tn HI dmm c at 3.748 dmf 200 np 21824 PROCESSING sw 2911.4 w t f 1le fb 1600 proc ft bs 16 f n not used tpwr 50 pw 5.5 werr dl 0 wexp tof -312.2 wbs nt 16 wnt ct 16 alock n ga 1 n not used FLAGS 1 1 n 1 n y dp y DISPLAY sp 690.6 wp 594.6 vs 800 sc 0 wc 250 hzmm 2 .38 1 s 500.00 rf 1 rfp 2440 . 0 2172 .4 IIs: th 8 1 ns 1. 000 nm cdc ph IIs: H-NMR spectrum of D9 (expanded) University of Ghana http://ugspace.ug.edu.gh APPENDIX III UC-NMR SPECTRA Page ilia. i;!C-NMR spectrum of PE6 ... ... ............................. 147 mb. nC-NMR spectrum of PEI 1 ... ................ 148 I lie, "C-NMR spectrum of PEI 1 (expanded) ................ 149 nid. L,C-NMR spectrum of D65 (triacontanyl ferulate) 150 Ille. nC-NMR spectrum of D9 151 mi. ' ’C-NMR spectrum of D9 (expanded) 152 lllg. ! !C-NMR spectrum of D9 (expanded) 153 146 University of Ghana http://ugspace.ug.edu.gh PE6 SAMPLE DEC. & VT exp2 stdl3c date Apr 23 01 df rq 300.061 solvent CDC 13 dn HI file exp dpwr 34 ACQUISITION dof 0 sf rq 75.458 dm yyy tn C13 dmm w at 1.815 dmf 9404 np 68096 PROCESSING sw 18761.7 lb 1.00 fb 10400 w t f i 1e bs 16 proc ft tpwr 54 f n not used pw 3.5 dl 2 . 000 werr tof 0 wexp nt 256 wbs ct 160 wnt alock n gal n not used FLAGS 11 n i n n dp y DISPLAY sp -1835 .3 wp 18761 .7 vs 500 sc 0 wc 250 hzmm 20.12 i s rf 1 500.00 7645 .6 Ilia: rfp 5810.2 th 20 i ns 1 .000 nm no ph C-NMR spectrum of PE6 co(N University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh SAMPLE DEC. & VT date Har 2 01 df rq 300.061 solvent 00013 dn HI file exp dpwr 34 ACQUISITION dof 0 sf rq 75 .458 dm yyy tn C13 dmm w at 1.815 dmf 9404 np 68096 PROCESSING sw 18761.7 1b 1 .00 fb 10400 w t f \ 1e bs 16 proc ft tpwr S4 f n not used pw 3.5 dl 2 .000 werr tof 0 wexp nt 256 wbs ct 256 wnt a lock n ga i n not used FLAGS i 1 n i n n dp y DISPLAY sp -1821.3 wp 18761.7 vs 200 s c 0 VC 250 hzmm 20 . 12 1 s 500.00 rf 1 1821.3 rfp 0 th 11 1 ns 1 .000 1UI1 1LU |JI1 TTTh- 1 University of Ghana http://ugspace.ug.edu.gh SAMPLE date Mar 2 01 solvent CDC13 file exp ACQUISITION sf rq tn 75.458 C13 df rq dn dpwr dof dm dmm VT 300 . 061 HI 34 0 yyyw 50 45 4 0 35 30 25 g t e a g HsSrr r t.i I I. a i - r: treK 'KWKBWSW.*1.* . * *- t - t - t - University of Ghana http://ugspace.ug.edu.gh 22 .7 45 O nIIIc: 13C-NMR spectrum of PE11 (expanded) 1 0 -5 ppm University of Ghana http://ugspace.ug.edu.gh SAMPLE date Aug 29 00 solvent CDC13 file exp ACQUISITION sf rq tn at npsw fb bs tpwr pw dl tof nt ct alock ga 1 n 11 1 n dp sp wp 75.458 C13 1 .815 68096 18761 .7 10400 16 54 3.52 . 0000 384 384 n not used FLAGS wc hzmm i s rf 1 rfp th 1 ns co e S DEC. & VT r«. r l£> df rq 300.061 1 "j dn HI - k dpwr 34 dof 0 dm yyy dmm w dmf 9404 PROCESSING lb 1 .00 w t f 1le proc ft f n not used werr wexp wbs wnt DISPLAY -1835.3 18761 .7 1 00 0 0 250 20.12 500.00 7645 .6 5810.2 26 . 000 Illd: no ph °C-NMR spectrum of D65 (triacontanyl ferulate) University of Ghana http://ugspace.ug.edu.gh Ille: 13C-NMR spectrum of D9 I '— r- i ' ------------r "— ^ — ' i ......... i— 1 ■ ■ ■ i 1 © 0 . 0 1 6 0 . 0 1 4 0 . 0 1 2 0 . 0 1 0 0 . 0 BO . 0 P P M University of Ghana http://ugspace.ug.edu.gh ^> 7 \| N I T RO B DAT E 6 - 3 - ! 31 S F 5 4 . 0 6 3 S Y 5 4 . 0 0 1 8 3 9 6 . 3 8 7 S I 3 2 7 6 8 TD 3 2 7 6 8 SW 1 2 5 0 0 . 0 0 0 H Z / P T 7 6 3 PW 5 . 2 RD 3 . 0 0 0 AQ 1 . 3 1 1 RG 40 0 NS 5 2 8 9 7 TE 2 9 7 FW 1 5 7 0 0 0 2 3 1 5 4 . 5 3 1 DP 1 8H CPD LB 1 . 0 0 0 GB 0 . 0 CX 20 . 00 CY 1 5 . 00 F I 1 8 6 . 1 8 0 P F 2 - . 9 1 8 P HZ/CM 5 0 5 . . 753 PPM/CM 9 . 3 5 5 SR 2 9 0 4 . 0 2 60 . O 4 0 . 0 University of Ghana http://ugspace.ug.edu.gh cuiajif—|i^ rvjjru fMjfu. C . 0 0 3 DATE 6 - 3 - 9 1 SF 5 4 . 0 6 3 SY 5 4 . 0 0 1 8 3 9 6 . 3 8 7 S I 3 2 7 6 8 TD 3 2 7 6 8 SW 1 2 5 0 0 . 0 0 0 HZ / P T 7 6 3 PW 5 . 2 RD 3 . 0 0 0 AQ 1 . 3 1 1 RG 4 0 0 NS 5 2 8 9 7 TE 2 9 7 FW 1 5 7 0 0 0 2 3 1 5 4 . 5 3 1 DP 18H CPO LB 1 . 0 0 0 GB 0 . 0 CX 20 . 00 CY 1 5 . 00 F I 1 8 5 . 0 0 9 P F 2 1 2 0 . 0 0 8 P HZ/CM 1 7 5 . 7 0 5 PPM/CM 3 . 2 5 0 SR 2 9 0 4 . 0 2 (N 1 1 I 1 I ’ ' “ I ' 1 ^ 1 ’ ' “T 1 8 0 1 7 0 . 0 1 6 0 . 0 1 5 0 . 0 1 4 0 . 0 1 3 0 . 0 P P M University of Ghana http://ugspace.ug.edu.gh 13C-NMR spectrum of D9 (expanded) C . 0 0 3 OATE 6 - 3 - 9 11 SF 5 4 . 0 6 3 S Y 5 4 . 0 01 8 3 9 6 . 3 8 7 S I 3 2 7 6 8 TD 3 2 7 6 8 SW 1 2 5 0 0 . 0 0 0 H Z / P T 7 6 3 PW 5 . 2 RD 3 . 0 0 0 AQ 1 . 3 1 1 RG 400 NS 5 2 8 9 7 TE 2 9 7 FW 1 5 7 0 0 0 2 3 1 5 4 . 5 3 1 DP 1 8H CPD LB 1 . 00 0 GB 0 . 0 CX 2 0 . 00 CY 1 5 . 00 F I 1 4 0 . 0 0 5 P F 2 1 2 0 . 0 2 3 P HZ /CM 5 4 . 0 1 6 PPM/CM 9 9 9 SR 2 9 0 4 . 0 2 University of Ghana http://ugspace.ug.edu.gh APPENDIX IV MASS SPECTRA Page IVa. Mass spectrum of D5 (triacon tano l) ................ ................ 155 IVb. Mass spectrum of D65 (triacontanyl ferulate)... ................ 156 IVc. Mass spectrum of D6(, (triacontanyl p-coumarate) 157 154 University of Ghana http://ugspace.ug.edu.gh ir CMT-L »-near 3 S c : 8 5 I n t . : ? 0 . 5 I 0 0 0 0 t o 6 0 0 . 0 2 5 0 Temp 2 4 1 . 8 d e g . C C u t L e v e l : 0 . 0 0 V. 739320100 7 0 - IVa: Mass spectrum of D5 (triacontanol) 4 2 0 392 321 336 350 ? III , llll , illyI hi, , i 4 10 lL 739328 320 34 0 3 B 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 0 0 5 0 0 5 2 0 5 4 0 5 6 0 97 5 8 0 6 0 0 m/z 155 University of Ghana http://ugspace.ug.edu.gh Temp . 2 6 8 . 9 d e g .C el : 0.QQ y. mm 50- IVb: Mass spectrum of D65 (triacontanyl ferulate) 177 137 83 97 ■il i„ ,ii 40! 60 194 20? 304 318 34 1 374 3 88 -t— ,--- j , n r~ 100 120 M 0 160 180 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 156 University of Ghana http://ugspace.ug.edu.gh : R e g u l a r t r f - L i n e a r ] S c ‘ n * • 1 6 9 % . m/z 164.0000 *n t • 1 l0 ‘, ' ?5 t p , " U range : 35.0000 to 000.0000 1098336 100 Temp . 2 6 9 . 0 d e g .C C u t L e v e l : 0 . 0 0 * 90 70 60 5 0 - IVc: Mass spectrum of D66 (triacontanyl p-coumarate) 584 425 ki i i »i 5 2 8i. ■ ?18S—1*—r 4 20 4 4 0 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0 5 6 0 5 8 9 6 0 0 6 2 0 6 4 0 GCG GO0 7 0 0 7 2 0 7 4 0 7 6 0 7 8 0 8 0 m/Z 1098336100 90 164 70 - 40 20 - jL jJ 83 UUL • S', 19-1 236 2G-I 2 9 2 3 J3 3 1 6 3 63 3 3 0 3 97 ■,0 6 0 0 0 10 0 | £ 0 | , io 16 0 ISO 2 0 0 3 J 0 J 8 0 3 0 0 3 iB 3 ' ifl 3 5 0 3 0 0 IGS 157 University of Ghana http://ugspace.ug.edu.gh APPENDIX V ULTRAVIOLET (UV) SPECTRA UV spectrum of D9 UV spectrum of E13 ... UV spectrum of PEI I ................ University of Ghana http://ugspace.ug.edu.gh !j H University of Ghana http://ugspace.ug.edu.gh Vb: UV spectrum of E13 University of Ghana http://ugspace.ug.edu.gh Vc: UV spectrum of PE11 University of Ghana http://ugspace.ug.edu.gh HOMONUCLEAR PROTON-PROTON SHIFT CORRELATION SPECTRA (COSY) Page Via. 1H-1H COSY spectrum of PEI I ................................................ 163 Vlb. 'H-'H COSY spectrum of D65 (triacontanyl ferulate) ... ... 164 Vic. 1H-1H COSY spectrum of D9 ... ... ... ... ... ... 165 APPENDIX VI 162 University of Ghana http://ugspace.ug.edu.gh PULSE SEQUENCE: relayh Relax, delay 1.000 sec COSY 90-45 A c q . time 0.190 sec Width 2695.7 Hz 20 Width 2695.7 Hz 4 repetitions 128 Increments OBSERVE HI, 300.0595294 MHz DATA PROCESSING ------ Sine bell 0.095 sec FI DATA PROCESSING Sine bell 0.024 sec FT Size 1024 x 1024 Total time 10 minutes Ambient temperature GEMINI-300BB "gemini" jl FI (ppm)-0 Via: COSY spectrum of PEI 1 6 - o 9O ■ 8- -i— ,— ,— 1 t i 5 4 F 2 ( p p m ) University of Ghana http://ugspace.ug.edu.gh FI CPPnO University of Ghana http://ugspace.ug.edu.gh PULSE SEQUENCE: relayh Relax, delay 1.000 sec COSY 9 0-90 A c q . time 0.176 sec Width 2911.4 HZ 20 Width 2911.4 Hz 4 repetitions 64 increments OBSERVE HI, 300.0595294 MHz OATA PROCESSING S1ne bell 0.088 sec ------ FI DATA PROCESSING Sine bell 0.011 sec FT size 1024 x 1024 Total time 5 minutes Ambient temperature GEMINI-300BB "gemini" University of Ghana http://ugspace.ug.edu.gh Vic: COSY spectrum of D9 0 osm O 0 \ 0 0 & S 8 l .-- .--]—.---1-- r4 F 2 C P P " > ) University of Ghana http://ugspace.ug.edu.gh Page The life cycle of the malaria parasite (Plasmodium falciparum)........................ 167 APPENDIX VII 166 University of Ghana http://ugspace.ug.edu.gh Life Cycle o f malaria Parasite {Plasmodium falciparum) (http://www.malariatest.com/cycle.html) 167 University of Ghana http://ugspace.ug.edu.gh Page Buffers and Solut ions . . . ... . . . . . . . . . . . . . . . . . . 169 APPENDIX VIII 168 University of Ghana http://ugspace.ug.edu.gh Buffers and Solutions (a) Washing Medium RPM I-1 6 4 0 500.0ml Gentamycin( lOmg/ml) 2.5ml / 500 ml RPMI L- glutamine 4.0ml / 500 ml RPMI (b) Complete Parasite Medium RPMI - 1640 Gentamycin(10mg/ml) L- glutamine NHS (A or O positive) 500.0ml 2.5ml / 500 ml RPMI 4.0ml / 500 ml RPMI 10% Filtered through 0.8 m pore filter (to start with and later 5%) Filter sterilize medium and store at 4°C (c) CPD Buffer, pH 5-6 17mM Citric acid 5.0 g 90mM Sodium citrate 26.47 g 175mM Glucose 31.54 g 16mM NaH3P04 2.21 g 2mM Adenin (6-Aminopurine) 0.27 g Distilled water up to 1.0 litre Filter sterilize medium and store at 4°C (d) Giemsa Buffer. pH 7.2 NaHP04 1.0 g K.H2PO4 0.7 g Distilled water up to 1.0 litre S to re at 4°C 169 University of Ghana http://ugspace.ug.edu.gh (e) Parasites Thawing Mix 3.5% sodium chloride in distilled water Filter sterilize and store at 4°C (f) Freezing Mix 4.2% Sorbitol in 0.9% sodium chloride Take 72 ml sorbitol solution and add 28 ml glycerol Stir well and filter sterilize Store at 4'’C 170 University of Ghana http://ugspace.ug.edu.gh