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Ethnopharmacological Assessment of Guibourtia Coleosperma and Diospyros Chamaethamnus Extracts As Alternative Treatment Options

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Ethnopharmacological Assessment of Guibourtia Coleosperma and Diospyros Chamaethamnus Extracts As Alternative Treatment Options ETHNOPHARMACOLOGICAL ASSESSMENT OF GUIBOURTIA COLEOSPERMA AND DIOSPYROS CHAMAETHAMNUS EXTRACTS AS ALTERNATIVE TREATMENT OPTIONS FOR MALARIA A DISSERTATION SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF NAMIBIA BY CHARWAN IWANETTE DU PREEZ (200631489) OCTOBER 2016 Main Supervisor: Dr. D.R. Mumbengegwi (Multidisciplinary Research Centre) Co-supervisor: Dr. R.A. Böck (Department of Biological Sciences) ii ABSTRACT Malaria, a parasitic infectious disease, remains one of the world’s foremost health concerns, even more so in developing countries. Much progress has been made in fighting the disease, particularly in Southern Africa where four countries (Namibia included) have targeted malaria elimination by 2020. Challenges such as the absence of a vaccine, resistance to insecticides, and particularly the emergence of resistance to current antimalarial treatment regimens threaten to undermine the current successes in malaria control efforts. Secondly, the lack of access and acceptance of conventional antimalarial treatment by populations in malaria endemic areas reduces the feasibility of eliminating and consequently eradicating malaria. Local communities in Namibia use plant-based medicines to treat malaria and malaria associated symptoms based on traditional observations or beliefs over decades. As to whether these plants are efficacious for the indication and cause toxicity is yet to be validated scientifically. The aims of this study were, therefore, to evaluate the biological activities of the extracts of two Namibian plant species to provide a scientific rationale for their traditional uses. Guibourtia coleosperma and Diospyros chamaethamnus, which are used to alleviate symptoms of malaria in Namibia, were investigated using phytochemical analyses, and in vitro and in vivo bioassays. Extracts were prepared by using solvent extraction of varying polarities to obtain a wide range of metabolites. Ground plant material was macerated in distilled water (aqueous extracts) and dichloromethane-methanol (1:1v/v) (organic extracts) respectively. The extracts were dried in vacuo, and examined for six classes of compounds known to have antiplasmodial activity using TLC. GC-MS was used to identify compounds iii in the plant extracts related to biological activity, using a BP5MS column. Radical scavenging abilities of the plant extracts were ascertained by the 2,2-Diphenyl-1-picryl- hydrazyl (DPPH) method. In vitro antimalarial activity was determined by reconstituting the plant extracts in water and DMSO at varying concentrations, and incubating extracts with Plasmodium falciparum D10 infected RBCs for 48 hours. Subsequently, growth inhibition of the P. falciparum parasites was determined using parasitaemia. In vitro assays to determine cytotoxic effects were conducted with the plant extracts using a fibroblast cell line (W138). In vivo inhibition of the growth of P. berghei in Swiss albino mice (20±4 g) was evaluated using optical microscopy on blood smears. Survival curves post-infection were also used to determine suppressive and prophylactic activities of extracts. The plant extracts were also evaluated for their toxicity in healthy mice using a dose escalation method with a starting dose of 300 mgkg-1. The crude extracts contained alkaloids, anthraquinones, flavonoids, steroids and terpenoids. Secondary metabolites with antiplasmodial, antibacterial and antioxidant properties were tentatively identified as 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4- one, phloroglucinol, stigmasterol, glycerol 2-hexadecanoate, α-amyrin, 9,12-octadecdenoic acid (Z,Z)-, hexadecanoic acid, oleic acid, lanosterol, spiculesporic acid, squalene, campesterol and vitamin E. Antioxidant activity results showed that the extracts of D. chamaethamnus yielded the highest antioxidant activities with IC50 values of ranging from 7.63 to 10.74 µg/mL, whilst the extracts G. coleosperma yielded antioxidant activities with IC50 values of ranging from 22.03 to 36.05 µg/mL. Moderate in vitro antiplasmodial activity (IC50 < 50 µg/ml) against P. falciparum D10 was observed for the two plants ranging from 18.30 to 31.61 µg/mL. All plant extracts showed no cytotoxicity with IC50 values above 100 iv µg/mL, except for the organic extract of D. chamaethamnus (CC50=29.73). The organic extracts for both D. chamaethamnus and G. coleosperma (800 mgkg-1) exhibited significant (P< 0.05) blood schizonticidal activity in the 4-day early infection test with parasite growth inhibition of 44.66 and 29.59 %, respectively. This dose also prolonged the survival of the mice by 50 and 58 % (i.e. with 6 and 7 days), respectively. The plant extracts also exhibited prophylactic activity in the mice inhibiting parasitaemia with 56.13 (D. chamaethamnus) and 55.48 % (G. coleosperma) at the highest dose (800 mgkg-1) and increasing survival by 155.6 and 22.2 % (i.e. with 14 and 3 days), respectively. Oral administration of crude extracts at the highest dose of 2000 mgkg-1 resulted in no mortalities or evidence of adverse effects, indicating that D. chamaethamnus and G. coleosperma extracts were non-toxic. The study showed promising antimalarial activities of D. chamaethamnus and G. coleosperma. The results show that the ethnomedicinal use of these plants to treat symptoms of malaria is rational and safe. This is a step in the right direction towards incorporating their use in mainstream health care policies as alternative treatment options for malaria. Identification of bioactive compounds to standardize extracts should the next step. Further studies should also include the optimization of doses to improve efficacy, and studies to assess the antiplasmodial activities of the two plants in combination treatments as used in an ethnomedicinal setting. Lastly, this study has shown that the plant extracts can also be used as a prophylactic. This is new knowledge should be shared with indigenous communities to maximize the medicinal use of these plants. v Key words: medicinal plants, Diospyros chamaethamnus, Guibourtia coleosperma, malaria, phytochemical screening, antimalarial compounds, in vitro antiplasmodial activity, in vivo antiplasmodial activity, cytotoxicity, acute oral toxicity vi TABLE OF CONTENTS ABSTRACT ...................................................................................................................... ii LIST OF FIGURES ......................................................................................................... x LIST OF TABLES ......................................................................................................... xv LIST OF ABBREVIATIONS ....................................................................................xviii ACKNOWLEDGMENTS ........................................................................................... xxii DECLARATION ......................................................................................................... xxiv CHAPTER 1: INTRODUCTION ................................................................................... 1 1.1 General introduction ............................................................................................... 1 1.2 What is malaria? .......................................................................................................... 3 1.3 Malaria, a health concern ............................................................................................. 5 1.4 Statement of the research problem ............................................................................... 6 1.5 Aims of the study ......................................................................................................... 7 CHAPTER 2: LITERATURE REVIEW ....................................................................... 9 2.1 Malaria: an infectious disease ................................................................................. 9 2.2 Developmental stages of the malaria parasites ..................................................... 14 2.3 Clinical manifestations of malaria ........................................................................ 16 2.3.1 Uncomplicated malaria ................................................................................. 17 2.3.2 Complicated malaria ..................................................................................... 19 2.3.3 Immunity and malaria ................................................................................... 20 2.4 Malaria burden ...................................................................................................... 22 2.4.1 The global malaria situation .......................................................................... 23 2.4.2 Malaria situation in Namibia ......................................................................... 27 2.5 Current efforts to control and eliminate malaria in Namibia ................................ 30 2.5.1 Control and prevention strategies .................................................................. 30 2.5.2 Diagnosis and treatment of malaria ............................................................... 31 2.6 Antimalarials ......................................................................................................... 33 vii 2.6.1 4-Aminoquinolines and 8-aminoquinolines .................................................. 34 2.6.2 Arylaminoalcohols ......................................................................................... 37 2.6.3 Artemisinins ..................................................................................................
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