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Addis Ababa University School of Graduate Studies ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES ANTIPLASMODIAL ACTIVITY OF EXTRACTS FROM DODONAEA ANGUSTIFOLIA L.F. AND BERSAMA ABYSSINICA FRESEN AGAINST PLASMODIUM BERGHEI IN MICE MODEL BERHAN MENGISTE A Thesis Submitted to the School of Graduate Studies of Addis Ababa University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Pharmacology MARCH, 2008 ANTIPLASMODIAL ACTIVITY OF EXTRACTS FROM DODONAEA ANGUSTIFOLIA L.F. AND BERSAMA ABYSSINICA FRESEN AGAINST PLASMODIUM BERGHEI IN MICE MODEL DEPARTMENT OF PHARMACOLOGY, FACULTY OF MEDICINE ADDIS ABABA UNIVERSITY Approval of the Board of Examiners Name Signature Date ______________________________________ ___________ ___________ Chairman, Department Graduate Committee ______________________________________ _____________ __________ Advisor ______________________________________ _______________ _________ Co-advisor ______________________________________ _______________ ________ External Examiner ______________________________________ ______________ ________ Internal Examiner CONTENTS PAGE TABLE OF CONTENTS ------------------------------------------------------------- i LIST OF ABBREVIATIONS --------------------------------------------------------- iii LIST OF TABLES -------------------------------------------------------------------- iv LIST OF FIGURES ------------------------------------------------------------------- v LIST OF PLATES--------------------------------------------------------------------- vi ACKNOWLEDGEMENT ------------------------------------------------------------- vii ABSTRACT --------------------------------------------------------------------------- viii 1. INTRODUCTION ------------------------------------------------------------------ 1 1.1. Epidemiology of Malaria -------------------------------------------------- 1 1.2. Etiology and Transmission of Malaria ----------------------------------- 2 1.3. Life cycle of malaria parasite --------------------------------------------- 3 1.4. Clinical features and Pathophysiology --------------------------------- 4 1.5. Malaria Diagnosis ---------------------------------------------------------- 5 1.6. Management of Malaria---------------------------------------------------- 6 1.7. Antimalarial drug resistance---------------------------------------------- 7 1.8. Prevention and control ----------------------------------------------------- 12 1.9. Rodent Model of Malaria for Antiplasmodial Activity Test------------ 14 1.10. Traditional Medicine in the Treatment of Malaria ------------------- 16 2. OBJECTIVE OF THE STUDY --------------------------------------------------- 19 2.1. General Objective ----------------------------------------------------------- 19 2.2. Specific objectives ---------------------------------------------------------- 19 3. MATERIALS AND METHODS --------------------------------------------------- 20 3.1. Plant material Preparation ----------------------------------------------- 20 3.2. Phytochemical Screening -------------------------------------------------- 21 3.3. In Vivo Antimalarial Screening with Crude Extracts ------------------ 21 3.3.1. Experimental Animals ----------------------------------------------- 21 3.3.2. Experimental Protocol ----------------------------------------------- 22 i 3.3.3. Parasite Inoculation ------------------------------------------------- 22 3.3.4. Extract Administration ---------------------------------------------- 23 3.3.5. Determination of Body weight change ---------------------------- 23 3.3.6. Measurement of Body Temperature ------------------------------- 23 3.3.7. Determination of Parasitemia -------------------------------------- 23 3.3.8. PCV Determination -------------------------------------------------- 24 3.3.9. Determination of Mean Survival Time ---------------------------- 25 3.4. Fractionation ----------------------------------------------------------------- 25 3.5. Antimalarial Activity Test with Fractions --------------------------------- 25 3.6. Acute Toxicity Study --------------------------------------------------------- 25 3.7. Data Management and Analysis ------------------------------------------- 26 4. RESULT ---------------------------------------------------------------------------- 27 4.1. Yields from Plant Materials ------------------------------------------------ 27 4.2. Phytochemical Screening--------------------------------------------------- 27 4.3. In Vivo Antimalarial Screening with Crude Extracts ------------------- 28 4.3.1. Effect of Crude Extracts on Body weight ------------------------- 28 4.3.2. Effect of Crude Extracts on Body Temperature ----------------- 29 4.3.3. Effect of Crude Extracts on Parasitemia and Survival Time --- 31 4.3.4. Effect of Crude Extracts on PCV ----------------------------------- 33 4.4. Yields of the Fractions of D. angustifolia aqueous extract ----------- 34 4.5. Effect of Fractions on Body Weight, Temperature, Parasitemia and mean survival time ---------------------------------------------------------- 34 4.6. LD50 of the Crude Extracts ------------------------------------------------ 38 5. DISCUSSION ---------------------------------------------------------------------- 40 6. CONCLUSION AND RECOMMENDATION ------------------------------------ 45 7. REFERENCES -------------------------------------------------------------------- 46 8. ANNEXES ------------------------------------------------------------------------- 54 ii LIST OF ABBEREVATIONS IC50 50% Inhibitory Concentration LD50 50% Lethal Dose WHO World Health Organization ACT Artimisinin Combination Therapy PCR Polymerase Chain Reaction RBC Red Blood Cells SP Sulphadoxine Pyrimethamine CQ Chloroquine DHFR Dihydrofolate Reductase PFCRT Plasmodium Falciparum Cross Resistance Transporter DHPS Dihydropteroate Synthase CQR Chloroquine Resistance DV Digestive Food Vacuole ITN Insecticide Treated Net IRS Indoor Residual Spray HM Herbal Medicine RPM Revolutions per Minute EHNRI Ethiopian Health and Nutrition Research Institute TSC Trisodium Citrate PCV Packed Cell Volume SPSS Statistical Packages for Social Science ANOVA Analysis Of Variance LSD Least Significant Difference SD Standard Deviation ATP Adinosine Triphosphate iii LIST OF TABLES Table Page 1: Geographical distribution of malaria drug resistance --------------------- 9 2: Experimental design of the study --------------------------------------------- 22 3: Percentage yields of crude extracts of D. angustifolia and B. abyssinica --------------------------------------------------------------- 27 4: Phytochemical screening of D. angustifolia and B. abyssinica ------------ 28 5: Effect of crude extracts of D. angustifolia seed on body weight of P. berghei infected mice --------------------------------------------------------- 29 6: Effect of crude extracts of B. abyssinica root bark on body weight of P. berghei infected mice ---------------------------------------------------------- 29 7: Effect of crude extracts of D. angustifolia seed on parasitemia and mean survival time of P. berghei infected mice ------------------------------- 32 8: Effect of crude extracts of B. abyssinica root bark on parasitemia and mean survival time of P. berghei infected mice ------------ ------------ 33 9: Percentage yields of fractions of D. angustifolia aqueous extract --------- 34 10: Effect of the fractions on body weight of P. berghei infected mice ------ 35 11: Effect of D. angustifolia seed aqueous extract fractions on parasitemia and mean survival time of P. berghei infected mice ---- 37 iv LIST OF FIGURES Figure Page 1: Global distribution of malaria in 2004 --------------------------------------- 2 2: Life cycle of malaria parasites in human and mosquito ------------------ 4 3: Effect of D. angustifolia seed (a) hydrolcoholic crude extract and (b) aqueous crude extract on rectal temperature of P. berghei infected mice ----------------------------------------------------------------------------------------- 30 4: Effect of B. abyssinica root bark (a) hydrolcoholic crude extract and (b) aqueous crude extract on rectal temperature of P. berghei infected mice --------------------------------------------------------------------------------- 31 5: Effect of D. angustifolia seed (a) hydrolcoholic crude extract and (b) aqueous crude extract on PCV of P. berghei infected mice on days 0 and 4 ----------------------------------------------------------------------------- 33 6: Effect of B. abyssinica root bark (a) hydrolcoholic crude extract and (b) aqueous crude extract on PCV of P. berghei infected mice on days 0 and 4 ------------------------------------------------------------------------------- 34 7: Effect of D. angustifolia seed (a) aqueous extract fractions and (b) butanol fraction on rectal temperature of P. berghei infected mice ------ 36 8: Effect of D. angustifolia seed (a) aqueous extract fractions and (b) aqueous extract butanol fraction on PCV of P. berghei infected mice on days 0 and 4 ----------------------------------------------------------------- 38 9: Probit Transformed Dose response of B. abyssinica LD50 ----------------- 39 v LIST OF PLATES Plate Page 1: Insecticide treated nets (ITN) as vector control strategy in malaria prevention and control ------------------------------------------------- 13 2: Intermittent preventive treatment with sulfadoxine- pyrimethamine in pregnant women ------------------------------------------- 14 3: Dodonaea angustifolia, L.F. ----------------------------------------------------- 18 4: Bersama abyssinica, Fresen ---------------------------------------------------
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