DETERMINANTS OF ABORTION PRACTICE IN ADDIS ABABA

112 Pages·2010·1.06 MB·English

-- .
e
v;.:,;.~
EVALUATION OF THE ANTIMALARIAL ACTIVITY OF SOME
ETHIOPIAN MEDICINAL PLANTS AGAINST
PLASMODIUM FALCIPARUM IN VITRO
A Thesis
presented to the School of Graduate Studies
Addis Ababa University
In partial fulfilment of the requirements
for the degree of
Master of Science in Biology
By
Mesfm Bogale
June, 1994.
ACKNOWLEDGEMENTS
I express my deepest gratitude to my research advisor Dr Beyene Petros who always
has had the time to guide me through the research work. I would also like to thank Dr
Ermias Dagne for his valuable advices and support of the work; the Natural Products
Research Network for Eastern and Central Africa (NAPRECA) for covering the research
and subsistence costs during my stay in Madagascar, and also for covering 25 % of the cost
of transportation, through its IPICS-supported Exchange of Researchers Scheme; the
Ethiopian Science and Technology COlllJlliSl'il,lI\ fqr, «oyt;npg .7,5%, 9~ ~~ ~o~t, <!f
transportation. ':, ,
P.j
I am indebted to Prof R. Ratsima~ang~" ~9f R4~o~(tlli,V6; ~IrR,. ~.filiJ~ona an:d
, ,
'"
(
Mrs B. Rafatro (Institut Malagache de Re:::herch~Ap~l,iqtiec;;, i~n:J\, M~~~M~car) who
. , ,
( , , , ( , ( , { ( ( ( ( ( ( , { ( , , ( , < " (
helped me with the work, and all the staff of IMRA who made my stay in Antananarivo
very p leasan t.
Dr Sebsibe Demissew and Dr Ensermu Kelbessa are thanked for identification of
the plants used in this study. My thanks are also due to Dr Wendimagegn Mammo, WIt
Senait Asmellash, Ato Melaku Alemu, Ato Tekabe Ayalew, Ato Mamuye Hadis and all
who helped me with one or another aspect of the research work.
The National Programme for the Control of Malaria and other Vector-Borne
Diseases is also thanked for allowing me access to its research facilities. I also thank all
its staff for the technical support they offered me.
n
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................... I
TABLE OF CONTENTS ................................... , n
LIST OF TABLES ....................................... m
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. V
ABSTRACT .......................................... vn
I. INTRODUCTION .................' ................... I
' . " , " , ' , " " " , . " " " " I
A . Malana ............. ',' ........ ' ................. .
, ' (- •• , • , , • ,'" ' " • c , , { ,
B. Medicinal plants against malaria '. .' ., .. co: .' ., .. :; ...... ; ...•. 8
i': .. :.)., .. :. ..
II. MATERIALS AND METHODS .: .. : . .' . .',.,.;. II
IJ
1. Plant materials . . . . . . . . . .' . . . .•. . .'. . .. , , :,... .' .' .' . ". . . .
, , " , '
1.1. Description of the plauts ..'...;.'...::. .. . . .• . . ' .. . . . II
-. , ,_ , , I : ( , , : _ , ( ( , : , , ( , , , , < , '
1.2. Extraction and fractionation ...................... 16
2. Antimalarial test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 17
2. I. Preparation of media. . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.2. Preparation of non-infected red blood cells ............. 18
2.3. Preparation of infected red blood cells . . . . . . . . . . . . . . . . 18
2.4. In vitro antimalarial test ......................... 20
3. Cytotoxicity test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1. Preparation of medium ......................... 21
3.2. Test Procedure .............................. 22
Ill. RESULTS........................................ 23
IV. DiSCUSSION...................................... 53
V. CONCLUSION AND RECOMMENDA nON .................. 59
VI. REFERENCES ..................................... 61
.'
- '.' ."
ill
LIST OF TABLES
1. Yields of petroleum ether, chloroform and
methanol extracts . . . . . . . . . . . . . . . . . ............. 23
2. Percentage inhibition of maturation of P. jaiciparum
at various concentrations of petroleum ether extracts . . . . . . . . . . . . . . . 25
3. Percentage inhibition of maturation of P. jaiciparum
at various concentrations of chloroform ether extracts .. . . . . . . . . . . . . 26
4. Percentage inhibition of maturation of P. jaiciparum
at various concentrations of methanol ether extracts ............... 27
5. Percentage inhibition of maturation of P. jaiciparum
at various concentrations of chloroquine .... .............. 28
.6. Kind and amount of organic solvents used,
and column-fractions obtained from chloroform
extract of leaves of W. somnijera .......................... 38
7. Kind and amount of organic solvents used,
and column-fractions obtained from methanol
extract of leaves of W. somnijera .......................... 39
8. Kind and amount of organic solvents used,
and column-fractions obtained from chloroform
extract of leaves of V. amygdaUna . . . . . . . . .............. 39
9. Percentage inhibition of maturation of P. jalciparum
at various concentrations of column-fractions
from chloroform extract of leaves of W. somnijera ............... 40
10. Percentage inhibition of maturation of P. jaiciparum
at various concentrations of column-fractions
from methanol extract of leaves of W. sOlllllijera .. . . . . . . . . . . . . . . . 41
'., , ._"_ .• '-'- .
IV
II. Percentage inhibition of maturation of P. !alcipanlln
at various concentrations of column-fractions
from chloroform extract of leaves of V. amygdaliTUl . . . . . . . . . . . . . . . 42
12. Inhibition of HeLa cell growth by chloroform and methanol
extracts of leaves of W. somnifera, and chloroform extract from
leaves of V. amygdalina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
'."
'.
v
LIST OF FIGURES
Figure ~
I. The life cycle of human malaria parasites ...................... 4
2a. Percentage inhibition of growth of P. jalciparum
by chloroquine and petroleum ether extracts . . . . . . . . . . . . . . . . . . . . 29
2b. Percentage inhibition of growth of P. jalciparum
by chloroquine and petroleum ether extracts . . . . . . . . . . . . . . . . . . . . 30
3a. Percentage inhibition of growth of P. jalciparum
by chloroquine and chloroform extracts . . . . . . . . . . . . . . . . . . . . . . . 31
3b. Percentage inhibition of growth of P. jalciparum
by chloroquine and chloroform extracts . . . . . . . . . . . . . . . . . . . . . . . 32
4a. Percentage inhibition of growth of P. jalciparum
by chloroquine and methanol extracts . . . . . . . . . . . . . . . . . . . . . . . . 33
4b. Percentage inhibition of growth of P. jalciparum
by chloroquine and methanol extracts . . . . . . . . . . . . . . . . . . . . . . . . 34
5. IC"" IC. . and MIC values for chloroquine and
!>
petroleum ether extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
6. IC,., Ie..; and MIC values for chloroquine and
chloroform ether extracts ............................... 36
7. IC"" Ie.., and MIC values for chloroquine and
methanol ether extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
8. Percentage inhibition of growth of P. jalciparum
by column-fractions obtained from chloroform extract
of leaves of W. somnifera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
9. Percentage inhibition of growth of P. jalciparum
by column-fractions obtained from methanol extract
of leaves of W. somnifera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
, • .'. ',<.
VI
10. Percentage inhibition of growth of P. Jalciparum
by column-fractions from chloroform extract
of leaves of V. amygdalina .............................. 46
II. IC"" IC. ." and MIC values for column-fractions
from chloroform extract of leaves of
W. somnifera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
12. IC"" Ie.." and MIC values for column-fractions
from methanol extract of leaves of
W. somnifera ............. . " " " " " " " " 48
13. IC,., Ie.." and MIC values for column-fractions
from chloroform extract of leaves of
V. amygdalina . . . . . . . . . . . . . . ................. 49
14. Percentage inhibition of groWth of HeLa cells by
chloroform and methanol extracts of leaves of
W. somnifera, and chloroform extract of leaves of
V. amygdalina . . . . . . . . . . . . . . . . . . . . . . . . ......... 51
15. IC"" Ie.." and MIC values for chloroform and methanol
extracts of leaves of W. somnifera, and chloroform
extract of leaves of V. amygdalina against HeLa cells . . . . . . . . . . . . . . 52
vn
ABSTRACT
Thirty-three crude extracts from nine Ethiopian medicinal plants that are used to
treat malaria were screened for antimalarial activity against a chloroquine-resistant FCM-29
strain of Plasmodiumjaiciparum in vitro. The measurement of radiolabelled hypoxanthine
incorporation by P. jaiciparum was used to assess inhibition of growth of the parasite.
Three extracts, namely, chloroform and methanol extracts of leaves of Withania somnifera,
and chloroform extract of leaves of Vernonia amygdalina, showed substantial antimalarial
activity with IC", and Ie.., values less than 20 and 100 pg/ml, respectively. The IC", and
Ie.., values of the remaining extracts were out of this range. The antimalarial activity of
the three crude extracts improved by several-folds upon column-fractionation. These three
crude extracts were also tested for cytotoxicity against human HeLa cells in vitro; all of
them showed high cytotoxic activity. However, the antimalarial and cytotoxic components
of the extracts have not been determined.
..
_ . i" • _. ~ . . . -' ."T",
I
I. INTRODUCTION
A. Malaria
Malaria is a protowal disease transmitted to man by female mosquitoes of the genus
Anopheles. It is characterized by a cyclic course of periods of febrile attacks.
Splenomegaly, anaemia and occasional severe lesions of the nervous system, kidneys, and
other organs also characterize the disease.
Malaria has been known since the beginning of civilization. In Ebers Papyrus (1500
B.C.) are mentioned 'rigors,' fevers, splenomegaly, and the use of an oil expressed from
Balanites trees as a mosquito repeliant (Loban and Polowk, 1985). The aetiology remained
a mystery until Laveran identified the causative agents of human malaria in 1880; it was
only in 1897 that the role of mosquitoes in the transmission of malaria was discovered by
Ronald Ross (Bruce-Chwatt, 1985).
Plasmodiumjalciparum, P. vivax, P. ovale and P. mawriae are the causative agents
of human malaria. They belong to the Phylum Protowa, Class Sporowa, Order
Haemosporidia, Family Plasmodiidae and Genus Plasmodium. The genus Pklsmodium is
further divide into two Subgenera: Subgenus Plasmodium, containing P. vivax, P. ovale
and P. maiariae; Subgenus Laverania, containing P. jaiciparum (Beaver et. at., 1984;
Black et. at., 1986).
The types of causative agents differ on the basis of their morphological
characteristics, virulence, duration of the incubation period, immunological and
epidemiological characteristics and their sensitivity to the effect of chemotherapeutic drugs.
Malaria occurrence is determined by the wne of prevalence of the vectors, female
mosquitoes of the genus Anopheles, and suitable ambient temperature ensuring the
2
completion of sporogony in the mosquito. Out of the four types of causative agents of
human malaria, the greatest dissemination has been achieved by P. vivax (TOR, 1988). In
some countries of temperate climate, vivax malaria has been the single or predominant
form. This is explained by the capacity of P. vivax to develop in the vector at lower
temperatures and also by the longer duration of the course of vivax malaria (Lysenko et.
al., 1978; Marcus, 1976). P. vivax can survive for a long time in the body of the
vertebrate host in the inactive dormant state in the form of hypnozoites (Markus, 1978. In:
Bruce-Chwatt, 1980; WHO, 1991).
In the tropical zone, particularly on the African continent, the predominant malaria
parasite is P. faldpamm (Bruce-Chwatt, 1985). However, in the equatorial zone of South
America, P. faldpamm is inferior to P. vivax in terms of geographical distribution (WHO,
1990). The geographical prevalence of P. mawriae and P. ovale is characterized by a
discrete and focal pattern (WHO, 1989). Despite its comparatively restricted geographical
area of incidence, P. faldparum is responsible for 50% of the morbidity and 98 % of the
mortality from malaria throughout the world (Bruce-Chwatt, 1985).
In Ethiopia, the two epidemiologically important species are P. faldparum,
comprising 60% of all the cases of malaria, and P. vivax, constituting nearly 40% of all
malaria cases (Assefa Nega, 1993). P. malariae comprises less than 1 % of all cases and
is most frequently reported from the Arba Minch area whereas P. ovale is rarely reported
(Assefa Nega, 1993). About three quarters of the total area of Ethiopia is estimated to be
malarious and about two thirds of the inhabitants of the country are at risk of infection
(Assefa Nega, 1993).
The sexual phase of the life cycle of malaria parasites begins with entry of the male
(micro) and female (macro) gametocytes into the alimentary tract of the female Anopheles
mosquito with its blood meal. The nucleus of the male gametocyte undergoes division to