UNIVERSITY OF GONDAR
COLLEGE OF MEDICINE AND HEALTH SCIENCES
DEPARTMENT OF PHARMACOLOGY
Antimalarial activity of crude hydromethanolic extract and solvent
fractions of leaves of Stephania abyssinica (Menispermaceae) against
Plasmodium berghei infection in mice
By: - MEKUANENT ZEMENE (B.PHARM)
A thesis submitted to the department of Pharmacology, School of
pharmacy, College of medicine and health sciences, University of Gondar
in partial fulfillment of the requirements for the degree of Master of
Science in Pharmacology
June, 2017
Gondar, Ethiopia
Antimalarial activity of 80% hydromethanolic extract and solvent
fractions of leaves of Stephania abyssinica (Menispermaceae) against
Plasmodium berghei infection in mice
Investigator;
Mekuanent Zemene(B.Pharm.)
Email; mekuanentzemene44@gmail.com
Tel; +2510918060682
Advisor:
Eshetie Melese (B.pharm, MSc, Assist. professor of Pharmacology)
Email; meshetie21@gmail.com,
Tel.: +2510910218939
Co-advisor:
Mestayet Geta (B. Pharm, M. Sc in Pharmacology)
Email; mestimengistie21@gmail.com
Tel.; +251913460324
June, 2017
Gondar, Ethiopia
UNIVERSITY OF GONDAR, COLLEGE OF MEDICINE AND HELATH
SCINECE, SCHOOL OF PHARMACY, DEPARTMENT OF PHARMACOLOGY
As a member of examining board of the final MSc open defense, we certified that we have
read and evaluated thesis prepared by Mekuanent Zemene entitled in antimalarial activity
of 80% methanolic crude extract and solvent fractions of leaves of Stephania abyssinica
against P.berghei infected mice. I recommended that it will be accepted as fulfilling the
thesis requirement for the Degree of Masters of Science in Pharmacology.
………………………………………
………………
………………………………………
……………
Name of examiners
………….
……………
Signature
Date
Final approval and acceptance of the thesis is contingent upon the submission of the final
copy of the thesis to the Department of Pharmacology, University of Gondar. I here by
certified that I have read this thesis prepared under my direction and recommend that it
will be accepted as fulfilling the thesis requirement.
…………………………
…………………
Principal advisor
Signature
…………………………
…………………
Co-advisor
Signature
………….
Date
…………..
Date
II
STUDENT’S CERTIFICATE
I hereby certify that my MSc thesis submitted to University of Gondar for the award of
Master‟s Degree in Pharmacology, based on my original research work carried out under
supervision of Mr. Eshetie Melese and Mrs. Mestayet Geta. I certify the material from
other sources referred to in my thesis hasbeen acknowledged in the list of
references/bibliography. I certify that there is no violation of patent and copy right while
incorporating materials derived from other sources.
Place: - Gondar, Ethiopia
Date ------------------Mekuanent Zemene
III
ACKNOWLEDGMENT
First, I would like to thank St. Mary next to the almighty God for giving me such power
and patience to accomplish this work
Second, I would like to take this opportunity to express my most sincere gratitude to my
advisors: Mr. Eshetie Melese and Mrs. Mestayet Geta for their invaluable constructive
comments and suggestions for conducting actual work and the write up of this thesis.
Third, I would like to thank Mr.Asnakew Asres(MSc. In pharmacology, lecturer of Teda
Health Science College) for his crucial comments for write up of thesis, Mr. Abreham
Degu (pharmacy laboratory technical assistant of Teda Health Science College) for his
technical supports; for all pharmacy laboratory technical assistants of Gondar University
for their help to perform different tasks in laboratory room, for animals‘ attendant of
school of pharmacy in University of Gondar for proper handling of animals.
Finally, I would like to take this opportunity to express my heartfelt and special thanks to
Amhara Regional Health Bureau and University of Gondar for giving educational
opportunity and financial support.
IV
ABSTRACT
Background: The appearance of drug resistant malaria especially first line antimalaria
drugs and insecticide resistant mosquitoes are the major obstacles for malaria control and
prevention programs. As a means of facing the challenges of searching for new antimalarial agents, the current study focused on evaluation of anti-malarial activity of extract
of leaves of S.abyssinica.
Methods: Chloroquine-sensitive rodent malaria parasite, Plasmodium berghei (ANKA
strain) was used to infect the male Swiss Albino mice( age 6–8 weeks and weight of rages
from 24-30 g ) in 4-day suppressive and prophylactic model. The crude hydromethanolic
extract and solvent fractions of leaves of S.abyssinica at100mg, 200, and 400 mg/kg doses
was administered to a group of five mice. level of parasitaemia, packed cell volume , mean
survival time, and body weight were determined and the significance of the differences
between mean values of the five groups and within groups was analysed by one-way
ANOVA followed by post hoc Tukey‘s ,and paired samples t test respectively .
Results: the hydro methanolic extract and the hexane , chloroform and ethyl acetate
fractions at 400mg/kg dose
shown 45.60% , 42.50%, 55.80% and 51.44% (<0.001)
significant difference parasite chemo suppressive activities respectively as compared to
negative control in chemosupresive model. In chemoprophylactic models, at 400 doses of
hydro methanolic extract and the chloroform fraction, suppressed the level of parasitaemia
significantly (p < 0.001) compared to the vehicle-treated groups, 54.41%, and 57.59% %,
suppression respectively and revealed positive effect on mean servival time, PCV, weight
and temperature drop due to parasite infection in both models.
Conclusions: The results collectively indicate that the plant has a promising
antiplasmodial activity against Plasmodium berghei, which upholds the earlier with the
invitro antimalaria test results and traditional claims.
Key words: antimalarial leaves of Stephania abyssinica, Plasmodium berghei, hearbal
medicine
V
ABSTRACT .......................................................................................................................................... V
LIST OF TABLES ............................................................................................................................ VII
LIST OF FIGURES ......................................................................................................................... VIII
ABBREVIATIONS AND ACRONYMS ......................................................................................... IX
1.
INTRODUCTION ................................................................................................................... 1
1.1.
Epidemiology of Malaria ........................................................................................................................... 1
1.2.
Etiology and Life Cycle of Plasmodium .................................................................................................... 2
1.3.
Pathophysiology of Malaria....................................................................................................................... 3
1.4.
Management of Malaria ............................................................................................................................ 4
1.4.1.
Enviromental Management and Vector Control ........................................................................................ 4
1.4.2.
Insecticide Resistances and mechanisms: ................................................................................................. 5
1.4.3.
Drug therapy of Malaria .......................................................................................................................... 5
1.4.4.
Drug Resistant Malaria ............................................................................................................................ 8
1.4.5.
Malaria Vaccine ...................................................................................................................................... 9
1.5.
Traditional Medicine in Malaria Treatment ........................................................................................... 10
1.6.
Traditional Claimed Antimalarial Plants in Ethiopia ............................................................................ 10
1.7.
Plants with Promising Invivo Antimalaria Activity ................................................................................ 11
1.8.
Experimental Plant (Stephania abyssinica) ............................................................................................ 11
1.9.
Justification of the study ......................................................................................................................... 14
2.
OBJECTIVE .......................................................................................................................... 15
2.1.
General objective:.................................................................................................................................... 15
2.2.
Specific objectives:................................................................................................................................... 15
3.
MATERIALS AND METHODS ......................................................................................... 16
IV
3.1.
MATERIALS .......................................................................................................................................... 16
3.1.1.
Equipments, chemicals, reagents and drugs ............................................................................................ 16
3.1.2.
Collection and identification of plant material ........................................................................................ 16
3.1.3.
Experimental animals ............................................................................................................................ 16
3.1.4.
Parasite ................................................................................................................................................. 17
3.2.
Methods ................................................................................................................................................... 17
3.2.1.
Extraction of crude plant material .......................................................................................................... 17
3.2.2.
Solvent fractionation of S.abyssinica hydromethanolic extract................................................................ 18
3.2.3.
Priminary phytochemical Screening ....................................................................................................... 18
3.2.4.
Grouping and dosing of animals ............................................................................................................ 18
3.3.
In vivo antimalarial activity screening .................................................................................................... 19
3.3.1.
Inoculation of parasite ........................................................................................................................... 19
3.3.2.
Chemo suppressive test ......................................................................................................................... 19
3.1.5.
Evaluation of prophylactic activity (repository test) ............................................................................... 20
3.1.6.
Evaluation parameters ........................................................................................................................... 20
3.4.
Quality control ......................................................................................................................................... 21
3.5.
Ethical consideration ............................................................................................................................... 22
3.6.
Statistical analysis.................................................................................................................................... 22
4.
RESULTS................................................................................................................................ 23
4.1.
Percentage Yield of Plant Extract and Solvent Fraction ........................................................................ 23
4.2.
Preliminary Phytochemical Screening .................................................................................................... 23
4.3.
Antimalarial Activity Evaluation ............................................................................................................ 24
4.3.1.
In vivo antimalarial activity of the hydromethanolic extract of the leaf of Stephania abyssinica on four
days suppressive test ............................................................................................................................. 24
4.3.2.
Invivo antimalarial activity of the solvent fractions of leaf of Stephanica abyssinica on four days
suppressive test ..................................................................................................................................... 27
4.3.3.
Prophylactic Effect of hydromethonolic extract and chloroform fraction of leaf of S.abyssinica .............. 35
5.
DISCUSSION ......................................................................................................................... 40
6.
CONCLUSION ...................................................................................................................... 45
V
7.
RECOMMENDATION ........................................................................................................ 46
REFERENCES ................................................................................................................................... 47
ANNEX 1: PREMINARY PHYTHOCHEMICAL SCREENING PROCEDURES ............... 54
VI
LIST OF TABLES
Table 1: Phytochemical screening of the leaves of crude extracts and solvent fractions of
S.abyssinica .............................................................................................................. 23
Table 2: Effect of crude extract the leaf of S. abyssinica on parasitemia level, % suppression
and mean survival time of P. berghei infected mice in four day suppressive. ............. 25
Table 3: Effect of crude extract of the leaf of S. abyssinica on packed cell volume and body
weight of infected mice in the 4 ay suppressive test. .................................................. 26
Table 4: Effect of crude hydromethanolicextract of the leaf of S. abyssinica on body
temperature of infected animals in the 4 day suppressive test .................................... 27
Table 5 Effect of solvent fractions of the leaves of S. abyssinica on parasitemia, % suppression
and mean survival time of P. berghei infected mice in four day suppressive. ............. 29
Table 6: Effect of solvent fractions of the leaf of S. abyssinica on packed cell volume and
body weight of infected mice in the 4 day suppressive test. ..................................... 32
Table 7: Effect of solvent fractions of the leaf S. abyssiniaon body temperature of infected
animals in the 4 day suppressive test ......................................................................... 34
Table 8: Effect of crude extract and cloroform fraction of the leaves of S. abyssiniaon
parasitemia, % suppression and mean survival time of P. berghei infected mice in
prophylactic tests ...................................................................................................... 36
Table 9: effect of packed cell volume and body weight of infected mice treated with crude
extract and chloroform fraction leaves of S. abyssinica in prophylactic tests. ............. 38
Table 10: Effect body temperature of infected animals treated with crude and solvent fraction
of the leaf S. abyssinica in the prophylactic tests. ...................................................... 39
VII
List of figures
Figure 1:The life cycle of Plasmodium falciparum(9) .............................................................. 2
Figure 2: photograph of leaf Stephania abyssinica ................................................................. 12
VIII
ABBREVIATIONS and ACRONYMS
ANOVA
Analysis of Variance
CF
Cloroform fraction
CQ
Chloroquine ,
D0
Day 0
D4
Day 4
D7
Day7
EF
Ethylacetate fraction
HF
Hexane fraction
HME
Hydrometanolic extract
IP
Intra Peritoneal
IRBC
Infected Red Blood Cell
IRS
Insecticide Residual Spray
ITNs
Insecticide Treated Nets
MST
Mean survive time
PCV
Packed Cell Volume
PfEMP-I
Plasmodium falciparum Erythrocyte Membrane Protein-I
RBC
Red Blood Cell
SEM
Standard Error of Mean
SPSS
Statistical Software for Social Science
WHO
World Health Organization
IX
1.
INTRODUCTION
1.1.
Epidemiology of Malaria
Malaria remains one of the most deadly infectious diseases affecting the world particularly those
living in tropical areas of the world. All segments of the population especially the most
vulnerable groups; children under the age of five years, pregnant women, people in emergency
situations and immunocompromized patients have been severely affected by malaria (1)
Malaria is a serious global public health problem that is responsible for million cases of acute
illness and disease in each year. According to WHO report, by 2015, it was estimated that 214
million malaria cases, and 438 000 deaths due to malaria infection (1). Most cases in 2015 was
estimated to occur in the African Region (88%), followed by South-East Asia Region (10%) and
the Eastern Mediterranean Region (2%). Similarly, it was estimated that, in 2015, most deaths
(90%) was happened in the African Region, followed by the South-East Asia Region (7%) and
the Eastern Mediterranean Region (2%) (1,2). Globally, 306 000-malaria deaths in children aged
less than 5 years was estimated in 2015. Notably, malaria remains a major killer of children,
particularly in sub-Saharan Africa(1).
In Ethiopia, 2, 118, 815 cases and 213 deaths were estimated in 2013 according to WHO report
by 2015. Major plasmodium species are Plasmodium falciparum (59%), Plasmodium vivax
(41%) ,and
Major anopheles species are anopheles arabiensis, anopheles pharoensis,
anopheles. funestus, and anopheles nili(1).In Ethiopia, over 50 million people are at risk.
Malaria accounts for proximity to breeding sites(2). Malaria transmission exhibits a seasonal
and unstable pattern, with transmission varying with altitude and rainfall. Currently, areas less
than 2,000 meters of altitude are considered malarious. In general, terms, 75% of the landmass of
Ethiopia is considered at risk of malaria, which corresponds to areas below 2000m. The western,
central and eastern highlands, as well as the highland-fringe areas along the Rift Valley are
especially vulnerable to epidemics altitude(1, 3). The burden of malaria has been increasing due
to a combination of large population movements, increasing large-scale epidemics, mixed
infections of P. vivax and P. falciparum, increasing parasite resistance to malaria drugs, vector
resistance to insecticides, low coverage of malaria prevention services, and general poverty.
Outpatient consultations, inpatient admissions and all in-patient deaths have risen by 21-23%
1
over the last five years. Overall, malaria accounts about 17% of outpatient consultations, 15% of
admissions and 29% of in-patient deaths(4).
1.2.
Etiology and Life Cycle of Plasmodium
Malaria is an infectious disease caused by the parasite plasmodia. There are five identified
species of the parasite causing human malaria, namely, P. vivax, P. falciparum, .ovale, P.
malariae and P. knowlesi(primarily a pathogen of monkeys)are human malaria species that are
spread from one person to another by female mosquitoes of the genus Anopheles. Malaria, due to
Plasmodium falciparum is the most dangerous and prominent of the malaria parasites. It causes
‗malignant‘ or cerebral malaria that can quickly progress to unconsciousness and death. (1, 5)
In P falciparum and P malariae infection, only one cycle of liver cell invasion and
multiplication occurs, and liver infection ceases spontaneously in less than 4 weeks. Thus,
treatment that eliminates erythrocytic parasites will cure these infections. In P vivax and P ovale
infections, a dormant hepatic stage, the hypnozoite, is not eradicated by most drugs, and
subsequent relapses can therefore occur after therapy directed against erythrocytic parasites.
Eradication of both erythrocytic and hepatic parasites is required to cure these infections. The
malaria parasite exhibits complex life cycle involving an Anophelesmosquito and a vertebrate
host (6, 7).
Figure 1: The life cycle
of Plasmodium falciparum(9)
The infection of a human with P. falciparum commences when a female Anopheles mosquito
takes a blood meal and injects infective sporozoites into the peripheral circulation then they are
2
carried by the circulatory system to the liver where they undergo asexual amplification,
producing infective merozoites. These are released by the hepatocyte into the blood circulation,
where they recognize and invade RBCs. The merozoites develop into early trophozoites known
as ―ring stages‖. These trophozoites further develop into schizonts, which divide into merozoites.
These are released from the RBCs in order to undergo a new replication cycle (7, 8). Some of the
trophozoites differentiate into female and male gametocytes. Ingestion of the mature
gametocytes by the blood-feeding mosquito induces the production of gametes in the mosquito
midgut(9). The motile flagellated microgametes fertilize the macrogametes to form a zygote.
Then the zygote develops into an invasive ookinete, which penetrates the gut epithelium and
develops into an oocyst. Asexual replication within the oocyst results in the production of
approximately 10,000 sporozoites, migrate to the salivary glands; here they mature and wait until
the mosquito bites a new host, thus spreading malaria (6-9).
1.3.
Pathophysiology of Malaria
Malaria presents as an acute febrile illness symptoms such as, chills and rigors, followed by
fever spikes up to 40° C, then profuse sweating.
There may alternate with relatively
asymptomatic periods, and are associated with extremely high levels of tumor necrosis factor-α
(TNF-α), which may originate from macrophages stimulated by glycosyl phosphatidylinositol
moieties or other substances released on schizont rupture. P. falciparum malaria is much more
acute and severe than malaria caused by other Plasmodiumspecies(7,8) . Almost all deaths
directly attributable to malaria are caused by severe manifestations of P. falciparum infection,
including cerebral malaria, severe anemia, respiratory failure, renal failure, and severe malaria of
pregnancy. Important contributory factors include metabolic acidosis, hypoglycemia, and
superimposed bacterial infections (10). An important features of the pathogenesis of P.
falciparumis its ability to sequester in the deep venous microvasculature. It involves a number of
processes including cytoadherence, resetting, reduced red cell deformability, and the collection
of infected erythrocytes within the proteoglycan matrix of placental spaces. P. falciparumsinfected erythrocytes sequester throughout the human body .Attachment points to endothelium
with knobs, on the surface of infected erythrocytes where variant cytoadherence proteins
(PfEMP-1 ;) are anchored. (7, 8, 10). Red blood cells with mature trophozoites stick to the
vascular walls of small blood veins. This causes a blockage of blood flow. Cerebral malaria
3
occurs when the blockage is in a vein of the brain. This complication has about a 20% mortality
rate(11,12) .
1.4.
1.4.1.
Management of Malaria
Enviromental Management and Vector Control
To ensure the prevention and control of malaria, it is important that all temporary or permanent
breeding sites with water are identified and eliminated. This malaria control strategy is effective
only when mosquitoes are interrupted from breeding and/or their population is substantially
decreased. This can be achieved in areas where only a limited number of fully identified
breeding sites exist(11).
Mosquito breeding sites malaria can be prevented and controlled by clearing bodies of water by
filling and leveling burrows and pits, and removing undesirable materials that contain water; in
dry seasons, intermittent rivers and streams that form stream beds, pools and side water pockets
can be filled, drained or connected to the main course of water(1, 11).
Larvicides: they can be used to apply on collected water. The most common water-soluble
chemical used to kill mosquito larvae in Ethiopia is temephos, which is safe for humans when
used in the recommended dosage, therefore, can be applied to drinking water. However,
considering its high cost, and the need for repeated applications, spray equipment and human
resources, temephos should be applied only for small breeding sites, and only if other control
measures are inapplicable (e.g. in towns, lowlands and agriculture-development areas with
irrigation systems (3, 11)
Indoor residual spraying (IRS): Indoor residual spraying (IRS) is the application of longacting chemical insecticides on the walls and roofs of all houses and domestic animal shelters in
a given area, in order to kill adult vector mosquitoes that land and rest on these surfaces. IRS is
one of the primary vector control interventions for reducing and interrupting malaria
transmission, and one of the most effective methods for obtaining rapid large-scale impact on
both vector populations and malaria morbidity/mortality. The primary effects of IRS towards
curtailing malaria transmission are: reducing the life span of vector mosquitoes so that they can
no longer transmit malaria parasites from one person to another; and reducing the density of
vector mosquitoes(3, 11).
4
Insecticide treated nets; Insecticide treated nets (ITNs) have been shown to reduce the contact
between the host and vector leading to significant reduction of malaria transmission. Currently,
only pyrethroid insecticides have been approved for use on ITNs. These insecticides have very
low mammalian toxicity but are highly toxic to insects and have a rapid knock-down effect, even
at low doses. Pyrethroids have a high residual effect, they do not rapidly breakdown unless
washed or exposed to sunlight (13).
1.4.2.
Insecticide Resistances and mechanisms:
There are different class of insecticides :organochlorines, organophospahtes , carbamates
,pyrethroids and etc.(1).Sodium channels modification and acetylcholinesterasemutuation are
mechanism of resistance for insectcides(12) as indicated below paragraphs.
Sodium Channels modification :Pyrethroids and DDT deliver their toxic, insecticidal effects
primarily by binding to the sodium channel, altering its gating properties, and keeping it open for
an unusually long time. Modifications in the sodium channel structure, in the form of either point
mutations or substitutions resulting from single nucleotide polymorphisms, lead to insensitivity
to DDT and pyrethroids in the sodium channels of the nervous system via a reduction in or an
elimination of the binding affinity of the insecticides to proteins (6,13).
Acetylcholinesterase mutuation :AChE1 and AChE2, encoded by the ace-1 and ace-2 genes,
respectively, have been identified in different species of mosquitoes, but only AChE1 has been
implicated in mosquito resistance to organophosphate and carbamate insecticides (12).
1.4.3.
Drug therapy of Malaria
Important attributes for the successful implementation of antimalarial drugs are good tolerability
and safety (especially in young children), affordability, availability in endemic countries and
short course regimens. Primarily to decrease the emergence of drug resistant parasites, almost all
antimalarials are now to be administered as part of a combination therapy, with each drug
targeting distinct mechanisms within the parasite (15).
Quinine, an aryl-amino alcohol, is one of the oldest antimalarial agents and has been used by the
native population of Peru for centuries in the form of pulverized bark of the cinchona tree to treat
fevers and chills; the active alkaloid from the bark was isolated and named quinine. Quinine is
an erythrocytic schizonticide; now used to treat severe cases of malaria and, as a second line
5
treatment, in combination with antibiotics to treat resistant malaria. Quinine has been
demonstrated to accumulate in the parasite‘s digestive vacuole (DV) and can inhibit the
detoxification of heme, an essential process within the parasite. The major adverse effect of
quinine is cinchonism a syndrome causing nausea, vomiting,tinnitus, and vertigo (13,15)
Piperaquine; it is bis-4-aminoquinoline it possesses an extended half-life of approximately
5weeks. Due to structure similarities with chloroquine, it has been postulated that piperaquine
has asimilar mode of action to chloroquine and used for chemophylaxis(13).
Primaquine is an 8-aminoquinoline that eradicates primary exoerythrocytic forms of P.
falciparum and P. vivax and the secondary exoerythrocytic forms of recurring malarias (P. vivax
and P. ovale). Primaquine is also gametocidal against human malaria species. It is used clinically
for standard therapy and terminal prophylaxis. Primaquine has a low incidence of adverse
effects, except for drug-induced hemolytic anemia in patients with genetically low levels of
glucose-6-phosphate dehydrogenase.(14).
Chloroquine is a 4-aminoquinoline that was introduced in the late 1940s and used on a massive
scale for malaria treatment and prevention. Its efficacy, affordability and safety, even during
pregnancy, made it the gold standard treatment of malaria for many years. Chloroquine has one
of the longest half-lives among antimalarials, which provides a chemoprophylactic effect during
the drug elimination phase. Chloroquine‘s mechanism of action has been an intense area of
research for decades and evidence supports that the principal target is the heme detoxification
pathway in the digestion vacule(DV), where the parasite degrades erythrocytic hemoglobin and
polymerizes the liberated toxic heme monomers to inert biocrystals of hemozoin(13,14,15).
Mefloquine: it is a synthetic 4-quinoline methanol. Its exact mechanism of action remains to be
determined, but like quinine, it can apparently damage the parasite's membrane. It has a long
half-life (17 days) because of its concentration in various tissues and its continuous circulation
through the enterohepatic and enterogastric systems (11). The combination of artesunate plus
mefloquine showed excellent antimalarial efficacy in regions of Southeast Asia with some
resistance to mefloquine, and this regimen is now one of the combination therapies
6
recommended by the WHO for the treatment of uncomplicated falciparum malaria and
mefloquine is effective in prophylaxis against most strains of P falciparum and probably all other
human malarial species (11, 14, 16).
Atovaquone is a lipophilic hydroxynaphthoquinone analog structurally related to ubiquinol (an
important coenzyme in the electron transport chain within the mitochondria). Molecular
evidence exists that atovaquone specificallytargets the cytochrome bc1 complex, located in the
innermitochondrial membrane, thereby inhibiting the respiratory chain (13). In P.falciparum, the
respiratory chain is required for the regeneration of ubiquinone, the electron acceptor for
dihydroorotate dehydrogenase, which is an essential enzyme for pyrimidine biosynthesis.
Atovaquone is
currently used in combination with proguanil (Malarone), mainly as a
prophylactic(13,16).
Artemisinin: it is derived from the qinghaosu plant, which has been used in Chinese medicine
for more than two millennia in the treatment of fevers and malaria. Artemisinin (or one of its
derivatives) is available for the treatment of severe, multidrug-resistant P. falciparum malaria. Its
antimalarial action involves the production of free radicals within the plasmodium food vacuole,
following cleavage of the drug's endoperoxide bridge by heme iron in parasitized erythrocytes. It
is also believed to covalently bind to and damage specific malarial proteins(14,17). Artemisinin
is insoluble and can only be used orally. Derivatives of artemisinin have been synthesized to
increase solubility and improve antimalarial efficacy. The most important of these analogs are
artesunate (water-soluble; useful for oral, intravenous, intramuscular, and rectal a dministration),
artemether (lipid-soluble; useful for oral, intramuscular, and rectal administration), and
dihydroartemisinin (water-soluble; useful for oral administration)(16).
Lumefantrine, an aryl alcohol related to halofantrine, is available only as a fixed-dose
combination with artemether (Coartem), which is now the first-line therapy for uncomplicated
falciparum malaria in many countries. Coartem should be administered with fatty food to
maximize antimalarial efficacy (5, 13).
The antifolate drugs used for malaria therapy are the sulfa drugs sulfadoxine and dapsone that
inhibit the dihydropteroate synthetase enzyme (PfDHPS), and pyrimethamine and proguanil,
7
which inhibit the dihydrofolatereductase (PfDHFR) activity .The drug combination sulfadoxine–
pyrimethamine (Fansidar); was a highly effective, cheap, well-tolerated drug combination with
good compliance rates due to being administered in a single dose. It is used for radical cure and
causal prophylaxis in malaria control(13, 16).
Tetracyclines and doxycycline; they are active against erythrocytic schizonts of all human
malaria parasites. They are not active against liver stages. Doxycycline is used in the treatment
of falciparum malaria in conjunction with quinine, allowing a shorter and better-tolerated course
of that drug. Doxycycline has also become a standard chemoprophylactic drug, espescially for
use in areas of Southeast Asia with high rates of resistance to other antimalarials, including
mefloquine(5, 16).
1.4.4.
Drug Resistant Malaria
Drug resistance is the degree to which a disease or disease causing organism remains unaffected
by a drug which was previously able to eliminate.The efficacy of many antimalarial drugs is
limited by drug resistance, and recent evidence suggests that parasites are becoming resistant to
the newest agents. These make the obstacle for achievement of effective for the treatment and
control of malaria (9, 17-19).
Chloroquine and Quinine: generally, resistance depends on the chemical class of the antimalarial
and its mode of action. Resistance to 4-aminoquinolines, Quinine and highly hydrophobic aryl
aminoalcohols, arises from mutations of genes encoding vacuolar trans-membrane proteins
which regulate the influx/efflux of the drug at the target and chloroquine resistance in P.
falciparumis primarily attributable to single nucleotide polymorphisms in pfcrt(CQ resistance
transporter). Mutations in P. falciparummultidrug resistance 1 (PfMDR1), the gene encoding the
P. falciparum Pglycoprotein homologue-1, seem to be the main cause of resistance to mefloquine
but are also implicated in CQ resistance(9, 17, 18).
Atovaquone;
it specifically inhibits cytochrome bc1 (cytbc1) complex, an important in the
electron transport system. The mitochondrial electron transport system in P. falciparum is tasked
with the purpose of regenerating ubiquinone, which in turn serves as an electron acceptor for
parasitic dihydroorotate dehydrogenase, an enzyme responsible for pyrimidine biosynthesis in
the parasite. The mechanism of resistance involves a point mutation of cytb gene.(9, 17, 18).
8
Anti-folates: resistance to antifolates is common worldwide and apparently depends on a
stepwise accumulation of single point mutations of genes pfdhps and pfdhpscoding the drug
targets, dihydropteroate synthase and dihydrofolatereductase, respectively (9, 17, 18).
Mefloquine: Resistance to mefloquine has been primarily attributed to amplification in Pfmdr-1
gene that encodes for an energy dependent p-glycoprotein pump This p-glycoprotein located on
the digestion vacuoles (DV) is responsible for extruding out anti-malarials(9, 17, 18).
Artemisinin and its derivatives: PfATP6 is the only SERCA-type (Sarcoplasma endoplasmic
reticulum calcium channel) Ca21-ATPase present in the malaria parasite. Inhibition of this
enzyme subsequently inhibits the action of artemisinin. There is no established mechanism
behind the development of artemisinin resistance. However, ongoing research has identified
mutations in the genes encoding for PfATP6 and amplification in the Pfmdr-1(9,17,18).
Thus, there is an urgent need for increased efforts in anti-malarial drug discovery especially in
Africa. In recent times, natural products of plant sources have been the center of focus as the
main source of new, safer and more effective bioactive compounds with medicinal properties.
1.4.5.
Malaria Vaccine
Candidate vaccine antigens from the pre-erythrocytic stages have been one of the targets of
antibodies that prevent sporozoite invasion of hepatocytes or the targets of cellular immune
responses that kill infected hepatocytes. A completely effective pre-erythrocytic vaccine would
inactivate the parasite before it left the liver, leading to sterile immunity and prevention of
disease. This goal is to decrease the incidence of new infections, and decrease the number of
merozoites exiting the liver, by decreasing the number of sporozoites entering the liver or killing
parasites within hepatocytes, leading to clinical benefits analogous to the direct effects of
insecticide treated bed nets. Clinical development of RTS,S/AS01E has been reviewed
extensively . This is by far the most advanced candidate malaria vaccine, is the only one in Phase
3 evaluation. RTS,S/AS01E has demonstrated 26-51% efficacy(95% confident interval ) in
reducing the rate of all episodes of malaria (20, 21). However, scientists have employed their
unlimited efforts to dig out antimalaria vaccine, still there is no licensed finished product found,
yet.
9
1.5.
Traditional Medicine in Malaria Treatment
In the early development of modern medicine, biologically active compounds from plants have
played a vital role in providing medicines to combat diseases. Plant-derived medicines continue
to occupy an important niche in the treatment of diseases worldwide(22). Medicinal plants are
important sources of traditional medicines for millions of people and additional inputs to modern
medicine in terms of exploring and producing new drugs. Medicinal plants are useful for curing
of human diseases because of the presence of photochemical constituents. cinchona tree was
used to treat malaria as early as the 1600‘s century in South America until it was developed to
quinine and artemisinin derivatives were isolated from leaf of Artemisia annuasince this plant
has been used as a traditional medicine in China for treatment of malaria(23, 24).
Ethiopian plants have shown remarkably effective medicinal values for many human and
livestock ailments according to research findings which have been conducted on the south,
southwest, central, north and northwestern parts of Ethiopia(25,26, 27).
Having potential rich, medicinal plants, in Ethiopia, are crucial for
pharmacological
investigation of medicinal properties of the plants (24, 28). Acceptance of traditional medicine
and limited access to modern healthcare facilities could be considered as the main factors for the
continuation of the practice (23).
1.6.
Traditional Claimed Antimalarial Plants in Ethiopia
Peoples in Ethiopia have knowledge and they have been practicing antimalarial plants for
treatment and prevention. Plants such as, leaf of Artemsia afra (Asteraceae) ,seeds of Pavetta
abyssinica ( Rubiaceaefugi) (29) ,Allium cepa (30), carica papaya (26), shoot apexof Croton
macrostachyus(Euphorbiaceae),
root of Phytolacca dodecandra (Phytolaccaceae) ,latex of
Euphorbiaceae abussinica , apex of jestica schimperiana (Weesacanthaceae) , roots and leaves
of Vernonia amygdalina (Asteraceae ) (31). lopididiumsativum ( Brassicaceae) (32), Allium
sativium (Alliacea) (33),leaves of Clematis simensis (Rananculaceae) , both stem and root of
Phoenix reclinata (34) have been used for management of malaria traditionally in different
places Ethiopia.
10
1.7.
Plants with Promising Invivo Antimalaria Activity
Leaves of Otostegia integrifolia exhibited
on hydroalcoholic
extractand solvent fractions
significant antimalarial activity(35). Similarly, a study on the leaves of Vernonia amygdalina
against P.berghei exhibited suppressive effect both of hydro alcoholic extract and chloroform
fraction. In the same manner ,the antimalarial activity of the hydromethanolic leaf extract of
Campurin aaurea was reported in a 4-day suppressive test in P. berghei-infected mouse model
(36). The crude aqueous extract, crude hydro methanolic extract,
n-hexane fraction of crude
hydro methanolic extract, chloroform fraction of crude hydromethanolic extract and aqueous
fraction of crude hydro methanolic extract of the leaf of Strychnos mitis revealed positive
effect(37). A 80% methanol extract of the stem bark of Syzygium guineense also showed
antimalaril activity significantly(38).
1.8.
Experimental Plant (Stephania abyssinica)
Stephania abyssinica,a vernacular name, ‟ye ayit hareg‟ or ,‟Este eyesus‟ (Amharic ), is a
climbing shrub within the family Menispermaceae and genus Stephania .It is found widely
distributed in tropical Africa, including Ethiopia. Its natural habitat includes grassland,
abandoned fields and roadsides, at elevations up to 3500 m. The members of genus Stephania are
slender, climbers with peltate and membranous leaves. The arrangements of flowers are in
umbelliform cymes, which arise from axils or from old leafless stems. S.abyssinica is a liana
wood at the base, 2-3 m hight. The leaves are peltate, broadly ovate, round base and obtuse or
acute base. Flowers are cream or reddish. Fruits are yellow or pinkish green round and 5-8 mm
in diameter. Its flowering seasons ranges from spring till early autumn (36, 39,40).
11
Figure 2: photograph of leaf Stephania abyssinica(by Mekuanent Zemene,May 23, 2017)
Traditionally, leaves of S.abyssinica has been used for treatment of different ailments such as
the menstrual disorders and child birth problems in Abeokuta south local government area of
Ogun state in Nigeria (41). Root has been used to treat malaria and internal parasites throughout
eastern Africa as well as in Uganda the leaves is believed to distract hunting dogs if they eat the
leaves, and disorientate hunters if they touch the plant (40).
The investigation focused on Stephania abyssinica which was identified during a survey of
traditional anti-malarial plants from southern Nyanza, Kenya (42) and where its aqueous root
extract has been used for malaria therapy throughout eastern Africa (43)
In Ethiopia, it is used in Ethiopia as a remedy, including anthrax, stomach problems,
miscarriage, rabies, syphilis and external tumor/swelling. The dried leaves of S.abyssinica has
been used for treatment of rabies by Sheko ethnic groups (45). Drinking juice of leaves and
stems
are used for headache and stomachache in Zegie people , in northern Ethiopia (46);
infused leaves is used for anthrax( kurba) and abdominal crump in Gondar town (25) , fresh
12
leaf for stomach ache and rabies in Mecha woreda in west Gojam (32); Southern Ethiopia leave
are used for dermal burn (29) .
For treatment of tumor, the root of the plant is crushed, squeezed and then spread on affected
part of the body. In confirmation of the antitumor activity reported, stephanine and stephavanine
isolated from S. abyssinica have been shown to exhibit antineoplastic activity (44). Root
decoction of the plant has been also used to treat jaundice, rabies and ascariasis in Bahirdar
zuria woreda, anthrax (47) and as an aphrodisiac (46). Fresh or dried root powder has been used
for leg ache, arthritis, rheumatism and the whole part of the plant used to treat common cold in
Wayu Tuka District, East Welega Zone (27). Roots are used for pneumonia, around Fiche
District, Central Ethiopia legache. arthirits, rheumatim fresh or poweder root(32).
Medicinal activities of S.abyssinica also were tested from body parts.
On invitro test,
antioxidant Activity(roots and rhizomes) was showed (48). It was also evaluated and showed as
the antidiarrheal and antispasmodic activities of the aqueous and methanol extract of the root and
leaf of S. abyssinica(43). The 80% methanol leaf extract of S. abyssinica showed analgesic and
anti-inlammatory activity on mice(49).
The solvent fraction S. abyssinica Walp leaves showed antimalarial activity in vitro test against
chloroquine sensitive and resistant laboratory adapted strains of P. falciparum (36), and aqueous
extract hepatoprotective effects (48).
It was also reported that it showed strong invitro anti-
plasmodial activity leaf of S.abyssinica (50, 51),however, still there is no invivo antimalaria
activities were not evaluated in both crude and solvent fractions on leaf of S.abyssinica .
13
1.9.
Justification of the study
Malaria continues to be a problem worldwide. Plasmodium will continue to develop resistance
against antimalarial pharmaceuticals. The parasite develops resistance much faster than the rate
of new pharmaceuticals is being developed. Currently there is a paucity of promising novel
antimalarial drugs under development and a loss of the artemisinins to resistance would be a
disaster for international malaria control(1). Due to cost , side effects and contra indication of
conventional
antimalarial drugs(14), absence of
challenges for malaria
to combat it.
vaccine(20,21) for
malaria is also still
As a result, searching for new safe, effective, and
accessible plant originated antimalarial drugs is still in homework for scientists.
Medicinal plants remain the focus by scientists and researcher as a noble source of lead
compound in the search and development of new antimalarial agents. Screening of plants for
antimalarial activity is of paramount importance to the effort made to develop new drugs. Plants
which were clamed as tradicional medicine, they have been the source of antimalarial drugs
such as quinine and artemisinin (23,24).
The Leaf of S.abyssinica has been traditionally claimed for malaria treatment and strong
antimalarial activity was also showed by invitro antimalarial screening(42,50) Therefore, this
study
was aimed at the evaluation of the claimed traditional use and reported in vitro
antimalarial activity of the plant material in animal models.
14
2. OBJECTIVE
2.1.
General objective:
To evaluate the antimalarial activity of 80% hydromethanolic crude extracts and solvent
fractions of leaves of S.abyssinica against P.berghei infected mice
2.2.
Specific objectives:
To perform preliminary phytochemical screening
of hydromethanolic crude extracts and
solvent fractions of the leaves of S.abyssinia.
To evaluate chemosupressive activities hydromethanolic crude extracts of leaves of
S.abyssinica against P.berghei infected mice.
To evaluate chemosupressive activities of n-hexane, chloroform and ethyl acetate fractions
of leaves of S.abyssinica against P.berghei infected mice
To evaluate the effect of the hydromethanolic crude extracts and solvent fractions on mean
survival time, packed cell volume ,body temperature and body weight of P. berghei infected
mice
To evaluate chemoprophylactic activity of hydromethanolic crude extracts and the most
active chemo suppressive solvent fraction of the leaves of S.abyssinica.
15
3. MATERIALS AND METHODS
3.1.
MATERIALS
3.1.1. Equipments, chemicals, reagents and drugs
Trisodium
citrate(Alpha
chem.,India),
Tween
80
(Avishkar
Lab
Tech
Chemicals,
India).,Absolute methanol(RANKEM , India ) , Absolute ethanol((Lobachemie ,India )
,Chloroquine 250 mg phosphate tablet( Addis pharmaceutical factory, Ethiopia ), Distilled
water( Gondar hospital lab) ,Normal saline(Ethiopia pharmaceutical factory, Ethiopia),
chloroform(ATCO,
india)
,
Giemsa
stain
10%
solution
(ScienceLab,
USA),
Microscope(OLYPUS cx21,Japan), N-hexane(ATCO, India), Ethyl acetate solution(ATCO,
india) , Mayer reagent (Avishkar Lab Tech Chemicals, India), Wagner‘s Reagent(BDH Ltd, UK
), sulfuric acid (Supertek, India)
, Glacial acetic acid (Lobe chemi, India), microscope
immersion oil (Neolab life science co, India),
ferric chloride (Fisher Scientific Co, USA), lead
acetate (BDH Ltd, UK), Ammonium solution (Techno. Pharm. Chem., India), Hydrochloric acid
(Supertek Chemicals, India) ,Disposable glove, Hemotocapillary tube(Drummond scientific,
USA) , Centrifuge, Desiccators, vials, rectal thermometer(TM01, Contronic manufacturing),
Sensitive balance, separatory funnel, beaker , Microscopes slides(Westmed proxiGmGH CoKG,
Germany), Erlenmeyer flasks, ball miller (FW100, China), in
hot air oven (YCO.IN010,
INDIA) ,measuring cylinder, cotton gauze ,Watt man paper((EXACL,India0),Oral gavages ,
insulin with needles(Hindustan syringes and medical device PLC, India) and scissors. All
chemicals and reagents were analytically graded and were procured from certified supplier.
3.1.2. Collection and identification of plant material
The fresh leaves of S. abyssinica were collected in January, 2017 in the adjacent rural kebeles
(Teda) of Gondar town, Ethiopia, which is 728 km far from Addis Ababa. The plant was selected
based on the indigenous knowledge of the local traditional healers. It was identified by a botanist
(Abiyu Enyew) and a voucher specimen of this plant is deposited in University of Gondar with
voucher no MZoo1.
3.1.3. Experimental animals
The healthy young male albino mice (of age 6–8 weeks and weight of rages from 24-30 g were
obtained from the University of Gondar. They were also be maintained under standard air
16
conditioned with at room temperature and 50-70% relative humidity at half day light and dark
cycle. They were fed standard diet, and water ad libitum. They were housed in a standard
plasticcages . Animals were acclimatized for one week to the experimental environment. All the
experiments were performed in accordance with the internationally laboratory animal use and
care guideline(52).
3.1.4. Parasite
The rodent malaria parasite, Chloroquine- sensitive Plasmodium berghei NK 65, was obtained
from Ethiopian Public Health Institute Addis Ababa, Ethiopia. The parasite was maintained alive
by intraperitoneal passage in mice on weekly bases from infected donor blood to health mice by
preparing 0.2ml infected blood via diluting with 0.9% normal saline solution treated by 0.5%
trisodium citrate as an anticoagulant, until it would be reached to 108 infected RBCs per ml.
Then it was transferred by injecting 0.2ml of diluted infected blood (52).
3.2.
Methods
3.2.1. Extraction of crude plant material
First, fresh leaves were washed with distilled water to remove dust and debris, dried with air at
normal temperature in the laboratory room for 2 weeks. Then, the dried specimens were crushed
into coarse powder using ball miller. After weighing by sensitive balance, the 1kg of specimen
powder was macerated with 80% methanol for three days with occasional agitation and stirring.
Next to that, it was filtrated with gauze and then with wattman paper No.1 with pore size 150
mm diameters into Erlenmeyer flasks. Again the mark was remacerated twice in the same
procedure . Then filtrates were combined and dried in hot air oven at a temperature of not
exceeding 400C. After the methanol was evaporated, the crude extract was farther concentrated
desiccators .Then the dried extract was transferred to vials and it was kept in desiccators a total
of 168 gm (16.8 %(W/W))s was obtained. The portion of the crude extracts were used to
evaluate the antimalarial activity in vivo model and phytochemical screening and the remaining
extract was subjected to n-hexan, chloroform, ethyl acetate and aqueous fractionation(53).
17
3.2.2. Solvent fractionation of S.abyssinica hydromethanolic extract
Hydromethanolic crude extract of leaf S.abyssinic was subjected solvent fractionation with nhexane, chloroform and ethyl acetate solvents. A total of 140 gm of 80% methanolic crude extract
of S.abyssinica was dissolved using separatory funnel in 350 ml distilled water, then fractionated
by adding n-hexane, chloroform and ethyl acetate solvents, in increasing order of their polarity.
The dissolved hydromethanolic extract was partitioned with 3 ×350 ml n-hexan. Then, the filtrate
was concentrated in hot air oven below 400C.The aqueous residue was further partitioned with
3x350ml chloroform and filtrates were dried on hot air oven less than 400C. The remaining
aqueous again residue also further partitioned with 3x 350ml ethyl acetate which was concentrated
with hot air oven similar to the hexane and chloroform fraction. The remainig aqueous fraction
was dried first in hot air oven, then with dedicator. All fractions were kept in tightly closed
containers in refrigerator at -200C until used (42, 53).
3.2.3.
Priminary phytochemical Screening
The 80% methanol and the solvent fractions(n-hexane, chloroform, ethyl acetate and aqueous s)
of S. abyssinia leaves were screened for the presence of secondary metabolites Hence, screening
tests for alkaloids, saponins, cardiac glycosides, flavonoids, terpenoids, steroids, phenols and
tannins were performed using standard procedures (54) as described in annex 1.
3.2.4. Grouping and dosing of animals
There were 120 male mice were grouped randomly (after inoculation for chemosuppressive test
and before inoculation for prophylactic test) into five groups of five mice each. In both the
chemosuppressive and chemoprophylactic models, group I mice were treated with10 ml/kg 4%
tweene-80 ( used as negative control), the group II III and IV mice mice were treated with 100,
200, and 400 mg/kg of crude extract, respectively. The rest group V was treated with the
standard drug (chloroquine, 25 mg/kg, served as positive control),
Previous toxicity studies made on hydromethanolic crude extract leaves of S.abyssinica
demonstrated that at 2000 mg/kg dose it was found safe and this served as base for the
determination of the current antimalarial activity test doses (100, 200 and400 mg/kg) of the
extract . For all groups the route of administration was oral using oral gavage
18
3.3.
In vivo antimalarial activity screening
3.3.1. Inoculation of parasite
Albino mice previously infected with Plasmodium berghei and having parasitemia level of 2030% were used as donor. The parasitaemia of the donor mice was first determined and
parasitized erythrocytes were obtained by scarification using ethyl ether as anesthesia and diluted
in physiological saline (0.9%). The dilution was made based on the parasitaemia of the donor
mice and the RBC count of normal mice in such a way that 1 mL blood contains 5 × 107 infected
erythrocyte. The amount of 0.9% normal saline solution per ml added calculated as:
VA-VB=VN
Where: - VA: Volume of blood after addition of 0.9% normal saline
VB: Volume of blood before addition of 0.9% normal saline
VN: Volume of 0.9% normal saline added
Each mouse was inoculated by intraperitoneal injection with a blood suspension (0.2 mL)
containing 1 × 107 parasitized erythrocytes (52).
3.3.2. Chemo suppressive test
This is the most widely used preliminary test, in which the efficacy of a compound is assessed
by comparison of blood parasitemia and mouse survival time in treated and control. Treatment of
infected mice was started after 3 h of infection on day 0 and continued daily for 4 days (i.e., from
days 0–3). On the fifth day (day 4) blood samples were collected from tail snip of each mouse.
Thin smears were prepared and stained with 10%Giemsa solution. Then, each stained slide was
examined under the microscope with an oil immersion objective of 100× magnification power to
evaluate the percent suppression of each extract with respect to the control groups (52). Average
percent parasitaemia and suppression was calculated by using the following formula:
Number of parasitized red blood cells (RBC) × 100
% Parasitaemia=
Total number of RBC count
% Suppression= (% Parasitemia of negative control − % Parasitaemia of treated group)x100
% Parasitaemia of negative control
19
3.1.5.
Evaluation of prophylactic activity (repository test)
The hydromethanolic crude extract and one of among solvent fraction (the most active in chemo
suppressive effect) were further tested using the residual infection procedure for prophylactic
activity. The vehicle, standard drugs and tested doses were administeredorally daily for four
days (D0-D3).
On the five day (D4), all the mice were inoculated with 0.2 ml standard
inoculums containing 1x 107P. berghei infected erythrocytes as indicated in title3.3.2. After 72 h
of infection (D7), thin blood smears were prepared from tail of each mouse and examined
microscopically for parasitaemia level, (52).
3.1.6.
Evaluation parameters
A. Determination of parasitemia
Thin blood smears were taken from tail snip of each mouse on the day 4 for 4-day suppressi ve
test and on day7 for prophylactic test on microscopic slides. The slides were dried and fixed with
absolute methanol. The slides were stained with 10% Giemsa at pH 7.2 for 10 minutes and then
washed gently using distilled water and air dried at room temperature and examined under
microscope with an oil immersion nosepiece of 100x magnification power. Percentage
parasitemia were calculated as described in title 3.3.2
B. Determination of mean survival time
The death of each mouse was observed and the number of days from the time of inoculation of
the parasite up to death of each mouse in the treatment and control groups throughout the follow
up period (30 days
for chemosupressive model and 21 days for prophylactic model) was
recorded. The mean survival time (MST) for each group was then calculated using the following
formula:
MST=Sum of survival time of all mice in a group (days)
Total number of mice in that group
C. Determination of packed cell volume (PCV)
Packed cell volume (PCV) was measured to predict the effectiveness of the test fractions in
preventing hemolysis resulting from increasing parasitemia associated with malaria. PCV is a
20
measure of the proportion of RBCs to plasma and measured before inoculating the parasite and
after treatment of all groups of mice. It was measured before inoculating the parasite (day 0 ) and
after treatment after 24 hour gap of the last dose in 4-day suppressive test but in case of
prophylactic tests, the PCV were measured before infection ,D4 and D7.
Blood were collected from tail of each mouse in heparinized microhaematocrit capillary tubes.
The capillary tubes were filled with blood up to 75% of their height; sealed and placed in a
microhematocrit centrifuge and centrifuged. The blood was centrifuged at 12,000 revolutions
per minute for 5 min. The tubes were then taken out of the centrifuge and PCV was determined
using apparatus of the standard Microhematocrit Reader.
PCV=Volume of erythrocytes in a given volume of blood
Total blood volume
D. Determination of body weight
The body weight of each mouse in all groups was measured before infection on day0 and on day
4 in the four-day suppressive test. For prophylactic test, body weight was taken on day 4before
inoculating parasite and on day 7; using a sensitive digital weighing balance. The mean
bodyweight per group were calculated using the formula as indicated below:
Mean body weight =Total weight of mice in a group
Total number of mice in that group
E. Measurement of body temperature
To predict the effectiveness of the fractions on temperature, rectal temperature of each mouse in
all groups were measured by a digital thermometer one hour before infection, and then on day4
day7 after infection for 4-day suppressive and prophylactic tests respectively.
3.4.
Quality control
The reliability of the results of the research was maintained by using standard analytical grade
reagents, more accurate measuring devices, accurate extracting procedure and performing tests
21
in triplicate (negative control, positive control groups and experimental group) with
randomization and blind techniques.
3.5.
Ethical consideration
The experiment protocols were requested to and approved by School of Pharmacy, College of
Medicine and Health Sciences, University of Gondar. The experiment was performed according
to the animal care and welfare guidelines (54).
3.6.
Statistical analysis
Data were analyzed using Windows SPSS 20 version software. One-way analysis of variance
(ANOVA) followed by Tukey‘s post-hoc test was used to determine statistical significance for
comparison of body weight, parasitemia percentage suppression, packed cell volume , rectal
temperature and survival time among groups. The Student‘s independent t-test was applied to
compare the PCV, rectal temperature differences, and body weight variations within a group
taking the same dose extracts before and after the treatment at D0 and D4 for chomosupressive
test and D4 and D7 for chemophylactic test. Results of the study was expressed as a mean plus
or minus standard error of mean (M ± SEM). The analysis was performed with 95% confidence
interval and P-values less than 0.05 is considered statistically significant.
22
4.
RESULTS
4.1.
Percentage Yield of Plant Extract and Solvent Fraction
The percentage yield of the crude hydro methanolic extract was found 16.80%. While the percentage
yield of successive solvent fractions of methanolic crude extract using hexane, chloroform, ethyl
acetate and aqueous were 11.43%, 9.14%, 7.43%, and 72.00%, respectively.
4.2.
Preliminary Phytochemical Screening
The preliminary phytochemical screening test of 80% methanolic crude extract and its solvent
fractions of the leaf of S.abyssinica revealed
the presence or absence of secondary
metabolites as it stated in table1.
Table 1: Phytochemical screening of the leaves of crude extracts and solvent fractions of
S.abyssinica
Phytochemical
Crude
Hexane
Chloroform
Ethyl
Aqueous
constituent
extract
fraction
fraction
acetate
fraction
fraction
Phenols
+
+
+
+
+
Flavonoids
+
+
+
+
+
Glycosides
-
-
-
-
-
Alkaloids
+
+
+
+
-
Saponins:
+
+
+
+
+
Steroids
-
-
-
-
-
Tannins:
+
+
+
+
+
Terpinoid
-
-
-
-
-
Anthraquinones
-
-
-
-
-
Key: (+) =present of phytochemical, (-) = absent of phytochemical during test
23
4.3.
Antimalarial Activity Evaluation
4.3.1. In vivo antimalarial activity of the hydromethanolic extract of the leaf of
Stephania abyssinica on four days suppressive test
A. Effects of crude extracts of the leaf of Stephania abyssinica on percentage of
suppression of parasitemia and mean survival time
After four day suppressive test, the present study shown that the percentage suppression of 80%
methanolic extract was 29.15%, 40% and 45.60% at 100mg/kg, 200mg/kg and 400mg/kg of the
extract, respectively in a dose dependent manner. All the doses of the 80% methanolic extracts
revealed statistically significant difference (P < 0.05) antimalarial activity when compared to the
negative control as shown in the table 2. The extract treated groups showed statistically
significant difference at the dose of 400mg/kg (p<0.001) and 200mg/kg (p<0.001) when
compared with 100mg/kg. In the same manner, the 80% methanolic extract treated groups at the
dose of 400mg/kg (p<0.01) showed statistically significant difference when compared to
200mg/kg dose treated group. The chloroquine treated group was completely free from the
parasites on day four as shown in table 2.
As regards to survival days, the extract treated group at the dose of 400mg/kg (p<0.001) showed
a statistically significance difference when compared to negative control and extract treated
group at the dose of 100mg/kg (p<0.01). However extract treated groups, at the dose of
200mg/kg and100mg/kg did not show statistically significant difference as compared to the
negative control. Chloroquine treated mice showed statistically significant (p<0.001) survival
time with respect to all the extract treated groups and the control group as shown in table 2.
24
Table 2: Effect of crude extract the leaf of S. abyssinica on parasitemia level, % suppression and
mean survival time of P. berghei infected mice in four day suppressive.
Treatments(mg/
kg, ml/kg)
Parasitaemia level
%suppression
MST(day)
Vehicle 10
39.40+0.68
0.00
6.80+.83
HME100
29.15 +0.66 a3d3e3
29.15
8.6+ .37e2
HME200
24.35+0.19 a3c3e2
40.00
9.80+51
HME400
21.43+0.77 a3c3d2
45.60
10.20+.68a3c2
CQ 25
0.00+0.00 a3c3d3e3
100.00
30.0 +0.00 a3c3d3e3
Values are expressed as Mean ± SEM; n = 5.Where a = as compared to negative control ( 4% Tween- 80
as a vehicle) 10ml/kg
; b = as compared positive control(chloroquine 25mg/kg; c= 100 mg/kg;
d=200mg/kg;e=400mg/kg;
1 = P < 0.05; 2 = P < 0.01; 3 = P < 0.001;CQ=chloroquine ,
HME=hydrometanolic extract ,MST=mean survive time.
B. Effect of crude extract of the leaf of S. abyssinica on packed cell volume and
body weight
At 400 mg/kg tested dose hydromethanolic extract showed significant (p<0.05) difference
prevention of PCV reduction as compared to the negative control. But both 200 and 100mg/kg
the extract revealed non statically significant prevention of PCV reduction as compared to
negative control. There were no statically significant differences in protection of PCV reduction
between Chloroquine treated group and all crude hydromethanolic extract treated groups. But,
positive controls shown stasticaly significant (p<0.01) in protection of PCV reduction as
compared to the negative control. When compared to PCV change between Do and D4 with in a
group, there was no significant differences in both the crude hydromethanolic extract treated
groups at 400 mg/kg and chloroquine treated groups. But, the rest groups showed statically
significant PCV reduction as compared to before treatment as shown in table 3.
In 4-day suppressive test, the 80% methanolic extract at all tested dose revealed statistically
nonsignificant (p>0.05) prevention effects of weight loss as compared to negative control.
25
Positive controls revealed significant difference (p<.05) in body weight loss prevention effect
when compared to negative control. When compared to weight change between day4 and day 0,
the negative control (p<0.01), 100 mg/kg dose (p<0.01) and 200mg/kg (p<0.05) dose treated
groups lost their weight with significant differences as compare to before treatment on D0.
Whereas, both 400mg/kg dose extract and chloroquine treated group did not reveal significant
differencse weight in weight changes as shown in table 3.
.
Table 3: Effect of crude extract of the leaf of S. abyssinica on packed cell volume and body
weight of infected mice in the 4 ay suppressive test
Treatments
(mg/kg,
ml/kg)
Vehicle 10
Packed cell volume (%)
54.20+.73
47.8.0+.73
-11.81
HME100
54.20+1.77
50.40+1.86
-7.01 β2
27.06+.27
25.59+.40
- 5.43 β2
HME200
55.00+.84
52.20+1.20
-5.09 β2
26.87+.88
25.87+.84
- 3.71 β1
HME400
54.60+1.03
53.40+.81 a1
-2.2
26.95+.48
26.44+.57
-0.69
CQ 25
54.40+.51
54.6.0+.68 a2
0.37
27.25+.78
27.56+1.11a1
1.14
D0
D4
Body weight(g)
% change
β3
D0
26.48+.40
D4
24.37+.57
%Change
- 8.00 β2
Values are expressed as Mean ± SEM; n = 5.Where a = as compared to negative control (4% tween- 80 as
a vehicle) 10ml/kg ; β= compare to before treatment within a group ; 1 = P < 0.05; 2 = P < 0.01;3=p<.001
CQ=chloroquine, HME=hydrometanolic extract ; D0, at day 0 and D4, at day 4.
C. Effect of crude extract of the leaf of S. abyssinica on body temperature of
infected animals in the 4 day suppressive test
Both 200 mg/kg and 400mg/kg dose extract significantly (p<.01) attenuated the reduction in
body temperature compared to negative control but at dose of100mg/kg, there was no significant
difference in temperature change as compared to negative control. When compare to the other
groups with positive control, chloroquine 25mg/kg dose prevented body temperature reduction
significantly with compare to negative control(p<0.001),100mg/kg extract(0.01), but, there were
no statistically significant with 200mg/kg,400mg/kg doses extract . Positive control shown a
26
statically significant dereferences in protection of body temperature reduction as compared to
100mg/kg treated groups (p<0.01), and negative control (p<0.001). Both negative control and
lowest dose hydro methanolic extract treated groups lose their weight at D4 with compare to
day0 before treatment (p<0.01) within the group but the other groups did not reveal body
temperature reduction as shown in table4.
Table 4: Effect of crude hydromethanolicextract of the leaf of S. abyssinica on body temperature
of infected animals in the 4 day suppressive test
Temperature (0c)
Treatments(m
g/kg, ml/kg)
D0
D4
%Change
37.06+.11
35.92+.21
-3.08 β2
HME100
36.94+.15
36.32+.21
-1.68 β2
HME200
37.18+.14
36.88+.07 a2
-0.81
HME400
37.00+.16
36.86+.17 a2
-0.38
CQ 25
36.98+.12
37.26+.09 a3c2
0.76
Vehicle 10
Values are expressed as Mean ± SEM; n = 5.Where a = as compared to negative control (4% tween- 80 as
avehicle) 10ml/kg ;b = as compared positive control (chloroquine 25mg/kg; c= 100 mg/kg; β= compare to
before treatment within a group ; 1; = P < 0.05; 2 = P < 0.01; 3 = P < 0.001, CQ=chloroquine
,HME=hydrometanolic extract ; D0, at day 0 and D4, at day4.
4.3.2. Invivo antimalarial activity of the solvent fractions of leaf of Stephanica
abyssinica on four days suppressive test
A. Effect of solvent fractions of the leaves of S. abyssinica on parasitemia, %
suppression and mean survival time of P. berghei infected mice in four day
suppressive
The result of 4-day suppressive test indicated that the percentage suppression of n-hexane
fraction on hexane fraction treated groups revealed 26.01 %, 36% and 42.50 % at 100mg/kg,
200mg/kg and 400mg/kg of the fractions respectively. Hexane fraction at dose of 400mg/kg
27
revealed significant percentage suppression with compared to 200mg/kg (p<0.05),100mg/kg
(p<0.01) of hexane fractions.
The percentage suppression of chloroform fractions, on chloroform fraction treated groups, was
34.83%, 48.08% and 55.8 % at 100mg/kg 200mg/kg and 400mg/kg dose of the fractions
respectively. Chloroform fraction at dose of 400mg/kg/ day (p<0.001), 200mg/kg (p<0.01),
revealed significant percentage suppression with compared to 100mg/kg of chloroform fractions.
On ethyl acetate treated groups, the percentage suppression of ethyl acetate fraction was 29.35
%, 38.82% and 51.44 % at 100mg/kg, 200mg/kg and 400mg/kg of the fractions respectively. At
dose of 400mg/kg , ethyl acetate fraction shown significant percentage suppression as compared
to at dose of 200mg/kg(p<0.05),100mg/kg(p<0.001) ethyl acetate fraction, but there was no
statically difference between 200mg,100mg/kg of its fraction in percentage suppression. All
hexane, chloroform and ethyl acetate fractions; shown high percentage suppression (p<0.001)
statistically significantly difference compared to that of the negative control. Positive control at
25mg/kg shown significant percentage suppression (p<0.001) with compared to all hexane,
chloroform, ethyl acetate fractions. The mice treated with CQ were completely free from the
parasites on day four as described in table 5.
The mean survival time of hexane fraction (9.4+.34 with p<0.01),chloroform (10.40+.60with
0.001) and ethyl acetate (10.4+.81 with p<0.01)fraction treated groups
reveled significantly
increasing survival time at 400mg/kg doses compared to negative control (6.60+.24). At dose of
200mg/kg , both chloroform (9.60+.40)and ethyl acetate (9.40+.40)fraction treated groups
did
have prolonged the survive time significantly(p<0.01) as compare to negative control but hexane
fraction treated groups (8.20+.49) at that dose did not reveal significant prolonged survival time
a as compare to negative control. At dose of 100mg/kg, all hexane fraction (7.80+.37),
chloroform fraction (8.40+.51) and ethyl acetate fraction (8.20+.20) group‘s prolonged survival
time non-significantly as compared to negative control. Positive controls prolonged mean
survive time greater than 30 days which was high significant difference (p<0.001) as compared
to other groups as shown in table 5.
28
Table 5 Effect of solvent fractions of the leaves of S. abyssinica on parasitemia, % suppression
and mean survival time of P. berghei infected mice in four day suppressive.
Treatments(mg/kg, Parasitaemia level
ml/kg)
%suppression
Vehicle 10
40.20+1.32
-
6.60+.24
HF100
29.73+.67 a3b3 e3
26.01
7.80+.37 b3
HF200
25.71+1.30 a3b3 e1
36.00
8.20+.49 b3
HF400
23.33+.1.05 a3b3 d1
42.50
9.40+.34 a2 b3
CF100
26.20+.86 a3b3d2e3
34.83
8.40+.51 b3
CF200
20.50+.55 a3b3c2
48.08
9.60+.40 a2 b3
CF400
17.75+1.18 a3b3c3
55.80
10.40+.60 a3 b3
EAF100
28.40+.51 a3b3 e3
29.35
8.20++.20 b3e1
EAF200
24.59+.51 a3b3 e1
38.82
9.40+.40 a2 b3
EAF 400
19.52+1.37 a3b3c3d1
51.44
10.40+.81 a3 b3c1
100
30.00+.00 a3c3d3e3
CQ 25
0.00+0.00 a3c3d3e3
MST(day)
Values are expressed as Mean ± SEM; n = 5.Where a = as compared to negative control ( 4% tween- 80 as a
vehicle) 10ml/kg
; b = as compared positive control(chloroquine 25mg/kg; c= 100 mg/kg; d=200mg/kg
;e=400mg/kg; 1 = P < 0.05; 2 = P < 0.01; 3 = P < 0.001CQ=chloroquine ; HF=n-hexane fraction; CF=chloroform
fraction ;EAF=ethyl acetate fraction.
B. Effect of solvent fractions of the leaf of S. abyssinica on packed cell
volume and body weight of infected mice in the 4 day suppressive test
The hexane fraction treatment group at400mg/kg dose level showed statically significant
differences on prevention against PCV reduction (p< 0.05) as compared to negative control. The
hexane fraction treated groups at dose of 200 and 100mg/kg did not
significantly (p>0.05) differences as compared to negative control on D4.
29
show PCV reduction
Chloroform fraction treated groups
at dose of 400 mg/kg (p<0.01), and 200mg/kg mg/kg
(p<0.05) shown significant difference protection against PCV reduction when compared to the
negative control, even though, lowest dose treated groups did not reveal significant difference
against PCV reduction as compared to the negative control treated groups.
Ethyl acetate fraction treated at dose of 400mg/kg (p<0.01), 200mg (p<0.05) and
100mg/kg(p<0.05) revealed statically significant protection
PCV reduction as compared to
negative control ,however, the lowest dose treated groups did not reveal significant difference
against PCV reduction as compared to the negative control treated groups.
when compared with positive control , at dose of all of hexane, chloroform and ethyl acetate
fraction treated groups did not reveal statically significant differences against PCV reduction
effect , however positive control revealed statically significant difference in protection of PCV
with compare to negative control(p<0.01).
When compared to PCV change between Do and D4 with in a group,
control(p<0.01),N-hexane fraction at
negative
dose of 200mg/kg (p<0.05)and 100mg/kg(p<0.01),
chloroform fraction at dose of 100mg/kg (p<0.01),and ethyl acetate fraction at 200mg/kg
(p<0.05)and 100mg/kg(p<0.01)
on D4 treated groups had significant PCV reduction as
compared to PCV level of the groups on D0(pretreated groups)
On D4, there were no statically significant differences in the PCV reduction of N-hexane
fraction at dose of 400mg/kg, chloroform fraction at dose of 400mg/kg and 200mg/kg, ethyl
acetate fraction at 400mg/kg, and chloroquine treated groups as compared to the PCV level in
D0 of the same groups. Hence the PCV reduction protective effects of the fraction depend on the
dose level.
All dose levels of all fractions treated groups did not reveal statically significance in weight
lose changes as compared to the negative control groups, however , there were non significant
increment of in percentage of weight changes as compared to negative control. Positive control
treated groups reveled statically significant (p<0.05) difference protection from body weight lose
as compared to the negative control. There were no statistical differences between positive
30
control and fractions treatment groups of D4 weight on weight loss protection, however the
positive control revealed increased body weight positively as compared to the fraction.
When compared to
weight changes between Do and D4 with in a group,
negative
control(p<0.001), the hexane fraction at dose of 200mg/kg (p<0.05)and 100mg/kg(p<0.01),
chloroform
fraction at
dose of 100mg/kg (p<0.05) and ethyl acetate fraction at
100mg/kg(p<0.01) on D4 treated groups revealed statically significant weight lose as
compared to measured weight of the same groups on D0(pretreated groups). At D4 the rest
groups: the n- hexane fraction at 400mg/kg dose, chloroform fraction at dose of 200mg/kg and
400mg/kg , Ethyl acetate fraction at 200mg/kg and 400mg/kg and chloroquine treatment groups
did not reveal a stastical difference in body weight change as compared to the weights of the
same group at D0.
Therefore, the above indicated different solvent fractions protected the weight lose in the dose
dependant manner as the dose level increased as shown in table 6
31
Table 6: Effect of solvent fractions of the leaf of S. abyssinica on packed cell volume and
body weight of infected mice in the 4 day suppressive test.
Treatments
Packed cell volume (%)
Body weight(g)
(mg/kg,
ml/k
D0
D4
% change
Vehicle 10
55.00+1.58
48.20+2.25
-12.36β2
HF100
55.80+.86
51.40+.81
HF200
55.00+.63
HF400
D0
D4
% change
27.06 +.59
24.96+.57
-7.62β3
-7.89 β2
27.12+.56
25.41+.51
-6.29β2
52.20+1.00
-5.09β1
26.87+.70
25.90+.51
-3.61 β1
55.40+.510
53.80+.58 a1
-2.89
26.90+.75
26.16+.66
-2.72
CF100
54.80+.37
51.40+.68
-6.20β2
27.62+.84
26.28+.81
-4.84 β1
CF200
54.20+.37
53.20+.49 a1
-1.85
27.13+.84
26.39+.48
-2.73
CF400
55.80+.93
55.00+.45 a2
-1.43
27.16+.47
26.84+33
-1.16
EAF100
54.40+.81
50.80+1.39
-6. 3β 2
26.87+.31
25.52+.45
-5.02β2
EAF200
54.60+.93
53.40+.89
-2.93β1
27.48+.81
26.44+.48
-2.93
EAF 400
55.80+
54.80+.58 a2
-1.79
26.93+.68
26.55+.80
-1.41
CQ 25
55.20+1.02
55.40+1.25 a2
0.36
27.26+.54
28.87+.61
2.23
g
a1
Values are expressed as Mean ± SEM; n = 5.Where a = as compared to negative control (4% Tween- 80 as a
vehicle) 10ml/kg, β= as compared with before treatment within the group,1 = P < 0.05; 2 = P < 0.01; 3 = P <
0.001 CQ=chloroquine; HF=n-hexane fraction; CF=chloroform fraction; EAF=ethyl acetate fraction; D0, at day
0; and D4, at day 4.
32
B. Effect of solvent fractions of the leaf S. abyssiniaon body temperature of
infected animals in the 4 day suppressive test
The effect of
all solvent fractions fraction at 400mg/kg dose shown statically significant
(p<0.05) activity on prevention against body temperature reduction as compared to negative
control.In all fractions at tested doses of 100 and 200 mg/kg did not show statically significant
difference effect on protection of body temperature reduction as compared to the negative
control. Hexane fraction at 400mg/kg dose revealed statically significant (p<0.05) preventive
effect on body temperature reduction as compare to hexane fraction at 100mg/kg dose. But the
rest; chloroform and ethylacetate treated groups did not reveal statically significant difference
in body temperatures change as compared to different dose treatment groups of the same solvent
fractions. The chloroquine treatment groups showed statically significant difference in the
protection of temperature reduction as compared to 100mg/kg dose treatment groups of all
fractions.
When compared the temperature change of the same group at D0and D4, both negative control
(p<0.01) and all 100mg/kg (p<0.01), and 200 mg/kg(p<0.05) solvent fractions tested groups
shown body temperature reduction at D4 with compare to D0 before treatment but the other all
400 mg/kg solvent faction dose tested groups and positive controls did not reveal significant
body temperature reduction
as compared to measured body temperature
treatment as described in table 7.
33
at Do
before
Table 7: Effect of solvent fractions of the leaf S. abyssiniaon body temperature of infected
animals in the 4 day suppressive test
Treatments(
mg/kg
,
ml/kg
Temperature (0c)
D0
D4
% change
Vehicle 10
37.00+.06
-3.08 β2
35.86+19
35.90+.27b3e1
-2.87 β2
HF100
36.96+.07
HF200
36.84+.10
HF400
36.94+.14
36.74+.13a1c1
-0.54
CF100
36.86+.18
36.12+.06b3
-2.22 β2
CF200
36.96+.14
36.68+13
-0.76 β1
CF400
36.86+.17
36.78+19a1
-0.22
EAF100
36.90+.12
36.02+.14b3
-2.38 β2
EAF200
36.94+.14
36.64+.14
-0.81 β1
EAF 400
36.92+.19
36.74+.19 a1
-0.49
CQ 25
37.20+.15
37.26+.09a3c3
0.16
36.50+.13
-0.92 β1
Values are expressed as Mean ± SEM; n = 5.Where a = as compared to negative control ( 4% tween- 80
as a vehicle) 10ml/kg ; b = as compared positive control(chloroquine 25mg/kg; c= 100 mg/kg; β= as
compared with before treatment within the group ,1 = P < 0.05; 2 = P < 0.01; 3 = P < 0.001
CQ=chloroquine ; HF=n-hexane fraction; CF=chloroform fraction ;EAF=ethyl acetate fraction;. D0, at
day 0 ; and D4, at day 4.
34
4.3.3. Prophylactic Effect of hydromethonolic extract and chloroform fraction of leaf
of S.abyssinica
A. Effect of crude extract and cloroform fraction of the leaves of S. abyssinia on
parasitemia, % suppression and mean survival time of P. berghei infected
mice in prophylactic tests
In a prophylactic test, effect of hydromethanolic crude extract treated groups
showed statistically
significant difference on the percentage parasite suppression at a dose of 400mg/kg(p<0.001), 200mg/kg
(p<0.001), and 100mg/kg (p<0.001) when compared to negative control. The extract treated group at the
dose of 100mg/kg and 200mg/kg did not showed statistically significant difference parasite suppression
as compared to each other. In the same manner, the extracted treated group at the dose of 200mg/kg and
400mg/kg did not showed statistically significant difference parasite suppression as compared to each
other. However, the extract treated group at the dose of 400mg/kg (P<0.001) showed statistically
significant difference when compared to the extract treated group at the dose of 100mg/kg as shown in
table 8.
The percentage parasite suppression of chloroform fractions on prophylactic test, revealed statistically
significant difference at the dose of 100mg/kg (P<0.001), 200mg/kg(P<0.001), and 400mg/kg(P<0.001)
when compared to the negative control as it did by the 80% methanolic crude extract treated groups. The
chloroform fraction treated groups at the dose of 200mg/kg (P<0.05) and 400mg/kg (P<0.001) revealed
statistically significant difference when compared to 100mg/kg treated group. The chloroform fraction
treated group at the dose of 200mg/kg and 400mg/kg did not revealed statistically significant difference
when compared to with each other. Both, the 80% methanolic crude extract and chloroform fraction
treated groups at all dose revealed statistically significant difference when compared to chloroquine
treated group(P<0.001) as shown in table 8.
The effect of both 80% methanolic crude extract and chloroform fraction on mean survival time on
prophylactic test, revealed statistically significant difference in the prolongation of mean survival time at
the dose of 400mg/kg (P<0.01) as compared to negative control. The 80% methanolic crude extract and
chloroform fraction at the dose of 100mg/kg and 200mg/kg did not revealed statistically significance
difference in the prolongation of mean survival time when compared to negative control. While both 80%
methanolic crude extract and chloroform treated groups at all doses revealed statistically significant
difference in the prolongation of mean survival time when compared to chloroquine treated groups
(P<0.001) as shown in table 8.
35
At dose of 400mg/kg, both the crude extract( 9.60+.51 day) and chloroform fraction(9.40+.51)
prolonged the survival time significantly(p<0.01) as compared to negative control
and
chloroquine treated groups survived more than 21days as shown in table 8.
Table 8: Effect of crude extract and cloroform fraction of the leaves of S. abyssiniaon
parasitemia, % suppression and mean survival time of P. berghei infected mice in
prophylactic tests
Treatments(m
g/kg, ml/kg)
Vehicle 10
HME100
HME200
HME400
CF100
CF200
CF400
CQ 25
Parasitaemia
level
22.03+.87
14.10+1.51
11.49+.97
a3b3e2
a3b3
10.03+1.41
a3b3c2
a3b3d1c3
13.67+1.51
10.55+.98
9.33+.92
a3b3c1
a3b3c3
0 .76+.10 a3c3d3e3
% suppression
0
35.91
47.77
54.41
37.86
50.77
57.59
96.55
MST(day)
6.80+.37
7.60+.51b3
8.60+51b3
9.60+.51a2b3
8.40+.40b3
8.80+.20b3
9.40+.51a2b3
>21.00a3c3d3e3
Values are expressed as Mean ± SEM; n = 5.Where a = as compared to negative control (4% twin- 80) 10ml/kg;
b = as compared positive control (chloroquine 25mg/kg; c= 100 mg/kg; d=200mg/kg; e=400mg/kg; 1 = P <
0.05; 2 = P < 0.01; 3 = P < 0.001. CQ=chloroquine , HME=hydrometanolic extract ; CF=chloroform fraction ,
MST=mean survival time
B. Effects on Packed cell volume and body weight of infected mice treated with
crude extract and chloroform fraction leaves of S. abyssinica in prophylactic
tests
As compared to the PCV level on D7,the chloroform fraction at 400mg/kg and positive controls
revealed significant prevention
activity against
PCV reduction (p<0.05) as compared to
negative control. All crude extract at dose of 100,200, and 400mg/kg did not reveal significant
protection of PCV reduction (p>0.05) as compared to negative control. The there were
nonstatically significant difference effect on PCV level among both crude hydromethanolic and
chloroform fractions as compared to based on respective different dose levels. There was no
significant difference between among crude hydromethanolic and chloroform fraction at 400,200
and100mg/kg tested dose as compared to the positive control as described in table 9.
36
When compared PCV differences between D4and D7, negative control(p<0.001),crude extract
at
200 mg/kg(p<0.05),and100mg/kg(p<0.01),
and chloroform
at 200mg/kg (0.05)and100
mg/kg (p<0.01)dose treated groups revealed significant PCV reduction on D7as compared to
the PCV level on D4 within the same group. Where both crude hydromrthanolic extract and
chloroform fractions at 400 mg/kg and positive control groups did not reveal significant PCV
reduction on D7 as compared to the PCV level on D4 as shown in the table 9.
Both crude hydromethanolic extract and chloroform fractions treated groups at all dose levels
did not reveal statically significance in weight changes as compared to the negative control
groups at D7, however, there were non significant increment of in percentage of weight changes
as compared to negative control. Positive control treated groups reveled statically significant
(p<0.05) difference body weight change as compared to the negative control. There were no
statistical differences between positive control and crude hydromethanolic extact and chloroform
fractions treatment groups of body weight change, however the positive control revealed
increased body weight positively as compared to both the fraction and the fraction.
When compared to
weight changes between D4 and D7 with in a group,
control(p<0.01), the crude hydromethanolic extract at
negative
dose of 200mg/kg (p<0.05)and
100mg/kg(p<0.01), and chloroform fraction at dose of 100mg/kg (p<0.01) treated groups at D7
revealed statically significant difference in weight lose as compared to measured weight of the
same groups on D4(pretreated groups). The crude hydromethanolic extract treated groups at
400mg/kg dose, chloroform fraction treated groups at dose of 200mg/kg and 400mg/kg, did not
reveal statically difference in body weight loss as compared to the weights of the same group at
D7. The chloroquine treated groups at D7 revealed statically significant difference in weight gain
as compared to measured weight of the same groups on D4 as shown in table 9.
37
Table 9: Effect of packed cell volume and body weight of infected mice treated with crude
extract and chloroform fraction leaves of S. abyssinica in prophylactic tests.
Treatments(
mg/kg, ml/kg)
Packed cell volume (%)
D4
D7
Body weight(g)
% change
D4
-10.11β3
26.41+.72
49.80+1.16
D7
%Change
24.63+.55
-6.70 β2
Vehicle 10
55.40+1.25
CQ 25
55.40+.51
HME100
54.20+1.72
50.60+1.5
-7.19β2
26.90+.37
25.76+.38
-4.25 β2
HME200
54.80+.66
52.40+1.24
-4.75β1
26.85+.55
26.14+.35
-2.66 β1
HME400
54.60+1.03
53.40+1.00
-2.20
26.93+.49
26.57+.55
-0.63
CF100
55.20+.86
52.00+1.10
-5.80 β2
26.85+.43
26.2+.43
-3.10 β2
CF200
54.80+.66
52.60+.93
-4.01β1
26.80+.61
26.18+.39
-2.31
CF400
56.00+.71
55.20+.58a 1
-1.43
26.86+.38
26.77+.92
-0.58
55.20+.80 a1
-0.36
26.78+.82
27.33+.78 a1
2.06 β 1
Values are expressed as Mean ± SEM; n = 5.Where a = as compared to negative control ( 4% tween- 80 as
avehicle) 10ml/kg ; b = as compared positive control(chloroquine 25mg/kg ; β=as compare to before treatment
within the group; 1 = P < 0.05; 2 = P < 0.01; 3 = P < 0.00 ;CQ=chloroquine ,HME=hydrometanolic extract ;
CF=chloroform fraction .D4, at day 4 and D7, at day 7.
C. Effect body temperature of infected animals treated with crude and solvent
fraction of the leaf S. abyssinica in the prophylactic tests.
Both hydromethanolic crude extract and chloroform fraction at dose of 400mg/kg reveled
statically significant(p<0.05 )protective effect from body temperature reduction as compared to
body temperature of negative control, however, at 100mg/kg and 200 mg/kg doses, both extract
and Chloroform fraction shown stastical non- significant(p>0.05) protective effect as compared
to the negative control. When compared to chloroquine treatment groups, both extract and
chloroform fractions at 200mg,400mg/kg shown comparable protective effects from rectal
temperature reduction ,but at 100mg/kg dose, chloroquine treatment shown significant difference
(p<0.05). The positive control produced preventive activity from temperature reduction statically
significant (P<0.01) difference when compared the negative control as shown in table10.
38
When compared to the body temperature difference between D4 (pretreatment) and D7 (after
treatment, all negative control, crude hydromethanolic and chloroform fraction treated groups at
200 and 100 mg/kg at D7 shown statically significant (p<0.05) body temperature reduction as
compared to the body temperature on D4 before treatment. whereas
both crude extract and
chloroform fraction 400mg/kg dose treated groups at D7 did not reveal significant temperature
reduction as compared to body temperature at day 4 and also the chloroquine treated groups did
not produce statically significant body temperature increment as compared to the body
temperature measured on D4 before treatment as shown in table10.
Table 10: Effect body temperature of infected animals treated with crude and solvent
fraction of the leaf S. abyssinica in the prophylactic tests.
Treatments(mg
/kg, ml/kg)
Vehicle 10
HME100
HME200
HME400
CF100
CF200
CF400
CQ 25
Temperature (0c)
D4
D7
37.00+.15
36.96+.17
37.06+.09
37.00+.16
36.88+.15
36.96+.10
36.94+.15
37.02+.13
%Change
36.48+.09
36.56+.16 b1
36.80+.15
36.82+.24 a1
36.50+.14 b1
36.74+.14
36.82+.16 a1
37.060+.18a2 c
-2.27β1
-1.08 β1
-0.81β1
-0.49
-1.03 β1
-0.60 β1
-0.33
+0.11
Values are expressed as Mean ± SEM; n = 5.Where a = as compared to negative control ( 4% tween- 80 as a
vehicle) 10ml/kg ; b = as compared positive control(chloroquine 25mg/kg; c= 100 mg/kg; β as compare to
before treatment within groups; 1 = P < 0.05; 2 = P < 0.01;;CQ=chloroquine , HME=hydrometanolic extract ;
CF=chloroform fraction .D3, at day 3 and D7, at day 7.
39
5. DISCUSSION
The spread and occurrence of resistant malaria to the front line antimalarial drugs (including
artemisinin) (1), toxicity of conventional antimalaria drugs ,absent of vaccine
and
the
appeareance of insecticide resistant mosquitos are the major challenges that overwhelm all
recent gains in malaria control and has major implications for public health(9,12, 17). Hence, the
scientific community is now underway to solve this problem by searching for new, safe,
affordable and effective antimalarial agents from medicinal plants and other sources (56, 57).
This study was conducted using in-vivo model in which the both crude hydromethanolic and
fractions were tested against P. berghei infected mice. The in-vivo model was employed because
it takes into account prodrug effect and possible involvement of immune system in eradication of
infection (52, 58).
Rodent models have been validated through the identification of several conventional
antimalarials, for example chloroquine, halofantrine, mefloquine and more recently artemisinin
derivatives (52). The common malaria parasite, chloroquine sensitive parasite, the P.berghie, is
with proven use in the prediction of treatment outcomes and remains a standard part of the drug
discovery pathway, hence, is the appropriate parasite for this study (52, 59).
For antimalarial drug screening, the 4-day suppressive test is the standard test commonly used P.
berghei infected mice are used for better prediction of antimalarial efficacy of drugs for human
use in many studies in search of antimalarial drugs (52, 58).
On preliminary phytochemical screening of hydromethanolic extract and n-hexane, chloroform,
ethyl acetate and aqueous solvent fractions leaf of S. abyssinica revealed that alkaloids(absent
in aqueous), flavonoids , tannins, phenol and saponins constituets were present in both hydro
methanolic crude extract as well as all solvent fractions. These phythochemicals have been wellknown for different pharmacological activities including antimalarial effects. Some alkaloids
such as, quinine prevent polymerization of heme in haemozoin in the digestive vacuole. Phenolic
compounds(flavonoids ,phenols and tannins,) act as antioxidant or free radical scavenger to
prevent/reduce oxidative stress induced by the parasite Antioxidants can inhibit also heme
polymerization and the unpolymerised heme is very toxic for the parasite compounds detected
40
in the leaf of Stephania abyssinica for their antioxidant.
membrane of the parasites.
The sapponincs affect the cell
Hence, these secondary metabolatites might be responsible for
antimalarial activitie of the leaf Stephania abyssinica against Plasmodium berghei infection in
mice (58, 59,60).
Four-day suppressive test assesses the potential schizontocidal activity of hydromethanolic
extracts and solvent fractions in early infection whereby the primary attack due to malaria can be
prevented or mitigated (52). The hydromethanolic extract, hexane, chloroform and ethylacetate
fractions at dose of 400mg/kg showed the maximum chemosupressive activities( 54.41%,
42.50%, 55.80% and 51.44% respectively )on parasitemia level .This might be due to the
presence of good concentrations of active compounds in higher dose.
Chemoprophylaxitic test is one of other secondary antimalaria screening tests in rodent malaria
(52). The chemoprophylactic activity of the crude extract and chloroform fractions were
evaluated using the residual infection procedure. The crude hydromethanolic extract (54.41%),
and chloroform fraction (57.59%) suppressions also showed chemoprophylactic activity against
Plasmodium berghei infection in mice at 400mg/kg dose. This might be due to delay of
metabolic clearance and tissue sequestration and redistribution of active photochemicals, which
are responsible for parasite suppression, present in both extract and fraction when administered
to the mice. It also might be due to the phytochemicals preventive effect of the parasites
adherence, cellular division on and/ or in the surface of erythrocytes or due to
immunomodulating effect in mice (37, 38).
Hence, hydromethanolic and all fractions can be considered to be active in their schizontocidal
activity against malaria. This finding supports the traditional use of the leaf of S.abyssinica and
invitro antimalaria activity results for malaria treatment by the peoples of eastern Africa (36, 42,
50). Since a compound is considered as active when percent, parasitemia inhibition is greater or
equal to 30% (52) which is in agreement with this study.
The antimalarial activity of might be due to the presence of active secondary metabolites present
in these extracts and fractions such as alkaloids, flavonoids ,tannins, phenol and saponins and
other untested constituents. These active secondary phytochemicals might inhibit or block folate
41
metabolism, protein synthesis, membrane transport system, haem polymerization, electron
transportsystem, free radicals cavengers and other mechanism or path way of the parasite which
might be similar to conventional antimalaria drugs and other unkown mechanisms (52,60, 61).
These methabolites might act singly or in synergy with one another to produce the observed
biological activities (60).
As different evidences were acquired from different literatures, S.abyssinica showed an
antiplasmodial activity (dichloromethane, IC50<3μg/ml, ethyl acetate IC50<5μg/ml and
methanol fraction, IC50<9μg/ml)
against Plasmodium falciparum strains in invitero test (42,
50). It also had antitumor (36), anti bacterial (36), antioxidant activity (48) in invitro
and
antiinflamatory and analgesic in invivo test (49).It can be convenced from invivo
chemosupresive effect because the leaf of the plant enriches in bisbenzylisoquinoline and
hasubanane alkaloids (42).
The positive control eliminated the parasite to non-detectable level and as a result led to survival
time of more than 30days by inhibiting the aminoacide production and toxic intermidet product
generation for parasite. The significantly lower parasitemia suppression by the extracts and
solvent fractions as compared to the positive control could be due to low level of active
compound(s) associated with the crude nature of the extract and fractions, non selectivity of the
extract or slow absorption and low bioavailability of these extracts (59).
The mean survival time is the second important parameter to test the antimalarial activity of
plant extracts (52). Both of the extracts and solvent fractions of S.abyssinica prolonged the
survival time of mice which might be due to the suppression of parasitemia and reduced the
overall pathologic effect of the parasite on the study mice (38, 62). The mean survival time of
hydromethanolic extract and solvent fractions at 400mg/kg revealed in significant prolongation
as compared to the negative control in both chemosupresive and chemophylactic mice models.
However, other lower doses of both methanolic extract and solvent fractions did not reveal
significant prolongation of means survival time as compared to negative control. It could be
related to the presence of active secondary metabolites in sufficient concentration unlike that of
the lower doses. This indicates that these doses suppressed P. berghei and thereby reduced
anemia and the overall pathologic effect of the parasite on the test groups (61, 38, 62).
42
Packed cell volume (PCV) is one of the third parameter that used to predict the effectiveness of
the test extract and fractions in preventing hemolysis resulting from increasing parasitemia
associated with malaria (10). Post infection of malaria, the rodents suffer from anaemia because
of RBC destruction by The underlying cause of anemia includes; loss of infected erythrocytes
through parasite maturation, destruction of uninfected red cells in the spleen and liver by
macrophages
activation
and/or
enhanced
phagocytosis,
reduced
erythropoiesis
and
dyserythropoiesis ( 38, 59,61).
At 400 mg/kg tested dose both hydromethanolic extract and fractions shown significant
protection against PCV reduction in both models. But the lowest dose did not protect against
PCV reduction due to plasmodium infection. This protection effect could be as a result of the
significant parasite suppression effect induced by active constituents in the administered doses of
crude extract and fractions at 400mg/kg were sufficient enough to prevent/delay RBC hemolysis
that could be linked to the higher concentration active constituents in the administered dose
(38,62).
The protective effect of both crude hydromethanolic extracs and fractions against PCV reduction
due to parasite might be due to destructive antiplasmodial effect of the extract and fractions
against the parasitized red blood cell and the causative parasite, thereby sustaining the
availability of the new red blood cells produced in the bone marrow (37, 62).
The fourth parameter in the evaluation of antimalaria screening is the effects of the plant on
malaria-induced weight lose. Anemia, body weight loss and body temperature reduction are the
highmarks of malaria infected mice. Weight decrement has been associated with decreased food
intake, disturbed metabolic function and hypoglycemia . So, an ideal antimalarial agents obtained
from plants are expected to prevent body weight loss in infected mice due to the rise in
parasitemia(37,63,64 ).
It was observed that the crude hydromethanolic extract and n- hexane fraction at 400mg/kg dose,
chloroform and ethyl acetate fraction fractions at dose of 200mg/kg and 400mg/kg protected
the body weight lost in chemosepresive test. And the crude hydromethanolic extract treated
group at 400mg/kg dose, chloroform fraction treated groups at dose of 200mg/kg and 400mg/kg
43
also revealed significant protective effect against body weight in chemoprophylactic test. These
indicate that these doses suppressed P. berghei and thereby reduced anemia and the overall
pathologic effect of the parasite on the test groups (38, 64, and 65).But the rest fraction and
crude extract doses did not keep body from weight lost in all models which might be insufficient
phytochemical concentration in lower doses. Therefore, the above indicated both crude and
different solvent fractions protected the weight lose in the dose dependant manner as the dose
level increased.
The last parameter is temperature change evaluation. A decrease in the metabolic rate of infected
mice occurred before death and was companied by a corresponding decrease in internal body
temperature. This implies that infected mice body temperature drops as parasite level grow
rapidly. This decrement in temperature has been associated with reduction in basal metabolic
rate and impact of anemia on heat production and/or heat conservation (38, 65,66, ).
Both crude hydromethanolic and solvent fractions significantly attenuated the reduction in body
temperature soundly at 400mg/kg comparable to chloroquin in both models. The lowest dose
hydro methanolic extract and all fractions at 100mg/kg and 200 mg/kg fractions did not
significant protect body temperature reduction. This might be due to availability of sufficient
active phytochemicals in higher doses that attacked against Plasmodium berghei and others
pathological abnormalities in mice due to infection (38, 62)
A cording to Deharo E et al, in vivo antiplasmodial activity can be classified as moderate, good
and very good if an extract displayed a percent parasite suppression equal to or greater than 50%
at a dose of 500, 250 and 100mg/kg body weight per day, respectively (67). Based on this
classification, both crude hydromethanolic and fractions leaf of S.abyssinica of had a moderate
antiplasmodial activity against P. berghei infection in mice on both four-day suppression and
prophylactic tests. The moderate antiplasmodial effect revealed by the hydromethanolic extract
and fractions with the longest survival time compared to negative control,preventive effect of
PCV reduction, weight lose and temperature reduction could be related with the presence of
active secondary metabolites in sufficient amount.
.
44
6. CONCLUSION
From this study, it can be concluded the leaf of S.abyssinica revealed moderate chemosupresive
and moderate chemophylactic antimalarial activities against Plasmodium berghei infection in
mice and uphold with the the invitro antimalaria test results and justified traditional claims made
by the peoples of Keynia. Accordingly, with the essence of further studies this plant could serve
as the potential source of new and novel antimalarial leads and/or drugs for the treatment and
preventionof malaria.
45
7.
RECOMMENDATION
Based on the present finings it could be recommended as follow:
Isolation and identification of the active constituents of leaf of Stephania abyssinica
should be done
The aqueous fraction evaluated in chemosupressive model, prophylactic and curative
models
The hydromethanolic extract and solvent fractions should be further evaluated in curative
test..
Further studies shall be carried out to determine the mechanism of action(s) responsible
for the antimalarial activities.
46
REFERENCES
1. WHO. World ssmalaria report Geneva,Netherland: 2015.1-55
2. Alelign A. , Dejene T. .Current Status of Malaria in Ethiopia: Evaluation of The Burden,
Factors for Transmission and Prevention Methods. Acta Parasitologica Globalis. 2016;7
(1):1-8.
3. National Malaria Control Team & Ethiopian Public Health Institute, Federal Ministry of
Health, World Health Organization ,Addis Ababa University and The INFORM Project
Department of Public Health ResearchKenya Medical Research Institute - Wellcome
Trust Progamme.Nairobi, Kenya .An Epidemiological Profile of Malaria in Ethiopia.
2014. 9-46
4. Adugna A. Vectored Infectious Diseases: Malaria in Ethiopia. 2014 1-17.Acessed on
12,12, 2016
5. WHO. Practice Handbook .Management of Sever Malaria 3rd ed. Geneva, Newzerland ;
2013. 1-56
6. Balarabe R Mohammed , Yayo M Abdulsalam, Deeni Y. Insecticide resistance to
Anopheles spp.mosquitoes (Diptera: Culicidae) in Nigeria: A review. International Journal
of Mosquito Research. 2015;2 (3):56-63.
7. Siciliano G. , Alano P. Enlightening the malaria parasite lifecycle: bioluminescent
Plasmodium in fundamenta land applied research. Frontiers Microbiology 2015;6:1-4.
8. CDC. malaria biology 2016. https://www.cdc.gov/Malaria/about/biology/index.html
acced on Feb.2017
9. Makoah N. Aminake , Pradel G. Antimalarial drugs resistance in Plasmodium falciparum
and the current strategies to overcome them. science, technology and education.
2013:269-74.
10. Autino B., Yolanda C., Francesco C. and Donatella T. Pathogenesis of Malaria in
Tissues and Blood. Mediterr Journal of Hematological
Infectious
Diseases. 2012;
4(1):2035-3006.
11. Fedral Democratic Republic Of
Ethiopia Ministery Of Health. National Malaria
Guidelines 3rd ed., 2012 ,1-110
47
12. Liu N. Insecticide Resistance in Mosquitoes: Impact, Mechanisms, and Research
Directions Annual Review of Entomology. Web of Science January 2015;60:: 537-59
13. Petersen I., Eastman R.,Lanzer M. Review Drug-resistant malaria: Molecular mechanisms
and implications for public health. Federation of European Biochemical Societies.
2011:1551–1562.
14. Whalen K. Lippincott Illustrated Reviews: Pharmacology 6 thed. University of
FloridaCollege of Pharmacy Gainesville, Florida: Wolters Kluwer; 2015.50-54
15. Gerald L. Mande, John E. Bennett , Dolin R. Principles and Practice of Infectious
Diseases. 6th.ed United States of America: Elsevier Inc; 2005, 272-280
16. Anthony J. Trevor , Bertram G. Katzung , Marieke Kruidering-Hall , Susan B. Masters
katzung basic and clinic al pharmacology McGraw-Hill Education.; 2015. 1281-1293
17. Bhattacharjee D,
Shivaprakash G. Drug Resistance In Malaria-In A Nutshell. Journal
Of Applied Pharmaceutical Science 2016;6 (3 ):137-43.
18. Cui L, Mharakurwa S., Ndiaye D,
Pradipsinh K.Rathod,
Philip J. Rosenthal.
Antimalarial Drug Resistance: Literature Review and Activities and Findings of the
ICEMR Network. The American Society of Tropical Medicine and Hygiene.
2015;93(Suppl 3):. 57–68.
19. Thomas L. a comprehensive review of malaria with an emphasis on plasmodium
resistance: University of Mississippi; 2014. 1-20
20. Olotu A. ,et‘al. Seven-Year Efficacy of RTS,S/AS01 Malaria Vaccine among Young
African Children. National England Journal of Medicine. 2016;374:2519-29.
21. Schwartz L. et‘al . A review of malaria vaccine clinical projects based on the WHO
rainbow table. Malaria Journal. 2012;11(11):5.
22. Prasad S . Traditional Medicine: The Goldmine for Modern Drugs. Biology & Medicine ;
2015;3( 1):2.
48
23. Samuels S.MekunenE.,AwasT. GidayM. The place of traditional medicine in the African
society:The science, acceptance and support. American Journal of Health Research. The
science, acceptance and support American Journal Of Health Research 2014 2(2):49-54.
24. Yadav R H. Medicinal plants in folk medicine system of Ethiopia. Journal of Poisonous
and Medicinal Plants 2013;1(1): 07-11.
25. Birhanu Z., Endale A., Shewamene Z. An ethnomedicinal investigation of plants used by
traditional healers of Gondar town, North-Western Ethiopia. Journal of Medicinal Plants
Studies. 2015;3( 2):36-43.
26. Birhanu T., Abera D. , Ejeta E. Ethnobotanical Study of Medicinal Plants in Selected
Horro Gudurru Woredas, Western Ethiopia. Journal of Biology, Agriculture and
Healthcare 2015;5(1). 84-90
27. Megersa M Asfaw Z. Kelbessa E , Beyene A , Woldeab B. . An ethnobotanical study of
medicinal plants in Wayu Tuka district, East Welega zone of oromia regional state, West
Ethiopia. . Journal of Ethnobiology and Ethnomedicine. 2013 9(1):100-110
28. Kalayu M., GebruT. , Teklemichae T. Ethnobotanical Study of Traditional Medicinal
Plants Used by Indigenous People of Gemad District, Northern Ethiopia. Journal of
Medicinal Plants Studies. 2013 1(4):32-3.
29. Maryo M, Nemomissa S, BekeleT. An ethnobotanical study of medicinal plants of the
Kembatta ethnic group in Enset-based agricultural landscape of Kembatta Tembaro
Zone,Southern Ethiopia. Asian Journal of Plant Science and Research,. 2015; 5(7):42-61.
30. Getanh S. , Girma Z.
an ethnobotanical study of medicinal plants in Debrelibanos
Wereda,Central EthiopiaS. African Journal of Medicinal Plant 2014;8(7):366-79.
31. Limenih Y , Umer S, Wolde-Mariam M..Ethnobotanical Study On Traditional Medicinal
Plants In Dega Damot Woreda, Amhara Region, North Ethiopia. International Journal Of
Research In Pharmacy And Chemistry. 2015;5(2) 2231-2781
32. Enyew A., Asfaw Z, Kelbessa E , Nagappan R. Ethnobotanical Study of Traditional
Medicinal Plants in and Around Fiche District, Central Ethiopia. Current Research Journal
of Biological Sciences. 2014;6(4):154-67
49
33. Alemayehu G. , Asfaw Z. .Kelbessa E. Ethnobotanical study of medicinal plants used by
local communities of Minjar-Shenkora District,North Shewa Zone of Amhara Region,
Ethiopia. Journal of Medicinal Plants Studies 2015;3(6):01-11.
34. Mengesha GG. Ethnobotanical survey of medicinal plants used in treating human and
livestock health problems inMandura Woreda of Benishangul Gumuz, Ethiopia.
Advancement in Medicinal Plant Research 2016 :4(1 ):11-26.
35. Endale A. Studies On Antimalarial Activity Of The Leaf Constituent Of Otostegia
integrifolia Benth. (Lamiacea) Against Plasmodium Berghei In Mice. Addis Ababa: Addis
Ababa University; 2012.26-33
36. Amuamuta A., Na-bangchang K .A review of ethnopharmacology of the commonly
usedantimalarial herbal agents for traditional medicinepractice in Ethiopia. African
Journal of Pharmacy andPharmacology. 2015;9(25):615-27.
37. Fantahun S. In-Vivo Anti-Plasmodium Activity Of Crude Extracts And Solvent Fractions
Of Strychnos mitis Leaves Inplasmodium Berghei Infected Mice. BMC Complementary
and Alternative Medicine 2017, 17(13 ), 2-10
38. Zeleke G. Antimalarial activity of 80% methanol extract of the stem bark of Syzygium
guineense (Willd.) DC. (Myrtaceae) in mice infected with Plasmodium berghei. Addis
Ababa: Addis Ababa University; 2015.36-47
39. De Wet H Van Wyk Be.. Taxonomic notes on the genus Stephania in southern Africa.
South African Journal of Botany. 2014;95: 146-51.
40. GraceO.Stephania
abyssinica
Walp.Prota.
2008;
11(1).
1-5(<Stephania
abyssinicaiprota4U.www.prota4u> acessed on december 2/2016
41. Kadiri M O, Onatade T N. Indigenous uses and phytochemical contents of plants used in
the treatment of menstrual disorders and after-child birth problems in Abeokuta south
local government area of Ogun state, Nigeria. Journal of Drug Delivery and Therapeutics.
2015;5(3):33-42.
42. Ruth A. Omole
et al . Bisbenzylisoquinoline and hasubanane alkaloids from Stephania
abyssinica (Dillon & A. Rich) (Menispermeceae). Science Direct. 2014;103:123–8.
50
43. Deneke T. Antidiarrheal And Antispasmodic Activities Of Stephania Abyssinica
(Minspermaseae) Used In Ethiopian Traditional Medicine: Addis Ababa Universityschool
Of Graduate Studies; 2010. 38-40
44. Abebe W. An Overview of Ethiopian Traditional Medicinal Plants Used for Cancer
Treatment. European Journal of Medicinal Plants. 2016;14(4):9.
45. Giday M, Asfaw Z, Woldu Z . Ethnomedicinal study of plants used by Sheko ethnic
group of Ethiopia. Journal of Ethnopharmacology. 2010;132:75–85.
46. Teklehaymanot T, Giday M. Ethnobotanical study of medicinal plants used by people in
Zegie Peninsula, Northwestern Ethiopia. Journal Ethnobio Ethno medical. 2007(3):12.-13
47. Reta H, Asfaw Z , Kelbessa E .Contribution of Traditional Farmers for Medicinal Plant
Conservation on the Farming Site in Gozamin District, Amhara Region, Ethiopia.
International Journal of Life Sciences. 2015;4(1):24-35.
48. Alemayehu P. Washe D . Hepatoprotective Activities and Bioactive Constituents of
Stephania abyssinica. British Journal of Pharmaceutical Research. 2016;10(6):5-6.
49. Leyikun T. Evaluation of the analgesic and anti-inflammatory activities of 80%methanol
leaf extract of Stephania abyssinica (Quart.-Dill. & A. Rich.) Walp. (Menispermaceae) in
mice. Addis Ababa, Ethiopia: Addis Ababa University; Complementary and Alternative
Medicine 2017 , 13-35
50. Anyango O R. Anti-Malarial Activity And Phytochemical Studies Of Cissampelos
mucronata And Stephania abyssinica. Kenya Kenyatta University; 2011.23-100
51. Semwala D K et al. Review: The genus Stephania (Menispermaceae): Chemical and
pharmacological perspectives. Journal of Ethnopharmacology. 2010;132: 369–83
52. Fidock DA. Antimalarial drug discovery: efficacy models for compound screening.
Nature Reviews Drug Discovery. 2004 3(6):509-520.
53. Tiwari P et al Phytochemical screening and Extraction: A Review. Internationale
Pharmaceutical Sciencie, 2014; 1 .98-104
51
54. Evans WC Trease and Evans Pharmacognosy. 15th ed.W.B. Saunders, London,2002. 3570
55. .Guide For The Care And Use Of Laboratoryanimals. USA , the National Academy of
Sciences; 8th ed. 2011. 1-50
56. Gamo FJ. Antimalarial drug resistance: new treatments options for Plasmodium.Drug
Discov Today Technol. 2014,11: 81-88.
57. Brian T. Grimberg and Rajeev K. Mehlotra. Expanding the Antimalarial Drug Arsenal—
Now, But How?: Pharmaceuticals, 2011, 4,, 681-712
58. Alli L. A. et al. Anti-plasmodial activity of aqueous root extract of Acacia nilotica:
African Journal of Biochemistry Research. July 20115(7): 214-219
59. Ebiloma U. Godwin, James O. .Aminu O .Rhoda : Suppressive, Curative and Prophylactic
potential of Moringalucid
Potentials of Morinda lucida against Erythrocytic Stage of
Mice Infective Chloroquine Sensitive Plasmodium berghei NK-65: British Journal of
Applied Science & Technology, 2011 1(3): 131-140
60. Abdulelah H. and Zainal-Abidin B. In Vivo Antimalarial Tests of Nigella sativa (Black
Seed) Different Extracts. American Journal of Pharmacology and Toxicology. 2007; 2 (2):
46-50.
61. Mengiste B., Makonnen E., Urga KIn-vivo antimalarial activity of Dodonaea Angustifolia
seed extracts against Plasmodium Berghei in mice model. MEJS, . 2012. 4 (1):47-63.
62. Bihonegn T., Yimer G., Giday M.: In Vivo Antimalarial Activity of Solvent Fractions of
the Leaf of Vernonia amygdalina (Asteraceae) against Plasmodium berghei in Mice:
Addis Ababa, Ethiopia: Addis Ababa University,January 2016 71-80
63. Lydia D. Ior, et al. In vivo assessment of the antimalarial activity of Cassia Singueana and
Cymbopogon Citrutus :Der Pharma Chemica, 2015, 7(3):272-278.
64. Abigail A. et al .Malarial anemia: of mice and men. Review in translational hematology
by The American Society of Hematology, May 30, 2017,110(1):18-25
65. Bantie L, Assefa S, Teklehaymanot T, Engidawork E. In vivo antimalarial activity of the
crude leaf extract and solvent fractions of Croton macrostachyus Hocsht. (Euphorbiaceae)
against Plasmodium berghei in mice. BMC. 2014 , 14(1):79.
66. Melese E. Biru, Geta M. and Gurmu A. Endale Antiplasmodial activity of
Indigoferaspicata root extract against Plasmodium bergheiinfection in mice: Malar J
(2017), 16:198 ,5-8
52
67. Deharo E., Bourdy G., Quenevo C., Munoz V., Ruiz G., Sauvain M., A search for national
bioactive compounds in Bolivia through a multidisciplinary approach: Part V. Evaluation
of the antimalarial activity of plants used by the Tecana Indians. J Ethnopharmacol, 2001.
77: 91–98.
53
Annex 1: PREMINARY PHYTHOCHEMICAL SCREENING
PROCEDURES
Test for Alkaloids; Crude extract and each solvent fractions were mixed with 2ml of 1% HCl
and heated gently. Mayer‟s and Wagner‘s reagents were then added to the mixture. Turbidity of
the resulting precipitate was taken as evidence for the presence of alkaloids.
Test for Terpenoids; Five ml of extracts and fractions dissolved in distilled water were mixed
in 2 ml of chloroform, and 3 ml concentrated sulfuric acid was carefully added to form a layer
and then observed a reddish brown coloration of the interface to detect the presence of
terpenoid.
Test for Steroids; About 2 ml of extract and individual different fractions were dissolved in 2
ml of chloroform and 2 ml concentrated sulfuric acid was added in extract and individual
different fractions. Then observation of a red color produced in the lower chloroform layer when,
indicates presence of steroids
Test for Flavonoids; Both extracts and fractions were dissolved in a mixture of 4%Tween. ssTo
2 ml of the extracts and fractions solution, few drops of 2 % lead acetate solution was added.
Then, it was observed whether it develops yellow or orange color, which indicates the presence
of flavonoids
Test for Tannin : About 2 ml of the extract and 2 ml solvent fractions were was stirred with 2
ml of distilled water and few drops of ferric chloride solution were added. The formation of
green precipitates indicates for the presence of tannins
Test for saponins
5 ml of extract and solvent fractions were shaken vigorously with 5 ml of
distilled water for 15 minutes in a test tube. The formation of stable foam was taken as an
indication of the presence of saponins.
Test for phenols;
About 0.5gm of extract and solvent fractions were dissolved in 5ml of
distilled water. Then few drops of 5% ferric chloride solution were added and observed for
formation of deep blue or black color.
Test for cardiac glycoside; Few drops of ferric chloride and concentrated sulfuric acid were
added in solution of the extract and fraction individually in glacial acetic acid. The formation of
reddish brown coloration at the junction of two layers and the bluish green color in the upper
layer indicates the presence of glycosides
54
Test of anthraquinones: about 0.5 g of the extract and fractions were taken into a dry test
tube and 5 mL of chloroform was added and shaken for 5 min. then, filtered and the filtrate
shaken with equal volume of 10% ammonia solution. Observing for a pink violet or red color
in the ammoniacal laye (lower layer) indicates the presence of this secondary metabolite.
55
Mekuanent Zemene