Article Type: Research Article Article Citation: Ibrahim M. K., Garga M. A., Mohammad A., Abdullahi H. A., Yusuf M. B.,
Ibrahim N., Garba I. L., Bako I., and Salisu B. (2020). IN VITRO ANTIMALARIAL ACTIVITY OF LEAVE
PETROLEUM ETHER EXTRACT OF FICUS SYCOMORUS PLANT. International Journal of
Research -GRANTHAALAYAH, 8(12), 279-283. https://doi.org/10.29121/granthaalayah.v8.i12.2020.2677 Received Date: 02 December 2020
Accepted Date: 31 December 2020
Keywords: Antimalarial Sycomorus Plant Petroleum In vitro anti-malarial activity of petroleum ether extracts of Ficus sycomorus leaves was investigated using Candle Jar method. The extractions were carried out through activity guided fractionation. Petroleum ether extract has the highest activity, at the concentration of 1mg and 0.5mg with percentage growth inhibition of (33.12 and 31.9%) respectively. These results suggest that the leaves of Ficus sycomorus plant have significant antimalarial activity and that the antiplasmodial agents might be as a result of the extract fractionated using the petroleum.
1. INTRODUCTIONMalaria is a
mosquito-borne infections disease caused by a eukaryotic protest of the genus
plasmodium. It is wide spread in tropical and subtropical regions, including parts
of the American, Asia and Africa. Each year there are approximately 350-500
million cases of malaria, killing between one and three million people, the
majority of whom are young children in sub-Saharan Africa (Snow et al.,
2005). Ninety percent of malaria related deaths occur in sub-Saharan Africa.
Malaria is commonly associated with poverty, but it also a cause of poverty
(Malaria: Disease impacts and long-run difference, 2008) and a major hindrance
to economic development. Five species of the
plasmodium parasites can infect humans; the most serious forms of the disease
are caused by Plasmodium falciparum. Malaria cause by Plasmodium
vivax, Plasmodium ovale and Plasmodium malariae causes milder disease in humans that is not
generally fatal. A fifth species, Plasmodium
knowlesi, is zoonoses that causes malaria in
macaque monkeys but can also infect humans (Fong et al., 1971) and
(Singh et al., 2004). Malaria is
naturally transmitted by the bites of a female Anopheles mosquito. When a
mosquito bites an infected person, a small amount of blood is taken, which
contains malaria parasites. These develop within the mosquito and about one
week later, when the mosquito takes the next blood meal, the parasites are
injected with the mosquito’s saliva in to the person being bitten. After a
period of between 2 weeks and several months (occasionally years) spent in the
liver, the malaria parasites start to multiply within red blood cells, causing
symptoms that include fever and headache. In severe cases, the disease worsens,
leading to coma and death (Snow et al., 2005). Malaria
transmission can be reduced by preventing mosquito bites with mosquito nets and
insect repellants or by mosquito control measures such as spraying insecticides
inside houses and draining standing water where mosquitoes lay their eggs.
Although many countries are under developed, the challenge of producing a
widely available vaccine that provides a high level of protection for a
sustained period is still to be met. 2. MATERIALS AND METHOD2.1. MATERIALSTable 2.1: List of
reagents
2.2. BLOOD SAMPLE COLLECTIONThe infected blood
sample used for the research was obtained at city campus, Pathology Department Usmanu Danfodio University
Teaching Hospital, Sokoto from patient suffering from malaria. Then the sample
was passed through malaria parasite (MP) test, which was found to be positive
(+ve). The uninfected
blood sample was obtained from one of my research colleague. The blood sample
were collected in Ethylene Diamine Tetra acetic acid (EDTA) bottles and kept in
refrigerator at 40C for further analysis. 2.3. PREPARATION OF INFECTED AND UNINFECTED RED BLOOD CELLSThe infected blood
sample was spinned for 5 minutes at 2500g to separate
the red blood cells from white blood cells and serum components. The uninfected
blood sample was spinned for 5 minutes at 2500g and
supernatant was decanted into another tube. The serum was inactivated by
heating. 2.4. PLANT MATERIALSThe plant used for
the research was obtained from Kebbi State and the plant was taken to Herbarium
section of Biological Science Department of Usmanu Danfodiyo Univesity Sokoto for
identification. A voucher number UDUH/ANS/0103 was obtained.
The voucher number was deposited at the herbarium unit. The leaves of Ficus sycomorus plant was used for the research. 2.5. PREPARATION OF THE PLANT MATERIALThe plant material
was air dried for one week and then pulverized in to powdered form. The powder
was then sieved to get fine powder. 60g of powdered
leaves of Ficus sycomorus were measured and soaked in 900mls of
distilled water for 24hours at room temperature respectively. The soaked plant
material were filtered and the residues were discarded. The filtrates were then
passed through activity guided fractionation. 2.6. ACTIVITY GUIDED FRACTIONATIONIn this process, an
equal proportion of the aqueous extract (filtrate) and organic solvent were
used. Aqueous extract of plant (100mls)
Aqueous extract of Plant + 100mls of petroleum ether
Petroleum ether extract (evaporate
40-500C) On addition of
organic solvent to aqueous extract of plant, there was thorough shaking and the
separating funnel was left for about 5 minutes for the immiscible layers to be
separated. First, 100mls of aqueous extract was placed inside the separating
funnel followed by the addition of 100mls of organic solvent (petroleum ether)
according to their polarity. These immiscible layers were separated gently in
to the weighted dried plates for drying in the drying cabinet for two days. The
extracts obtained after drying was weighted on the weighting balance and kept
inside the refrigerator for further use. The percentage yield was calculated
using this formula; % yield = Weight
of extract (g) x 100 Weight of plant material used (g) 2.7. METHODThe method used is
the cabled jar method of in vitro test for anti-malarial activity (Trager and
Jensen 1976). Principle The principle of
the “candle jar” method is to maintain the infected erythrocytes in a
relatively simple culture medium, with the addition of human serum in an
atmosphere of relatively high CO2 and low O2 contents.
Fresh red blood cells can be added for the continuation of the growth, division
and multiplication of plasmodia (Trager and Jensen 1976). 2.8. PREPARATION OF THE MALARIA PARASITE CULTURE MEDIUMFirstly, all the
apparatus were disinfected in an autoclave. The medium was prepared by
dissolving 9.8g of RPMI 1640 powder in 900mls of redistilled water and stirred
until it dissolved. Seven points eight one gram (7.81g) of sodium dihydrogen
orthophosphate was added as buffer and 2.0g of sodium bicarbonate was added.
The solution was stirred together, filtered through sterilized Whattman number 1 filter paper and stored at 40C. The blood medium
mixture (BMM) was prepared by mixing 0.9ml of RPMI with 100µ1 of malaria
parasite infected blood sample. 0.01mg/mol of gentamicin was added in to the
RPMI 1640 to inhibit the bacteria contamination of the parasite culture. One and zero point
five milligram (1mg and 0.5mg) concentration of petroleum ether extract was put
into different culture plates. As the negative control plate, the parasites
were cultured without any extract or drug (100% growth) and the positive
control plate contained 20µ1 of artesunate (100% inhibit). The mixture was
mixed in order to ensure maximum mixing; the mixing was carried out near flame
to minimize bacterial contamination. The culture plates
were covered with a lid and incubated at 370C for 48hours. 2.9. PREPARATION OF FILMThe blood films
were prepared on the microscope slides. The films were air dried and the films
were fixed with methanol which was poured off after 30seconds. The freshly
prepared Giemsa stain was used to stain slide films for 20-30 minutes. The
slide films were flushed with tap water and stand to dry. 2.10. MICROSCOPIC EXAMINATION OF BLOOD FILMS AND PARASITES COUNTThe dried slide
films were examined under 100x magnification (oil immersion) for the presence
of schizonts. An area of the film where the total number of erythrocytes is
approximately 120 per field was viewed. The number of parasitized erythrocytes
in 10 fields were counted and recorded. 2.11. DETERMINATION OF PERCENTAGE GROWTH INHIBITIONThe percentage
growth inhibition was calculated using the formula; Percentage growth
inhibition = Control parasitemia – Extract parasitemia x 100 Control parasitemia Parasitemia is the
number of schizonts obtained after counting. 3. RESULTSTable 3.1: Shows the
yield and percentage yield of plant material after extraction.
The results of
extraction of leaves of Ficus Sycomorus obtained through activity guided
fractionation are shown above (Table 3.1) with yield and percentage yield of
plant material of petroleum ether. Table 3.2: Number of
infected cells for each 10 fields counted on the light microscope of different
extracts concentration and percentage growth inhibition.
The result of in
vitro anti-malarial activity of leaves of Ficus sycomorus is shown in (Table 3.2). From The results
above, the lower the number of infected cells the higher the inhibition and the
higher the number of infected cells, the lower the inhibition. The results
obtained shows that, petroleum ether, which has 33.12% at concentration of
1mg/ml and 31.9% at concentration of 0.5mg/ml, however, the extract shows
anti-malarial activity. 4. DISCUSSIONSIn sub-saharan African were malaria is endemic and in other
parts of the world, plants are extensively used for treating periodic malaria
disease. The spread of multi drug-resistance P. falciparum has highlighted the urgent need to develop new
antimalarial drugs preferably inexpensive drugs that are affordable for
developing countries, where malaria is prevalent. In this study four
organic extract were obtained from a plant (Ficus sycomorus leaves) that
are used for the treatment of fever and malaria were tested In vitro against P. falciparum using
Candle Jar Method. Table 3.1 shows the percentage yield of petroleum ether extract as
against the gram of plant sample used which is 60g. Table 3.2 shows the
percentage growth inhibition at different concentration of the extract.
Petroleum ether extract exhibit highest growth inhibition of 33.12 and 31.9 at
concentration of 1mg/ml and 0.5mg/ml respectively. This indicates that, the anti-plasmodic agent
in petroleum ether fraction may be more than the other fractions of hexane,
chloroform and water extract. 5. CONCLUSIONThe current work
confirms the traditional used of Ficus sycomorus as an effective
antimalarial preparation. The research also indicated that, the active antiplasmodic agent of F. sycomorus may be
fractionated in the petroleum ether fraction. Also, petroleum ether had shown
to have the highest percentage inhibition when compared to the other solvents. SOURCES OF FUNDINGThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. CONFLICT OF INTERESTThe author have declared that no competing interests exist. ACKNOWLEDGMENTNone. REFERENCES [1] Singh B., Kim Sung L., Matusoc A. (2004). “A large focus of naturally acquired
plasmodium infection in human beings Lancet, 363 (9414). [2] Snow R. W., Guerra C. A., Noor A.
M., Myint H. Y., S. I. (1995). The global
distribution of clinical episodes of plasmodium falciparum malaria”. Nature
434(7030):214-7. [3] Trager W., Jensen J. B. (1976).
“Human malarial parasites in continuous culture” Science 193(4254):673-675. [4] Fong Y.L., Cadigon
F.C., Coatney G.R. (1971) “Presumptive case of
naturally occurring Plasmodium knowlesi malaria in
Malaysia.” Trans R. Soc trop Med Hyg 65. [5] Malaria: Disease impacts and
long-run income difference” Study of Lasor Retrieved 2008-12-10.
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