FUNGI AS POTENTIAL SOURCE OF ANTIMALARIAL AGENTS

Full text

Katsayal et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 135– 142
135
Nigerian Journal of Pharmaceutical Sciences
Vol. 8, No. 1, March, 2009, ISSN: 0189-823X
All Rights Reserved
FUNGI AS POTENTIAL SOURCE OF ANTIMALARIAL AGENTS
*1Katsayal U. A., 1Abdurahman E. M., 1Abubakar M. S., 1Musa K. Y. 2Ambali S. F. and 3M. B. Jahun
1Department of Pharmacognosy and Drug Development
Faculty of Pharmaceutical Sciences
2Department of Veterinary Pharmacology and Physiology
Faculty of Veterinary
3Veterinary Teaching Hospital
Ahmadu Bello University, Zaria, Nigeria
*Author for Correspondence: (uakatsayal@abu.edu.ng, katsayal2000@yahoo.com,
+234–803–674–5451, +234–805–343–1090)
ABSTRACT
Recent developments in the field of biological control of malaria mosquitoes have shown that certain fungi are
virulent to adult Anopheles mosquitoes. This was indicated by practical delivery of two entomopathogenic fungi,
Beauveria bassiana and Metarhizium anisopliae, which infected and killed adult Anopheles gambiae, the Africa’s
main malaria. The present study reports the antiplasmodial properties of aqueous extract of a fungus, Chlorophyllum
molybdites in vivo in Swiss albino mice, using suppressive, curative and prophylactic procedures. The extract at a
dose of 200 mgkg-1day-1 given orally, for four days was able to inhibit the growth of Plasmodium berghei in mice
for up to 42.6 %, 42.0 % and 44.4 % when concurrently compared with standard chloroquine at a dose of 5 mgkg-
1day-1 which completely cleared the parasites. The result of this study indicates the potentials of macrofungi as a
source of antimalarial compounds.
Key words: Malaria, Chlorophyllum molybdites, aqueous extract acute toxicity, antiplasmodial, phytochemical
constituents.
INTRODUCTION
Throughout the history of drug
development, natural products have
provided a fundamental source of drugs for
fighting infection, inflammation and cancer
in humans. In the case of malaria,
leveraging biodiversity in the natural
environment has been one of the most
effective ways of combating the disease.
Despite intense research, there is no doubt
that plant-derived compounds have outlived
many of the synthetic drugs (Sianne and
Fanie, 2002; Jacques et al., 2008). Malaria
remains the world most devastating human
parasitic infection, afflicting more than 500
million people and causing about 2.5million
death each year. Over 75% of the estimated
2.5 million deaths each year occur in
children living in Sub-Saharan Africa is
attributed to malaria (WHO, 2006). This
region is particularly affected by malaria

Katsayal et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 135– 142
136
due to a unique combination of economic
and ecological factors. In Nigeria over 100
million people are at risk of malaria every
year and it is estimated that about 50
percent of the adult population experience at
least one episode of the disease yearly,
while children of under five years old have
up to 2 to 4 attacks of the disease annually
(FMOH, 2005).
Chlorophyllum molybdites (G. Mey Fr.)
Massee, Family: Agaricaceae. Agaricaceae
is a family of macro-fungi belonging to the
order Agaricales. C. molybdites is a
saprotrophic, poisonous fungus commonly
found in humus-rich soil, such as farm
lands, lawns, garden beds and park lands,
throughout the period of rainy season,
which makes it amenable to artificial
cultivation. This fungus has a large fruit-
body and occurs solitary or in small groups
of fairy rings. It has a large cap, measuring
10 – 30 cm in diameter, which is whitish to
brown in colour, broadly conical often with
margins slightly upturned, and covered with
concentric circles of pinkish-brown to
chocolate-brown scales (Ryvarden et al.,
1994; Queesnland Health, 2006). The
medicinal uses of mushrooms have a very
long tradition, and have found extensive as
antimicrobial, morphological, physiological,
antitumor, biochemical and genetic
applications (Giovanni, 1985). The potential
of this mushroom was therefore investigated
by evaluating its antiplasmodial properties
in vivo in Swiss albino mice in the present
study. This is a because attention is
presently being focused on fungi and
ocean’s microorganisms for the discovery of
natural products as a source of human
therapeutics (Jacques et al., 2008), with aim
of developing a new lead for a yet another
antimalarial agent(s).
MATERIALS AND METHODOLOGY
Fungus Material
Sample of Chlorophyllum molybdites (G.
Mey Fr.) Massee was obtained from a lawn,
in the National Institute for Transport
Technology (NITT) Zaria, Nigeria, in the
month of August 2007. The identity of this
fungus was authenticated using the
descriptions given in the Mushroom Rook
(Thomas and Anna, 1996). It was further
confirmed by Dr. S. K. Jonathan of the
Department of Botany, University of Ibadan,
Nigeria.
Experimental Animals
Swiss albino mice of both sexes, weighing
between 20.5 g - 25.5 g obtained from
Animal House, Faculty of Pharmaceutical
Sciences, Ahmadu Bello University, Zaria,
Nigeria were used as experimental animals
for this study. Their experimental usage was
according to the National Institutes of
Health (NIH) Guide for the Care and Use of
Laboratory Animals (NIH Publication, No.
83 - 23), (revised 1978). These guidelines
are consistent with guidelines of Ahmadu
Bello University for animal handling and
welfare.
Parasites
Sample of chloroquine sensitive
Plasmodium berghei (NK–65 strain)
obtained from National Institute of Medical
Research (NIMR), Yaba, Lagos, Nigeria
was used for this study.
Drugs
Standard chloroquine (Diphosphate salt –50-
63-5 EC No. 200-055-2) and pyrimethamine
obtained from Sigma-Aldrich Company
POB 1450B, Louis MD 63178, United State
of America (USA) were employed as
standard reference drugs in this study.
Extraction of the Fungus Material
The fresh fungus material was immediately
pounded using ceramic pestle and mortar
after collection and then extracted with

Katsayal et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 135– 142
137
aqueous ethanol (70% v/v). The extract after
concentrating at reduced pressure was
suspended in water and partitioned with
chloroform to obtained polar (aqueous) and
non polar (chloroform) portions, referred to
as aqueous extract and chloroform extract,
respectively. However, in the present study,
we reported only the findings of the aqueous
extract.
Determination of the Constituents of the
Extract
The presence of the constituents in the
extract was determined using thin layer
chromatographic technique on fluorescent
pre-coated silica gel plates, developed in
chloroform:methanol (4:1) for 45 minutes,
sprayed with vanillin-H2SO4 reagent and
heated at 105o C for 15 minutes (Krishnan,
et al., 2005).
Determination of Acute Toxicity of the
Extract
The extract was subjected to acute toxicity
testing to determine its median lethal dose
(LD50) orally (p.o.) in Swiss albino mice
using the method described Lorke (1983) at
different geometrical doses (10 - 5000
mgkg-1) in two phases. The animals were
kept and observed under the same condition
for 72 hours within which signs of toxicity
and mortality were observed and recorded.
Determination of Antiplasmodial
Properties of the Extract
The extract together with the standard drug
were subjected to standard anti-malarial
screening in vivo orally in Albino mice
using suppressive, curative and prophylactic
procedures. The experiments were carried
out according to the procedure described by
Bulus et al., 2003; Katsayal and Obamiro
2003; Elufioye and Agbedahunsi, 2004; and
Jude, et al., 2006. Briefly, in the suppressive
test, the extract at doses of 50 mgkg‾1, 100
mgkg‾1 and 200 mgkg‾1, and the standard
chloroquine at a dose of 5 mgkg‾1 orally
were screened against the early infection of
the parasites in the mice to evaluate the in-
vivo schizontocidal activity of the
extract/standard drug. In the curative test,
the extract and the standard drug at the doses
described above were screened against the
established infection of the parasites in the
mice to evaluate the in-vivo schizontocidal
activity of the extracts/standard drug. In the
prophylactic test, the extract at the doses
described above and the standard
pyrimethamine at a dose of 1.2 mgkg‾1
orally were screened against the residual
infection of the parasites in the mice to
evaluate the in-vivo pre-erythrocytic activity
of the extracts/standard drug. The mice in
the negative group were however,
administered 0.2 mL of normal saline orally.
At the end of each study, thin blood films
were prepared from the blood obtained from
the caudal vein of the mice and stained with
Giemsa. The percentage parasitaemia was
determined by counting the number of
parasitized erythrocytes out of 200
erythrocytes in random fields of the
microscope. Average percentage
chemosuppression was calculated as:
100[(A−B)/A], where A is the average
percentage parasitaemia in the negative
control group and B is the average
parasitaemia in the test/standard group.
Statistical Analysis
The results generated from this study were
statistically analyzed using one-way
Analysis of Variance (ANOVA) with
Dunnett's post-test. This was performed by
using GraphPad Prism software, version
4.00 for Windows, from GraphPad Software
Company, San Diego California,
USA, (www.graphpad.com). Values of P < 0.05 were considered significant.

Katsayal et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 135– 142
138
RESULTS
Determination of the Constituents of the
Extract
The result of phytochemical screening of
aqueous extract o C. molybdites revealed the
presence of terpenoids compounds.
Determination of Acute Toxicity of the
Extract
The median lethal dose (LD50), of the
aqueous extract of C. molybdites was
estimated to be greater than 5000 mgkg‾1
orally in Albino mice. No mortality was
recorded, even in the group administered
with the highest dose (5000 mgkg‾1). The
observed signs of toxicity were decreased
limb tone and watery stool.
Determination of Antiplasmodial
Properties of the Extract
The aqueous extract of C. molybdites was
found to demonstrate a dose dependent
chemosuppression effect at the different
doses of the extract employed (50, 100, and
200 mgkg‾1) and administered orally
causing significant (P < 0.05)
chemosuppressions when compared to the
negative control, which produced on effects.
The standard drugs, chloroquine and
pyrimethamine, were however, caused a
significant higher chemosuppression than
that of the extract treated groups (Table 1-
3).
Table 1: Suppressive effects of C. molybdites aqueous extract (Chm) on the early infection
of P. berghei in mice with chloroquine (Chq) as positive control and normal saline (Nms) as
negative control
Treatment
(Extracts/drug)
Dose
(mgkg‾1day‾1, p.o.)
Mean parasitaemial
level
Percentage
inhibition
ChmAE
50
26.8 ± 0.7
18.3*
ChmAE
100
21.8 ± 1.9
33.5*
ChmAE
200
18.8 ± 1.2
42.6*
Chq
5
00.0 ± 0.0
100.0
Nms
0.2
32.8 ± 2.0
00.0
Table 2: Curative effects of C. molybdites aqueous extract (Chm) on the established
infection of P. Berghei in mice with chloroquine (Chq) as positive control and normal
saline (Nms) as negative control
Treatment
(Extracts/drug)
Dose
(mgkg‾1day‾1, p.o.)
Mean parasitaemial
level
Percentage
inhibition
ChmAE
50
32.4 ± 1.1
9.5*
ChmAE
100
27.4 ± 1.0
23.5*
ChmAE
200
20.8 ± 0.5
42.0*
Chq
5
00.0 ± 0.0
100.0
Nms
0.2
35.8 ± 0.6
00.0

Katsayal et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 135– 142
139
Table 3: Prophylactic effects of C. molybdites aqueous extract (Chm) on the residual
infection of P. berghei in mice with pyrimethamine (Pmt) as positive control and
normal saline (Nms) as negative control
Treatment
(Extracts/drug)
Dose
(mgkg‾1day‾1, p.o.)
Mean parasitaemial
level
Percentage
inhibition
ChmAE
50
33.8 ± 0.2
6.1*
ChmAE
100
25.2 ± 0.5
30.0*
ChmAE
200
20.0 ± 0.6
44.4*
Pmt
1.2
00.0 ± 0.0
100.0
Nms
0.2
36.0 ± 0.5
00.0
i
Figure 1: Blood smears from mice infected with P. berghei showing the effects of C.
molybdites aqueous extract (Chm) with chloroquine (Chq) as positive control and normal
saline (Nms) as negative control in the established infection.
Note the red blood cells (arrowed)
i: treated with Chm (200 mgkg‾1day‾1)
ii: treated with Chq (5 mgkg‾1day‾1)
iii: treated with Nms (0.2 mLday‾1)
ii
iii

Katsayal et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 135– 142
140
DISCUSSION
The new interest in the strategies on malaria
treatment and control is to investigate the
folkloric medicine in the search for potent
antimalarial drug. For example, the now
prominent artemisinin was isolated from the
herb Artemisia annua, which has been in use
in traditional Chinese medicine as remedy
for chills and fever for more than 2000 years
(Klayman, 1985). In the present study, the
acute toxicity evaluation of the C.
molybdites aqueous extract revealed that, the
extract is safe. The method of Lorke (1983)
was adopted for the median lethal dose
determination (LD50) of the extract, which
was estimated to be greater than 5000
mgkg‾1 orally in Albino mice, and no
motility was recorded, even in the group
administered with the highest dose (5000
mgkg‾1), the observed signs of toxicity were
decreased limb tone and watery stool. This
indicates that the experimental doses (50,
100, 200 mgkg‾1) used were safe
(Homburger, 1989). The results of the
present study indicate that the extract
possesses significant blood schizontocidal
activity (P < 0.05) as evident from the
chemosuppression obtained during the early
infection test (Table 1). A significant
curative effect was also recorded during
established infection test, although the doses
employed could not produce cure
comparable to that of the standard drug
(chloroquine, 5 mgkg‾1) (Table 2). The
extract also exhibited a considerable
repository activity, which was also observed
to be lower than that of the standard drug
(pyrimethamine mgkg‾1) (Table 3). The
healing effects of the extract of this fungus
on the infected red blood cells (RBCs) were
also found to be lower than the standard
chloroquine (Fig. 1). Higher doses of the
Figure 2: Thin-layer chromatogram of the aqueous extract of C. molybdites (Chm)
developed in CHCl3: CH3OH (4:1) for 45 minutes, sprayed with vanillin-H2SO4 reagent
and heated at 105oC for 15 minutes, Rf value: 0.80

Katsayal et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 135– 142
141
extract could therefore be employed in order
to achieve a better antiplasmodial activity,
since it was observed to be safe even at 5000
mgkg‾1 as evident in the acute toxicity test.
Although the mechanism of action of this
extract has not been elucidated, some plant
extracts were known to exhibit
antiplasmodial activities either by causing
elevation of red blood cell oxidation (Etkin,
1997) or by inhibiting protein synthesis
(Kirby et al., 1989). This extract could have
therefore elicited its action through either of
the two mechanisms mentioned above or by
some other unknown mechanism. Thin layer
chromatography of the aqueous extract of C.
Molybdites revealed a distinct spot with Rf
value of 0.80 when the TLC was carried out
on commercially prepared silica gel pre-
coated flexible plates and developed in
chloroform: methanol (4:1) for 45 minutes,
sprayed with vanillin-H2SO4 reagent and
heated at 105o C for 15 minutes (Fig. 2).
Vanilline-sulphuric acid reagent was found
to be suitable in detecting terpenoids
compounds and all constituents of isoprene
unit origin (Wagner and Bladt, 1996). These
compounds were reported to exhibit
antiplasmodial properties (Milijaona et al.,
2003). Antiplasmodial screenings of plants
have also implicated alkaloids, terpenes and
flavonoids in such activity (Philipson and
Wright, 1990; Christensen and Kharazmi,
2001). Some of these compounds were
found to be present in the extract studied and
may be responsible for the observed
antiplasmodial activity of the extract, though
the active principle is yet to be identified.
CONCLUSION
The result of the present study shows that
the aqueous extract of C. molybdites
exhibited a significant antiplasmodial
activity; this fungus could therefore be
considered as a potential agent for both
malaria treatment and control. Further work
is therefore suggested to use higher doses, to
isolate, identify and characterize the active
constituents present in C. molybdites for
possible development of yet another
antimalarial agent.
ACKNOWLEDGEMENT
The authors are grateful to Mal. Bala
Muhammad of the Veterinary Parasitology
Laboratory, Ahmadu Bello University,
Zaria, Nigeria for his technical assistance.
REFERENCES
Bulus A., Joseph A., Habiba V. and
Karniyus G. (2003). Studies on the Use of
Cassia singueana in Malaria
Ethnopharmacology. Journal of
Ethnopharmacology, 88: 261 – 267.
Christensen, S.B., Kharazmi, A., 2001.
Antimalarial Natural Products Isolation,
Characterization and Biological Properties.
In: Tringali, C. (Ed.), Bioactive Compounds
from Natural Sources: Isolation,
characterization and Biological Properties.
Taylor & Francis, London, Pp. 379–432.
Elufioye, T. O. and Agbedahunsi, J. M.
(2004). Antimalarial Activities of Tithonia
Diversifolia (Asteraceae) and Crossopteryx
febrifuga (Rubiaceae) on Mice In-vivo. J.
Ethnopharmacology, 93: 167 – 171.
Etkin, N.L., 1997. Antimalarial plants used
by Hausa in Northern Nigeria. Tropical
Doctor, 27, 12–16.
FMOH (2005). National Antimalarial
Treatment Guidelines. Federal Ministry of
Health, National Malaria and Vector Control
Division, Abuja-Nigeria, P. 25.
GraphPad Prism software, version 4.00 for
Windows, from GraphPad Software
Company, San Diego California, USA,

Katsayal et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 135– 142
142
(www.graphpad.com).
Homburger, F., 1989. In vivo testing in the
study of toxicity and safety evaluation. In:
Marquis, J.K. (Ed.), A Guide to General
Toxicology. Karger, New York, p. 198.
Jacques, P., Eric, M., Nadia, P., Stephane,
B., William, F., Paul, J. and Karine, L. R.
(2008). Marine Actinomycetes: A New
Source of Compounds against the Human
Malaria Parasites. PloS ONE, 3(6), e2335.
Jude, E. O., Aniekan, E. U. and Garace, A.
E. (2006). Antimalarial Activity of Mammea
africana. Afr. J. Trad. CAM 3 (4): 43 – 49.
Katsayal, U. A. and Obamiro, K. O. (2007).
In-vivo Antiplasmodial Activity and
Phytochemical Screenin of Ethanolic Extract
of the Leaves of Cissampelos mucronata.
Nig. J of Pharm. Sci.,Vol. 6(2): 109 – 103.
Kirby, G.C., O’Neil, M.J., Philipson, J.D.,
Warhurst, D.C., 1989. In vitro Studies on the
Mode of Action of Quassinoids with
Activity against Chloroquine Resistant
Plasmodium falciparum. Biochemical
Pharmacology, 38, 4367–4374.
Klayman, D. L. (1985). Quinghausu
(artemisinin): An Antimalarial Drug from
China. Science, 228: 1049 – 1055.
Krishnan, P., Kruger, N. J. and Ratcliffe, R.
G. (2005). Metabolite Fingerprinting and
profiling in plants using NMR. Journal of
Experimental Botany, Vol. 56: No. 410, 255
– 265.
Lorke, D. (1983). A New Approach to
Practical Acute toxicity Testing. Achieves of
Toxicology, 54, 275 – 287.
Milijaona, R., Valérie, T. R., Harison, R.,
Peter, K. C, Michel, R. Ratsimbason,
Dulcie, A. M. and Philippe, M. (2003).
Plants Traditionally Prescribed to Treat Tazo
(Malaria) in the Eastern Region of
Madagascar. Malaria Journal, 2, 1 – 9.
NIH (1978). Guide for the Care and Use of
Laboratory Animals (Revised), Institute for
NIH Publication No. 83 - 23.
Philipson, J. D. and Wright, C. W., 1990.
Antiprotozoal Compounds from Plants
Sources. Planta Medica, 57: 553–559.
Queesnland Health (2006). Plants and
Mushrooms: Green-spored parasol. The
State of Queensland (Queensland Health).
Ryvarden, L., Piearce, G. D. and Masuka, A.
J. (1994). An Introduction to the Larger
Fungi of South Central Africa. Baobab
Books, P. O. Box 567, Harare, Zimbabwe,
Pp. 63 – 70.
Sianne, S. and Fanie, R. H. (2002).
Antimalarial Activity of Plant Metabolites.
Nat. Prod. Rep., 19, 675 – 692.
Thomas, L. and Anna, D. C. (1996). The
Mushroom Book: Practical Know-how on
Identifying, Gathering and Cooking Wild
Mushrooms and other Fungi. Dorling
Kindersley Limited, London, P. 139.
Wagner, H. and Bladt, S. (1996). Plants
Drug analysis: A Thin Layer
Chromatography Atlas (2nd Edt). Berlin,
Springer, 306 – 364.
WHO (2006). Guidelines for the Treatment
of Malaria. World Health Organization 20,
Avenue Appia, 1211 Geneva 27,
Switzerland.