The Potency of African Locust Bean Tree as Antimalarial
This study was aimed to evaluate the antiplasmodial and antipyretic activities of the stem bark of African locust bean tree. The stem barks of African locust bean tree were extracted with methanol to obtain methanol extract. The antipyretic, acute toxicity, chemical constituents, antioxidant properties as well as the trace metallic content of this extract were determined. The extract was also subjected to column chromatography to obtain four fractions, these fractions were preliminary tested for antiplasmodial potency and methanol fraction which gave the most potent effect was fully evaluated. Flavonoids, tannins, terpenes, saponins, sterols, phenols and reducing sugars as well as Mg, Ni, Ca, Fe, Zn, Na, K and Cu were detected in the extract. It also showed a strong free radical scavenging activity on DPPH (2,2-diphenyl-2-picrylhydrazyl). An oral median lethal dose (LD50) greater than 5 g kg-1 in mice was established for the crude extract and a significant dose dependent antipyretic and inhibition of parasitaemia in suppressive, curative and prophylactic tests. The antiplasmodial and antipyretic activities of the extract were tracked to the methanol fraction when evaluated with rodent malaria model Plasmodium berghei berghei and clinical isolates of Plasmodium falciparum. studies have established sufficient evidence collaborating the antimalarial activities of the stem bark of African locust bean tree, though the active principles are yet to be identified, further studies to elucidate these are ongoing.
April 06, 2012; Accepted: September 05, 2012;
Published: September 14, 2012
Malaria is a potentially deadly tropical disease characterized especially by
cyclical bouts of fever, chills, shaking and sweating often accompanied by muscle
aches and headache. It is a mosquito-borne disease of humans caused by a protozoan
of the genus Plasmodium. Most of the lethal cases are caused by Plasmodium
falciparum, the most virulent of the four Plasmodia species that
infect humans. Infestation with P. falciparum is responsible for hundreds
of millions of cases and more than one million deaths each year (White,
The increased attention on malaria is mainly due to the continuing high mortality
and morbidity caused by this disease and has been related to both increased
resistance of the vector to most of the available insecticides and the causative
parasites to the commonly used antimalarial drugs (Marsh,
1998). To stem the challenge of resistance to antimalarials there has been
an intensified drive to develop new chemical entities with antimalarial potential
and the plant kingdom remains an in exhaustive source of biochemical entities
with useful pharmacological activities. A review of the medicinal plants used
in the northern central and southwestern part of Nigeria for the treatment of
malaria reveals the rich floral diversity of Nigeria (Idowu
et al., 2010).
African locust bean tree popularly known as the Parkia biglobosa
(Jacq.) R.Br. ex G. Don belongs in the family Fabaceae formerly Leguminosae
and the subfamily Mimosoideae have been used traditionally as food and medicine
and are of high commercial value in the south west and northern parts of Nigeria.
Many traditional medicine practitioners in Nigeria have identified the stem
bark and leaf of African locust bean tree as a condiment of great value in the
treatment of malaria (Shao, 2002; Builders
et al., 2011a). The stem bark is boiled and taken orally in form
of decoction for malaria fever; there are also reports on the use of the bark
of African locust bean tree, for the treatment of fever in Ghana (Asase
et al., 2005; Igoli et al., 2005; Kayode
et al., 2009). The establishment of its anti-inflammatory and analgesic
activities only partially collaborate the claimed efficacy by the Traditional
Medicinal Practitioners in Nigeria for the treatment of malaria (Gronhaug
et al., 2008).
The aim of this study is to evaluate the antiplasmodial and antipyretic activities of the stem bark of African locust bean tree so as to authenticate its wide spread use by Traditional Medicinal Practitioners, especially in north central Nigeria for the treatment of malaria.
MATERIALS AND METHODS
Sample collection and identification: The stem barks of African locust bean tree were collected in February, 2009 in Chaza village in Niger state of Nigeria. The identification and authentication were done by (Ethno botanist) Mallam Muazam Wudil of department of Medicinal Plant Research and Traditional Medicine of National Institute for Pharmaceutical Research and Development, (NIPRD), Abuja, Nigeria where a voucher specimen (NIPRD/H/6228) was deposited at the herbarium for reference. The stem barks were cleaned, dried at 50°C in a hot air oven (Salvis ISG 160, Germany) for 72 h and milled into a coarse powder.
Chemicals and reagents: All chemicals were purchased from Sigma-Aldrich, USA.
Extraction of plant materials
Methanol extract: A 200 g quantity of the coarse powder was extracted
with 2 L methanol for 48 h using a Soxhlet apparatus (Quicket, UK). The extract
was filtered through Whatman No. 1 (Whatman International Ltd., Maidstone, UK)
paper and evaporated under reduced pressure using a rotary evaporator to a yield
of 20.17% w/w referred as crude extract. The dried extract was stored at 4°C
Fractionation of the methanol extract: The methanol extract was fractionated
successively with hexane, ethyl acetate and methanol. A total of 34 fractions
(100 mL each) were collected and combined into four main groups (MF1 to MF4)
on the basis of their Thin Layer Chromatography (TLC) profiles. These combined
fractions (MF1 to MF4) were concentrated over water bath and allowed to evaporate
to dryness at room temperature. The fractions were preliminary tested for antiplasmodial
potency and MF4 (methanol fraction) which gave the most potent effect was fully
evaluated (Adzu et al., 2007).
Identification of the chemical constituents: The methanol extract and
the methanol fractions of the African locust bean stem bark were subjected to
qualitative chemical screening for the identification of the various classes
of chemical constituents using the method described by Builders
et al. (2011b). Identification of tannins and flavonoids was further
carried out by TLC using pre-coated silica gel 60G F254 Merck plates
with different eluting systems (Wagner and Bladt, 2004).
Antioxidant potential: Ascorbic acid was used as the antioxidant standard
and the methanol extract with methanol fraction were used for the antioxidant
assessment. The extract, methanol fraction and ascorbic acid at equal concentrations
of, 0.175, 0.25, 0.5, 1 and 2 m mL-1 in methanol were prepared. The
radical scavenging activities of the extract and methanol fraction against 2,
2-Diphenyl-1-picrylhydrazyl radical were determined by UV spectrophotometry
at 517 nm (Ayoola et al., 2008), 1 mL of the
extract or methanol fraction was placed in a test tube and 3 mL of methanol
was added followed by 0.5 mL of 1 mM DPPH in methanol. A blank solution was
prepared containing the same amount of methanol and DPPH. The radical scavenging
activity presented as percentage inhibition was calculated using Eq.
where, Ab is the absorbance of the blank sample and Aa is the absorbance of the drug (methanol extract or methanol fraction or ascorbic acid).
Elemental analysis: The dried extracts 1.0 g was ashes in oven electric
muffle furnace maintained at 400 and 450°C, for about 6-7 h to destroy all
organic materials present in the sample. The crucible containing pure ash was
dried in a desiccator. Thereafter the ash was digested with triple mixture acid:
sulfuric acids: sulphuric: perchloric acid (11:6:3) to obtain a clear solution,
2.5 mL 6 M nitric added to ensure complete dissolution. The solution was then
made to 25 mL with double distilled water and read up with a flame absorption
spectrophotometer (Hitachi Model 80-80 polarize Zeeman Atomic, USA). Sodium
and potassium was determined by flame fluorimeter (Miroslave,
In vivo antiplasmodial study
Animals: Swiss male albino mice (20-25 g body weight) were obtained
from the animal facility centre of the Department of Pharmacology and Toxicology,
National Institute for Pharmaceutical Research and Development (NIPRD), Abuja,
Nigeria and used for the study. The animals were fed ad libitum with
standard feed and had free access to water (Abuja Municipal water Supply). They
were also maintained under standard conditions of humidity, temperature and
12 h light/ darkness cycle. The animals were acclimatized for two weeks before
the commencement of the study. A standard protocol was drawn up in accordance
with the Good Laboratory Practice (GLP) regulations of the ENV (ENV/MC/CHEM,
1998). The principle of laboratory animal care was also followed in this
study (NIH Publication No. 85-23, 1985). The acute toxicity
of the extract was determined by evaluating its median lethal dose (LD50)
using the Lorke method (Lorke, 1983).
Antipyretics studies: The effect of yeast-induced pyrexia on albino
Wistar rats was evaluated by determining the body temperatures of the rats by
measuring Rectal Temperature (RT) at predetermined intervals (Al-Ghamdi,
2001). Fever was induced in the rats by injecting 15%w/v suspension of Brewers
yeast (Saccharomyces cerevisiae) at a dose of 1 mL kg-1 body
weight subcutaneously. The rectal temperature of each rat was again determined
after 24 h of yeast administration. Rats that did not show a minimum increase
of 0.5°C in temperature 24 h after yeast infection were discarded. Thirty
five selected rats were grouped into five and immediately treated as follows:
group 1 received normal saline, group 2 received acetaminophen (150 mg kg-1
b.wt. p.o), while groups 3, 4 and 5 were received extract (25, 50 and 100 mg
kg-1 b.wt. p.o), respectively. The rectal temperatures of all the
rats were then determined by inserting a digital thermometer (Omron Digital
fever Thermometer, Omron Health Care, China) into the rectum of each rat at
30 min intervals for 120 min, similar protocol was employed for methanol fraction
at 12.50, 25 and 50 mg kg-1 doses.
Parasite inoculation: The malaria parasite used was a chloroquine-sensitive
strain of Plasmodium berghei berghei (NK-65), obtained from the National
Institute for Medical Research (NIMR), Lagos, Nigeria and kept at the Department
of Pharmacology and Toxicology, NIPRD, Idu, Abuja, Nigeria. The parasites were
maintained by serial blood passage in mice (Adzu et al.,
2007). Parasitized erythrocytes were obtained from a donor- infected mouse
by cardiac puncture in heparin and made up to 20 mL with normal saline. Animals
were inoculated intraperitoneally with infected blood suspension (0.2 mL) containing
107 parasitized erythrocytes on day zero. Infected mice with parasitaemia
of 5-7% were allocated to five groups of six mice each (Ishih
et al., 2004).
In vivo antimalarial assay: A series of experiments were carried
out to evaluate the in vivo anti-malarial activities of the methanol
extract of African locust bean tree stem bark at 25, 50 and 100 mg kg-1
doses as compared to control groups treated with 0.2 mL of normal saline and
reference groups treated with standard drugs (Chloroquine diphosphate 25 mg/kg/day).
Malaria infection was first established in male mice by the intraperitoneal
(i.p.) administration of donor male Swiss albino mouse blood containing about
1x107 parasites. The three different methods of treating malaria
infections, i.e., 4-Day suppressive test, curative and prophylactic methods
were applied according to Chung et al. (2009),
Chandel and Bagai (2010) and Okokon
et al. (2005), respectively. The laboratory tests were started with
oral administrations of the compound in the 4-day suppressive tests (early malaria
infection) and further screened for their curative (established malaria infection)
and prophylactic (residual malaria infection) activities. Thick blood smears
were prepared and blood films were fixed with methanol. The blood films were
stained with Giemsa and then microscopically examined with 100-x magnification.
The percentage suppression of parasitaemia was calculated for each dose level
by comparing the parasitaemia in infected controls with those of treated mice.
Similar protocol was employed for methanol fraction at 12.50, 25 and 50 mg kg-1
Patients selection: Three fresh blood specimens were collected
from three patients suffering from fever and other malaria symptoms with confirmed
infection by P. falciparum. Already prepared dried -in-acridine orange-stained
thin smears were examined for Plasmodium species identification. The
parasite density was determined by counting the number of infected erythrocytes
among 20,000 erythrocytes from each patient, 4 mL of venous blood was collected
in a tube coated with EDTA. Samples with monoinfection due to Plasmodium
falciparum and a parasite density between 1 and 2% were used for the
in vitro antimalarial tests (WHO, 2001).
In vitro test: The assay was performed in duplicate in a 96-well
microtiter plate, according to WHO method in vitro micro test (Mark III)
that is based on assessing the inhibition of schizont maturation. RPMI 1640
(Sigma Company, USA) was the culture medium used for cultivation of P. falciparum.
Dilutions were prepared from the methanol extract, MF4 and drug concentrations
in the wells ranged from 100-3.125 μg mL-1 for the methanol
extract, from 50 to 1.594 μg mL-1 for methanol fraction, from
0.1 to 0.03 μg mL-1 for chloroquine phosphate. Fifty micro liters
from blood mixture media was added to each well in plate and incubated in a
candle jar (with gas environment of about 3% O2, 6% CO2
and 91% N2) (Karou et al., 2003; Ogunlana
et al., 2009) at 37.0°C for 24-30. After incubation, contents
of the wells were harvested and stained for 5 min in an already prepared dried
-in-acridine orange reagent. The developed schizonts were counted in five fields
of vision (>200 total cells) using a fluorescence microscope (Partec cyscope
fluorescence microscope, Germany) at a magnification of 40.
Statistical analysis: Data were expressed as the mean±standard error of mean (SEM). Statistical analysis of data was carried out using one-way analysis of variance (ANOVA). The IC50 values were determined graphically on a log dose-response curve (log concentration versus percent inhibition curves) by interpolation while those values for the antioxidant activities were calculated from the linear regression of plots of concentration of the test compounds (mg mL-1) against the percentage of inhibition of (2,2-Diphenyl-1-picrylhydrazyl).
Phytochemical tests: The result of the phytochemical screening of the crude methanol extract of African locust bean tree and methanol fraction is presented in Table 1. The analysis revealed the presence of saponins, tannins, terpenes, flavonoids and phenols, reducing sugars and sterols in the crude methanol extract while tannins, terpenes and flavonoids were found in the methanol fraction. However cardiac glycosides, resins, volatile oil and anthraquinones were absent.
Thin layer chromatography: The presence of tannins and flavonoids was further confirmed by thin layer chromatography and their Rf values have been presented (Table 2). Different screening systems were used to obtain better resolution of the components.
|| Phytochemical analysis of the methanol extract and methanol
fraction of African locust bean tree stem bark
|-: Compound not detected, +: Compound detected
|| TLC screening of methanol extract and methanol fraction of
African locust bean tree stem bark
|*:Catechin (tannin) and Rutin (flavonoid)
|| Elemental analysis of the methanol extract of African locust
bean tree stem bark
|Value are Mean±SE
||Antioxidant properties of methanol extract and methanol fraction
of African locust bean tree in relation to ascorbic aid
Antioxidant potential: Figure 1 shows the antioxidant potential of the crude extract of African locust bean tree and methanol fraction as determined by their inhibition of the free radical activities of DPPH. Ascorbic acid was used as the prototype antioxidant agent. The antioxidant activities of the methanol extract and methanol fraction were concentration dependent, increasing with concentration. The sensitivity of the antioxidant activity of the methanol fraction was higher than that of the crude un-fractionated methanol extract.
Elemental analysis: Table 3 shows the metallic elements
detected in the methanol extract of African locust bean tree. Magnesium was
the most abundant macro element (300.8 mg/100 g) closely followed by Calcium
(60.40 mg/100 g) and the least was Nickel (3.01 mg/100 g). Amongst the micro
elements, manganese was most abundant (18.10 mg/100 g) while the contents of
zinc and iron were (9.55 to 10.10 mg/100 g). The Arsenium and lead contents
were very low, Cadmium was not found in the extract.
Acute toxicity tests: There was no mortality in animals at all doses of the extract up to 5000 mg mL-1. The absence of death at doses up to 5 g extract/kg showed that LD50 of the methanol extract of African locust bean tree is greater than 5 g kg-1 p.o. Rubbing of nose and mouth on the floor of the cage and restlessness were the only behavioral signs toxicity shown by the animals, these disappeared within 24 h of extract administration.
Antipyretic studies: The methanol extract caused a reduction dependent decrease in rectal temperature at the highest dose of 100 mg kg-1, the effect became significant at 30 to 90 min. The reduction was comparable with that of acetaminophen. Methanol extract caused inhibition of the pyrexia induced yeast after 30 min at variable doses (25-50 mg kg-1, p.o). Methanol fraction exhibited a significant reduction in the yeast induced elevated temperature after treatment from 60-120 min, the inhibition was comparable with that of acetaminophen (Table 4).
In vivo antiplasmodial study: Figure 2 shows
the antiplasmodial activities of the crude methanol extract and methanol fraction
of the stem bark of African locust bean tree in relation to chloroquine. The
crude extract and methanol fraction exhibited dose dependent reduction in parasitaemia
at the different doses administered. Methanol fraction showed higher inhibition
(93.29±1.2%, 96.28±1.4% and 100±1.0) of parasitaemia.
|| Antipyretic properties of methanol extract and methanol fraction
of African locust bean tree stem bark
|M.: Methanol, PCM: Acetaminophen, *Significantly different
from the control at p<0.05, BBT: Basal body temperature
||In vitro suppressive, curative and prophylactic antiplasmodial
activities of methanol extract (ME) and methanol fraction (MF) of African
locust bean tree
||Mean survival period of mice treated with methanol extract
and (ME) methanol fraction (MF) of African locust mean tree
||Photomicrograph of the in vitro antiplasmodial activities
of the methanol extract and methanol fraction, (a) Complete RPMI medium,
(b) Untreated RPMI medium with O. falciparum, (c) RPMI medium treated
with methanol extract (d) and RPMI medium treated with methanol fraction
Mean survival time: Figure 3 shows the mean survival time for the extract and fraction treated mice were dose dependent and ranged from 23.4±1.6-30.0±1.0 days. Mice treated with 50 and 100 mg kg-1 extract survived the total duration of the study, similar observation was observed with mice treated with 50 mg kg-1 of the fraction.
In vitro antiplasmodial activity: The photomicrographs of the
in vitro antiplasmodial activities of the methanol extract and methanol
fraction are presented in Fig. 4. The crude methanol extract
and methanol fraction showed concentration dependent growth inhibition of P.
falciparum (Fig. 5).
||In vitro antiplasmodial activities of methanol (M.)
extract and methanol fraction of African locust bean tree
||In vitro antiplasmodial activities of methanol extract
(ME) and methanol fraction (MF) of African locust bean tree versus chloroquine
The IC50 of the crude extract and methanol fraction were 12.92 and
0.12 μg mL-1, respectively. The maximum plasmodia inhibitions
were 90±1.2% and 96.7±1.0 for the crude extract and methanol fraction
respectively and 98.5±1.0% for chloroquine phosphate (Fig.
Extraction was carried out to remove impurities or recover a desired product
by dissolving the plant materials in methanol which have certain selectivity
for the extracted materials. (Scholz et al., 2000;
Parekh et al., 2006). With sequential solvents
extraction by fractionation, which involves successive extraction with solvents
of increasing polarity from a non polar (hexane) to a more polar solvent (methanol),
a wide polarity range of compound was extracted (Kuntal
et al., 2010), it therefore possible to isolate the most active fraction
Traditionally used medicinal plants have recently attracted the attention of
the biological scientific communities. This has involved the isolation and identification
of secondary metabolites produced by plants and their use as active principles
in medicinal preparations (Taylor et al., 2001).
Therefore, the traditional use of the stem bark of African locust bean tree
for the treatment of malaria could be attributed to the presence of certain
phytochemicals that constitute the bioactive principles in the plant. Numerous
plants containing a wide variety of phytochemicals as their bioactive principle
have shown antiplasmodial activities (Francois et al.,
1996; Kim et al., 2004; Monbrison
et al., 2006).
Thin layer chromatographic studies showed the confirmation of active principles like tannin and flavonoids on 0.97 and 0.54 Rf values for methanol fraction which were more close to standards 0.97 and 0.54 Rf with prominent blue and greenish blue coloration in both.
Many secondary plant substances had been assessed either for in vitro activity
against P. falciparum or in vivo activity against P. berghei
(Saxena et al., 2003; Fidock
et al., 2004). Tannins, flavonoids and terpenes (Jimoh
and Oladiji, 2005; Alshawsh et al., 2007)
are the classes of compounds possessing antimalarial activities.
The presence of flavonoids and tannins in the African locust bean is likely
to be responsible for the free radical scavenging effects observed. Flavonoids
and tannins are phenolic compounds and plant phenolics are a major group of
compounds that act as primary antioxidants or free radical scavengers (Ayoola
et al., 2008; Al-Adhroey et al., 2010).
The antioxidant effect of the African locust bean may represent another mechanism
that contributes to its antimalarial activity.
Elemental analysis of the extract indicated the presence of pharmacologically
useful trace metal elements with established usefulness various body functions.
These elements are used extensively in chemotherapy and are essential in human
and animal health. The mineral Mg detected in this plants are involved in over
300 enzymatic reactions of the body involving glycolysis, Krebs cycle, nucleic
acid synthesis, amino acid activation, muscle regulations and protein synthesis.
Thus due to high content of Mg, African locust bean tree may have the potential
to cure malaria (Moses et al., 2002; Koche,
WHO recommends that medicinal plants which form the raw materials for the finished
products may be checked for the presence of heavy metals, further it regulates
maximum permissible limits of toxic metals like arsenic, cadmium and lead, which
amount to 1.0, 0.3 and 10 ppm, respectively (WHO, 1998),
these values are within the range found in our sample. Quinine- Zinc complex
had been confirmed to have 3 times antimalarial potency over Quinine sulphate
(Ogunlana et al., 2009) Iron, Manganese and copper
are important trace elements in the human body, they play crucial roles in haemopoiesis,
control of infection and cell mediated immunity (Anhwange
et al., 2004; Oluyemi et al., 2006),
therefore the presence of these minerals contributes to the antimalarial activity
of African locust bean tree.
The acute toxicity of African locust bean tree has been investigated to determine
any adverse effect that may arise as a result of a single contact or multiple
exposures in a short time within 24 h period. Though African locust bean tree
has been used by TMPs without any mortality due to toxicity, this claim has
been authenticated by the lack of death at oral treatment of over 5000 mg kg-1
b.wt. of the extract. The results thus suggest that the methanol extract of
the stem bark of African locust bean tree is has low toxicity (Subhan
et al., 2008; Builders et al., 2012).
The ability of African locust bean tree to reduce the experimentally elevated
body temperature shows that African locust bean tree possesses significant antipyretic
effect on yeast induced pyrexia. The reduction in yeast induced fever by African
locust bean tree might be due to its influence on the prostaglandin biosynthesis
since it is involved in the regulation of body temperature. In general, it is
believed that several mediators and multiple processes play a vital role in
the pathogenesis of fever. Inhibition of any of these mediators is said to bring
about antipyresis and as to how they interfere with PG synthesis is not clearly
established (Aronoff and Neilson, 2010).
Methanol fraction exerted similar suppressive, prophylactic and curative antiplasmodial activities with chloroquine by the extent of inhibition of parasitemia, the African locust bean tree extract also indicated similar antiplasmodial activities however to a lower potency. The extract with its fraction also exhibited dose dependent chemo-suppressive and curative activities and also enhanced the mean survival time period of the treated mice particularly the group administered with the 100 mg/kg/day of the extract.
The IC50 of the methanol extract and methanol fraction of African
locust bean tree were determined as 12.92 and 0.12 μg mL-1.
According to the norm that active extract has IC50< 5 μg
mL-1 and moderate active extract 5 μg mL-1<
IC50<50 μg mL-1 (Rasoanaivo
et al., 1992), the methanol fraction of African locust bean tree
could be considered active while the methanol extract is moderately active when
compared with that of the standard, chloroquine phosphate (0.050 μg mL-1).
The antiplasmodial activity of the plant was found to reside majorly in methanol
fraction, this may be indicative of a significant potential for isolating purer
The phytochemical assessments of the extract and purified fraction of the stem bark of African locust bean tree showed the presence of phytochemicals with established antimalarial activities. The high value of its LD50, antipyretic as well as its effective in vivo and in vitro antiplasmodial activities explains its safety and effectiveness in its use for the treatment of malaria. Our studies have established sufficient evidence collaborating the antimalarial activities of the stem bark of African locust bean tree though the active principles are yet to be identified, further studies to elucidate these are in progress in our laboratories.
This study was carried out in laboratories of Department of Medicinal Plant Research and Traditional Medicine, Department of Pharmacology and Toxicology and Department of Microbiology and Biotechnology (National Institute for Pharmaceutical Research and Development [NIPRD]), Idu Industrial area, Abuja, Nigeria. The authors are grateful to Prof. Osunkwo, U. A., for providing support and encouragement.
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