Tropical Journal of Natural Product Research Original ResearchArticle Antimalarial Activities of Leaf Extract and Fractions of Setaria megaphylla(Willd.) Loes. in Plasmodium berghei Infected Mice

Abstract

The paper highlighted the in vivo antimalarial activities of leaf extract and fractions of Setaria megaphylla

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Tropical Journal of Natural Product Research
Available online at https://www.tjnpr.org
Original ResearchArticle
Antimalarial Activities of Leaf Extract and Fractions of Setaria megaphylla(Willd.)
Loes. in Plasmodium berghei Infected Mice
Ndanti B. William1,Augustine L. Bassey2, Akaninyene O. Daniel3,Otuekong Ekong3, Jude E. Okokon1*
1Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria
2Department of Clinical Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, University of Uyo, Uyo, Nigeria
3Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria
Introduction
About 229 million malaria cases have been estimated to have
occurred in 2019 in 87 malaria endemic countries globally.1Despite a
significant decline in mortality due to malaria, the highest mortality rate
of all malaria deaths globally in 2019 was recorded in Nigeria. This
shows that malaria still threatens most countries of Africa particularly
Nigeria despite the successes achieved in the global fight against
malaria over the last two decades. Medicinal plants, therefore serve as
an excellent reservoir for antimalarial remedies with the advantage of
being safer and providing many therapeutic effects. Setaria megaphylla
(Steud) Dur&Schinz (Poaceae), a perennial grass found in tropical and
subtropical areas of the World,2 is traditionally used for the treatment
of diverse ailments including malaria and diabetes among others.3
Preliminary reports of antiplasmodial activity on the leaf have been
published3,4 The leaf extract also possesses antidiabetic and
hypoglycaemic,5 anti-inflammatory, analgesic,6 cytotoxic,
immunomodulatory and antileishmanial,7 antidepressant,8 inhibitory
effect on α-amylase and α-glucosidase9 activities. Phytochemical
analysis of the leaves extract revealed that the leaves extract
contains specialized secondary metabolites such as flavonoids,
carbohydrate, terpenes, saponins, tannins, anthraquinones, cardiac
glycosides (Z,Z,Z)-8,11,14-eicosatrienoic acid, phthalic acid, diisooctyl
ester, vitamin E, ᵞ-elemene, urs-12-ene, bicyclogermacrene, α-
muurolene, germacrene-A, and guaiol3,7.
*Corresponding author. E mail: judeefiom@yahoo.com;
Tel: 08023453678; 08064465654
Citation: William NB, Bassey AL, Daniel AO, Ekong O, Okokon JE.
Antimalarial Activities of Leaf Extract and Fractions of Setaria megaphylla
(Willd.) Loes. in Plasmodium berghei Infected Mice. Trop J Nat Prod Res.
2022; 6(9):1504-1510. http://www.doi.org/10.26538/tjnpr/v6i9.28
Official Journal of Natural Product Research Group, Faculty of Pharmacy,
University of Benin, Benin City, Nigeria.
1-triacontanal, 1-triacontanol, 1-dotriacontanol, 1-triacontyl cerotate,
and stigmasterol have also been isolated from the plant leaves.5 We
report the antimalarial potentials of the leaves extract and fractions of
Setaria megaphylla in Plasmodium berghei-infected mice.
Materials and Methods
Collection and identification of plant material
The leaves of Setaria megaphylla were collected from bushes in the
Uruan area of Akwa Ibom State, Nigeria in July, 2020. The plant was
identified by Prof. Margaret Bassey, a taxonomist in the Department of
Botany and Ecological Studies, University of Uyo, Uyo, Nigeria. A
voucher specimen (UUPH. 221 d) of the plant was deposited in the
Department of Pharmacognosy and Natural Medicine herbarium at the
University of Uyo, Nigeria.
Extraction
The leaves were washed and shade dried for two weeks. The dried
leaves were cut into smaller pieces and pulverized to powder using
electric grinder. The leaves powder was divided into two parts. One
part (1.5 kg) was macerated in ethanol (7.5 L) for 72 hours, while the
remaining part (1.5 kg) was successively and gradiently macerated for
72 hours in 7.5 L each of, n-hexane, dichloromethane, ethyl acetate and
methanol respectively, which is along their polarities to give the
corresponding gradient extract for each solvent. The liquid filtrates of
the extract and fractions were concentrated and evaporated to dryness
in vacuo 400C using a rotary evaporator. The different yields were
calculated and the extract and fractions refrigerated at -40C, until used
for the proposed experiments.
Microorganism (parasite)
The chloroquine-sensitive strain of Plasmodium bergheiANKA strain
was obtained from the National Institute of Medical Research
(NIMER), Yaba Lagos, Nigeria and maintained by subpassage of
blood from infected to healthy mouse once every 7-8 days.
ARTI CL E I NF O
ABSTRACT
Article history:
Received 07 July 2022
Revised 15 September 2022
Accepted 19 September 2022
Published online 01 October 2022
Setaria megaphylla (Steud) Dur&Schinz (Poaceae), a perennial grass used traditionally in the
treatment of various diseases such as malaria was screened for antiplasmodial activity.The
leaves extract (200–600 mg/kg) and fractions (hexane, dichloromethane, ethyl acetate and
methanol; 400 mg/kg) were investigated for suppressive, prophylactic, and curative antimalarial
activities against chloroquine-sensitive Plasmodium berghei infections in Swiss albino
mice.Chloroquine (5 mg/kg) and pyrimethamine (1.2 mg/kg) were used as positive
controls.Thin films made from tail blood of each of the mice were used to assess the level of
parasitaemia of the mice. The extract/fractions progressively reduced parasitaemia induced by
chloroquine-sensitive P. berghei infection in suppressive (11.12–46.94%), prophylactic (19.20
–47.96%) and curative (20.65–75.06%) models in mice. These reductions were statistically
significant
(
p<
0.01–0.001).
They also improved significantly
(
p<
0.01–0.001)
the mean survival
time
(MST)
from 13.33 to 18.60 d in suppressive, 10.33 to 26.00 in prophylactic and 12.50 to
22.66 days in curative models relative to respective controls. The activities of extract/fractions
were not comparable to that of the standard drugs used (chloroquine and pyrimethamine) in all
the models.The leaf of S. megaphylla may possess antimalarial effect which may in part be
mediated through the chemical constituents of the plant.
Keywords:Setaria megaphylla, Antimalarial, Malaria, Plasmodium berghei.
Copyright: © 2022 Williamet al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.

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Parasite inoculation
The inoculum consisted of 5 x 107 P. berghei infected erythrocytes per
milliliter prepared by diluting serially with normal saline, parasitized
blood erythrocytes collected from an infected mouse with 20-30%
parasitaemia to make a suspension of P. berghei parasitized
erythrocytes.10 Each mouse used in the experiment was inoculated
intraperitoneally with 0.2 mL of infected blood containing about 1 x
107P. berghei parasitized erythrocytes. Parasitemia was monitored by
standard methods; thin blood smears were made on glass slides, fixed
using methanol, and stained with Giemsa stain. Parasitemia was
counted using a microscope and was calculated as a percentage of
infected red blood cells (RBCs) relative to the total number of cells in
a microscopic field at ×100 magnification according to the formula
given below11:
Experimental animals
Male and female Swiss albino mice, each weighing 21-32 g, were
obtained from the University of Uyo’s animal house. They were kept in
standard cages and acclimatized for 10 days before use in the
experiments. The mice were fed on a standard pelleted diet and water
ad libitum. All animals were kept at room temperature in cross
ventilated rooms. The care and use of animals were conducted in
accordance with the National Institute of Health Guide for the Care and
Use of Laboratory Animals (NIH Publication, 1996). Approval for the
study was obtained from the University of Uyo’s Animal Ethics
Committee (UU/FP/AE/21/035).
Drug administration
The extract, fractions, chloroquine and pyrimethamine that were used in
the antimalarial study, were administered orally with the aid of a
stainless metallic feeding cannula.
Evaluation of the in vivo antimalarial activities of leaf extract and
fractions of Setaria megaphylla:
Evaluation of suppressive activities of the leaf extract and fractions of
Setaria megaphylla (4-day test)
This test was used to evaluate the schizontocidal activity of the crude
extract and fractions as well as chloroquine against early P. berghei
infection in mice. This was done as described by Ali et al.11Forty-five
mice were randomly divided into nine groups of five (5) mice each. On
the first day (D0), the mice were infected with the parasite and
randomly divided into various groups. These were administered the
crude extract, fractions, chloroquine and distilled water. Based on
previously determined LD50 by Okokon et al..3 the mice in groups 1-3
were given 200 mg/kg, 400 mg/kg and 600 mg/kg of crude extract
respectively, while groups 4, 5, 6, 7 were administered 400 mg/kg of n-
hexane, dichloromethane, ethyl acetate, and methanol fractions
respectively, group 8 was given 5 mg/kg of chloroquine (positive
control) and group 9 was given 10 mL/kg of distilled water (negative
control) for four consecutive days (D0-D3) between 8am to 9am. On
the fifth day (D4), thin film smears were made from the tail blood of
each mouse. The films were stained with Giemsa stain to reveal
parasitized erythrocytes out of 500 in a random field of the microscope.
The average suppression of parasitemia was calculated as follows:
The mean survival time of the mice in each treatment group was
determined over 29 days (D0-D28), as follows:
Evaluation of prophylactic activities of the leaf extract and fractions of
Setaria megaphylla
This was evaluated using the method described by Okokon et al.,12. The
mice were randomly divided into nine groups of 5 mice per group.
Groups 1-3 were given 200, 400, and 600 mg/kg of crude extract
respectively, groups 4, 5, 6, and 7 were given 400 mg/kg of n-hexane,
ethyl acetate, dichloromethane, and methanol fractions respectively,
group 8 was given 1.2 mg/kg of pyrimethamine (positive control) and
group 9 was given 10 mL/kg of distilled water (negative control).
Administration of the extract and fractions continued for three
consecutive days (D0-D2). On the fourth day (D3), the mice were
inoculated with 0.2 mL of infected blood containing about 1 x 107 P.
berghei parasitized erythrocytes. The parasitemia level was assessed by
blood smears 72 hours later. The mean survival time of the animals was
calculated over 29 days.
Evaluation of the curative activities of the leaf extract and fractions of
Setaria megaphylla
This test was used to evaluate the schizontocidal activity of the extract,
fractions and chloroquine in established plasmodial infection. This was
conducted according to the methods described by Evinemi et al.,13.
Ninety (90) mice were injected intraperitoneally with 0.2 mL of
infected blood containing about 1 x 107P. berghei parasitized
erythrocytes on the first day (D0). Seventy-two hours later (D3), the
mice were divided into nine groups of ten mice per group. Groups 1-3
were given different doses of extract, 200, 400, and 600 mg/kg
respectively, groups 4-7 were given 400 mg/kg of n-hexane, ethyl
acetate, dichloromethane, and methanol fractions respectively. Group 8
was given 5 mg/kg chloroquine (positive control) and group 9 was
given 10 mL/kg distilled water (negative control). The crude extract,
fractions and chloroquine were administered once daily for 5 days.
Giemsa stained thin smears were prepared from tail blood samples
collected on each day of treatment to monitor the parasitemia level. The
rectal temperature of the mice was taken on days 0, 3, 5, and 7 to
monitor changes in the body temperature of the mice. On the sixth day,
five mice from each group were sacrificed under light diethyl ether
vapour. Blood samples were collected by cardiac puncture into EDTA
bottles and used for haematological analysis. The mean survival time
(MST) of the mice in each group was determined over 29 days (D0-
D28).
Hematological study
Blood samples collected from each mouse into ethylene diamine tetra-
acetic acid (EDTA) – coated sample bottles were used to determine the
effect of the extract/fractions on hematological parameters such as Red
blood cell count (RBC), hemoglobin, (Hb), packed cell volume (PCV),
platelet concentration (PLC) and total and differential white blood cell
count (WBC). These parameters were analyzed using automatic
hematological system (Sysmex Hematology – Coagulation system,
Model MO-1000 I, Trans Asia, Japan).
Gas chromatography-Mass spectrometry analysis
GC-MS was carried out on ethyl acetate fraction on an Agilent 7890A
gas chromatograph, coupled with an Agilent MS model 5975C MSD
with triple axis detector (Agilent Technologies, USA). The system was
equipped with a HP5-MS column 5 % phenyl-methyl polysiloxane, 30
m × 0.25 mm × 0.25 µm (Agilent Technologies, USA). The carrier gas
was helium with a gas flow under a constant pressure of 10 psi. The
injector temperature was set at 280 °C. The initial oven temperature
was 160 °C and increased to 320 °C at 10 °C/min, and the final
temperature was held for 6min at 320 °C. The mass spectrometer was
operated in the electron ionization mode at 70 eV. The 0.2-0.4 mg of
each compounds were dissolved in CHCl3 (5 mg/mL) to attain a final
concentration of 5 mg/ml for 20min at room temperature before
injection of 1 uL to GC-MS system.5 The compounds were identified
by comparison of spectral data and fragmentation pattern with
reference compounds in the NIST 2011 database.
Statistical analysis
Data obtained from this work were analysed statistically using ANOVA
(one –way) followed by a post test (Tukey-Kramer multiple

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comparison test). Differences between means were considered
significant at a 5% level of significance ie p≤ 0.05
Results and Discussion
The percentage yields of the extract and fractions were; crude-7.38%,
n-hexane- 0.15%, DCM -0.31%, ethyl acetate -0.24%,methanol -1.25% The results of the suppressive activity of the leaf extract are shown in
Table 1. The extract showed a dose-dependent and significant
(p<0.001) reduction in parasitaemia levels of the treated mice at the
different doses employed when compared to the control group in the
study. The chemosuppression percentages were; 24.29, 30.76 and
46.94% for 200, 400 and 600 mg/kg of extract respectively. The
standard drug, chloroquine showed relatively higher chemosuppression
(92.78%).
The leaves fractions (n-hexane, DCM, ethyl acetate and methanol; 400
mg/kg); exerted prominent reductions in parasitaemia levels of the
treated mice. The chemosuppressions were 11.12, 23.96, 20.81 and
41.00% for n-hexane, dichloromethane, ethyl acetate and methanol
respectively. There was a significant (p<0.05-0.001) decrease in
parasitaemia levels of groups treated with DCM, ethyl acetate and
methanol fractions when compared to the control with the methanol
fraction showing the highest activity. This was however lower than that
of chloroquine, the standard drug as shown in Table 1. The extract and
fractions exerted prominent protection to the animals as demonstrated
in the significantly (p<0.05-0.001) prolonged MST of the treated mice
with the highest dose (600 mg/kg) and methanol fraction having higher
MST values of 18.6±0.66d and 17.6±0.88d respectively (Table 1).
The leaf extract showed a dose-dependent chemotherapeutic effect in
the different doses used in this test with chemosuppressions of 19.20,
24.37 and 26.06% respectively for 200, 400 and 600 mg/kg doses of the
extract. The reduction in parasitaemia was statistically significant
(p<0.01) in all doses when compared to the control, as shown in Table
2, but less than that of the standard drug, pyrimethamine.The leaf
fractions (400 mg/kg) showed significant (p<0.001) reductions in
parasitaemia levels with 11.56±0.89, 10.66±0.85, 15.02±0.65, and
10.46±1.12% for n-hexane, dichloromethane, ethyl acetate and
methanol respectively, and111 2.15±0.95 % for pyrimethamine.
There were significant (p<0.001) decreases in the parasitaemia level of
all the fractions-treated groups compared to the control with the
methanol fraction showing the highest activity though less than that of
the standard drug, pyrimethamine, as shown in Table 2. The extract and
fractions further demonstrated prominent protection on the treated
infected mice with dichloromethane and methanol fractions treated
group having elongated MST of 26.00±1.04 and26.00±2.00
respectively (Table 2). Progressive reductions in parasitaemia levels of
the extract/fractions-treated groups of infected mice were observed in
the study from day 0 to day 7 (Figure 1). The extract showed dose-
dependent and statistically significant (p<0.001) chemotherapeutic
effects when compared to control at 400 and 600 mg/kg doses of the
extract on day 7. These reductions were, however, less than that of the
standard drug (chloroquine) as shown in Figure 1.
Significant decreases (p<0.05-0.001) in parasitaemia levels of the
infected mice were also observed on day 7 following treatment with
the fractions (400 mg/kg) with ethyl acetate fraction showing the
highest activity, compared to the control, though lower than that of the
standard drug (chloroquine), as shown in Figure 1. There was a dose-
dependent increases in the mean survival time of the leaf extract-treated
groups. The extract increased the MST from 12 to 16 days when
compared with the control. However, it was shorter when compared to
that of the standard drug, chloroquine (28.50 days) as shown in Table
3.The leaf fractions (400 mg/kg) increased the MST from 12-22 days
with the ethyl acetate fraction showing the highest activity compared to
the control but lower than that of chloroquine the standard drug (28.50
days) as shown in Table 3.Administration of the leaf extract and
fractions as well as chloroquine did not cause any significant difference
(p>0.05) in the rectal temperatures of the treated mice when compared
with that of control on days 5 and 7 (Table 4). Administration of the
leaf extract and fractions of S. megaphylla to P. berghei-infected mice
caused significant (p<0.05-0.001) increases in RBC, lymphocytes and
platelets counts, Hb concentration and PCV percentages and MCH
levels when compared to untreated infected mice though non-dose
dependently. While the elevated WBC counts, neutrophils percentages,
MCV and MCHC percentages in the untreated infected animals were
reduced significantly (p<0.05-0.001) in the treated infected animals
when compared statistically (Table 5).The results of GCMS analysis of
the ethyl acetate fraction revealed that it contains some bioactive
compounds such as (E)-β-ocimene, P-cymene, D:A-friedooleannan-3-
ol,(3a)-, Bicyclo[2.2.1] heptan-2-ol,4,7,7 trimethyl, Stigmastone-3,6-
dione,(5a)- and Phenol 2,6-dimethoxy among others Table 6).The
leaves of S. megaphylla are used in Ibibio traditional medicine as
malaria remedy and this work was designed to confirm and authenticate
its antimalarial potential to provide the scientific basis for its usage as
an antimalarial plant.The leaf extract and fractions of S. megaphylla
were investigated for antimalarial activity against rodent malaria
parasite, P. berghei infection in mice using standard in vivo models. It
was found that the extract and fractions significantly reduced the
parasitaemia in prophylactic, suppressive and curative models in a
dose-dependent fashion with methanol fraction exhibiting the highest
suppressive and prophylactic activities, while ethyl acetate fraction
followed by DCM fraction exerted the highest curative effect
confirming the antimalarial potential of this extract. These varying
potencies of the fractions could have resulted from the activities of the
various phytochemical constituents of each fraction. However, based on
the classification of Tchatat et al,14 their activities cannot be considered
to be very good. The extract and fractions also prolonged the MST of
the mice suggesting that they were able to offer a certain degree of
protection to the mice. This activity could have resulted from
plasmodicidal or plasmodistatic activity of the extract and fractions as
reported earlier by Okokon et al.,4 The results of this study confirm and
corroborate earlier report by Okokon et al,3 in which significant
antimalarial activity was preliminarily reported on the leaves extract,
and also provide information on the most active fraction(s) where the
active principles may likely be localised.
Table 1: Suppressive activities of leaf extract and fractions of S. megaphylla during early Plasmodium berghei infection in mice
Treatment
Dose (mg/kg)
Parasitaemia
Chemosuppression (%)
MST
Control
-
29.12 ±0.56
-
13.3 ±0.98
Extract
200
22.04±1.20c
24.29
16.0±0.57a
400
20.16±0.98c
30.76
16.66±0.33c
600
15.45±1.15c
46.94
18.6±0.66c
n-hexane
400
25.88 ±1.54
11.12
14.02±0.33b
Dichloromethane
400
22.14±0.33a
23.96
15.0±0.57b
Ethyl acetate
400
23.06±0.96c
20.81
14.6±0.60c
Methanol
400
17.18 ± 0.78c
41.00
17.6±0.88b
Chloroquine
5
2.10 ± 1.38c
92.78
30.00 ±0.00c
Values are expressed as mean ± SEM. Significant relative to control. ap<0.05; bp<0.01; cp<0.001. n = 6

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Table 2: Prophylactic activities of leaf extract and fractions of S. megaphylla
Treatment
Dose (mg/kg)
Parasitaemia
Chemosuppression (%)
MST
Control
-
20.10 ± 0.86
-
10.33 ± 0.33
Extract
200
17.24 ± 1.26b
19.20
11.33 ± 0.33
400
15.20 ± 1.42b
24.37
12.0 ± 0.57
600
14.86 ± 1.50b
26.06
13.33 ± 0.66
n-hexane
400
11.56 ± 0.89c
42.48
19.30 ± 1.09
Dichloromethane
400
10.66 ± 0.85c
46.96
26.00 ± 1.04a
Ethyl acetate
400
15.02 ± 0.65c
25.27
12.66 ± 0.33
Methanol
400
10.46 ± 1.12c
47.96
26.00 ± 2.00a
Pyrimethamine
1.2
2.15 ± 0.95c
89.30
25.04 ± 0.29c
Values are expressed as mean ± SEM. Significant relative to control. ap<0.05; bp<0.01; cp<0.001. n = 6.
Table 3: Mean survival time of mice treated with leaves extract
and fractions of S. megaphylla during established Plasmodium
berghei infection in mice
Treatment
Dose (mg/kg)
Mean Survival Time (Days)
Control
-
12.50 ±0.28
Extract
200
12.75±0.75b
400
14.50±0.28c
600
16.75±1.43c
n-hexane
400
16.75±2.17c
Dichloromethane
400
14.05±0.75c
Ethyl acetate
400
22.66±4.34c
Methanol
400
17.75±1.03c
Chloroquine
5
28.50±1.19c
Values are expressed as mean ± SEM. Significant relative to control.
ap<0.05; bp<0.01; cp<0.001. n = 6.
Figure 1: Effect of leaf extract and fractions of Setaria
megaphylla on established Plasmodium berghei infection in
mice
Thus, validating the use of the leaves extract decoction as malarial
remedy. However, it was observed that lower chemosuppression
percentages were recorded especially in the suppressive model in this
study compared to previous reports; this could have been due to
different seasons of collection of the plant materials which might have
affected the composition of the phytochemical constituents. However,
in vitro studies by Okokon et al.4 carried out to evaluate the activities of
the leaf extract and fractions against human malaria parasite,
chloroquine-resistant strain of P. falciparum (Pf3D7) and chloroquine-
resistant strain (Pf INDO), had reported the highest activity against
both strains of P. falciparum from ethyl acetate fraction with IC50 of
8.15±1.10 µg/mL (Pf3D7) and 8.94±1.26 µg/mL (PfINDO).
Chloroform fraction was found to be more active against Pf INDO with
IC50 of 8.05±0.12 µg/mL (PfINDO), probably suggesting the
localization of the active compounds in these fractions. However, ethyl
acetate fraction was outstandingly more active during the in vitro test
corroborating the in vivo studies in which ethyl acetate was most active
in curative test, while methanol fraction was the most active in
suppressive and prophylactic tests. The findings of the in vivo study
corroborate the previous report of in vitro activity.4 The slight variation
in the activities of the fractions suggests the involvement of
immunostimulating activity which may be due to the phytochemical
constituents in these fractions especially those that were active during
in vivo study. The reported phytochemical screening and GC-MS
analysis of n-hexane, DCM, and ethyl acetate fractions revealed the
presence of some pharmacologically active compounds such as
tannins, flavonoids, alkaloids, terpenes, triterpenes like squalene,
phenolics, β-sitosterol and polyunsaturated fatty acids (PUFAs), (E)-
β-ocimene, P-cymene, D:A-friedooleannan-3-ol,(3a)-, Bicyclo[2.2.1]
heptan-2-ol,4,7,7trimethyl, Stigmastone-3,6-dione, (5a)- and
Phenol,2,6-dimethoxy among others.4,7 These compounds are likely to
be responsible for the observed activities of the extract and fractions.
Some secondary metabolites of plants such as alkaloids, flavonoids and
triterpenoids have been reported previously to have antiplasmodial
properties.15 Polyunsaturated fatty acids such as hexadecanoic acid,
methyl ester, 9,12-octadecadienoic acid methyl ester (linoleic acid),
9,12,15-octadecatrienoic acid, methyl ester (linoleic acid), and 9-
octadecenoic acid have been found in the active antiplasmodial
fraction especially n-hexane fraction and other fractions. These PUFAs
mentioned above have been implicated in antiplasmodial activity and
this activity has been reported to increase with the degree of
unsaturation.16 Also, an earlier study by Okokon et al.7 had shown the
presence of ᵞ-Elemene, Urs-12-ene, Bicyclogermacrene, α-muurolene,
Germacrene- A, and Guaiol in the leaf fractions, which are mono and
sesquiterpene compounds implicated in antiplasmodial activity of
many plants.17 These compounds mentioned above to be present in the
extract and the active fraction maybe responsible for the observed
antiplasmodial activities.
The findings of this study further suggest that leaf extract and fractions
of S. megaphylla possess antimalarial activity which is due to the
activities of its phytochemical constituents. This confirms and
authenticates its use as malarial remedy in folkloric medicine. Fever is
one of the cardinal symptoms of malaria especially in humans.
However, P. berghei infection in mice is reported to be associated with
hypothermia rather than pyrexia18.
Results of rectal temperatures of the infected mice in this study
(curative test), showed that there was no significant difference between
the mean temperature values of both the treated and untreated infected
mice before and after treatment, suggesting that the mice were
hypothermic.

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Table 4: Effect of leaf extract and fractions of Setaria megaphylla on rectal temperatures of mice infected with Plasmodium berghei during established infection
Treatment
Dose (mg/kg)
Rectal Temperature (˚C)
D0
D3
D5
D7
Control
-
34.86 ± 0.46
35.22 ± 0.02
35.22 ± 0.02
35.75 ± 0.06
Extract
200
35.47 ± 0.07
35.52 ± 0.04
35.53 ± 0.02
35.42 ± 0.07
400
35.47 ± 0.07
35.43 ± 0.02
35.33 ± 0.02
35.30 ± 0.04
600
35.50 ± 0.04
35.32 ± 0.02
35.32 ± 0.02
35.23 ± 0.02
n-hexane
400
35.47 ± 0.04
35.40 ± 0.02
35.30 ± 0.02
35.50 ± 0.04
Dichloromethane
400
35.52 ± 0.04
35.30 ± 0.06
35.40 ±0.04
35.33 ± 0.06
Ethyl acetate
400
35.52 ± 0.02
35.45 ± 0.04
35.33 ±0.06
35.30 ± 0.06
Methanol
400
35.00 ± 0.20
35.30 ± 0.10
35.30 ± 0.10
35.31 ± 0.04
Chloroquine
5
35.18 ± 0.27
35.37 ± 0.07
35.37 ± 0.07
35.22 ± 0.08
Values are expressed as mean ± SEM.
TABLE 5: Effect of Setaria megaphylla leaf extract/fractions on haematological parameters of P. berghei-infected mice
Treatment
WBC
(x109/L)
LYM
(x109/L)
NEUT
(x109/L)
RBC
(x1012/L)
HGB
(g/dL)
PCV
(%)
MCV
(fL)
MCH
(pg)
MCHC
(g/dL)
PLT
(x109/L)
Control
15.70 ± 4.30
32.53 ± 2.45
65.57 ± 1.52
5.34 ± 0.35
8.43 ± 0.96
38.07 ± 2.43
75.07 ± 0.62
11.83 ± 0.09
30.13 ± 0.33
454.33 ± 64.32
Extract(200mg/kg)
6.46 ± 0.90c
54.85 ± 10.5c
39.95 ± 11.5c
7.62 ± 0.38
10.75 ± 1.25
41.85 ± 2.85
54.85 ± 0.95b
14.05 ± 0.95
25.60 ± 1.30
674.50 ± 86.5a
Extract (400mg/kg)
7.95 ± 1.78b
40.0 ± 2.60
54.80 ± 1.78
8.24 ± 0.3a
12.70 ± 0.10
49.0 ± 0.60a
49.0 ± 0.60b
19.50 ± 1.70a
25.95 ± 0.55
696.01 ± 83.0a
Extract (600mg/kg)
5.66 ± 0.06c
51.15 ± 14.5a
45.0 ± 14.60b
7.66 ± 0.31
12.95 ± 0.15a
54.55 ± 6.35b
71.65 ± 11.0
17.0 ± 0.90
24.10 ± 2.50
968.56 ± 86.5b
Hexane fraction
10.96 ± 2.88a
45.90 ± 3.90a
49.70 ± 3.80b
8.41 ± 1.2a
13.50 ± 0.20a
50.50 ± 2.45a
61.20 ± 11.0a
16.40 ± 2.60
27.0 ± 1.00
673.0 ± 53.00a
Dichloromethane fraction
6.89 ± 0.90b
51.2 ± 6.30a
44.20 ± 6.40c
8.29 ± 0.0a
12.65 ± 0.15a
48.55 ± 1.85a
58.50 ± 2.90b
15.30 ± 0.40
26.05 ± 0.75
702.5 ± 32.50b
Ethyl acetate fraction
12.26 ± 2.83
41.30 ± 2.70
53.0 ± 1.60a
8.60 ± 0.7b
14.55 ± 2.05a
59.90 ± 9.10b
69.35 ± 4.55
16.90 ± 0.90
24.40 ± 0.30
539.0 ± 11.00
Methanol Fraction
9.06 ± 4.30a
44.85 ± 1.15
60.15 ± 3.05
7.47 ± 0.17
10.30 ± 0.20
40.90 ± 3.50
54.95 ± 5.95b
13.80 ± 0.60
25.30 ± 1.60
1065.0 ± 25.0c
Chloroquine
9.58 ± 1.39
40.66 ± 0.90
46.34 ± 0.39
8.53 ± 0.2a
12.94 ± 0.70a
48.72 ± 1.22a
59.40 ± 0.84b
18.88 ± 0.27a
25.68 ± 0.71
631.40 ± 44.7a
All values are presented as mean±S.E.M. for six rats in each group.compared with control group a p<0.05, b p<0.01, c p<0.001.WBC-white bllod cell, RBC-Red blood cell, HGB-Hemoglobin, PLT-Platelets, PCV-
Packed cell volume, LYM-Lymphocytes, NEUT-Neutrophils, MCH-Mean corpuscular hemoglobin, MCV-Mean copuscular volume, MCHC-Mean corpuscular hemoglobin concentration.

Trop J Nat Prod Res, September 2022; 6(9):1504-1510 ISSN 2616-0684 (Print)
ISSN 2616-0692 (Electronic)
1509
© 2022 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License
TABLE 6: GC–MS analysis of ethyl acetate fraction of Setaria megaphylla
S/No.
Name of compound
Retention
Index
Mol.wt
g/mol
Chemical formula/Chemical Structure
1.
2-propenoic acid, 3-(2
hydroxy-phenyl,(E)-
101
164
C9H8O3
2.
Phenol,2,6-dimethoxy
280
154
C8H10O3
3.
(E)-β-ocimene
810
136
C10H16
4.
P-metha-1(7),8-diene
999
136
C10H16
5.
D:A-friedooleannan-3-ol,(3a)-
1011
428
C10H16
6.
Stigmastone-3,6-dione,(5a)-
1012
428
C29H48O2
7.
Bicyclo[2.2.1]heptan-2-
ol,1,7,7-trimethyl
282
154
C10H18O
8.
P-cymene
1012
134
C10H14

Trop J Nat Prod Res, September 2022; 6(9):1504-1510 ISSN 2616-0684 (Print)
ISSN 2616-0692 (Electronic)
1510
© 2022 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License
This hypothermia in mice may have resulted from the serious
physiological and metabolic effects of the malaria parasite on the host,
leading to body heat loss and ultimately death of mice19. The
extract/fractions however, were unable to attenuate these processes
and hence the resultant hypothermia. Alterations in hematological
indices of the infected mice such as decreases of RBC count, Hb level,
PCV, and mean haemoglobin concentration levels were observed in
infected animals which are common signs of anaemia.20 The invasion
of host’s RBC by Plasmodium during malaria infection, shortens the
lifespan of RBC through the digestion of Hb using glucose, oxygen
and hemozoin formation and finally the bursting of the erythrocytes
during the development of their asexual blood stage. 21 The treatment
of animals with the leaf extract and fractions significantly improved
the haematological parameters, portraying the leaf extract and
fractions potentials to inhibit the parasites growth as confirmed by the
decreased parasitaemia and thus, protecting animals from death.
WBC was observed to increase significantly in the infected mice.
This can be attributed to the immunogenic effects induced by the
parasite and their pigment (hemozoin).22 Decreased parasitaemia due
to extract/fractions treatment caused corresponding reduction in WBC.
The platelets counts of the treated infected mice were significantly
improved compared to untreated P. berghei-infected mice. The
treatment of infected mice with the S. megaphylla extract and
fractions must have offered some degree of protection to the infected
mice from the immune cells and platelets dysregulation, through the
activities of its phytochemical constituents.
Conclusion
The results of this study show that the leaf extract and fractions of
Setaria megaphylla possess antimalarial potentials which maybe
attributed to the activities of its phytochemical constituents.
Conflict of Interest
The authors declare no conflict of interest.
Authors’ Declaration
The authors hereby declare that the work presented in this article is
original and that any liability for claims relating to the content of this
article will be borne by them.
Acknowledgements
The authors are grateful to Mr. Nsikan Malachy of Pharmacology and
Toxicology Department for providing technical assistance.
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