Exploring the Antimalarial Efficacy of Globimetula oreophila Leaf Fractions in Plasmodium berghei-Infected Mice: In Vivo Approach
Dauda Garba★ , Bila Hassan Ali, Bashar Bawa, Abdulrazaq Sanusi, Yahaya Mohammed Sani, Muhammad Garba Magaji, Musa Isma’il Abdullahi, Aliyu Muhammad Musa, Hassan Halimatu Sadiya
Malaria is a
major public health concern leading to morbidity and mortality, which occurs in
all six World Health Organization regions (1). In 2021, an estimated 247
million cases and 619,000 fatalities occurred worldwide, with sub-Saharan
Africa accounting for the vast majority of cases and deaths—roughly (1).
According to the WHO, 26.6% of all malaria cases and 31.3% of deaths worldwide
occurred in Nigeria in 2021 (1). The population of people most vulnerable to
malaria are pregnant women and children under the age of five. In 2021, 76.8%
of all the world's malaria deaths were of children under the age of five (1).
In 2021, an estimated 1.3 million women were exposed to malaria during
pregnancy, which resulted in 961,000 children with low birth weight (1). The
development and reemergence of parasite resistance to first-line antimalarial
drugs, particularly ACTs, as well as the disruption of humanitarian
initiatives, have led to a sustained rise in malaria-related death and
morbidity despite all efforts (1).
According to Sivakrishnan (2018), “a medicinal plant” is any plant
in which one or more of its organs contain substances that can be used for
therapeutic purposes or are precursors for the synthesis of effective drugs.
Medicinal plants contain diverse phytochemicals with known or unknown
biological activities. Plant-derived substances are believed to be more
accessible, in line with patients' preferences, and safer and more
cost-effective treatments. Examples of these substances include tropane
alkaloids (atropine, hyoscine, scopolamine, and hyoscyamine), opium alkaloids
(paperverine, codeine, and morphine), flavonoids (stilbenes, chalcones,
luteolin, quercetin, rutin, apigenin, and kaemferol), terpenoids (stigmasterol,
sitosterol, betulinic acid, lupeol, and olenolic acid), and essential oils
(from caraway and peppermint) (2, 4). High concentrations of phytochemical
constituents with antiplasmodial properties found in medicinal plants aid in treating
malaria. For example, quinine from the bark of the Cinchona tree, a
member of the Rubiaceae family of plants, is well known for treating malaria (5,
6). Alternatively, Artemisia annua, another ancient medicinal plant, is
a significant source of the antimalarial drug artemisinin, a sesquiterpene
lactone (, ).
Declarations
Conflict of Interest
The authors declare no conflicting interest.
Data Availability
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to [ethical concerns about plagiarism].
Ethics Statement
This study was conducted by the Declaration of Helsinki and approved by Ahmadu Bello University Zaria, Committee on Animal Use and Care (ABUCAUC) with an approval number: ABUCAUC/2023/061.
G. oreophila
is a plant species in the family of Loranthaceae (8, 9, 10), a large family of
about 75 genera and over 900 species (11). It is a hemiparasitic plant commonly
called mistletoe, found growing on several dicotyledonous trees, using them as
a host for its root-like structure called haustoria (10, 12). G. oreophila
is widely distributed in tropical Africa, particularly in Nigeria, Cameroon,
Gabon, Congo, and the Central African Republic (10, 12). Studies have revealed
that the host plant can significantly affect the chemical composition and,
thus, the biological activity of the hemiparasite mistletoe (13, 14). G.
oreophila has been used to cure various ailments, including diarrhea,
stomachache, headache, and fever. As a treatment for these ailments, the leaves
and stems are boiled, and the resulting decoction is drunk (15). G.
oreophila (Loranthaceae), growing on a host plant of Azadirachta indica,
is mainly used in northern Nigeria to manage malaria. Previous phytochemical
reports showed that the genus Globimetula contains several secondary
metabolites, such as triterpenes, tannins, glycosides, alkaloids, flavonoids,
and saponins (9, 16, 17), which have been reported to possess antimalarial
activity (18, 19). Flavonols, which belong to the class of flavonoids, are the
major biological markers of the genus Globimetula, which have been shown
to have antimalarial activity. The antiplasmodial potential of the ethanol leaf
extract of G. oroephila has been previously reported (9, 10). In
previous studies, we reported the isolation of a prenylated quercetin from the
ethyl acetate fraction of this plant (10). Subsequent in-silico analysis of
this compound against seven P. falciparum enzymes indicates its
potential to competitively inhibit enzyme activity by targeting co-factor
binding sites, particularly in specific proteases. These findings highlight the
promise of prenylated quercetin as a potent inhibitor for therapeutic applications
in the prevention and treatment of malaria (20), elemental analysis of the crude ethanol
extract (21), and qualitative and quantitative phytochemical profiling of this
plant (22). In the present study, we now report on the antiplasmodial effect of
fractions of G. oreophila growing on A. indica via in vivo
models in mice, considering its wide acceptability as a material remedy in most
of Nigeria.
Experimental
Section
Materials
Collection, Identification, and
Preparation of Plant Material
G. oreophila comprising the leaves and fruits was
collected from Sokoto Metropolitan, Nigeria, in July 2019 and authenticated by
a Taxonomist (Musa Muhammad) of the Herbarium Section, Department of Plant
Biology, Ahmadu Bello University, Zaria-Nigeria by comparison with herbarium
specimen having Voucher number ABU0886. The leaves were air dried under shade,
reduced in size manually using mortar and pestle, and stored for further use.
Chemicals
All organic solvents employed for extracting and fractionating plant
material were of laboratory grade (Merck Millipore). The solvents utilized for
column chromatography, the crystallization of compounds, and the chemicals used
to assess antimalarial properties were of analytical grade. The standard
chloroquine powder was purchased from Sigma Aldrich, St. Louis, MO, USA.
Extraction and Fractionation
The powder plant material (900 g) was macerated with 8 L of 70% ethanol for 3 days with occasional shaking. The extract was then concentrated in vacuo,affording 74.55 g (8.28%) of a sticky dark green semi-solid mass called the crude ethanol extract (CEE). First, 74.55 g of CEE was partitioned successfully with n-hexane, chloroform, ethyl acetate, and n-butanol in sequential order of increasing polarity to obtain the following yield of fractions: hexane fraction (HF; 21.78 g), chloroform fraction (CF; 9.83 g), ethyl acetate fraction (EF; 6.96 g); and n-butanol fraction (nBF; 17.19 g), which were subjected to further studies (23).
Source
and Maintenance of Experiment Animals
Locally bred adult Swiss Albino mice of either sex (19–30 g
body weight) were obtained from the Animal House Facility of the Department of
Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria. The
animals were fed with standard rodent commercial feed and water ad libitum and maintained under
standard laboratory conditions in a polypropylene cage at room temperature
throughout the study. All experimental protocols were approved by Ahmadu Bello
University Zaria, Committee on Animal Use and Care (ABUCAUC) with an approval number
ABUCAUC/2023/061 and the “principle of laboratory animal care” (NIH publication No. 85-23, 1985)
guidelines and procedures were followed.
Rodent Malaria Parasite
A mouse-infected chloroquine-sensitive
strain of P. berghei NK-65 was
obtained from the National Institute of Medical Research, Lagos (NIMR). The
parasites were kept alive by continuous intra-peritoneal passage in mice at the
Department of Pharmacology and Therapeutics Ahmadu Bello University Zaria,
Kaduna State of Nigeria.
Acute
Oral Toxicity Study
The hexane fraction (HF), chloroform fraction (CF), ethyl
acetate fraction (EF), and butanol fraction were evaluated for their acute oral
toxicity in non-infected Swiss albino mice of 6–8 weeks old and weighing 27–32
g according to Organization for Economic Cooperation and Development (OECD)
guidelines in mice (OECD Test Guideline 425, 2008) (24). The mice were fasted
overnight and weighed before the test. Limit tests of 2000 and 5000 mg/kg were
used. Limit test of 2000 mg/kg: Six mice were used; two mice were dosed orally
with the fractions' 2000 mg/kg body weight. After administration of the
fractions, food was withheld for a further 2 h period. Death was not observed
in the first 24 h. Then, four more mice were given the same extract dose (2000
mg/kg).
Observation included changes in skin and fur, eyes and
mucous membranes, and respiratory and behavior patterns. The mice were then
observed for signs and symptoms of toxicity and mortality over 14 days. The 5000
mg/kg limit test was also employed using the same procedure stated above.
Preparation of Parasite Inoculation
A
carrier mouse with about 30% parasitemia was euthanized with diethyl ether, and
the blood was collected using a cardiac puncture into a heparinized vacutainer
tube containing 0.5% trisodium citrate. The blood was then diluted with
physiological saline (0.9%) until an inoculum containing approximately 107
infected erythrocytes was obtained. The inoculum was appropriately preserved
(through a refrigerator) for further study.
In Vivo Antimalarial Activity
Animal Grouping and Dosing
This
study used thirty mice of both sexes for the grouping. The prophylactic study
used pyrimethamine (1.2 mg/kg) as the positive control. For the suppressive and
curative studies, the positive (Group IV) and negative (Group V) controls were
administered with distilled water (10 mL/kg) and chloroquine (5 mg/kg),
respectively, through the oralroute.
Prophylactic Test
The
method described by Ryley and Peters (1970) was used to assess the repository
effect of the fractions (25). Five groups of six mice each were respectively
administered orally with 125, 250, and 500 mg/kg of the test fractions (test
groups), 1.2 mg/kg pyrimethamine (positive control), and 10 mL/kg distilled
water (negative control) once daily for 4 consecutive days. On the 5th day, the
mice were intraperitoneally inoculated with 0.2 mL standard inoculum containing
approximately 1 × 107P.
berghei infected erythrocytes. After 72 h of infection, the parasitemia
level was assessed by studying the slides with thin blood smears under a
microscope.
Suppressive Test
To
establish the suppressive activity of G.
oreophila fractions against chloroquine-sensitive P. berghei infection in mice, a method previously described by
Peter et al. (1975) was adopted (26). Thirty mice were grouped into five groups,
each containing six. An inoculum of 0.2 mL containing
approximately 1 × 107P.
berghei-infected erythrocytes
was given to each mouse via the intraperitoneal route. 3 h post-infection,
doses of the fractions at 125, 250, and 500 mg/kg were orally administered to
groups 1, 2, and 3, respectively, which served as the treatment groups once
daily for 4 days (0 to 3). A parallel test was conducted with 5 mg/kg standard
chloroquine and 10 mL/kg distilled water as vehicles in groups 4 and 5,
respectively. On day 4, thin smears were made from the tail blood. The slides
were then fixed with methanol, stained with 10% Giemsa solution for 30 min, and
examined in 10 fields under a microscope. Average suppression was calculated
using Equation 1.
%Suppression=ACAC−AG×100
Equation 1
Where AC is average parasitemia in control group and AG is average parasitemia in tested group.
Curative Test (Test
on Established Infection)
The schizontocidal effect in established infection was assessed using the Rane test, as described by Ryley and Peters (1970) (25). Thirty mice were inoculated with 0.2 mL standard inoculum via the i.p. route. Seventy-two hours post-infection, the mice were randomly divided into five groups of six each. Graded doses of 125, 250, and 500 mg/kg of the fraction were administered to treatment groups 1, 2, and 3, respectively. Positive and negative control groups 4 and 5 received 5 mg/kg standard chloroquine and 10 mL/kg distilled water. The fractions were given once daily for four days, and on day seven, the schizontocidal effect was evaluated through a microscopic examination of Giemsa-stained thin blood smears across 10 fields of each slide.
Statistical Analysis
Data from the antiplasmodial study were analyzed using
Statistical Package for Social Sciences (SPSS), IBM version 20. The result was
presented as mean ± standard error of the mean (SEM) and percentages. Data were
analyzed using one-way Analysis of Variance (ANOVA) followed by Dunnett's post
hoc test for multiple comparisons. Values of p<0.05 were considered significant.
Fractions
Treatment (mg/kg)
Average Parasitaemia ± SEM
% Suppression
Distilled water
10 mL/kg
25.20 ± 0.86
-
Hexane (HF)
125
17.04 ± 0.50 *
32.38
250
15.88 ± 0.26 *
36.98
500
14.52 ± 0.53 *
42.38
Chloroform (CF)
125
13.48 ± 0.46 *
46.51
250
12.84 ± 0.71 *
49.05
500
13.12 ± 0.70 *
47.94
Ethyl acetate (EF)
125
15.08 ± 0.90 *
40.16
250
15.32 ± 0.81 *
39.21
500
8.22 ± 0.55 *
67.38
Butanol (BF)
125
18.15 ± 0.24 *
27.98
250
17.34 ± 0.94 *
31.19
500
15.88 ± 0.47 *
36.98
Pyrimethamine
1.2
6.00 ± 0.86 *
76.19
Note: values
are presented as mean ± SEM; Data analyzed by one-way ANOVA followed by Dunnett's post hoc test; n = 6, (*) shows
a significant difference to the distilled water group (p<0.001).
Results
Acute Toxicity Test of Globimetula oreophila Leaf Fractions
Using the oral route and the OECD technique, the acute toxicity (LD50) of the G. oreophila leaf fractions, such as the hexane fraction (HF), chloroform fraction (CF), ethyl acetate fraction (EF), and butanol fraction (BF), were assessed. The results indicated that the LD50 for each fraction was more than 5000 mg/kg. Following treatment, the animals showed no changes in their food or water intake, nor did they exhibit any changes in their behavior or autonomic functions (pupillary size, lacrimation, salivation, defecation, or urination). The animals did not show any signs of poisoning or mortality.
In Vivo Antimalarial Studies
Prophylactic Activity of Fractions of Globimetula oreophila Leaf Extract in Plasmodium Berghei-Infected Mice
At all the studied doses, the administration of the hexane fraction (HF), chloroform fraction (CF), ethyl acetate fraction (EF), and butanol fraction (BF) resulted in a significant decrease in parasitemia levels. However, the positive control (pyrimethamine) produced a higher reduction in parasitemia level with 76.19% chemosuppression (Table 1).
Suppressive
Activity of the Fractions of G. oreophila Leaf Extract in Plasmodium
Berghei-Infected Mice
In comparison to the negative control, the
fractions (HF, CF, EF, and BF) exhibited statistically significant (p<0.001) chemosuppression at the
tested doses. The HE and EF of the extract at a dose of 500 mg/kg afforded
59.39% and 60.15% chemosuppression, respectively. The highest percentage of
inhibition activity was seen with EF compared to HF, CF, and BF in a
dose-dependent manner at p<0.001
(Table 2).
Table
2. Suppressive
effect of fraction of Globimetula oreophila in early malarial infection in
mice.
Fractions
Treatment (mg/kg)
Average Parasitaemia ± SEM
% Suppression
Distilled water
10 mL/kg
26.40 ± 0.60
-
Hexane
125
16.4 ± 0.46 *
37.89
(HF)
250
11.4 ± 0.71 *
56.82
500
10.72 ± 0.55 *
59.39
Chloroform
125
16.12 ± 0.31 *
38.94
(CF)
250
14.12 ± 0.19 *
46.52
500
11.60 ± 0.54 *
56.06
Ethyl acetate
125
15.12 ± 0.83 *
42.73
(EF)
250
12.88 ± 0.58 *
51.21
500
10.52 ± 0.78 *
60.15
Butanol
125
16.42 ± 0.30 *
37.80
(BF)
250
11.74 ± 0.51 *
55.53
500
14.76 ± 0.85 *
44.09
Chloroquine
5
6.72 ± 0.69 *
74.55
Note: values
are presented as mean ± SEM; Data analyzed by one-way ANOVA followed by Dunnett's post-hoc test; n = 6, (*) shows
a significant difference to the distilled water group (p<0.001).
Curative Activity of the
Fractions of G. oreophila Leaf Extract in Plasmodium Berghei-Infected
Mice
All tested doses of the fractions significantly (p<0.001) reduced parasitemia levels compared to the negative control. The greatest reduction was observed in the group receiving 500 mg/kg, achieving approximately 65.97% suppression. Chloroquine, used as the standard drug, achieved 83.40% suppression. At a 500 mg/kg dose, both the CF and EF fractions demonstrated a stronger curative effect than the other extracts, although similar activity levels were observed at the highest doses tested across the groups (Table 3). This indicates that CF and EF may have enhanced efficacy at higher concentrations.
Table 3.
Curative effect of Globimetula oreophila leaf extract fractions in
established infection in mice.
Fractions
Treatment
(mg/kg)
Average
Parasitaemia ± SEM
%
Suppression Effect
Distilled water
10 mL/kg
28.80 ± 1.93
-
Hexane (HF)
125
16.80 ± 0.76 *
41.67
250
12.68 ± 0.66 *
55.97
500
12.72 ± 0.94 *
55.83
Chloroform (CF)
125
16.38 ± 0.56 *
43.13
250
17.12 ± 0.67 *
40.56
500
11.08 ± 0.89 *
61.53
Ethyl acetate (EF)
125
16.0 ± 0.11 *
44.44
250
10.80 ± 0.62 *
62.50
500
9.80 ± 0.49 *
65.97
Butanol (BF)
125
16.48 ± 0.42 *
42.78
250
15.72± 0.16 *
45.42
500
11.60± 0.60 *
59.72
Chloroquine
5
4.78 ± 0.41 *
83.40
Note: values are
presented as mean ± SEM; Data analyzed by one-way ANOVA followed by Dunnett's
post-hoc test; n = 6, (*) shows a significant difference to the distilled
water group (p<0.001).
Discussions
The LD50 is a metric
utilized to assess the difference between a safe, effective dose and a
potentially harmful or lethal dose (9). In our study, the oral LD50
for all fractions of G. oreophila was determined to be over 5000 mg/kg in mice,
suggesting that these fractions are practically non-toxic. Additionally, we
observed no signs of toxicity in the animals, including changes in skin, eyes,
and mucous membranes, as well as no alterations in behavior, trembling,
diarrhea, or fur loss.
In vivo
models are usually employed in antimalarial studies because they consider the
possibility of the prodrug effect and the probable involvement of the immune
system in eradicating the parasite (27). The most accurate metric in
antimalarial investigations is typically the evaluation of the percentage
suppression of parasitemia (27). It has been suggested that lowering
parasitemia levels is essential for an organism to recover from symptomatic
malaria (28). The ability to lower the parasitemia levels of the target
organism has been shown by medicinal plant extracts with high chemosuppressive
action (29). Using prophylactic, suppressive, and curative tests in P.
berghei-infected mice at graded dosages of 125, 250, and 500 mg/kg, the in
vivo antiplasmodial potential of HF, CF, EF, and BF was assessed.
Prophylactic
antimalarials act at the pre-erythrocytic stage (initial phase of the parasite)
of the parasite's life cycle (30). This also shows that the substance possibly
acts as tissue schizontocides. Secondary metabolites in the fractions of the G.
oreophila plant may be acting singly or in synergy in inhibiting enzymes at
the pre-erythrocytic stage, such as P. falciparum dihydrofolate
reductase-thymidylate synthase (DHFR-TS), P. falciparum cysteine
proteases (falcipain-2 and falcipain-3), and glutathione S-transferase (GST) (31).
In the
suppressive study, except for the butanol fraction at a dose of 500 mg/kg (p<
0.001), all the fractions showed a significant dose-dependent reduction in
parasitemia levels when compared to the negative control
(distilled-water-treated group). This further confirms the claim's validity
about using plants to manage malaria. Dauda et al. studied the crude
ethanol extract of the plant (9). Since secondary metabolites have been known
to have antimalarial activity, it is possible that the presence of
phytochemicals that are present in each of the fractions, such as
carbohydrates, steroids, triterpenes, glycosides, saponins, tannins, flavonoids,
and alkaloids, may have brought about the antimalarial activity observed (9).
Thus, the antimalarial activity of G. oreophila fractions in P.
berghei-infected mice might be due to the presence of one or a combination
of these phytochemical constituents acting singly or in synergy in inhibiting
enzymes such as heme oxygenase-1 (HO-1), serine repeat antigen (SERA),
calcium-dependent protein kinase (CDPK), dihydrofolate reductase (DHFR), and
aspartic and cysteine proteases, which have been studied for their potential to
act on suppressive in vivo models of malaria (32, 33).
In the curative study, all the
fractions exhibited a statistically significant inhibition in a dose-dependent
manner, except for hexane and chloroform fractions, in which a non-does
dependent parasitemia chemosuppression (p<0.001 in all cases) was
seen when compared with the negative control (distilled-water-treated group),
probably due to non-selectivity of the fractions to the proliferation process
of the parasite. The observed inhibition of plasmodial growth could be
attributed to the fraction's ability to inhibit malaria protease enzymes
required for parasite survival. These enzymes, such as aspartic protease,
dihydrofolate reductase (DHFR), cysteine protease enzymes, and proteasome,
which are potential targets for antimalarial drug discovery, act on the
erythrocytic stage of the parasite life cycle (31). Suppressive and curative antimalarials act at the
erythrocytic stage (the parasite's life cycle that causes the clinical
symptoms), of which chloroquine is a potent suppressive and curative
antiplasmodial agent (34). The mechanism of action of chloroquine is through
the formation of a heme-chloroquine complex that caps hemozoin molecules and
prevents further bio-crystallization of toxic heme produced within the parasite's
digestive vacuole (35). Although the mechanism of action of antimalarial
activities of flavonoids has not been fully established, inhibition of the
fatty acid biosynthesis (FAS II) of the parasites and inhibition of the influx
of L-glutamine and myoinositol into the infected erythrocytes are some proposed
mechanisms (36).
The acidic character of flavonoids (due to phenolic -OH
groups) prevents them from entering the acidic food vacuole of parasites,
unlike basic quinoline-based antimalarials (37, 38). As a result,
flavonoid-based compounds do not interfere with degrading hemoglobin, which is
the only site of action for most current medications against which malaria
parasites have developed resistance (37, 38). Hence, the exhibited
chemosuppressive and chemo-prophylactic effects may be due to these secondary
metabolites in the plant, which have been reported to have antimalarial
activity (39, 40). When an extract exhibits a percentage chemosuppression equal
to or more than 50% at dosages of 250, 500, and 1000 mg/kg body weight per day,
in vivo antiplasmodial activity can be categorized as moderate, good,
and very good (41, 42). According to this classification, at doses of 250 and
500 mg/kg, the hexane, chloroform, ethyl acetate, and n-butanol fractions
displayed a good suppressive and curative effect.
