Infectious Diseases and Herbal Medicine 2025; volume 6:431

In vivo antimalarial property of Markhamia lutea (Benth.) K. Schum leaf

and stem-bark crude extracts and spectrophotometric evaluation of
alkaloids, flavonoids, and phenolic contents
Casim Umba Tolo,1,2 Clement Olusoji Ajayi,2,3 Rapheal Wangalwa,1 Bruhan Kaggwa,4 Joshua Kiptoo,3 Martin Amanya5
1Department of Biology, Mbarara University of Science and Technology, Mbarara; 2Pharm-Biotechnology and Traditional Medicine
Centre, Mbarara University of Science and Technology, Mbarara; 3Department of Pharmacy, Mbarara University of Science and
Technology, Mbarara; 4Department of Pharmacy, College of Health Sciences, Makerere University, Kampala; 5Department of
Pharmacology, Mbarara University of Science and Technology, Mbarara, Uganda

Correspondence: Casim Umba Tolo, Department of Biology, Mbarara Abstract

University of Science and Technology, P.O. Box 1410, Mbarara, Uganda.
Tel: +256772837055. Malaria is endemic to developing countries despite several
E-mail: tolocas@must.ac.ug efforts from the World Health Organization (WHO) and other
organizations, and this has been attributed to many factors, includ-
Key words: antimalarial, parasitaemia, chemosuppression, phytochemicals, ing resistance to first-line antimalarial drugs. This study, therefore,
Markhamia lutea.
evaluated phytochemical levels and antimalarial properties of
Contributions: CUT and COA conceived and designed the study; CUT, COA, Markhamia lutea (Benth.) K. Schum leaf and stem-bark extracted
and RW performed the experiment; CUT, COA, RW, BK, JK, and MA with various solvents as potential future alternative antimalarial
analysed the data and contributed to the manuscript write-up; CUT and COA drug development. Aqueous, ethanol, ethyl acetate, and methanol
reviewed and edited the manuscript. All authors read and approved the final extracts from M. lutea leaf and stem-bark were evaluated for alka-
version of the manuscript, and agreed to be held accountable for all aspects of
the work.
loid, flavonoids, and phenolic levels using an ultraviolet-visible
spectrophotometer. The extracts were further evaluated for anti-
Conflict of interest: the authors declare no potential conflict of interest. malarial properties at 100-400 mg/kg on Plasmodium berghei
NK65-induced mice using a 4-day test. The ethyl acetate leaf
Funding: this research study was supported by the Pharm-Biotechnology and extract (50.8 µg/mg) gave the highest Quercetin Equivalent of
Traditional Medicine Centre, Eastern and Southern Africa Higher Education
Center of Excellence II (ACE II), Mbarara University of Science and
Flavonoids (QEF), followed by ethanol and methanol leaf extracts
Technology, Uganda. with 46.2 and 47.5 µg/mg QEF, respectively. Methanol exhibited
the highest level of Gallic Acid Equivalent of Phenolics (GAEP)
Ethics approval and consent to participate: this study was carried out in strict with 213.4 µg/mg, while aqueous leaf extract was the lowest with
accordance with the recommendations in the Guide for the Care and Use of
96.1 µg/mg GAEP. The results showed that aqueous and methanol
Laboratory Animals of the National Institutes of Health. The study protocol
was approved by the MUST Research Ethics Committee (MUREC1/7).
leaf extract, ethanol, and methanol stem-bark extract possessed
antimalarial activity with the lowest ED50 of 237.5 and 240.6,
Informed consent: not applicable. 233.8, and 236.6 mg/kg, respectively. The extracts of Markhamia
lutea leaf and stem-bark demonstrated antimalarial properties with
Patient’s consent for publication: not applicable.
high contents of phenolic and flavonoid components, while the
Availability of data and materials: all data generated or analyzed during this
extracts showed no acute toxicity at the tested doses in the animals
study are included in this published article. studied.

Acknowledgments: the authors appreciate BEI Resources, NIAID, NIH for

donating Plasmodium berghei NK65 strain. We also acknowledge the techni-
cal support of Musa Kasone Wakabi of the Directorate of Government Introduction
Analytical Laboratory (DGAL), Kampala, Uganda for the GCMS analysis.
The burden of malaria is still immense in Africa despite the
Received: 15 January 2025. World Health Organization’s (WHO) efforts, which regis-
Accepted: 30 March 2025. tered 619,000 mortalities in the World Malaria Report recently.
The African region is the most affected, with 234 million morbidi-
©Copyright: the Author(s), 2025
Licensee PAGEPress, Italy ties and 593,000 deaths in 2021 alone. According to the WHO,
Infectious Diseases and Herbal Medicine 2025; 6:431 malaria control efforts are still facing many challenges, including
doi:10.4081/idhm.2025.431 a rise in resistance to insecticide-treated mosquito nets, resistance
to antimalarial drug remedies, and drops in the primary malaria-
This work is licensed under a Creative Commons Attribution-NonCommercial
fighting tools effectiveness in Africa.1 A recent report on malaria-
4.0 International License (CC BY-NC 4.0).
endemic countries showed that Uganda was among the four coun-
Publisher's note: all claims expressed in this article are solely those of the tries that accounted for almost half of all morbidities, with 1.7 mil-
authors and do not necessarily represent those of their affiliated organiza- lion morbidities between 2019 and 2021.1 Recent reports showed
tions, or those of the publisher, the editors and the reviewers. Any product a significant increase in malaria morbidities and mortalities
that may be evaluated in this article or claim that may be made by its manu-
despite steady declines after some decades. This has been attribut-
facturer is not guaranteed or endorsed by the publisher.
ed to the malaria mosquito and parasite adaptation to drugs, mak-

[Infectious Diseases and Herbal Medicine 2025; 6:431] [page 19]

Article

ing them resistant to treatments.1 With a recent increase in the ties and appropriate solvent with optimum activity based on the
malaria resistance to first-line drugs as reported by the WHO, with extracted constituents. Therefore, this study has presented the anti-
a greater than 10% failure rate in the activities of Artemisinin- malarial effects M. lutea leaf and stem-bark extracted with ethanol,
based combination therapies in Uganda, Angola, Burkina Faso, the ethyl acetate, methanol, and water to establish the best solvent-
Democratic Republic of the Congo, there is an urgent need to bearing the most active antimalarial properties for future drug
search for alternative drugs that are safe, less toxic, cheap and development and also the alkaloidal, flavonoids, and phenolic lev-
available. Many plants are reportedly used in Uganda for malaria, els of each extract.
among which Markhamia lutea (Benth.) K. Schum leaf and stem-
bark are included, but there is no information on the antimalarial
efficacy of this plant whatsoever.
Markhamia species roots, barks, stems, and leaves are being
Materials and Methods
used in folklore in the treatment of different ailments, including Quercetin was purchased from Targetmol (Boston, USA) while
anemia, parasitic diseases, backache, diarrhea, intercostal pain, gallic acid (CAS No. 5996-86-8), atropine sulphate anhydrous,
sore eyes, external skin diseases, rheumatoid arthritis, pulmonary anhydrous sodium carbonate (CAS No. 497-19-8), vanillin (CAS
troubles, scrotal elephantiasis.2-4 M. lutea (Bignionaceae) is among No. 121-33-5) Folin–Ciocalteu reagent, aluminium chloride (CAS
the listed plants commonly used for different treatments, including No. 7784-13-6), sodium acetate, sodium hydroxide (CAS No.
malaria in Uganda.2,4 1310-73-2), bromocresol green (CAS No. 76-60-8), methanol AR,
It is reported to have antiplasmodial, antiviral, anti-leishma- methanol HPLC grade, disodium hydrogen phosphate (CAS No.
nial, antimalarial, antimicrobial antiprotozoal, antiviral, anti- 7558-79-4), acetic acid, citric acid (CAS No. 77-92-9), and
cancer, anti-trypanosomal, and antioxidant properties.5-7 The plant Whatman paper No. 1 were purchased from Sigma Aldrich
was reported to contain alkaloids, saponins, tannins, terpenes, car- (Steinheim, Germany).
bohydrates, quinones, and phenols.6-9 M. lutea leaf ethyl acetate
crude extract was reported to have significant anti-parasitic in vitro Collection of plant material
activity and low cytotoxicity on MRC5 and KB cells with identi- The leaf and stem-bark of Markhamia lutea (Benth.) K. Schum
fied components including musambins A-C, musambiosides A-C, were collected from Rukararwe, Bushenyi district, Southwestern
and cycloartane triterpenoids.10 Uganda, with coordinates of 0031.47889’S 300 12.99765’E as
Despite its general use for different ailments and its use for shown in Figure 1, generated from ArcMap 10.8, before their iden-
malaria, there has been no study on its in vivo antimalarial activi- tification and authentication was done by Dr. Eunice A. Olet of the

Figure 1. Map indicating the sample site of Markhamia lutea (Benth.) K.Schum at Rukararwe, Bushenyi district, Uganda (ArcMap 10.8
generated).

[page 20] [Infectious Diseases and Herbal Medicine 2025; 6:431]

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Department of Biology, Mbarara University of Science and methanol was added to 1 mL each of 1 mg/mL concentration of
Technology (MUST), Mbarara, Uganda before their voucher spec- M. lutea leaf and stem-back extracts in a 10 mL volumetric flask,
imens were deposited at the Makerere University Herbarium, agitated before the addition of 0.2 mL of 10% AlCl3 solution and
Kampala with assigned voucher number: 51269. The plant was fur- 0.2 mL of 1M sodium acetate. The solution was made to the mark
ther subjected to confirmation on the Plants of the World Online with methanol before the incubation. Thereafter, the incubation of
(POWO) website (https://powo.science.kew.org/taxon/urn: the solution was done in the dark at room temperature for 30 min-
lsid:ipni.org:names:110020-1). The M. lutea stem-bark and leaves utes. The absorbance of the resulting solution was then taken at
were oven-dried at 50 and 40°C, respectively, for 48 h and there- 420 nm using a Jenway 6705 UV-VIS spectrophotometer. Working
after pulverized mechanically using an electric grinder, and sepa- solutions of 10-100 µg/mL of quercetin standard were prepared
rately stored in amber bottles. from the standard concentration of 1 mg/mL and were used to pre-
pare a calibration curve with methanol substituting for the sample
Extraction process in the blank and the linear regression equation of y=0.0092x-
Aqueous extract of powdered stem-bark and leaves of 50 g 0.0083; r2=0.9982 generated was used to determine the content in
each were separately prepared in 500 mL using decoction and infu- the samples. The total flavonoid content was expressed as micro-
sion methods, respectively, while ethyl acetate, methanol, and 70% gram Quercetin Equivalence of Flavonoids (QEF)/mg crude
ethanol of both parts were separately prepared using the macera- extract.
tion method. The ethanol and methanol extractions were achieved
in 72 h, while ethyl acetate was done within 3 h at room tempera- Total phenolic content determination of M. lutea leaf
ture. The extracts were all separately filtered using Whatman no. 1 and stem-bark extracts
filter paper and thereafter concentrated in vacuo at 45°C except for A modified Folin-Ciocalteau method described by Baba and
the aqueous that was lyophilized and were coded as AQL and Malik 14 and Wangalwa et al.15 was used to determine the total
AQSB for aqueous leaf and stem-bark, EtOHL and EtOHSB for phenolic content of the sample. One milliliter (1 mL) concentration
ethanol leaf and stem-bark, EtOAcL and EtOAcSB for ethyl of 0.5 mg/mL M. lutea leaf and stem-bark extracts was pipetted
acetate leaf and stem-bark, MeOHL and MeOHSB for methanol into a measuring cylinder, and 2 mL of 10% (v/v) Folin-Ciocalteu
leaf and stem-bark, respectively. The percentage yield was deter- reagent was added together with 2 mL of 7.5% (w/v) Na2CO3 solu-
mined using the formula below, and the yields for AQL, AQSB, tion with incubation of the solution done in 30 minutes at 40ºC.
EtOHL, EtOHSB, EtOAcL, EtOAcSB, MeOHL, and MeOHSB The absorbance of the solution was taken at 760 nm using a
were 21.9, 24.1, 19.9, 15.5, 2.9, 9.5, 0.2 and 0.9%, respectively. Jenway 6705 UV-VIS spectrophotometer. Also, working solutions
Thereafter, the samples were screened for the presence of phyto- of 1-100 µg/mL gallic acid were prepared and used to develop a
chemicals using the method of Balamurugan et al.11 calibration curve for the standard and the linear equation of
y=0.0137x+0.0545; r2=0.972 generated was used to determine the
Determination of total alkaloid levels in M. lutea leaf gallic acid concentration in the samples. The total phenolic content
and stem-bark extracts in M. lutea leaf and stem-bark was expressed as microgram Gallic
Total alkaloidal content was determined by using the modified Acid Equivalence/mg (GAE).
methods of John et al.12 and Patel et al.13 To prepare bromocresol
green (BCG) solution, 6.98 mg BCG was heated with 0.3 ml of 2N High-Performance Liquid Chromatography analysis of
NaOH and 0.5 ml distilled water at 50°C for 15 min to attain dis- M. lutea leaf and stem-bark extracts
solution of the components which was further made up to 1 L with High-Performance Liquid Chromatography conditions
distilled water in a measuring cylinder. A phosphate buffer solution The High-Performance Liquid Chromatography (HPLC) anal-
of pH 4.7 was prepared from 2 M Na2HPO4 and 0.2 M citric acid.12 ysis was performed on a UFLC Prominence Shimadzu chromato-
graph (Japan) at the Analytical and Pharmaceutical Laboratory,
Preparation of atropine standard curve MUST, Uganda. The HPLC machine comprised SIL-20AC HT
Atropine standard concentration (1 mg/mL) was prepared autosampler, column oven (CTO-20AC), UV-visible detector
using methanol, from which the working solution of 10-100 (SPD-20A), LC 20 AD pumps, and an online degassing unit
µg/mL was obtained. One milliliter (1 mL) of the solution was (DGU-20A). For each M. lutea leaf and stem-bark extract (aque-
measured into a separating funnel before 5 mL each of phosphate ous, ethanol, ethyl acetate, methanol), 1 mg/mL concentration was
buffer (pH 4.7) and bromocresol green were separately added and prepared and filtered with 0.22 μm. A reversed-phase HPLC assay
shaken vigorously. The complex formed was extracted serially into was carried out using isocratic elution with a flow rate of 0.8
1, 2, 3, and 4 mL of CHCl3 in a 10 mL measuring cylinder that was mL/min at a column temperature of 35°C, injections volume of 20
adjusted to volume with CHCl3. Thereafter, the absorbance of the μL, a mobile phase of 1% acetic acid/methanol in ratio 7:3 deliv-
chloroform containing alkaloidal component was taken at 415 nm ered by pump A and B, respectively and detected under the wave-
using a Jenway 6705 UV-VIS spectrophotometer against the blank length of 230 and 254 nm. The acquisition time for each injection
prepared, and the linear regression equation of y=0.0092x-0.003; was 40 minutes.
r2=0.9929 generated was used to determine the content in the sam- Solvents and deionized water were prior filtered through a 0.45
ples. The total alkaloidal content was expressed as microgram μm nylon membrane with the aid of Buckner, enhanced by ILM-
Atropine Equivalence of Alkaloids (AEA)/mg crude extract. VAC GmbH vacuum pump. All solvents were of HPLC grades.
Data were processed with LC-Solution Software.
Total flavonoid content determination of M. lutea leaf
and stem-bark extracts Gas Chromatography-Mass Spectrometric of M. lutea
The total flavonoid content of the sample was determined leaf and stem-bark extracts
using a modified AlCl3 colorimetric method as described by Baba Gas Chromatography-Mass Spectrometric (GC-MS) analysis
and Malik14 and Wangalwa et al.15 Three milliliters (3 mL) of was carried out in a Shimadzu GCMS-QP2020 NX with an RXI-

[Infectious Diseases and Herbal Medicine 2025; 6:431] [page 21]

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Sil MS capillary column measuring 30 meters in length, 0.25 mil- tive) strain was obtained through the United States BEI Resources,
limeters in internal diameter, and possessing a 0.25- µm film thick- NIAID, NIH, as contributed by Thomas F. McCutchan before it
ness, with cross bonds similar to 5% diphenyl/95% dimethyl silox- was activated in mice at the Animal Facility Laboratory, MUST,
ane was utilized. Helium was used as the carrier gas at a flow rate Uganda.17 The mice were infected with standard inoculum pre-
of 1.60 milliliters per minute, and the injector temperature was set pared from donor mice 2 h before the drug administration (post-
at 250°C. Column interfaced with 5675C Inert MSD with Triple- infection), and the mice were randomly assigned to 26 groups of 3
Axis detector. Helium gas was used as carrier gas and was adjusted mice per group (2 females and 1 male). The administration of
to a column velocity flow of 1.0 mL/min. aliquot to animals was conducted daily for 4 days as follows:
Other GC-MS conditions are ion-source temperature, 230°C; Groups I, II, and III received AQL extract at 100, 200, and 400
interface temperature, 250°C; pressure, 100:1 kPa; Solvent Cut mg/kg. Groups IV, V, and VI received AQSB extract at the same
Time: 3 minutes; and Injection Mode: Spitless with injection tem- doses; likewise, groups VII, VIII, and IX received EtOHL extract,
perature of 250°C. The column temperature started at 50°C for 5 while groups X, XI, and XII received EtOHSB extract. Groups
min and changed to 150 V at the rate of 4°C/min. The temperature XIII, XIV, and XV received EtOAcL; groups XVI, XVII, and
was raised to 250°C at the rate of 20°C/min and held for 5 min. The XVIII received EtOAcSB; groups XIX, XX, and XXI received
total elution time was 30 min. The relative percent amount of each MeOHL; and groups XXII, XXIII, and XXIV received MeOHSB
component was calculated by comparing its average peak area to extract. Also, groups XXV and XXVI were administered with
total areas. One milligram of each extract was dissolved in 1 mL artemether-lumefantrine at 4 mg/kg (positive control) and water at
of hexane and filtered with 0.22 μm before the sample was loaded 10 mL/kg (negative control). On day five, the smears were pre-
for injection into the machine. pared by collecting blood from the tail of each animal, fixed with
methanol, and stained with 10% Giemsa-stain before the para-
Acute oral toxicity of M. lutea leaf and stem-bark sitemia levels were examined by counting both the parasitized and
extracts non-parasitized erythrocytes in eight random views under a light
The median lethal dose (LD50) of each sample was determined microscope at 100× oil immersion objective lens. From this count,
in vivo using the ‘up-and-down’ method of the Organisation for the percentage parasitemia levels and chemosuppression were
Economic Co-operation and Development.16 This method is guid- determined as follows;
ed by the ‘aot 425 software’. Five healthy female Swiss albino
mice (Mus musculus L.) weighing 20-22 g were used for each Percentage parasitemia = {Na/Nb}100 (1)
extract (8 extracts totaling 40 animals). The mice were fasted for
3-4 hours before dosing, while the food and water were withheld where “Na” is the total number of parasitized red blood cells, while
for 1 hour after treatment administration. In this method, the 1st “” is the total parasitized and non-parasitized red blood cells.
animal was administered with the herbal supplement at 175 mg/kg
as predicted by the software and observed for any sign of toxicity Percentage chemosuppression = {A-B/A}100 (2)
for 48 h (short-term outcome), during which the animals were
observed for loss of appetite, reduced mobility, ruffled fur, signs of where “A” is the negative control group percentage parasitaemia
dizziness or mortality before the next predicted dose of 550 mg/kg level, and “B” is the test group percentage parasitaemia levels.
was administered to the 2nd animal. This was also observed for 48 Thereafter, the animals were observed for 28 days post-inocu-
h before the 3rd animal was dosed with aliquot at 2000 mg/kg, and lation to monitor their survival. The animals that showed signs of
this same dose was administered to the 4th and 5th animals before loss of appetite, reduced mobility, ruffled fur, or signs of dizziness
the software indicated ‘stop dosing’. Thereafter, the dosed animals during this period were removed and euthanized with halothane
were subjected to normal lives for an additional 12 days (long-term and those that reached day 28 were all euthanized using halothane.
outcome) to make 14 days for each animal. The LD50 was automat- The carcasses of the animals were appropriately disposed of by
ically generated by the software on day 14, while the animals that incineration.
reached day 14 post-administration were all humanely euthanized.
This study was carried out in strict accordance with the recommen- Data management and analysis
dations in the Guide for the Care and Use of Laboratory Animals All quantitative data were expressed as mean ± Standard
of the National Institutes of Health. All euthanasia was performed Deviation (SD). The effective doses were determined using
under halothane anesthesia, and all efforts were made to minimize Microsoft Excel 2016, while the variation in the data set was ana-
suffering. lyzed through one-way analysis of variance. The means variation
was considered at a 95% confidence level using Tukey’s Multiple
Antimalarial study on M. lutea leaf and stem-bark Comparison post-hoc Test through Graph Pad Prism10 software
extracts 2023 version.
In vivo antimalarial activity of M. lutea leaf and stem-bark
extracts was assessed through a 4-day suppressive test as described
in a previous study by Ajayi et al.17 A total of 90 healthy Swiss
albino mice of both sexes (18-22 g) were obtained from the MUST Results and Discussion
Animal Research Facility, Uganda. They were kept separately in Phytochemicals in M. lutea leaf and stem-bark
different cages under a 12-hour light/dark cycle and were fed with The extracts of M. lutea (Benth.) K. Schum showed the pres-
commercial food pellets with free access to water. This was main- ence of various phytochemicals in different solvents, with saponin
tained for two weeks for acclimatization before the assay. absent in ethyl acetate extract. Also, anthraquinone was absent in
all the extracts tested. Alkaloids were present in all the extracts
Preparation of inoculum and antimalarial assessment aside from ethyl acetate stem-bark extract, which was absent,
Plasmodium berghei NK65 (Chloroquine-sensitive, CQ-sensi- while a traceable amount was observed in ethanol and ethyl acetate

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leaf extracts. Steroids were present in aqueous ethanol, methanol Though the stem-bark extracted with ethanol and methanol
stem-bark extracts, and aqueous leaf extract with traceable gave comparable amounts of respective 0.8 and 0.6 µg/mg atropine
amounts in ethyl acetate and methanol leaf extract, whereas it was equivalence of alkaloidal content, the amount is significantly dif-
absent in ethanol leaf extract. ferent from that of the leaf part (p<0.0001). The extracts of all the
solvents gave a considerable amount of phenolic contents, with
Alkaloids, flavonoids, and phenolic contents in M. lutea stem-bark extracted with methanol having the highest content of
leaf and stem-bark 213.4 µg/mg crude extract, while the lowest was recorded in
Chemical contents in M. lutea leaf and stem-bark showed dif- ethanol leaf extract, as shown in Table 1. Generally, phenolic con-
ferent yields. Flavonoid content in the leaf extracted with ethyl tent was high in the stem-bark compared to the leaf part (Table 1).
acetate was the highest at 50.8 µg/mg, which was significant com-
pared to the rest; in addition, ethanol and methanol extracts gave High-Performance Liquid Chromatography fingerprint
46.2 and 47.5 µg/mg, respectively, in the leaf, which showed that of M. lutea leaf and stem-bark
the leaf is rich in flavonoids than the stem-bark (Table 1). The HPLC fingerprint is one of the most sensitive, reliable, and
aqueous extract of the stem-bark was the lowest, with a significant reproducible means of identification of component(s) or extracts.
content of 4.3 µg/mg. The ethanol and methanol extracts showed In this study, the HPLC fingerprint of M. lutea leaf fingerprint at
quantifiable amounts of alkaloids, with ethanol having the highest 230 nm showed characteristic peaks at 4.9 and 13.8 mins that are
content of 21.6 µg/mg in the leaf, whereas methanol extracts had peculiar to aqueous, ethanol, and methanol extracts (Figure 2a).
13.5 µg/mg in the same part, as shown in Table 1. Also, M. lutea stem-bark at 230 nm wavelength showed character-

Figure 2. HPLC fingerprints of M. lutea leaf (a) and stem-bark (b) @ 230 nm wavelengths AQ, Aqueous; EtOH,Ethanol; MeOH,
Methanol; EtOAc,Ethyl Acetate

Table 1. Phytochemical contents of M. lutea leaf and stem-bark.

Phytochemicals QE flavonoids (µg/mg) AE alkaloids (µg/mg) GAE phenolics (µg/mg)
EtOH leaf 46.19±0.33 21.60±0.19 86.25±0.084
EtOH sb 9.09±0.063 0.80±0.5a 137.40±0.00
AQ leaf 5.69±0.00 0.00 96.08±0.30
AQ sb 4.34±0.063 0.00 135.10±0.29
MeOH leaf 47.50±0.063 13.48±0.063 103.00±0.084
MeOH sb 7.68±0.063 0.62±0.11a 213.40±0.59
EtOAc leaf 50.79±0.20 0.00 107.70±0.84
EtOAc sb 8.37±0.16 0.00 197.50±0.69
Data are expressed as mean ± Standard Deviation, SD; same superscripted letter within the columns are comparable (p=0.05). QE, Quercetin Equivalent; AE, Atropine Equivalent; GAE, Gallic Acids Equivalent.

[Infectious Diseases and Herbal Medicine 2025; 6:431] [page 23]

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istic peaks at 6.2 and 14.4 mins in all the extracts. In addition, stem-bark extract, sitosterol was the most abundant (Figure 4c)
aqueous, ethanol, and methanol extracts showed peaks at 22.02, with 12.75% followed by pentadecanoic acid with 11.67% and 2-
26.02, and 34.2 mins, which are diagnostic, as shown in Figure 2b. (Isobutoxymethyl)oxirane with 7.73% appearing at retention times
At 254 nm wavelength, M. lutea leaf showed a unique peak at of 24.61, 13.75 and 8.83 minutes, respectively as shown in
4.9 min, which was prominent in aqueous extracts but also Supplementary Table 1.
observed in ethanol and methanol, as shown in Figure 3a. There
was another characteristic peak at 34.5 min, which was prominent Acute toxicity of M. lutea leaf and stem-bark
in ethanol and methanol extracts and diagnostic (Figure 3a). The The acute toxicity study on all the extracts (aqueous, ethanol
fingerprint of M. lutea stem-bark showed characteristic peaks at ethyl acetate, and methanol) of M. lutea leaf and stem-bark showed
4.6 and 25.9 min, which were peculiar to aqueous, ethanol, and no mortality at all the doses tested up to 2000 mg/kg, and there was
methanol extracts but missing in ethyl acetate extract (Figure 3b). no sign of distress aside from the calmness at the highest dose of
There was a peak at 22.9 min, which was peculiar to ethanol 2000 mg/kg. Hence, the LD50 generated by the ‘aot software’ gave
extract alone and could be used for identification of the extract, as above 2000 mg/kg.
shown in Figure 3b.
Antimalarial efficacy of M. lutea leaf and stem-bark
Gas Chromatography-Mass Spectrometric analysis of There was a reduction in the parasitaemia levels of all the test-
the active M. lutea extracts ed extracts at 100 to 400 mg/kg from 17.1 to 5.1%. At 100 mg/kg,
The extracts of ethanol stem-bark, methanol leaf, and stem- aqueous leaf extract showed a significant reduction in parasitaemia
bark were analysed using Gas Column Chromatography to identify level of 7.7% which was comparable to that of ethyl acetate leaf
the components, and 35 compounds from each of the three extracts with 8.6% parasitaemia level (p=0.26), methanol leaf (8.5% para-
were identified. Ethyl alpha-d-glucopyranoside (19.4%) appearing sitaemia level) (p=0.48) and stem-bark (8.1% parasitaemia level)
at a retention time of 10.43 min (Figure 4a) was the most abundant (p=0.97) extracts while ethyl acetate stem-bark was the least with
in the ethanol stem-bark extract followed by E,E,Z-1,3,12-nonade- 11.3% parasitaemia though the result was significant compared to
catriene-5, 14-diol with 18.8% at 15.16 min and ethyl 13-methyl- the negative control (p<0.0001) as shown in Table 2. At 200
tetradecanoate with 12.1% at 15.39 min while ketone, methyl 2- mg/kg, ethanol stem-bark extract gave the highest reduction of
methyl-1,3-oxothiolan-2-yl with 0.14% at 7.40 min was the least 5.6% parasitaemia level, which was comparable to that of the
abundant as shown in Supplementary Table 1. aqueous leaf (6.2% parasitaemia level) (p=0.79), and methanol
In methanol leaf extract, phytol was the most abundance stem-bark (6.4% parasitaemia level) [extracts while ethanol leaf
(13.92%), followed by vitamin E (11.89%), squalene (10.52%), extract was the gave the lowest parasitaemia level of 8.9% that was
and 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- significant (p<0.0001) compared to the negative control (Table 2).
(9.07%) appearing at retention times of 14.63, 22.66, 20.32 and At 400 mg/kg, there was a further reduction in the parasitaemia
5.95 mins, respectively as shown in Figure 4b. Also, in methanol level of animals dosed with ethanol stem-bark (5.1%) extract, a

Figure 3. HPLC fingerprints of leaf (a) and stem-bark (b) @ 254 nm wavelengths. AQ, Aqueous; EtOH,Ethanol; MeOH, Methanol;
EtOAc, Ethyl Acetate.

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reduction that was comparable to that of the aqueous leaf (6.0% comparable to ethyl acetate leaf (p=0.13), methanol leaf (p=0.29)
parasitaemia level) (p=0.099), methanol leaf (5.6%) (p=0.75) and and stem-bark (p=0.92) extracts with 49.6, 50.4 and 52.5% chemo-
stem-bark (5.7%) (p=0.58) extracts whereas ethyl acetate leaf suppression, respectively, though the activity was significantly dif-
extract exhibited the lowest parasitaemia reduction of 7.8% as ferent from that of positive control with 91.6% chemosuppresion
shown in Table 2. Percentage suppression established that showed (p=0.0001) while ethyl acetate (33.9%) and ethanol (35.6%) stem-
aqueous leaf has the highest at 100 mg/kg with 54.8% which was bark gave the lowest chemosuppression as shown in Table 2. The

Figure 4. Gas Chromatography-Mass Spectrometric (GC-MS) chromatograms of M. lutea ethanol stem-bark (a), methanol leaf (b) and
methanol stem-bark (c) extracts.

Table 2. Antimalarial properties of M. lutea leaf and stem-bark extracts on P. berghei-infected mice at 100-400 mg/kg.
Extracts 100 mg/kg 200 mg/kg 400 mg/kg
% Parasitaemia % Chemosuppression % Parasitaemia % Chemosuppression % Parasitaemia % Chemosuppression

Negative control (10 mL/kg) 17.12±0.72 0.00 17.12±0.72 0.00 17.12±0.72 0.00
AQ leaf 7.74±0.090a,c 54.81±0.53n 6.22±0.15e 63.69±0.88r,s 5.99±0.060i,j 64.99±0.35v
AQ SB 9.04±0.20b 47.20±1.18m 8.19±0.25f,f 52.16±1.48o,p,q 7.30±0.066k 57.37±0.38t,u
EtOAc leaf 8.64±0.083a,b 49.55±0.48m,n 7.72±0.35f 54.89±2.02o,p,q 6.81±0.16j,k 60.20±0.93u
EtOAc SB 11.32±0.29d 33.89±1.66l 8.37±0.20f 51.09±1.15o,p 7.71±0.24k 54.94±1.43t
EtOH leaf 9.39±0.43b 45.17±2.51m 8.86±0.074f 48.23±0.43o 7.38±0.028k 56.90±0.16t,u
EtOH SB 11.03±0.27d 35.59±1.59l 5.59±0.48e 67.33±2.83r 5.14±0.37i 69.96±2.15w
MeOH leaf 8.49±0.60b,c 50.41±3.48m,n 7.09±0.94f,g,h 58.58±5.49q,s 5.61±0.27i 67.23±1.60v,w
MeOH SB 8.13±0.67b,c 52.52±3.93m,n 6.40±0.35e,h 62.60±2.05r,s 5.69±0.48i 66.78±2.83v,w
ACT (4 mg/kg) 1.43±0.069 91.63±0.40 1.43±0.069 91.63±0.40 1.43±0.069 91.63±0.40
Data are expressed as mean ± Standard Deviation, SD; the same superscripted letter within the column means p=0.05; SB, Stem-Bark; AQ, Aqueous; Etoac, Ethyl Acetate, Etoh, Ethanol; Meoh, Methanol; ACT,
Artemisinin-Based Combination Therapy.

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medium dose of 200 mg/kg showed an increase in the activity of The result on phytochemicals was in accordance with a review
ethanol stem-bark with the highest chemosuppression of 67.3%, of Genus Markhamia phytochemicals and pharmacology by
which was comparable to the activities of aqueous leaf (p=0.64) Ibrahim et al.,7 who reported that the species are known for the
and methanol stem-bark (p=0.33) extracts with 63.7 and 62.6%, presence of biologically active substances like flavonoids,
respectively. At 400 mg/kg, ethanol stem-bark extract gave the best saponins, steroids, terpenes, and terpenoids, phytosterols, tannins,
activity with 70.0% reduction, which was comparable to those of phenols, coumarins, and quinones. Likewise, it was reported that
aqueous leaf, methanol leaf, and stem-bark with 65.0, 67.2 and the flowers, leaves, and stem-bark of M. lutea contain alkaloids,
66.8%, respectively, but the activity was significantly different quinones, saponins, tannins, phenols, and terpenes.6,8 The com-
from that of positive control with 91.6% chemosuppression (Table pounds identified by GC-MS are rich in esters and steroids, and
2). The effective doses with 50 and 90% activity (ED50 and ED90) these groups have been reported for antiplasmodial activity, for
results showed that aqueous and methanol leaf, ethanol, and instance, a report showed glycosides of stigmasterol inhibited
methanol stem-bark gave the lowest ED50 of 237.5 and 240.6, Plasmodium falciparum growth using 3D7 strain by schizont inhi-
233.8, and 236.6 mg/kg, likewise gave lowest ED90 of 427.6 and bition maturation assay.18 With the presence of stigmasterol in this
433.0, and 420.9 and 425.9 mg/kg, respectively. Meanwhile, ethyl study and other steroids, possibly there could be the presence of
acetate stem-bark gave the lowest ED50 and ED90 of 291.5 and sugar moiety in the extract that has contributed to the antimalarial
531.2 mg/kg, respectively, as shown in Figure 5. activity in addition to the other components that the GC-MS could
This study screened the phytochemicals present in Markhamia not identify due to the high temperature involved.
lutea leaf and stem-bark extracts of aqueous, ethanol, ethyl acetate, The acute toxicity result showed that all the extracts tested
and methanol, quantified the total flavonoid, and phenolic con- were acutely safe. It was reported that the extract of this plant can
tents, evaluated the acute toxicity level and evaluated their anti- cause the regeneration of tissue, which shows that the plant can
malarial effects on Plasmodium berghei NK65 infected mice. The lead to curative properties against degeneration.9
results, as indicated, presented the presence of a range of phyto- Alkaloids, flavonoids, and phenolics levels reported in this
chemicals, including saponins, alkaloids, flavonoids, and ter- study have been reported to have many therapeutic values, includ-
penoids in most of the extracts, in particular ethanol and methanol ing antimalarial properties, and this could have contributed to the
extracts. There was an appreciable amount of alkaloids, antimalarial properties exhibited in ethanol and methanol. Some of
flavonoids, and phenolic contents in the extracts, with ethanol and these components could have possibly exhibited the activity syner-
methanol giving the highest levels. The HPLC fingerprint of the gistically. Some terpenoidal and steroidal, indole, isoquinoline,
extracts of M. lutea leaf and stem-bark was carried out for identi- benzylisoquinoline, hasubanane, naphthoisoquinoline, phenan-
fication of the extracts, and the results are reproducible following throindolizine, etc. Alkaloids have been reported for antimalarial
the condition. There was a dose-dependent in the antimalarial activities, and the research continues on these promising phyto-
activities of all the extracts with a significant reduction in the par- chemical groups.19-21 In addition, several flavonoids, including
asitaemia levels of the animal dosed with aqueous leaf, ethanol flavones, have been reported for either antiplasmodial or anti-
stem-bark, methanol leaf, and stem-bark extracts, and they pos- malarial properties.22-24 The activity expressed in the ethyl acetate
sessed the highest percentage chemosuppression with lowest ED50 extract could have been due to the presence of flavonoids, and it
and ED90. was established that flavonoids are phenolic, which was reported

Figure 5. Effective doses (ED50 and ED90) of M. lutea leaf and stem-bark extracts. Data are expressed as Mean ± Standard Deviation, SD,
same superscripted letter means p=0.05; SB, Stem-Bark; AQ, Aqueous; EtAc, Ethyl Acetate, EtOH, Ethanol; MeOH, Methanol.

[page 26] [Infectious Diseases and Herbal Medicine 2025; 6:431]

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to possess antimalarial activity.23 The higher antimalarial proper- curative anti-inflammatory activity attenuates paclitaxel toxic-
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Online supplementary material.

Supplementary Table 1. Gas Chromatography-Mass Spectrometric (GC-MS) profile of M. lutea active extracts.

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