In Vitro Antimalarial Susceptibility of Plasmodium falciparum and Plasmodium berghei Isolates to Selected Antimalarial Agents, Column Chromatographic Subfractions of Glyphaea brevis Leaves Extract and FTIR and GCMS of SF8

Malaria still remains a life-threatening parasitic disease with universal targets set for control and elimination. This study aimed to evaluate the in vitro antimalarial susceptibility of Plasmodium falciparum isolates and Plasmodium berghei to selected antimalarial agents and column chromatographic subfractions of Glyphaea brevis leaves extract and FTIR and GCMS of SF8. Trager and Jensen as well as World Health Organisation (WHO) standardised in vitro micro-test system methods were used to determine susceptibility on the patients’ blood samples; Column chromatographic procedure was carried out to obtain 11 pooled fractions; FTIR and GCMS were used to determine functional groups and phytochemicals respectively. In vitro anti-plasmodial activity against P. falciparum clinical isolates had IC50 range of 1.03 μg/mL–7.63 μg/mL while their IC50 against P. berghei ranges from 4.32 μg/mL–7.89 μg/mL. Subfraction 8 (SF8) had the least IC50 of 4.32 μg/mL. The FTIR spectrum showed the presence of isoprenoid, alcohol, phenol, alkane, alkenes, ester, carboxylic acids, aromatics and nitro compounds while GCMS identified dodecanoic acid, methyl ester; carotol; hexadecanoic acid, methyl ester; 9-octadecenoic acid (Z)-, methyl ester (oleic acid); methyl stearate; heptadecanoic acid, 16-methyl-, methyl ester; all with their antimalarial reported activities. In conclusion, G. brevis has a great potential for drug development against malaria parasite since it inhibited schizont growth and possesses phytocompounds with antimalarial report.


INTRODUCTION
Malaria still remains one of the major and most devastating human parasitic infectious diseases in the world, responsible for high rate of morbidity and mortality in developing countries (Tindana et al. 2021).An estimate of 241 million cases of malaria and 627,000 malaria deaths occurred globally in 2020 (WHO 2022).Millions of deaths attributable to malaria are still being recorded with a huge epidemiologic burden in Africa and continues to cripple the economic development in the region.In Nigeria, malaria is responsible for 60% outpatient visits to health facilities, 30% childhood death, 25% of death in children under one year and 11% maternal death.Also, the financial loss due to malaria annually is estimated to be about by 132 billion Naira (USD318 million) in form of treatment costs, prevention, loss of man-hours and so on (Federal Ministry of Health Nigeria 2017); globally, it has been estimated that USD5.6 billion is required annually to remain on track toward WHO global malaria strategy targets (Oladipo et al. 2022) The constant evolution of the malaria parasite has rendered the cheapest and most widely available antimalarial treatments ineffective, especially with the reports on the increasing resistance of Plasmodium falciparum to currently used artemisinin-based compounds (Cui et al. 2012;Nassirou et al. 2015).There is great concern that malaria parasite may soon develop total resistance to modern approach, thereby making medicinal plants to generate recent attention in the treatment of malaria.There is an urgent need to explore the naturally endowed rich biodiversity of communities through research that could translate to benefits for mankind (Olasehinde et al. 2014).
Glyphaea brevis (G.brevis) belongs to the family Tiliaceae; it is a spreading shrub, climber or small tree up to 8 m high.It is very common in undergrowth of closed forest, secondary jungle and on riverbanks, lowlands to sub-mountain and widespread in tropical Africa and South America.It is considered a vegetable in some cultures (Anjuwon et al. 2015;Okafor et al. 2016).Traditionally G. brevis is used in Africa and South America to treat various disease conditions of man including fevers, gonorrhoea, dysentery, stomach troubles, lung troubles, parasitic infections, convulsions and constipation.N-butanol fraction of methanol crude extract (NBFx-MExt) of G. brevis leaves has showed suppressive antiplasmodial activity against Plasmodium berghei infected mice (Anjuwon et al. 2015).
In recent years, G. brevis has come under the limelight of researchers in various parts of the world due to its broad ethno-medicinal uses.Its crude extracts and phytochemicals showed a wide spectrum of activity including anti-infective, antioxidant, anti-allergic, anti-inflammatory, anticonvulsant, hypocholesterolaemic activity, weight control and blood glucose control activity, glycosidase inhibitory activity and hepatoprotective effect (Osafo & Boakye 2016).
Therapeutics based on combinations with artemisinin and derivatives (ACTs), perhaps the most effective treatment against falciparum malaria recommended by the World Health Organisation (WHO) emerged as the best practical solution in overcoming the resistance of select strains (WHO 2008).The multidrug-resistant strains of Plasmodium represent a great threat in relation to the treatment and prevention of the disease leading to limitations in drugs application and current primary cause of control failure (Akuodor et al. 2017).This infectious disease is highly fatal, and may predispose the patient to opportunistic infections unless diagnosed and treated in a timely fashion (Nardos & Makonnen 2017).
The use of medicinal plants in modern medicine is less valued due to the fact that though hundreds of plants are used in the world to prevent or to cure diseases, scientific evidence in terms of modern medicine is lacking in some cases.Since its N-butanol fraction has been shown to have suppressive antiplasmodial effect (Anjuwon et al. 2015), further purification method is necessary if the plant is to be considered for drug design and development.

METHODOLOGY Plant Sample Collection, Extraction and Chromatographic Processes
Fresh leaves of G. brevis was collected from Irun Akoko, Ondo State and was brought for identification at the herbarium unit of Botany Department, Ahmadu Bello University, Zaria where a voucher number 2634 was obtained and specimen deposited; the air dried and powdered G. brevis leaves extraction procedures have been earlier reported (Anjuwon et al. 2019) to obtain N-butanol fraction of methanol crude extract (NBFx-MExt).
A preliminary thin layer chromatography (TLC) was carried out on the NBFx-MExt of G. brevis leaves to determine the best solvent system that would give the highest number of distinct band of components.A pre-coated aluminium chromatographic plate with silica gel was used.The NBFx-MExt was dissolved in the solvent used for extraction and was applied several times on the plate using a micro haematocrit capillary tube until the load quantity was adjusted sufficiently for the experiment.The plate was placed in a chromatographic tank and eluted with a varying ratio of the different solvent system as the mobile phase in order to get the suitable solvent.Thereafter the plate was removed, dried in air and developed using 20% sulphuric acid in methanol to identify most suitable solvent system.
Column chromatography of the NBFx-MExt, as a follow up from the TLC plate result, was carried out.The column was conditioned using best solvent system with distinct bands (from the TLC analysis previously carried out) to pack the silica gel of 50 µm-200 µm mesh size.The NBFx-MExt (2.0 g) of G. brevis leaves was emptied into a porcelain mortar, 5 g of silica gel was added and a pestle was used to macerate the mixture to homogeneity.The mixture was carefully packed on top of the column chromatography and was then eluted with the varying solvents system.Approximately 50 mL/18 min flow rate was maintained.The collected elutes (fractions) were evaporated to near dryness.Each of the fractions was weighed and spotted on TLC plate, it was viewed under UV radiation.The plate was further sprayed with 20% sulfuric in methanol solution and dried for 15 min at 110°C (Ejele et al. 2012).This TLC procedure enabled similar fractions to be pooled together based on the pattern and retention factor (R f ) values of the spots on the TLC plate into main fractions respectively.The R f values were calculated using this formula: R f = Dc /Ds where Dc is distance travelled by component and Ds is distance travelled by solvent front.
The combined yield of fractions with the same R f value and combined yield as percentage (%) of the NBFx-MExt fractionated were calculated.The intermediate pooled fractions were kept in a refrigerator for further analysis.

Malaria Patients Blood Sample Collection and Study Protocol
This study was carried out at Department of Pharmaceutical Microbiology, A.B.U Zaria, Nigeria.The clinical isolates of P. falciparum were obtained from patients attending A.B.U Medical Service Centre.Blood samples of volunteered patients presented to the parasitology laboratory for malaria test with symptoms of uncomplicated malaria, mono infection with P. falciparum and parasitaemia level (1,000-80,000) asexual parasites per micro litre of the blood were recruited for the in vitro study were collected via venipuncture by Hospital Clinicians/Laboratory Scientist and dispensed in sterile EDTA tubes.The collected clinical blood samples were transported immediately to Pharmaceutical Microbiology laboratory, A.B.U Zaria for further analysis.The in vitro susceptibility test was carried out according to the standard culture techniques of Trager and Jensen (1976) and Mark III microtest (Singh et al. 2015;WHO Malaria Unit 2001) with some modifications under strict aseptic condition.
Plasmodium berghei [susceptible (NK-65) strain] was obtained from National Institute of Medical Research (NIMR), Lagos, Nigeria.P. berghei has been used for in vitro assay as reported by Longley et al. (2015).The berghei parasite was passage into an albino mouse and continuous check and parasite maintenance were ensured.

Stock Solutions of Selected Antimalaria Drugs, N-butanol Fraction and Sub-Fractions Preparation
The N-butanol fraction and sub-fractions samples were dissolved in sterile distilled deionised water to obtain the highest working concentration of 160 µg/mL according to reported IC 50 3.60 µg/mL (Lawal et al. 2015).Stock solutions of Chloroquine, Artemether, Artemether-Lumefantrine (Coartem®) and Artemisinin-piperaquine (Artequick®) (standard controls) were prepared in 70% ethanol and distilled deionised water, respectively.All stock solutions were sterilised by filtration through 0.22 µm microfilters.

Susceptibility Assay
Antimalarial drug susceptibility assay was carried out using the method of (Singh et al. 2015) adopted from WHO Malaria Unit ( 2001) with slight modification.For in vitro assay, 10 µL of the antimalarial agents was dispensed into wells B-F of graded concentration (well A was without drug).The solutions were left to dry to powder.Ninety microliters (90 µL) of complete culture medium (RPMI 1640 and O + serum) was added into each well (A-F), then finally 10 µL of the parasitised erythrocytes dispensed into each of the wells.
For the extract, graded solutions of the G. brevis subfractions was prepared in sterile distilled deionised water and 10 µL each was also loaded into the microtiter well plate.Well B-F had the graded concentration of the extracts in the increasing doubling concentration from B to F, where A is the control wells that contain the patients' blood and complete culture medium in ratio 1: 9 (10 µL: 90 µL) after blood samples were standardised and the extract well had the mixture of graded concentrations of the extracts, parasitised blood samples and complete culture medium in ratio 1:1:8 (10 µL:10 µL:80 µL) from well B through F. All these were done under strict aseptic condition.
All the plates were shaken gently without lifting it from the laboratory bench to properly mix the contents of the wells.The plates were incubated (5% CO 2 ) at 37°C for 30 h (Trager & Jensen 1976).After incubation, the red cells decant were transferred to a clean grease free microscope slide by removing the supernatant from each well.The smears were allowed to dry for 24 h and stained with 5% Giemsa for 30 min.

Microscopic Examination
Thick and thin films were prepared for microscopic examination of the Plasmodium species.Thin films were fixed with methanol for 60 sec, rinsed gently and allowed to dry.Both blood films were stained with 10% Giemsa of pH 7.1 for 30 min (WHO Malaria Unit 2001).Films were allowed to air-dry and were observed microscopically using x100 (oil immersion) objective lens as described by Cheesbrough (2000).The thin films were used to identify the parasites species while the thick films were used to determine the parasite density.Average parasite counts per 10 microscopic fields were determined and multiplied by a factor of 500 to estimate the number of parasite per microliter of blood (Greenwood & Armstrong 1991).The parasitaemia level within 1,000-80,000/µL of samples were recruited for the in vitro antimalarial susceptibility test (WHO Malaria Unit 2001).

Post Culture Slides Examination
The stained thick films were examined under oil immersion objective (×100) lens and parasite densities (number) were counted and related with the parasite density in the control well.Schizont growth inhibition per 200 asexual parasites was counted in 10 microscopic fields.The control parasites culture freed from G. brevis subfractions or drugs were considered as 100% growth.The percentage inhibition per concentration was calculated using the formula (Ngemenya et al. 2006; WHO Malaria Unit 2001): % inhibition per concentration = (% parasitaemia in control wells -% parasitaemia of the control) × 100 The inhibitions were fed into the IC 50 values, the concentration required to inhibit schizont growth by 50% were determined by linear regression analysis using Microsoft Excel 2016 from the schizont growth inhibition curves (Log of concentration versus percent inhibition) generated from each parasite-plant fraction interaction (Mustofa et al. 2007).Drug resistant P. falciparum parasites were identified with IC 50 values greater than the peak plasma concentration of the antimalarial drugs.

Fourier Transform Infrared Spectrophotometer (FTIR) Analysis
The IR transmission spectra of the SF8 were obtained using an FTIR spectrophotometer (Shimadzu, Japan).The powdered sample was loaded into FTIR spectroscope and the characteristic peaks were recorded using the FTIR spectrophotometer (Dwivedi et al. 2021).

Gas Chromatography Mass Spectroscopy (GC-MS) Analysis
The subfraction 8 of G. brevis leaves extract with the least IC 50 was accurately weighed and dissolved in methanol as the solvent.GC-MS experiment was then carried out using standard procedure (Waheed 2012).

Ethical Consideration
Ethical clearance was obtained from A.B.U Zaria Health Service Ethical committee.
Written informed consent was obtained from patients prior to collection of their blood samples for this study.

In Vitro Susceptibility Pattern of Antimalarial Agents
The screening of the N-butanol fraction and the 10 subfractions obtained from the column chromatography of the fraction was done using Plasmodium falciparum and P. berghei to determine their in vitro susceptibility pattern.
The IC 50 , i.e., the concentration of the test antimalarial agent that inhibited 50% of the parasite were calculated and compared their peak plasma concentration to determine the susceptibility of each drug and subfractions.Eight (8) blood samples obtained from patients were first screened to obtain three (3) chloroquine (CQ)-resistant clinical isolates.The three (3) CQ-resistant clinical isolates had peak plasma concentrations 6.56, 5.11 and 6.95 µmol as against 4.47 µmol for CQ as presented in Table 2.
The subfractions of N-butanol fraction of G. brevis leaves showed good anti-plasmodial activity in vitro against P. falciparum clinical isolates with IC 50 range of 1.03 μg/mL-7.63 μg/mL while their IC 50 against P. berghei ranges from 4.32 μg/L-7.89μg/L as presented in Table 3. Subfraction 8 (SF8) had the least IC 50 of 4.32 µg/mL, hence, it was considered for the FTIR and GCMS studies.

Fourier Transform Infrared (FTIR) Spectroscopy Peak Values and Functional Groups of SF8 of G. brevis Leaves Extract
The SF8 of G. brevis leaves extract was analysed for functional groups identification using the FTIR spectroscopy.The FTIR spectra of the SF8 of G. brevis leaves extract as presented in the Table 4 shows the absorption peak characteristic of SF8 at a particular wavenumber, the functional groups and the corresponding bonds with movement.The FTIR spectrum showed the presence of isoprenoid, alcohol, phenol, alkane, alkenes, ester, carboxylic acids, aromatics and nitro compounds.

Gas Chromatography Mass Spectrometry Analysis of the Sub-Fraction 8 of N-Butanol Fraction of G. brevis Leaves Extract
The results of the GC-MS analysis identified compounds that are present in the SF8 of N-butanol fraction of G. brevis leaves extract.The various components, their peak area and retention time (RT) as well as their reported biological activities are presented in the

DISCUSSION
Malaria still remains a life-threatening parasitic disease that has been the highpoint of many nations of the world's declarations, with universal targets set for control and elimination since more than two decades (Teng et al. 2019).
Screening of plants has become an attractive way of recognising new lead candidates for drug development against parasitic diseases.Plants are used as bases to the development of new synthetic/semi-synthetic drugs (Banzragchgarav et al. 2021).
This research explored in vitro antimalarial activity of the various column chromatographic subfractions (SFs) obtained from N-butanol fraction of G. brevis leaves extract against P. falciparum and P. berghei infection.Of all the subfractions that were evaluated, SF8 had the minimum inhibitory concentration (IC 50 ) of 4.32 µg/mL.The subfractions of N-butanol fraction of G. brevis leaves showed good in vitro anti-plasmodial activity against P. falciparum clinical isolates with IC 50 range of 1.03 µg/mL-7.63 µg/mL which is classified under active (0.03 < IC 50 < 2.5) to moderately active (2.5 < IC 50 < 10); their IC 50 against P. berghei ranges from 4.32 µg/mL-7.89µg/mL as moderately active (2.5 < IC 50 < 10) (Eseyin et al. 2021).These IC 50s obtained in this research were very similar but lower than the reported values in Laryea et al. (2021).
G. brevis leaves N-butanol fraction and subfractions showed inhibitory effects on chloroquine resistant isolates of the malarial parasite hence giving a clear indication that the plant extract showed anti-plasmodial effects.This concurred with a study done by Marie et al. (2018) that presented three plants extracts having noteworthy anti-plasmodial activities, with good selectivity against chloroquine-sensitive and -resistant strains of P. falciparum.
Phytochemical investigations on medicinal plants like G. brevis have shown that it contains phenolic compounds, such as flavonoids, flavones and lignans, have been frequently investigated for their antimalarial activities and found to exhibit both in vitro and in vivo effectiveness against Plasmodium strains (Mamede et al. 2020;Shen et al. 2020).Thus, the expected outcome obtained from the study, being therapeutic in their effects against malaria parasites; agreeing with the research of Banzragchgarav et al. (2021).
FTIR spectroscopy is a rapid, non-destructive and high-resolution analytical technique used for the identification of various functional groups or chemical bonds found in plant extracts (Kalaichelvi & Dhivya 2017).The wavelength of light absorbed was the relevant feature of chemical bond as can be seen in the annotated spectrum.By interpreting the infrared absorption spectrum, the chemical bonds in a compound can be determined.Moreover, FTIR spectroscopy is an established time-saving method to characterise and identified functional groups (Kalaichelvi & Dhivya 2017).
Thus, in this research, FTIR technique was used to assess the IR fingerprint in SF8 of G. brevis leaves extract.
FTIR analysis revealed the presence of hydroxyl, phenols, alkanes, esters, carboxylic acids and nitro compounds that are vital functional groups in majority of pharmaceutically active phyto-constituents as also reported in the study of Dwivedi et al. (2021).The analysis revealed the presence of polyphenols and flavonoids due to O-H stretching, terpenes due to C-H group (Maobe et al. 2013); the presence of alkanes confirm the presence of alkaloids (Pawar & Kamble 2017) in G. brevis.All these compounds belong to secondary plant metabolites.The existence of characteristic functional groups of carboxylic acids, alcohols, phenols, esters, etc. could be responsible for the various medicinal properties of G. brevis (Baltacıoğlu et al. 2021;Kalaichelvi & Dhivya 2017).Studies also reported that these groups are present in plants such as leaves extract of V. negundo L. (Dwivedi et al. 2021;Janakiraman & Jeyaprakash 2015;Pawar & Kamble 2017).
The chemical constituents of SF8 of N-butanol fraction of G. brevis leaves extract were analysed by GC-MS, revealed the presence of 12 components.The GC-MS running time was 38.44 min, and 12 components were detected.The GC-MS spectra showed high concentration of fatty acids.Unsaturated fatty acids are not produced in humans but have been shown to possess diverse pharmacological properties and aid in general functioning of the human body.In the study of Orabueze et al. (2020), they reported on antimalarial properties of polyunsaturated fatty acids, especially the essential fatty acids and their bioactivity increases with their degree of unsaturation.Polyunsaturated fatty acids such that were identified in SF8 (Hexadecanoic acid, methyl ester; 9-octadecenoic acid methyl ester and 9-Octadecenoic acid (Z)-, methyl ester (Oleic acid)) were among the identified compounds in their active antiplasmodial fractions of their plant extract (Orabueze et al. 2020).Specifically, 9-Octadecenoic acid (Z)-, methyl ester and other ester compounds have been reported to possess anti-gastric, anti-(breast) cancer, antioxidant, anti-inflammatory, antiandrogenic and anemiagenic properties (Duraisamy & Selvaraju 2020;Lalitha & Palani 2017).For Oleic acid, it has been reported to have an anti-inflammatory potential through the activation of different pathways of immune competent cells, they also lower heart attack risk, artherosclerosis, and improved the effectiveness of herceptin which aids in cancer prevention; it also produces a suppression of lymphocyte proliferation, an inhibition of cytokine production and a reduction in Jurkat cells activity (Oche et al. 2017).It has been suggested that oleic acid causes disruptions of the plasma membrane, as well as depolarization and consequent breaching and activates an influx of calcium into the cells (Zahara et al. 2022).
Carvone, has been chosen as starting material for the synthesis of antimalarial agent Artemisinine, a new lactone sesquiterpene containing one peroxide binding (Araujo et al. 1991).G. brevis may possibly be a good source for its extraction.Also, carotol is sesquiterpene alcohol and previous study has shown that carotol may be involved in allelopathic interactions expressing activity as an antifungal, herbicidal and insecticidal agent (Fard et al. 2020).
It can be concluded that G. brevis is a good medicinal plant that may be considered as a potential for malaria drug development considering the phytocompounds present in it as well as their impressive antiplasmodial activity.It is therefore recommended that further purification and identification of pure (single) compound can be researched upon for furtherance increase in the alternative options of malaria chemotherapy.

Table 1 :
Column chromatography of N-butanol subfractions of Glyphaea brevis leaves extract.
Notes: SF = subfraction; PPC = peak plasma concentration; * = Resistant (at peak plasma level of the drugs and agents.Sample 1 to 3 are P. falciparum isolates while sample 4 is P. berghei isolate.

Table 4 :
FTIR peak values and functional groups of SF8 of G. brevis leaves extract.

Table 5 :
Reported activity of compounds identified in sub-fraction 8 of N-butanol fraction of G. brevis leaves extract.