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Trop Life Sci Res
Trop Life Sci Res
Tropical Life Sciences Research
Tropical Life Sciences Research
1985-3718
2180-4249
Penerbit Universiti Sains Malaysia

10.21315/tlsr2024.35.2.8
tlsr-35-2-167
Articles
Bioassay-Guided Fractionation of Acetone and Methanol Extracts of Quercus infectoria Galls with Antimalarial Properties
Hamidon Nurul Hammizah Conceptualization Methodology Investigation Formal analysis Writing - original draft Writing - review & editing 12
Dona Anjana Chamilka Thuduhenage Investigation Formal analysis Writing - original draft Writing - review & editing 1
Zin Nik Nor Imam Nik Mat Investigation Formal analysis Writing - original draft Writing - review & editing 12
Nordin Nurul Izza Conceptualization Methodology Writing - review & editing 2
Sulaiman Shaida Fariza Conceptualization Methodology Writing - review & editing 3
Abu-Bakar Nurhidanatasha Conceptualization Methodology Writing - review & editing 1*
1 School of Health Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
2 SIRIM BERHAD, Institute of Biotechnology Research Centre, Block 19, No. 1, Persiaran Dato Menteri, Section 2, 40700 Shah Alam, Selangor, Malaysia
3 School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM Pulau Pinang, Malaysia
* Corresponding author: natashaa@usm.my
7 2024
31 7 2024
35 2 167185
18 6 2023
03 1 2024
© Penerbit Universiti Sains Malaysia, 2024
2024
https://creativecommons.org/licenses/by/4.0/ This work is licensed under the terms of the Creative Commons Attribution (CC BY) (http://creativecommons.org/licenses/by/4.0/).
The antimalarial properties of crude extracts from Quercus infectoria galls were investigated through bioassay-guided fractionation. Acetone (QIA) and methanol (QIM) crude extracts have been reported to have promising antimalarial activity against Plasmodium falciparum (3D7 strain). These extracts were subjected to fractionation using automated preparative high-performance liquid chromatography (prep-HPLC) to identify the most active fractions. Nine fractions were isolated from each extract, of which the fractions QIA11 and QIM16 showed antimalarial activity, with IC50 values of 17.65 ± 1.82 μg/mL and 24.21 ± 1.88 μg/mL, respectively. In comparison, the standard antimalarial drug artemisinin has an IC50 value of 0.004 ± 0.001 μg/mL). Through high-resolution liquid chromatography coupled with mass spectrometry (HR-LCMS) analysis of the fractions, four known compounds were successfully identified: gallic acid, ellagic acid, 1,3,6-tris-o-(3,4,5-trihydroxybenzoyl)-beta-d-glucose and 1-O,6-O-digalloyl-beta-D-glucose.

Abstrak

Sifat-sifat antimalaria ekstrak-ekstrak mentah dari biji Quercus infectoria dikaji melalui fraksinasi berpandukan bioasai. Ekstrak mentah aseton (QIA) dan metanol (QIM) dilaporkan sebelum ini mempunyai aktiviti antimalaria terhadap Plasmodium falciparum (strain 3D7). Ekstrak-ekstrak ini melalui fraksinasi menggunakan persediaan automatik kromatografi cecair berprestasi tinggi (prep-HPLC) untuk mengenal pasti fraksi yang paling aktif. Sembilan fraksi telah diasingkan daripada setiap ekstrak, di mana fraksi QIA11 dan QIM16 menunjukkan aktiviti antimalaria dengan nilai IC50 17.65 ± 1.82 μg / mL dan 24.21 ± 1.88 μg/mL, masing-masing dibandingkan dengan ubat antimalaria standard, artemisinin (IC50 = 0.004 ± 0.001 μg/mL). Analisa menggunakan kromatografi cecair beresolusi tinggi bersama dengan spektrometri jisim (HR-LCMS) berjaya mengenal pasti empat sebatian: asid gallic, asid ellagic, 1,3,6-tris-o-(3,4,5-trihydroxybenzoyl)-beta-dglukosa dan 1-O, 6-O-digalloyl-beta-D-glukosa.

Quercus infectoria
Antimalarial Activity
Plasmodium falciparum
Preparative-HPLC
HR-LCMS
Kata kunci

Quercus infectoria
Aktiviti Antimalaria
Plasmodium falciparum
Persediaan-HPLC
HR-LCMS
Ministry of Higher Education Malaysia for providing the Fundamental Research Grant SchemeFRGS/1/2019/STG03/USM/03/3
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pmcHighlights

Acetone (QIA) and methanol (QIM) crude extracts were reported to have promising antimalarial activity against Plasmodium falciparum (3D7 strain).

The antimalarial properties of crude extracts from Quercus infectoria galls were investigated through bioassay-guided fractionation and identification of compounds through high-resolution liquid chromatography coupled with mass spectrometry (HR-LCMS) analysis.

Four known compounds were successfully identified which was isolated from two most active fractions, fractions QIA11 and QIM16.

INTRODUCTION

Malaria is a parasitic disease that has a negative impact on global health (Ferguson 2018). In many developing countries, the disease causes major complications leading to high morbidity and mortality (Nigussie & Wale 2022). The five Plasmodium species responsible for human malaria are P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi (Nigussie & Wale 2022). P. falciparum is the most threatening species in terms of morbidity and mortality (Ouji et al. 2018). According to the World Health Organisation (WHO), an estimated 247 million cases of malaria occurred worldwide in 2021, compared with 245 million cases in 2020 (WHO 2021), which resulted in 619,000 deaths. The WHO African Region accounted for the majority of malaria cases (95%) and deaths (96%), with children under five years of age (80%) being those mainly affected by the disease (WHO 2021).

Plants are an excellent source of novel natural products. Medicinal plants have long been used as a source of therapeutic agents and they have demonstrated beneficial uses in a variety of applications (Yuan e t al. 2016). Despite intense competition from synthetic compounds, numerous bioactive substances found in plants have been shown to be significantly important in advancing human health (Tahir et al. 2022). Between 1981 and 2014, the Food and Drug Administration (FDA) approved 1,562 drugs in the United States, of which 4% were unaltered natural products, 9% were botanical drugs, 21% were natural derivatives and 4% were synthetic drugs containing natural pharmacophores (Newman & Cragg 2020). These findings have increased the interest in herbal medicine (Che & Zhang 2019).

There are numerous examples of effective contemporary drugs based on medicinal plants, especially with the discovery of new drug candidates for the treatment of many infectious diseases like malaria (Nakalembe et al. 2019; Atanasov et al. 2015). Medicinal plants have enormous potential for use in the effective management of various strains of malaria parasites, including those resistant to the available antimalarial drugs (Shah et al. 2014). In the absence of viable malaria vaccinations, accurate diagnosis and treatment remain the best hope of avoiding serious consequences. Several antimalarial medications have been discovered for this purpose, including mefloquine, chloroquine, quinine, proguanil, atovaquone, sulfadoxine-pyrimethamine and artemisinin (Arya et al. 2021). Artemisinin, which was discovered by Tu Youyou in the 1970s, originally came from the plant Artemisia annua, which was commonly used in Chinese medicine (Tu 2011). Artemisinin-based combination therapies (ACTs) are recognised for their effectiveness to swiftly reduce the number of Plasmodium parasites in the blood of patients with malaria (Ouji et al. 2018). However, P. falciparum resistance to ACTs has emerged, posing a threat to the global elimination of malaria. Due to this phenomenon, the development of new antimalarial drugs especially derived from medicinal plants is urgently needed (Shah et al. 2014).

Quercus infectoria, commonly referred to as the gall oak tree, is a small shrub 4–6 feet tall which originates mainly from Greece, Asia Minor and Iran (Abdul Haque et al. 2016). The galls of the plant are formed when the wasp species Adleria gallae-tinctoria or Cynips gallae-tinctoria deposit their eggs on the branches of young trees. The subsequent enzymatic reaction results in the appearance of hard galls (Wan Nor Amilah et al. 2022). These globular-shaped galls, known as majuphal or machakai in India and manjakani in Indonesia and Malaysia (Samuelsson 1999; Fatima et al. 2001), are 0.8 cm–2.5 cm in diameter and hard in consistency. They have a rough surface and a greyish-brown to brownish-black colour (Shaikh Imtiyaz et al. 2013).

Traditional uses of Q. infectoria galls have prompted researchers to investigate and validate their biological activities and therapeutic uses. Extracts, fractions and single compounds of the galls have been shown to have various pharmacological activities, including antioxidant, anti-inflammatory, antitumoural, antibacterial, antiviral, antifungal and antimalarial activities (Wan Nor Amilah et al. 2022; Morales 2021; Iylia Arina & Harisun 2019; Basri et al. 2012; Nik Mat Zin et al. 2019a; 2019b). The phytochemicals of the Q. infectoria galls highlight the abundance of phenolic compounds belonging to the pyrogallol, quercetin, tannins, gallic acid and ellagic acid classes (Kheirandish et al. 2016; Dash et al. 2016; Tayel et al. 2018; Ma et al. 2020; Kamarudin, Nik Salleh, et al. 2021; Kamarudin, Muhamad, et al. 2021). The phenolic compounds of the galls have been hypothesised to have antimalarial effects on haemoglobin degradation and haem detoxification in the digestive vacuole of P. falciparum (Tajuddeen & Van Heerden 2019; Mamede et al. 2020).

The preliminary study revealed that aqueous, ethanol, methanol and acetone crude extracts from Q. infectoria galls had antimalarial activity against the chloroquine-sensitive strain (3D7) of P. falciparum, with IC50 values of 30.95, 20.00, 10.31 and 5.85 μg/mL, respectively (Nik Mat Zin et al. 2020). These extracts were non-toxic to normal kidney epithelial cells (Vero) and mildly toxic to normal embryo fibroblast cells (NIH/3T3). According to Berthi et al. (2019) and Jonville et al. (2008), the antimalarial activity of an extract can be considered very active with an IC50 < 5 μg/mL, promising with an IC50 of 6 μg/mL–15 μg/mL, moderate with an IC50 of 16 μg/mL–30 μg/mL, low with an IC50 of 31 μg/mL–50 μg/mL, and inactive if the IC50 > 50 μg/mL. Thus, the antimalarial activity of the acetone and methanol extracts in this study was classified as promising. Here, bioassay-guided fractionation was used to identify potential active compounds in the acetone and methanol crude extracts of Q. infectoria galls, as well as evaluate the antimalarial activity of their fractions.

MATERIALS AND METHODS

Chemicals and Reagents

Acetonitrile, acetone, methanol, formic acid and trifluoroacetic acid were HPLC-grade with 99% purity and purchased from Fisher Scientific (Malaysia) Limited. Ultrapure water with 18.2 Ώm was used for gradient elution in the prep-HPLC and HR-LCMS.

Plant Material and Extraction Procedure

Q. infectoria galls were purchased from a local market in Kota Bharu, Kelantan, Malaysia and authenticated at the Natural Medicinal and Product Centre, International Islamic University Malaysia (voucher specimen: PIIUM 0229-1). The galls were washed and dried at 50°C before being ground to obtain a powder. The acetone crude extract, which exhibited the highest antimalarial activity (Nik Mat Zin et al. 2020), was prepared by soaking 100 g of the powdered material in 500 mL of 100% acetone. The maceration technique was used at room temperature for 72 h and the extracts were filtered and concentrated using a rotary evaporator. The same process was followed to prepare the methanol crude extract using methanol (100%).

Fractionation of Gall Crude Extracts

Fractionation was achieved using prep-HPLC (GX-281 Purification System) combined with a Supelco RP-C18 preparative column (10 mm × 250 mm, 5 μm particle size, Merck, Germany). The extracts were dissolved in either acetone or methanol (100%) to achieve a concentration of 100 mg/mL. Subsequently, 100 μL of this solution was injected into the liquid chromatography. Then, 0.0085% trifluoroacetic acid (TFA) in deionised water (A) and acetonitrile (B) was used as the mobile phase. Peaks were detected at a wavelength of 254 nm and eluted with a gradient system mixture for 36 minutes. The gradient elution procedure was as follows: B was increased from 0% to 5% at 0 to 5 min and B was increased from 5% to 95% at 5 to 27 min. Next, B was flowed at 95% for 3 min and B was decreased from 95% to 5% for 6 min at a flow rate of 2.8 mL/min. The chromatographed fractions of acetone and methanol were collected individually and vacuum-dried below 45°C. The fractions were subjected to analysis of the antimalarial activity and compound identification using HR-LCMS (Harborne 1998).

Culture of the Malaria Parasite

A chloroquine-sensitive strain (3D7) of P. falciparum was kindly provided by the Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia (USM). It was maintained in culture flasks containing a complete culture medium (CCM) and washed type O+ human erythrocytes at 2% haematocrit, based on a previously devised protocol (Mohd-Zamri et al. 2017). Human blood was acquired from healthy donors who gave informed consent and were recruited at the School of Health Sciences, Health Campus, USM. The nature and risks of the study were approved by the Human Research Ethics Committee, USM (USM/JEPeM/18050263). Donors were notified of this before being recruited.

Synchronisation of the Malaria Parasite

The parasites were mainly at the ring stage (2% parasitaemia) upon confirmation by Giemsa-stained thin blood smears. They were synchronised through sorbitol treatment at a ratio of 100 μL of cell pellets per 1,000 μL of 5% D-sorbitol (w/v; Sigma Aldrich, Missouri, USA) to kill the mature-stage parasites (trophozoite and schizont stages) (Ibrahim & Abu-Bakar 2019). Synchronised ring-stage parasite-infected erythrocytes (2 h post-synchronisation) were used in the antimalarial activity assay.

Malarial SYBR Green I Fluorescence-based (MSF) Assay

The antimalarial activity of the acetone and methanol fractions was assessed through MSF assay using a previous method (Mohd-Zamri et al. 2017). Stock solutions of each fraction were diluted in the complete culture medium (CCM) at ten concentrations of two-fold dilutions into 96-well microtitre plates, and 20 μL aliquots of the fraction concentrations were transferred into individual wells in other plates containing 180 μL suspensions of synchronised ring-stage parasite-infected erythrocytes (2% parasitaemia, 2% haematocrit). Artemisinin (Sigma Aldrich, Missouri, USA) was used as a standard control, infected erythrocytes devoid of the fractions were used as a negative control and 100% DMSO was used as a positive control. Parasite plates were incubated for 48 h at 37°C in 5% CO2 incubator. After incubation, 180 μL aliquots of the cell suspensions were dispensed into new plates containing 20 μL solutions of 20× SYBR Green I (Invitrogen, Waltham, Massachusetts, USA), wrapped in aluminium foils and incubated for 1 hour at room temperature (Nik Mat Zin et al. 2019a; 2019b). The total fluorescence (TF) signal was measured with a microplate reader at the excitation (490 nm) and emission (530 nm) wavelengths. The percentage of parasite inhibition of each concentration was calculated as follows:

The mean of three half-maximal inhibitory concentration (IC50) values of the fractions was determined using probit regression analysis with GraphPad Prism software (Version 9).

LC-MS/MS Analysis of the Fractions

Analysis of the compounds present in the fractions with good antimalarial activity was performed using Dionex Ultimate 3000 RS UPLC with a Thermo Scientific Q Exactive Orbitrap Hybrid Tandem Mass Spectrometer. The column used was HSS XSelect Waters C18 (4.6 mm × 250 mm, 5 μm). The gradient was linear with water (A) and acetonitrile (B), both of which were buffered with 0.1% formic acid, starting at 5% B and equilibrated for 5 min. The gradient was increased to 95% after 30 min and held for 5 min. The gradient was equilibrated for 5 min before the next injection. The flow rate of 0.8 mL/min and the column temperature of 35°C were adjusted. The sample injection used was 3 μL. The mass detection was performed in both positive and negative atmospheric pressure ionisation-electrospray source modes. The drying gas temperature (250°C), gas flow (11 L/min), nebuliser pressure (110 psig), nebuliser assistant gas temperature (350°C), capillary voltage (400 V) and collision energy (30 eV) were set. Compound Discoverer 3.1 software was used to analyse the known and unknown compounds. The software was equipped with known online libraries (mzCloud and ChemSpider) and mzLogic algorithm was applied to rank the ChemSpider results.

Statistical Analysis

The dose-response curves from the experimental data were analysed using the GraphPad Prism 9 software package for Windows (San Diego, California USA). The data were further analysed using one-way ANOVA using the same software, with a significant difference at p < 0.05. Data from three different experiments were reported as mean and standard deviation (SD) values undertaken in triplicate.

RESULTS AND DISCUSSION

The Yield of Fractionated Extracts

The methanol crude extract produced the highest yield of dry powder (51.64%, w/v) from 100 g of the Q. infectoria gall powder, followed by the acetone crude extract (50.85%, w/v). The yields produced from these extracts were similar (P > 0.05) because their polarity indexes of methanol and acetone were the same (P’ = 5.1). The extraction of active chemical compounds depends greatly on the solvent’s polarity, mainly because polar molecules are easily extracted using polar solvents (Goli et al. 2005). Therefore, the solvent used for bioactive chemical extraction must be strategically chosen because it will affect the quantity and quality of the final extract (Zhang et al. 2018)

The crude extracts were then fractionated through a semi-preparative HPLC C18 column eluted with acetonitrile at gradient concentrations ranging from 5%–95%. The fractionation yield of the methanol and acetone extracts is shown in Table 1. The fractions QIA11 (58.88%, w/v) and QIM15 (29.80%, w/v) demonstrated the highest yield percentages for the acetone and methanol extracts, respectively. QIA11 had the highest yield due to the chromatographic co-elution between the peaks, as shown in Fig. 1. This was caused by closely eluting peaks of two or more compounds in the same fraction that were not chromatographically separated (Dworkin 2011). Peak resolution can be improved by optimising the method to increase the selectivity and efficiency of the chromatography (Vink 1972). The parameters that can be set are the chemistry of the mobile phase, the stationary phase, the temperature and the column particle size (Kanu 2021). Applying detection techniques such as mass spectrometry is the best way to distinguish co-eluting compounds that cannot be resolved through this analysis, as explained in Section 3.4 (Alseekh et al. 2021).

Prep-HPLC Analysis of the Gall Crude Extracts

Both methanol and acetone crude extracts underwent the fractionation process for the isolation and semi-purification of the extracts. Fractions were collected every 1 min based on the elution of the peaks at UV absorbance values of 210 nm and 254 nm. In total, 33 fractions were collected from the methanol extract, but only nine fractions proceeded for analysis of the antimalarial activity and compound identification (as shown in Fig. 1). As for the acetone extract, 18 fractions were collected, but only nine fractions were subjected to further analysis (as shown in Fig. 2). The peaks eluted in the chromatograms represent the nine fractions of the acetone and methanol extracts, respectively. Compounds present in the fractions with chromophores that adsorbed UV at wavelengths of 210 nm and 254 nm were investigated (Shukla et al. 2017; Walker 2009). The chromophore is a conjugated pi-electron system that absorbs light in the region of 200 nm–800 nm (Joshi 2012); thus, compounds or molecules analysed using UV must contain pi-bonds (Shukla et al. 2017; Joshi 2012) to perform peaks.

Some peaks were fully eluted with good separation in fraction 06 for the methanol extract (Fig. 1) and fraction 08 for the acetone extract (Fig. 2). Although prep-HPLC is widely used for separation and detection in many applications (Kanu 2021), when it comes to complexity, plant extracts like Q. infectoria gall extracts are considered some of the most complex matrices. Similar to detecting pesticide residues, carbendazim and carbaryl in several paprika samples would be difficult to detect due to the interference of other compounds like indimethoate, carbofuran, imidacloprid, methomyl, spinosad and methamidophos (Ferrer Amate et al. 2010). The more complex the sample, the greater the challenge of developing chromatographic strategies to obtain isolated molecules (Oldoni et al. 2021). Sample complexity means the number of compounds in a sample is abundant; thus, the separation of sample components becomes progressively challenging. The probability of successfully separating complex samples can be improved by altering the pack capacity, which means changing the column dimension, particle size and flow rate (Snyder & Dolan 2007). This indicates that additional isolation using various techniques or different chromatographic methods (Kanu 2021; Snyder & Dolan 2007) is required to successfully achieve good separation for fractions of Q. infectoria.

Antimalarial Activity of the Fractions

Malarial SYBR Green I fluorescence-based (MSF) assay was conducted to determine the antimalarial activity of the fractions in terms of the value of half-maximal inhibitory concentration (IC50). In our previous study, we revealed that acetone (IC50 = 5.85 ± 1.64 μg/mL) and methanol extracts (IC50 = 10.31 ± 1.90 μg/mL) of Q. infectoria galls were active against the 3D7 parasite (Nik Mat Zin et al. 2020). Fractionation was then performed on these extracts, followed by the in vitro antimalarial activity of the fractions against the parasite. For both extracts, only four of the nine fractions were analysed (Table 2). Fractions with IC50 values greater than 100 μg/mL were not statistically analysed as there was no antimalarial activity. When tested as a control, the artemisinin had an IC50 value of 0.004 ± 0.001 μg/mL (Fig. 3C). The methanol fraction QIM16 (IC50 = 24.21 ± 1.88 μg/mL) and acetone fraction QIA11 (IC50 = 17.65 ± 1.82 μg/mL) exhibited the highest antimalarial activity when compared to other fractions, as shown in Figs. 3A and 3B. The antimalarial activity of the acetone and methanol fractions was, however, lower than that of the acetone (IC50 = 5.85 ± 1.64 μg/mL) and methanol extracts (IC50 = 10.31 ± 1.90 μg/mL) reported previously (Nik Mat Zin et al. 2020). This finding aligns with the result reported by Ekasari et al. (2022), whereby 96% ethanol extract (IC50 = 1.88 μg/mL) from the leaves of Sauropus androgynous showed higher in vitro antimalarial activity against P. falciparum compared to its fraction (IC50 = 2.042 μg/mL). The reduction in antimalarial activity in the fractions compared to the crude extracts was also demonstrated by Ochieng et al. (2010), who reported that crude extracts from the aerial part of Gardenia ternifolia exhibited potent in vitro antimalarial activity against P. falciparum, compared to their fractions and pure isolates. The potent in vitro activity of the G. ternifolia crude extracts against the malaria parasite was possibly due to the synergistic effects of the flavonoid components (Ochieng et al. 2010).

To our knowledge, these are new findings regarding the comparison of the antimalarial activity of the Q. infectoria gall extracts and fractions, with the extracts displaying more promising antimalarial activity than their fractions against P.falciparum. The extracts and fractions of the Q. infectoria galls exhibited antimalarial activity, which could be attributed to the presence of various secondary metabolites, particularly phenolic compounds such as pyrogallol, ellagic acid, gallic acid, tannins and quercetin (Hamid et al. 2005; Shrestha et al. 2014; Tayel et al. 2018). The antimalarial action of these secondary metabolites has been demonstrated in their ability to inhibit the breakdown of haemoglobin and detoxify haem in the digestive vacuole of P. falciparum (Tajuddeen & Van Heerden 2019; Mamede et al. 2020). Bioactive chemicals such as phenolic compounds have been proven to suppress bacteria by disrupting cellular membranes, resulting in the loss of cellular components and finally, death. It is conceivable due to the presence of the hydroxyl (-OH) group in phenolic compounds, which has been associated with antimicrobial properties including antimalarial activity (Kumar & Goel 2019; Othman et al. 2019; Tajuddeen & Van Heerden 2019). The reduction in the antimalarial activity of the fractions compared to the crude extracts might be affected by the loss of synergistic activity between different compounds in the fractions (Rasoanaivo et al. 2021). Some compounds are inactive on their own but may act synergistically with other constituents. Comparing QIA11 and QIM16 to all other fractions, it shows that these fractions had the most compelling antimalarial activity since they isolated various phytochemical constituents. The same mechanism is applied to the Q. infectoria gall extracts. However, the differences in antimalarial activity might be attributed to variations in the amounts of secondary metabolites (Mazid et al. 2011). This indicates that the gall extracts possess a higher abundance or quality of effective antimalarial phytochemicals as compared with the fractions. Thus, antimalarial activity typically declines or ceases when the compounds become separated. In this case, the crude extracts were far more active than their fractions (Rasoanaivo et al. 2021). In order to further the investigation into the antimalarial properties of these active extracts, it is recommended to design an in vivo study using Q. infectoria gall extracts. This study will expedite and enhance the creation of more effective antimalarial drugs. By employing high-throughput and high-content in-vivo research, it is possible to accelerate the identification of new compounds, thus significantly expediting the discovery of novel antimalarial drugs.

Identification of Chemical Constituents from the Fractions Using HR-LCMS

The different magnitudes of the antimalarial effects of the extracts and fractions may be affected by varying phytochemical compositions (Uzor et al. 2021). Thus, HR-LCMS analysis was run for the most prominent fractions, QIM16 and QIA11. The analysis was conducted in positive and negative modes, but only compounds with negative ions were detected. Adding 0.1% formic acid in the mobile phase often leads to formate adducts in negative mode and produces ion suppression when HR-LCMS methods are developed, especially during analysis in negative-ionisation mode (Núñez & Lucci 2014). This occurs as a result of the electrospray ionisation (ESI) mechanism in the HR-LCMS, which was affected by the excess negative charge formed either by the reduction of the mobile phase used in the analysis (i.e., water and acetonitrile) or by the presence of a weak acid (i.e., 0.1% formic acid) in the mobile phase (Kebarle & Verkerk 2010). As an acidic modifier, 0.1% formic acid provides additional protons that facilitate reduction, making it simpler for the spray droplets to carry excess negative charge (Rayleigh 1882). This negative charge excess likely accumulates on the surface of the droplet as a result of electric repulsion during negative ion ESI, increasing the pH on the surface of the droplet and creating a local environment in which deprotonation of the analytes occurs more readily than in the bulk solution (Blades et al. 1991). They can be strong and reproducible, and they can fragment well in MS analysis (Wu et al. 2004). Tables 3 and 4 show the major compounds identified in the fractions, in which the phenolic groups of gallotannin, simple phenols and tannins were identified.

Gallic acid (1) and 1,3,6-tris-o-(3,4,5-trihydroxybenzoyl)-beta-d-glucose (3) were the major compounds identified in both the methanol and acetone fractions. Gallic acid was detected at a retention time of 10.15 min with the mass found at m/z 171.9927. Three fragment ions were obtained in a negative mode [M-H]−, 68.95251, 115.05408 and 169.07555, confirming the availability of gallic acid in the fractions QIM16 and QIA11. Meanwhile, 1,3,6-tris-o-(3,4,5-trihydroxybenzoyl)-beta-d-glucose, a tannin compound in the form of galloyl glucose, was detected at 13.34 min at m/z 635.0865. In line with the results obtained in both this study and the previous study (Kamarudin, Nik Salleh, et al. 2021; Kamarudin, Muhamad, et al. 2021), tannin and gallotannin were identified in the Q. infectoria gall crude methanol and aqueous extracts using HPLC. Gallotannin, also known as tannic acid, is in the hydrolysable tannins category. It consists of a central glucose molecule esterified by gallic acid units. Although the gallotannin content in the Q. infectoria aqueous extract (72.0 μg/mL) was higher than in the methanol extract (46.8 μg/mL) of the same plant ((Kamarudin, Nik Salleh, et al. 2021; Kamarudin, Muhamad, et al. 2021; Abdullah et al. 2017), the compound was still identified in both the acetone and methanol fractions in this study.

The peak at m/z 483.0761 in the fraction QIA11 was 1-O,6-O-digalloyl-beta-D-glucose (4). This trigalloyl glucoside was also identified in negative mode and eluted at 11.71 min. Its availability was confirmed by the MS/MS spectrum ion fragmentation of 271.04473, 169.01276 and 211.02335. This compound is another tannin compound in the form of galloyl glucose that was isolated from the fraction QIA11 after 1,3,6-tris-o-(3,4,5-trihydroxybenzoyl)-beta-d-glucose (3).

Another compound abundant in Q. infectoria galls is ellagic acid (2). However, ellagic acid (C14H6O8) was only detected in the methanol fraction QIM16. The [M-H]− ion of this compound was identified at m/z 300.9989 with a retention time of 14.78 min. The MS/MS spectrum of the peaks gave the characteristic fragments at m/z 125.02329, 169.01332 and 107.01264. A study by Abdullah et al. (2017) proved that ellagic acid was detected in a semi-purified fraction with 6.22%, a similar finding to our study on fractionation. In a review by Tajudeen and Van Heerden (2019), ellagic acid was the most active metabolite isolated from Anogeissus leiocarpus Combretaceae) methanol bark extracts, and it exhibited antiplasmodial activity against the 3D7 parasite, with an IC50 value of 18.8 μg/mL. The full HR-LCMS analysis of the fractions QIM16 and QIA11 revealed the presence of four major compounds, gallic acid, 1,3,6-tris-o-(3,4,5-trihydroxybenzoyl)-betad-glucose, 1-O,6-O-digalloyl-beta-D-glucose and ellagic acid, which could have potency as new antimalarial drug candidates. Nevertheless, further purification is needed to isolate pure compounds from the QIM16 and QIA11 fractions that exhibit higher antimalarial activity than other fractions. Purification may result in the discovery of other pure compounds in Q. infectoria gall with higher antimalarial activity than these semi-fractions.

CONCLUSION

The bioassay-guided fractionation of the Q. infectoria gall methanol and acetone crude extracts resulted in the isolation of two active fractions QIM16 and QIA11 with varying degrees of antimalarial activity against the 3D7 strain of P. falciparum. The study further revealed the presence of four major compounds, identified as gallic acid (1), ellagic acid (2), 1,3,6-tris-o-(3,4,5-trihydroxybenzoyl)-beta-d-glucose (3) and 1-O,6-O-digalloyl-beta-D-glucose (4). The findings of this study confirm the ethnobotanical use of Q. infectoria galls as a herbal treatment for malaria, which may lead to further research on the purification of Q. infectoria fractions or compounds.

ACKNOWLEDGEMENTS

The authors would like to thank the Ministry of Higher Education Malaysia for providing the Fundamental Research Grant Scheme (FRGS/1/2019/STG03/USM/03/3). The authors would like to acknowledge Dr. Khairul Mohd Fadzli Mustaffa from Institute for Research in Molecular Medicine (INFORMM), USM, Kubang Kerian, Kelantan, Malaysia for giving the 3D7 parasite and access to the laboratory, equipment and cell culture facilities.

Figure 1 The prep-HPLC chromatogram of the methanol extract at a wavelength of 254 nm.

Figure 2 The prep-HPLC chromatogram of the acetone extract at a wavelength of 254 nm.

Figure 3 Log concentration-response curve of the: (A) acetone fractions; (B) methanol fractions; and (C) artemisinin against the chloroquine-sensitive (3D7) strain of P. falciparum. The horizontal dashed line corresponds to the approximate mean IC50 value from three independent experiments done in triplicates.

Table 1 The yield (w/v, %) of the fractions of the Q. infectoria gall crude extracts.

Q. infectoria gall extract	Fraction	Yield (w/v,%)	
Methanol (QIM)	06	11.31	
07	1.81	
11	20.49	
12	5.82	
15	29.80	
16	9.24	
17	2.84	
18	3.68	
19	15.00	
Acetone (QIA)	06	2.91	
07	4.01	
08	1.19	
09	6.35	
10	10.17	
11	58.88	
12	9.86	
13	4.12	
14	2.51	

Table 2 The antimalarial activity of the fractions of the Q. infectoria gall extracts.

Q. infectoria gall extract/drug	Fraction	IC50 (μg/mL)	P-value	F (DFn, DFd)	
Methanol (QIM)	06	70.71	< 0.0001	F (8, 18) = 32.94	
07	> 100*	-		
11	> 100*	-		
12	> 100*	-		
15	> 100*	-		
16	24.21	0.0100		
17	36.74	0.0002		
18	54.63	< 0.0001		
19	> 100*	-		
	
Acetone (QIA)	06	> 100*	-		
07	> 100*	-		
08	24.55	0.0089		
09	> 100*	-		
10	77.95	< 0.0001		
11	17.65	0.0786		
12	60.48	< 0.0001		
13	> 100*	-		
14	> 100*	-		
Artemisinin		0.004	0.001		
Notes:

* Data with IC50 values more than 100 μg/mL do not possess antimalarial activity.

The data were expressed as mean (SD) of three independent experiments. Mean values were tested for normality before proceeding to the parametric test; one-way ANOVA followed by Dunnett’s multiple comparisons at 95% confidence. Value of P < 0.05 was statistically significant. All methanol and acetone fractions were known as treated groups. Artemisinin was known as a control group. All fractions were statistically significant except for QIA11 was not significant with comparison of artemisinin (P = 0.078). Fractions with IC50 values more than 100 μg/mL were not statistically analysed as there were no antimalarial activity. DFn = degree of freedom numerator; DFd = degree of freedom denominator.

Table 3 Compounds identified in the methanol fraction QIM16 by HR-LCMS.

Compound name	Molecular structure	Retention time (min)	Found mass (m/z)	m/z values for fragment ions	Ionisation mode	
Gallic acid (C7H6O5)	
1	10.15	171.9927	68.95251, 115.05408, 169.07555	Negative	
Ellagic acid (C14H6O8)	
2	14.78	300.9989	125.02329, 169.01332, 107.01264	Negative	
1,3,6-tris-o-(3,4,5-trihydroxybenzoyl)-beta-d-glucose (C27H24O18)	
3	13.34	635.0865	483.07592, 331.06565, 465.06540	Negative	

Table 4 Compounds identified in the acetone fraction QIA11 by HR-LCMS.

Compound name	Molecular structure	Retention time (min)	Found mass (m/z)	m/z values for fragment ions	Ionisation mode	
Gallic acid (C7H6O5)	
1	10.15	171.0283	68.95251, 115.05408, 169.07555	Negative	
1,3,6-tris-o-(3,4,5-trihydroxybenzoyl)-beta-d-glucose (C27H24O18)	
3	13.34	635.0865	271.04473, 169.01283, 313.05531	Negative	
1-O,6-O-digalloyl-beta-D-glucose (C20H20O14)		11.71	483.0761	271.04473, 169.01276, 211.02335	Negative	

AUTHORS’ CONTRIBUTIONS: Nurul Hammizah Hamidon: Conceptualised the research, designed the experiments, collected the data, performed the analysis, wrote the paper and participated in the revisions of it.

Anjana Chamilka Thuduhenage Dona: Collected the data, performed the analysis, wrote the paper and participated in the revisions of it.

Nik Nor Imam Nik Mat Zin: Collected the data, performed the analysis, wrote the paper and participated in the revisions of it.

Nurul Izza Nordin: Conceptualised the research, designed the experiments and participated in the revisions of it.

Shaida Fariza Sulaiman: Conceptualised the research, designed the experiments and participated in the revisions of it.

Nurhidanatsha Abu Bakar: Conceptualised the research, designed the experiments, wrote the paper and participated in the revisions of it.

All authors have read and agreed to the published version of the manuscript.
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