Trends Sci. 2025; 22(10): 10576

Malaria remains a major global health concern due to its high morbidity and mortality, particularly in subtropical and tropical regions. This burden necessitates sustained strategic interventions. The World Health Organization (WHO) 2023 report highlights the magnitude of the global malaria problem, with 263 million cases recorded in 83 endemic countries and a death toll of around 608,000 [1]. In Indonesia, malaria cases exceeded 369,000, with notable increases in regions such as Gorontalo, East Nusa Tenggara, and Papua [2]. Plasmodium falciparum and Plasmodium vivax are the 2 main Plasmodium species that cause malaria in humans. Compared to other species, these parasites have the highest incidence of infection, as well as the highest rates of complications and mortality. Alarmingly, resistance to most available antimalarial agents, including artemisinin and its derivatives, has been confirmed in both species. The emergence of artemisinin resistance, initially documented in Cambodia in 2007 [3], and its subsequent rapid spread across Southeast Asia, has jeopardized the progress achieved in malaria control programs. Consequently, the emergence of new antimalarial drug candidates, particularly from herbal medicines, has become imperative.

The genus Cratoxylum has a history of traditional use for malaria treatment [4]. Several active antimalarial compounds have been isolated from C. mangiayi, C. cochinchinense, and C. glaucum [5,6]. A chemotaxonomic approach highlights the need to explore other species within this genus, including C. sumatranum. This study evaluated the in vitro inhibitory activity of C. sumatranum twig extract against P. falciparum strains 3D7 and Dd2, followed by assessing its cytotoxicity and analyzing metabolite profiles to identify compounds responsible for antimalarial activity. C. sumatranum (Jack) Bl., commonly known as Geronggang, is endemic to Kalimantan and West Sumatra, is a member of the Hypericaceae family, and has traditional medicinal value for treating various conditions, including fever, stomach ailments, dysentery, and burns [4,7,8]. Previous studies have also reported the presence of such metabolites in C. sumatranum extracts. C. sumatranum stem barks, twigs, and roots harbor diverse bioactive compounds, including xanthones, flavonoids, anthraquinones, and benzophenones [9,10]. The dichloromethane extract from C. sumatranum stem bark has successfully isolated caged xanthones, including Cochinchinone D and Cochinchinoxanthone, which exhibited IC₅₀ values of 4.79 and 4.41 mM, respectively [9,10]. Dichloromethane, a solvent with intermediate polarity, effectively extracts semi-polar compounds such as xanthones, flavonoids, and anthraquinones, many of which are known for their antimalarial properties. In this study, the dichloromethane extract of C. sumatranum twigs (Cs-T-D) was found to contain several xanthones, including Gerontoxanthone I, Macluraxanthone, and γ-Mangostin. These compounds may contribute individually or synergistically to the extract’s potent in vitro antimalarial activity against P. falciparum strains 3D7 and Dd2 [10,11]. Based on these results, our findings support the potential of C. sumatranum twigs as a promising source of antimalarial agents.

Materials and methods

Plant material

The twigs of C. sumatranum were collected from the Environmental and Forestry Instrument Standard Implementation Center, Samboja, Balikpapan, East Kalimantan, Indonesia. A licensed botanist at Purwodadi Botanical Garden Growth Conservation Center, East Java, Indonesia (1048/IPH.06/HM/IX/2019) confirmed the identity of the plant material, where voucher specimens (collection number: Cs-T-D) were deposited at the Institute of Tropical Disease, UNAIR.

Extraction of plant material

Cratoxylum sumatranum twigs were dried at room temperature (around 27 °C) without exposure to direct sunlight for 14 days and were mechanically powdered. A maceration process was conducted using successive extractions on 1 kg of the dried powder with 3 L of dichloromethane solvent. The extract underwent filtration, concentration using a rotary evaporator, and subsequent drying in an oven maintained at 40 °C to yield a dry extract. This process produced a single extract called Cratoxylum sumatranum-twigs dichloromethane extract (Cs-T-D). The extract was stored in glass bottles and kept at 4 °C in the refrigerator for future analysis.

P. falciparum culture

Plasmodium falciparum strains 3D7 and Dd2, obtained from Eijkman Institute for Molecular Biology, Jakarta and Department of Biomedical Chemistry, The University of Tokyo, Bunkyo-ku, Japan (resistant to chloroquine, pyrimethamine, and mefloquine), maintained at NPMRD-ITD Universitas Airlangga, was cultured in vitro following a previously described method [10]. Parasite cultures were maintained in human O-type red blood cells at 37 °C in a controlled atmosphere (5% O₂, 5% CO₂ and 90% N₂) using RPMI-1640 medium (Gibco, Thermo Fisher Scientific, USA) containing 50 µg/mL hypoxanthine, 25 mM HEPES, 2 g/L NaHCO₃, 0.3 g/L L-glutamine, 11 mM glucose, 25 mM NaHCO₃, 2.5 µg/mL gentamicin, and 0.5% (w/v) Albumax II (SIGMA-Aldrich). Parasite cultures were synchronized to the ring stage using sorbitol treatment. The level of parasitemia was quantified by microscopic examination, counting the number of infected erythrocytes in a sample of 1,000 total erythrocytes.

Antimalarial activity test by LDH assay

The lactate dehydrogenase (LDH) assay evaluated the Cs-T-D extract’s antimalarial properties against P. falciparum strains 3D7 and Dd2. The extract was dissolved in DMSO at a 5 µg/mL concentration and tested for initial screening. Extracts exhibiting > 50% inhibition of parasite growth were selected for IC₅₀ determination. Eight 2-fold serial dilutions of these extracts and the standard drug chloroquine diphosphate were prepared and added to 96-well plates, with concentrations of 50 to 0.01 μg/mL for the extracts and 10 to 0.001 μg/mL for chloroquine diphosphate. Assays were performed in triplicate. Subsequently, 100 μL of Following the addition of parasitized red blood cells to each well, the plates were incubated at 37 °C under 5% oxygen, 5% carbon dioxide, and 90% nitrogen for 72 h. Following incubation, plates were harvested and stored at −30 °C. The LDH activity was then measured by adding 90 µL of prepared substrate to each well, incubating on a flatbed shaker at 650 rpm at room temperature for 30 min in the dark. Absorbance was read at 650 nm using a multiscan sky-high microplate spectrophotometer (Thermo Fisher Scientific). IC₅₀ values were calculated using non-linear regression curve analysis with GraphPad PRISM 7.0 software (GraphPad Co., Ltd., San Diego, CA, USA).

Cytotoxicity test by resazurin assay

The method used to measure the cytotoxicity of the samples was the resazurin-based cell viability assay [12] on BHK-21 normal cell lines. Cells were cultured in D-MEM (High Glucose) enhanced with L-Glutamine, Phenol Red, NaHCO₃, 10% FBS, and 1% Penicillin-Streptomycin. For the assay, BHK-21 cells were seeded in 96-well plates at densities of 1×10⁴ cells/well and exposed to various concentrations (100, 50, 25, 12.5 and 6.25 μg/mL in DMEM) of the extracts and fractions. After a 44-hour incubation period at 37 °C under 5% CO₂, 10 μL of a 0.5 mM resazurin solution was added to each well. Following a 4-hour reduction period, fluorescence was quantified using a Nivo plate reader (PerkinElmer) with wavelengths set at 530 and 595 nm, respectively. Cytotoxic concentrations (CC₅₀) were analyzed using non-linear regression curve analysis in GraphPad PRISM 7.0 software (GraphPad Co. Ltd., San Diego, CA, USA).

Sample preparation for LC-MS/MS analysis

The dichloromethane extract from C. sumatranum twigs (20 mg) was accurately weighed and dissolved in a solvent mixture of acetonitrile and ultra-pure water (98:2, v/v%). Before analysis, the solution was filtered through a 0.45 μm PTFE membrane. The TMstrata® C18-E column (Phenomenex) was preconditioned with ultra-pure water and acetonitrile (3 mL each), followed by an additional conditioning step using 3 mL of acetonitrile and ultra-pure water (98:2, v/v%). The prepared sample (20 mg/mL) was loaded onto the column, after which 2 mL of the same solvent mixture was added. The collected eluate was subsequently concentrated using a rotary evaporator at 40 °C, yielding 20 mg of purified twig extract.

Further analysis of the pre-treated C. sumatranum twig sample was conducted using an Orbitrap mass spectrometer. Separation was performed chromatographically using an Accucore^TM Vanquish^TM C18 column (100×2.1 mm, 1.5 μm, Thermo Scientific, Lithuania), where both solvents were supplemented with 0.1% formic acid. The elution gradient was set as follows with a mobile phase comprising ultra-pure water (solvent A) and acetonitrile (solvent B): 0 - 35 min, from 40% to 100% B; 20 - 25 min, held at 100% B; 25 - 29 min, from 100% to 40% B; and 29 - 35 min, maintained at 40% B. The analysis was run in positive ion mode (1 μL injection, 1,000 ppm, 0.2 mL/min flow rate) using the established UHPLC gradient. The mass spectrometric analysis was conducted at resolutions of 60,000 and 15,000 in full scan and MS/MS modes, respectively. Electro-spray ionization (ESI) was utilized as the ion source, operating at a spray voltage of 3,500 V. Additional parameters included an ion transfer tube temperature of 300 °C, sheath gas at 35 arb, auxiliary gas at 7 arb, vaporizer temperature at 275 °C, and RF lens setting at 60%. The scan range spanned from m/z 100 to 1,000. Thermo Scientific Xcalibur software version 4.2.47 was employed to acquire and process data.

Database export

The LOTUS: Natural Products Online (https://lotus.naturalproducts.net) database retrieved metabolite data associated with the genus Cratoxylum and the Hypericaceae family. The retrieved data were exported as an Excel (.xls) formatted file containing essential details such as chemical names, synonyms, molecular formulas, accurate masses, and SMILES representations. The SMILES data underwent manual curation to remove inconsistencies before being saved as a comma-separated values (CSV) file. The processed file was subsequently analyzed using DataWarrior software to verify and refine the structural representations of the identified metabolites [13].

Analysis of MS data using MZmine

Thermo raw data from the mass spectrometer were initially converted into mzML format using MSConvert from ProteoWizard. The resulting mzML files were subsequently imported into MZmine 3 for further processing. Before data analysis, the project was saved in the MZmine-specific format. The crude chromatogram was visualized using blank and crude sample data to assess the noise level before proceeding with mass spectrometry (MS) detection for [M+H]⁺ (MS¹) and product ions m/z (MS²) levels. A centroid mass detector was employed with polarity set to positive mode. The noise thresholds were configured at 2.0E6 for MS¹ and 0.0E0 for MS². Following this, chromatogram construction and resolution steps were performed, applying an isotope filter, alignment, and gap-filling procedures. The final processed dataset was exported as an mgf file for further structural elucidation using Sirius 5.8.3 software [13].

Phytochemical analysis

An academic account for Sirius was initially registered to facilitate the dereplication process. The procedure commenced by importing the mgf file into Sirius software, ensuring a stable connection to the web server. Custom databases were generated by incorporating curated SMILES representations of metabolites derived from Hypericaceae into the software. Before computation, all detected features were assigned the adduct type [M+H]⁺. The Sirius analysis was initiated by activating the corresponding function, with molecular formula constraints limited to hydrogen (H), carbon (C), nitrogen (N), and oxygen (O). The mass spectrometry instrument settings were configured for an Orbitrap system with an MS² mass accuracy threshold of 15 ppm. The MS/MS isotope scoring was set to “SCORE”.

Annotation was performed utilizing all available databases integrated within Sirius, including Biocyc, CHEBI, COCONUT, HMDB, KEGG, KEGG Mine, KNApSAcK, MeSH, NORMAN, Natural Products, PubChem, PubMed, YMDB, YMDB Mine, ZINC bio, SuperNatural, alongside the 2 custom-developed databases. Following this, the Zodiac function was activated, which was succeeded by fingerprint prediction, structure database search, and compound classification using CANOPUS. The computation process was finalized by selecting the compute function, allowing the system to complete the data analysis.

Results and discussion

The genus Cratoxylum (Hypericaceae) is known for its diverse pharmacological properties, including antimalarial potential. Phytochemical studies on Cratoxylum species have identified bioactive compounds such as xanthones, flavonoids, and anthraquinones, many exhibiting antimalarial activity [5,7,9,14]. This ethnopharmacological evidence highlights the promise of C. sumatranum as a source for antimalarial drug discovery, warranting further bioassay-guided research to validate its efficacy and identify active constituents.

Percentage yield of dichloromethane extract of C. sumatranum twigs (Cs-T-D)

The dichloromethane extract of C. sumatranum twigs (Cs-T-D) was produced with a dark green sticky solid with 14.73 g for a 1.473% (w/w) yield. In this study, dichloromethane was chosen as the extraction solvent for C. sumatranum based on previous studies on other Cratoxylum species, where dichloromethane extracts showed significant antimalarial activity compared to extracts obtained with more polar (methanol extract) and non-polar (hexane extract) solvents [6,15]. Given the structural similarity of secondary metabolites across the genus, dichloromethane is considered a suitable solvent for selectively extracting bioactive compounds.

Antimalarial activity and cytotoxicity of the dichloromethane extract of C. sumatranum twigs

Dichloromethane extract of C. sumatranum twigs showed significant antimalarial potential against Plasmodium falciparum sensitive to chloroquine (3D7 strain) and resistant to chloroquine (Dd2 strain) with IC₅₀ values of 0.28 ± 0.55 and 0.66 ± 0.02 µg/mL, respectively. Although the antimalarial activity against sensitive strains is higher than against resistant strains, the extract is still classified as very active against both test strains, based on the classification established in previous literature [12,14]. Statistical analysis revealed a significant difference in the extract efficacy between the 2 strains, with greater potency observed against 3D7 than Dd2 (p < 0.05). Nonetheless, the extract maintained a very active classification against both strains. Compared to the standard chloroquine diphosphate, the extract demonstrated significantly lower antimalarial activity in both strains (p < 0.05). Despite this, its strong inhibitory profile supports its potential as a promising natural antimalarial candidate.

Furthermore, an in vitro toxicity evaluation on BHK-21 cells using the Resazurin assay showed that the dichloromethane extract was non-toxic according to previously defined criteria, with a CC₅₀ value exceeding 100 µg/mL [16]. The data is presented in Table 1. The potent antimalarial activity, coupled with the favorable safety profile of this multi-component extract from C. sumatranum twigs, highlights the potential for synergistic effects among its constituents. Subsequent isolation and identification of individual compounds could lead to the discovery of novel, effective, and safe antimalarial agents with activity comparable to chloroquine diphosphate.

Evaluating the selectivity index (SI) is essential in herbal drug research to determine the feasibility of further investigation. To determine the selectivity index (SI), the toxic concentration is divided by the effective bioactive concentration. An ideal drug exhibits high toxicity and low active concentrations [17]. A selectivity index of SI ≥ 2 is recommended as the acceptance criterion for selecting bioactive samples for further study [18]. Table 1 indicates good selectivity for the extract, which showed selectivity towards malaria parasites, as its selectivity indices were more significant than 2.

Table 1 Antimalarial activity (IC₅₀), Cytotoxicity (CC₅₀), and SI of dichloromethane extract from Cratoxylum sumatranum twigs.

Cs: Cratoxylum sumatranum, T: Twigs, D: Dichloromethane extract. Chloroquine diphosphate is the standard drug used in this study. *Mean ± SD of 3 replicates. Selectivity Index (SI) was calculated as: SI = CC₅₀/IC₅₀

Phytochemical analysis

Cratoxylum is widely recognized for its rich content of bioactive secondary metabolites with antimalarial potential. In this study, LC-MS/MS analysis of the dichloromethane extract from C. sumatranum twigs identified 41 compounds, including xanthones (17), flavonoids (10), terpenoids (4), coumarins (2), chalcones (2), anthraquinones (2), phenylpropanoids (2), lignans (1), and amino acids (1). The data is presented in Figure 2. The Total Ion Chromatogram (TIC) provided a comprehensive profile of these chemical constituents (Figure 1). Table 2 lists selected metabolites based on abundance, structural uniqueness, and reported bioactivity. Compound identification was achieved through MS/MS fragmentation pattern analysis and comparison with curated databases, including LOTUS, PubChem, and COCONUT. The complete compound list is available in Supplementary (Table S1)

Figure 1 Total Ion Chromatography (TIC) of the dichloromethane extract from C. sumatranum twigs.

Among the identified metabolites (Table 2), several compounds have previously been isolated from other plant species and reported to exhibit antiplasmodial activity. Notably, xanthones such as Gerontoxanthone I from C. mangiayi, Macluraxanthone from C. cochinchinense, γ-Mangostin from Garcinia mangostana, and Trapezifolixanthone from Chrysochlamys tenuis demonstrated significant in vitro inhibitory activity against Plasmodium falciparum, with IC₅₀ values ranging from 2.52 to 15.9 µM [5,19,20]. These compounds are known to affect parasite survival by inhibiting enzymes in the parasite’s food vacuole, such as plasmepsin-II, falcipain-3, and M1-alanyl aminopeptidase, as well as by disrupting heme polymerization [10,19]. Chalcone compounds such as Pinocembrin chalcone have also exhibited activity against Plasmodium berghei [20]. In addition, the terpenoid Caryophyllene, isolated from Copaifera reticulata, effectively inhibited P. falciparum W2 and 3D7 strains with IC₅₀ values of 1.66 and 2.54 μg/mL, respectively [21]. Furthermore, the anthraquinone Vismione B demonstrated strong antimalarial activity, with an IC₅₀ value of 0.66 μg/mL [5]. According to Widyawaruyanti et al. [22], the flavonoid group, particularly prenylated flavones and chalcones, has exhibited strong antimalarial activity. Bilia et al. [23] stated that flavonoids exert their mechanism of action by inhibiting nutrient transport and blocking the degradation and detoxification of hemoglobin in Plasmodium. Flavonoids inhibit the influx of L-glutamine and Myo-inositol into P. falciparum-infected erythrocytes [24]. The presence of these compounds in the dichloromethane extract of C. sumatranum twig is believed to play a major role in the extract’s strong antimalarial activity, as indicated by IC₅₀ values of 0.28 µg/mL against the 3D7 strain and 0.66 µg/mL against the Dd2 strain of Plasmodium falciparum.

Table 2 Representative metabolites with reported antimalarial activity identified in the dichloromethane extract of C. sumatranum twigs.

*Compound numbers correspond to Figure 1

Importantly, several other metabolites detected in the extract remain biologically uncharacterized. These include various xanthones and flavonoids with unknown bioactivity, as well as minor classes such as phenylpropanoids, lignans, and amino acid derivatives. Although not directly linked to antimalarial effects, these compounds may play auxiliary roles by enhancing pharmacokinetic properties, providing antioxidant or anti-inflammatory benefits, or modulating host-parasite interactions [25,26]. Their presence may contribute to the overall efficacy and safety profile of the extract through indirect or synergistic mechanisms [27,28]. The alignment between the phytochemical content and antimalarial activity observed in vitro highlights the value of further bioassay-guided fractionation. Future studies focusing on the isolation, structural elucidation, and biological evaluation of these uncharacterized compounds are warranted to uncover their roles and potential as novel therapeutic agents [29].

Figure 2 The metabolite classes in the dichloromethane extract of C. sumatranum twigs with potential antimalarial activity.

Conclusions

The dichloromethane extract of Cratoxylum sumatranum twigs displayed antimalarial activity against Plasmodium falciparum strains 3D7 and Dd2, with minimal cytotoxicity against BHK-21 cells. LC-MS/MS profiling revealed a diverse array of metabolites, predominantly xanthones and flavonoids, many of which have known or potential antimalarial properties. These results suggest that the observed bioactivity is likely due to the combined effects of multiple constituents. This study highlights the potential of C. sumatranum as a promising source of antimalarial agents. Future research should focus on bioassay-guided isolation of individual active compounds, evaluation of their mechanisms of action, and in vivo efficacy studies to validate their potential as lead candidates in the development of antimalarial drugs.

Acknowledgments

This research was funded by Universitas Airlangga through the Ministry of Research and Technology, Republic of Indonesia, for the “Penelitian Tesis Magister (PTM),” with contract no 1666/B/UN3.LPPM/PT.01.03/2023. The authors were grateful to the Center for Natural Product Medicine Research and Development (cNPMRD), Institute of Tropical Disease, Universitas Airlangga, for the support facilities and to The Faculty of Pharmacy, Universitas Airlangga, which has provided funding for outbound studies to the Atta-ur Rahman Institute of Natural Product Discovery, Universiti Teknologi MARA, Malaysia.

Declaration of Generative AI in Scientific Writing

The authors acknowledge the use of generative AI tools (e.g., ChatGPT by OpenAI) in the preparation of this manuscript, specifically for language editing and grammar correction. No content generation or data interpretation was performed by AI. The authors take full responsibility for the content and conclusions of this work.

CRediT Author Statement

Maylisa Natalia Corry: Investigation, Writing-original manuscript, Visualization.

Hilkatul Ilmi: Methodology, Formal analysis, Writing-review & editing manuscript

Lidya Tumewu: Data curation and Writing-review & editing manuscript

Hanifah Khairun Nisa: Methodology and Data curation

Firman Wicaksana: Data Curation and Writing-original manuscript

Irfan Rayi Pamungkas: Data Curation and Writing-original manuscript

Supriatno Salam: Resources and Validation

Hadi Kuncoro: Resources and Validation

Suciati: Conceptualization, Methodology, Validation, Writing - Review & Editing, Supervision

Che Puteh Osman: Methodology, Validation, and Writing - Review & Editing manuscript

Achmad Fuad Hafid: Conceptualization, Validation, and Supervision

Aty Widyawaruyanti: Conceptualization, Methodology, Validation, Writing - Review & Editing, Supervision, Project Administration, Funding Acquisition.

References

1. World Health Organization. World malaria report 2021. World Health Organization, Geneva, Switzerland, 2021.

2. P Li. Annual report 2022. AIMS Energy 2023; 11(1), 135-139.

3. N Mishra, RS Bharti, P Mallick, OP Singh, B Srivastava, R Rana, S Phookan, HP Gupta, P Ringwald and N Valecha. Emerging polymorphisms in falciparum Kelch 13 gene in Northeastern region of India. Malaria Journal 2016; 15(1), 4-9.

4. CY Bok, J Low, E Kat, D Augundhooa, H Ariffin, YB Mok, KQ Lim, S Le Chew, S Salvamani, KE Loh, CF Loke, B Gunasekaran and SA Tan. Comprehensive review of Cratoxylum genus: Ethnomedical uses, phytochemistry, and pharmacological properties. Pertanika Tropical Agricultural Science 2023; 46(1), 213-241.

5. S Laphookhieo, W Maneerat and S Koysomboon. Antimalarial and cytotoxic phenolic compounds from Cratoxylum maingayi and Cratoxylum cochinchinense. Molecules 2009; 14(4), 1389-1395.

6. S Suryanto, L Tumewu, H Ilmi, AF Hafid, S Suciati and A Widyawaruyanti. Antimalarial activity of Cratoxyarborenone E, a prenylated xanthone, isolated from the leaves of Cratoxylum glaucum Korth. Pharmacia 2024; 71(1), 1-7.

7. MLG Dapar. Cratoxylum sumatranum (Jack) Blume Hypericaceae. In: FM Franco (Ed.). Ethnobotany of the mountain regions of Southeast Asia. Springer, Cham, Switzerlands, 2021, p. 333-337.

8. A Arnida, ER Sahi and S Sutomo. Aktivitas antiplasmodium in vitro dan identifikasi golongan senyawa dari ekstrak etanol batang manuran (Coptosapelta tomentosa Valeton ex K.Heyne) asal kalimantan Selatan (in Indonesian). Jurnal Ilmiah Ibnu Sina 2017; 2(2), 270-278.

9. C Tantapakul, W Maneerat, T Sripisut, T Ritthiwigrom, RJ Andersen, P Cheng, S Cheenpracha, A Raksat and S Laphookhieo. New benzophenones and xanthones from Cratoxylum sumatranum ssp. neriifolium and their antibacterial and antioxidant activities. Journal of Agricultural and Food Chemistry 2916; 64(46), 8755-8762.

10. F Wicaksana, FY Wardana, H Ilmi, L Tumewu, T Widiandani, AF Hafid and A Widyawaruyanti. Antimalarial activity of caged xanthone isolated compounds from Cratoxylum sumatranum stem bark: In vitro and in silico approaches. Journal of Advanced Pharmaceutical Technology & Research 2024; 15(4), 352-358.

11. L Tumewu, FY Wardana, H Ilmi, AA Permanasari, AF Hafid and A Widyawaruyanti. Cratoxylum sumatranum stem bark exhibited antimalarial activity by Lactate Dehydrogenase (LDH) assay. Journal of Basic and Clinical Physiology and Pharmacology 2021; 32(4), 817-822.

12. MF Dolabela, SG Oliveira, JM Nascimento, JM Peres, H Wagner, MM Póvoa and AB de Oliveira. In vitro antiplasmodial activity of extract and constituents from Esenbeckia febrifuga, a plant traditionally used to treat malaria in the Brazilian Amazon. Phytomedicine 2008; 15(5), 367-372.

13. HA Abd Karim, NE Rasol, NH Ismail, C Wiart and CP Osman. Metabolites annotation of dichloromethane extract of Kibatalia maingayi woods using orbitrap high-resolution mass spectrometry. Malaysian Journal of Chemistry 2022; 24(4), 161-172.

14. S Laphookhieo, JK Syers, R Kiattansakul and K Chantrapromma. Cytotoxic and antimalarial xanthones. Chemical and Pharmaceutical Bulletin 2006; 54, 745-747.

15. DK Sari, G Jeelani, H Ilmi, L Tumewu, R Wahyuni, A Widyawaruyanti, T Nozaki and AF Hafid. Therapeutic potential of Indonesian plant extracts in combating malaria and protozoan neglected tropical disease. BMC Complementary Medicine and Therapies 2024; 24(1), 416.

16. P García-Huertas, A Pabón, C Arias and S Blair. Evaluation of cytotoxic effect and genetic damage of standardized extracts of Solanum nudum with antiplasmodial activity. Biomédica 2013; 33(1), 78-87.

17. G Indrayanto, GS Putra and F Suhud. Validation of in-vitro bioassay methods: Application in herbal drug research. Profiles of Drug Substances Excipients and Related Methodology 2021; 46, 273-307.

18. B Kwansa-Bentum, K Agyeman, J Larbi-Akor, C Anyigba and R Appiah-Opong. In vitro assessment of antiplasmodial activity and cytotoxicity of Polyalthia longifolia leaf extracts on Plasmodium falciparum strain NF54. Malaria Research and Treatment 2019; 2019(1), 6976298.

19. M Zakiah, RA Syarif, M Mustofa, J Jumina, N Fatmasari and EN Sholikhah. In vitro antiplasmodial, heme polymerization, and cytotoxicity of hydroxyxanthone derivatives. Journal of Tropical Medicine 2021; 2021(1), 8866681.

20. Y Melaku, M Solomon, R Eswaramoorthy, U Beifuss, V Ondrus and Y Mekonnen. Synthesis, antiplasmodial activity and in silico molecular docking study of pinocembrin and its analogs. BMC Chemistry 2022; 16(1), 36.

21. GAG de Souza, NC da Silva, J de Souza, KRM de Oliveira, AL da Fonseca, LC Baratto, ECP de Oliveira, F de Pilla Varotti and WP Moraes. In vitro and in vivo antimalarial potential of oleoresin obtained from Copaifera reticulata Ducke (Fabaceae) in the Brazilian Amazon rainforest. Phytomedicine 2017; 24, 111-118.

22. A Widyawaruyanti and NC Zaini. Mekanisme dan aktivitas antimalaria dari senyawa flavonoid yang diisolasi dari cempedak (Artocarpus Champeden) (in Indonesian). Jurnal Bina Praja 2011; 13(2), 67-77.

23. AR Bilia, P Melillo de Malgalhaes, MC Bergonzi and FF Vincieri. Simultaneous analysis of artemisinin and flavonoids of several extracts of Artemisia annua L. obtained from a commercial sample and a selected cultivar. Phytomedicine 2006; 13(7), 487-493.

24. JB Lekana-Douki, JB Bongui, SLO Liabagui, SEZ Edou, R Zatra, U Bisvigou and P Druilhe. In vitro antiplasmodial activity and cytotoxicity of 9 plants traditionally used in Gabon. Journal of Ethnopharmacology 2011; 133(3), 1103-1108.

25. CW Wright. Traditional antimalarials and the development of novel antimalarial drugs. Journal of Ethnopharmacology 2005; 100(1-2), 67-71.

26. K Kaur, M Jain, T Kaur and R Jain. Antimalarials from nature. Bioorganic & Medicinal Chemistry 2009; 17(9), 3229-3256.

27. B Gilbert and L Alves. Synergy in plant medicines. Current Medicinal Chemistry 2005; 10(1), 13-20.

28. R Verpoorte, YH Choi and HK Kim. Ethnopharmacology and systems biology: A perfect holistic match. Journal of Ethnopharmacology 2005; 100(1-2), 53-56.

29. AL Harvey. Natural products in drug discovery. Drug Discovery Today 2008; 13(19-20), 894-901.

Supplementary Materials

Table S1 Identified metabolites in the dichloromethane extract of C. sumatranum twigs