**Background:** The emergence and spread of antimalarial drug resistance, including resistance to artemisinin-based combination therapies, poses a significant global health challenge. Natural products, particularly traditional herbal formulations, offer a promising source for novel antimalarial agents due to their diverse chemical compositions and potential for multi-target activity. Chan-Ta-Lee-La (CTLL) and Pra-Sa-Chan-Dang (PSCD) are traditional Thai formulations used for fever relief, but their antimalarial properties had not been previously investigated. This study aimed to evaluate the in vitro and in vivo antimalarial activity and toxicity of CTLL and PSCD formulations and their constituent plants.
**Methods:** Crude extracts of CTLL and PSCD formulations and their individual plant ingredients were prepared using 95% ethanol and distilled water. In vitro antimalarial activity was assessed against the chloroquine-resistant Plasmodium falciparum K1 strain using the Plasmodium lactate dehydrogenase (pLDH) assay, with IC50 values calculated. Cytotoxicity was evaluated on Vero and HepG2 cells using the MTT assay, determining CC50 values. The selectivity index (SI) was calculated as CC50/IC50. The ethanolic extracts of CTLL and PSCD formulations, which showed the highest SI values (>10), were selected for in vivo studies. Male ICR mice infected with Plasmodium berghei ANKA were used for Peter's 4-day suppressive test, curative (Rane's) test, and prophylactic (repository) test, with doses of 200, 400, and 600 mg/kg body weight. Acute oral toxicity was assessed at 2,000 mg/kg. Phytochemical profiling was performed using gas chromatography-mass spectrometry (GC-MS).
**Key Results:** In vitro, the ethanolic extracts of CTLL and PSCD formulations showed high antimalarial activity with IC50 values of 4.88 ± 0.36 μg/mL and 4.19 ± 0.33 μg/mL, respectively, and low cytotoxicity (CC50 > 42 μg/mL on both cell lines). Among individual plants, the aqueous extract of Eurycoma longifolia (CTLL ingredient) had the highest activity (IC50 = 2.12 ± 0.13 μg/mL), while the ethanolic extract of Mammea siamensis (PSCD ingredient) showed the most potent activity (IC50 = 0.99 ± 0.18 μg/mL). In the 4-day suppressive test, CTLL extract at 600 mg/kg achieved 89.80 ± 2.96% suppression, and PSCD extract at 600 mg/kg achieved 78.36 ± 3.46% suppression, both dose-dependent and significant (P < 0.05) versus control. In the curative test, CTLL extract at 600 mg/kg showed 35.94 ± 1.07% suppression, while PSCD extract at 600 mg/kg showed 29.65 ± 1.74% suppression. In the prophylactic test, PSCD extract at 600 mg/kg demonstrated the highest suppression (65.82 ± 2.23%), comparable to chloroquine (66.02 ± 3.25%), while CTLL extract at 600 mg/kg showed 24.59 ± 4.33% suppression. Acute oral toxicity testing at 2,000 mg/kg revealed no mortality, behavioral changes, or significant alterations in body weight, liver enzymes (AST, ALT, ALP), or kidney function (BUN, creatinine) except for a significant increase in ALP in the CTLL group. Histopathological examination of liver and kidney tissues showed normal architecture. GC-MS analysis identified major compounds in CTLL extract including linderol (13.14%), isoborneol (7.88%), β-eudesmol (6.52%), linoleic acid (5.93%), and oleic acid (3.85%). In PSCD extract, major compounds included ethyl 4-methoxycinnamate (5.32%), 3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-chromen-4-one (1.97%), flamenol (1.59%), oleic acid amide (1.37%), and linoleic acid (1.28%).
**Clinical Implications:** This study provides scientific evidence supporting the traditional use of CTLL and PSCD formulations for malaria-like symptoms. The ethanolic extracts demonstrated potent in vitro and in vivo antimalarial activity with a favorable safety profile at therapeutic doses. CTLL extract showed strong suppressive and curative effects, while PSCD extract exhibited notable prophylactic activity, suggesting potential for different therapeutic applications. The identified phytochemicals, such as linoleic acid, β-eudesmol, and ethyl 4-methoxycinnamate, may contribute to the observed antimalarial effects through single or synergistic mechanisms. These findings warrant further research to isolate active compounds, elucidate mechanisms of action, and evaluate chronic toxicity, paving the way for development of novel antimalarial agents from these traditional formulations.