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INTRODUCTION
Malaria remains a life-threatening infectious disease caused by protozoan parasites of the genus Plasmodium. It is characterized by fever, anemia, and splenomegaly due to intraerythrocytic invasion and destruction.[1] In 2022, there were an estimated 249 million cases globally, with the WHO African Region accounting for 94% of cases and 95% of deaths.[1] Ghana is among the top 15 high-burden countries, contributing significantly to morbidity and mortality despite ongoing interventions such as insecticide-treated nets, indoor residual spraying, and artemisinin-based combination Therapies (ACTs).[2]
The effectiveness of ACTs is increasingly threatened by resistance. Resistance to dihydroartemisinin-piperaquine has been reported in Southeast Asia, accompanied by delayed clearance after artesunate treatment.[3, 4] Worryingly, recent reports of ACT failures in travellers returning from Africa suggest the early emergence of resistance on the continent.[5, 6] These developments highlight the need to identify and optimize new antimalarial scaffolds.
Natural products remain indispensable in antimalarial drug discovery, with quinine and artemisinin serving as classical examples.[7] Plant-derived secondary metabolites often possess diverse structures and bioactivities, making them attractive leads against resistant parasites.[9] Within this context, the Rubiaceae plant Aidia genipiflora (Hook.f.) Dandy has gained increasing pharmacological interest due to its bioactive constituents, including oleanonic acid, a pentacyclic oleanane-type triterpenoid.[9]
Oleanonic acid has been reported to exhibit antibacterial, resistance-modulatory, and anti-inflammatory activities.[10, 11] Its immunomodulatory potential is relevant because inflammation and elevated cytokines are critical in malaria pathogenesis.[12] While Irungu et al[13] demonstrated antiplasmodial activity of oleanonic acid from Ekebergia capensis, no study has yet established its in vivo efficacy when isolated from A. genipiflora. This presents both a phytochemical novelty, introducing A. genipiflora as a new source, and a pharmacological contribution by validating its in vivo antimalarial potential.
The present study, therefore, investigated the in vitro and in vivo antiplasmodial activities of A. genipiflora stem bark extract and oleanonic acid, alongside cytotoxicity evaluation, to provide preclinical evidence supporting its potential as a lead compound in malaria drug discovery.
MATERIALS AND METHODS
Plant Material Collection and Authentication
Fresh stem bark of Aidia genipiflora (Hook.f.) Dandy was collected from the wild in the Central Region of Ghana (06°36.704′N, 000°42.659′W). The plant was authenticated by comparison with herbarium specimens deposited at the Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi. A voucher specimen (UCC/PHM/AG/2023/SB01) was deposited at the Department of Pharmacognosy and Herbal Medicine, University of Cape Coast, for future reference.[9]
Extraction, Isolation, and Characterization of Oleanonic Acid
Air-dried and pulverized stem bark (3 kg) was Soxhlet-extracted with chloroform-methanol (1:4 v/v). The concentrated extract (AG extract) was fractionated over silica gel (70-230 mesh) using gradient elution with petroleum ether, ethyl acetate, and methanol. Fractions were pooled based on TLC profiles, and the ethyl acetate fraction was subjected to further purification to afford oleanonic acid. Structural elucidation was confirmed by 1D/2D NMR and MS data compared with literature.[9, 13]
Parasite Strains and Animals
Plasmodium falciparum strains (chloroquine-sensitive 3D7 and chloroquine-resistant Dd2) and Plasmodium berghei ANKA strain were obtained from Noguchi Memorial Institute for Medical Research (NMIMR), Ghana. Healthy ICR mice (18-22 g) were housed under standard conditions with free access to food and water. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC), University of Cape Coast (Approval No. UCC/IACUC/2023/AG01).
In vitro Antiplasmodial Assay
Synchronized P. falciparum parasites were cultured in O⁺ human red blood cells at 2% haematocrit using RPMI 1640 supplemented with 25 mM HEPES, 0.5% AlbuMAX II, 0.2% sodium bicarbonate, and 50 μg/mL hypoxanthine, under a mixed gas atmosphere (90% N₂, 5% CO₂, 5% O₂) at 37ºC.[14, 15]
Antiplasmodial activity was assessed using the SYBR Green I fluorescence assay, which measures parasite DNA content as a proxy for viability.[16, 17] Briefly, parasites at 1% parasitaemia and 2% haematocrit were incubated with serial dilutions of AG extract or oleanonic acid (0.39-100 μg/mL) for 72 hr in 96-well plates. Artesunate and dihydroartemisinin served as positive controls, and 0.5% DMSO as vehicle control. Fluorescence was read at 485 nm excitation and 530 nm emission.
In vivo Antiplasmodial Assay
The 4-day suppressive test was performed following standard.[18, 19] Mice were inoculated intraperitoneally with 1.2 × 10⁶ P. berghei-infected RBCs. After 72 hr, they were randomized into groups (n = 5) and treated orally for four days with AG extract (50-800 mg/kg), oleanonic acid (3-150 mg/kg), or vehicle (10% Tween-80 in saline). Dihydroartemisinin (0.5-8 mg/kg) was used as a positive control.
Parasitaemia was determined from Giemsa-stained thin smears prepared on day 8. Percent parasitaemia and chemosuppression were calculated relative to controls:[20]
Cytotoxicity Assays
Cytotoxicity of oleanonic acid was evaluated on human RBCs (for haemolysis) and HepG2 liver cells using the MTT assay.[21, 22] RBC haemolysis was quantified spectrophotometrically at 540 nm after 48 hr incubation, and HepG2 viability was measured after 72 hr exposure to serial dilutions of the compound. The Selectivity Index (SI) was calculated as:
Data Analysis
IC₅₀ and CC₅₀ values were obtained by nonlinear regression of concentration-response curves (GraphPad Prism v9.1.5). Results were expressed as Mean±SEM of at least three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test, with p≤0.05 considered significant.
RESULTS AND DISCUSSION
In vitro Antiplasmodial Activity of Oleanonic Acid
Oleanonic acid (Figure 1), a pentacyclic triterpenoid isolated from Aidia genipiflora, demonstrated concentration-dependent inhibitory effects against the chloroquine-resistant Plasmodium falciparum Dd2 strain. Using the SYBR Green I fluorescence assay, the compound yielded IC₅₀ values of 10.00 µg/mL and 7.34 µg/mL in two independent experiments, giving a mean IC₅₀ of 8.67 µg/mL. In comparison, the reference drug artesunate exhibited markedly higher potency, with IC₅₀ values of 2.75 ng/mL and 0.64 ng/mL, giving a mean IC₅₀ of 1.70 ng/mL (Figure 2 and Table 1).
| Compound | IC50 (Trial 1) | IC50 (Trial 2) | Mean IC50 |
|---|---|---|---|
| Artesunate | 2.75 ng/mL | 0.64 ng/mL | 1.70 ng/mL |
| Oleanonic acid | 10.00 µg/mL | 7.34 µg/mL | 8.67 µg/mL |
In vivo Antiplasmodial Activity
Effect of Aidia genipiflora Stem Bark Extract
Administration of the methanolic extract of A. genipiflora significantly suppressed parasitaemia in Plasmodium berghei-infected mice in a dose-dependent manner. Suppression ranged from 17.5% at 50 mg/kg to 94.2% at 800 mg/kg when compared with the vehicle-treated group (Table 2). These findings demonstrate the potent in vivo efficacy of the crude extract.
| Dose (mg/kg) | Parasitaemia (%) | Chemosuppression (%) |
|---|---|---|
| 50 | 11.89 | 17.45 ± 8.49 |
| 100 | 6.4 | 55.56 ±1.40 |
| 200 | 3.28 | 77.26 ±1.69 |
| 400 | 2.1 | 85.42 ±1.69 |
| 800 | 0.83 | 94.18 ±1.51 |
| Vehicle | 14.4 | - |
Effect of Oleanonic Acid
Oleanonic acid (3-150 mg/kg) produced significant, dose-dependent suppression of parasitaemia in P. berghei-infected mice p<0.0001). The compound exhibited an ED₅₀ of 14.76 mg/kg, while dihydroartemisinin (DHA), used as a reference drug, was more potent with an ED₅₀ of 3.96 mg/kg (Figures 3A and 3B).
On day 8, endpoint analysis showed that oleanonic acid induced a clear dose-dependent chemosuppression, though consistently lower than DHA at equivalent doses (Figures 4A and 4B)
These results reinforce the in vivo efficacy of both the extract and its constituent, suggesting possible additive or synergistic effects of triterpenoids, flavonoids, and alkaloids found in the crude extract.[9] These findings also indicate that oleanonic acid is effective in suppressing parasite multiplication in vivo and supports its potential as a scaffold for developing antimalarial agents, particularly in combination therapies.
Cytotoxicity and Selectivity Index
To assess safety, cytotoxicity of oleanonic acid was evaluated in human Red Blood Cells (RBCs) and HepG2 liver cells using the MTT assay. Oleanonic acid showed no toxicity to RBCs (CC₅₀>100 µg/mL), but moderate toxicity to HepG2 cells with a CC₅₀ of 5.08 µg/mL (Table 3). These results correspond to a high selectivity index (SI>11.53) for RBCs and a low SI of 0.59 for HepG2 cells. According to the Medicines for Malaria Venture (MMV) criteria, an SI≥10 is desirable for lead compounds.[25] Although the HepG2 result indicates moderate cytotoxicity, the strong selectivity for RBCs suggests preferential activity toward parasitized erythrocytes, making oleanonic acid a promising, albeit partially selective, antiplasmodial candidate.
| Cell type | Cell line | CC50 (µg/mL) | Parasite IC₅₀ (µg/mL) | SI |
|---|---|---|---|---|
| Hepatic | HepG2 | 5.08 | 8.67 | 0.59 |
| Erythrocytic | Human RBCs | >100 | 8.67 | >11.53 |
DISCUSSION
This study demonstrates that both the crude stem bark extract of Aidia genipiflora and the isolated triterpenoid oleanonic acid possess antiplasmodial activity. Although oleanonic acid exhibited only moderate in vitro potency (IC₅₀ = 8.67 µg/mL) compared with artesunate, it produced significant in vivo efficacy in Plasmodium berghei-infected mice (ED₅₀ = 14.76 mg/kg), with dose-dependent chemosuppression. Such differences between in vitro and in vivo activity are frequently observed in natural product pharmacology and may be attributed to improved bioavailability, host-immune interactions, or metabolism in vivo.[23, 24]
The crude extract showed stronger parasitaemia suppression at higher doses, suggesting that multiple phytoconstituents contribute additively or synergistically to the overall activity. This aligns with the traditional use of multi-component plant remedies and underscores the therapeutic potential of A. genipiflora as a source of structurally diverse secondary metabolites.[7, 25] Such synergism is of particular interest in malaria therapy, where combination effects can help delay or overcome resistance development.
The cytotoxicity results provide important lead-optimization insights. Oleanonic acid showed no toxicity toward RBCs (CC₅₀>100 µg/mL; SI>11.5), indicating good selectivity for parasitized erythrocytes-a desirable property for antimalarial agents.[26, 27] However, the moderate toxicity observed against HepG2 cells (CC₅₀ = 5.08 µg/mL; SI = 0.59) suggests possible hepatotoxic risk. While this raises caution, it does not invalidate the compound’s potential; rather, it highlights the need for structure-activity relationship (SAR) studies, semi-synthetic analogues, and detailed toxicity profiling to expand its therapeutic window. Importantly, many frontline antimalarials, including artemisinin derivatives, also required optimization to balance efficacy and safety before clinical use.[28, 29]
From a broader perspective, this work makes three key contributions. First, it provides the first in vivo evidence of the antiplasmodial efficacy of oleanonic acid when isolated from A. genipiflora, thereby adding species-specific pharmacological relevance. Second, it highlights the potential value of A. genipiflora as a source of antiplasmodial natural products. Third, it demonstrates that triterpenoids such as oleanonic acid, despite moderate in vitro activity, may possess favourable in vivo efficacy profiles, making them worthy of further optimization within modern hit-to-lead frameworks.[30] Taken together, the results establish a scientific basis for further investigation of A. genipiflora and its constituents in antimalarial drug discovery. Oleanonic acid in particular represents a promising, though partially selective, scaffold that could be refined through medicinal chemistry approaches and tested in combination therapies to enhance efficacy and safety.
CONCLUSION
The stem bark extract of Aidia genipiflora and its isolated triterpenoid oleanonic acid exhibited significant antiplasmodial activity. While oleanonic acid showed moderate in vitro potency, its strong in vivo efficacy provides the first evidence of antiplasmodial activity from this species. The compound was non-toxic to erythrocytes but moderately toxic to HepG2 cells, warranting further optimization. These findings position A. genipiflora as a promising source of antimalarial agents and justify future studies on mechanism, structure-activity relationships, and combination therapies.