Exploring the antimalarial potential of (+)-2,2'-epicytoskyrin A: in vitro and in vivo studies of a bioactive metabolite from endophytic fungus Diaporthe sp. GNBP-10

Malaria remains a major global parasitic disease, increasingly complicated by resistance to frontline antimalarial drugs such as chloroquine and artemisinin. This challenge underscores the urgent need for novel antimalarial agents with alternative mechanisms of action. Endophytic fungi constitute a promising yet underexplored source of structurally diverse bioactive metabolites. This study investigated the antimalarial potential of ( +)-2,2′-epicytoskyrin A (Epi-A), a bisanthraquinone isolated from Diaporthe sp. GNBP-10, an endophyte of Uncaria gambir (Hunter) Roxb., was utilized in both in vitro and in vivo models.

In vitro antimalarial activity was assessed against chloroquine-sensitive Plasmodium falciparum 3D7 using microscopic examination of Giemsa-stained thin blood smears. At the same time, cytotoxicity was evaluated in MCF-7 cells by measuring absorbance at 450 nm and calculating CC₅₀ values using GraphPad Prism. In vivo efficacy was evaluated in Plasmodium berghei–infected Swiss Webster mice. Animals were assigned to five groups: oral Epi-A (12.5, 25, and 50 mg/kg BW), artemisinin (20 mg/kg BW), and an untreated infected group. Treatment was administered for four consecutive days after the mean parasitaemia reached 10%. Histopathological analysis of the liver, spleen, kidney, and intestine was performed on day 8 pi using one mouse per group, while the remaining mice were monitored until day 15 pi for parasitaemia, survival, clinical manifestations, and body weight changes.

Epi-A demonstrated potent in vitro activity, with an IC₅₀ of 0.24 µM, a CC₅₀ of 4.4 µM, and a selectivity index of 18.33. In vivo, on day 8 pi, histopathology revealed haemozoin deposition with mild tissue alterations in the liver, spleen, kidney, and intestine. Parasitaemia did not differ among Epi-A–treated groups but was significantly lower than in the negative control. Epi-A achieved > 50% inhibition (63.6%, 77.4%, and 50% at 12.5, 25, and 50 mg/kg BW, respectively), classifying it as a very good antimalarial activity. No post-treatment weight loss was observed, and only mild clinical signs were noted, compared with the severe manifestations observed in the negative control.

Epi-A demonstrates potent in vitro and very good in vivo antimalarial activity, with low toxicity and potential organ-protective effects, supporting its promise as a candidate for further mechanistic studies.