Leishmaniasis remains one of the most burdensome parasitic diseases worldwide, presenting in cutaneous, mucocutaneous and visceral forms. The visceral form can be fatal if untreated, while the cutaneous form causes disfiguring skin lesions and lasting stigma. Existing therapies rely heavily on pentavalent antimonials such as Glucantim, the meglumine antimoniate formulation used as the comparator in this study, along with newer agents like miltefosine and amphotericin B. All of these carry significant drawbacks: antimonials require injections over weeks, cause cardiac and hepatic toxicity, and have lost effectiveness in some endemic regions where resistant parasite strains circulate. The limitations of the current pharmacopoeia are precisely why the Iranian team turned to natural toxins, a class of molecules evolution has refined to disrupt cells with remarkable efficiency.

The study compared two test compounds against the standard drug. The first was crude venom collected from the western honey bee, Apis mellifera, a complex cocktail of peptides and enzymes including melittin, apamin and phospholipase A2. The second was purified bee venom phospholipase A2, abbreviated bvPLA2, an enzyme that hydrolyzes phospholipids in cell membranes and is a major allergenic and pharmacologically active component of the venom. By testing both the whole venom and its isolated enzyme, the researchers could ask whether the anti-parasitic effect resides in the phospholipase itself or in the broader mixture, a question that matters greatly for any future effort to develop a tolerable medicine.

Methodologically, the team worked entirely in vitro, using the promastigote stage of L. major, the motile flagellated form that lives in the sand fly vector, and the amastigote stage, the round intracellular form that resides inside host macrophages and causes disease in humans. They also assessed toxicity toward RAW 264.7 macrophage cells, a standard murine macrophage line, using the MTT assay, a colorimetric test in which metabolically active cells convert a yellow tetrazolium salt into a purple formazan product, allowing viability to be quantified by absorbance. From these measurements the researchers calculated the CC50, the concentration killing half of the host cells, and the IC50, the concentration inhibiting half of the parasites, across 24, 48 and 72 hour exposure windows, with statistical analysis performed in GraphPad Prism.

The cytotoxicity results revealed a striking difference between the two compounds. Crude bee venom proved highly toxic to macrophages, with a CC50 of just 2.17 micrograms per milliliter and complete host-cell mortality at the later 48 and 72 hour time points. Purified bvPLA2, by contrast, was far gentler on the host cells, with CC50 values of 45.89, 36.22 and 86.01 micrograms per milliliter at 24, 48 and 72 hours respectively. This separation matters because a useful anti-parasitic drug must kill the parasite at concentrations that leave host cells largely unharmed, a balance expressed as the selectivity index. The enzyme’s much wider margin between parasite inhibition and host toxicity suggests that the crude venom’s other constituents, most likely membrane-lytic peptides such as melittin, drive the host-cell killing rather than the phospholipase itself.

Against the parasites, both compounds showed dose-dependent inhibition. On promastigotes, the inhibitory effect was evident from the lowest tested concentration at both time points examined. On the intracellular amastigotes, the clinically relevant stage, crude bee venom produced no significant inhibition at lower concentrations, whereas bvPLA2 significantly suppressed amastigote viability at both incubation times. At higher concentrations, however, viability fell more steeply in the crude venom treatments, indicating that the full venom mixture retains potent but indiscriminate lethal activity. Interestingly, when the researchers compared inhibition at constant concentrations between 24 and 48 hours, the effect either diminished or lost statistical significance at the longer exposure, an unexpected pattern that the authors note may reflect compensatory parasite responses or assay dynamics and that warrants further investigation.

The headline numbers came at the 48 hour mark. Crude venom achieved IC50 values of 4.89 micrograms per milliliter against promastigotes and 6.07 against amastigotes, while bvPLA2 recorded 5.76 and 9.03 micrograms per milliliter respectively. Both figures were significantly lower than the IC50 of Glucantim, the standard antimonial drug, with a probability value below 0.0001. In plain terms, on a pure potency basis, the bee-derived compounds outperformed the frontline therapy in this assay system. The researchers also observed an anomaly in the bvPLA2 data: MTT absorbance values rose above baseline at the first four enzyme concentrations after 48 hours, a pattern that could indicate a proliferative or metabolic stimulation of host cells at low enzyme doses and that the team flags as deserving of dedicated follow-up.

Mechanistically, the findings fit a broader literature on secretory phospholipase A2 enzymes as anti-parasitic agents. Leishmania parasites depend heavily on membrane phospholipid dynamics for host-cell invasion, intracellular survival and immune evasion, and their plasma membranes are rich in substrates that bvPLA2 can hydrolyze. Enzymatic disruption of these membranes could compromise the parasite’s integrity or its ability to remodel the parasitophorous vacuole it inhabits inside macrophages. Snake venom phospholipases have shown similar anti-leishmanial effects in earlier studies, and the present work extends that logic to the bee-derived enzyme. Because the purified enzyme is less cytotoxic than the whole venom, it offers a more plausible starting point for medicinal chemistry, where enzymatic activity could be tuned, or synthetic peptide analogues developed, to maximize parasite selectivity.

The authors are careful about scope. This is an in vitro study, and the leap from a culture dish to a patient involves pharmacokinetics, immunogenicity, delivery to infected macrophages and the formidable allergenic potential of bee venom components, since bvPLA2 is a principal trigger of bee-sting anaphylaxis. The anomalous MTT signals and the waning of inhibition at longer exposures also mean the dose-response picture is not yet fully resolved. Still, the study, which was extracted from the thesis of lead author Pouria Kavousi and funded by the School of Medicine at Shahid Beheshti University of Medical Sciences, establishes a concrete benchmark: the first documented anti-leishmanial activity of bvPLA2, with potency exceeding the standard drug in vitro and a cytotoxicity profile far more favorable than that of the crude venom. The researchers suggest this opens new avenues for exploring the pharmacological and biotechnological potential of the compound, and it adds honey bee venom to the growing list of venom sources, from snakes to scorpions to cone snails, being mined for next-generation anti-parasitic leads.

For the global health community, the significance lies in the direction of travel rather than any immediate therapy. Roughly a billion people live in areas endemic for leishmaniasis, and the World Health Organization counts hundreds of thousands of new cases annually, yet no vaccine exists for humans and the drug pipeline is thin. Natural product screening of this kind, pairing crude extracts with purified constituents and rigorous host-cell toxicity controls, is exactly the kind of low-cost, hypothesis-generating work that can seed future drug discovery programs. If subsequent studies can confirm the mechanism, improve the selectivity index further and demonstrate efficacy in animal models, the enzyme that honey bees use to defend their hive may one day contribute to treating a disease that has defied conventional chemotherapy for decades.

Subject of Research: Anti-leishmanial activity of honey bee venom and its phospholipase A2 enzyme against Leishmania major in vitro

Article Title: A step forward to exploring the inhibitory effects of crude honey bee venom (Apis mellifera) and its derived phospholipase A2 (bvPLA2) against Leishmania major in vitro

Article References: Kavousi, P., Lasjerdi, Z., Tabaei, S. J. S., Taghipour, N., Koosha, M., Koochaki, A., & Vaziri, V. M. (2026). A step forward to exploring the inhibitory effects of crude honey bee venom (Apis mellifera) and its derived phospholipase A2 (bvPLA2) against Leishmania major in vitro. BMC Complementary Medicine and Therapies. https://doi.org/10.1186/s12906-026-05572-3

Image Credits: AI Generated

DOI: 10.1186/s12906-026-05572-3

Keywords: Leishmania major, bee venom, phospholipase A2, leishmaniasis, Apis mellifera, antiparasitic agents, promastigotes, amastigotes, MTT assay, cytotoxicity, natural toxins, drug discovery