Prunus amygdalus var. amara seed extract enhances the antileishmanial activity of miltefosine.

BACKGROUND

Leishmaniasis, an infectious disease transmitted via sand flies is caused by the protozoan parasite of Leishmania spp. The treatment of this disease is quite challenging due to the high cost, resistance, and toxicity of conventional drugs. Various research studies have demonstrated that plant based drug possess least toxicity, anti-inflammatory and anti-oxidant properties. Here, evaluation of anti-leishmanial activity of methanolic Prunus amygdalus var. amara seed extract was conducted and found that it inhibited L. donovani proliferation and cause apoptosis. Moreover, its combinations with miltefosine enhanced antileishmanial effects. GC-MS analysis confirmed the presence of various phytochemicals in the extract that contributed pharmacological efficacy. These findings highlighted the potential of herbal products as a valuable source of new treatments for leishmaniasis.

METHODS

The antileishmanial effect was determined by promastigote and amastigote assays. Parasite load was evaluated by staining L. donovani-infected macrophages with modified Giemsa stain. Cytotoxicity of seed extract was estimated by MTT (3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay. In addition, Pro-apoptotic events were inferred using RT-PCR and qRT-PCR. Further characterization of phytoconstituents was evaluated by gas chromatography and mass spectrometry.

RESULTS

The extract promoted a dose-dependent reduction in growth of promastigotes (IC50 = 43.12 ± 3.03 μg/ml) and amastigotes (IC50 = 49.65 ± 3.34 μg/ml). Further, extract in combination with miltefosine showed enhanced antileishmanial activity against both forms of the L. donovani, promastigotes (IC50 = 4.547 ± 1.2 μg/ml) as well as amastigotes (IC50 = 19.54 ± 2.4 μg/ml). Early-stage apoptotic events were also observed in promastigote forms by determining the increased expression of LdMetacaspase and PARP1. The cytotoxic potential on THP-1 differentiated macrophages was assessed and indicated insignificant cytotoxicity of different doses of the extract (CC50 = 799.19 ± 134.59 μg/ml) and in combination with miltefosine (CC50 = 384.16 ± 177.47 μg/ml). Furthermore, the presence of phytocompounds like chaulmoogric acid and hydnocarpic acid was described, for the first time, in Prunus amygdalus var. amara seed extract.

CONCLUSION

The findings indicated that EPA plays a significant role in combating leishmaniasis and holds promise as a potential treatment for this disease. Moreover, when combined with miltefosine, EPA demonstrated increased effectiveness against leishmaniasis. Therefore, the combination of EPA and miltefosine presents a more promising outlook as a potential therapy for leishmaniasis.