Protulišmanijski učinak iscrpka biljke Peganum harmala na in vitro rast promastigota Leishmania major u odnosu na trovalentne pripravke antimona

Parasites of the genus Leishmania are transmitted by sandflies that ingest the parasite in the amastigote stage resident within macrophages, then inoculate the promastigote stage into other hosts. Peganum harmala, or Syrian Rue, has pharmacologically active compounds including several alkaloids with antiprotozoal properties, which are found especially in the seeds and the roots. In this research, Leishmania major were cultured in vitro, then by using a MTT assay, the biological activity of P. harmala extract in comparison to potassium antimonyl tartrate [Sb(III)] on L. major promastigotes was assessed. P. harmala extract and Sb(III) solutions for biological testing were prepared in PBS at 5000-20000 μg/mL and 62.5-500 μg/mL, respectively. All experiments were repeated at least three times in duplicate. For P. harmala extract and Sb(III), the concentration-response curve was plotted, from which IC50 values were determined. Both P. harmala extract and Sb(III) inhibited the growth of promastigote forms of L. major in vitro after 72 h. of incubation and had an IC50 of 1832.65 ± 89.72 μg/ mL and 17.87 ± 2.05 μg/mL, respectively. Statistical analysis of the results (optical density and inhibitory percentage) of the different concentrations of P. harmala extract and Sb(III) showed that there was no significant difference between P. harmala extract and Sb(III) (P>0.05) but with a concentration increase of P. Harmala extract or Sb(III), optical density decreased significantly, while inhibitory percentage increased. The different concentrations resulted in different optical densities or inhibitory percentages (P<0.05) so that P. Harmala extract is effective against L. major in vitro.Parazite roda Leishmania prenose papatači preko makrofaga zaraženih amastigotima, a nakon razvoja nastale promastigote prenose na druge domaćine. Biljka Peganum harmala ili sirijska rutvica posjeduje farmakološki aktivne sastojke uključujući nekoliko alkaloida s protuprotozojskom učinkovitošću, koji se osobito nalaze u sjemenkama i korijenu biljke. Za potrebe ovog istraživanja, protozoon Leishmania major uzgojen je in vitro. Učinak iscrpka biljke P. harmala na promastigote L. major u odnosu na kalijev antimoniltartarat [Sb(III)] određen je MTT metodom. Iscrpak P. harmala bio je pripravljen u PBS-u u koncentraciji od 5.000-20.000 μg/mL. Antimonski pripravak bio je pripravljen u PBS-u u koncentraciji od 62,5-500 μg/mL. Svaki pokus in vitro bio je ponovljen najmanje tri puta. Dobiveni rezultati prikazani su krivuljom učinkovitosti kojom su određene vrijednosti inhibitorne koncentracije (IC50). Oba su pripravka kočila rast promastigota in vitro nakon 72 sata. Vrijednost IC50 za iscrpak P. harmala iznosila je 1832,65 ± 89,72 μg/mL. Vrijednost IC50 za antimonski pripravak iznosila je 17,87 ± 2,05 μg/mL. Statističkom obradom rezultata (optička gustoća i postotak inhibicije) dobivenim inkubiranjem protozoa u različitim koncentracijama pripravaka nisu utvrđene značajne razlike (P>0,05). Više koncentracije iscrpka P. harmala imale su za posljedicu značajno smanjenje optičke gustoće te istovremeno povećanje postotka inhibicije. S obzirom da su različite koncentracije rezultirale različitom optičkom gustoćom ili postotkom inhibicije (P<0,05) zaključuje se da iscrpak P. harmala djeluje in vitro na vrstu L. major

The effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Leishmania tropica to meglumine antimoniate

Pentavalent antimonials are the standard treatment for cutaneous leishmaniasis (CL) with low efficacy and resistance is emerging. CL is increased significantly in respect to incidence rate and expanding to new foci. In the present study, the effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Leishmania tropica to meglumine antimoniate (MA, Glucantime) was evaluated using colorimetric assay (MTT) and in a macrophage model, respectively. Verapamil, as a calcium channel blocker, affects drug uptake by preventing of drug efflux from the cells. In promastigote form, several concentrations of MA with or without verapamil showed significant decrease (P<0.05) in optical density. The overall mean IC50 value with combination of MA plus verapamil (IC50=116.03 μg/ml) was significantly less than MA (IC50=225.14 μg/ml) alone (P<0.05) for promastigote stage. Similarly, the amastigote stage was more susceptible to treatment with MA plus verapamil to that of MA alone (P<0.05). Analysis of overall effect of different concentrations of MA alone, compared with combination of MA plus verapamil by mean infection rate of amastigotes in each macrophage showed a significant difference (P<0.05).These findings indicated some degree of synergistic effects between MA and verapamil on in vitro susceptibility of L. tropica to MA. Further works are required to evaluate this synergistic effect on animal model or volunteer human subjects