The potential role and apoptotic profile of three medicinal plant extracts on Leishmania tropica by MTT assay, macrophage model and flow cytometry analysis

Mozhde Ilaghi; Iraj Sharifi; Fariba Sharififar; Fatemeh Sharifi; Razieh Tavakoli Oliaee; Zahra Babaei; Manzume Shamsi Meimamandi; Alireza Keyhani; Mehdi Bamorovat

doi:10.1016/j.parepi.2021.e00201

The potential role and apoptotic profile of three medicinal plant extracts on Leishmania tropica by MTT assay, macrophage model and flow cytometry analysis

Parasite Epidemiol Control. 2021 Jan 10:12:e00201. doi: 10.1016/j.parepi.2021.e00201. eCollection 2021 Feb.

Authors

Mozhde Ilaghi¹, Iraj Sharifi¹, Fariba Sharififar², Fatemeh Sharifi³, Razieh Tavakoli Oliaee¹, Zahra Babaei¹, Manzume Shamsi Meimamandi⁴, Alireza Keyhani¹, Mehdi Bamorovat¹

Affiliations

¹ Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
² Herbal and Traditional Medicines Research Center, Department of Pharmacognosy, Kerman University of Medical Sciences, Kerman, Iran.
³ Research Center of Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran.
⁴ Physiology and Pharmacology Department, Kerman Medical School, Kerman University of Medical Sciences, Kerman, Iran.

Abstract

Introduction: Treatment of leishmaniasis with conventional synthetic drugs is a major global challenge. This study was designed to explore the leishmanicidal activity and apoptotic profile of three leaf extracts on Leishmania tropica stages.

Methods: The plants of Quercus velutina, Calotropis procera and Nicotiana tabacum were gathered from Anbarabbad county, in the southeastern part of Kerman province and extracted by maceration method using methanol alcohol. Various concentrations of the extracts (1, 10, 100 and 1000 μg/mL) were used against L. tropica stages to evaluate the inhibitory effect by colorimetric assay, macrophage model and flow cytometry. The MTT assay was conducted to determine the IC₅₀ and CC₅₀ values in promastigotes and J774-A1 macrophages, respectively. For intra-macrophage amastigotes, the leishmanicidal activity was evaluated by calculating the mean number of amastigotes in each macrophage and also IC₅₀ values. The promastigote or amastigote stages with no drug and complete medium without organisms were considered as positive and negative controls, respectively. Meglumine antimoniate (Glucantime) was also used as standard drug. Also, annexin V was used to assess the apoptotic profile. All treatment settings were incubated for a standard time of 72 h in triplicates. Data were analyzed by t-test and ANOVA.

Results: The findings showed that all plant extracts inhibited the proliferation rate of promastigotes and amastigotes (P ˂ 0.001); especially, Q. velutina represented the lowest IC₅₀ in both stages. Besides, Q. velutina showed the least number of amastigotes in each macrophage compared to the other groups (4.5 μg/mL). The percentage of parasitic apoptosis at 1000 μg/mL of Q. velutina, C. procera, N. tabacum and Glucantime® were 37.4, 18.6, 8.5 and 52.4, respectively. Amastigotes (clinical stage) were significantly more susceptible to extracts and also Glucantime® than promastigotes (P < 0.001).

Conclusions: This study revealed that all three extracts of Q. velutina, C. procera and N. tabacum exhibited an effective antileishmanial activity and induced apoptosis against the L. tropica promastigotes. Further investigations are essential to isolate and analyze the chemical compositions and their biological properties.

Keywords: Apoptosis role; Leishmania tropica; Leishmanicidal activity; MTT assay; Medicinal plants.