Evaluation of the in vitro effect of alcoholic extract of Nepeta ispahanica L. leaves on Leishmania major promastigote stage by colorimetric assay

Document Type : Research Paper

Authors

1 M. Sc. Student, College of Agriculture and Natural Resources, University of Hormozgan, Bandar Abbas, and Shahid Bahonar University of Kerman, Kerman, Iran.

2 Department of Agricultural Biotechnology Engineering, College of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran.

3 Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.

Abstract

Objective
Leishmaniasis is one of the most prevalent parasitic unicellular diseases worldwide, which is caused by the Leishmania genus. Currently, pentavalent antimony compounds such as Glucantime and amphotericin B are used to treat cutaneous leishmaniasis. In many cases, herbal remedies have no adverse effects and are both affordable. Therefore, finding an herbal medicine to treat leishmaniasis is one of the global objectives. This study aimed to evaluate the anti-leishmanial activity of Nepeta ispahanica L. leaves alcoholic extract on promastigote stage of Leishmania major by in vitro colorimetric method.
 
Materials and methods
Different concentrations of Nepeta ispahanica L. leaf extract were used against L. major promastigotes compared to amphotericin B. L. major promastigotes were cultured in RPMI-1640 medium supplemented with 10 Percent FBS. Optical absorption (OD) was measured by the ELISA method to determine the 50 Percent inhibitory concentration (IC50) of drugs. Promastigotes were added to the 96-well plate and incubated for 72 h with different concentrations of bay laurel leaf extract and amphotericin B (800, 600, 400, 200, 50, 25, and 10 μg mL-1). Flow cytometry was used to evaluate apoptosis. All concentrations were incubated in the MTT assay for 3 min and the flow cytometric assay for 2 h. Statistical data were analyzed by Duncan, ANOVA, and SAS software at P≤0.05.
 
Results
In N. ispahanica L. leaf extract treatments, the greatest amount of apoptosis (42.47 Percent) was related to treatment of 800 μg mL-1 concentration. Maximum amount of apoptosis (97.35 Percent) in amphotericin B treatments as positive control was related to treatment 800 μg mL-1 concentration.  CC50 of Amphotericin B and leaf extract of bay laurel treatments was obtained in 129.6 and 162 μg mL-1 concentrations, respectively. Inhibitory concentration (IC50) of amphotericin B and N. ispahanica L. leaf extract treatments were 18.5 and 2989.5 μg mL-1 on L. major promastigotes.
 
Conclusions
The results showed an increase in dose-dependent apoptosis based on flow cytometric analysis.  Also, the proximity of N. ispahanica L. leaf extract and amphotericin B treatments in apoptosis-like cell death, which increases with increasing drug concentration. According to the IC50 results of N. ispahanica L. leaf extract and its high apoptotic efficiency, further studies are needed to evaluate of N. ispahanica L. leaf extract on Leishmania parasite and also, to design a clinical setting for this herbal drug.

Keywords

References

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Agricultural Biotechnology Journal
Volume 17, Issue 4
November 2025
Pages 51-70

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