Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo. Tomo 56(2). ISSN (en línea) 1853-8665. Año 2024.

Original article

 

In vitro efficacy testing of citronella grass oil against Tritrichomonas foetus trophozoites

Evaluación in vitro de la eficacia del aceite de citronela contra trofozoítos de Tritrichomonas foetus

 

María Rosana Ramirez1,

Estefanía Sereno Bruno2,

Debora Manuale3,

Juan Carlos Yori3,

Jorge Oyhenart2*

 

1Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Instituto Universitario de Ciencias de la Salud, Fundación H. A. Barceló. Buenos Aires. Argentina.

2Universidad Nacional de La Pampa. Facultad de Ciencias Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). La Pampa. Argentina.

3Universidad Nacional del Litoral. Instituto de Investigaciones en Catálisis y Petroquímica. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Santa Fe. Argentina.

 

*jorgeoyhenart@gmail.com

 

Abstract

Tritrichomonas foetus, a sexually transmitted parasitic protozoan, causes abortion in cattle. Nitroimidazoles, such as metronidazole, treat bovine trichomonosis, but their use is precluded. Plant extracts might have antiparasitic effects. This study aimed to assess Cymbopogon nardus oil activity against T. foetus as an alternative to metronidazole. T. foetus trophozoites were incubated in culture medium containing serial dilutions of C. nardus oil. Cytotoxicity was assessed 24 hours later. C. nardus oil killed T. foetus cells. Half maximal effective concentration (EC50) was 0.4 μg/mL. These findings suggest that C. nardus oil could be exploited for discovery and compound isolation of plant-derived phytopharmaceuticals for bovine trichomoniasis and other protozoan diseases.

Keywords: disease, venereal, trichomonas, trichomoniasis, antimicrobial, dose-response

 

Resumen

Tritrichomonas foetus es un protozoo parásito de transmisión sexual que provoca abortos en el ganado. Los nitroimidazoles, como el metronidazol, pueden ayudar en el tratamiento de la tricomonosis bovina, pero no se aconseja su uso. Los extractos de plantas pueden tener un efecto antiparasitario. El estudio actual tuvo como objetivo evaluar la actividad del aceite de Cymbopogon nardus contra T. fetus como alternativa al metronidazol. Se incubaron trofo­zoítos de T. fetus en medio de cultivo con diluciones seriadas de aceite de C. nardus. La cito­toxicidad se evaluó a las 24 horas. El aceite de C. nardus mató las células de T. fetus. La concen­tración efectiva media máxima (CE50) fue de 0,4 μg/ml. Estos hallazgos sugieren que el aceite de C. nardus podría servir para el descubrimiento y aislamiento de fitofármacos para el tratamiento de la tricomoniasis bovina y de otras enfermedades protozoarias.

Palabras clave: enfermedad, venérea, trichomonas, trichomoniasis, antimicrobiano, dosis-respuesta

 

Originales: Recepción: 19/02/2024 - Aceptación: 13/06/2024

 

 

Introduction

 

 

Tritrichomonas foetus is a parasite protozoan that causes bovine trichomonosis (2). This venereal disease causes premature abortion and extended intercalving seasons. Nitroimidazoles are antibiotics used to treat infections such as trichomonosis (10). However, strain resistance and potential toxicity to meat consumers limit their use.

Bovine trichomonosis vaccines are not available and culling TF-infected animals is recommended. Between 2015 and 2019, more than 3,800 T. foetus-carrying bulls were culled in the United States (11). Every year in La Pampa, Argentina, more than 300 diagnosed bulls must be slaughtered (13). So far, testing and elimination programs have rarely succeeded in their purpose of eradicating T. foetus from cattle populations (13, 16).

New therapies for T. foetus-infected animals consider plant extracts as alternatives to commercial drugs. Extracts obtained from black tea, green tea, grape and pomegranate inhibit pathogenic trichomonads (9). Among natural extracts, essential oils are commonly used in traditional medicine and some studies have addressed their effect against protozoa (1, 5).

Citronella essential oil (EO) is extracted from the perennial herb Cymbopogon nardus (7). C. nardus is cultivated in tropical and subtropical regions, including northern Argentina. The oil has several biological activities, including insect-repellent, fungicidal, and bactericidal properties (7, 9, 12). To the best of our knowledge, the anti-trichomonas activity of this compound has not yet been investigated (4).

This study aimed to investigate the efficacy of C. nardus essential oil against the parasite T. foetus in vitro, in comparison with metronidazole. Increasing concentrations of C. nardus essential oil should significantly decrease T. foetus trophozoites viability.

 

 

Materials and methods

 

 

Plant material was obtained from existing monoculture plantations from El Soberbio, Misiones, Argentina (27°17’43.76” S, 54°11’46.84” W). Approximately 1,000 g of aerial parts were hydrodistilled. The chemical composition of C. nardus oil was determined by capillary gas chromatography on a 2014 Shimadzu GC equipped with a FID detector and a capillary column (30 m × 0.25 mm internal diameter) J&W INNOWax 19091 N-213. Nitrogen was used as carrier gas. Samples (1 μl) were diluted with acetone and injected at a 50:1 ratio. Operating conditions considered oven temperature maintained at 60°C for 8 min and gradually raised by 3°C per min, reaching 180°C, and kept for 5 min. Temperature at injection and detector ports was 250°C. C. nardus oil main components were citronellal (40.1%), citronellol (12.7%) Geraniol (25.3%), limonene (3.7%), trans-PMD (3.6%), cis-PMD (2.6%) and isopulegols (0.3%).

The clone T. foetus B1 obtained from a cow with pyometra, was used for conducting parasite-killing assays (14). Trophozoites were maintained in trypticase-yeast-maltose (TYM) medium supplemented with 1 g/L streptomycin, 1,000,000 IU/L ampicillin and 10% v/v heat-inactivated horse serum. Cultures were maintained at 37°C in sterile 2.0 mL tubes filled with media and tightly capped. Subcultures were made in 2-3 days intervals maintaining cell concentrations below 8 105 trophozoites/mL.

Pure cultures in antibiotic-free medium were seeded at a density of 5.105 T. foetus cells/mL in 10-fold triplicate serial dilutions containing C. nardus oil between 0-800 ug/mL or metronidazole (Sigma Aldrich, M1547) dilutions from 0 to 100 ug/mL. Vehicles and drugs diluted in water or 70% ethanol accounted for 1% of final volume. Incubation proceeded for 24 h at 37°C under microaerophilic conditions. All experiments were performed three times. Compound toxicity was assessed by Trypan blue exclusion on hemocytometer slides. GRmetrics’ 4-parameter non-linear regression model was used to calculate IC50 and EC50 values as well as to generate dose-response curves (3). T. foetus growth rates were plotted against logarithmically transformed drug concentrations.

 

 

Results

 

 

Figure 1 shows best-fitted dose-response curves depicting T. foetus B1 cells response to C. nardus oil and metronidazole control.

 

 

Untransformed growth rate versus log-transformed concentration showing effects of C. nardus oil compared to metronidazole. Standard curves were built with Grmetrics. GR: growth rate.

Tasa de crecimiento no transformada versus concentración logarítmica transformada para mostrar el efecto del aceite en comparación con el metronidazol. Las curvas estándar se construyeron con Grmetrics. GR: tasa de crecimiento.

Figure 1. Dose-response curve for Cymbopogon nardus oil effect on Tritrichomonas foetus.

Figura 1. Curva dosis-respuesta del efecto del aceite de Cymbopogon nardus sobre Tritrichomonas foetus.

 

Over 24 hours, mock-treated cells doubled 4.6 times. At the same time, the vehicle (0.7% ethanol) had no visible effect on cell division or morphology. In contrast, treatment with metronidazole (positive kill control) produced a standard S-shaped curve, indicating strain sensibility. The EC50 for metronidazole was 0.016 μg/mL (16 μM). The concentration at which relative cell count was 0.5 of maximal value (IC50) was also 0.016 μg/mL (table 1).

 

Table 1. Cymbopogon nardus oil inhibition values.

Tabla 1. Valores de inhibición del aceite de Cymbopogon nardus.

r2 coefficient of determination indicating goodness of fit; GR50, concentration at which the effect reaches a growth rate value of 0.5 based on interpolation; EC50, the concentration at which relative cell count was 0.5 of maximal values; IC50, the concentration at half-maximal effect; h, Hill coefficient of the fitted (traditional) dose-response curve and the p-value of a F-test comparing curve fit to a horizontal line fit. Concentrations are expressed in ug/mL.

Se indican: r2, el coeficiente de determinación para indicar bondad de ajuste de la curva; GR50, la concentración a la cual el efecto alcanzó un valor de tasa de crecimiento de 0,5 basado en la interpolación de la curva ajustada; EC50, la concentración a la que el recuento relativo de células fue 0,5 de los valores máximos; IC50, la concentración a la mitad del efecto máximo; h, el coeficiente de Hill de la curva dosis-respuesta ajustada (tradicional) y el valor p de una prueba F que compara el ajuste de la curva con un ajuste de línea horizontal. Las concentraciones se expresan en ug/mL.

 

C. nardus oil also exhibited dose-dependent inhibition, with all cells killed after 24 hours at high oil concentrations. The EC50 value for C. nardus oil was 0.391 μg/mL (table 1). Oil concentration required to achieve a growth rate of 0.5 was 0.65 μg/mL (table 1). Fitted curves explain over 95% of observed variation (figure 1).

 

 

Discussion

 

 

Several plant compounds have in vitro anti-trichomonal activity (6). Data presented here indicate that C. nardus oil can kill T. foetus in a dose-dependent manner. C. nardus oil could be used for local treatment. Additionally, C. nardus oil could constitute a basic resourse for plant-derived drugs for protozoan diseases. The main constituents of C. nardus oil (geranial, citronellol and elemol) show activity against Trypanosome brucei (9). In addition, several components have been found active against fungal strains (citronellal and linalool), Gram-positive and Gram-negative bacterial species (elemol, citronellol, citronellal) (7, 8, 15).

 

 

Conclusion

 

 

C. nardus oil affects growth and viability of T. foetus in vitro. C. nardus oil constitutes a useful resource for the discovery of plant-derived drugs for treating bovine trichomonosis and other protozoan diseases.

 

Acknowledgments

This work has been supported by PIP0833 and PIP0054 CONICET.

 

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Conflict of interests

The authors declare that there is no conflict of interest.