First Insights into the Biological Activity and Molecular Docking of Citral (3,7-Dimethyl-2, 6-Octadienal) Against Trichomonas vaginalis

Abstract

The increasing resistance of Trichomonas vaginalis to the only approved chemical family of drugs for treatment, the 5-nitroimidazoles, has prompted the exploration of new therapeutic agents against this prevalent non-viral sexually transmitted infection. Natural products have emerged as a significant source of novel treatments for trichomoniasis. The aim of this study was to evaluate the anti-T. vaginalis activity of citral (3,7-dimethyl-2,6-octadienal), the main constituent of the essential oil of Cymbopogon species, commonly known as lemongrass. Our findings indicate that citral exhibits a minimum inhibitory concentration (MIC) of 100 μM, effectively inhibiting the growth of T. vaginalis trophozoites within 12 h of exposure, and a 50% inhibitory concentration (IC₅₀) of approximately 40 μM after 24 h. Furthermore, the evaluation of nitric oxide (NO) levels suggests that citral possesses antioxidant properties. Molecular docking studies reveal a weak interaction with three parasite proteins: thioredoxin reductase (TvTrxR), purine nucleoside phosphorylase (TvPNP), and methionine gamma lyase (TvMGL). The present study highlights the potential of citral as a candidate for the development of no-nitroimidazole drugs, offering new avenues for trichomoniasis treatment and underscoring the importance of further investigation into citral’s mechanism of action.

1. Introduction

More than one million new cases of curable sexually transmitted infections (STIs) occur daily, with the four most common pathogens being Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum and Trichomonas vaginalis [1]. In 2018, the World Health Organization (WHO) estimated that 354 million cases of these four STIs occurred in individuals between 15 and 49 years old, of whom 156 million were associated with T. vaginalis. This infection exceeds the number of cases of chlamydial infection and syphilis [2]. This parasite is of significant public health importance, as it is associated with an increased risk of acquiring and transmitting other STIs, including HIV and human papillomavirus (HPV), among others. Additionally, trichomoniasis can cause adverse effects on pregnant women, such as preterm delivery and low-birth-weight infants [3,4].

Metronidazole (2-methyl-5-nitroimidazole-1-ethanol) was the first drug approved for the treatment of anaerobic pathogens, including T. vaginalis. However, clinical cases of resistance have been reported since 1962 [5,6]. Subsequently, only tinidazole (1-(2-ethylsulfonylethyl)-2-methyl-5-nitroimidazole) and secnidazole (2-dimethyl-5-nitro-1H-imidazole-1-ethanol) were approved by the FDA for trichomoniasis in 2004 and 2021, respectively [7]. As the treatment of trichomoniasis has relied primarily on 5-nitroimidazole drugs, effective alternatives are lacking for patients who experience side-effects, hypersensitivity, or resistance. Consequently, when infections do not clear, the current strategy is to either repeat the same treatment or increase the dose of the 5-nitroimidazole drug. This approach may inadvertently promote the selection of more resistant isolates [8].

Natural products have long been the main source of therapeutic agents. In this context, plants and their derivatives have been investigated for their potential as trichomonacidal agents, yielding promising results [9]. Citral (3,7-dimethyl-2,6-octadienal) is a monoterpene composed of two isomeric compounds: alfa-citral (geranial) and beta-citral (neral) [10]. The composition of essential oils can be influenced by environmental factors and geographic location; however, lemongrass (Cymbopogon species) shows a remarkable consistency in its composition, with citral being a predominant and stable constituent in different variants of this plant species [11].

In light of the ongoing search for novel products derived from natural resources and the need for alternatives to 5-nitroimidazoles drugs, the present study aims to evaluate the trichomonacidal activity of citral, the main compound of Cymbopogon species.

2. Materials and Methods

2.1. Parasite and Cell Culture

Trichomonas vaginalis JH31A#4 (registration number 30236^TM) from the American Type Culture Collection (ATCC, Manassas, VA, USA) was axenically grown in trypticase–yeast extract–maltose (TYM) medium at pH 6 supplemented with 10% sterile heat-inactivated bovine serum and 5 mg/mL of streptomycin solution to avoid contamination [12]. Trophozoites with more than 95% mobility and normal morphology were sub-cultured every 48 h and incubated at 37 °C and 5% CO₂ [13].

2.2. Compounds

Citral and metronidazole (MTZ) were purchased (Sigma-Aldrich, Darmstadt, Germany) and dissolved in dimethyl sulfoxide (DMSO) prior to use. The final concentration of DMSO in cultures never exceeded 0.6%.

2.3. In Vitro Determination of Trichomonacidal Activity

Anti-T. vaginalis activity was determined following the methodology described by Sena-Lopes et al. [14]. Briefly, assays were executed in 96-well microtiter plates with a density of 2 × 10⁶ trophozoites/mL and incubated with the compounds at 37 °C and 5% CO₂ for 24 h. Citral was evaluated at different concentrations (100 μM, 80 μM, 60 μM, 40 μM, and 20 μM), and MTZ was used as the positive control at 100 μM. Negative controls with trophozoites and a DMSO control (0.6%) were also included in every plate. Trichomonas vaginalis motility and morphology were analyzed in a Neubauer chamber by light microscopy. Later, to determine the minimum inhibitory concentration (MIC), the trophozoites present in the wells were inoculated in fresh TYM medium and incubated at 37 °C with 5% CO₂ in microtiter plates, as mentioned above. A kinetic growth curve was established to obtain a more accurate profile for citral activity against T. vaginalis. Growth analysis was performed at 1, 6, 12, 24, 48, 72, and 96 h by a trypan blue dye (0.4%) exclusion assay. The tests were performed independently three times in triplicate, and the results are expressed as the percentage of viable trophozoites.

2.4. Nitric Oxide (NOx) Levels

Nitric oxide (NO) is a highly unstable reactive species that is rapidly oxidized to its metabolites nitrate and nitrite. To investigate whether citral treatment could lead to NO production and/or accumulation by T. vaginalis, NO metabolite (NOx) levels were measured using the Griess reaction method adapted from Birmann et al. (2020) [15]. Briefly, parasites (5 × 10⁵ trophozoites/mL) were seeded in a 96-well plate and treated with citral at different concentrations for 24 h at 37 °C, 5% CO₂. A negative control of untreated trophozoites was included in all plates. An equal volume (100 μL) of Griess reagent (1:1) was then added to the wells for 5 min at 25 °C. The NOx concentration was determined by measuring the optical density at 462 nm and plotted against a NaNO₂ standard curve [15]. The results are expressed as µmol NOx/10⁵ trophozoites.

2.5. Molecular Docking

To investigate the potential mechanism of action of citral, molecular docking was performed using three essential proteins for the survival of T. vaginalis. The protein structures of thioredoxin reductase (TvTrxR; PDB 2F51), purine nucleoside phosphorylase (TvPNP; PDB 1Z36), and methionine gamma lyase (TvMGL; PDB 1E5F) were retrieved from RSCB Protein Data Bank (http://www.rcsb.org/ (accessed on 6 May 2024)). Prior to docking, the protein structures were prepared by removing water molecules and other ligands using CHIMERA 1.5.3 software [16]. A grid box was set to encompass the active site residues of each protein. Citral was constructed and optimized with Avogadro 1.1.1 [17]. The lowest binding energy (binding affinity) of citral with each protein was calculated using Autodock Vina 1.1.2. with default parameters [18], and the docking poses of citral were visualized using Accelrys Discovery Studio 3.5.

2.6. Statistical Analysis

The screening test results are presented as percentages and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-test for carrying out multiple comparisons between all treatments. Tukey’s post-test was conducted to identify significant differences between the negative control and the means of the different concentrations evaluated. The significance level was p < 0.001.

The results of NOx are expressed as the mean ± standard error (S.E). The results were analyzed by one-way ANOVA following the Newman–Keuls post hoc test. A value of p ˂ 0.05 was considered significant. The statistical analysis was accomplished using GraphPad Prism version 7.0 for Windows, Graph Pad Prism Software version 8 (San Diego, CA, USA).

3. Results

3.1. In Vitro Screening Assay

The anti-T. vaginalis assays revealed that, at the highest concentration assayed, citral was able to reduce the 100% viability of trophozoites (MIC₁₀₀ = 100 µM). Activity data at lower concentrations showed a remarkable activity of citral, maintaining a reduction in parasite viability in cultures of 99.1% and 94.1% at 80 and 60 µM, respectively. The IC₅₀ value was 34.4 μM. The vehicle (DMSO) did not affect parasitic cultures, as demonstrated by the absence of significant differences between the control group with DMSO (0.6%) and the negative control (TYM medium and trophozoites), as shown in Figure 1.

The kinetic growth curve demonstrated that citral at 100 μM had a significant impact on trophozoite growth. A notable decline in trophozoite motility was observed after one hour of incubation, with a further reduction to 50% after 6 h and a complete cessation of growth after 12 h of treatment. However, a divergent pattern was observed with MTZ, with 18% of trophozoites remaining viable after 12 h of incubation. All trophozoites were only inactivated after 24 h of exposure with the reference drug (Figure 2).

3.2. Nitric Oxide (NOx) Levels

The antioxidant activity of citral was evaluated after 24 h of treatment. Decreased NOx levels were detected when trophozoites were cultured with different concentrations of citral when compared of the control (Figure 3).

3.3. Molecular Docking

To elucidate the mechanism of action of citral against T. vaginalis, a molecular docking experiment was conducted. The estimated binding free energy (ΔGbind) was determined, and the optimal docking pose of citral with each protein was identified. As illustrated in Figure 4A, citral exhibits an interaction with amino acids in proximity to the active site of thioredoxin reductase (TvTrxR), showing ΔGbind = −3.8 kcal/mol. Citral forms a hydrogen bond with LYS59 and interacts via covalent interactions with PHE31 and CYS38. Van der Waals interactions with other amino acids contribute to the stabilization of the interaction between TvTrxR and citral. The binding mode of citral in the purine nucleoside phosphorylase (TvPNP) active site (ΔGbind = −5.1 kcal/mol) is depicted in Figure 4B. The model suggests that citral interacts via hydrogen bonds with ANS161 and ALA167. The maintenance of citral in the active site of TvPNP is facilitated by covalent interactions (PHE159, VAL178, and ILE206) and van der Waals interactions. As demonstrated in Figure 4C, citral interacts with methionine gamma lyase (TvMGL), exhibiting a ΔGbind of −5.0 kcal/mol. Citral forms a covalent hydrogen bond with PRO290 and several covalent and van der Waals interactions with other residues.

4. Discussion

Cymbopogon with more than 55 species possesses several biological activities including antiprotozoal, antibacterial, and antifungal properties [19,20,21,22,23]. These microbicidal properties are attributed to the high content of citral (3,7-dimethyl-2,6-octadienal). This key component has been previously isolated and studied in vitro against the human trypanosomatid protozoan parasites Trypanosoma cruzi and Leishmania spp. [24,25,26].

The present study reports for the first time the inhibitory effects of this major compound against the human sexually transmitted parasite T. vaginalis. The mechanism responsible for the antiprotozoal activity detected in our study is still unexplained and can probably be justified by the capacity of this compound to promote cell membrane damage and disrupt the integrity and permeability of the membrane, as has been observed in antifungal assays [27]. Essential oil (EO) extracts are known for their lipophilic properties, which enable their penetration through cell membranes. Upon interaction with components like polysaccharides, fatty acids, and phospholipids within the membrane, a series of events are triggered, ultimately leading to cell death [28]. Furthermore, the mode of action of EOs appears to be multi-target-oriented, which enhances their potential application in different diseases and resistant pathogens [29].

The inhibitory effect of citral has been observed in vitro against T. cruzi, reducing the epimastigote and trypomastigote forms and acting on the differentiation process of the protozoa [24,25]. The same antiprotozoal activity has been demonstrated against promastigotes and intracellular forms of L. amazonensis, with a dose-dependent effect consistent with that observed in our results [26].

Our data are consistent with those found in the literature, which point to the antiprotozoal effects of citral, which, due to its lipophilic properties, could be responsible for the morphological changes of the parasite caused by the rupture of the plasma membrane, interfering with the process of cell division and promoting the death of the parasite [13,28]. However, citral has also exhibited a remarkable cytotoxic profile against Vero cells in previous studies. Baccega et al. (2024) evaluated the cytotoxic effect of citral on Vero cells, detecting a marked reduction in the viability of mammalian cells (CC₅₀~30 µM) after 24 h of contact with the compound [12]. This could explain the antiparasitic effect of citral against kinetoplastid and trichomonadid protozoans reported in other studies [12,26]. According to these data, citral does not exhibit an adequate selectivity index; nonetheless, considering the urgent need for new compounds with chemical structures distinct from 5-nitroimidazoles, citral remains a promising scaffold for structural modification. Its cytotoxicity may be mitigated through pharmaceutical strategies or combination therapy with existing drugs, allowing dose reduction and a multi-target mechanism of action.

In the present study, it was observed that citral possesses the capacity to inhibit the generation of reactive oxygen species (ROS). In iron-deficient situations, T. vaginalis uses a NO-dependent regulatory network for hydrogenosome modulation, a mitochondrial homolog responsible for producing energy and essential for protozoan proliferation and survival [30]. In view of this fact, the decrease in NO levels may have contributed to the antiprotozoal activity observed, as NO is essential to the viability of this parasite in iron-deprived conditions. A similar behavior was observed in melanoma cells, where the high levels of NO and nitric oxide synthase ensure the survival and proliferation of the cells [31].

In addition, molecular docking with T. vaginalis enzymes was performed to investigate the possible molecular mechanism responsible for the anti-T. vaginalis activity of this compound, revealing a weak interaction of citral with the three enzymes. TvTrxR is the isoenzyme responsible for the reduction in thioredoxin, which in turn protects the parasite from oxidative stress and contributes to the maintenance of intracellular redox homeostasis. This important isoenzyme is the target of nitroimidazole drugs [32]; however, in our study, the citral did not present a significant interaction with TvTrxR. Setzer et al. (2017) proposed TvPNP as an interesting protein for the study of new trichomonacidal agents, concluding that several polyphenolic compounds showed selective docking to this isozyme, as well as to TvMGL [32]. Trichomonas lacks the de novo synthesis of purine nucleotides and relies on purine salvage. TvPNP is involved in this pathway to replenish purine nucleotide pools. The characteristic purine auxotrophy of T. vaginalis makes PNP an attractive chemotherapeutic target [33]. Regarding TvMGL, this enzyme is involved in the degradation of sulfur-containing amino acids through methionine catalysis [34]. Its pivotal function in amino acid synthesis and sulfur homeostasis identifies it as a compelling pharmacological target. Nevertheless, citral has shown a weak interaction with the active site of TvPNP and does not interact with the active site of TvMGL. Based on this molecular docking approach, we suggest that the mechanism of action of citral should be associated with other pathways that deserve further investigation.

In relation to the findings of preceding studies, citral has also been reported to exert cytotoxic and genotoxic effects on hematopoietic human cells and leukocytes, showing a significant decrease in cell viability [12,35]. Consequently, citral has attracted considerable attention for its chemotherapeutic effect against several cancer cell lines, yielding noteworthy results [36,37,38]. In light of its antiparasitic activity, the development of a new compound series using citral as the lead structure—with the aim of improving its antiparasitic selectivity—or its incorporation into a nanostructured lipid-based system [39,40] represents promising strategies to overcome its cytotoxic limitations and enhance its therapeutic potential. Thus, the nanocarrier could be an alternative to the use of citral in lower concentrations or even in association with MTZ, with high antiparasitic activity and a reduced cytotoxic effect. Due to the long-standing traditional use of medicinal plants and the promising results observed in this in vitro study, citral in nanostructured form should be evaluated against T. vaginalis, in order to establish it as a safe compound to therapeutic use.

5. Conclusions

The increase in global STI rates, drug resistance, and health consequences has made trichomoniasis an emerging public health problem. Based on the experimental data obtained, citral has interesting properties to inhibit the proliferation of T. vaginalis, making it a suitable candidate for further studies, particularly in drug delivery systems. In addition, more research is needed to better identify the mechanism of action of citral in order to develop new trichomonacidal drugs that are not derived from nitroimidazoles, to address cases of resistance to current treatment.