ABSTRACT

Objective

Due to the limitations of current therapeutic approaches in treating parasitic diseases, there is a growing need for new and effective products, prompting interest in alternative approaches such as medicinal plants. Flavonoids, including luteolin, have shown promise in the treatment of many diseases due to their natural properties and pharmacological effects. This study aimed to investigate the in vitro activity of luteolin against Acanthamoeba castellanii, Entamoeba histolytica, and Leishmania tropica.

Methods

The reference parasite strains were tested for antiparasitic activity using luteolin concentrations ranging from 200 to 1.5 µg/mL. Positive controls included chlorhexidine, metronidazole, and glucantime, while dimethyl sulfoxide and parasite specific culture medium served as negative controls. Parasite mortality was assessed XTT (2,3-bis(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide) and trypan blue dye exclusion assays. Minimum inhibitory concentration (MIC) and median lethal dose (LD₅₀) values were determined via non-linear regression analysis.

Results

Luteolin exhibited significant activity, with MIC values of 100 µg/mL for A. castellanii and E. histolytica, and 12.5 µg/mL for L. tropica. LD₅₀ analysis revealed effective concentrations of 3.125 µg/mL for E. histolytica and 1.5 µg/mL for A. castellanii, while L. tropica displayed an LD₅₀ below 1.5 µg/mL, indicating the highest sensitivity.

Conclusion

Luteolin demonstrated potent antiprotozoal effects in vitro, with L. tropica being the most susceptible, followed by A. castellanii and E. histolytica. Notably, luteolin’s anti-leishmanial activity was comparable to glucantime. In conclusion, luteolin demonstrates significant potential as a broad-spectrum antiparasitic agent, and comprehensive in vivo studies are recommended to further evaluate its therapeutic efficacy.

INTRODUCTION

Millions of individuals, predominantly in low- and middle-income countries, are affected by parasitic infections, particularly those caused by protozoans such as Entamoeba spp., Acanthamoeba spp., and Leishmania spp., which contribute to significant morbidity and mortality globally (1-3).

Amoebiasis, caused by Entamoeba histolytica (E. histolytica), is a notable parasitic disease, leading to an estimated 55,500 deaths and 2.237 million disability-adjusted life years annually, as reported by the Global Burden of Disease 2010 Study (4). The prevalence of E. histolytica infection can reach up to 50% in some developing regions, including parts of South and Central America, Africa, and Asia. In Türkiye, the incidence varies regionally, with an average rate of 2.27% (1). Although the disease is often asymptomatic, it can present with symptoms ranging from diarrhea to dysentery, and in severe cases, it may disseminate to other organs such as the liver, lungs, and brain, leading to abscess formation. The standard pharmacological treatment includes nitroimidazoles like metronidazole (MTZ), but these drugs have limitations, including adverse effects such as neurotoxicity with prolonged use and the emergence of drug-resistant strains (5).

Acanthamoeba castellanii is an opportunistic, free-living amoeba found in soil and water, associated with approximately 2.9 cases per million globally. It causes two primary clinical conditions: Acanthamoeba keratitis (AK) and granulomatous Acanthamoeba encephalitis. AK begins with the attachment of trophozoites to the corneal epithelium, potentially progressing to stromal invasion and vision loss or blindness (2). Current treatments for Acanthamoeba infections include 0.02% chlorhexidine and 0.02% polyhexamethylene biguanide, though prolonged use of these topical agents can result in corneal toxicity (6).

Leishmaniasis, caused by over 20 pathogenic Leishmania species, is transmitted by various species of female phlebotomine sandflies (3). The disease is endemic in more than 102 countries/regions bordering the Mediterranean and the Black Sea, including Türkiye, with thousands of new cases reported annually (7). Clinical manifestations vary based on the Leishmania species and the host’s immune response. Treatment options include antimony (first line), amphotericin B (second line), imidazole’s, miltefosine, paromomycin, and liposomal amphotericin B. However, these drugs are expensive, exhibit toxic side effects, and are often rendered ineffective by drug-resistant strains (8).

Considering the challenges associated with current treatments for these parasitic diseases, there is growing interest in exploring medicinal plants for alternative therapies. Flavonoids, a diverse group of polyphenolic compounds found in various plants, are gaining attention due to their pharmacological properties (9). With over 9,000 types identified, flavonoids are characterized by their C6-C3-C6 structural backbone and include several subgroups based on structural variations (10). Luteolin, a prominent flavonoid found in numerous plants used in traditional medicine, has demonstrated a range of biological effects, including anti-diabetic, anti-allergic, and anti-cancer activities. Despite extensive research on its antibacterial and antiviral properties, its antiparasitic activity remains underexplored (11-13). This study aims to evaluate the in vitro activity of luteolin against A. castellanii, E. histolytica, and L. tropica, contributing to the search for novel therapeutic agents.

METHODS

Ethical Approval

No clinical material or data were used in this study. Therefore, ethics committee approval is not required.

Preparation and Storage of Luteolin Stock Solution

Luteolin was purchased from Sigma-Aldrich (St. Louis, MO). A stock solution of one hundred millimolars in dimethyl sulfoxide (DMSO) was ready and held on at -20 °C.

In Vitro Cultures of A. castellanii, E. histolytica and L. tropica

Axenic cultures of A. castellanii trophozoites (ATCC 30010) were propagated in protease peptone-yeast extract-glucose medium. This medium contained 0.75% (w/v) protease peptone, 0.75% (w/v) yeast extract, and 1.5% (w/v) glucose, supplemented with penicillin G (500 U/mL) and streptomycin (50 µg/mL) to maintain sterility and promote optimal axenic growth. Cultures were maintained in 25 mL cell culture flasks (Sigma), refreshed weekly, and incubated at 30 °C. For cell harvesting, the culture medium was removed by centrifugation at 1500 rpm for 5 minutes, followed by three washes in phosphate-buffered saline to eliminate any remaining medium components. To detach the trophozoites adhering to the flask walls, the flasks were gently agitated on ice for 30 minutes.

The E. histolytica strain (ATCC 30459) was generously provided by Dr. Charles Graham Clark from the London School of Hygiene and Tropical Medicine. E. histolytica trophozoites were axenically grown in LYI (liver digest, yeast extract, iron) medium, which included 880.0 mL of LYI broth, 20.0 mL of a vitamin mixture, and 100.0 mL of heat-inactivated adult bovine serum. The medium was further supplemented with penicillin G (500 U/mL) and streptomycin (50 µg/mL). To ensure continuous growth and viability, trophozoites were routinely subculture into screw-capped test tubes containing 7 mL of LYI medium.

L. tropica promastigotes (ATCC 50129) were cultured at
26 °C in RPMI-1640 medium (Sigma), supplemented with 10% heat-inactivated fetal bovine serum sourced from Cegrogen, Stadtallendorf, Germany. This enriched medium provided the necessary nutrients for promastigote growth and development. The cultures were maintained in 25 mL flasks, ensuring optimal conditions to support the promastigotes’ stationary phase proliferation.

In Vitro Antiparasitic Test of Luteolin

A. castellanii, E. histolytica, and L. tropica were seeded into 96-well microtiter plates (Greiner, Germany), with luteolin applied in serial concentrations ranging from 200 µg/mL to 1.5 µg/mL. For this, the trophozoite densities of A. castellanii and E. histolytica were adjusted to 5×10⁴ cells/mL and 1×10⁶ cells/mL, respectively, and L. tropica promastigotes were standardized to 1×10⁵ cells/mL. The trophozoites of A. castellanii and E. histolytica were given 20 minutes to adhere to the wells, a process that was confirmed under a Leica inverted microscope (Leica, Wetzlar, Germany). The plates were then incubated at 30 °C for 24 hours for A. castellanii, at 26 °C for 72 hours for E. histolytica, and at 37 °C for 48 hours for L. tropica.

Two distinct assays were employed to assess the antiprotozoal activity of luteolin in vitro. The anti-leishmanial effect was evaluated using the XTT cell proliferation kit from Roche Molecular Biochemicals (Mannheim, Germany), following the manufacturer’s instructions (14). A viability assay was conducted for A. castellanii and E. histolytica, which involved staining the cells with 0.1% trypan blue [(TB) 0.4%] at a 1:1 ratio. Live cells remained unstained, while alive cells were stained, and both were counted using a hemocytometer (15). The percentage of parasite mortality was calculated using the formula: % mortality = (negative control-test sample) × 100/negative control.

Parasite mortality was determined as 100% when no motile parasites were observed. Minimum inhibitory concentration (MIC) and LD₅₀ values were determined, with MIC representing the lowest concentration that fully inhibited parasite growth and LD₅₀ representing the dose required to kill 50% of the parasites. To verify trophozoite and promastigote viability, the samples were reinoculated into fresh media and monitored over 24, 48, and 72 hours for regrowth. LD₅₀ values were calculated by fitting a non-linear sigmoidal dose-response curve (four-parameter logistic regression) to the mortality data using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). The 95% confidence intervals (CI) were determined from the regression model. Analyses were performed separately for each protozoan species.

Each experiment included appropriate controls. Negative controls consisted of DMSO (final concentration <1%), which was used as the solvent for luteolin, and parasite specific culture medium appropriate for each organism without luteolin. For A. castellanii, the negative control medium was PYG, for E. histolytica, LYI medium; and for L. tropica, RPMI-1640. These media served as parasite maintenance controls to ensure that any observed mortality was due solely to the treatment and not to culture conditions. The positive controls consisted of MTZ (Specia Rhone Poulenc Rorer, Paris, France) for E. histolytica, chlorhexidine (Sigma) for A. castellanii, and N-methyl meglumine (Glucantime™, Rhone Poulenc, France) for L. tropica. All assays were conducted three times in triplicate to ensure reliability and reproducibility of the results.

Statistical Analysis

The mean, degrees of freedom, and t-value (t) were calculated. Data on the antiprotozoal activity were analysed for statistical significance by using the two-tailed Student’s t-test for unpaired samples. A p-value of 0.05 or less was considered indicative of a statistically significant difference. LD₅₀ regression was conducted using GraphPad 10 (GraphPad Software, San Diego, CA, USA). LD₅₀ values were determined by fitting the data to a non-linear regression model (four-parameter logistic, variable slope). The goodness-of-fit (R²) and 95% CIs for each LD₅₀ estimate were calculated to assess model reliability. Dose response curves were plotted for each parasite species to visualize differences in susceptibility. All experiments were performed in triplicate and repeated three independent times, and the results are presented as mean ± standard error.

RESULTS

According to the Acanthamoeba TB dye exclusion assays, mortality in the negative control group was minimal, with a maximum value of 8%, while significantly higher mortality rates were recorded in both the luteolin and chlorhexidine groups, reaching 100%. The highest mortality was determined at concentrations of 100 µg/mL and 200 µg/mL for both luteolin and chlorhexidine, with 100% mortality. At the lowest concentration (1.5 µg/mL), the mortality rate decreased to 30% for luteolin, while it remained 98% for chlorhexidine. Overall, an increase in mortality rates was observed as the concentration increased (Picture 1a). The in vitro effect of luteolin on Acanthamoeba was determined to be significant and dose dependent. A statistical relationship was not found between luteolin and the positive control (p=0.0172; t=2.7026; df=14).

The TB dye exclusion assays demonstrated that luteolin achieved complete effectiveness (100% mortality) against E. histolytica trophozoites at concentrations of 100 µg/mL and 200 µg/mL. At 50 µg/mL, luteolin induced 98% mortality, while at lower concentrations, the parasites were affected but their viability remained higher compared to the positive control. The number of motile trophozoites increased as the drug concentrations decreased (Picture 1b). No statistical relationship was detected between luteolin and the positive control (MTZ) (p=0.0953; t=1.7886; df=14).

According to XTT analysis, luteolin induced 100% mortality at concentrations ranging from 200 to 12.5 µg/mL against the promastigote form of L. tropica. Mortality rates between 95% and 98% were determined at the three lowest concentrations (6.25, 3.125, and 1.5 µg/mL). No significant differences were detected between luteolin and glucantime (p=0.1108; t=1.8249; df=7).

The MIC of luteolin was determined as 100 µg/mL for A. castellanii and E. histolytica, while a value of 12.5 µg/mL was noted for L. tropica. Histogram graphs showing the in vitro activity of luteolin against A. castellanii, E. histolytica, and L. tropica are presented in Graphics 1, 2, 3, respectively.

LD₅₀ regression analysis, performed using a non-linear dose-response model, revealed that luteolin exhibited the highest potency against L. tropica, with an LD₅₀ value below 1.5 µg/mL (95% CI: X-Y) (Graphic 4). For A. castellanii and E. histolytica, the LD₅₀ values were calculated as 1.5 µg/mL (95% CI: X-Y) and 3.125 µg/mL (95% CI: X-Y), respectively (Graphic 4a and 4b). The steep slope of the dose–response curve for L. tropica indicates a rapid decline in viability with small increases in concentration, suggesting greater susceptibility compared with the other protozoa. In contrast, E. histolytica demonstrated the least sensitivity to luteolin within the tested range, as reflected by the higher LD₅₀ value.

DISCUSSION

The decreasing effectiveness of conventional therapeutic agents in treating parasitic diseases has led to delays and failures in treatment. Moreover, long-term use of these agents often results in toxicity and adverse side effects (16). Accordingly, there has been a shift towards the discovery of natural medicinal compounds with antiparasitic properties. Medicinal plants, commonly used as raw materials across various sectors, including medicine and pharmacy, produce phytoalexins in response to microbial invasion. These compounds, derived from secondary metabolites, are mainly found in flavonoids (17). Many studies have demonstrated that flavonoids possess a wide array of biological activities, such as antioxidant, antimutagenic, antibacterial, antiangiogenic, anti-inflammatory, antiallergic, enzymatic regulatory, and anticancer effects (10, 18, 19).

Luteolin and its glycosides are significant flavonoids with demonstrated antimicrobial activity. Several studies have reported their effectiveness against bacteria, viruses, and fungi. However, studies on the antiparasitic activity of luteolin are limited. For instance, research has shown that flavonoids, when used alone or in combination, are effective against Cryptosporidium parvum and Encephalitozoon intestinalis (20). One study revealed that luteolin inhibits the transformation of Plasmodium falciparum rings into more advanced stages, without affecting the sensitivity to chloroquine or artemisinin (21). In addition, luteolin was shown to markedly inhibit the growth of Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, starting from an initial parasitemia of 1%. Furthermore, at a dosage of 5 mg/kg, luteolin resulted in a 77.5% reduction in the growth of Babesia microti in BALB/c mice, indicating its potential applicability in the treatment of babesiosis. (22).

Although research on luteolin’s effects against Acanthamoeba is scarce, it has been suggested that luteolin could play an important role in developing alternative treatments for Acanthamoeba infections. Luteolin has demonstrated cytotoxicity against mouse macrophages (J774A.1) and has been reported to induce programmed cell death in A.castellanii (23). However, luteolin’s effects appear to vary among species. For example, in a study evaluating the anti-amoebic activity of 18 flavonoids against A. castellanii, A. polyphaga, and Naegleria fowleri (N. fowleri), luteolin was highly effective against N. fowleri but less so against A. castellanii. Notably, A. polyphaga was found to be more sensitive to luteolin than A. castellanii, with the former exhibiting higher sensitivity index values (>11) and lower IC₅₀ values (<30 µM) (24). In the present study, the highest mortality rate of luteolin on A. castellanii was 100% at concentrations of 200 µg/mL and 100 µg/mL. The MIC and LD₅₀ values were determined to be 100 µg/mL and 3.125 µg/mL, respectively. However, luteolin’s efficacy at lower concentrations was less pronounced compared to chlorhexidine, highlighting the need for further in vitro and in vivo studies to fully understand luteolin’s effects on Acanthamoeba species.

Recent research has increasingly focused on the potential of medicinal plants as complementary or specific treatment strategies with amoebicidal properties against Entamoeba species, given the limitations of current therapeutic agents (25). Notably, natural compounds have been observed to induce morphological changes in amoebae, including chromatin condensation and cytoskeletal protein rearrangement (26). In one study, ten flavonoids and four iridoids were evaluated for their anti-amoebic activity and cytotoxicity against MT-4 cells. Among them, kaempferol (IC₅₀=10.3±2.3 µg/mL), apigenin (IC₅₀=12.7±4.3 µg/mL), and luteolin (IC₅₀=17.8±4.3 µg/mL) exhibited stronger activity than their glycoside counterparts (27). In the current study, luteolin demonstrated a potent effect against E. histolytica at higher concentrations, with 100% mortality at 200 and 100 µg/mL and 98% mortality at 50 µg/mL. The mortality effect decreased dose-dependently, with an MIC value of 100 µg/mL and an LD₅₀ of 1.5 µg/mL. Although MTZ proved more effective at lower concentrations, these results indicate that luteolin effectively eliminates half of the trophozoites at the lowest concentration tested.

Studies have also suggested that flavonoids could serve as food supplements in the treatment of leishmaniasis due to their low IC₅₀ values (28). In one study, it was noted that the side effects of quercetin and luteolin on cutaneous wounds caused by Leishmania species were less severe than those caused by meglumine (29). While quercetin exhibited non-specific effects on normal human T-cells, luteolin was found to be non-toxic and a strong candidate for anti-leishmanial drug development (30). Additionally, in vivo studies have demonstrated that luteolin has greater cytotoxicity against lymphocytes and is a more potent inhibitor of L. tropica amastigotes than luteolin-4’-O-β-D-glucopyranoside (12). Similarly, another study evaluated the cytotoxicity of 105 compounds on mammalian L6 cells and their antiparasitic activities, identifying fisetin, 3-hydroxyflavone, luteolin, and quercetin as the most potent anti-leishmanial agents, with IC₅₀ values of 0.6, 0.7, 0.8, and 1.0 µg/mL, respectively (31). In the present study, luteolin exhibited strong anti-leishmanial activity against L. tropica, comparable to glucantime. According to the XTT analysis, luteolin induced 100% mortality at concentrations ranging from 200 to 12.5 µg/mL, with mortality rates of 95-98% at the lower concentrations. These findings are consistent with glucantime’s effect, which induced 100% mortality at concentrations of 200-12.5 µg/mL and 98-99% at the lower concentrations. No meaningful distinction was found between luteolin and glucantime (p=0.1108; t=1.8249; df=7). The MIC value of luteolin was 12.5 µg/mL, and its LD₅₀ value at even lower concentrations underscores its potential as an effective anti-leishmanial agent.

CONCLUSION

This study highlights the significant antiparasitic potential of luteolin against E. histoytica, A. cestallanii and L. tropica. Luteolin demonstrated comparable effectiveness to established treatments like MTZ, chlorhexidine, and glucantime under in vitro conditions. The lack of significant statistical differences between luteolin and these standard treatments suggests that luteolin may possess similar antiparasitic properties. Furthermore, its effective performance, particularly against L. tropica, supports the potential for luteolin to be explored further as an anti-leishmanial agent. Our results may form the basis for future studies to evaluate the anti-parasitic activity of luteolin in potential therapeutic applications.