Effect of aqueous extract of Eucalyptus Microtheca on Acanthamoeba genotype T5, an in vitro study

Abstract

Background

Acanthamoeba is a resilient protozoan that causes serious diseases such as Acanthamoeba Keratitis (AK). Due to the ineffectiveness of many drugs against this parasite, plant extracts provide a promising alternative in the treatment process. This study aimed to assess the effectiveness of the aqueous extract of Eucalyptus microtheca against Acanthamoeba trophozoites and cysts in vitro.

Methods

In the current experimental study, Acanthamoeba isolate was cultured in a non-nutritive agar (NNA) medium. Aqueous extracts of Eucalyptus microtheca were prepared at concentrations of 10, 20, 40, and 80 mg/ mL, with exposure times of 30, 60, 90, and 120 min for trophozoites and 24, 48, and 72 h for cysts. Chlorhexidine (0.02%) and PBS served as positive and negative controls, respectively. Viability was assessed using 1% eosin dye.

Results

Our study revealed that the aqueous extract of Eucalyptus microtheca significantly reduced the number of both trophozoites and cysts. At 80 mg/ mL, the extract completely eliminated cysts (100%) after 24 h and reduced trophozoites by 99.33% within 120 min.

Conclusion

This study indicates that Eucalyptus microtheca aqueous extract possesses significant anti-Acanthamoeba properties, with effects strongly associated with concentration and exposure time (p < 0.001).

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-025-04869-z.

Background

Acanthamoeba is a free-living protozoan that thrives in diverse environments, including surface water, seawater, pool water, and soil. It is widely prevalent in Iran and globally, demonstrating remarkable tolerance to environmental changes, such as variations in osmolarity, temperature, salinity, and pH [1, 2]. The parasite exists in two forms: the cystic resistant form and the invasive trophozoite form. Infections caused by Acanthamoeba include granulomatous amoebic encephalitis, a chronic central nervous system infection, and Acanthamoeba keratitis, a severe eye infection. Other complications include chronic granulomatous skin infections, tissue damage, and osteomyelitis [3–5]. While Acanthamoeba keratitis primarily affects healthy individuals, there has been a marked increase in cases since 1985, particularly among contact lens users [3, 6]. Various factors, including the diverse virulence exhibited by different isolates, pose challenges in establishing a direct correlation between in vitro and in vivo efficacy [7, 8]. Biguanides, such as polyhexamethylene biguanide (PHMB), are recognized as potent topical agents against both trophozoites and Acanthamoeba cysts, even at low concentrations, although they exhibit toxicity to human corneal cells [9–11]. Recently, the FDA and the European Medicines Agency approved Akantior (polyhexanide) for treating Acanthamoeba keratitis in Europe. A 0.08% polyhexanide concentration has demonstrated enhanced efficacy over other therapies, often achieving resolution without surgery [12, 13]. Chlorhexidine is also effective and non-toxic at low concentrations [9, 14]. Given the side effects of chemical drugs and increasing interest in natural products, medicinal plants have gained popularity as alternative treatments [15, 16]. Several studies have demonstrated the amoebicidal properties of plant extracts, both in vitro and in vivo [10, 17–19]. For instance, methanolic extracts of fenugreek (Trigonella foenum-graecum) and its seeds exhibited trophicidal and cysticidal activity [19]. Ornithogalum sigmoideum leaves, flowers and stem also exhibited trophicidal and cysticidal effects of Acanthamoeba [20]. Also, the application of nanoparticles, especially those produced through green chemistry using herbal extracts, has shown encouraging outcomes in combating Acanthamoeba infections. Studies suggest that nanoparticles combined with herbal extracts demonstrate improved effectiveness at reduced doses [21, 22].

Eucalyptus, a member of the Myrtaceae family, is recognized for its diverse medicinal properties, including antibacterial, anti-malarial, and anthelmintic effects [23–26]. The essential oil derived from Eucalyptus exhibits antiseptic properties, while its monoterpene components possess antibacterial activity. Given the well-established anti-parasitic properties of certain plant extracts, Eucalyptus stands out as one of the most prominent medicinal plants. Eucalyptus leaf extract contains various compounds such as cineole, citronellal, limonene, and betapinene, with eucalyptol being the primary compound contributing to its anti-parasitic and antifungal properties [27, 28]. This study evaluates the effects of Eucalyptus microtheca aqueous extracts on Acanthamoeba trophozoites and cysts in vitro.

Methods

Extraction of Eucalyptus aqueous extract by cold method

Sampling was conducted after obtaining the necessary permissions prior to collection. Leaves of E. microtheca were collected from the Kowsar research station in Gareh Bygone Plain (28◦36′ 09.9"N and 53◦55′ 26.6” E), Fars province, Southwest of Iran. The samples were authenticated by Dr. Hamid Mahmoodpour at the Herbarium of the Pharmacognosy Department, School of Pharmacy, Shiraz University of Medical Sciences, Iran (Voucher No. 3065).The dried leaves were powdered, and stored in dark glass containers. Fifty grams of the powder were mixed with distilled water and subjected to magnetic stirring for 48 h. The solution was filtered and freeze-dried to produce a powdered extract, stored at 4 °C until use [28].

Genotyping and phylogenetic analysis and Acanthamoeba cultures

An environmental Acanthamoeba strain provided by Prof. Maryam Niyatti (Shahid Beheshti University of Medical Sciences) was cultured in NNA medium with Escherichia coli as a nutrient source at 26 °C for 72 h. Subsequently, DNA extraction was performed to confirm the sample and determine the parasite genotype by amplifying the 18 S rRNA gene using a DNA extraction kit (Yekta Tajhiz Co, Iran), following the kit’s protocol [29]. The PCR product was sequenced (Pishgam Co, Iran). To demonstrate the precise position of Acanthamoeba genotypes and compare them with similar and standard cases registered in the global gene bank, a phylogenetic tree was constructed using MEGA5 software based on Maximum Likelihood analysis.

Plates containing trophozoites were washed twice with sterile saline solution from 72- to 96- hours cultures, followed by centrifugation at 1500 rpm for 5 min, repeated twice. Similarly, plates containing cysts were washed with sterile saline solution three-week-old cultures and centrifuged twice at 2000 rpm for 5 min. The number of trophozoites and cysts was determined using eosin staining and direct counting with a Neubauer slide. The final concentration was 25 × 10⁴ trophozoites and cysts per milliliter, which were immediately used in the experiment [30, 31].

Effects of the extract on trophozoite and cystic stages

To evaluate the amoebicidal activity, Acanthamoeba trophozoites and cysts were incubated with different concentrations of E. microtheca extract (10, 20, 40, and 80 mg/mL). Under sterile conditions, 100 µl of trophozoite and cyst suspension (calibrated at 25 × 10⁴ /mL) obtained from NNA medium was added to each 1.5 milliliter microtube. Then, 100 µl of each concentration of the Eucalyptus aqueous extract was added to the microtubes containing parasites. The tubes were sealed and incubated at 30 °C for various incubation periods, with each experiment performed in triplicate. All experiments included parasite treated with 0.02% chlorhexidine gluconate as a reference drug control and parasite with PBS as a non-treated control.

The effects of extract on Acanthamoeba were evaluated at different time; 30, 60, 90, and 120 min for trophozoites, as well as 24, 48, and 72 h for cysts. After each incubation period, 20 µl of the test and control solutions were mixed with an equal volume of eosin dye and incubated at room temperature for 3 min. Counting was performed using a Neubauer slide. Viable parasites remained unstained, while nonviable parasites were stained [32, 33].

Statistical analysis

An ANOVA test was performed to identify significant differences using GraphPad Prism (version 8.0.2) (Bioz, San Diego, CA, USA). For all evaluated data, a p-value of < 0.001 was considered to indicate a statistically significant difference.

Results

Phylogenetic analysis

The results of phylogenetic analysis showed that the studied Acanthamoeba belongs to genotype T5 of the species A. lenticulata, which is classified within its own clade V/Exclusive Group 5 (Accession number; PQ559813, marked with an asterisk *) compared to other Acanthamoeba genotypes (Fig. 1).

Fig. 1.

Maximum Likelihood Phylogenetic Tree and the Relationship between Different Acanthamoeba Genotypes using MEGA v5.05 software. The examined Acanthamoeba sp, was identified as A. lenticulata (T5 genotype; Accession number; PQ559813)

Effect of E. microtheca extract

The comparison of the viability rate of Acanthamoeba trophozoites exposed to different concentrations of E. microtheca extract at various exposure times is shown in Fig. 2. The aqueous extract of E. microtheca demonstrated significant efficacy in reducing both the average number of trophozoites and cysts, with notable effects observed in relation to exposure time and concentration. Our findings reveal that E. microtheca at the concentrations of 10 mg/mL exhibited more than a 50% amoebicidal effect after 120 min of exposure. With increasing concentration, the inhibitory effect also increased, with the most potent amoebicidal effect recorded by 80 mg/mL after 120 min. Additionally, in the positive control group no viable trophozoites were observed after 30 min.

Fig. 2.

Mortality rate of Acanthamoeba trophozoites after exposure to different concentrations of aqueous extract of E. microtheca. Positive and negative controls included 0.02% chlorhexidine and phosphate-buffered saline (PBS), respectively

The comparison of the viability rate of Acanthamoeba cysts exposed to different concentrations of the E. microtheca extract at various exposure times is shown in Fig. 3. The aqueous extract of E. microtheca at a concentration of 40 mg/mL demonstrated approximately 50% mortality of cysts after 72 h. At a concentration of 80 mg/mL, no viable parasites were observed after 24 h (Fig. 4). Additionally, in the positive control group, no viable parasites were observed after 24 h.

Fig. 3.

Mortality rate of Acanthamoeba cyst after exposure to different concentrations of aqueous extract of E. microtheca. Positive and negative controls included 0.02% chlorhexidine and phosphate-buffered saline (PBS), respectively

Fig. 4.

Eosin-stained Acanthamoeba cysts before and after exposure to the extract

Discussion

Over the last few decades, there has been an increase in infections caused by Acanthamoeba [1, 4]. Invasions by Acanthamoeba sp. pose significant challenges for chemotherapy, with most cases resulting in patient death and only limited successes achieved using highly toxic disinfectant drugs, such as chlorhexidine derivatives. Effective treatment for immunocompetent individuals requires early combined therapy, while late-stage cases are often resistant to most therapeutics. Given the limitations and health risks associated with current drugs, the discovery of new anti-amoebic agents targeting both the cyst and trophozoite stages is crucial [34–36]. Medicinal plants have been utilized since ancient times due to their affordability, accessibility, and the cultural rapport they foster between physicians and patients. They support self-sufficiency in drug production and facilitate scientific research. In recent years, there has been a growing global interest in medicinal plants because of their historical significance in medicine. Iran’s favorable geographical location and climate have made it one of the richest countries in terms of plant diversity [16, 37]. In the present study, the effect of varying concentrations of E. microtheca extract on both trophozoites and Acanthamoeba cysts were investigated. To our knowledge, this study is the first to examine the amoebicidal effects of this medicinal plant on Acanthamoeba cysts and trophozoites. The findings revealed that the lethality percentage of trophozoites and cysts increased with higher extract concentrations and longer exposure times. Specifically, at the highest concentration of 80 mg/mL, the cysts mortality rate reached 100% after 24 h, while same mortality rate was observed for trophozoites after 120 min. These results indicate that the anti-amoebic activity of E. microtheca extract is dependent on both dosage and duration and parasite stage, aligning with findings from other studies. Several studies have explored the therapeutic potential of Eucalyptus in various conditions. For instance, Hassani et al.. investigated the effect of various extracts of Eucalyptus camalrolensis on the parasite Trichomonas vaginalis in culture medium. Their study found that the ethyl acetate extract, at a concentration of 12.5 mg/mL, inhibited growth by 100% within the first 24 h, while the water extract, at a concentration of 50 mg/mL, achieved 100% growth inhibition after 72 h [38]. In another study conducted by Algabani et al., the effects of methanol extracts of clove, ginger, garlic, and Eucalyptus essential oils on Entamoeba histolytica and Giardia lamblia were evaluated. It was observed that Eucalyptus essential oil exhibited 100% inhibition of growth in both parasites [39]. Mirzaalizadeh et al.. studied the effects of methanolic extracts Eucalyptus on toxoplasmosis under laboratory conditions. Their research demonstrated the anti-Toxoplasma gondii activities of Eucalyptus both in vitro and in vivo, as well as its antioxidant activity which was associated with a higher survival rate [40]. Mahmoudpour et al.. investigated the effects of hydroalcoholic extract of Eucalyptus microtheca on Echinococcus granulosus protoscolices. The study results indicated that the hydroalcoholic extract of E. microtheca killed all protoscolices (100%) at concentrations of 10 and 12.5 mg/mL [28]. Additionally, Sadatrasoul et al. examined an oil-in-water emulsion formulated with Eucalyptus leaf extract to prevent the attachment and replication of the influenza virus in laboratory conditions. Their findings revealed that the oil-in-water emulsions containing 2% Eucalyptus leaf extract completely inhibited virus replication [41]. Studies have identified over 700 different species of Eucalyptus worldwide, of which 500 are known to produce essential oils. The primary components of these oils include 1,8-cineole (34.0%), p-cymene (12.4% ) α-pinene (10.7%), β-pinene (10.5%) and virdiflorene (5.2%) [27]. The high concentration of 1, 8-cineole in Eucalyptus oil is particularly notable for its inhibitory effects on certain types of experimental inflammation in rats. Additionally, 1,8-cineole has demonstrated antimicrobial, anti-inflammatory, and antinociceptive properties [25, 42]. Al-Rahimy et al. conducted a study on the effects of a cold-water extract from Eucalyptus leaves, finding that a concentration of 5 mg/mL caused morphological deformities and the highest mortality rates in fourth instar larvae and pupae of Culex molestus. At a concentration of 10 mg/mL, the extract completely stunted the development of fourth instar larvae compared to the control group, which exhibited a deformity rate of 0% [43]. Furthermore, additional studies have investigated the antimicrobial properties of Eucalyptus, providing broader insights into its potential efficacy. Al-Taai et al. demonstrated that Eucalyptus extract exhibited significant activity against Pseudomonas at a concentration of 0.6 mg/mL and against Citrobacter at 20 mg/mL. The minimum bactericidal concentration (MBC) of Eucalyptus was lowest at 0.3 mg/mL against Pseudomonas and Enterococcus, and highest at 10 mg/mL against Staphylococcus and Proteus. However, no effects were observed against Escherichia coli and Citrobacter [44]. Seyyednejad et al.. reported notable antibacterial activity against both gram-positive and gram-negative bacteria, with Escherichia coli and, P. aeruginosa showing the highest sensitivity and S. aureus the least [24]. Similarly, Omer Ishag et al. found that leaf extracts of E. camaldulensis and E. microtheca effectively inhibited the growth of S. aureus, P. aeruginosa, Escherichia coli, K. pneumoniae, and C. albicans. The aqueous and ethanolic extracts of both plants exhibited the highest antimicrobial activity at concentrations of 100 and 50 mg/mL, while no inhibitory effects were detected at lower concentrations (25 and 12.5 mg/mL) [45]. These findings underscore the significant impact of Eucalyptus aqueous extract on Acanthamoeba trophozoites and cysts, with 1,8-cineole a major component of Eucalyptus showing promising anti-Acanthamoeba effects. As we acknowledge, this research represents an initial step toward fully demonstrating the effectiveness of the extract in treating Acanthamoeba infections. Looking ahead, we propose the following future directions to expand our understanding of the extract’s potential: Investigate the possible toxic effects of the extract on non-Acanthamoeba cells, such as corneal cells, to ensure its safety for potential therapeutic use. Evaluate the effectiveness of the extract on cell lines infected with Acanthamoeba to gain insights into its therapeutic potential in more complex biological environments. Conduct animal studies to further assess the pharmacokinetics and pharmacodynamics of the extract, thereby providing a clearer picture of its efficacy and safety.

Conclusion

Our findings highlight the significant amoebicidal activity of Eucalyptus aqueous extract against Acanthamoeba. However, we acknowledge that this research represents an initial step in a broader investigation. We anticipate that the proposed future studies will deepen our understanding of the extract’s potential and pave the way for its therapeutic applications in combating Acanthamoeba infections.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Study concept and design: M.H.K, Acquisition of data: F.A, H.A, Analysis and interpretation of data: M.H.K, E.A, M.M.O, A.S, H.M, Drafting of the manuscript: M.H.K, Critical revision of the manuscript for important intellectual content: E.A, M.H.K, Statistical analysis: M.H.K, Administrative, technical, and material support: M.M.O, A.S.

Funding

The study was supported by immunology research center, Tabriz University of medical sciences (number 71823).

Data availability

The data are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC.1402.090). Sampling was conducted on public land, and we obtained the necessary permissions from the Ministry of Jihad Agriculture prior to collection.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.