Anti-Amoebic Activity of Clitoria ternatea L. Flower Extract against Acanthamoeba culbertsoni Genotype T4

Acanthamoeba spp. are opportunistic free-living amoebae capable of causing severe infections such as Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE) particularly among contact lens users and immunocompromised individuals [1]. The pathogenicity of Acanthamoeba is driven by its ability to transition between proliferative trophozoites and highly resistant cysts via encystation and excystation, processes that underlie therapeutic failure and recurrence [2]. Current treatment regimens, which include biguanides and diamidines remain limited by cyst resilience and significant host cytotoxicity, highlighting an urgent need for safer and more effective dual-stage anti-amoebic agents.

Recent research has explored novel anti-Acanthamoeba agents beyond conventional antimicrobials. For example, Mangifera indica leaf extract demonstrated amoebicidal, anti-adhesive activity and low cytotoxicity in Acanthamoeba spp., underscoring the therapeutic potential of plant-derived compounds [3]. Likewise, Camellia sinensis extracts induced morphological and chemical changes in trophozoite and cyst forms of Acanthamoeba castellanii, indicating that phytochemicals can affect both life stages [4]. Nano-formulations combining quercetin with zinc–copper nanoparticles exhibited significant activity against trophozoites, cysts, encystation and excystation, and reduced amoeba-mediated cytopathogenicity where a promising multi-targeted strategy [5]. Additionally, aqueous leaf extracts of Rosa gallica and Picea orientalis showed trophozoiticidal effects but limited cysticidal activity and significant cytotoxicity, illustrating persistent challenges in plant extract efficacy and safety [6]. These studies collectively demonstrate that while phytochemicals hold promise, a few have achieved both potent dual-stage activity and acceptable host safety profiles, and many have not been evaluated across trophozoite, encystation or excystation and cyst endpoints in a single comprehensive framework.

Clitoria ternatea L. (Fabaceae), commonly known as the Asian pigeonwings or bunga telang, is rich in anthocyanins, flavonoids and other secondary metabolites with established antioxidant, antimicrobial, anti-inflammatory and cytoprotective activities [7,8]. Although various parts of C. ternatea have been reported to possess antibacterial and antifungal activities [9,10], its anti-amoebic potential particularly against Acanthamoeba spp, which was remains largely unexplored. Notably, C. ternatea extracts have not been systematically evaluated for their effects on amoeba encystation and excystation processes or assessed for cytotoxicity using relevant human cell models.

In the pursuit of novel therapeutic candidates against A. culbertsoni, this study represents the first (1) to evaluate the anti-amoebic potential of C. ternatea flower extract against both trophozoite and cyst stages of A. culbertsoni genotype T4, (2) to assess the effects of the extract on encystation and excystation processes, which were critical for parasite survival and recurrence as well as (3) to determine the cytotoxicity of the extract using human keratinocyte (HaCaT) cells to determine its safety profile toward host cells. This integrated approach enabled a comprehensive evaluation of both efficacy and biocompatibility, thereby providing novel insight into the dual-stage anti-amoebic potential of C. ternatea and supporting its development as a promising alternative therapeutic candidate for Acanthamoeba infections.

Plant Materials

The blue flowers of Clitoria ternatea L. (Asian pigeon wings) used in this study were selected based on their documented medicinal relevance and high abundance of bioactive anthocyanins reported in floral tissues. Fresh, mature, and disease-free blue petals were collected from Jenderam Hulu Village, Dengkil, Selangor, Malaysia, during the flowering season in November 2022, a period associated with optimal phytochemical accumulation. The plant material was identified using standard morphological criteria and authenticated by Dr. Fadzureena Jamaludin, Research Officer at the Forest Research Institute Malaysia (FRIM). A voucher specimen was prepared and deposited in the FRIM Herbarium, Selangor, Malaysia, under the accession number SBID: 023/22, ensuring traceability and taxonomic reliability. An aqueous extraction approach was employed in this study to reflect traditional medicinal practices and to prioritise biocompatibility for potential therapeutic application. Water was selected as the extraction solvent due to its safety, non-toxicity and suitability for isolating polar phytochemicals such as anthocyanins, flavonoids and phenolic compounds, which have been implicated in antimicrobial and anti-amoebic activities. Furthermore, the use of an aqueous extract minimizes solvent-related cytotoxicity and enhances translational relevance for future biomedical applications, particularly in ocular and topical formulations.

Extraction and Isolation

A total of 1 kg of C. ternatea flowers was collected and processed for extraction. The collected flowers were thoroughly cleaned and only healthy, undamaged blue petals were selected for subsequent freezing at –80°C. The frozen samples were then subjected to freeze-drying using a Scanvac Coolsafe system (Biopharmaceutic Laboratory, Atta-Ur-Rahman Institute for Natural Product Discovery, AuRIns) at –100°C for three days, following the procedure described by Muhammad et al. [11]. The dried samples were weighed to determine moisture loss before being pulverized using a Waring Commercial 8011G lab blender (Waring Commercial, HGB2NTGA model, USA) yielding 122.56 g of fine flower powder.

Extraction was carried out using the maceration technique with distilled water as the solvent at a ratio of 1:30 (w/v). The mixture was stirred continuously with a magnetic stirrer at room temperature for three days. The resulting extract was filtered through Whatman™ filter paper (110 mm) and the filtrate were concentrated under reduced pressure using a rotary evaporator (Heidolph Instruments, Germany) to obtain 10 g of dried extract, corresponding to an 8.16% yield. The solvent-free extract was transferred into universal bottles and stored at 4°C until further analysis.

Culture and Maintenance of Acanthamoeba culbertsoni

1. culbertsoni (genotype T4; ATCC MH791017), originally isolated from contact lens paraphernalia was employed in this study. Trophozoites were cultured monoxenically on 1.5% non-nutrient agar (NNA) plates (Sigma Aldrich, A7002, USA) overlaid with Escherichia coli and incubated at 30°C for three days. Cyst induction was performed using approximately 100 late-log-phase trophozoite cultures on 1.5% NNA medium, following the method described by Anwar et al. [12]. Briefly, trophozoite cultures were observed daily and harvested after three to four days, corresponding to the exponential growth phase. Two millilitres of Page’s Amoebic Saline (PAS) were added to each plate, and trophozoites were gently scraped off using a sterile L-shaped spreader. The harvested cells were centrifuged at 3500 rpm for 10 minutes and fresh trophozoite suspensions were prepared prior to microscopic examination.

For cyst preparation, trophozoite cultures were maintained on NNA plates for up to two weeks at 30°C. Cultures were examined daily and harvested after 14 days, when approximately 95% of the cells had transformed into mature cysts. Cysts were collected as described above, the supernatants were discarded and the pellets were washed twice in PAS (3000 rpm for 5 minutes). The cyst suspensions were stored at 4°C and used for experiments within 14 days.

Both trophozoite and cyst populations were quantified using a haemocytometer (WITEG-Glasgeräte, Germany). Each sample was counted in triplicate and the mean value was used to determine the cell density per millilitre. Stock suspensions containing 5 × 10⁵ cells/mL were prepared for subsequent anti-amoebic, encystation and excystation assays. Experimental assays were conducted using a final concentration of 1 × 10⁵ trophozoites or cysts/mL, as previously described by Mohd Hussain et al. [13].

Anti-Amoebic Evaluation

The amoebicidal activity of C. ternatea flower aqueous extract against A. culbertsoni was evaluated to determine its effects on trophozoite viability. The assay was carried out following the procedure described by Siddiqui et al. [14], with minor modifications. Briefly, A. culbertsoni trophozoites were cultured in 96-well microplates (at a density of approximately 1 × 10⁵ cells/mL in Page’s Amoeba Saline (PAS). A total of 100 µL of trophozoite suspension was dispensed into each well, followed by the addition of 100 µL of C. ternatea extract to achieve final concentrations of 5, 10, 15, 25 and 30 mg/mL. The total volume in each well was adjusted to 200 µL. PAS was used as the negative control, while 0.02% chlorhexidine (Sigma) served as the positive control due to its established anti-amoebic efficacy. Distilled water was included as the solvent control.

After incubation, the well contents were gently resuspended and 50 µL of the suspension was transferred to a new 96-well plate. Subsequently, an equal volume (50 µL) of 0.4% Trypan blue solution prepared in 1X phosphate-buffered saline (PBS) was added. Cell viability was determined using a haemocytometer, where viable trophozoites excluded the dye and remained unstained, whereas nonviable cells displayed blue staining due to compromised membranes. The assays were performed in triplicate and data were presented as the mean±standard error (SE) from at least three independent experiments conducted in duplicate. Inverted microscopic images were captured at 100X magnification using an inverted microscope.

1. culbertsoni exhibits a biphasic life cycle comprising trophozoite and cyst stages. Under favourable conditions, the organism exists as an active trophozoite. However, exposure to adverse environmental factors induces the transformation of trophozoites into double-walled dormant cysts, a process known as encystation. When environmental conditions become favourable, the trophozoites re-emerge from cysts through excystation, thereby completing the life cycle [15].

Encystation Assay

The encystation assay was performed to evaluate the inhibitory effect of the C. ternatea flower aqueous extract on the phenotypic transformation of A. culbertsoni trophozoites into cysts. The procedure was adapted from Anwar et al. [16], with minor modifications. Briefly, A. culbertsoni trophozoites (1 × 10⁵ cells) were counted and exposed to varying concentrations of the C. ternatea extract (5, 10, 15, 25 and 30 mg/mL). Each concentration (100 µL) was added to 96-well microplates containing 100 µL of Page’s Amoeba Saline (PAS), resulting in a total reaction volume of 200 µL per well.

The plates were incubated at 30°C for 72 h without agitation. Following incubation, the contents of each well were gently resuspended with sterile pipette tips and 50 µL of the suspension was transferred to new 96-well plates. An equal volume of 0.4% Trypan blue prepared in 1X PBS was added to each well. Trophozoites incubated solely in PAS served as the negative control, whereas those treated with water and 0.02% chlorhexidine (Sigma) were used as solvent and positive controls, respectively.

Mature, double-walled cysts were enumerated using a haemocytometer under a light inverted microscope, as described by Dudley et al. [17]. The assays were performed in triplicate and data were presented as the mean±standard error (SE) from at least three independent experiments conducted in duplicate. Representative micrographs were captured to illustrate the morphological effects of the extract on trophozoite-to-cyst transformation.

Excystation Assay

The effect of C. ternatea flower aqueous extract on the transformation of A. culbertsoni cysts into trophozoites was evaluated using an encystation assay adapted from Anwar et al. [16] with minor modifications. Briefly, A. culbertsoni cysts (1 × 10⁵ cells) were counted and exposed to various concentrations of the extract (5, 10, 15, 25 and 30 mg/mL). The treatment was performed in 96-well microplates containing 100 µL of Page’s Amoeba Saline (PAS) and 100 µL of the respective extract concentration, resulting in a total reaction volume of 200 µL per well.

For controls, cysts incubated with PAS alone served as the negative control, while those treated with sterile water and 0.02% chlorhexidine (Sigma) were used as solvent and positive controls, respectively. The plates were incubated at 30°C for 72 h without agitation. After incubation, the contents of each well were gently resuspended with sterile pipette tips and 50 µL of the suspension was transferred to new 96-well plates for observation. The assays were performed in triplicate and data were presented as the mean±standard error (SE) from at least three independent experiments conducted in duplicate.

Phenotypic transformation from cysts to trophozoites was examined under a light inverted microscope following the procedure described by Dudley et al. [17]. An equal volume of 0.4% Trypan blue solution prepared in 1X PBS was added to each well and viable trophozoites were counted using a haemocytometer under a light inverted microscope. Representative images from each treatment were captured to document the morphological changes and the effects of the extract on cyst-to-trophozoite transformation.

Culture and Maintenance of Human Keratinocyte Cells (HaCaT)

The HaCaT human keratinocyte cell line (CVCL-0038, CLS 300493) was procured from Cell Line Service (CLS, Germany). As no primary human tissues or identifiable human data were involved, ethical approval was not required for this study in accordance with institutional and international guidelines for the use of established cell lines in in vitro research [Ethics Review Exemption: REC/06/2024 (PG/EX/28)]. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HiMedia Laboratories, India) supplemented with 10% fetal bovine serum (FBS) (^©Capricorn Scientific, Germany) and 1% penicillin–streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin) (HiMedia Laboratories, India) as described by Anwar et al. [12]. Cultures were maintained at 37°C in a humidified incubator with 5% CO₂ until a confluent monolayer was formed. Upon reaching confluence, the culture medium was aspirated and the cells were detached using 2 mL of trypsin solution. The cell suspension was centrifuged at 3500 × g for 5 min and the pellet was resuspended in 25 mL of fresh growth medium. Subsequently, 200 μL of the cell suspension was seeded into each well of a 96-well microplate and incubated for 24 h under standard culture conditions to allow uniform monolayer formation, which was confirmed microscopically. The established monolayer was then used for the cytotoxicity assay.

Cell Cytotoxicity Assay

The cytotoxic potential of the C. ternatea flower aqueous extract toward host cells was evaluated using the MTT assay, following the method described by Muniandy et al. [18] with minor modifications. Human keratinocyte (HaCaT) cells were employed as the model for host cell toxicity assessment. Briefly, HaCaT cells were seeded at a density of 1 × 10⁴ cells per well in 96-well microplates and incubated for 24 h to allow cell adherence. The confluent monolayers were subsequently exposed to 100 µL of C. ternatea extract at concentrations of 5, 10, 15, 25 and 30 mg/mL and incubated for an additional 24 h. After treatment, the culture medium was discarded and the cells were gently washed with phosphate-buffered saline (PBS) (Sigma Aldrich, USA) to remove residual extract. A fresh culture medium containing 0.5% (w/v) MTT reagent was then added to each well and incubated for 3 h to allow the formation of formazan crystals by viable cells. Untreated cells cultured in complete DMEM served as the negative control, while cells treated with 0.02% chlorhexidine served as the positive control and sterile distilled water was used as the solvent control. The MTT solution was carefully removed and the resulting crystals were solubilized in dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA). Absorbance was recorded at 570 nm using a using a Spectrostar Nano microplate reader (BMG Lab Tech, Germany) at 570 nm., where higher absorbance values corresponded to greater cell viability. The assays were performed in triplicate and data were presented as the mean±standard error (SE) from at least three independent experiments conducted in duplicate. The percentage of cell survival was calculated using the following equation:

Survival % = (ABt / ABu) x 100

ABt and ABu denote the absorbance values of treated and untreated cells, respectively [19].

Statistical Analysis

All experimental results are presented as the mean±standard error (SE) of at least three independent replicates. Statistical analyses were performed using Microsoft Excel (Microsoft Excel version 2021, Microsoft Corporation, USA). Differences among multiple treatment groups were assessed using IBM SPSS Statistics version 29.0.2.0 (IBM Corp., Armonk, NY, USA) such as Tukey’s post hoc test, while pairwise comparisons were evaluated using a two-tailed unpaired Student’s t-test. A p-value of less than 0.05 was considered statistically significant. The half-maximal inhibitory concentration (IC₅₀) values were determined by nonlinear regression analysis using Microsoft Excel (Microsoft Excel version 2021, Microsoft Corporation, USA), based on dose–response curves of treated versus control samples.

Ternatea Extract Showed Anti-Amoebic Activity against A. culbertsoni

The anti-amoebic assay was performed to assess the effects of C. ternatea flower aqueous extract on viability of A. culbertsoni. The free-living amoeba was treated with various concentrations of extract ranging from 5 mg/mL to 30 mg/mL. Overall, the results revealed that tested extract showed a potent activity against A. culbertsoni. All tested concentrations significantly reduced viability dose-dependent. The reduction in trophozoite viability was statistically significant (p<0.05, with Tukey’s post hoc test while pairwise comparisons were evaluated using a two-tailed unpaired Student’s t-test). As shown in (Figure 1A), the number of trophozoites was maintained at 1 x 10⁵ in the negative control. At 5 mg/mL, the concentration showed a significant (p = 0.001) activity against A. culbertsoni as compared to solvent control. However, at 25 mg/mL, the extract produced a 94% inhibitory effect on the viability of A. culbertsoni trophozoites and showed an IC₅₀ of 14.2 mg/mL. The most potent activity was recorded at 30 mg/mL, an upon treatment, 100% reduction in viability was recorded (Figure 1A). As shown in (Figure 1B), the images of individual wells from a 96-well plate recorded at 100X, showing the effects of various C. ternatea extract on the viability of A. culbertsoni.

Figure 1: Anti-Amoebic Effect of C. Ternatea Flower Aqueous Extract Against A. Culbertsoni Trophozoites. (A) Percentage Viability of Trophozoites Following Treatment with 5–30 Mg/Ml of C. Ternatea Extract. Data are Expressed as Mean±SE (N = 3 Independent Experiments). (^***) P<0.05 Compared To Solvent Control. (B) Representative Inverted Microscope Images Showing Trophozoite Morphology after Treatment. Scale Bar = 100 µm. (I) Negative Control, (Ii) Solvent Control (Aqueous), (Iii) Positive Control (0.02% Chlorhexidine), (Iv) 5 Mg/Ml Extract, (V) 10 Mg/Ml Extract, (Vi) 15 Mg/Ml Extract, (Vii) 20 Mg/Ml Extract, (Viii) 25 Mg/Ml Extract And (Ix) 30 Mg/Ml Extract

The life cycle of A. culbertsoni is composed of two stages: an active trophozoite and a dormant cyst stage. The tested C. ternatea extract was also assessed for its possible effects on the phenotypic alteration of A. culbertsoni.

Ternatea Extract Showed Significant Effects on Encystation of A. culbertsoni

The encystation assay was performed to assess the inhibition of the phenotypic transformation of A. culbertsoni trophozoites into cysts. As shown in (Figure 2A), the C. ternatea L. flower aqueous extract significantly reduced the encystation at all tested concentrations (5 mg/mL to 30 mg/mL) in a dose-dependent manner. The reduction in cyst viability was statistically significant (p<0.05, with Tukey’s post hoc test while pairwise comparisons were evaluated using a two-tailed unpaired Student’s t-test). Preliminary results showed that C. ternatea extract significantly inhibited the phenotypic transformation of A. culbertsoni. The result demonstrated a statistically significant (p = 0.001) inhibition of trophozoite to cyst transformation at concentration 5 mg/mL as compared to solvent control, indicating that the extract effectively disrupts encystation under in vitro conditions. At 15 mg/mL, the extract inhibited 98% of the trophozoites from undergoing encystation and showed an IC₅₀ of 11.7 mg/mL. While no viability was recorded at 20, 25 and 30 mg/mL, suggesting that the extract interferes with the physiological or molecular mechanisms required for encystation. The typical images of a 96-well plate showing the viability of cysts inhibited by C. ternatea extract are shown in (Figure 2B).