Can In Vitro Cell Cultures of Eryngium planum, Lychnis flos-cuculi, and Kickxia elatine Be an Alternative Source of Plant Biomass with Biological Antimicrobial and Anti-Acanthamoeba Activities?

Abstract

The sustainable production of plant bioactive compounds is increasingly important as natural habitats decline. This study investigates whether in vitro cell cultures of Eryngium planum, Lychnis flos-cuculi, and Kickxia elatine can serve as alternative sources of biologically active biomass with antimicrobial and anti-Acanthamoeba properties. Callus cultures were established under optimized and controlled conditions, and metabolomic profiling was completed using UPLC-HRMS/MS. In silico analysis, using a molecular docking approach, was applied to understand the interaction between target compounds and Acanthamoeba profilin and identify possible targets for antimicrobial properties. Untargeted metabolomic analysis confirmed the presence of valuable compounds in the callus cultures of the studied species. Biological activity was assessed through anti-Acanthamoeba and antimicrobial assays. Lychnis flos-cuculi and Kickxia elatine callus extracts showed significant inhibitory effects on Acanthamoeba trophozoites, with 87.5% and 80.1% inhibition at 10 mg/mL, respectively. In contrast, E. planum extract stimulated amoebic growth. The anti-Acanthamoeba activity correlated with the presence of ferulic acid and p-coumaric acid in L. flos-cuculi extract, and acteoside in K. elatine extract. Antibacterial testing revealed moderate activity of E. planum and K. elatine extracts against Staphylococcus spp., while Gram-negative bacteria and fungi were largely resistant. These findings highlight the potential of in vitro cultures—particularly those from L. flos-cuculi and K. elatine—as promising, sustainable sources of anti-Acanthamoeba and antimicrobial agents, warranting further investigation into their pharmacologically active constituents.

1. Introduction

The limited availability of Eryngium planum L., Lychnis flos-cuculi L., and Kickxia elatine (L.) Dumort from natural habitats poses a significant challenge to their sustainable use. Overharvesting, habitat destruction, and environmental changes threaten wild populations, making alternative production methods necessary. Plant cell cultures offer an efficient and sustainable solution, ensuring a stable supply of bioactive compounds without depleting natural resources. Unlike wild-harvested plants, cell cultures provide a continuous and reliable source of secondary metabolites, independent of seasonal and environmental fluctuations. Natural plant material often varies in chemical composition due to environmental influences. In vitro cultures offer consistent phytochemical profiles, ensuring reproducibility. Cultivation in controlled laboratory conditions prevents overexploitation of endangered species and helps conserve biodiversity. Specific conditions in in vitro cultures can be optimized to enhance the production of secondary metabolites in these species. Moreover, the absence of environmental pollutants and microbial contamination improves the quality and safety of the obtained extracts. Plant cell cultures have a shorter production cycle compared to traditional cultivation, allowing rapid biomass accumulation. Moreover, suspension cultures can be scaled up in bioreactors, enabling large-scale industrial production of medicinal compounds [1,2,3,4].

The plant species Eryngium planum, Lychnis flos-cuculi, and Kickxia elatine are rich sources of bioactive compounds. Their diverse phytochemical profiles contribute to a wide range of pharmacological activities. The presence of phenolic acids and flavonoids enhances the ability to scavenge free radicals, reducing oxidative stress and potentially preventing degenerative diseases. All three species have demonstrated anti-inflammatory effects by inhibiting key mediators involved in inflammatory pathways, supporting their traditional use in wound healing [5,6,7]. E. planum and L. flos-cuculi exhibit antibacterial and antifungal activity, suggesting potential applications in infection management [8,9,10]. The phytochemical complexity and pharmacological potential of these species make them promising candidates for further research in drug development. Their diverse bioactive compounds support their traditional medicinal uses and provide a basis for modern therapeutic applications.

Eryngium planum L. (flat-sea eryngo, from the Apiaceae family) is known for its complex chemical composition, including triterpenoid saponins, flavonoids (quercetin and kaempferol derivatives), coumarins, polyacetylenes, phenolic acids, essential oil, and phytosterols (mostly sesquiterpene and monoterpene hydrocarbons) [11,12]. Among its bioactive compounds, barrigenol-type triterpenoid saponins have been identified, exhibiting strong cytotoxic activity [11]. Phenolic acids such as rosmarinic acid, chlorogenic acid, and caffeic acid contribute to its antioxidant properties [13,14]. Pharmacologically, extracts from E. planum demonstrate anti-inflammatory, antibacterial, and cytotoxic effects [5,6,9,12]. Furthermore, the plant has been traditionally used for wound healing and respiratory conditions [11].

Lychnis flos-cuculi L. (ragged robin, from the Caryophyllaceae family) is rich in phytoecdysteroids, flavonoids (C-glycosyl and O-glycosyl derivatives), saponins, and phenolic acids (benzoic and trans-cinnamic acid derivatives) [15,16]. Phytoecdysteroids, particularly those structurally related to ecdysterone, have been associated with adaptogenic and anti-stress effects. The plant also contains quillaic acid and gypsogenic acid derivatives, known for their biological activity [8]. The pharmacological effects of L. flos-cuculi include antioxidant, anti-inflammatory, and antimicrobial activities [8,16,17]. The presence of caffeic acid and flavonoids enhances its anti-inflammatory properties, supporting traditional uses in wound healing [18].

Kickxia elatine (L.) Dumort (sharpleaf cancerwort), a member of the Plantaginaceae family, contains a variety of metabolites, including phenylethanoid glycosides (such as acteoside, isoacteoside, echinacoside, and lavandulifolioside), phenolic acids (caffeic acid, trans-4-coumaric acid), fatty acids (azelaic acid, linoleic acid), and peptides [19,20,21,22,23,24]. Recent research highlights its cytotoxic potential, particularly against melanoma cells [23]. K. elatine was used for internal bleeding, menstruation, and nosebleeds. In the Balkans and India, it aids wound healing, while in Serbia, it treats bedwetting. In Italy, its leaves help with foot hyperhidrosis [25,26].

Acanthamoeba is a genus of free-living amoebae that are widely distributed in the environment, inhabiting soil, air, and various water sources, including freshwater, seawater, swimming pools, and even contact lens solutions. These protozoa can exist in two distinct life stages: an active, feeding trophozoite stage and a dormant, resistant cyst stage. This ability to form cysts makes Acanthamoeba highly resilient to adverse environmental conditions and contributes to its persistence in both natural and artificial habitats. Although Acanthamoeba species are typically non-pathogenic, certain strains can act as opportunistic pathogens, causing serious human diseases such as Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Acanthamoeba keratitis is a severe and potentially sight-threatening corneal infection that primarily affects contact lens users, often resulting from improper lens hygiene or exposure to contaminated water. On the other hand, granulomatous amoebic encephalitis is a rare but fatal central nervous system infection that predominantly occurs in immunocompromised individuals. Additionally, Acanthamoeba has been implicated in skin infections, sinusitis, and pulmonary complications, further emphasizing its medical significance. The treatment of Acanthamoeba-related infections remains challenging due to their ability to encyst, which makes them highly resistant to conventional therapies. Current pharmacological treatments often involve a combination of diamidines, biguanides, and antifungal agents, but their efficacy is limited, and prolonged treatment durations are required. Given these challenges, there is a growing interest in exploring alternative therapeutic agents, particularly those derived from natural sources, which may offer new avenues for effective Acanthamoeba control [27,28,29]. In this context, previous studies have reported strong amoebicidal activity of E. planum and L. flos-cuculi from in vitro organ culture extracts, supporting their potential as promising and alternative sources for anti-Acanthamoeba therapy [8,30]. To the best of our knowledge, there have been no studies on the effect of K. elatine extract for Acanthamoeba treatment.

This study aims to investigate the anti-amoebic potential of compounds from plant cell culture extracts of E. planum, L. flos-cuculi, and K. elatine, focusing on their effectiveness against Acanthamoeba trophozoites. Antimicrobial activity is further assessed using in vitro and in silico approaches. By evaluating plant-derived extracts and secondary metabolites, this research seeks to contribute to the development of novel therapeutic strategies for combating Acanthamoeba and other microbial infections.

2. Materials and Methods

2.1. Plant Material and In Vitro Cultures

Generally, the seeds were surface-sterilized using ethanol (70%, 30 s) followed by sodium hypochlorite (1–2% for 10–15 min) and rinsed three times with sterile distilled water. The sterilized explants were cultured on Murashige and Skoog (MS) medium, supplemented with plant growth regulators for general callus induction, high biomass production, and enhanced metabolite synthesis. The cultures were incubated at 20–25 °C, under a 16 h photoperiod (light intensity: ~40 µmol m²/s). Callus formation was observed within 14–20 days, and calluses were subcultured onto fresh media every 20–30 days to maintain growth and prevent browning. Actively growing callus (~500 mg) was transferred to liquid MS media containing the same plant growth regulators as used for callus induction and growth. The flasks containing the suspension culture were placed on a shaker (Orbitron, Infors HT, Hamburg, Germany) at 100 rpm for proper aeration and nutrient distribution. Subculturing was performed every 14–20 days by transferring 5–10 mL of the suspension into fresh media.

Sterile stem fragments of E. planum were placed on MS medium with dicamba (3.0 mg/L, Sigma-Aldrich, St. Louis, MO, USA) + thidiazuron (0.3 mg/L, Sigma-Aldrich, St. Louis, MO, USA), then subcultured every 28 ± 2 days until a stable callus line was obtained. Callus from L. flos-cuculi was generated from in vitro stem cultures on MS medium with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma-Aldrich, St. Louis, MO, USA) and subcultured every 28 ± 2 days until sufficient material was obtained. Callus induction of K. elatine was initiated using selected explants from in vitro-grown seedlings on solidified MS medium containing 1.0 mg/L 2,4-D (Sigma-Aldrich, St. Louis, MO, USA) + 1.0 mg/L 3,6-dichloro-2-methoxybenzoic acid (dicamba, Dic; Sigma-Aldrich, St. Louis, MO, USA) until sufficient material was collected. Starting from each stable callus, cell suspension biomass of E. planum was initiated into liquid MS media supplemented with 0.5 mg/L 2,4-D + 0.1 mg/L Kinetin, L. flos-cuculi cell suspension was initiated and maintained in 1.0 mg/L 2,4-D + 1.0 mg/L kinetin, and the cell suspension of K. elatine was cultured in MS containing 1.0 mg/L 2,4-D + 1.0 mg/L Dic.

2.2. Phytochemical Evaluation

L. flos-cuculi callus biomass (~100 mg) was pulverized using an MM 400 Mixer Mill (Retsch GmbH, Haan, Germany). Samples were prepared in Eppendorf tubes with 100% methanol and 100 μg/mL Formononetin (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard for UPLC-MS. After vortex mixing for 20 min and sonication for 15 min (Bandelin Sonorex, Bandelin Electronic GmbH and Co., Berlin, Germany), samples were centrifuged at 12,000 rpm for 10 min. The supernatant (~800 μL) was collected, vacuum-dried (Savant SC100 SpeedVac, Farmingdale, New York, NY, USA), and reconstituted in 1 mL of 0.1% formic acid in water solution. Following another 10 min sonication and 5 min centrifugation at 15,000 rpm, methanolic extraction of L. flos-cuculi biomass was purified using Oasis HLB Cartridges (1 cc, 30 mg of solid phase, Waters Corp., Milford, MA, USA). Three biological replicates were analyzed.

E. planum and K. elatine cell biomass were pulverized and then added to 0.1% Formononetin (Sigma-Aldrich, St. Louis, MO, USA) and 100% and 80% methanol, respectively. The mixtures were then vortexed for 15–20 min and sonicated at room temperature for 15 min. The samples were centrifuged at 15,000 rpm for 10 min, followed by filter-extraction using a Kinesis KX Syringe Filter PTFE 13 mm pore size 0.45 µm (Kinesis Scientific Expert, Cole Parmer, St. Neots, UK). Three biological replicates were used in the analysis.

Extracts were analyzed via Waters ACQUITY UPLC (Milford, CT, USA) coupled with a PDA detector and HRMS/MS (Qexactive, Thermo Fisher Scientific, Bremen, Germany). A 5 µL of extract was separated on an Acquity BEH Shield column (2.1 × 150 mm, 1.7 µm, Waters, Milford, MA, USA) at 40 °C. A 0.1% of formic acid in water (A, LC-MS grade, Merck, Darmstadt, Germany) and acetonitrile (B, LC-MS grade, Merck) gradient was applied: 5–75% B (1–14 min), 75–99% B (14–16 min), 99% B isocratic (16–19 min), and 5% B re-equilibration (19–20 min) at a 400 µL/minute flow rate. UV–Vis spectra were recorded at a wavelength of 250–550 nm and a scan rate of 20 Hz. The HESI-II operated at −3.5 kV in negative ion mode, with an ion transfer tube temperature of 350 °C. Nitrogen was used as a sheath, auxiliary, and sweep gas with flow rates of 35, 10, and 3 arbitrary units, respectively. An auxiliary gas temperature was set at 400 °C and an S-lens RF level of 50. Full MS scans (120–1800 m/z) were acquired at 70,000 FWHM with a 200 ms ion-trap time, while ddMS2 scans (TopN = 5) were collected at 17,500 FWHM with a 100 ms ion-trap time and 30% of normalized collision energy in the HCD cell.

Putative annotation of selected peaks and the raw data from UPLC-HRMS/MS in negative ion mode were processed using MS-DIAL software (version 5.5) [31], with the following parameters. Data collection was performed with 0.01 Da and 0.025 Da tolerance for MS1 and MS2, respectively. Furthermore, the retention time was set from 2 to 17 min, MS1 ranging between 120 and 1800 Da, and a range of 50–2000 Da for MS/MS. Peak detection was applied with a minimum 10,000 amplitude and 0.1 Da mass slice width. Simple moving average level 5 and a minimum of 7 scans of peak width were set in the advanced settings. Deconvolution parameters were set with a value of 0.5 for the sigma window and an MS/MS abundance cutoff of 1 amplitude. Identification and annotation of the detected peaks were completed using ESI(-)-MS/MS from standards + bio + in silico and RIKEN PlaSMA bio-MS/MS from plant tissues from the MS-DIAL metabolomics MSP spectral kit. Alignment was performed with 0.2 min of retention tolerance and 0.015 Da of MS1 tolerance. Additionally, the 0.4 and 0.6 factors were set for retention time and MS1, respectively.

2.3. Preparation of Cell Biomass Extract for Investigations of Their Biological Activities

Air-dried cell biomasses for E. planum, K. elatine, and L. flos-cuculi were powdered and weighed, and then subjected to extraction three times using 70% ethanol (v/v) in an ultrasonic water bath for one hour each. The procedure was repeated for each extract and then evaporated to dryness at a temperature of 40 ± 1 °C using a rotary evaporator. The obtained extracts were then used for biological activity assays

2.4. Anti-Acanthamoebic Assay

The dried plant material was ground into a fine powder, extracted using 70% ethanol three times, and concentrated under reduced pressure at 40 °C. The study utilized Acanthamoeba sp. trophozoites obtained from an axenic culture system (Figure 1). The strain was maintained in a liquid medium containing 2% Bacto-Casitone at 28 °C. The trophozoites were periodically observed under an inverted microscope to assess viability and morphological integrity. Prior to experimental procedures, cultures were adjusted to a standardized density of 5 × 10⁴ cells/mL to ensure uniformity across trials. Trophozoites of Acanthamoeba were plated on 2% non-nutritive agar plates coated with E. aerogenes bacteria, and the plates were incubated at 28 °C. To evaluate the effects of the plant extracts on Acanthamoeba trophozoites, a Thoma hemocytometer chamber was used for cell counting. Paper discs were soaked with test substances at a concentration ranging from 1 mg/mL to 10 mg/mL and standard substances at a concentration of 0.1 mg/mL. The soaked discs were plated on non-nutritive agar with amoeba culture. Amoeba growth was assessed using a microscope for 3 consecutive days at 24 h intervals. Each sample was tested in triplicate. After 3–5 days, the number of amoebae was observed and examined under an inverted microscope at ×200. The plates were monitored microscopically for up to 14 days for the growth of Acanthamoeba trophozoites or the presence of cysts (until 100% cysts were obtained). Control cultures without extracts were maintained under identical conditions. The half-maximal inhibitory concentration (IC₅₀) was calculated by plotting the percentage of growth inhibition against extract concentration using a nonlinear regression model. Statistical significance was determined using one-way ANOVA followed by Duncan’s multiple range test, with a significance threshold of p < 0.05. All experiments were conducted in 12–18 replications, and data are presented as mean values with standard deviations (±SD). Statistical comparisons were performed using the agricolae version 1.3-7 package in R [32].

2.5. Antimicrobial Activity

The in vitro antimicrobial activity of E. planum, K. elatine, and L. flos-cuculi cell biomass extracts was assessed against Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 35984, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 10231. Each bacterium was inoculated into 4–5 mL of Mueller–Hinton broth (MHB; bacteria) or RPMI 1640 medium (RPMI; fungi) and incubated overnight at 36 °C until it reached approximately 1.5 × 10⁸ CFU/mL. A dilution of the inoculum suspension was prepared by adding 100 µL of inoculum into 19.9 mL fresh MHB so that the final concentration of bacteria was 5 × 10⁶ CFU/mL. Similarly, before the antifungal assay, C. albicans inoculum was diluted in RPMI to obtain 2.5 × 10⁶ CFU/mL.

The antimicrobial activity was studied using the microbroth dilution method by EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines. The studied extracts were first diluted in water and sterilized using a Millex^®-GV 0.22 µm filter unit (Millipore, Carrigtwohill, Ireland). Twofold serial dilutions of the extracts were performed, yielding a concentration range starting at 100 mg/mL. Aliquots of 100 µL of fresh MHB/RPMI and 90 µL of each plant extract dilution were distributed in 96-well plates. Each well was added to 10 µL of the bacteria/fungi suspension. Growth control and media sterility control were performed simultaneously. The plates were then incubated for 24 h at 36 °C. The minimum inhibitory concentration (MIC) was determined as the lowest concentration at which observable bacteria/fungi growth is inhibited. Gentamycin and Nystatin were used for antibacterial and antifungal positive control, respectively.

Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) assays were performed to determine the lowest concentration of plant extracts, which resulted in the death of 99.9% of the inoculum. The assay was performed after the MIC test and data collection. From each well of the 96-well plates, 10 µL was taken and inoculated onto tryptic soy agar (bacteria) or Sabouraud dextrose agar (fungi) for further microbial observation. The plates were incubated at 36 °C overnight. All tests were duplicated, and the antimicrobial activity was represented as the mean values.

2.6. Molecular Docking

Molecular docking was carried out to understand the interaction between target compounds and Acanthamoeba profilin and identify possible targets for antimicrobial properties from extracts of E. planum, K. elatine, and L. flos-cuculi cell biomass. Acteoside, chlorogenic acid, p-coumaric acid, ferulic acid, and rosmarinic acid were chosen for in silico analysis based on the compounds used in this study (Figure 2). Chemical structures were drawn using ChemSketch software (2024.2.0; Advance Chemistry Development, Inc., Toronto, ON, Canada). On the other hand, acteoside, leucosceptoside A, rosmarinic acid, chlorogenic acid, p-coumaric acid, and ferulic acid were chosen based on prior research for an antibacterial and antifungal computational assay [12,15,23]. Chemical structures were acquired from the open chemistry database NCBI PubChem (https://pubchem.ncbi.nlm.nih.gov/ (accessed on 28 March 2025)) and RCSB Protein Data Bank (https://www.rcsb.org/ (accessed on 28 March 2025)).

Acanthamoeba castellanii profilin 1B (PDB 1ACF) was used for anti-Acanthamoeba docking [33]. Profilin 1B is a common anti-Acanthamoeba target, as it is a crucial protein for cell shaping through the synthesis and degradation of actin microfilaments [34]. The chosen proteins for antibacterial investigation from bacteria, Gram-positive and Gram-negative, were dihydropteroate synthase (PDB 1AD4, 5V7A) and gyrase B (PDB 4URN, 1KZN). These proteins have been previously assessed as targets for antibacterial treatments [35]. Additionally, sterol 14-demethylase (PDB 5TZ1) was chosen as the protein target for antifungal examination against C. albicans. Sterol 14-demethylase regulates ergosterol production, which is crucial for fungal cell permeability, fluidity, and the efficacy of membrane-associated proteins, in which some antifungal agents were developed to specifically target it [36].

The selected target proteins were then subsequently brought to the PyMOL Molecular Graphics System, Version 3.0 Schrödinger LLC. (New York, NY, USA), for water molecules, ions, and complex co-structure removal [37]. Using AutoDockTools in MGLTools 1.5.7 [38], polar hydrogen atoms, Kollman charges, and Gasteiger charges were assigned to target proteins and then converted into PDBQT format. At the active site, a 60 × 60 × 60 cubic grid box was visualized for each protein. Simulations were carried out in AutoDock 4.2.6 [39] with the Lamarckian genetic algorithm for docking parameters. Post-docking visualization and analysis were performed using BIOVIA Discovery Studio 2025 [40].

3. Results

The identified metabolites of E. planum callus include various organic acids, saccharides, phenolic acids, flavonoids, and triterpenoid saponins (

Table 1. Main identified metabolites in the callus of Eryngium planum L. using LC-MS/MS in negative ion mode.

RT (min)	Observed m/z	Theoretical m/z	Error [ppm]	Formula	MS/MS Fragmentation	Metabolite Name	Ontology
2.29	191.0188	191.0190	−1.05	C6H8O7	111.0072, 129.0178, 173.0078, 191.0187	Citric acid	Carboxylic acid
2.44	339.1295	339.1250	13.27	C13H24O10	101.0227, 207.0866, 339.1295	Disaccharides	Saccharides
3.7	167.0337	167.0338	−1.16	C8H8O4	83.0120, 95.0122, 108.0202, 123.0435, 138.9277, 152.0102, 167.0337	Vanillic acid	Methoxybenzoic acids and derivatives
4.47	353.0877	353.0878	0.28	C16H18O9	135.0436, 161.0229, 173.0443, 179.0338, 191.0550, 353.1698	Neochlorogenic acid	Quinic acid derivatives
5.80	179.0337	179.0340	−1.68	C9H8O4	135.0436, 179.0337	Caffeic acid (or isomer)	Hydroxycinnamic acid
5.39	353.0876	353.0867	2.66	C16H18O9	191.0550	Chlorogenic acid	Quinic acid derivatives
6.29	367.1033	367.1035	−0.54	C17H20O9	111.0435, 175.0443, 191.0551	3-O-Feruloylquinic acid	Quinic acid derivatives
6.91	163.0388	163.0389	−0.84	C9H8O3	119.0487, 163.0388	trans-Coumaric acid	Hydroxycinnamic acid
7.05	193.0497	193.0506	4.66	C10H10O4	134.0358, 149.054, 178.0260, 193.0495	Ferulic acid	Hydroxycinnamic acid
7.16	593.1509	593.1511	−0.34	C27H30O15	284.0326, 384.9893, 593.1503	Kaempferol-3-O-rutinoside	Flavonoid glycoside
7.49	447.0932	447.0933	−0.22	C21H20O11	284.0325, 447.0931	Kaempferol-3-O-glucoside	Flavonoid glycoside
7.50	549.0884	549.0886	−0.36	C24H22O15	178.9969, 300.0275, 345.0374, 463.0880, 505.1010	Quercetin 3-O-malonylglucoside	Flavonoid glycoside
7.71	359.077	359.0761	2.46	C18H16O8	161.0231, 179.0338, 197.0446	Rosmarinic acid	Hydroxycinnamic acid
7.79	179.0338	179.0338	−0.57	C9H8O4	135.0436, 179.0338	Caffeic acid (or isomer)	Hydroxycinnamic acid
8.23	263.1287	263.1289	−0.76	C15H20O4	151.0750, 204.1146, 219.1383	Abscisic acid	Abscisic acids derivatives
8.39	1119.5593	1119.5581	1.02	C54H88O24	751.4631, 795.4525, 913.5151, 957.5065, 1055.692, 1119.5581	Triterpenoid saponin (Eryngioside C)	Triterpenoid saponin
9.39	1251.6019	1251.6004	1.19	C59H96O28	101.0229, 161.0443, 221.0657, 296.9634, 500.9339, 608.2999, 719.4329, 881.4872, 1043.5446, 1205.6021	Unknown triterpenoid saponin I	Triterpenoid saponin
9.49	1057.5229	1057.5214	1.46	C52H82O22	113.0229, 119.0333, 131.0331, 149.04441, 157.0130, 169.8532, 316.0179, 408.9600, 503.3569, 610.5472, 655.4232, 683.4191, 831.3508, 877.3508, 877.4583, 895.4639, 925.4744, 1057.5229	Unknown triterpenoid saponin II	Triterpenoid saponin
9.64	925.4802	925.4791	1.17	C47H74O18	113.0228, 157.0132, 179.0553, 655.4217, 745.6167, 795.4150, 843.4360, 925.4805	Unknown triterpenoid saponin III	Triterpenoid saponin
11.47	909.4845	909.4853	−0.88	C47H74O17	539.4251, 909.4849	Unknown triterpenoid saponin IV	Triterpenoid saponin

). Among the notable compounds are maleic acid and methylmalonic acid, both classified as dicarboxylic acids and derivatives. The presence of citric acid, a widely known carboxylic acid, indicates a role in primary metabolism. Phenolic acids such as caffeic acid, trans-caffeic acid, chlorogenic acid, trans-coumaric acid, neochlorogenic acid, trans-coumaric acid, ferulic acid, and rosmarinic acid were also detected in the callus extract. Additionally, vanillic acid, a methoxybenzoic acid derivative, contributes to the phenolic profile. Furthermore, several triterpenoid saponins, whose specific structures remain unidentified, are present, suggesting a role in plant defense and bioactivity. The flavonoid glycosides present in this species include kaempferol-3-O-rutinoside and 3-O-malonylglucoside. Overall, the metabolite profile of E. planum callus culture highlights a diverse array of bioactive compounds, many of which are associated with biological activities.

Table 2. Main identified metabolites in the callus of Lychnis flos-cuculi L. using LC-MS/MS in negative ion mode.

RT (min)	Observed m/z	Theoretical m/z	Error [ppm]	Formula	MS/MS Fragmentation	Metabolite Name	Ontology
4.00	167.0338	167.0338	−0.34	C8H8O4	167.0338	Vanillic acid	Phenolic
4.62	341.0879	341.0867	3.91	C15H18O9	163.0389	1-O-caffeoylglucose	Phenolic
4.78	163.0389	163.0389	−0.28	C9H8O3	163.0389	cis-p-coumaric acid	Phenolic
5.10	193.0498	193.0495	1.40	C10H10O4	193.0498	Ferulic acid	Hydroxycinnamic acid
5.12	621.2035	621.2025	1.75	C26H37O17	459.1514, 283.1038, 193.0498, 175.0391, 134.0360	Ferulic acid derivative	Hydroxycinnamic acid
5.32	179.0338	179.0339	−0.23	C9H8O4	135.0450, 179.0339	Caffeic acid	Hydroxycinnamic acid
5.57	355.1039	355.1024	4.20	C16H20O9	85.0279, 134.0360, 160.0155, 175.0392, 193.0499, 209.0297	1-O-feruloylglucose	Phenolic
5.75	137.0231	137.0233	−1.49	C7H6O4	93.0331, 137.0231	4-hydroxybenzoic acid	Phenolic
6.33	593.1498	593.1501	−0.43	C27H30O15	225.0667, 269.0572, 293.0460, 413.0885	Kaempferol 3-rutinoside	Flavonoid glycoside
6.65	577.1553	577.1552	0.16	C27H29O14	293.0455, 311.0560, 323.0561, 413.0877	Vitexin O-rhamnoside	Flavonoid glycoside
6.88	609.1454	609.1450	0.62	C27H30O16	241.0612, 271.0346, 300.0277, 301.0357	Rutin	Flavonoid
7.73	163.0389	163.0389	−0.37	C9H8O3	-	trans-p-coumaric acid	Phenolic
8.67	987.4813	987.4795	2.18	C48H75O21	119.0336, 439.3219, 663.3750	Triterpene saponin VIII (hydroxygypsogenic acid derivative)	Triterpenoid saponin
8.69	967.4548	967.4533	2.07	C48H71O20	414.8124, 437.3048, 499.3075, 521.2425, 553.2910, 613.3378, 675.3400	Triterpene saponin IX (GA or QA derivative)	Triterpenoid saponin
8.74	1277.581	1277.5797	−0.09	C60H93O29	101.0227, 423.3267, 647.3798	Triterpene saponin X (GA or QA derivative)	Triterpenoid saponin
9.37	1247.570	1247.56914	1.23	C59H91O28	405.3152, 485.3299, 643.3488, 761.4094, 823.4127	Triterpene saponin XXI (GA or QA derivative)	Triterpenoid saponin
13.84	269.0457	269.0445	3.68	C15H9O5	-	Apigenin	Flavonoid

presents the main metabolites identified in the callus of L. flos-cuculi (Table 2). The identified compounds include hydroxycinnamic acid derivatives—ferulic acid, caffeic acid, p-coumaric acid, and other phenolic compounds, e.g., 1-O-caffeoylglucose, 1-O-feruloylglucose, and 4-hydroxybenzoic acid. Several triterpenoid saponins are detected, though their specific structures remain unidentified. The table also includes vitexin O-rhamnoside, kaempferol 3-rutinoside, rutin, and apigenin, which are classified as flavonoids. Furthermore, some unknown compounds are categorized as carboxylic acids or phenolic/flavonoid derivatives, indicating the presence of yet-to-be-characterized metabolites. Overall, the table highlights a diverse metabolite profile in L. flos-cuculi callus cultures, including organic acids, flavonoids, and saponins.

Table 3. Main identified metabolites in callus of Kickxia elatine (L.) Dumort using LC-MS/MS in negative ion mode.

RT (min)	Observed m/z	Theoretical m/z	Error [ppm]	Formula	MS/MS Fragmentation	Metabolite Name	Ontology
2.78	164.0704	164.0717	−7.92	C9H11NO2	72.0075, 103.0535, 147.0439, 164.0705	Phenylalanine	Phenylalanine and derivatives
3.22	218.1029	218.1034	−2.29	C9H17NO5	59.0123, 88.0400, 116.0703, 146.0810, 218.1030	Pantothenate	Secondary alcohols
3.89	203.0818	203.0826	−3.93	C11H12N2O2	72.0074, 116.0490, 142.0648, 159.0914, 186.0547, 203.0816	Tryptophan	Indolyl carboxylic acids and derivatives
4.50	175.06	175.0612	−6.85	C7H12O5	85.0643, 115.0386, 131.0700, 157.0494, 175.0602	2-Isopropylmalic acid	Hydroxy acids
4.64	431.1556	431.1559	−0.69	C19H28O11	59.0122, 89.0228, 101.0228, 119.0336, 149.0443, 191.0552, 233.0671, 299.1137, 336.2642	Darendoside A	Phenylpropanoids
5.38	179.0339	179.035	0.20	C9H8O4	135.0437, 179.0339	Caffeic acid	Hydroxycinnamic acids
5.84	415.1614	415.161	0.96	C19H28O10	59.0122, 71.0122, 89.0228, 101.0229, 113.0229, 131.0335, 147.0284, 161.0442, 173.0078, 191.005, 228.2101, 269.1042, 301.8495, 415.1274	(2R,3S,4S,5R,6R)-2-[[(2S,3R,4R)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxymethyl]-6-(2-phenylethoxy)oxane-3,4,5-triol	Phenylpropanoids
6.19	785.2514	785.251	0.51	C35H46O20	161.0232, 461.1668, 623.1983	Echinacoside	Phenylpropanoids
6.31	755.2402	755.2404	−0.26	C34H44O19	161.0232, 447.1517, 593.2087, 755.2401	Lavandulifolioside	Phenylpropanoids
6.67	623.1978	623.1981	−0.48	C34H44O19	161.0231, 315.1089, 461.1667, 623.1978	Acteoside	Phenylpropanoids
6.71	217.1074	217.1082	−3.68	C10H18O5	155.1066, 171.1016, 199.0968, 217.1077	Hydroxysebacic acid	Medium-chain hydroxy acids and derivatives
6.92	163.0389	163.0401	−7.36	C9H8O3	119.0487, 163.0338	trans-4-Coumaric acid	Hydroxycinnamic acids
7.08	623.1979	623.1981	−0.32	C29H36O15	161.0232, 315.1091, 461.1670, 623.1969	Isoacteoside	Phenylpropanoids
7.19	637.2136	637.2127	−1.41	C30H37O15	175.0390, 461.1668, 637.2114	Leucosceptoside A	Phenylpropanoids
7.39	187.0966	187.0976	−5.34	C9H16O4	57.0329, 94.0642, 125.0956, 143.1062, 169.0858, 187.0695	Azelaic acid	Medium-chain fatty acids
9.82	329.2335	329.2333	0.61	C18H34O5	171.1014, 211.1331, 229.1439, 329.2333	(Z)-5,8,11-trihydroxyoctadec-9-enoic acid	Long-chain fatty acids
10.61	309.2074	309.2071	0.97	C18H30O4	137.0958, 171.1015, 251.1649, 291.1969, 309.2076	FA 18:3+2O	Lineolic acids and derivatives
13.24	295.2278	295.2279	−0.34	C18H32O3	171.1015, 276.8783, 295.2279	9-HODE	Lineolic acids and derivatives
14.77	297.2434	297.2435	−0.34	C18H34O3	155.1065, 171.1017, 279.2328, 297.2434	FA 18:1+1O	Lineolic acids and derivatives
14.91	279.2327	279.2329	−0.72	C18H32O2	96.9584, 138.3048, 232.9239, 279.2330	Linoleic acid	Lineolic acids and derivatives

presents the main metabolites identified in the callus of K. elatine (Table 3). The table includes phenylalanine and tryptophan. Pantothenate is listed under secondary alcohols, while 2-isopropylmalic acid falls under hydroxy fatty acids. Several phenylpropanoids have been identified, including echinacoside, lavandulifolioside, acteoside, isoacteoside, and leucosceptoside. Additionally, caffeic acid and trans-4-coumaric acid are classified as hydroxycinnamic acids. The table also lists hydroxysebacic acid and azelaic acid. Long-chain fatty acids include (Z)-5,8,11-trihydroxyoctadec-9-enoic acid, while linoleic acids and their derivatives, such as FA 18:3+2O, 9-HODE, and FA 18:1+1O, are categorized under linoleic acids and derivatives. Overall, the metabolite profile of K. elatine callus culture includes amino acids, phenylpropanoids, hydroxycinnamic acids, and fatty acids, highlighting its diverse composition.

Table 4. Effect of rosmarinic acid and chlorogenic acid on inhibition of Acanthamoeba trophozoites during three days of treatment.

Extracts Concentration	1st Day		2nd Day		3rd Day
	MN ± SD	% GS/GI	MN ± SD	% GS/GI	MN ± SD	% GS/GI
Rosmarinic acid
control	4.28 ± 2.02 ab	0	8.66 ± 2.36 ab	0	10.31 ± 2.68 a	0
0.01 mg/mL	5.61 ± 2.03 a	31.07 ST	6.05 ± 2.39 c	31.34 IN	9.33 ± 2.81 b	9.51 IN
0.05 mg/mL	5.50 ± 2.01 a	28.50 ST	7.33 ± 2.49 bc	15.36 IN	8.19 ± 2.92 b	20.56 IN
0.1 mg/mL	3.67 ± 1.25 b	14.26 IN	9.44 ± 2.33 a	9.00 ST	10.61 ± 2.75 a	2.91 ST
Chlorogenic acid
control	5.28 ± 2.02 a	0	8.66 ± 2.36 a	0	10.31 ± 2.68 a	0
0.01 mg/mL	4.44 ± 1.50 b	15.91	5.33 ± 2.05 b	38.46	8.71 ± 1.66 a	15.52
0.05 mg/mL	4.72 ± 1.91 b	10.61	5.83 ± 1.64 b	32.68	8.35 ± 2.54 a	19.02
0.1 mg/mL	4.83 ± 1.89 b	8.53	6.15 ± 1.16 b	28.99	9.00 ± 3.21 a	22.71

MN ± SD—mean ± standard deviation; % GS/GI—percentage of growth stimulation or inhibition; ST—stimulation; IN—inhibition; mean values within a row sharing the same letter are not significantly different at p < 0.05, as determined by Duncan’s multiple range test. The first letter of the alphabet denotes the highest values, while subsequent letters indicate statistically significant decreases.

,

Table 5. Effect of extract from Eryngium planum callus culture on inhibition of Acanthamoeba trophozoites during three days of treatment.

Extracts Concentration	1st Day		2nd Day		3rd Day
	MN ± SD	% GS	MN ± SD	% GS	MN ± SD	% GS
control	6.27 ± 2.43 a	0	8.59 ± 2.43 b	0	18.08 ± 2.75 b	0
1 mg/mL	6.35 ± 2.19 a	1.28	10.33 ± 1.63 a	20.26	19.63 ± 4.53 b	8.57
5 mg/mL	6.57 ± 1.40 a	4.78	10.76 ± 1.70 a	25.26	28.60 ± 6.86 a	58.19
10 mg/mL	6.92 ± 1.69 a	10.37	9.89 ± 2.35 ab	15.13	28.81 ± 5.60 aa	59.35

MN ± SD—mean ± standard deviation; % GS—percentage of growth stimulation; mean values within a row sharing the same letter are not significantly different at p < 0.05, as determined by Duncan’s multiple range test. The first letter of the alphabet denotes the highest values, while subsequent letters indicate statistically significant decreases.

,

Table 6. Effect of ferulic acid and p-coumaric acid on inhibition of Acanthamoeba trophozoites during three days of treatment.

Extracts Concentration	1st Day		2nd Day		3rd Day
	MN ± SD	GI [%]	MN ± SD	GI [%]	MN ± SD	GI [%]
Ferulic acid
control	3.17 ± 1.01 a	0	2.72 ± 1.73 a	0	2.94 ± 1.16 a	0
0.05 mg/mL	2.06 ± 1.47 b	5.07	2.44 ± 0.96 a	10.30	2.71 ± 1.56 a	7.83
0.1 mg/mL	1.56 ± 0.76 b	28.12	2.28 ± 1.27 a	16.18	2.44 ± 2.00 a	17.01
0.2 mg/mL	1.94 ± 0.85 b	10.60	1.88 ± 0.68 a	30.89	2.00 ± 1.41 a	31.98
p-coumaric acid
control	3.17 ± 1.01 a	0	2.72 ± 1.73 a	0	2.94 ± 1.16 a	0
0.05 mg/mL	1.72 ± 1.04 b	45.75	2.28 ± 1.18 a	16.18	2.41 ± 1.42 a	18.03
0.1 mg/mL	1.67 ± 1.45 b	47.32	2.11 ± 1.66 a	22.43	2.24 ± 1.00 a	25.81
0.2 mg/mL	1.50 ± 1.21 b	52.69	2.00 ± 1.20 a	26.48	2.17± 1.26 a	26.20

MN ± SD—mean ± standard deviation; % GI—percentage of growth inhibition; mean values within a row sharing the same letter are not significantly different at p < 0.05, as determined by Duncan’s multiple range test. The first letter of the alphabet denotes the highest values, while subsequent letters indicate statistically significant decreases.

,

Table 7. Effect of extract from Lychnis flos-cuculi callus culture on inhibition of Acanthamoeba trophozoites during three days of treatment.

Extracts Concentration	1st Day		2nd Day		3rd Day
	MN ± SD	GI [%]	MN ± SD	GI [%]	MN ± SD	GI [%]
control	3.56 ± 1.57 a	0	5.89 ± 1.41 a	0	10.25 ± 2.38 a	0
1 mg/mL	2.11 ± 1.20 bc	40.74	3.67 ± 0.94 b	37.70	4.63 ± 1.56 b	54.83
5 mg/mL	2.41 ± 1.03 b	32.31	2.83 ± 1.38 b	51.96	2.78 ± 1.58 c	72.88
10 mg/mL	1.28 ± 0.99 c	64.05	1.11 ± 1.05 c	81.16	1.28 ± 0.99 d	87.52

MN ± SD—mean ± standard deviation; % GI—percentage of growth inhibition; mean values within a row sharing the same letter are not significantly different at p < 0.05, as determined by Duncan’s multiple range test. The first letter of the alphabet denotes the highest values, while subsequent letters indicate statistically significant decreases.

,

Table 8. Effect of acteoside on inhibition of Acanthamoeba trophozoites during three days of treatment.

Extracts Concentration	1st Day		2nd Day		3rd Day
	MN ± SD	GI [%]	MN ± SD	GI [%]	MN ± SD	GI [%]
control	1.61 ± 1.41 a	0	3.61 ± 1.38 a	0	6.00 ± 1.33 a	0
0.01 mg/mL	2.44 ± 1.44 a	6.52	2.75 ± 1.19 b	23.83	4.00 ± 2.47 b	43.33
0.1 mg/mL	2.42 ± 1.54 a	7.28	2.61 ± 0.95 b	27.71	3.76 ± 1.70 b	37.34
0.2 mg/mL	2.17 ± 1.57 a	16.86	2.17 ± 0.96 b	39.89	2.95 ± 0.91 b	50.84

MN ± SD—mean ± standard deviation; % GI—percentage of growth inhibition; mean values within a row sharing the same letter are not significantly different at p < 0.05, as determined by Duncan’s multiple range test. The first letter of the alphabet denotes the highest values, while subsequent letters indicate statistically significant decreases.

and

Table 9. Effect of extract from Kickxia elatine callus culture on inhibition of Acanthamoeba trophozoites during three days of treatment.

Extracts Concentration	1st Day		2nd Day		3rd Day
	MN ± SD	GI [%]	MN ± SD	GI [%]	MN ± SD	GI [%]
control	2.61 ± 1.57 a	0	3.50 ± 2.09 a	0	5.33 ± 3.61 a	0
1 mg/mL	1.25 ± 0.56 b	52.11	2.00 ± 1.15 b	42.86	3.94 ± 2.17 b	26.08
5 mg/mL	1.39 ± 0.68 b	46.75	1.89 ± 0.94 b	46.00	1.82 ± 1.38 b	65.86
10 mg/mL	1.56 ± 0.93 b	40.33	1.22 ± 0.85 b	65.15	1.06 ± 1.13 b	80.12

MN ± SD—mean ± standard deviation; % GI—percentage of growth inhibition; mean values within a row sharing the same letter are not significantly different at p < 0.05, as determined by Duncan’s multiple range test. The first letter of the alphabet denotes the highest values, while subsequent letters indicate statistically significant decreases.

present the inhibitory/stimulatory effects of the reference compounds (Figure 2) and callus extracts of E. planum, L. flos-cuculi, and K. elatine on Acanthamoeba trophozoites over a three-day treatment period. The table includes mean values (MN ± SD) and the percentage of growth inhibition or stimulation (% GS/GI) at different concentrations compared to the control.

Chlorogenic acid exhibits a stronger inhibitory effect than rosmarinic acid, particularly on day 2, where inhibition increases significantly before gradually decreasing. Rosmarinic acid’s effectiveness declines over time, and at higher concentrations (0.1 mg/mL), it even leads to growth stimulation, indicating a possible hormetic effect (low doses inhibit, while higher doses promote growth). While both compounds exhibit inhibitory effects on Acanthamoeba, chlorogenic acid demonstrates a more stable and prolonged inhibitory action, whereas rosmarinic acid’s effect diminishes over time, with higher doses even stimulating growth (Table 4).

Both chlorogenic acid and rosmarinic acid (Figure 2D,E), tested individually in Table 4, are also present in the callus extract of E. planum analyzed in Table 5. These comparisons suggest that the presence of chlorogenic and rosmarinic acids alone is insufficient to inhibit Acanthamoeba growth and that other compounds in the E. planum extract may either counteract or mask any weak inhibitory effect, potentially even stimulating growth. The callus extract, which contains both of these acids, caused a pronounced stimulation of trophozoite growth, especially at higher doses. The fact that both the individual compounds (Table 4) and the complex extract containing them (Table 5) show trophozoite proliferation suggests that rosmarinic and chlorogenic acids may contribute to, or synergize with, other compounds in the extract to enhance growth rather than inhibit it.

The effect of Eryngium planum callus culture extract on Acanthamoeba growth is positive for all the tested concentrations (Table 4). The stimulation of Acanthamoeba growth is dose- and time-dependent, increasing with both concentration and duration of treatment, reaching 59.35% stimulation at 10 mg/mL on day 3. Given its growth-promoting effects rather than inhibition, E. planum extract does not appear to be a viable candidate for combating Acanthamoeba infections. Further studies would be needed to understand the underlying mechanism of this stimulation and assess its potential implications.

Table 6 evaluates how two hydroxycinnamic acids—ferulic acid and p-coumaric acid (Figure 2B,C)—influence the growth of Acanthamoeba trophozoites over a 3-day period (Table 6). The results demonstrate that both compounds exhibit moderate to strong inhibitory effects, which are dose-dependent and generally increase over time. Ferulic acid displays moderate inhibition, with effectiveness improving with dose and time. Interestingly, the strongest inhibition for p-coumaric acid occurs early (day 1) and slightly decreases over time, although it remains significant. p-Coumaric acid is more potent than ferulic acid, particularly at lower doses, suggesting rapid initial action but with less cumulative effect over three days. The extract from Lychnis flos-cuculi callus culture shows strong growth inhibition (Table 7). Growth inhibition increases over time for all concentrations. The highest inhibition (87.52%) occurs on day 3 at 10 mg/mL, suggesting a cumulative effect of the extract. Even at lower concentrations (1 mg/mL, 5 mg/mL), inhibition trends upward each day, indicating sustained effectiveness. This extract appears to be a promising candidate for anti-Acanthamoeba applications, as it effectively suppresses growth with increasing efficacy over time.

Both the individual compounds (Table 6) and the whole callus extract (Table 7) of L. flos-cuculi demonstrate significant inhibition of Acanthamoeba trophozoite growth over a 3-day period. The anti-Acanthamoeba activity observed in the callus extract can be partly attributed to the presence of ferulic acid and p-coumaric acid, as demonstrated in their isolated form in Table 6. This supports the idea that these phenolic acids contribute substantially to the extract’s bioactivity.

Table 8 evaluates the anti-amoebic activity of phenylethanoid glycoside, namely acteoside (Figure 2A), by measuring the impact on Acanthamoeba trophozoite growth over three days at different concentrations (Table 8). Inhibition increases with higher concentrations, especially by day 3. Inhibition becomes more pronounced over time. Even at the lowest dose (0.01 mg/mL), growth inhibition rises from 6.52% (day 1) to 43.33% (day 3). Acteoside exhibited inhibitory activity against Acanthamoeba trophozoites, with a stronger and consistent dose- and time-dependent effect. These results support the potential role of phenylethanoid glycosides in developing plant-based anti-Acanthamoeba treatments.

The extract of Kickxia elatine exhibits a dose-dependent inhibition of Acanthamoeba growth (Table 9). The inhibition pattern is not strictly linear across time. The highest inhibition is achieved on day 3 at 10 mg/mL (80.12%), indicating a sustained and strong inhibitory effect at the highest dose. The extract is a promising candidate for anti-Acanthamoeba activity, though higher concentrations are required for sustained inhibition.

Lychnis flos-cuculi shows the strongest inhibitory effect, reaching 87.52% inhibition on day 3 at 10 mg/mL. K. elatine also performs well, reaching 80.12% inhibition. E. planum does not inhibit growth; instead, it stimulates Acanthamoeba growth.

Acteoside (Table 8; Figure 2A) and the whole callus extract of K. elatine (Table 9) demonstrate significant inhibition of trophozoite growth, confirming that the active constituent, acteoside, likely contributes to the observed biological activity of the extract. The isolated compound and the full extract show increased effectiveness over time, which was particularly evident by day 3. This pattern indicates a cumulative anti-amoebic effect, whether from individual glycosides or the complex mixture within the extract. Given that acteoside is present in K. elatine and shows individual anti-amoebic properties, its presence likely contributes significantly to the strong inhibitory effects observed with the whole callus extract.

The strong anti-Acanthamoeba activity seen in the K. elatine callus extract (Table 9) mirrors the effects of acteoside when tested individually (Table 8). The datasets confirm that phenylethanoid glycoside is a key contributor to the extract’s bioactivity. The dose- and time-dependent inhibition observed in both tables reinforces the idea that K. elatine is a promising source of natural anti-amoebic compounds.

Molecular docking is an in silico analysis of protein–ligand interactions to provide insight into its binding affinity at the active site and potency. This approach has been used to help with drug design and discovery, or to provide an understanding of disease treatment [41].

Table 10. Docking ΔG (kcal/mol) and inhibitory constant scores of selected main compounds from cell biomass of Eryngium planum, Kickixa elatine, and Lychnis flos-cuculi on the active site of Acanthamoeba profilin 1B protein (PDB 1ACF).

	ΔG (kcal/mol)	Inhibitory Constant
Acteoside	−2.86	8.02 mM
Chlorogenic acid	−4.58	437.19 µM
p-coumaric acid	−4.33	667.05 µM
Ferulic acid	−3.80	1.64 mM
Rosmarinic acid	−4.58	483.33 µM

and Figure 3 present the docking results between selected compounds against the Acanthamoeba profilin 1B protein. This protein is a crucial regulator of the actin cytoskeleton, which participates in cellular activities, including filament assembly [33]. Based on the docking analysis, chlorogenic acid and rosmarinic acid have the most negative ΔG (−4.58 kcal/mol) and a low inhibitory constant (437.19 and 483.33 µM, respectively), suggesting a strong binding affinity of these compounds for the profilin protein. p-coumaric acid was proposed to give strong binding affinity and high potency with −4.33 kcal/mol and a 667.05 µM inhibitory constant. Meanwhile, ferulic acid and acteoside showed moderate to low binding affinity for the Acanthamoeba profilin protein.

As shown in

Table 11. Minimum inhibitory concentration (MIC, mg/mL) values of methanolic extracts from the callus, cell suspension cultures, and in vitro shoot culture of Eryngium planum, Lychnis flos-cuculi, and Kickxia elatine tested against S. aureus, S. epidermidis, P. aeruginosa, and C. albicans.

Extract	S. aureus ATCC 25923		S. epidermidis ATCC 35984		P. aeruginosa ATCC 27853		C. albicans ATCC 10231
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MFC
Eryngium planum
in vitro shoot culture	12.5	50	12.5	25	100	>100	>100	>100
callus culture	50	100	50	100	>100	>100	>100	>100
cell suspension culture	100	100	>100	>100	>100	>100	>100	>100
Kickxia elatine
in vitro shoot culture	100	100	25	100	100	100	>100	>100
callus culture	>100	>100	25	100	100	>100	>100	>100
cell suspension culture	>100	>100	>100	>100	>100	>100	>100	>100
Lychnis flos-cuculi
in vitro shoot culture	25	50	>100	>100	100	>100	>100	>100
callus culture	>100	>100	>100	>100	>100	>100	>100	>100
cell suspension culture	>100	>100	>100	>100	100	>100	>100	>100
Gentamycin	0.125	0.25	-	-	0.125	0.125	-	-
Nystatin	-	-	-	-	-	-	0.125	0.25

, the callus cultures are more effective than suspension cultures in inhibiting bacterial growth. The suspension cultures exhibit little to no antimicrobial activity across all tested microorganisms. Gram-positive bacteria (Staphylococcus species) are more sensitive to the extracts compared to Gram-negative bacteria and fungi. Pseudomonas aeruginosa and Candida albicans are resistant to all the tested plant extracts. The E. planum and K. elatine callus cultures show moderate antibacterial potential, while the Lychnis flos-cuculi extracts are mostly ineffective. The E. planum suspension culture shows weak activity against S. aureus (MIC = 100 mg/mL) but is ineffective against all other microbes. The K. elatine and L. flos-cuculi suspension cultures are ineffective (MIC > 100 mg/mL for all strains). The suspension cultures are consistently less effective than their callus counterparts. The plant extracts require much higher concentrations (≥25 mg/mL) to have an antibacterial effect, making them far less potent than standard antibiotics. Further testing on purified bioactive compounds from E. planum and K. elatine is necessary to enhance antibacterial effects. Combination studies with standard antibiotics are needed to assess potential synergistic effects.

The molecular docking results on the molecular interaction between selected phytochemicals sourced from E. planum, K. elatine, and L. flos-cuculi to target proteins (dihydropteroate synthase, gyrase B, and sterol 14-demethylase) are presented in

Table 12. Docking ΔG (kcal/mol) scores of selected main compounds from cell biomass of Eryngium planum, Kickixa elatine, and Lychnis flos-cuculi on the active site of each protein target.

	Dihydropteroate Synthase		Gyrase B		Sterol 14-Demethylase (PDB 5TZ1)
	Gram + (PDB 1AD4)	Gram − (PDB 5V7A)	Gram + (PDB 4URN)	Gram − (PDB 1KZN)
Acteoside	−4.80	−3.48	−5.10	−4.83	−7.94
Azelaic acid	−4.24	−4.82	−3.42	−2.74	−5.90
Chlorogenic acid	−6.35	−6.33	−5.12	−5.49	−7.89
p-Coumaric acid	−5.43	−5.49	−5.45	−4.65	−5.46
Ferulic acid	−4.84	−4.95	−5.64	−4.75	−5.79
Leucosceptoside A	−3.21	−4.50	−4.53	−4.68	−6.63
Rosmarinic acid	−7.13	−6.50	−5.76	−6.78	−7.77

and visualized in Figure 4, Figure 5, Figure 6 and Figure 7, respectively. The in silico analysis results indicated that rosmarinic acid showed the strongest predicted binding to dihydropteroate synthase with ΔG −7.13 and −6.50 kcal/mol and inhibition constants of 5.95 and 17.14 µM (Gram-positive and -negative, respectively. Chlorogenic acid also displays a substantial affinity for this target, registering a ΔG of −6.35 and −6.33 kcal/mol along with 21.97 and 22.92 µM inhibition constant scores for Gram-positive and Gram-negative, respectively (Table 1, Figure 4 and Figure 6). Both compounds also showed potent interactions with gyrase B, with ΔG values around −6 kcal/mol for both Gram-positive and Gram-negative. On the other hand, leucosceptoside A demonstrated the weakest interaction with dihydropteroate synthase and azelaic acid with gyrase B (Table 12, Figure 5 and Figure 7).

Acteoside, chlorogenic acid, and rosmarinic acid exhibited the most negative ΔG values with −7.94, −7.89, and −7.77 kcal/mol and inhibition constant scores of 1.5, 1.66, and 2.0 µM, respectively. The results suggested a strong binding affinity of these compounds towards the sterol 14-demethylase active site, thus promising antifungal agents from these species by inhibiting fungal cell wall biosynthesis and their growth. Surprisingly, leucosceptoside A showed a strong bonding affinity against sterol 14-demethylase with a ΔG of −6.63. Meanwhile, moderate binding affinity was presented by azelaic acid, ferulic acid, and p-coumaric acid with a ΔG within −5 kcal/mol.

4. Discussion

Acanthamoeba infections present a persistent clinical challenge. Therefore, the search for effective, less toxic, and, ideally, novel treatments is crucial. Chemotherapy has been the gold standard for treating Acanthamoeba infections. Yet, animal-based and plant-based products have been reported to be a promising alternative for novel anti-amoebic agents [42]. This study investigated the anti-amoebic potential of E. planum, L. flos-cuculi, and K. elatine callus culture extracts against Acanthamoeba trophozoites. The findings demonstrate a species-dependent effect, ranging from stimulation (E. planum) to strong inhibition (L. flos-cuculi).

Contrary to expectations, E. planum callus extract stimulated trophozoite proliferation at all tested concentrations over the three-day experiment. The growth stimulation increased with both dose and time, reaching 59.35% stimulation at 10 mg/mL on day 3. These results align with previous studies showing that flavonoid-rich extracts can have unexpected proliferative effects on microorganisms. The stimulatory effect may be attributed to phenolic acids and flavonoids, which, in Eryngium species, have been linked to antioxidant and immunomodulatory properties rather than antimicrobial activity [30]. Additionally, studies on Acanthamoeba suggested that this organism has a vast range of oxidative stress-counteracting machinery, making it resistant to most oxidative-based therapy [43,44]. Other studies confirm that Eryngium extracts possess various pharmacological effects, including diuretic, expectorant, and anti-inflammatory properties, but their anti-amoebic potential remains questionable [30].

In contrast, L. flos-cuculi callus culture extract exhibited the strongest growth inhibition, achieving 87.52% inhibition at 10 mg/mL on day 3. The inhibitory effect was dose- and time-dependent, with higher concentrations yielding more significant reductions in trophozoite counts [8]. K. elatine callus culture extract demonstrated moderate inhibition of trophozoite growth, reaching 80.12% inhibition at 10 mg/mL on day 3. However, the inhibition pattern was inconsistent—while higher doses resulted in greater inhibition over time, lower doses sometimes exhibited fluctuating effects. Compared to L. flos-cuculi, K. elatine appears to have a weaker but still significant amoebicidal effect. Previous studies highlight the presence of flavonoids and iridoid glycosides in K. elatine [20,21,24], which may contribute to its anti-amoebic potential. The observed effects can be contextualized within a broader scope of plant-derived anti-amoebic agents.

In silico anti-amoebic analyses were conducted between the tested compounds and Acanthamoeba profilin 1B protein. This protein is involved in the synthesis and degradation of actin microfilaments, which makes it crucial for cell shaping. Thus, it is one of the anti-Acanthamoeba targets [34]. Comparative analyses, including in silico and in vitro studies on chlorogenic and p-coumaric acids, further support the potential of plant-derived compounds as anti-amoebic agents. Both compounds exhibited potent inhibition against Acanthamoeba and possessed high predicted binding affinity for the profilin protein. In parallel, Lonicera japonica and its major active constituent, chlorogenic acid, significantly reduced the viability by 49.2% after 48 h of 1.5 mg/mL fraction treatment and 100% reduction in the cysts/trophozoite ratio after 24 h of treatment with 1 mg/mL chlorogenic acid [45]. Furthermore, there are agreements between both analyses on ferulic acid and acteoside as moderate and the least potent anti-Acanthamoeba activities. However, the most significant discrepancy is observed in rosmarinic acid. As the in silico analysis proposed, this compound is a potent inhibitor compared to the other tested compounds; yet the result is inconsistent with the in vitro data. There is a possibility that Acanthamoeba is able to degrade or metabolize rosmarinic acid, leading to the reduction in the effective concentration. Nonetheless, this study only focuses on the interaction with a single profilin protein and a single molecule, which could not explain other factors that are present at the cellular level. Siddiqui et al. (2022) reported low inhibitory potency of rosmarinic acid isolated from Rinorea yaundensis and Salvia triloba against A. castellanii, as a 90% viable amoeba was still observed after treatment (100 µg/mL, overnight) [46]. Kikowska et al. (2022) proposed that anti-amoebic activity from the Eryngium species does not come from the polyphenol contents but rather from its triterpenoid saponin content [30]. This genus has been extensively studied for its oleanane triterpenoid saponins as the dominant component [47,48]. This is consistent with the study by Sifaoui et al. (2019) that pentacyclic triterpenoids, ursolic acid, and its derivatives are potent anti-amoebic agents that induce programmed cell death via the mitochondrial pathway [49].

The observed anti-amoebic activity in E. planum, L. flos-cuculi, and K. elatine cell biomass extract probably arises from the synergistic interactions of their constituent compounds. Studies on other medicinal plants, such as Chaenomeles japonica [50] and Plantago media [51], show similarly strong inhibitory effects on Acanthamoeba trophozoites. For example, C. japonica extracts inhibited 95.4% of Acanthamoeba trophozoites at 5 mg/mL after three days, an effect attributed to pentacyclic triterpenoids and polyphenols [50]. Plantago media extracts, rich in phenylethanoid glycosides, exhibited strong inhibition (63.89%) at 5 mg/mL [51]. The alkaloid and pentacyclic triterpenoid content of Pericampylus glaucus presented potent inhibition to A. triangularis. At 100 µg/mL, the alkaloid extract demonstrated potent anti-amoebic activity, inhibiting 99% of trophozoite growth over 72 h. Notably, periglaucine A (the major alkaloid constituent), exhibited the same level of inhibition (99%) within 24 h at the same concentration. Furthermore, the isolated betulinic acid (pentacyclic triterpenoid) inhibited 94.6% of trophozoites’ survival within 48 h [52]. These findings suggest that the chemical composition of plant extracts plays a crucial role in their anti-amoebic potential.

The combination of in vitro studies with molecular docking (in silico) approaches can aid in predicting target proteins and ligands, as well as their interactions, which are important for understanding bioactivity properties. Sterol 14-demethylase is an enzyme that regulates ergosterol synthesis, which is crucial for fungal cell permeability, fluidity, and activity of membrane-associated proteins. Therefore, many antifungal agents were developed to target this protein [53]. The antibacterial property was evaluated against dihydropteroate synthase (PDB 1AD4 and 5V7A) and gyrase B (PDB 4URN and 1KZN) of Gram-positive and Gram-negative bacteria, respectively. These proteins have been studied as targets for antibacterial treatments [35]. Dihydropteroate synthase is an enzyme that catalyzes 7,8-dihydropteroate production from para-aminobenzoic acid and dihydropterin pyrophosphate, which are crucial for bacterial growth [54]. Gyrase B catalyzes the cleavage of ATP in the ATP hydrolysis reaction and is coupled with gyrase A, which is involved in DNA breakage during negative supercoiling of plasmid and chromosomal DNA [55].

The molecular docking findings suggested that rosmarinic acid and chlorogenic acid, which are present in E. planum, are potent antimicrobial inhibitors with the possibility of targeting proteins responsible for growth and genetic multiplication (Table 12). However, this property could also happen due to the synergistic effect of multiple compounds or other compounds that are not included in this study. Nevertheless, the saponin–phenolic acid fraction and the saponin fraction from E. planum demonstrated prominent antimicrobial activity with MICs of 1–2 mg/mL and 2.5 mg/mL, respectively [56]. It is hypothesized that the antibacterial and antifungal activity of saponins were found to be dependent on their carbohydrate-attached aglycone, which may interact with the microbial cell membranes [57].

Previous studies postulated that ecdysteroid derivatives were responsible for antimicrobial properties [8,17]. However, ecdysteroid was not identified in the callus of L. flos-cuculi. Therefore, the possibility lies in ferulic acid and p-coumaric acid as potential antimicrobial agents in L. flos-cuculi, in addition to the presence of triterpenoid saponins in callus culture, as shown by the results. The variation in activity shown among several forms of culture (shoot, callus, cell suspension) indicates that the production of active compounds is quite dependent on the culture systems. Data obtained from in vitro experiments suggest poor antimicrobial activities of all the samples, suggesting a low concentration of active compounds within the extracts. Further testing on purified bioactive compounds in the cell biomass might be necessary to enhance antibacterial effects.

These findings emphasize the potential for natural plant extracts as alternative anti-amoebic agents. Given that conventional treatments (e.g., biguanides, diamidines) are often toxic and cause irritation, plant-derived compounds could offer safer therapeutic options. Further studies should focus on isolating the specific bioactive compounds responsible for the inhibitory effects in L. flos-cuculi and K. elatine, testing combinations of plant extracts with existing anti-amoebic drugs to explore synergistic effects, and investigating cytotoxicity on human cells to ensure safety for potential therapeutic use.

5. Conclusions

The results support the potential of Lychnis flos-cuculi and Kickxia elatine cell biomass as sustainable sources for promising anti-Acanthamoeba agents, and potentially antimicrobial agents. The in vitro and molecular docking analyses have confirmed that these bioactivities are supported by the presence of phenolic acids or phenylethanoid glycosides, in this case ferulic acid, p-coumaric acid, and acteoside. Overall, these results confirm the pharmacological potential of specific in vitro plant and cell cultures as an alternative source. While the results highlight the biotechnological potential of plant cell cultures, further studies are needed to optimize cultivation conditions and to quantify metabolite yields to fully evaluate their production efficiency.