Ethical approval was not required for this study because all parasite materials were obtained from abattoir-derived cattle organs collected during routine slaughterhouse inspections, and no live animals were directly handled, restrained, or experimentally manipulated during the study. The samples used in this investigation consisted exclusively of post-mortem biological materials collected from cattle slaughtered for commercial purposes at a municipal abattoir. Therefore, the study did not involve experimental animal procedures or interventions requiring ethical clearance under institutional animal welfare regulations.

In addition, parasite specimen collection was conducted in accordance with standard biosafety and laboratory handling procedures to ensure the safe processing of biological materials. The use of post-mortem samples was considered exempt from formal animal ethics committee review, in accordance with institutional guidelines and national recommendations regarding the use of slaughterhouse-derived specimens for laboratory-based parasitological research.

The study was conducted from July to December 2024. Adult worms and eggs of F. gigantica were collected from naturally infected cattle slaughtered at the municipal abattoir of Ende Regency, East Nusa Tenggara, Indonesia. Samples were obtained from multiple naturally infected cattle of the same species (Bos taurus) during routine post-mortem inspection to minimize host-specific sampling bias. In vitro ovicidal and anthelmintic assays were conducted at the Animal Quarantine Laboratory, Agricultural Quarantine Agency of East Nusa Tenggara.

Preparation of green tea leaf powder and turmeric rhizome powder was carried out at the Faculty of Animal Science, Universitas Padjadjaran, Indonesia. Extraction of plant materials was performed at the Pharmacology Laboratory, Faculty of Veterinary Medicine, Universitas Airlangga, Indonesia. Histological slide preparation was conducted at the National Veterinary Standard Testing Center, Bogor, Indonesia, whereas histological examination was carried out at the Animal Quarantine Laboratory, East Nusa Tenggara, Indonesia. SEM analysis was performed at the Integrated Science Area Laboratory, National Research and Innovation Agency, Cibinong, Indonesia, and at the Integrated Research and Testing Laboratory, Universitas Gadjah Mada, Indonesia. The study was conducted from July to December 2024.

This study was conducted as an in vitro laboratory-based experimental study to evaluate the ovicidal and anthelmintic activities of combined ethanolic extracts of green tea and turmeric against eggs and adult worms of F. gigantica. The experiment employed a completely randomized design with six treatment groups, including negative and positive controls, and four replicates per treatment. The experimental design was established to assess concentration-dependent effects of the combined extracts on egg degeneration, adult worm mortality, histopathological alterations, and ultrastructural damage.

Adult F. gigantica worms were recovered from the livers of naturally infected cattle during post-mortem inspection. Morphological identification was based on the typical elongated leaf-like body shape, prominent cephalic cone, broad shoulders, and body proportions consistent with standard taxonomic descriptions of F. gigantica. Worms were washed repeatedly with physiological saline (0.9% NaCl) to remove host debris. Eggs were collected from the gall bladder of infected cattle and processed for ovicidal assays [27].

Green tea extract: Dried green tea leaves were ground into a powder and extracted by maceration with 50% ethanol at a 1:8 (w/v) ratio. The mixture was allowed to stand with occasional stirring, filtered, and concentrated using a rotary evaporator (IKA Rotary Evaporator RV 10, IKA Works GmbH & Co. KG, Staufen, Germany) at 70°C–80°C to obtain a viscous extract [28]. The extract yield was recorded as a percentage by weight (% w/w) relative to the initial dry plant material.

Turmeric extract: Dried turmeric rhizomes were pulverized and macerated with 96% ethanol at a ratio of 100 g powder to 800 mL solvent for 24 h with intermittent stirring. The filtrate was separated from the residue and concentrated using the same rotary evaporator (IKA Works GmbH & Co. KG) at 70°C–80°C to obtain a thick extract [29]. The final yield (% w/w) was also documented to support reproducibility.

Nitroxynil (Fluconix-340®, MSD Animal Health, Boxmeer, the Netherlands) was used as a positive control. Nitroxynil was selected instead of triclabendazole because it is a commercially established fasciolicidal agent with proven efficacy against adult Fasciola spp. and is widely available in the local veterinary setting, making it an appropriate comparative reference for the present study. The working solution (10%) was prepared by diluting the commercial formulation with physiological saline, in accordance with the manufacturer’s specifications and previous studies. Physiological saline (0.9% NaCl) served as the negative control [30].

Gall bladder contents containing F. gigantica eggs were diluted with tap water and homogenized. The suspension was centrifuged at 1,184 × g for 10 min, and the supernatant was discarded. This washing step was repeated until a clear suspension was obtained. Egg concentration was adjusted to a minimum of 1000 eggs/mL and stored at 4°C for no longer than 24 h before use in ovicidal assays [31].

Six experimental groups were established as follows:

P0: Physiological saline (negative control)

All concentrations represent final concentrations (w/v) in the incubation medium. The selected concentrations were based on previous literature reports and preliminary pilot observations indicating observable adulticidal and ovicidal activities without immediate non-specific precipitation or medium instability. Each treatment was performed in four replicates, determined using the Federer formula.

Equal volumes (1 mL) of egg suspension and treatment solution were mixed in test tubes, covered with aluminum foil, and incubated at 28°C for 14 and 16 days. The incubation temperature of 28°C was selected because it falls within the standard embryonation range reported for Fasciola spp. eggs (26°C–28°C). On day 14, samples were exposed to artificial light for 2 h under standardized laboratory illumination conditions to stimulate miracidial development.

Egg morphology was examined microscopically on day 16. Eggs were classified as degenerated based on eggshell damage, nuclear disintegration, or premature operculum opening. A minimum of 100 eggs per replicate was evaluated microscopically, resulting in at least 400 eggs per treatment group. Ovicidal activity was expressed as the percentage of damaged eggs [32].

Adult F. gigantica worms (five worms per Petri dish) were immersed in treatment solutions. Worm mortality was assessed at 5, 10, 20, 40, 80, 160, and 320 min. These time points were selected to capture both rapid and delayed mortality kinetics and to enable comparison with the rapid onset of the positive control.

Worms were considered dead when no muscular response was observed following mechanical stimulation. Mechanical stimulation was performed by gently probing the anterior and mid-body tegument using a sterile blunt dissecting needle for approximately 5 s per worm. Absence of visible contraction or movement after stimulation was recorded as mortality. Dead worms were collected for histological and ultrastructural analyses [27].

Dead worms were fixed in 10% buffered formalin, processed using routine histological techniques, and stained with hematoxylin and eosin. At least one representative worm from each replicate was processed for histological examination. Tissue sections were examined under a light microscope to assess structural alterations in the tegument and internal organs [33].

For SEM analysis, worm samples were fixed in 2.5% glutaraldehyde for 3 h, washed with phosphate-buffered saline (pH 7.4), post-fixed with osmium tetroxide for 1 h, dehydrated through a graded ethanol series, and dried using critical point drying. Samples were coated with carbon and examined using a SEM to evaluate surface ultrastructural changes [34]. Representative worms from each treatment group were selected for SEM evaluation.

Percentage egg degeneration data and adult worm mortality data were analyzed using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test for multiple comparisons. Descriptive observations of egg morphology are presented to distinguish descriptive and analytical results. Statistical analyses were performed using SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA). Histopathological and SEM findings were evaluated descriptively. Statistical significance was considered at p < 0.05.

Microscopic evaluation of F. gigantica eggs after 16 days of incubation revealed distinct morphological alterations among treatments. Eggs in the negative control group (physiological NaCl) showed normal development and hatching, indicated by operculum opening and larval emergence. Similar developmental patterns were observed in the lowest extract combination (0.15% C. sinensis + 0.12% C. longa).

In contrast, eggs exposed to nitroxynil and higher concentrations of combined extracts exhibited severe structural damage, including eggshell rupture, membrane degeneration, lysis of egg contents, abnormal egg shape, and failure to develop. The most pronounced ovicidal effect was observed in the 0.30% C. sinensis + 0.24% C. longa treatment (Figure 2).

The proportion of damaged eggs increased significantly with increasing extract concentration (Tables 1 and 2). Nitroxynil induced complete egg damage (100%), whereas physiological NaCl showed the lowest damage rate.

At the highest extract concentration (P5: 0.30% green tea + 0.24% turmeric), the mean percentage of egg damage reached 94.03 ± 2.38%, representing the highest ovicidal activity among all extract-treated groups. Among the extract combinations, the highest egg damage was observed in the 0.30% C. sinensis + 0.24% C. longa group.

One-way ANOVA demonstrated a significant effect of treatment on egg damage (p < 0.05). Duncan’s multiple range test indicated significant differences between the control group and all extract-treated groups, as well as dose-dependent differences among extract combinations (Table 2). For clarity, Table 1 presents descriptive morphological egg outcomes, whereas Table 2 summarizes the inferential statistical analysis of total egg damage percentages.

Adult fluke mortality increased progressively with exposure time and extract concentration (Table 3). The positive control, nitroxynil, induced 100% mortality within the first 5 min of exposure. In contrast, the combined extracts exhibited gradual and time-dependent mortality, with increasing efficacy at later observation points.

The highest concentration (P5) produced 90% mortality at 160 min and 100% mortality at 320 min, approaching the efficacy of nitroxynil only at prolonged exposure times. No complete mortality was observed in the NaCl control.

At 160 min, mortality values in P2 (80%), P3 (70%), P4 (70%), and P5 (90%) were substantially higher than at earlier time points. Based on post hoc analysis, the lack of significant difference from nitroxynil was specifically observed in P2 and P5, whereas P3 and P4 showed numerically similar but statistically distinguishable values under the applied Duncan’s multiple range test.

This finding indicates that although the combined extracts exhibited slower onset kinetics than nitroxynil, higher concentrations achieved near-complete adulticidal activity at later time points.

Histological examination revealed progressive structural damage correlated with extract concentration (Figures 3–5).

Control worms exhibited intact tegument, normal spines, and preserved intestinal lumen. In contrast, nitroxynil and higher extract concentrations caused tegument separation, spine deformation, and intestinal lumen rupture with villi loss.

The most severe histological damage was consistently observed in P5 and the nitroxynil-treated group, supporting the quantitative mortality findings presented in Table 3.

SEM analysis demonstrated severe ultrastructural damage in extract-treated worms, including tegument erosion, spine distortion, oral and ventral sucker deformation, and surface exfoliation (Figures 6–9). Damage severity increased with extract concentration and closely resembled that induced by nitroxynil. Notably, P5 demonstrated marked tegumental peeling, spine swelling, and surface erosion, consistent with its higher adulticidal activity at 160–320 min.

The present study demonstrated that the combined ethanolic extracts of green tea and turmeric exhibited significant ovicidal and anthelmintic activities against F. gigantica in vitro. Although the combined extracts showed marked biological activity, their efficacy was lower and their onset slower than that of the commercial anthelmintic nitroxynil, particularly at the early observation time points (Table 3). These effects were evidenced by marked egg damage, increased adult fluke mortality, and pronounced histological and ultrastructural alterations. Collectively, the findings support the potential of this herbal combination as a natural alternative to synthetic anthelmintics and warrant further in vivo investigation before practical application can be recommended.

The ovicidal activity observed in this study was characterized by eggshell rupture, membrane degeneration, abnormal morphology, and lysis of egg contents (Figure 2; Table 1). These alterations are consistent with previous studies evaluating plant-based anthelmintics against F. gigantica eggs, in which damage to the eggshell and embryonic degeneration were considered indicators of ovicidal efficacy [35]. The similarity of egg damage patterns between extract-treated groups and the nitroxynil control suggests that the combined extracts may act through comparable structural disruption of the egg envelope [36].

The ovicidal activity of the extracts is likely attributable to the combined, potentially additive, effects of tannins from green tea and curcuminoids from turmeric. Tannins are known to bind structural proteins in the eggshell, impair membrane integrity, alter permeability, and interfere with embryogenesis, ultimately preventing larval development [37]. Meanwhile, curcumin, a major bioactive curcuminoid in turmeric, has been reported to induce oxidative stress, membrane destabilization, mitochondrial dysfunction, and cellular degeneration in helminth eggs [38]. In addition, curcuminoids may disrupt intracellular redox balance and impair key enzymatic pathways necessary for embryonic development. The concentration-dependent increase in egg damage observed in this study (Table 2) further supports a direct chemical interaction between these phytochemicals and egg structures [39].

The 30.04% egg damage observed in the NaCl negative control (Table 2) was relatively high and may reflect the inherent variability of field-collected egg samples obtained from naturally infected abattoir-derived material. Variations in egg age, pre-existing developmental status, and minor handling-associated mechanical stress during processing may have contributed to baseline degeneration. Therefore, this finding should be interpreted as biological variability rather than treatment-related activity.

The incubation period (16 days at 28°C) was selected to coincide with the known embryonation window of Fasciola spp. eggs. Increased ovicidal efficacy at later observation points aligns with previous reports indicating progressive egg vulnerability during embryonic development [40]. These findings suggest that the combined extracts could potentially be applied as natural environmental disinfectants to interrupt parasite transmission in endemic areas [41].

Adult fluke mortality increased with both extract concentration and exposure time, indicating a time-dependent anthelmintic effect (Table 3). Although nitroxynil induced rapid mortality within 5 min (100%), the combined extracts demonstrated substantial efficacy, achieving complete mortality within 320 min at all tested concentrations [42]. Therefore, the extract activity should be interpreted as approaching or becoming comparable to nitroxynil only at prolonged exposure times rather than across all observation points.

Importantly, at later time points (160–320 min), mortality induced by higher extract combinations, particularly P5, approached the efficacy of nitroxynil, suggesting comparable efficacy under prolonged exposure [40]. This distinction is important because the kinetic profiles differed considerably between the herbal extracts and the positive control.

The delayed onset of action observed in extract-treated groups compared with nitroxynil may reflect differences in pharmacodynamic properties. Synthetic anthelmintics typically exert rapid neuromuscular paralysis, whereas plant-derived compounds may require longer exposure to disrupt metabolic, neuromuscular, oxidative, or membrane-associated processes [43]. Nevertheless, the progressive increase in mortality across extract combinations indicates that even lower concentrations possess measurable bioactivity against adult flukes [44].

The use of physiological NaCl as a negative control confirmed that the mortality observed in the treated groups was attributable to the extracts rather than to environmental stress alone. Although limited mortality was observed in the control group at later time points (20% at 320 min; Table 3), this is likely attributable to prolonged in vitro maintenance conditions rather than toxic effects [45].

Tegumental damage is widely recognized as a critical indicator of anthelmintic efficacy in trematodes. In the present study, histological examination revealed tegument separation, spine deformation, and intestinal lumen disruption in extract-treated worms (Figures 3–5). These alterations compromise nutrient absorption, osmoregulation, and host–parasite interactions, ultimately leading to parasite death [46].

SEM analysis provided detailed confirmation of these structural changes, revealing tegument erosion, spine swelling, spine loss, and deformation of oral and ventral suckers (Figures 6–9). Such damage likely facilitates increased permeability of the tegument, allowing deeper penetration of bioactive compounds and exacerbating internal tissue damage [47]. Similar ultrastructural disruptions have been reported following exposure to both synthetic anthelmintics and plant-derived compounds, supporting the hypothesis that tegumental integrity is a primary target of anthelmintic action [48].

The observed spine distortion and loss may also impair parasite attachment and locomotion, further contributing to mortality. Previous studies have reported that spinal damage is associated with osmotic stress and disruption of the tegumental syncytium, which may explain the progressive deterioration observed as extract concentration increases [49].

The combined use of green tea and turmeric extracts appeared to show enhanced activity compared with several reports in the literature describing the use of individual plant extracts. However, because this study did not include single-extract treatment groups tested in parallel, a true synergistic interaction cannot be conclusively demonstrated. Therefore, the observed effect is more appropriately described as a potential additive or enhanced combined effect rather than confirmed synergy. This clarification is important in interpreting the biological interaction between the two plant extracts.

The enhanced activity may result from complementary mechanisms of action, including protein binding by tannins and oxidative, metabolic, or membrane-disrupting effects of curcuminoids [50]. Such combined mechanisms support the rationale for using multi-plant formulations in helminth control strategies [51].

A further limitation of the present study is the absence of phytochemical profiling of the final extracts. Quantitative confirmation of key bioactive constituents, such as epigallocatechin gallate and curcuminoids, would strengthen the mechanistic interpretation of the observed biological effects and improve reproducibility [52].

This study was conducted exclusively under in vitro conditions, which may not fully replicate the complexity of host–parasite interactions in vivo. Therefore, further studies are needed to evaluate in vivo efficacy, pharmacokinetics, safety, and optimal dosing strategies. Future studies should also include single-extract control groups, dose–response modeling, and phytochemical quantification to better define the contribution of each extract component.

Additionally, molecular investigations targeting parasite genes involved in metabolism, proteolysis (e.g., cathepsin L), and tegument maintenance would provide valuable insights into the precise mechanisms underlying the observed effects [53].

Despite these limitations, the findings highlight the promise of combined green tea and turmeric extracts as environmentally friendly, accessible, and cost-effective alternatives to synthetic anthelmintics, particularly for small-scale livestock systems in endemic regions.