Rumen flukes, or paramphistomes, are parasites found in cattle rumen. The main rumen fluke species, belonging to the superfamily Paramphistomoidea, are Paramphistomum epiclitum, Calicophoron calicophorum, P. cervi, Gastrothylax crumenifer, Cotylophoron cotylophorum, Fischoederius elongatus, and Fischoederius cobboldi (Sey, 1991). The life cycle of rumen flukes includes a snail as an intermediate host, whereas adult flukes live in the rumen of cattle. Rumen flukes are widely distributed globally, including in Thailand (Hajipour et al., 2021; Nak-on and Chontananarth, 2020; Tookhy et al., 2022). A common species of rumen fluke in Thailand is P. epiclitum; they are approximately 1–2 cm long and have a pink color (Nak-on and Chontananarth, 2023; Nak-on et al., 2022). Because of independent rearing, cattle may drink water containing infective metacercaria of helminths, resulting in rumen fluke infection. Heavy infection can cause symptoms such as diarrhea, weight loss, and poor body condition, resulting in a decrease in milk and meat production, with substantial impacts on livestock production (Huson et al., 2017).

There are some previously developed molecular detection tools for paramphistomes (Anuracpreeda et al., 2017; Khan et al., 2021; Nak-on and Chontananarth, 2023). The rapid diagnosis in routine monitoring of parasite epidemic is the beginning of drug treatment consideration. Regarding the recent situation, the most effective anthelmintic drugs and alternative treatments still be interesting to study and evaluate the anti-parasite activity, especially for natural products or extracted solutions. Although the use of anthelmintic drugs such as albendazole, oxyclozanide, or mebendazole is an effective treatment method, drug resistance in hosts may occur (García-Dios et al., 2020; Veerakumari and Munuswamy, 2000; Wolstenholme et al., 2004). Therefore, medicinal plants have frequently been used as alternative drugs, employing various plant species (Atjanasuppat et al., 2009; Veerakumari, 2015). In addition, utilizing medicinal plants can be a cost-effective approach with low environmental impact compared to synthetic anti-helminthic drugs.

Scanning electron microscopy (SEM) is a useful tool to study tegument changes. In trematodes, the tegument is the outermost tissue layer and in direct contact with the host tissue and body fluids. Therefore, it can absorb and exchange molecules in host body fluids, making it important when developing anthelmintic substances (Anuracpreeda et al., 2012; Panyarachun et al., 2010, 2013). In addition, morphological analysis under SEM is useful for investigation the in vitro effect of natural compound to against paramphistome by tegumental change observation (Shalaby et al., 2010).

Previous studies have reported tegument changes induced by plant extract treatment. For example, Saowakon et al. (2013) reported that plumbagin (PB) extract affected P. cervi. The damage involved swelling, blebbing (after 6 h of incubation with 10 μg/mL of PB), disruption blebbing (after 12 h of incubation with 10 μg/mL of PB), erosion (after 3 h of incubation with 100 μg/mL of PB), and lesions (Saowakon et al., 2013). Rutin, isolated from Terminalia catappa, was tested against Gastrothylax crumenifer. At concentrations of 75 and 100 μg/mL, the surface was swollen, with deep grooves, erosion, and large lesions after 3 h of incubation; it exhibited drastic destruction and extreme deformity over the tegumental surface after 12 h of incubation (Minsakorn et al., 2021). Minsakorn et al. (2019) reported that for Carmyerius spatiosus, the tegument changes after treatment with plant extracts for 24 h, including heartwood ethyl acetate extract of Cassia siamea, root n-butanol extract of Plumbago zeylanica, root hexane, ethyl acetate, n-butanol extract of Plumbago indica, and leaf n-butanol and water extract of T. catappa, were swelling of ridges and folds, blebbing, rupturing of the blebs, erosion, lesion, and disruption of the tegument (Minsakorn et al., 2019). Tegument changes in Cotylophoron cotylophorum, treated with alcoholic extract of Allium sativum (garlic), were studied by Radwan et al. (2012), who observed moderate changes in the tegument at a concentration of 1 mg/L for 8 h (Radwan et al., 2012).

Bombax ceiba L. belongs to the family Bombacaceae, and its flower contain β-D-glucoside of β-sitosterol, free β-sitosterol, hentriacontane, and hentriacontanol. In previous studies, flowers of Bombax ceiba showed antioxidant effects and antiproliferative activity against human cancer cell lines (Chaudhary and Khadabadi, 2012; Chaudhary and Tawar, 2019; Huang et al., 2023). Black pepper (Piper nigrum), belonging to the family Piperaceae, is used as a flavoring in foods. It has antibacterial, antifungal, antidiarrheal, anti-inflammatory, and insecticidal activity. The most abundant and active alkaloid in pepper is piperine, along with compounds such as piperamide, piperamine, pipericide, and sarmentosine (Ahmad et al., 2012). In the light of knowledge in the major component and safety of extract solution for animal, it could be further applied for veterinary treatment as alternative drugs, bioavailable activity products, and medical consumables.

Although several plant extracts have been evaluated for their activities against adult rumen flukes, studies using the helminth P. epiclitum are still scarce. Tegumental damages and changes between treatment and; control group are useful qualitative indicators to determine the antihelminth activity of the interested treatment according to the previous studies (Minsakorn et al., 2019, 2021; Saowakon et al., 2013). In the present work, black pepper seeds and B. ceiba flowers were used regarding their impacts on the tegument surface, which is the tissue that is most immediately exposed to anthelmintics. This study investigated the changes in the surface morphology of the tegument of adult P. epiclitum following subjection to black pepper extract and B. ceiba flower extract at various concentrations and exposure times, using SEM.