Pa ge 1 Pa ge 1 American Journal of Life Science and Innovation (AJLSI) In VitroIn Vitro Antihelmintic Activity of the Hydroethanolic Leaf Extracts of Avicennia marinaAvicennia marina (White Mangroves) on Eudrilus eugeniaeEudrilus eugeniae (African Nightcrawlers) Malacad1, Magallanes1, Felizarte1, Fenete1, Guillermo1, Jaspe1, Jordan1, Legada1, Losañes1, Marañon1, Esteva1, Palmos1, Barrido1, Leong-on1* Volume 4 Issue 2, Year 2025 ISSN: 2833-1397 (Online) DOI: https://doi.org/10.54536/ajlsi.v4i2.5052 https://journals.e-palli.com/home/index.php/ajlsi Article Information ABSTRACT Received: April 12, 2025 Accepted: May 16, 2025 Published: June 25, 2025 Research is needed to determine the Avicennia marina hydroethanolic leaf extract is effective against adult E. eugeniae, despite research showing that it can eliminate helminths such as Pheretima posthuma. The purpose of this study was to ascertain the hydroethanolic leaf extracts of A. marina antihelmintic effectiveness against adult E. eugeniae in vitro. Using E. eugenie adults (6–8 cm in length and 0.2–0.3 cm in breadth), the percentage vermicidal activity of three concentrations of dried crude hydroethanolic leaf extract of A. marina, 25, 50, and 75 mg/mL of distilled water, was assessed every three hours for 12 hours. Five replicates, three trials, distilled water as a negative control, and mebendazole as a positive control were all examined. According to the findings, the hydroethanolic leaf extracts of A. marina have shown antihelmintic efficacy against E. eugenie adults in vitro at doses of 50 mg/mL and 75 mg/mL utilizing Kruskal-Wallis and LSD on the 3, 6, 9, and 12 hours, with a p-value of less than .05. In both concentrations, 50 and 75mg/ml, until the 12-hour exposure to the extract, there was no discernible difference between them. In vitro antihelmintic activity was best demonstrated at 50 mg/mL for 3 hours (mean = 91.1; sd = 26.6), and the in vitro antihelminthic activity was significantly superior to that of the positive control for the same duration (mean = 2.22; sd = 8.61). The results scientifically demonstrated the antihelmintic efficacy of hydroethanolic leaf extract of A. marina against E. eugeniae and provided the foundation for testing against Ascaris, Trichuris trichiura, and hookworms. Keywords Antihelmintic, Avicennia Marina, Eudrilus Eugeniae, Hydroethanolic Extract, Vermicidal Activity 1 Medical Laboratory Science, College of Pharmacy and Medical Technology, University of San Agustin, Iloilo City, Philippines * Corresponding author’s e-mail: mleongon@usa.edu.ph INTRODUCTION According to estimates, 1.5 billion people, or 24% of the world’s population, are affected by helminth infections, making it one of the most common infections in the world (World Health Organization, 2023). It is especially common in third-world countries, where poor sanitation and hygiene create the perfect conditions for parasitic helminth infection and extensive transmission. Between 33.8% to 75.9% of Filipinos were afflicted with soil- transmitted helminth (STH) infections in the Philippines, with schoolchildren being the most susceptible (Mationg et al., 2021). The roundworms Ascaris, Trichuris trichiura, and hookworms are the most frequent causes. Numerous health problems, such as stomach pain, diarrhea, loss of blood and protein, rectal prolapse, and physical and cognitive growth retardation, can be caused by significant helminthic infections in humans (CDC, 2022). Over the past few decades, numerous anthelmintic studies have been developed as a result of the increased incidence and prevalence of helminthic illnesses. Using roundworm species that are morphologically similar to parasitic species is one strategy. Ogen et al. (2017) proposed using the common soil earthworm Pheretima posthuma as test subjects because of its morphological similarity to intestinal roundworms. In the Philippines, E. eugeniae (African nightcrawlers) are especially common and make excellent subjects for anthelmintic research. Additionally, they are helminths, which and intestinal roundworms have comparable anatomy (Pueblos et al., 2015; Asiimwe et al., 2023; Hogade et al., 2014). Commercial medications such as Albendazole, Mebendazole, Levamisole, and Pyrantel Pamoate were frequently used to treat helminth infections in humans (Moser et al., 2017). Despite their effectiveness, these medications have a number of drawbacks when used over extended periods of time. In their research, Kotze et al. (2019) observed that helminth parasites have a propensity to become resistant to some drugs following prolonged therapy. Agricultural research, which focused on natural chemicals with anthelmintic action, was therefore very interested in novel natural alternatives. Other plants with comparable phytochemical components, including Leucaena leucocephala (Lead Tree), Carica papaya (Pawpaw), and Cayriata auriculata (ear-leafed Cayratia), have been the subject of numerous studies and have been shown to be effective anthelmintic agents (Kancherla et al., 2019; Widaad et al., 2022). In particular, the synergistic anthelmintic effects of flavonoids and tannins mostly functioned by making the nematode cuticle stiffer, which inhibited growth and encouraged death (Greiffer et al., 2022). The anthelmintic activity of A. marina is one of the plants that can be investigated. This kind of mangrove can be found in rivers, streams, coastal regions, and the intertidal zones of seabeds. For coastal barangays, it was a natural defense, particularly in remote, impoverished Pa ge 2 https://journals.e-palli.com/home/index.php/ajlsi Am. J. Life Sci. Innov. 4(2) 1-5, 2025 areas. The effectiveness of A. marina tree extracts against helminths, including the earthworm Pheretima posthuma, has been the subject of a limited number of studies (Kondepudi, 2018). Furthermore, previous research had shown that phytochemicals like phenolic compounds, alkaloids, flavonoids, sugars, carotenoids, aliphatic alcohols, amino acids, hydrocarbons, fatty acids, tannins, saponins, and terpenes were present (Wilfred et al., 2023). In support of this, Mughal et al. (2016) also found that A. marina contains phytochemicals proteins, amino acids, flavonoids, phenols, carbohydrates, and alkaloids. Accordingly, the phytochemical substances saponins, flavonoids, and tannins were nematicidal chemicals according to research by Wilfred et al. (2023) and Mughal et al. (2016); D’Addabbo et al. (2022). The use of A. marina leaf extract hydroethanolic against adult E. eugeniae has not been studied, despite the fact that it has been scientifically demonstrated to kill helminths like P. posthuma. The overall goal of this work was to ascertain the hydroethanolic leaf extracts of A.marina’s antihelmintic effectiveness against E. eugeniae in vitro. The study’s specific goal was to ascertain the hydroethanolic leaf extracts of A.marina’s in vitro antihelmintic activity against adults of E. eugeniae using varying concentrations (25, 50, and 75 mg/mL) and time (3, 6, 9, and 12 hours) in terms of percent vermicidal activity (Va) in comparison to the negative control, distilled water and the positive control, Mebendazole (25 mg/mL). In terms of percent vermicidal, the study identified the optimal concentrations (25, 50, and 75 mg/mL) and times (3, 6, 9, and 12 hours) that demonstrated the hydroethanolic leaf extracts of A. marina’s in vitro anthelmintic activity against E. eugeniae adults. The study’s findings gave A. marina’s vermicidal action against E.eugeniae a scientific foundation. MATERIALS AND METHODS A quantitative post-test, fully randomized approach was employed in the investigation. The % vermicidal (mortality) effects of A.marina at three different doses (25, 50, and 75 mg/mL) against E. eugeniae adults were evaluated. The study was reviewed by a private university’s recognized Ethics Review Committee. In addition, the study received biosafety and ethics clearance. E. eugeniae were gathered from a vermicomposting region in Antique Province, A. marina were gathered from the riverbanks in Dumangas, Iloilo. The test was carried out in Antique between February and April of 2024. Identification of Samples The pictures of trees, fruits, and plant leaves (Figure 1) were sent to the Department of Environment and Natural Resources for verification. This sample’s taxonomic classification is as follows: Kingdom: Plantae Phylum: Tracheophyta Class: Magnoliopsia Order: Lamiales Family: Acanthaceae Genus: Avicennia Species name: marina Common name: api-api or white/gray mangrove Figure 1: Images of the plant A. marina: A. yellowish leaf abaxial surface; B. edge of the leaf (adaxial surface); C. arrangement of leaves (opposite); D. roots (pneumatophores that resemble pencils); E. trunk of a tree (lenticels and smooth bark); F. The crown of a tree Figure 2: Photograph of A. marina whole plant Collection of plant material A. marina leaves were gathered in Dumangas, Iloilo. Samples of plants were gathered 100 meters from the road. The leaves were picked between 6:00 and 7:00 a.m. Leaves that were not budding or decaying were used. Leaf samples were free of insect bites, trauma, and irregularities. Collection was avoided within 3 days after rainfall in the area. Identification and Collection of E. eugeniae A veterinarian identified the worm. A veterinarian verified the authenticity of E. eugeniae worms that were gathered from vermicomposting facilities in the province of Antique. According to the research of Akande et al. (2018), the earthworms were measured to be 6–8 cm long and 0.2–0.3 cm wide. When the earthworms were collected, they were alive and moving. Following the Pa ge 3 https://journals.e-palli.com/home/index.php/ajlsi Am. J. Life Sci. Innov. 4(2) 1-5, 2025 confirmation of the roundworm species, the E. eugeniae worms were cleaned of dirt and debris using a standard saline solution. Preparation of A. marinaA. marina Extract Following collecting, the leaves were carefully cleaned with tap water and then rinsed with distilled water. The leaves were allowed to air dry at room temperature for an hour without exposure to sunlight in a screen bag. The leaves were allowed to air dry before being ground into a fine powder in a blender. One gram of powdered leaves was mixed with five milliliters of 95% ethanol for a duration of seventy-two hours in order to homogenize the leaves. After that, a muslin cloth was used to filter the homogenized leaves. The ethanol was then extracted from the filtrate using a rotary evaporator set to 45 °C. The filtrate was dried in an oven set to 45 °C in order to recover either the powdered or sticky form.To be sure there was no ethanol in the extract, a flame test was conducted. An anthelmintic test was performed on the dried, crude hydroethanolic extract against adult E. eugeniae. Ibrahim et al. (2022) modified this process and used it in their studies. Antihelmintic Assay Preparation The Kondepudi (2018) method was used to perform the anthelmintic assay. The ethanolic leaf extract was made into a stock solution at varying doses (25, 50, and 75 mg/ mL). A positive control solution containing 25 mg/mL Mebendazole and a negative control solution consisting of distilled water were made. Each disposable plastic petri dish with a perforated lid and a 5.9-inch diameter was filled with three adult E. eugeniae. Each treatment’s 5 mL solution is used to soak them. In accordance with the experiment carried out by Bestari et al. (2020), the percentage vermicidal of the helminth was measured at 3-hour intervals for 12 hours. Validity and Reliability A certified medical technologist verified the study’s findings. To ensure the study’s reliability, three concentrations—25 mg/mL, 50 mg/mL, and 75 mg/ mL—were used in five replicates with three trials. To guarantee the precision of the tests conducted, the devices were also appropriately calibrated. Waste Disposal The adult E. eugeniae samples, both dead and alive, were disposed of appropriately by filling the petri plates with a 10% hypochlorite solution and securing the arrangement with tape. To guarantee the organisms’ total demise, the sample was submerged for a whole day. After being put in the biohazard bag, the petri plates were disposed of in the proper garbage receptacle. Statistical Analysis The mean and SD of the results were displayed. Using varying quantities of A.marina hydroethanolic leaf extract, the study compared the mean results of vermicidal counts of E. eugeniae taken every three hours for 12 hours using the Kruskal-Wallis test. Statistical significance was defined as a p-value of 0.05. To determine whether there were any notable variations or differences between the treatments, a post-hoc Fisher’s least significant difference (LSD) test was also employed. RESULTS AND DISCUSSIONS Table 1 demonstrates that A. marina hydroethanolic leaf extracts at 50 and 75 mg/mL demonstrated antihelmintic efficacy against E. eugeniae. Vermicidal activity (Va) showed p< 0.05 for E. eugeniae adults on the 3, 6, 9, and 12 hours using Kruskal-Wallis and LSD. The antihelmintic effects of the 50 mg/mL and 75 mg/5mL doses of A. marina extract did not significantly change from the 3 to the 12 hour. There was no antihelmintic effect from the 25 mg/ ml. 50 mg/mL was the optimal dose and duration that demonstrated in vitro antihelmintic action for the three hours of exposure (mean = 91.1; sd = 26.6), and it was noticeably superior to the positive control during that period (mean = 2.22; sd = 8.61). Table 1: Anthelmintic activity of A. marina hydroethanolic leaf extract against E. eugeniae taken at different hours using different concentrations in terms of % vermicidal activity(Va) Treatments Time (hours) 3 6 9 12 %Va %Va %Va %Va m sd M sd m sd m Sd 25 mg/mL a8.89 15.4 a20a 24.6 a33.3a 30.9 a46.7a 35.2 50 mg/mL b91.1a 26.6 b93.3a 25.8 c95.6a 17.2 c97.8a 8.61 75 mg/mL b91.1 a 26.6 b93.3 a 25.8 c93.3 a 25.8 c100 a 0.0 Negative-Control (Distilled Water) a0.0 a 0.0 a0.0 a 0.0 a0.0 a 0.0 a15.6b 30.5 Positive-Control Mebendazole) 25 mg/mL a2.22 a 8.61 a6.67 a 13.8 b51.1 ab 39.6 b84.4ab 27.8 Note: letters before the numbers mean comparison with the treatments; while letter after number means comparison among the hours Pa ge 4 https://journals.e-palli.com/home/index.php/ajlsi Am. J. Life Sci. Innov. 4(2) 1-5, 2025 Discussions Significant anthelmintic activity was demonstrated by the hydroethanolic leaf extract of A. marina at concentrations of 50 and 75 mg/mL in terms of percent vermicidal. Significant anthelmintic action was demonstrated by concentrations of 50 mg/mL and 75 mg/mL in terms of percent vermicidal, with the peak activity occurring after 12 hours. Our results suggest that the phytochemical compounds present in A. marina, including alkaloids, flavonoids, sugars, carotenoids, aliphatic alcohols, amino acids, hydrocarbons, fatty acids, phenolic compounds, tannins, saponins, and terpenes, may have contributed to the anthelmintic activity (Wilfred et al., 2023; Mughal et al., 2016). Greiffer et al. (2022) found that flavonoids and tannins particularly inhibit helminth proliferation and cause helminth mortality. In addition to A. marina, several plants were reported to possess these chemicals, including L. leucocephala (Widaad et al., 2022). Because of its physical similarity to diseased helminths, E. eugeniae was used in the study as a reference for creating synthetic anthelmintic treatments. Notably, the strongest anthelmintic activity against E. eugeniae was obtained at concentrations of 50 mg/mL and 75 mg/mL. The results of Kondepudi’s (2018) investigation are consistent with the observed trend, which shows that higher concentrations exhibit stronger anthelmintic activity. The highest level of anthelmintic activity of A. marina was similarly seen at a concentration of 75 mg/mL to P. posthuma. As demonstrated by the notable impacts on mortality employing varying doses of hydroethanolic leaf extracts of A. marina, the results gave a scientific foundation for the vermicidal actions of A.marina against E. eugeniae. For future research on helminths or organisms that share a biological morphology with African nightcrawler worms, such as hookworms, Trichuris trichiura, and roundworms (Ascaris suum), a concentration of 50 mg/mL may be utilized as a reference. Given the persistently high incidence of helminth infections in the Philippines, the experimental findings are positive about the best use of readily available plants with anthelmintic qualities. The results show that natural products are reliable and strong enough to serve as a pointer to other possible sources of helminth infection treatment. This is especially true for higher concentration therapies. Furthermore, by placing more significance and focus on the product’s source, this may also have positive economic effects. Last but not least, our research may help address the ongoing concern about the spread of drug-resistant parasites (Fissiha & Kinde, 2021) by introducing a novel antidote that significantly slows the development of undesired resistance. Only the hydroethanolic leaf extract of A. marina at concentrations of 25 mg/mL, 50 mg/mL, and 75 mg/ mL and the anthelmintic assay method produced these results. Different results can be obtained by using a different extractant, concentration, technique, or part of the plant. CONCLUSION Comparing the extract concentrations at 50 mg/mL and 75 mg/mL to the negative control (distilled water), there was notable antihelmintic activity based on the percentage of vermicidal activity, from 3 to 12 hours. On the 3rd hour, the optimal concentration and duration of in vitro anthelmintic action were 50 mg/mL. On the 3rd hour, the extract doses of 50 mg/mL and 75 mg/mL exhibited similar vermicidal effectiveness to the positive control, Mebendazole. The results scientifically demonstrated the antihelmintic efficacy of hydroethanolic leaf extract of A. marina against E. eugeniae and provided the foundation for testing against Ascaris, Trichuris trichiura, and hookworms. Since the study demonstrated the plant’s effectiveness against E. eugenia in vitro, it is advised that a study on in vitro human helminths be carried out utilizing the crude ethanolic extract of A. marina leaves. The phytochemical of A. marina leaves should be used to identify or isolate the particular components that give it its anthelmintic effects. REFERENCES Akande, A. A., Aboaba, S. A., & Flamini, G. (2018). Constituents and anthelmintic activity Evaluation of Albizia adiantifolia (Schumach) W.F. Wright Essential Oils FromNigeria. International Journal of Chemistry, 10(2), 10. https://doi.org/10.5539/ijc.v10n2p10 Asiimwe, E., Ondari, E. N., Odongo, G. A., & Kimanje, R. K. (2023). 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