Molluscicidal Activity of the Crude Extract and Fractions of Myrsine parvifolia

Abstract

As the second most common infectious parasitic disease in the world, schistosomiasis is present in Brazil, mainly in the Northeast and Southeast regions. Currently, the recommended form of prevention is controlling this disease’s intermediate host, mollusks of the Biomphalaria genus, using the chemical molluscicide Bayluscide WP 70^®. This synthetic molluscicide is expensive, has low selectivity for Biomphalaria glabrata species, and is toxic to the environment. In this context, the use of natural products such as molluscicides represents a sustainable control method. The objective of this study was to evaluate the molluscicidal effects of a crude ethanolic extract of the leaves and stems and fractions of the species Myrsine parvifolia on the mollusk Biomphalaria glabrata. Methods: The methodology was adapted from standards defined by the World Health Organization (WHO), where the molluscicidal activity of Myrsine parvifolia was investigated as an alternative for the population control of Biomphalaria glabrata and That’s right. environmental toxicity was evaluated using the Physella sp. The results revealed that the stem ethanolic crude extract exhibited activity after 24 h of exposure, with an LC₅₀ of 207.4 mg/L and an LC₉₀ of 256.2 mg/L. Conclusions: Myrsine parvifolia can be used as a sustainable biological alternative for the population control of Biomphalaria glabrata snails, especially for poor populations and inaccessible regions.

1. Introduction

Schistosomiasis is a parasitic infection caused by trematodes of the genus Schistosoma, which affects approximately 240 million people worldwide, with more than 700 million living in areas considered at risk for disease transmission [1,2]. The species Schistosoma mansoni, responsible for the hepatointestinal form of schistosomiasis, represents a serious threat to public health, putting at risk approximately 780 million people in endemic regions, such as sub-Saharan Africa, the Middle East, the Caribbean, Brazil, Venezuela, and Suriname [3,4,5,6]. In the Americas, Brazil has the largest endemic area on the continent, with predominance in the states of the Northeast and Southeast regions [3,4]. The migration of individuals from endemic areas, frequently infected by S. mansoni, to regions previously free of the disease has contributed to its reintroduction and dissemination, especially in contexts of socioeconomic vulnerability [3,4].

Infection occurs in aquatic environments through the penetration of the active cercariae of Schistosoma mansoni into human skin. Cercariae are fork-tailed larval forms that emerge from intermediate host mollusks of the genus Biomphalaria, including Biomphalaria tenagophila, Biomphalaria straminea, and Biomphalaria glabrata. Among these, the species B. glabrata stands out for its greater susceptibility to infection and wide distribution. One way to control this disease is to manage these mollusks, thus interrupting the parasite’s transmission cycle [7].

Currently, the standard molluscicide on the market is the synthetic drug Bayluscide WP 70^®, which has a high cost, low selectivity, and is highly environmentally toxic [8,9]. Therefore, discovering and developing new molluscicides with high effectiveness and reduced toxicity is necessary.

Plant tests have been explored as biotechnological and sustainable alternatives for disease control, as natural products are biodegradable, more accessible, and less aggressive to the environment [9,10]. Arpairin et al. (2024) [11] investigated the effects of extracts of symbiotic bacteria, such as P. laumondii, which induced histopathological changes in Biomphalaria glabrata, an intermediate host of schistosomiasis. In addition, Araújo et al. (2024) [12] evaluated the use of silver nanoparticles associated with Croton urucurana plants, which resulted in mortality, developmental inhibition, and morphological changes in the same hosts. These approaches reveal different natural and sustainable alternatives for the control of the intermediate host of schistosomiasis.

The present study investigated several species of Myrsine parvifolia, which is a plant known for its diverse biological activities [13,14]. M. parvifolia, a native shrub of the Primulaceae (Myrsinaceae) family, is commonly called “capororoca” or “pororoca”. Previous research has highlighted its anti-inflammatory and antivenom properties [13]. In 2017, Corrêa et al. [13] reported the presence of sesquiterpene compounds in the essential oils of the aerial parts of this species, with β-caryophyllene as the predominant compound. Additionally, Côrrea et al. (2019) [14] reported that flavonoids are the main metabolic class found in the genus Myrsine. The chemical compounds were identified via chromatographic and spectroscopy techniques. The analysis of the crude ethanolic extract of M. parvifolia leaves revealed the presence of phenolic compounds, tannins, and flavonoids.

We evaluated the molluscicidal activity of the Myrsine parvifolia species using the ethanolic crude extract of the leaves and stems and the fractions in dichloromethane, ethyl acetate, butanol, and hexane obtained from the stem.

2. Materials and Methods

2.1. Plant Collect

The stems of M. parvifolia were collected in the state of Parque Nacional da Restinga de Jurubatiba, Carapebus, Rio de Janeiro, in May 2013. The plant material was collected with authorization for scientific activities (13659-2) granted by IBAMA/SISBIO. The botanist, Dr. Marcelo Guerra Santos, identified the plant species, and a specimen was deposited in the Herbarium of the Teacher Training College of the State University of Rio de Janeiro under the registration M.G. Santos 2253.

2.2. Obtention of the Extracts and Fractions for the Bioassay

The stems of M. parvifolia (1200 g) were dried in an oven with forced ventilation, pulverized in a knife mill, and subjected to an exhaustive extraction process with 96% ethanol using the static maceration method. The plant material was filtered, and the solvent was removed on a rotary evaporator under reduced pressure, thus obtaining the crude ethanolic extract (200 g) at an approximately 16.6% yield. The resulting crude ethanolic extract of the stems was subjected to liquid-liquid partitioning with solvents of increasing polarity, resulting in fractions of hexane, dichloromethane, ethyl acetate, and butanol partitions. The obtained fractions were stored in a desiccator in a freezer until use.

2.3. Molluscicidal Activity Assay with Specie Biomphalaria Glabrata

The molluscicidal assay was performed according to the methodology adapted from the World Health Organization (WHO) [15] using a 24-well plate as described by Santos et al. (2017) [16] and B. glabrata (10–12 mm) (coordinates S 22°52′33′′ and W 43°14′46′′), which was created in a laboratory and free from infection. In this work, 3 mollusks were used per test on at least three different days [16].

The crude ethanol extract and its fractions were tested in aqueous solutions at 100 mg/L, 150 mg/L, 200 mg/L, 250 mg/L, and 300 mg/L. As a positive control, we used Bayluscide WP 70^® at 1 mg/L, and as a negative control, we used distilled water and 1% DMSO. The final volume for the tests and controls was 2 mL per well. Mollusk mortality was observed at 6 h, 24 h, and 48 h after the beginning of the experiment. The absence of the retraction of the mollusks into their shells and the release of hemolymph were the criteria used to verify the death of the mollusks [17].

2.4. Molluscicidal Activity Assay with the Specie Physella sp.

The molluscicidal assay was performed according to the methodology adapted from the WHO [15] using a 24-well plate as described by Santos et al. (2017) [16] and Physella sp. (7–9 mm) (coordinates S 22°52′45″ and W 43°14′45″). This species was collected in drainage channels at Fundação Oswaldo Cruz—Fiocruz, Manguinhos campus, Rio de Janeiro, RJ. In this work, 3 mollusks were used per test on at least 3 different days [16].

The crude ethanolic extract and its fractions were tested in aqueous solutions at concentrations of 60 mg/L, 80 mg/L, 100 mg/L, 120 mg/L, and 250 mg/L. As positive controls, we used Bayluscide WP 70^® at a concentration of 1 mg/L, and distilled water and 1% DMSO were used as negative controls. The final volume for the tests and controls was 2 mL per well. Mollusk mortality was observed at 6 h, 24 h, and 48 h after the beginning of the experiment. The absence of the retraction of the mollusks into their shells and the release of hemolymph were the criteria used to verify the death of the mollusks [17].

2.5. Statistical Analysis

Statistical analysis of the data from the experiments was performed using the Prism 8 GraphPad program (GraphPad software 8.0) and two-way ANOVA, with a significance level of p < 0.0001 compared to the negative control (H₂O). Lethal concentrations (LC₅₀ and LC₉₀) were calculated using the Statgraphics Program 19.5.01.

3. Results

The crude ethanolic extract obtained from the stem of M. parvifolia showed molluscicidal activity of more than 50% when using 200 mg/L, reaching 100% lethality at a concentration of 250 mg/L after 48 h and 300 mg/L after 24 h of exposure (Figure 1 and Figure 2). At concentrations below 150 mg/L, the crude extract showed no molluscicidal activity. At the end of the test, the crude extract presented the following lethal concentrations: an LC₅₀ of 207.4 mg/L and an LC₉₀ of 256.2 mg/L (

Table 1. Lethal concentrations of the mollusk Biomphalaria glabrata after 24 and 48 h of exposure.

Substance	Time (h)	LC50 (mg/L)	LC90 (mg/L)
Crude ethanolic extract	24	223.6	281.5
	48	207.4	256.2
Niclosamide	48	0.06	0.2

).

The fractions in hexane, dichloromethane, ethyl acetate, and butanol had no molluscicidal effects on adult B. glabrata mollusks (10–12 mm) after 48 h at a concentration of 300 mg/L. Bayluscide WP 70^® treatment at a concentration of 1 mg/L caused 100% mortality within 24 h (Figure 3).

The crude ethanolic extract of M. parvifolia was 100% lethal at a concentration of 250 mg/L (Figure 4 and Figure 5). The lethal concentrations obtained were an LC₅₀ value of 118 mg/L and an LC₉₀ value of 136.5 mg/L for Physella sp. mollusks after 48 h of exposure (

Table 2. Lethal concentrations after 48 h of exposure to the mollusk Physella sp.

Substance	Time (h)	LC50 (mg/L)	LC90 (mg/L)
Crude ethanolic extract	48	118	136.5
Niclosamide	24	0.106	0.2

). Bayluscide WP 70^® treatment at a concentration of 1 mg/L caused 100% mortality within 48 h.

4. Discussion

The World Health Organization (WHO) recommends that molluscicide product testing be conducted in 250 to 500 mL vials. However, this standard approach can limit the evaluation of a wide range of natural and synthetic drugs, as many of these substances require large quantities of plant or synthetic materials. In contrast, the present study adopted the methodology described by Santos et al. (2017) [16] and utilized 24-well plates for testing. This alternative approach offers several advantages. Notably, the methodology of using 24-well plates reduces the volume of the substance required for testing from 250 to 500 mL to just 2 mL per test, making this method more efficient in terms of material use and cost. Additionally, the number of animals required for the tests is considerably lower; only three mollusks are used per concentration compared to the ten typically needed in traditional testing methods. This not only represents considerable savings in resources but also minimizes the ethical impact by reducing the number of animals used in research, aligning with the principles of sustainability and ethical responsibility in scientific experimentation [16].

Compared with other molluscicides, Bayluscide WP 70^® requires relatively low concentrations to effectively exert molluscicidal activity. However, niclosamide, which is the active ingredient of Bayluscide WP 70^®, remains highly toxic to the environment and is particularly harmful to nontarget species. The environmental risks associated with niclosamide have become more concerning over time, especially considering its prolonged use over more than five decades. This extensive use has not only raised ecological concerns but has also led to the development of resistance in mollusk populations, particularly Biomphalaria species exposed to Bayluscide WP 70^®. This growing resistance emphasizes the need to explore alternative control strategies, particularly the use of natural products. Natural molluscicides may offer a more sustainable solution, with reduced environmental toxicity and the potential to circumvent resistance mechanisms, as observed for synthetic chemicals such as Bayluscide WP 70^® [12,18].

Plants exhibit a variety of biological activities, including molluscicidal properties, and offer significant advantages, such as biodegradability, low cost, and ease of application [19,20]. These characteristics make them promising alternatives for the control of mollusk populations. The present study demonstrated the molluscicidal activity of Myrsine parvifolia on Biomphalaria glabrata through extracts obtained from plant stems. While the dichloromethane, ethyl acetate, butanol, and hexane fractions did not show molluscicidal activity, the crude ethanolic extract, which retained all the compounds from the individual fractions, exhibited a potent molluscicidal effect. These findings suggest that the combined presence of these compounds increases the overall molluscicidal activity of the extract.

Although molluscicidal activity was observed, the specific compound responsible for this effect could not be identified. This emphasizes the need for further studies to isolate and characterize the active compounds involved. Among the metabolic classes found in Myrsine species, flavonoids are of particular interest. These compounds may have induced cell membrane disruption and a reduced heart rate in the snail, similar to the effects observed in other snail species. For example, flavonoids in Lymnaea stagnalis and Cornu aspersum have been shown to interfere with synaptic communication, suggesting a possible mechanism of action that may also be at play in B. glabrata [17].

In a related study, Ibrahim et al. (2018) [21] evaluated the molluscicidal effects of the aqueous extract of Anagallis arvensis (family Myrsinaceae) on Biomphalaria alexandrina, which is a species of the genus Biomphalaria. The results revealed that the aqueous extract significantly reduced the survival and reproduction rates of B. alexandrina, with an LC₅₀ of 37.9 mg/L and an LC₉₀ of 48.3 mg/L. Histopathological changes, including the degeneration of eggs and sperm, were also observed in the reproductive glands of mollusks exposed to sublethal concentrations of the extract. These findings, together with those of the present study on Myrsine parvifolia, highlight the potential of Myrsinaceae plants in mollusk population control. Despite the differences in the chemical compositions of Myrsine parvifolia and Anagallis arvensis, both species exhibit molluscicidal properties, suggesting that the Myrsinaceae family may contain several bioactive compounds with potential for mollusk control. This correlation highlights the importance of exploring the diverse phytochemical profiles of this family further to better understand the mechanisms underlying their molluscicidal activity and identify the most effective compounds for practical applications.

Thoa and Son (2024) [22] reported the isolation of 134 substances from extracts of Myrsine species, highlighting the diverse chemical compositions of plants. The majority of the compounds identified were flavonoids, with 58 distinct chemical structures described. In addition to flavonoids, other significant chemical classes were identified, including mono-phenols (23 compounds), terpenoids (5 compounds), steroids (5 compounds), saponins (11 compounds), quinones (8 compounds), megastigotes (8 compounds), lignans (6 compounds), benzofurans (3 compounds), chromanes (1 compound), anthraquinone (2 compounds), phthalides (1 compound), and naphthols (1 compound). This rich chemical profile underscores the substantial phytochemical diversity among Myrsine species and opens avenues for further exploration of their bioactive potential.

Similarly, in an earlier study by our group, Correa et al. (2019) [14] focused on the chemical composition of Myrsine parvifolia, further contributing to the understanding of the bioactive properties of Myrsine species. In this study, the extract of Myrsine parvifolia contained a variety of flavonoids, including myricetin, myricetin-3-O-β-arabinopyranoside, quercetin, and kaempferol. Additionally, compounds such as γ-tocopherol (vitamin E), lupenone, lupeol, and various fatty acid esters were identified. These findings align with those of Thoa and Son (2024) [22] reinforce the importance of flavonoids and other bioactive compounds in Myrsine species. The presence of these compounds suggests that Myrsine parvifolia, like other Myrsine species, could possess significant antioxidant and anti-inflammatory properties, providing further justification for the continued investigation of both its individual and synergistic bioactivity.

Abou El-Nour (2021) [23] evaluated the molluscicidal activity of Origanum majorana, Salvia fruticosa, and Ziziphus spina-Christi, which are species belonging to the Lamiaceae and Rhamnaceae families, respectively, and reported the following lethal concentrations 90 (LC₉₀): Origanum majorana, LC₉₀: 172,000 mg/L; Ziziphus spina-Christi, LC₉₀: 374,000 mg/L; and Salvia fruticosa, LC₉₀: 315,000 mg/L. These results indicate the need for relatively high concentrations to achieve the molluscicidal effect. However, the present study demonstrated molluscicidal activity at considerably lower concentrations, which can be attributed to the chemical constituents that were present and the synergistic effect of the compounds in the crude ethanolic extract of Myrsine parvifolia (LC₅₀ = 207.4 mg/L and LC₉₀ = 256.2 mg/L). According to Hostettmann et al. (1982) [24], when classifying molluscicidal plant species as active or inactive, those with a 90% lethal concentration (LC₉₀) below 400 mg/L are considered bioactive. Thus, the results obtained for Myrsine parvifolia can be considered bioactive since its lethal concentrations are below the established limit, reinforcing the efficacy of the extract tested in the present study.

Other plant species belonging to the Primulaceae (Myrsinaceae) family, such as Anagallis arvensis (aqueous extract of leaves) [25], Maesa lanceolata [26], and Cyclamen purpurascens [27], have already demonstrated molluscicidal activity against mollusks of the genus Biomphalaria. However, our study reinforces the need for further investigations involving different stages of the parasite and host life cycle. Future studies should include the evaluation of the effects of the extracts on young mollusks, embryos, and cercariae, in order to understand in greater depth the mechanisms of action throughout the different stages of the disease.

A concentration of 1 mg/L Bayluscide WP 70^® had a comparable effect on B. glabrata survival compared to that of the 250 mg/L M. parvifolia extract. At this concentration of the M. parvifolia extract, we observed 100% P. acuta death. This is a warning against the use of this extract, and new formulations may be necessary to reduce undesirable effects. Additionally, Bayluscide WP 70^® also exhibited potent toxic effects on P. acuta, as expected [28,29].

5. Conclusions

We conclude that the present study demonstrated that Myrsine parvifolia has potential as a molluscicide for controlling the population of Biomphalaria glabrata, the intermediate host of schistosomiasis, given the mortality caused by low concentrations of its crude extract. Importantly, its lethal concentration can potentially be reduced by the use of pharmaceutical alternatives as new formulations. Furthermore, additional tests are needed to assess its activity on other stages of this mollusk, as well as on Schistosoma mansoni cercariae.

We also emphasize the importance of conducting field trials and further studies on the rich diversity of plants in the country to explore their full potential.