Essential oils of the leaves of Syzygium cumini (L.) Skeels and fruit peels of Hymenaea courbaril (L.) var. courbaril as molluscides against Biomphalaria glabrata Óleos essenciais das folhas de Syzygium cumini (L.) Skeels e cascas do fruto de Hymenaea courbaril (L.) var. courbaril como moluscicidas contra Biomphalaria glabrata Aceites esenciales de hojas de Syzygium cumini (L.) Skeels e Hymenaea courbaril (L.) var peels. courbaril como molusquicidas contra Biomphalaria glabrata

This study evaluated the molluscicidal activity of essential oils (EOs) of Syzygium cumini L. and Hymenaea courbaril L. Hydrodistillation was used to extract the EOs. The chemical characterization was performed by Gas Chromatography coupled to mass spectrometry. The bioassay of Artemia salina Leach was used for the toxicity test. The molluscicidal assay tested concentrations of 10-80 mg L of the EOs against Biomphalaria glabrata. The major constituent of The EO of S. cumini was isokaryophyllene and H. courbaril the Germacreno-D. The toxicity assay classified The EOs as nontoxic with LC50 412.10 mg L -1 for S. cumini and Research, Society and Development, v. 9, n. 10, e1239108215, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8215 3 LC50 354.80 mg L -1 for H. courbaril. The molluscicidal assay purchased LC50 44.76/ LC90 77.20 for S. cumini and LC50 37.34 mg L / LC90 73.24 mg L -1 for H. courbaril. The results showed that both EOs can be used as natural molluscicides in combating the snail Biomphalaria glabrata.


Introduction
Schistosomiasis, popularly known as schist, snail disease or water belly, is a parasitic disease caused by helminth Schistosoma mansoni that has as its definitive host man and intermediate the mollusc of the Biomphalaria glabrata (Rocha et al., 2013). Historically, the nature of parasite-host relationships has attracted much attention from the scientific community, especially parasites that affect human populations, given the medical interest in controlling their transmission (Gashaw et al., 2015).
The transmission of parasitosis occurs through the human count with contaminated water in places where there are molluscs of the genus Biomphalaria spp. infected with the verme fences (Alves, 2018). Biomphalaria spp., are pulmonary and hermaphrodite organisms, which inhabit freshwater collections. Known for more than 140 million years, these molluscs have survived various environmental pressures and currently occupy large territorial tracts inhabiting preferably lentic water regions. In general, they are found in several collections such as rivers, ponds, ditches, streams, reservoir banks or artificial collections ( Baron et al., 2013;Saúde, 2014;Standley, Goodacre, Wade, & Stothard, 2014).
According to WHO estimates, schistosomiasis is established in 76 countries, with an estimated 779 million people being at risk of infection and 207 million infected worldwide container, about 64 mg of Artemia salina cysts were added, taking care that they did not cross the partition. The part of the system containing artemia saline cysts was covered with aluminum foil, so that the organisms, at birth, were attracted by light on the other side of the system, forcing them to cross the partition. This procedure aims at homogenizing the physical conditions of the test organisms. Incubation was performed for a period of 48h. Throughout the test the temperature was monitored.
For the evaluation of the lethality of Artemia salina Leach, a stock saline solution of each EO was prepared at the concentration of 10,000 mg L -1 and 0.02 mg of Tween 80 (active tense). Aliquots of 5, 50 and 500 μL of this were transferred to test tubes and completed with saline solution previously prepared up to 5 mL, obtaining concentrations of 10, 100 and 1000 mg L -1 , respectively. All tests were performed in triplicates, where ten larvae in the nauplium phase were transferred to each of the test tubes.
For white control, 5 mL of saline solution was used for positive control K2Cr2O7 and for negative control 5 mL of a solution 4 mg L -1 of Tween 80. After 24 hours of exposure, the live larvae were counted, considering those that did not move during observation or with the slight agitation of the bottle. The criterion established by Dolabela(1997) was adopted to classify the toxicity of The EOs, being considered highly toxic when LC50 ≤ 80 mg L -1 , moderately t vbboxic to 80 mg L -1 ≤ LC50 ≥ 250 mg L -1 and mildly toxic or nontoxic when LC50 ≥ 250 mg L -1 .
The statistical analysis of the data for the LC50 was performed according to the method Reed&Muench (1938), from the table containing the mortality data for each concentration tested, a graph is constructed where a curve is observed for the accumulation of dead animals in each concentration log and another curve for the accumulation of survivors. The point of intercession between the curves is the Lethal Concentration 50% (LC50), because at this point the number of surviving animals is equal to the number of dead animals. The confidence interval was calculated according to method Pizzi (1950) in which a graph of the percentage of dead versus log of the concentration is constructed.

Total phenolics
The determination of the total phenolic compounds of the EOs was performed with adaptation of the Folin-Ciocalteu method (Waterhouse, 2002). 5 mg of the essential oil diluted in 1 mL of ethanol was used. To this solution was added 3 mL of distilled water, 500 μL of folin-ciocalteu reagent and 2.0 mL of sodium carbonate at 20%. The solution formed was Development, v. 9, n. 10, e1239108215, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8215 8 taken to the water bath at 50 ºC for 5 min, removed and left to cool; and then, the reading was performed in a manual spectrophotometer, in a length of 760 nm. The standard curve was expressed in mg L -1 of tannic acid.

Antioxidant activity
The determination of antioxidant activity was performed by the ABTS method [2,2azinobis-(3-ethylbenzothiazolin-6-sulfonic)], according to the methodology suggested by Re et al. (1999). The ABTS•+ radical was prepared by the 5.0 mL reaction of a 3840 μg mL -1 solution of ABTS with 88 μL of the 37,840 μg mL -1 potassium persulfate solution, the mixture was left in a dark environment for 16 hours. After radical formation, the mixture was diluted in ethanol until absorbance of 0.7±0.01 to 734 nm was obtained.
From the concentrations of The EOs (5 to 150 μg mL -1 ) the reaction mixture with the radical cation ABTS was prepared. In a dark environment, an aliquot of 30 μL of each concentration of The EO was transferred in test tubes containing 3.0 mL of the radical Cation ABTS and homogenized in a tube agitator and after 6 minutes the absorbance of the reaction mixture was performed in spectrophotometer in length of 734 nm.
The capture of the free radical was expressed as a percentage of inhibition (%I) of the radical cation ABTS according to Equation 1 (Babili et al., 2011), where ABSABTS represents the absorbance of the ABTS radical solution and ABSAM represents the absorbance of the sample.

Eq. 01
From the obtained data, the efficient concentrations IC50 and IC90 were calculated, defined as the concentration of the sample necessary to kidnap 50% of the ABTS radicals.

Obtaining and cultivating snails
Samples of snails of the species Biomphalaria glabrata were captured in rainy periods, in areas with low sanitation in the neighborhood Sá Viana, São Luís-MA. The collection technique was performed according to a proposal from Brazil(Epidemiológica, 2008) performing a scan with a shell in the submerged areas and the captured snails were Research, Society and Development, v. 9, n. 10, e1239108215, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx. doi.org/10.33448/rsd-v9i10.8215 9 collected in a glass container with lid, with water from the breeding site itself. Their search was carried out at various points in each breeding site, and then sent to the molluscum of the Laboratory of Research and Application of Essential Oils (LOEPAV/UFMA).
The snails were kept in the laboratory for 30 days and analyzed every 07 days to confirm the absence of Infection by Schistossoma mansoni. For this, 05 snails were placed in transparent glass containers with 25 mL of dechlorinated water, that is, 5 mL/snail, exposed to light (60 W lamps) for one hour with a distance of 30 cm to stimulate the release of the fences and taken to be analyzed, through visualization with the aid of a stereoscopic magnifying glass (8x), those that were parasitized (positive) were labeled and separated for future individual analysis and those who showed no signs of trematoid infection in the period of 30 days were selected for the molluscicidal activity test.

Evaluation of molluscicide activity
For the evaluation of molluscicide activity, the technique recommended by the World Health Organization(1983) was used, where two tests were performed in triplicate. In the first, called a pilot test, a solution of the oil under study was prepared in a volume of 500 mL at a concentration of 100 mg. L -1 and 0.15 mL of Tween 80 (active tense), where 10 adult snails were placed, negative for Schistossoma mansoni, obtaining at the end a ratio of 50 mL/snail and feeding them with hydroponic lettuce. They were exposed in the solution for 24 h, at room temperature, removed from the solution, washed twice with dechlorinated water, placed in a glass container containing 500 mL of dechlorinated water, feeding them with hydroponic lettuce and observed to every 24 hours for 4 days to assess mortality.
In the second test, called lethal concentration (LC50), solutions of each oil were prepared in a volume of 500 mL at concentrations 100, 75, 62.5, 50, 20, 10, 5 and 2 mg L -1 and 0.15 mL of Tween 80 (surfactant), using the same methodology of the pilot test. For the negative control, two tests were also used, in the first we placed 500 mL of dechlorinated water and 10 snails in a glass container and in the second 10 snails immersed in a solution with 0.15 mL of Tween 80 in 500 mL of distilled water, feeding both with hydroponic lettuce and the analysis also performed in the previous tests.
The lethal concentration LC90 of the bioassay was determined by linear regression, obtaining the concentration versus mortality ratio of molluscs (Colegate&Molyneux, 2007).
Mortality rates were obtained by averaging dead individuals as a function of the logarithm of Research, Society and Development, v. 9, n. 10, e1239108215, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx. doi.org/10.33448/rsd-v9i10.8215 10 the tested dose. The statistical analysis of the data for the LC50 was performed according to the Reed&Muench method (Reed&Muench, 1938). The confidence interval was calculated according to method Pizzi (1950) in which a graph of the percentage of dead versus log of the concentration is constructed.    Research, Society and Development, v. 9, n. 10, e1239108215, 2020 (CC BY 4. According to Pereira et al. (2007), the composition of the EO of a species of this plant is related to environmental aspects, such as: the vegetative cycle of the plant; the process of obtaining essential oil; the environment in which the species develops, the type of cultivation, temperature, relative humidity, the amount of water and nutrients in the terrain, among others.

Chemical constituents
This dependence on the composition of essential oil from environmental conditions can be   Source: Authors (2020). Research, Society and Development, v. 9, n. 10, e1239108215, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx. doi.org/10.33448/rsd-v9i10.8215 13 According to Table 2, the chemical composition of the EO of S. cumini leaves showed that the major components present were isokaryophyllene (18.01%), followed by naphthalene   Development, v. 9, n. 10, e1239108215, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8215 the Folin-Denis method, obtaining 335.0 mg EAT g -1 . Veggi et al. (2014) by analyzing the content of maximum total phenolic compounds in the extracts of the barks of H. courbaril, the quantification was observed in 335.00 mg of TAE / g of extract. Vecanto et al. (2016) found in the aqueous extract of the bark the value of 516.89 EAG g -1 . For the EO of S. cumini, the total phenolics were 578,453 mg EAT g -1 , according to Table 3. Thus, this study obtained superior results in relation to the analyzed one.

Antioxidant activity
The chemical complexity of EOs, usually a mixture of dozens of compounds with different functional groups, polarity and antioxidant activity that can lead to scattered results, depending on the test employed (Sacchetti et al., 2005), that's why we prefer to use ABTS and DPPH, widely used. Currently, there is no official method for determining antioxidant activity Research, Society and Development, v. 9, n. 10, e1239108215, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx. doi.org/10.33448/rsd-v9i10.8215 16 of plant origin and its derivatives due to the variety of antioxidant mechanisms that may occur and the diversity of bioactive compounds. The literature reports several methods for evaluating antioxidants, each with a different principle using free radicals and/or different patterns. Thus, studies aimed at evaluating the antioxidant properties of plant extracts need to use more than one method to accurately conclude that the extracts analyzed may also be able to combat the harmful effects of free radicals on the human body (Sousa et al., 2011).   antioxidants (Maisuthisakul et al., 2007). Syzygium sp. species are reported to be very rich in tannins, flavonoids, EOs, anthocyanins and other phenolic constituents (Reynertson et al., 2008;Sharma et al., 2003) . Fifteen polyphenols and two acylated flavonal glycosides have been isolated from leave S. cumini (L) Skeels (Mahmoud et al., 2001).
Antioxidant agents reduce oxidative stress one of the main mechanisms involved in the pathogenesis and progression of chronic diseases such as cancer, cardiovascular and inflammatory diseases (Krishnaiah et al., 2011). In this sense, the indications for popular use of H. courbaril bark to treat inflammatory processes, ulcers, arthritis and rheumatism (Fernandes et al., 2015;Jayaprakasam et al., 2007) seem to be linked to its antioxidant potential and its chemical constitution.

Toxicity
Lethality tests are performed in toxicological tests and the median lethal concentration (LC50) can be obtained, which indicates death in half of a sample (Bednarczuk et al., 2010).
Artemia salina is a microcrustacean used in fish feeding and is widely used in toxicological studies due to low cost and easy cultivation. Figure Research, Society and Development, v. 9, n. 10, e1239108215, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx. doi.org/10.33448/rsd-v9i10.8215  Toxicity studies, as in this study, are conducted as a way to evaluate or predict toxic effects on biological systems and the relative toxicity of compounds to the environment, through a previous ecotoxicology assay on living organisms (Silva et al., 2016). Several species should be used as test organisms, such as the microcrustacean Artemia sp., fish and plants. Because it is relatively simple in the laboratory and widely distributed, Artemia sp. has been widely used as an indicator in acute toxicity tests (Silva et al., 2016). Therefore, this bioassay occurs quickly, with low economic cost and high reproducibility (Nunes et al., 2006). The toxicity of plants used in traditional medicine is cited by Miao et al. (2009).
Toxicity in molluscicide s and larvicidal agents is explained by Luna et al. (2005) and insecticide by Uchida et al. (1965). The tests on Artemia salina are also used to express the safety of EOs or products of plant origin with molluscicidal activity, as it indicates the possibility of toxicity against non-target organisms, such as fish and small crustaceans, which occur in the same place as the snails (Carvalho, 2018). Although natural molluscicides are biodegradable, at certain concentrations, within the values required by who, their derivatives may present risks. For this reason, it is necessary to perform tests that evaluate the toxicities of potentially molluscicide plants (Santos et al., 2007;Luna et al., 2005).

Molluscicidal activity
The death of the snails by molluscicide was evidenced by the retraction of the cephalopodal mass into the shell, release of hemolymph or a swelling with consequent prolongation of its cephalopodal mass out of the shell due to the breakdown of osmotic balance that is under neurohormonal control (McCullough et al., 1980). The results obtained for action of the EOs are presented in Table 6 for H. courbaril and Table 7 for S. cumini.