Larvicidal activity of hydroalcoholic extracts of Persea americana Mill. Seeds against Aedes aegypti Atividade larvicida dos extratos hidroalcóolicos das sementes de Persea americana Mill. frente Aedes aegypti Actividad larvicida de extractos hidroalcohólicos de semillas de Persea americana Mill. delantero Aedes aegypti

This study aimed to evaluate the phytochemical composition of hydroalcoholic extracts from the seeds of Persea americana (avocado) and evaluate the larvicidal activity against Aedes aegypti. The extracts were obtained by maceration in a 70% PA ethanol extractant solvent in 1:4/1:6/1:8 hydromodules (7 days), with subsequent concentration in a rotaevaporator. The larvicidal activity was performed by the method recommended by the WHO Lethal Concentration 50% against Aedes aegypti larvae with statistics by the Probit method. Toxicity to non-target organisms was verified by the Artemia salina test. Activity was observed against Aedes aegypti larvae with LC50 ranging from 181.72-401.96 mg L-1 with a 95% confidence interval. Finally, it states that the extract of P. americana has potential for larvicidal activity and does not present toxicity to target organisms, showing itself to be a sustainable alternative for the control and combat of Aedes aegypti larvae.


Introduction
The Aedes mosquito is a vector that transmits arboviruses, has a wide geographic distribution and its expansion is influenced by several factors, including: its behavior, the large size of the urban population, the precariousness of the good proportion of households in relation to the water supply, inadequate disposal of waste, the increase in the number of nonbiodegradable containers and inadequate disposal of solid waste. Arboviruses are still considered infections with a great impact on public health, and their prevention and control is one of the main challenges (Moyses&Bonatti, 2021).
The re-emergence of these diseases caused by arboviruses and transmitted by mosquitoes is observed, with emphasis on Dengue, Chikungunya Fever, Yellow Fever and Zika Fever in several countries in the Americas and in other tropical regions. The entry of these viruses in Brazil, a country already endemic for Dengue, represents a major challenge for public health, as they are all susceptible to infections and there are still no specific antivirals, nor vaccines for prevention free from side effects (Viana et al., 2018 ).
In the absence of dengue drugs and the variable safety and efficacy of the dengue vaccine, Dengvaxia (Thomas&Yoon., 2019), vector control remains the mainstay of Dengue control programs (Chang et al., 2011). Several plants have been used by humanity on all continents to control different diseases and pests, in addition to representing an important source of biologically active natural products, many of which constitute a model for the synthesis of a large number of drugs (Simões et al. 2017). Therefore, the search for new larvicides from natural resources has always been the criterion for finding alternatives for mosquito control. The advantages of natural insecticides are non-polluting to the environment and are safe for human health. Several natural products have been shown to have mosquito repellent, larvicides, pupicide and ovicidal activities (Shaalan et al., 2005;Nerio et al., 2009;Pavela, 2015;Kishore et al., 2011;Ghosh et al., 2012).
Within this, the search for new pesticides from natural resources has been the criterion for finding alternatives for the control of arbovirus vectors. The advantages of natural biolarvicides include the fact that they are environmentally friendly and safe for human health. Thus, natural products have shown potential in the action of controlling and combating larvicide, pupicide and ovicide (Shaalan et al., 2005;Ghosh et al., 2012;Nerio et al., 2010;Kishore et al., 2011;Pavela, 2015).
Several plants, despite being consumed as functional foods and/or nutraceuticals, are used for bioprospective research, which means the search for chemical products with biological or pharmacological properties that can be used to treat various diseases. Research, Society andDevelopment, v. 10, n. 10, e89101018144, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18144 3 Among these, stands out Persea americana Mill. (Lauraceae), being an evergreen tree found and cultivated in the tropical and subtropical region of the world. It's called the avocado fruit and it's edible. In traditional medicine, the plant is reported for a variety of diseases such as heart, skin problems, hypertension, diabetes, bronchitis and diarrhea (Yasir et al., 2010;Dabas et al., 2013). Therefore, considering the importance of finding natural biolarvicides, this study aimed to evaluate the larvicidal activity of hydroalcoholic extracts from P. americana seeds against Aedes aegypti.

Botanical material
Fruits of Persea americana were collected from a site located in the Ipem Turu, in the municipality of São Luis, MA in November 2020. These were dried at 40ºC in an air-circulation oven and pulverized in a knife mill with moderately coarse powder particle size. . Exsiccates were made with flowering branches and sent to the Ático Seabra Herbarium, at the Center for Biological and Health Sciences at the Federal University of Maranhão, for confirmation of their botanical identification.

Obtaining hydroalcoholic extracts
To obtain the hydroalcoholic extracts, the maceration technique was used using an ethanol extracting solvent 70% PA in hydromodules of 1:4, 1:6 and 1:8 (m/v), in 70% ethanol. The maceration will be carried out for 10 days, under agitation and filtration ( Figure 1). The resulting extracts, observed in Figure 1, were concentrated in a rotaevaporator ( Figure 2) and stored in a refrigerator at 4ºC in amber colored flasks (Matos, 2009). Research, Society and Development, v. 10, n. 10, e89101018144, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18144 The extracts yields were calculated by gravimetric technique and the physicochemical parameters: density and refractive index were determined according to the techniques recommended by the Brazilian Pharmacopoeia (2019).

Phytochemical analysis
The extracts were subjected to chemical tests based on the methodology presented by Matos (2009). The tests performed were: Salkowsk test (steroids), Mayer test (alkaloids), flavonoids, glycosides, saponins test, keller kiliani test (cardiac glycosides), ferric chloride test (phenols) and lead acetate test ( tannins). And the total phenolic content was quantified through the spectrophotometric method of Folin-Ciocalteau and the total flavonoid through the spectrophotometric method of complexation with aluminum (Lugasi et al., 1998;Oliveira et al., 2009).

Collection of Aedes aegypti eggs
The collection of Aedes aegypti eggs was carried out using traps called ovitraps, with the aid of Eucatex palettes. The Eucatex palettes were properly inspected, sanitized and dried before setting up the traps. The traps were installed at various points on the campus of the Universidade Federal do Maranhão, Recanto dos Vinhais (São Luís, MA) and Alemanha (São Luís, MA) under shelter from the sun and rain. The collected eggs were hatched in mineral water and fed until the stage where the experiments were carried out.

Larvicide activity against Aedes aegypti
The tests for larvicidal activity were carried out according to the adapted methodology proposed by Silva (2006).
Initially, a 500 mg L -1 stock solution of each of the hydroalcoholic extracts was prepared and diluted in a 2% Tween 80 solution. From this solution, serial dilutions were prepared at concentrations 100-400 mg L -1 . At each concentration, 10 larvae were added at the rate of 2 ml per larva. After 24 hours the count of live and dead was carried out, and larvae that did not react to touch after 24 hours of the Research, Society and Development, v. 10, n. 10, e89101018144, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18144 5 beginning of the experiment were considered dead. To quantify the efficiency of the extracts, the Probit statistical test (Finney, 1952) was applied.

Toxicity assessment against Artemia salina
This test was performed according to the methodology described by Meyer et al. (1982). To assess the lethality of Artemia salina Leach, a stock saline solution of each hydroalcoholic extract was prepared at a concentration of 10,000 mg L -1 and 0.02 mg of Tween 80 (active tension). Aliquots of this were transferred to test tubes and supplemented with saline solution previously prepared up to 5 mL, obtaining in the end concentrations of 1000-10 mg L -1 , respectively, where ten larvae in the nauplial stage were transferred to each of the tubes of rehearsal.
For the blank, 5 mL of saline solution was used, for the positive control K2Cr2O7 and for the negative control 5 mL of a 4 mg L -1 solution of Tween 80. After 24 hours of exposure, the count of live larvae was performed , considering dead those that did not move during the observation or with the slight shaking of the flask.
The statistical analysis of the data for the toxicity test was carried out according to the method of Reed&Muench (1938). The intersection point between the curves is the 50% Lethal Concentration (LC50), since at this point the number of surviving animals is equal to the number of dead animals (Colegate;Molyneux, 2007).

Phytochemical parameters and phytochemical screening
The physicochemical parameters are important for determining aspects of biological applications and are presented in Table 1. In a study conducted by Ferrari (2015) evaluating the oil refraction index of P. americana seed, it verified a variation of 1.4620 to 1.4625 nD 25°. In another analysis by Jorge (2014) of the pulp oil of this species, 1.4615 nD 25°. Results close to the one found in this search can be found. There is a scarcity of data in the literature regarding the density of extracts from the studied vegetable.
The classes of secondary metabolites identified in the extracts obtained from Persea americana are shown in Table 2. Through the phytochemical study, the following metabolites were identified in the researched extracts: steroids, flavonoids, glycosides, cardiac glycosides, saponins, phenols and tannins, presented in Table 2   Similar results were described by Rotta et al. (2016) when quantifying the content of phenolics in dehydrated P.
americana bark and resulted in a quantity of 108.48 ± 1.62 mg GAE g -1 ). In another study by Athayde et al. (2019) with hydroalcoholic extracts of P. americana seeds, the authors verified a total phenolic content quantified in 366.79 ± 5.05 mg EAG g -1 . Table 4 shows the total flavonoid content identified in the extracts of P. americana. Similar results were also evidenced by Rotta et al. (2016) the flavonoid content of 136.034 ± 1.88 mg EQ g -1 in Research, Society andDevelopment, v. 10, n. 10, e89101018144, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18144 7 dehydrated bark of P. americana, there is a result well above that identified in our study. Athayde et al. (2019) when evaluating P. americana seed extracts recorded 28.09 ± 0.64 mg EQ g -1 . Gobbo-Neto et al. (2007) claim that there are factors that influence the content of secondary metabolites, among them, one of the most relevant is the period of the year in which the vegetable was collected. Another important factor is the age and development of the plant, as well as the temperature of its habitat, the water supply of the soil in which the plant is grown, exposure to ultraviolet radiation, rainfall and altitude. Therefore, these causes can cause a variation of secondary metabolites in plants.  Note: a LC50 was calculated by Reed&Muench (1938); b 95% confidence interval; no dead larvae in the negative control, composed of 4 mg L -1 Tween 80; the positive control, K2Cr2O7, had 100% larval mortality; c Dolabela (1997). Source: Authors (2021).

Lethality against Artemia salina Leach.
The relationship between the degree of toxicity and the mean lethal concentration (LC50) presented for medicinal plant extracts on Artemia salina larvae was established by the criteria adopted by Dolabela (1997) for classification of the toxicity of natural products. As observed in Table 5, all extracts obtained presented LC50 above 250 mg L -1 , thus being defined as nontoxic, and their application was encouraged.
The nontoxicity profile found in this study is confirmed by Valente (2020)  Persea americana species, where the LC50 found was 204.95 mg L -1 . Therefore, it is important to emphasize the use of seed extract in practical applications, aiming at its atoxicity potential, inferring its selectivity to target organisms. Table 6 presents the results regarding mortality Aedes aegypti against the action of the hydroalcoholic extract of the seeds of P. americana. Note: a LC50 was calculated by Probit analysis (Finney, 1952); b 95% confidence interval; no dead larvae in the negative control, composed of 2% DMSO solution; the positive control, 1 mg L -1 temephos, had 100% larval mortality. Source: Authors (2021).

Lethality against Aedes aegypti
As shown in Table 6, it is observed that the lowest LC50 was observed for EHPA1:4, quantified at 181.72 mg L -1 .
However, an action similar to EHPA1:8 is observed with a statistically similar LC50 of 181.76 mg L -1 .
To analyze the efficiency of larvicidal activity of natural products, some studies suggest some criteria, as there is still no standard concentration range for determining the efficiencies of natural products. It should be considered that these criteria are related to the time of exposure to the bioproducts and the origin of the larvae, which can change the LC50 values.
According to Komalamisra et al. (2005), LC50 between 100 and 750 mg L -1 are effective, up to 48 hours of exposure. Thus, the LC50 obtained for P. americana seed extracts obtained in this study can be correlated to effective alternatives in the control of Aedes aegypti.
The larvicidal action observed was also described in the study by Torres et al. (2014) in locations in the Philippines, in which the authors used two extracting solvents (ethanol and hexane), and LC50 was observed in P. americana seed extracts, respectively, at intervals from 16.48 mg L -1 to 20.61 mg L -1 and 9.82 mg L -1 to 13.39 mg L -1 . Already in larvicidal trials performed by Agrela et al. (2014), in which methanol was adopted as solvent in P. americana seed extracts, the LC50 were recorded for Rockerfeller and Mario Briceno Iragorry strains, respectively, 5.7 mg L -1 and 9.9 mg L -1 . In a research carried out by Louis et al. (2020), in which the rind of the fruit of the same plant species was subjected to methanol extract, the LC50 detected for 3rd stage larvae was 7.12 mg L -1 .
Comparing with the LC50 results identified in the present study, it is noted that larvicidal activity was evidenced, however with a lower lethality potential, requiring a higher extract concentration to obtain the death of 50% of the larvae.
It is noteworthy that the results obtained with plant extracts may vary according to the period of collection, temperature, type of soil cultivated, plant species and others. For this reason, the isolation of the active compound from extracts is essential for standardization as a method of larval control (Gupta et al., 2011).
The solvent used can directly contribute to the variation of larvicidal activity, since it has been shown that the extraction of active biochemicals from plants requires the polarity of the solvents used (Ghost et al., 2012). The favorable results obtained encourage the continuation of the study, aiming at the isolation of the active principle and, mainly, ways that enable its practical use for the control of Aedes aegypti.
It was not yet possible through the study to specify which component or which components are responsible for larvicidal activity, because the isolation of such has not yet been verified. It is also necessary to perform the chemical characterization by chromatographic techniques of the extracts studied. Thus, it will be feasible to identify the metabolites to be attributed such larvicide potentials, being possible to generate bioproducts with bioprospecting activity for development of larvicide agents of plant origin.

Conclusion
The seeds of the species Persea americana presented a heterogeneity of secondary metabolites, encouraging future studies with other biological potentials. The tested extracts show activity against Aedes aegypti larvae, still revealing an atoxicity in the bioassay with Artemia salina against the non-target organism, showing selectivity against target organisms.
Finally, the potential for using this product to control the vector responsible for the propagation of important arboviruses with high morbidity and mortality is confirmed and efficient, and should be explored in its control and combat.