Larvicide potential of essential oils from Brazilian plants against Aedes aegypti

The arboviruses Dengue, Chikungunya and Zika virus are present in several tropical regions and are transmitted by the Aedes aegypti mosquito. The containment of these diseases is done by fighting the vector, usually using chemical insecticides, such as organophosphates and organochlorines. These provoke the resistance of the transmitter, have a high accumulation rate in the body of non-target populations, and promote the contamination of ecosystems. The application of materials of natural origin with larvicidal activity, such as essential oils, is a promising alternative to replace the use of chemical insecticides. In this systematic review, we sought to present the larvicidal properties of essential oils from botanical species of Brazilian flora against Ae. aegypti. The search resulted in 36 papers selected as articles of interest. The 65 plants described in the selected articles showed larvicidal activity mostly excellent (27 were classified as strongly active) or satisfactory (13 were moderately active, and 24 were effective), while only one was inactive. The species that showed the highest larvicidal activity were: Anacardium occidentalis L. (0.01 ppm); Copaifera langsdorffii Desf. (0.04 ppm); Carapa guianensis Aubl. (0.06 ppm); Cymbopogon winterianus Jowitt. (0.10 ppm); Ageratum conyzoides L. (0.15 ppm); Tagetes minuta L. (0.21 – 0.25 ppm); and Siparuna guianensis Aubl. (1.76, 0.98 and 2.46 ppm). Studies on the essential oils of Brazilian plants are of great relevance to combat arboviruses. The Brazilian flora, despite its vast biodiversity, is still little known and explored, possessing a huge potential for the development of eco-friendly, environmentally safe, and low-cost products.


Introduction
Dengue, Chikungunya and Zika viruses are the main arboviruses present in tropical countries, transmitted by the Aedes aegypti mosquito. Due to its great epidemiological relevance, efforts have been made by the government to promote the control of the transmission agent. The usual forms of mosquito control occur through chemical insecticides, such as organochlorines and organophosphates (Moreira et al., 2012). Due to the high environmental persistence, the tendency to accumulate in organisms, the high degree of toxicity to animals and the emergence of resistance in insects (Sucen, 2001), organochlorines have had their use reduced or even discontinued in many countries. Organophosphates have a higher acute toxicity for mammals, are chemically unstable and biodegradable, have a short persistence in soil, and need to be replaced periodically (Nascimento & Melnyk, 2016), and have been widely used in the health area.
Given the need to develop alternative ways to combat the vector, the use of plant extracts and essential oils with larvicidal properties has shown promise because it is an easy method to obtain, low production cost and low residual effect. Cavalcanti et al. (2004) proved the larvicidal activity of nine Brazilian species against Ae. aegypti, such as Alpinia zerumbet and Hyptis suaveolens. Essential oils are complex natural mixtures that contain about 20-60 components in different concentrations (Bakkali et al., 2008), being composed predominantly of terpenic hydrocarbons and terpenoids. They are characterized by two or three main components in reasonably high concentration (20% -70%) compared to other components present in trace amounts (Koul et al., 2008). Essential oils can be produced in all parts of the plant, such as in barks, stems, flowers, leaves, fruits, branches, roots, seeds (Bizzo et al., 2009), and are stored in secretory cells, cavities, channels, epidermal cells or glandular trichomes (Carréra, 2016).
The Brazilian flora is characterized by its vast biodiversity. Of a total of more than 46,000 species, 14,776 are in the Amazon Forest, 5,865 in the Caatinga, 13,566 in the Cerrado domain, 1,588 in the Pantanal biome, 2,096 in the Pampa and 20,174 in the Atlantic Forest (Guatimosim, 2020). Of these, 43% are endemic to the national territory, placing Brazil as the country with the highest plant richness in the world (Jacques, 2016), being identified annually, on average, 250 species (Fioravanti, 2016). Despite its richness and potential, Brazilian biodiversity is still little known, and its use has been greatly neglected (Coradin et al., 2018). Therefore, it is essential to intensify the investment in research in the search for a better use of this natural heritage (Coradin et al., 2011). In this review, we sought to gather articles proving the larvicidal potential of the essential oil of botanical species verified in the national territory against Ae. aegypti.

Methodology
This systematic review article addressed the topic: "Larvicidal properties of essential oils from plants occurring in Brazil against Ae. aegypti", seeking to answer the following question: "Is there evidence of the effectiveness of using essential oils from plants occurring in Brazil to combat Ae. aegypti? The search was conducted in the following databases: Portal de Periódicos CAPES/MEC; PubMed.gov; SciELO.org; ScienceDirect (Elsevier); and Web of Science -Core Collection (Clarivate Analytics), using the descriptors: Aedes aegypti; Brazil; Brazilian plants; essential oil; larvicidal. These were selected based on the terms suggested by the descriptor locator in Health Sciences DeCS/MeSH Finder for the theme addressed. The values of the median lethal concentration (LC50)the dose of the substance needed to kill 50% of the test populationwere observed, as well as the larval instar (L1 to L4), botanical material collected, and exposure time.
The inclusion criteria used to select the articles were: articles on Brazilian plants (or with occurrence in Brazil) used as larvicide to combat Ae. aegypti; articles in English, Portuguese, or Spanish; full text available. The reading order for the choice of articles was title, abstract and content. The temporal inclusion criterion for the articles comprised the publication date between 2000 and 2020. We did not consider articles about seaweed used as larvicide; articles that use plant extract instead of essential oil, or the mixture of essential oils from different plants, or the association of essential oil with chemical insecticides; articles whose results did not demonstrate the efficacy of the essential oil; and review articles. The analysis and data collection were carried out by three fixed evaluators, and when there was disagreement, a fourth evaluator was recruited. This systematic review was based on the article published by Marmitt et al. (2015).

Results
The search resulted in the identification of 395 publications that, after checking eligibility following the inclusion criteria, 134 were discarded for not meeting the criteria 'full article', 'year of publication' and 'language'. After reading the sections 'title', 'abstract' and 'full article' (Figure 1), 36 papers were selected as 'articles of interest'. The main characteristics of the articles discarded in the reading stage corresponded to research conducted using seaweed, essential oil mixtures, isolated oil compounds, plant extracts, nanoemulsions, nanosuspensions and oleoresins. Following these criteria, there was disagreement among the evaluators in the selection of articles regarding the inclusion of the latter three, which was resolved through the opinion of an external evaluator, who recommended their exclusion. Research, Society andDevelopment, v. 11, n. 2, e53211226140, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i2.26140 4 In Figure 1, the ScienceDirect (17) and Capes (16) databases had the most articles selected, while the PubMed (1), SciELO (1), and Web of Science (1) databases had only one article selected.
The relevant information from each research was summarized in Table 1, which presents the median lethal concentration (LC50) values obtained in the analyzed articles, highlighting the major constituents present in the chemical composition of the essential oil of each species. The lethality data were converted to the same unit of measurement, enabling comparison of their efficacy against Ae. aegypti larvae. It was considered that 1 mg/l = 1 μg/mL is approximately equivalent to 1 ppm.

Discussion
Terpenes represent the largest class of secondary metabolites, with recognized antimicrobial activity ( (Felipe et al., 2016).
Thus, the relationship between the structural form of the molecules and their biological properties becomes evident (Strub et al., 2014).
The analysis of the chemical composition of the essential oils presented in Table 1 shows the majority presence of the secondary metabolites monoterpenes, sesquiterpenes and phenylpropenes, such as limonene and 1,8-cineole, (E) and (β)caryophyllene, and eugenol, respectively. The terpenes 1,8-cyenol (9.23%) and limonene (7.69%) and the phenylpropene eugenol (7.69%) had the highest frequency of appearance in the papers as major constituents of the essential oil. Limonene is the main constituent of the essential oil of the peels of citrus fruits (genus Citrus), such as lemons (C. limonia Osbeck) and oranges (C. sinensis (L.) Osbeck) and is responsible for the characteristic odor that these fruits present. The 1,8-cineol or eucalyptol is found in the essential oil of the leaves of botanical species of the genus Eucalyptus, being also reported to occur in plants of the genus Hyptis, as in H. fruticosa Salzm. (Silva et al., 2007), H. martiusii Benth. (Costa et al., 2005) and H.
suaveolens Poit. (Cavalcanti et al., 2004). The caryophyllene is present in the composition of many essential oils used as spices, especially in clove, rosemary, and black pepper. Eugenol is the predominant component of the essential oil of clove (Syzygium aromaticum (L.) Merr. & Perry), as reported by Araujo et al. (2016), Costa et al. (2005), and Santos et al. (2020).
To classify the larvicidal potential of essential oils, the literature provides different criteria, and there are no standardized median lethal concentration values for determining the efficiency of the analyzed substances. According to Kiran et al. (2016), the larvicidal effect is considered significant in essential oils with LC50 less than 100 ppm under 24h of exposure.
Another methodology is proposed by Komalamisra et al. (2005), according to which substances that present LC50 less than 50 ppm are considered strongly active, LC50 between 50 and 100 ppm, moderately active, and LC50 between 100 and 750 ppm, effective, while those with LC50 values higher than 750 ppm are considered inactive, under 48h of exposure. Table 2 shows the classification of the studied plants adopting the second specification presented. For items that present different larvicidal potentials for the same plant, depending on the botanical material used, place of larval collection, and time of exposure to the essential oil, the lethality considered corresponds to the arithmetic mean of the LC50 values.
Of the species mentioned above, the identification of the chemical composition of the essential oil was performed only for S. guianensis. The main components were dependent on the botanical material used for essential oil extraction, being β-myrcene (79.71%) for stems, β-myrcene (26.91%) for leaves, and 2-tridecanone (38.75%) for fruits. This fact was not observed in the study of Autran et al. (2009), where the essential oil of Piper marginatum Jacq. showed the same composition ((Z) and (E)-asarone and patchouli alcohol) when extracted from leaves, stem, and inflorescences, varying only the percentage with respect to the total composition. The chemical composition of volatile oils varies between parts of the same plant and, when extracted from the same organ of the same plant species, can vary significantly according to age and stage of development, season and time of collection, weather and soil conditions, and their properties depend on the extraction technique used (Burt, 2004;Morais, 2009 Note that such species correspond to a small percentage (17.86%) in relation to the total number of species analyzed, which shows the lack of knowledge of the existing native flora, although Brazil is a country of great biodiversity.
The occurrence of species exclusive to the Northeast region of Brazil and the Caatinga biome is also observed. Croton tetradenius Baill. is endemic to the Caatinga biome and is frequently found in most states of the Northeast region of Brazil (Carvalho et al., 2016). Lippia gracilis Schauer is endemic to Northeast Brazil and is widely distributed in the Caatinga (Gomes et al., 2011). Vitex gardneriana Schauer is a native and endemic species of Brazil, having its distribution restricted only to the Northeast region (Soares, 2017).

Conclusion
Unlike chemical insecticides, which are harmful to the environment and, when unstable, require periodic application to the soil, essential oils are an eco-friendly and economically viable alternative in combating arboviruses transmitted by Ae.
aegypti. Brazil has a vast natural wealth, with about 46 thousand plant species in the most diverse biomes, 43% of which are endemic to the national territory. The essential oils from different botanical species of Brazilian flora proved to be efficient in  Komalamisra et al. (2005). Thus, it was proven the ability to use plants grown in the national territory as an alternative in combating Ae. aegypti. Therefore, studies on the essential oils of Brazilian plants are of great relevance, not only to combat arboviruses, but also due to their potential for medicinal applications, showing other relevant properties.
As suggestions for future work, we recommend the analysis of the larvicidal activity of essential oils from Brazilian plants against Ae. aegypti with different approaches, prioritizing the species that occur exclusively in the Northeast region and the Caatinga biome, to explore the vast biodiversity and the enormous potential existing in the native flora of this region.