Chemical composition, in vitro larvicidal and antileishmanial activities of the essential oil from Citrus reticulata Blanco fruit peel

Abstract Numerous studies have investigated the chemical composition and biological activities of essential oils from different Citrus species fruit peel, leaves and flowers. This paper aims to investigate the chemical composition, larvicidal and antileishmanial activities of essential oil from Citrus reticulata fruit peel (CR-EO). CR-EO was obtained by hydrodistillation in a Clevenger-type apparatus and its chemical composition was analyzed by GC-MS and GC-FID. Limonene (85.7%), ɣ-terpinene (6.7%) and myrcene (2.1%) were identified as its major components. CR-EO showed high activity against promastigote forms of Leishmania amazonensis (IC50 = 8.23 µg/mL). CR-EO also exhibited high larvicidal activity against third instar Aedes aegypti larvae at a lethal concentration (LC50 = 58.35 µg/mL) and 100% mortality at 150 µg/mL. This study suggests, for the first time, the potential use of CR-EO against this important mosquito-borne viral disease caused by the genus Aedes.


Introduction
Concern for the control and fight against Aedes aegypti, the mosquito that is the main vector of a severe hemorrhagic disease known as dengue, has been latent (Wankhar et al., 2015). The number of dengue cases has reached about 400 million per year. In the last 50 years, this disease has been endemic in 128 countries, while 36 countries are considered dengue-free ones. The highest prevalences are found in countries in the Americas, Asia and Africa, i. e., 3.97 billion inhabitants are exposed to the risk of infection (Brady et al., 2012). In addition to the dengue virus, the vector may transmit other diseases known all over the world as chikungunya and zika (Neves et al., 2017).
Leishmaniasis is another worrisome disease worldwide which is also caused by the infected vector known as "straw mosquito" . The estimate is that 2 million new cases of leishmaniasis occur annually and that between 15 and 20 million people have got the disease in the world (Moreira et al., 2019). In the search for plants indices on an Rtx-5MS (30 m × 0.25 mm; 0.250 µm) capillary column under the operating conditions used for GC relative to a homologous series of n-alkanes (C 8 -C 20 ). Structures were computer-matched with Wiley 7, NIST 08, and FFNSC 1.2, and their fragmentation patterns were compared with literature data (Adams, 2007).

Larvicidal assay
Larvae of A. aegypti were obtained from the Laboratório de Patologia Tropical e Saúde Pública that belongs to the Universidade Federal de Goiás, UFG, Brazil. The larvicidal assay was performed in agreement with a previously reported method (Mesquita et al., 2018), as follows: larvae were kept in plastic trays under controlled temperature (26 ± 2 °C) and humidity (70-80%) until they reached the third final instar stage. Afterwards, 10 larvae were transferred to 50-mL plastic cups, each containing 10 mL mineral water and ground fish food (TetraMin Tropical Flakes), followed by the addition of 100 µL solution of CR-EO in dimethyl sulfoxide (DMSO) (25-500 µg/mL). After 24 hours, the number of dead larvae was counted and the lethal percentage was calculated. All experiments were carried out in quintuplicate, including a negative control treatment with DMSO, mineral water, larvae and ground fish food. Permethrin was used as a positive control. Larvicidal activities were reported as lethal concentration at 50% (LC 50 ), representing the concentration in micrograms per milliliter that caused 50% larval mortality, with 95% confidence interval. Mortality data were assessed by probit analysis (Finney, 1971). Mortality data were treated by the Polo plus® software (Robertson et al., 2003) with 95% confidence interval and values of P < 0.05 were considered statistically significant.
with therapeutic potential, species of the genus Citrus not only have economic importance, but also produce bioactive essential oils (EOs) which have high value in perfume, food and beverage industries (Dosoky and Setzer, 2018).
EOs and extracts from Citrus reticulata fruit peel and leaves have several biological applications, such as antimicrobial, antioxidant, anti-inflammatory, anticancer, antiproliferative, anti-pulmonary fibrosis, hypoglycemic and insecticidal ones, besides being useful in skin care (Hamdan et al., 2016;Apraj and Pandita, 2016). Therefore, this study aimed to evaluate the chemical composition, larvicidal and anti-Leishmania amazonensis activities of essential oil from C. reticulata fruit peel (CR-EO). So far, larvicidal activity of CR-EO against third instar A. aegypti larvae has not been investigated in the literature.

Plant material
Fruits were collected in Rio Verde (17°99.4'63.2"S and 51°05.2'44.6"W), a city located in Goiás state, Brazil, on January 18th, 2019, at 9 a.m. The plant was identified by the botanist Luzia Francisca de Souza and a voucher specimen of Citrus reticulata was deposited in the herbarium in Rio Verde, at the Instituto Federal Goiano (IFGOIANO) under identification number #4488.

Essential oil extraction
Essential oil (EO) was extracted from C. reticulata (CR-EO) fruit peel by hydrodistillation for 3 h in a Clevenger-type apparatus. Hydrodistillation was performed in triplicate. To this end, fruit peel was divided into three 500-g samples and 500 mL distilled water was added to each sample. After manual collection of EO, remaining traces of water were removed with anhydrous sodium sulfate. Filtration was then carried out. EO was stored in an amber bottle and kept in a refrigerator at 4 °C until analysis (Carneiro et al., 2017).

Chemical identification of essential oil from C. reticulata fruit peel (CR-EO)
CR-EO was dissolved in ethyl ether and analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS), with the use of Shimadzu QP5000 Plus and GCMS2010 Plus (Shimadzu Corporation, Kyoto, Japan) systems. The temperature of the column in GC-FID was programmed to rise from 60 to 240 °C at 3 °C/min and was held at 240 °C for 5 min; the carrier gas was H 2 at a flow rate of 1.0 mL/min. The equipment was set to operate in the injection mode; the injection volume was 0.1 µL (split ratio of 1:10), while injector and detector temperatures were 240 and 280 °C, respectively. Relative concentrations of components were obtained by normalizing peak areas (%). Relative areas consisted of the average of triplicate GC-FID analyses. GC-MS conditions and the identification of essential oils have been previously reported (Lemes et al., 2018). Identification of volatile components of essential oil from C. reticulata (Table 1) was based on their retention Essential oil from C. reticulata fruit peel

Antileishmanial assay
To evaluate antileishmanial activity, L. amazonensis promastigote forms (MHOM/BR/PH8) were maintained in RPMI 1640 (Gibco) culture medium supplemented with 10% fetal bovine serum, penicillin (100 UI/mL) and streptomycin (100 µg/mL). Subsequently, about 1 x 10 6 parasites were distributed on 96-well plates, while CR-EO previously dissolved in 100% dimethylsulfoxide (DMSO, stock solution 100 mM) (Synth) was added to the cultures at concentrations of 6.25 to 100 µg/mL. Amphotericin B (Sigma Aldrich, 97% purity), at concentrations ranging from 0.011 to 0.19 µg/mL, was added to cultures and used as positive control. Cultures were incubated in a BOD (Quimis) incubator at 25 °C for 24 h and antileishmanial activity was determined by verifying whether growth of promastigote forms was inhibited. It was carried out by counting the total number of live promastigotes in the Neubauer (Global Glass -Porto Alegre, BR) chamber on the basis of flagellar motility. RPMI 1640 medium (Gibco) containing 0.1% DMSO (Synth) (highest concentration) was used. Results were expressed as the average of the percentage of growth inhibition relative to the negative control (0.1% DMSO) (Cabral et al., 2020).
Previous reports of EOs from fruit peel from other C. reticulata specimens showed that terpenes limonene, sabinene, linalool, ɣ-terpinene, octanal and capraldehyde were its major constituents (Hamdan et al., 2016;Martins et al., 2017). EOs from different Citrus species are chemically similar with the predominance of the monoterpene limonene, characteristics that are pointed out by Bozkurt et al. (2017). High limonene concentration (74.38%) was also identified in C. reticulata peel grown in Spain (Espina et al., 2011). A recent study of ten Citrus species should be highlighted, since it reinforces limonene and aldehyde compounds in CR-EO (González-Mas et al., 2019).
CR-EO had its larvicidal activity investigated against third instar A. aegypti larvae. Initially, larvae in contact with CR-EO showed accelerated movement. However, after prolonged exposure, they began to exhibit tremor and slow or lethargic movement, even when artificially stimulated. CR-EO also caused darkening of the entire larval body. Doses of 12.5, 25, 50 and 100 µg/mL resulted in 10.2, 25.1, 51.7 and 85.3% of dead larvae, respectively, while 150 µg/mL ensured 100% mortality. LC 50 of CR-EO was 58.35 µg/mL. Even though EOs from several Citrus species have already been tested against A. aegypti larvae, this is the first promising report of C. reticulata. Larvicidal and insecticidal activities have already been evaluated in EOs from C. sinensis, C. limon, C. grandis, C. aurantifolia, C. hystrix, C. maxima and C. medica (Araújo et al., 2016;Gomes et al., 2019;Sarma et al., 2017Sarma et al., , 2019Soonwera, 2015). High larvicidal activity could be due to the high limonene concentration (85.7%), a botanical insecticide which has been patented as an active agent of larvicidal formulations (Dias and Moraes, 2014).
Several authors have reported that EOs whose values are IC 50 < 10 µg/mL are highly active Almeida et al., 2020). The range of IC 50 values that considers EOs highly active has been described by Estevam et al. (2016), who studied EOs from C. limonia and C. latifolia fresh leaves. It should be highlighted that results found by the study reported by this paper do not agree with the ones reported by Monzote et al. (2019), since the IC 50 value of commercially obtained CR-EO was 70.7 µg/mL. This fact leads to questioning the nature of EOs that are commercialized with no compliance with regulations. On the other hand, high antileishmanial activity of CR-EO found by this study may also be justified by the amount of limonene, a monoterpene that exhibits this activity against parasites that belong to the genus Leishmania (Arruda et al., 2009).

Conclusion
This study described the chemical composition of CR-EO, in which thirteen compounds were identified, and showed their high larvicidal activity against Aedes aegypti larvae and anti-Leishmania amazonensis promastigote forms. Limonene, as the major compound in CR-EO, could explain their high larvicidal and antileishmanial activities. This communication is the first report of larvicidal evaluation of CR-EO.