Bioinsecticide potential of Curcuma zedoaria rhizome essential oil

In this study  the potential bioinseticide of the essential oil (OE) extracted from the rhizomes of the species Curcuma zedoaria (Zingiberaceae) was evaluated. The rhizomes were collected during dormancy (winter) and budding (summer). The EO was obtained by hydrodistillation (2h) and identified by GC/MS. In addition, a multivariate exploratory analysis was done to determine the analysis of the major compounds (PCA). The EO yield in dormancy was 0.61± 0.07 (%) and in budding 0.55 ± 0.08 (%). The bioassays on Aedes aegypti larvae and pupae were done by immersion test at different EO concentrations which ranged from 500.00 to 0.003 mg mL-1 (v/v). The results on the larvae and pupae indicated LC99.9 of (0.01 and 1.38 mg mL-1) for EO in dormancy, and (0.08 and 2.63 mg mL-1) for EO during budding, respectively. The action mechanism of EOs in both periods was determined by autobiographic method evaluating the inhibitory potential on the acetylcholinesterase enzyme, indicating greater inhibition of the EO enzyme during dormancy (0.039 mg mL-1) when compared to the EO during budding (0.156 mg mL-1). The projection representation of the EO chemical classes in both evaluated periods indicated that oxygenated sesquiterpenes are the major compound class (46.99% in dormancy) and (43.59% in budding). The projection of major chemical compounds of EOs presented three compounds with greater mass flow distancing: epicurzerenone (18.20% and 12.10%); 1.8 cineole (15.76% and 12.10%) and β-elemene (4.43 and 0.01%) that are found in greater amounts in the dormancy EO when compared to budding, respectively. These results corroborate with the greater potential on Ae. aegypti larvae and pupae found for the dormancy EO. The results are promising because they show in which vegetative cycle phase C. zedoaria EO presents greater bioinsecticide potential.


INTRODUCTION
The species Aedes aegypti L. is the main vector transmitting Dengue, Chikungunya fever and Zika virus, which are responsible for significant human morbidity and mortality in a lot of countries, (BRASIL, 2015;WORLD HEALTH ORGANIZATION (WHO), 2016).
According to the epidemiologic report of the Health Ministry-from December 31, 2017 to March 10, 2018) -51,980 cases of dengue, 12,261 cases of Chikungunya fever were reported, and in 2018 there must have been 1,174 probable cases of fever by Zika virus (BRASIL, 2018). In Brazil, the high incidence of these transmitting insects is due to the accelerated and disorderly urbanization process as well as the climatic and social conditions that favor their quick proliferation (OLIVEIRA; BIAZOTO, 2012;BUSATO et al., 2014).
The strategies to eradicate Ae. aegypti consist of the application of synthetic insecticides, mainly organophosphates and pyrethroids (BRAGA; VALLE, 2007). However, the utilization of these insecticides must be careful to avoid slowing down the development Bioinsecticide potential of… OLIVEIRA, H. L. M. et al. of resistance to them (SMITH; KASAI; SCOTT, 2016), toxicity to humans as well as the impact that they cause to biodiversity (NICOLAU, 2013). Therefore, research studies utilizing biomolecules such as plant essential oils have been done (COSMOSKI et al., 2015) because these chemical structural groupings of these compounds or their combination can have, and also intensify, the larvicidal potential (SANTOS et al., 2010). Considering this, the evaluated plant, Curcuma zedoaria (Zingiberaceae), is an exotic plant from Southern and southeastern Asia, which is well-adapted to the Southern region of Brazil, especially the northeastern region of Paraná state. Popularly known as zedoaria, vick and fake saffron, this species has been studied due to its therapeutic potential with several scientific results that confirm its traditional and popular medicinal use (LORENZI;2002).
Its cultivation is easy and its rhizomes are rich in essential oil, mainly consisting of pinene, camphene, cineol, camphor and borneol (GUENTHER, 1950). However, this composition varies and is influenced by the plant vegetative phase and abiotic factors such as radiation, temperature, rainfall, winds, altitude, soil and cultivation implementation location (MORAIS, 2000;GOUINGUENÉ;TURLINGS, 2002). Research studies on its EO larvicidal potential are scarce and, therefore, caused our research group to deepen the investigations on the probable action against Ae. aegypti larvae and pupae.
Thus, this study aimed to evaluate the larvicidal potential of Curcuma zedoaria essential oil obtained in budding and dormancy periods against Ae. aegypti larvae and pupae.

Botanical identification and vegetal material preparation
The cultivation of Curcuma zedoaria is found in the Medicinal Garden of the Paranaense University -Umuarama -PR. An exsiccata is deposited in the Educational Herbarium of Paranaense University -HEUP, under the registration number 2400. The botanical identification was done by Prof. Dr. Ezilda Jacomassi. This species is registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under the registration number ACFC9FF The vegetal material was dried, powdered and submitted to hydrodistillation process for two hours. The oil was withdrawn with a Pasteur pipette, filtered with Na2SO4 and stored at -10ºC (LAI et al., 2004).

Chemical composition analysis of Curcuma zedoaria essential oil
The chemical composition analysis was done using a gas chromatographer (Agilent 7890 B) coupled to mass spectrometer (Agilent 5977 A) equipped with an Agilent HP-5MS (30m x 0.250mm x 0.25mµ) under the following conditions: injector temperature at 250°C, injection volume 1mL at a 1:2 rate (splitless mode), initial temperature of the column at 80°C with gradual heating until 260°C and a ramp of 4°C/min. The carrier gas (helium) flow was fixed at 1 mL/min. The transfer line temperatures and quadrupole ion sources were 250, 230, and 150°C, respectively. The mass spectra were obtained in a 40-500 (m/z) interval provided by scanning with solvent permanence time of 3 min. The compounds were identified by comparing their retention indices (RI) obtained from a series of n-alkanes (C8-C25) (LAI et al., 2004). Moreover, EI-mass spectra were compared to the spectra obtained from Wiley's Spectral Library 275L, and according to the literature (ADAMS, 2012).

Major Compound Analysis (PCA)
Complementarily, a multi-varied exploratory analysis was also done to determine the main compound analysis (PCA) which allowed the evaluation of the major chemical compounds and chemical class of all compounds found in the essential oil obtained in three analyzed periods (vegetative, flowering and fructification). The analysis result was graphically presented (Biplot), helping the characterization of the analyzed variable groups (MOITA NETO;MOITA, 1998).
For each sample of essential oil obtained during these three periods (vegetative, flowering and fructification), the identified major chemical compounds and their respective chemical classes, as well as the area amount in (%) ( Table 1), were plotted in Excel spreadsheets. These data were transformed in orthogonal latent variables called major components that are linear combinations of original variables created with the eigenvalues of the data covariance matrix (HAIR, 2005).
Kaiser's criterion was utilized to choose the main components. An eigenvalue preserves the relevant information when it is greater than the unit. This analysis was carried out in two ways: the former contained only data referring to the chemical composition of the major compounds obtained in the three periods, and the latter analyzed the grouped chemical classes to which those compounds belong to. Both analyses were done utilizing Statistica 7 program (STATSOFT, 2001).

Larvicidal activity against Aedes aegypti
The larvicidal activity was done by Larval Immersion Test (LIT) against Aedes aegypti L. larvae and pupae from the Center for Vector transmitted Endemics -Secretary of Sanitary Surveillance of Umuarama, PR. The EO was tested at different concentrations: 500, 400, 300, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.390, 0.195, 0.097, 0.048, 0.024, 0.012, 0.006, 0.003 mg mL -1 (v/v), diluted in an aqueous polysorbate 80 solution at 2.0%. Five third-stage Ae. aegypti larvae and five pupae were separated using a Pasteur pipette and placed in assay tubes containing 1.00 mL of different concentrations of EO (COSTA et al., 2005;CAVALCA;LOLIS BONATO, 2010). For the negative control, an aqueous polysorbate 80 solution at 2% was utilized whereas for the positive control, a commercial Temephós ® solution at a concentration of 400 mg L -1 was used (CAMARGO et al., 1998). The larvae were exposed to EO at different concentrations for 24 hours (CARVALHO et al., 2003). They were considered dead when presented absence of movements and were irresponsive to stimuli (CAVALCA; LOLIS; BONATO, 2010). The data on the number of live and dead larvae and pupae were found through an average of three replications for each one of the tested concentrations.

Anticholinesterase activity of essential oil
The anticholinesterase activity was determined by the bioautographic method described by Marston et al. (2002) with modifications (YANG et al., 2009). The essential oil of C. zedoaria rhizome was tested starting from the initial concentration of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1562, 0.078, 0.039, 0.019 mg mL -1 , diluted in methanol. The samples were plotted in aluminum chromo plates (10 x 10 cm, 0.2 mm-thick 60 F254 silica gel). After plotting, the plates were dried and sprayed with an acetylcholinesterase enzyme solution in buffer solution; next, they were sprayed with an α-naftyl acetate solution. The plates were kept at 37 °C for 20 minutes. After this period, the chromo plates were sprayed with Fast Blue B salt colorimetric reagent, resulting in a purple color surface. The anticholinesterase activity of C. zedoaria rhizome EO was determined by the emergence of white stains after 10 minutes, showing the inhibitory action of the evaluated concentrations on the enzyme activity, contrasting with the purple color of the colorimetric reagent (COLLINS et al., 1997) like the positive standard of the larvicide (Temephós ® ).

Statistical analysis
The experiments were done in triplicate and the mortality percentage (%) of Ae. aegypti larvae was obtained by calculating the mean ± standard deviation and coefficient variation utilizing Microsoft Excel ® program (Excel ® Version 2010). The values of Lethal Concentration (LC50 and LC99.9) and their respective confidence intervals (CI) were calculated by Probitos analysis (ED 50 Plus version 1.0). The obtained data were submitted to analysis of variance (ANOVA) and the differences between the averages were determined by Scott-Knott's test (p ≤ 0.05).

RESULTS AND DISCUSSION
The essential oil of Curcuma zedoaria rhizome has lilac coloring, with greater yield in the dormancy period (0.61 ± 0.07 %) compared to the budding period (0.55 ± 0.08 %). This difference in the yield is justified because in this period the buds are in the peak of their maturation (DING; NILSSON, 2016), and the found values are in accordance with Angel et al. (2014), who found yields ranging from 0.38 to 1.4 (%) for C. zedoaria species.
The chemical identification of the essential oil obtained during the dormancy and budding periods was done by a gas chromatographer coupled to a mass spectrometer and whose results are shown in Table 1   According to the data in Table 1, the rhizome EO from the dormancy period presented 120 compounds, and from the budding period 119 compounds. According to Figure 1 and Table 1, the predominant class was oxygenated Sesquiterpenes (43.30% and 43.89%), followed by oxygenated Monoterpenes (25.74 % and 24.57%) and hydrocarbon sesquiterpenes (21.96% and 22.36%), respectively. In Figure 2, grouping by PCA was done and major compounds were identified in EO in dormancy and budding. Three of these compounds presented a greater mass flow distancing : epicurzerenone with 18.20% in dormancy EO and 14.05% in budding EO, 1,8cineole (15.76% and 12.10%) and β-elemene (4.43 and0.01%),respectively. Moreover, it was possible to verify the formation of other group: camphor (5.61 and 5.83), curzerene (4.68; 4.78), and velleral (4.78; 3.91) respectively, found in both periods, at higher concentrations than 3.5%. The differences observed in the chemical composition in both evaluated periods (Table 1 and Figures 1 and 2) occurred in function of C. zedoaria vegetative cycle which is well defined in this species. It starts with the growth of vegetative sprouts (buds) in the summer. Next, growth ceasing, bud formation, senescence and leaf abscission occur in the autumn, and bud dormancy in the winter (RINNE et al., 2010;DING;NILSSON, 2016). This cycle is dynamically modulated by intrinsic environmental and hormonal factors, and according to Horvath et al. (2003), the presence of the hormones ethylene and auxin and phytochrome protein act directly on the bud dormancy stage while gibberellic acid, abscisic acid and cytokine play antagonistic roles in dormancy, regulating specific components of the cellular cycle (ANDERSON et al., 2001). Regarding the influence of intrinsic and environmental factors (photoperiod and water deficit), the vegetative and reproductive meristems of several perennial plants in temperate climate remain in a latent state without growth during the cold period of the autumn and winter, within the buds, guaranteeing an optimal protection against low and dry temperatures (RIOS et al., 2014).
C. zedoária EO was evaluated against Ae. aegypti larvae and pupae by calculating the Lethal Concentrations (LCs) that are necessary to eliminate 50.0% (LC50) and 99.9% (LC99.9). The results are described in Table 2. The essential oil of C. zedoaria obtained in the periods of dormancy and budding killed Ae. aegypti larvae and pupae with LC99.9 of (0.01 and 1.38 mg mL -1 ) and (0.08 and 2.63 mg mL -1 ), respectively ( Table 2). The greatest activity was observed in dormancy EO, corroborating the results found in the chemical analysis in which it was possible to verify that the compounds epicurzerenone, 1,8 cineole and β-elemene are found in greater amounts. From these three compounds, 1,8 cineole and β-elemene can be responsible for the larvicidal action because, similarly in C. zedoaria, these compounds were the major ones in EOs of some species. One of the major compounds of EO from Croton jacobinensis (Euphorbiaceae) leaves and inflorescences was 1,8 cineole (16.90%), and when tested against Ae. aegypti, it presented LC50= 0.0793 mg mL -1 for the leaves and LC50= 0.0658 mg mL -1 for the inflorescences (PINTO et al., 2016). Regarding βelemene, it is one of the major compounds of the EO of both species that were tested against Ae.  (MAHESWARAN et al., 2016). In Murraya exotica L (Rutaceae) EO, (7.56%) was found with LC99= 0.152 mg mL -1 (KRISHNAMOORTHY et al., 2015). Epicurzerenone presents high antimicrobial (PRAKASH et al., 2018) and antitumor activities (SEPTANINGSIH et al., 2018); however, there have been no reports showing its larvicidal potential.
We understand that because they are major compounds of this species, it would be important to isolate them to investigate if these compounds were responsible for the mortality of Ae. aegypti larvae and pupae.
Another point to be discussed is the differentiated action of EOs against larvae and pupae.
The obtained results showed greater potential against larvae because the bio compounds acted on the interaction of the larval cell wall as well as in the ingestion and absorption by their gastrointestinal tract - (PROCÓPIO et al., 2015). However, in pupae, because they do not feed themselves, the biocompound penetration was more difficult (CHAUBEY 2012;PROCÓPIO et al., 2015;PIETA et al., 2017).
The aim of our study was to verify how C. zedoaria EO killed larvae and pupae. Therefore, the inhibitory potential of anticholinesterase enzyme, responsible for the transmission of nervous impulses, was determined by bioautographic method, and the results are shown in Table 3. The dormancy EO presented greater inhibition on the acetylcholinesterase enzyme (0.039 mg mL -1 ) when compared to the budding period (0.156 mg mL -1 ), probably due to the presence of a greater amount of 1,8-cineol in dormancy EO as it presents larvicidal action already reported in the literature (LOBO et al., 2009;LIU et al., 2012;PINTO et al., 2016).
Comparing the LCs found for larvae (Table  2) and a smaller concentration that inhibited acetylcholinesterase enzyme (Table 3), it was evident that the in vitro test (bioautographic) showed lower effectiveness than the in vivo test against Ae. aegypti larvae. It can be suggested that this observed difference can be related to more than one action mechanism of the EO chemical compounds, besides the inhibition of the acetylcholinesterase enzyme. It is known that the conventional chemical larvicides, such as pyrethroids, act through different action mechanisms, which act on the neural sodium channels, interfering in their opening and closing, and extending the entry of Na + ions into the cell (SANTOS et al., 2007) while organophosphates inhibit acetylcholinesterase enzyme (BRAGA; VALLE, 2007). These results open new perspectives because our objective it to provide natural bioinsecticide that can act in synergism with conventional larvicides.

CONCLUSIONS
The essential oil (EO) of Curcuma zedoaria rhizome obtained in the dormancy and budding periods were evaluated against Ae. aegypti larvae and pupae.
The chemical analysis by GC/MS and grouping by PCA indicated the presence of epicurzerenone (18.20%); 1,8 cineol (15.76%) and β-elemene (4.43) with greater amount in dormancy period, indicating that these compounds can be responsible for the action against larvae and pupae.
The results were promising because they establish in which period of the vegetative cycle C. zedoaria EO presents greater bioinsecticide potential.