Chemical Composition and Insecticide Activity of Essential Oil of Mesosphaerum suaveolens Against Nauphoeta cinerea

Essential oils are compounds produced by secondary plant metabolites and are found in leaf trichomes, moreover, they are characteristic because they present strong odors and present biological activities. Thus, the objective of this work was to evaluate the insecticidal action of Mesosphaerum suaveolens essential oil against nymphs of Nauphoeta cinerea as well as to characterize the chemical constituents present in it. The EO was extracted by means of hydrodistillation and its chemical characterization was done by Gas Chromatography coupled to Mass Spectrometry (GC/MS). For the biological assay against cockroaches, nymphs of 20 days old were selected for which they were submitted to different concentrations (50 1000 μg/mL oil per air) and mortality was evaluated over 48 hours of EO exposure. As a positive Journal of Agricultural Studies ISSN 2166-0379 2020, Vol. 8, No. 2 http://jas.macrothink.org 353 control, ethanol (C2H6O) was used. Have been identified 44 compounds in EO, with β-Caryophyllene (18.57%), sabinene (15.94%) and spatulenol (11.09%) being the major compounds of EO. This showed no activity against the nymphs at any of the concentrations, whereas the positive control caused mortality at all concentrations tested. Thus, the essential oil of M. suaveolens does not present insecticidal properties in low concentrations against the cockroach.


Introduction
Essential oils are compounds produced by secondary plant metabolites and are found in leaf trichomes, moreover, they are characteristic because they present strong odors and present biological activities (Bezerra et al., 2017). Usually they are mainly composed of mono and sesquiterpenes, however it is common to find other types of terpenes such as diterpenes (Bezerra et al., 2018). Among the biological activities, the main ones are antimicrobial, larvicidal, allelopathic and insecticidal actions .
Regarding the insecticidal activity, some plants developed chemical constituents with repellent and/or insecticidal properties during the evolution, so that insects did not interfere in the ecological succession of the species, mainly annual species, which are more susceptible4. Among the annual species with insecticidal potential is Mesosphaerum suaveolens (L.) Kuntze, belonging to Lamiaceae, a botanical family rich in volatile terpenes (Bezerra et al., 2017;Harley, 2016).
In addition to diptera and insects of the order Coleoptera, organisms of the order Blattodea, which are included cockroaches, cause serious economic damages to the feeding sectors. So these insects must be controlled in order not to cause damage. As a model in insecticide studies for this order, the cockroach Nauphoeta cinerea has been worked. This species has several advantages for toxicological studies, since they include an easy handling, a rapid proliferation and growth and it is exempt from the ethics committee (Rodrigues et al., 2013;Rugg;Rose, 1984).
Thus, the objective of this work was to evaluate the insecticidal action of M. suaveolens essential oil against nymphs of N. cinerea as well as to characterize the chemical constituents present in it.

Plant Material
As for the leaves of M. suaveolens, these were collected in the morning (09:00 hrs) in 2015 under coordinates of Lat: -6°14'22.40", Long: -39°16'14.29'' and altitude of 193 m of altitude ( Figure 1). Fertile branches were treated according to the collection procedures, and later deposited in the same herbarium, under voucher #12.104.

Extraction of Essential Oil
The essential oil of M. suaveolens was extracted from dried leaves, submitted to hydrodistillation in Clevenger apparatus. After collection, the leaves were crushed into small pieces (150 g) and filled into a 1 L volumetric flask, where 2 L of distilled water was added. The flask was coupled to the Clevenger apparatus under the heating mantle and the temperature adjustment was carried out until the water boiled. After boiling, the 2 h time of the extraction cycle was started. At the end of each extractive cycle, the oil contained in the apparatus was collected with the aid of a pipette and stored in amber and refrigerated bottles. After extraction, sodium sulphate was used to remove the aqueous phase present in the essential oil (Bezerra et al., 2017;Matos, 2009).

Gas Chromatography-Mass Spectrometry (GC-MS)
GC-MS analyses were performed on a Agilent Technologies AutoSystem XL GC-MS system operating in the EI mode at 70 eV, equipped with a split/splitless injector (220°C). The transfer line temperature was 220 °C. Helium was used as carrier gas (1.0 mL/min) and the capillary columns used were an HP 5MS (30 m x 0.35 mm; film thickness 0.50 µm) and an HP Innowax (30 m x 0.32 mm i.d., film thickness 0.50 µm). The temperature programmer was the same as that used for the GC analyses. The injected volume was 1 μL of the essential oil diluted in chloroform (1:10).

Identification of the Components
Identification of the constituents was performed on the basis of retention index (RI), determined with reference of the homologous series of n-alkanes, C7-C30, under identical experimental conditions, comparing with the mass spectra library search (NIST and Wiley), and with the mass spectra literature date Adams (Adams, 1995). The relative amounts of individual components were calculated based on the CG peak area (FID response).

Inventory and Creation of Nauphoeta Cinerea
The cockroaches, N. cinerea were obtained from the Federal University of Santa Maria -UFSM provided by Professor Dr. Joã o Batista Teixeira da Rocha. They were created and maintained at the Laboratory of Microscopy -LABOMIC, of the Regional University of Cariri -URCA, under temperature conditions of 25±5 °C and relative humidity of 50%. The diet of adult cockroaches and nymphs consisted of dog food and water at will.

Insecticidal Test Against Nauphoeta Cinerea
For the insecticidal assay the essential oil of L. montevidensis was contacted with filter paper and then attached to the 330 mL volume flask lid. Subsequently, 40 nymphs per group with 20 days of age were submitted to different concentrations of essential oil (50 -1000 μg/mL oil per air) and mortality was evaluated over 24 hours of OE exposure. The assay was accompanied by a negative control and a positive control. In this case, for the positive control ethanol (C2H6O) was used, since it is toxic and volatile, the ethanol was used at the same concentration. The protocol followed the methodology (Bezerra et al., 2017).

Statistical Analysis
Statistical analyzes were performed using the software GraphPad Prism 6, using One-Way Variance Analysis (ANOVA), followed by the Tukey test at 95% reliability (p<0.0001).

Chemical Composition
According to table 1, the essential oil of M. suaveolens presents a heterogeneous chemical composition, since it had a total of 44 components. Among these, the main constituents are β-Caryophyllene (18.57%), the sabinene (15.94%) and spathulenol (11.09%), which together account for 45.65% of the composition. Relative proportions of the essential oil constituents were expressed as percentages.
RI a Retention indices experimental (based on homologous series of n-alkane C7-C30).

Insecticide Activity Against Nauphoeta Cinerea
As shown in Figure 2, contrary to the hypothesis of this work, the essential oil of M. suaveolens did not present insecticidal action in 24 hours against the nymphs of N. cinerea. There was only mortality in the positive control group from the lowest concentration (50 μg/mL).

Discussion
Phytochemically, the essential oil of M. suaveolens presents variations of study for study, this is common for this species since it has a great distribution throughout the terrestrial globe, consequently the environments that this species is present have their characteristics (Mishra et al., 2011). In this study the collection was carried out in the Caatinga, a seasonally dry tropical forest of Northeast Brazil, and showed as main components β-Caryophyllene (18.57%). In the study (Wangrawa et al., 2018) the major compound is 1.8 Cineole (26.4%), and the species was collected in the African continent in a Sudanian Savanna area which is a large tropical savannah belt that runs to the east and west across the African continent, from the Atlantic Ocean to the west to the western lowlands in the east (Savadogo et al., 2007).
Generally, the major compound determines the biological property of the essential oil, and in this study the main constituent was β-Caryophyllene (C15H24), this is a bicyclic sesquiterpene present in other essential oils such as Syzygium aromaticum (L.) Merr (Prasha; Locke; Evans, 2006) and Cannabis sativa L. (Wangrawa et al., 2018). This terpene is a sweet and dry tasting compound that can be found in a number of food items such as allspice, fig, pot marjoram, and roman camomile, which makes β-caryophyllene a potential biomarker for the consumption of these food products. Biologically, this compound presents some properties as larvicide in Anopheles subpictus, Aedes albopictus and Culex tritaeniorhynchus (Govindarajan et al., 2016).
The oil of the species under study has an insecticidal activity against the fly Drosophila melanogaster justifying that such action can be attributed to a synergistic action of the constituents, since the oil has a heterogeneous composition (Bezerra et al., 2017). While in our study there was no insecticidal action, this fact can be justified because they are different biological organisms. ISSN 2166-0379 2020 Usually the essential oils present antioxidant actions, however some research shows that at the cellular level in eukaryotic organisms, oils can act as pro-oxidants, affecting cell membranes and organelles like mitochondria. Furthermore, depending on the concentration, they may exhibit cytotoxic effects, thus these oils can be used in the formulation of bioinsecticide (Bakkali et al., 2008).

Conclusion
In our study, the oil of M. suaveolens did not present an insecticidal action against the cockroach, however the oil may present an insecticidal action in higher concentrations.