Volatile oils from Philodendron meridionale Buturi & Sakur: chemical composition and in vitro effect against Ctenocephalides felis and leukemia cells

Essential oils are an important natural source of pesticides and can be suitable used in ectoparasite control. The goal of this paper was to investigate the chemical composition of the volatile oils obtained from leaves and stems of Philodendron meridionale Buturi & Sakur by gas chromatography/mass spectrometry and to evaluate their insecticidal effect against Ctenocephalides felis Bouché adult insects and their cytotoxicity on human Jurkat leukemic T-cells by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoluim bromide (MTT) reduction method. The main compound was ent-kaur-16-ene at concentrations of 20.78 and 22.46% for volatile oils from leaves and stems, respectively. These volatile oils provided insecticidal effect against C. felis with an average knockdown time of 9.61 and 7.35 min for fumigant test performed with the leaves and stems samples, respectively. Considering the topical application, these P. meridionale volatile oils extracted from leaves and stems demonstrated average knockdown values of 14.70 and 20.0 min, respectively. A very low cytotoxic effect of 9.48 and 11.32% was observed for P. meridionale volatile oils from leaves and stems even at the highest tested concentration of 400 μg.mL, respectively. Therefore, it is possible to propose a biological selectivity to the volatile oils under study, which guarantees, at the same time, effect against this insect with no evident damage to human biological system.


Introduction
Essential oils are complex mixtures of volatile and natural compounds with strong odor. They consist of secondary metabolites produced by plants. In nature, essential oils play an important role in protecting plants and serving as antibacterial, antiviral, antifungal, and insecticidal substances. They also act against herbivores by reducing their interest in eating them. Furthermore, they are able to attract certain insects, which disperse pollen and seeds as well as repel other undesirable insects (Bakkali et al., 2008;Ellse & Wall, 2014;Barros, Garcia & Andreotti, 2019;Betim et al., 2019;Rodrigues et al., 2020;Apolinário et al., 2020). Research on essential oils has been significantly intensified in order to discovery new active molecules and formulations. In addition, researchers are interested in precursors to be used in new medicines for human and animal use with higher efficacy and safety. Many essential oils demonstrate strong pharmacological properties as antiinflammatory, antimicrobial, anticytotoxic, antioxidant, antifungal, antinociceptive, antipyretic, healing and antileishmanial activities (Bakkali et al., 2008;Cardile et al., 2009;Ribeiro et al., 2010;Pereira et al., 2011;Sousa, 2012;Ract et al., 2015;Miranda et al., 2016;Damasceno et al., 2017;Pereira et al., 2017;Kauffmann et al., 2019).
Philodendron meridionale Buturi & Sakur is a plant native to Brazil (Buturi, Temponi & Sakuragui, 2014). To the best of our knowledge, this is the first study on the chemical composition of volatile oils from P. meridionale that investigates their in vitro insecticidal and cytotoxic activities. Based on this purpose, Ctenocephalides felis Bouché was chosen because it is the most common flea among dogs and is responsible for a variety of veterinary dermatological problems. It can also be a vector for bacteria (such as Bartonella sp., responsible for rickettsial diseases) and an intermediate host for Filarioidea, Cestoda, other parasites, and viruses (Lans, Turner & Khan., 2008;Batista et al., 2016). Considering the effect of volatile compounds on human cells (Chkhikvishvili et al., 2013;Pereira et al., 2017), tests were performed using Jurkat cells, a human T lymphocytes widely used to study leukemia, in order to determine the P. meridionale cytotoxic potential.

Botanical material
Plant material was collected following a previous authorization from the Biodiversity Authorization and Information System (SISBIO), an organization associated with the Brazilian Ministry of Environment (MMA), and Chico Mendes Institute for Biodiversity Conservation (ICMBi) numbered 44970-1. The botanical material was collected at the Botanical Garden Campus of Federal University of Paraná, Brazil (25°26′58′′ S, 49°37′12′′ W -906 m altitude) in June, 2014. All plant material was obtained from the same sample in a sterile phase. A specimen was stored at the Municipal Botanical Museum of Curitiba, numbered 390207 and then identified by an expert botanist, Dr. M.C.S.

Obtaining the Essential oil
Extraction was carried out by hydrodistillation for 7 h in dark conditions using Clevenger apparatus (USP XXV, 2002) and dry leaves (extraction 1) or dry stems (extraction 2) from P. meridionale, ground in a knife mil. The oil was collected into an Eppendorf tube containing anhydrous sodium sulfate and kept at 4 ± 0.5 ºC.

Identifying the essential oil constituents
The samples were analyzed by chromatography using a GC/MS-QP 2010 Plus Shimadzu equipped with an Rtx-5MS capillary column (30 m x 0.25 mm x 0.25 µm). Ten microliters of essential oils were dissolved in 1 mL of n-hexane for each oil sample, and 1 µL of the sample solution was injected. The injector was set to splitless mode at 250°C, while the interface and the ion source were set to 300°C. Helium at a flow rate of 1 mL/min was used as the carrier gas. An injection ramp method was used, with an injector temperature of 250ºC and a column pressure of 20 psi. The starting temperature was set at 50ºC for the first 5 min. The temperature was then increased to 200 ºC at a rate of 5ºC/min. Triplicate injections were made for each sample. Mass spectra were recorded at 70 eV in a scan mode from m/z 40 to 350.
The volatile compounds from P. meridionale oils were identified by comparing their relative retention indices to the literature (Adams, 2007). The raw percentage from the peak area of each compound was obtained in full-scan GC/MS analyses. Further standardization was not carried out, since our aim was to identify the volatile oil compounds.

Insecticidal activity of P. meridionale volatile oils against C. felis
Adult insects were collected from well-fed infected dogs in Ponta Grossa, Paraná by combing the dog hair and transferring the specimens to plastic Falcon tubes labelled with collection data. The insects were kept in groups of six and identified by the specialist Dr. I.F.B. After confirming specimen mobility, they were transferred to Petri glass dishes of 10 cm in diameter, where they were immediately subjected to testing. They were kept in a controlled environment at 28 ± 1°C and 60 ± 5% of relative humidity.

Fumigant activity
The fumigant analysis was performed in triplicate using a total of 216 non-sexed adult insects. For each test and sample (n = 4), the insects were divided into three groups of six insects each. The assay was carried out as suggested by Toloza et al. (2006). A closed chamber system consisting of a Petri dish and its cover that allowed for the formation of vapors was used. A drop (50 μL) of each volatile oil was placed on a glass cover inside the dish. Three groups of six adult insects were monitored at 10-min intervals for 1 h. The results were expressed as average knockdown time (KT50). Melaleuca essential oil (Via Farma, São Paulo, Brazil) was used as a positive control, while water was used as a negative control.

Topical action
The topical analysis (Yang et al., 2004) was also performed by dividing 216 non-sexed adult insects as reported in 2.3.1. Each test was carried out in a closed chamber consisting of a Petri dish and its corresponding cover, using 30 μL of each volatile oil or positive control diluted to a 10% concentration in a mixture of propylene glycol and ethyl alcohol (1:4). This volume of solution was then placed on each insect. Three groups of six adult insects were monitored at 10-min intervals for 1 h. Results were expressed as KT50. Melaleuca essential oil (Via Farma, São Paulo, Brazil) was used as a positive control, while a mixture of propylene glycol and ethyl alcohol (1:4) was used as a negative control.

Cell viability by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
A stock solution (10 mg/mL) was prepared by dissolving each volatile oil in a mixture of propylene glycol and ethyl alcohol (1:4) (Virador et al., 1998). The stock solution was diluted in RPMI 1640 with 10% sodium dodecyl sulfate to achieve concentrations of 0, 50, 100, 200, and 400 μg.mL -1 shortly before beginning the experiments (Sylvestre et al., 2006;Cardile et al., 2009). The Jurkat cells were then seeded in plates containing 96 wells, at concentrations of 1.5 x 10 4 cells per well. They were then treated with each volatile oil at different concentrations for 72 h to investigate cell viability by MTT reduction. After this time interval, the cells were transferred to 2 mL tubes and centrifuged at 1500 rpm for 5 min. The supernatant was discarded and 200 L of a 1:10 mixture of MTT in RPMI with 10% FBS was added to each tube. The cells were kept in an incubator for 30 min and centrifuged again at 2500 rpm for 5 min. The supernatant was discarded and 200 L of DMSO was added in order to solubilize the purple formazan dyes resulting from the MTT reduction. The color of the supernatant was measured using a UV/Vis Shimadzu-1601 spectrophotometer at 550 nm. Absorbance values were used to calculate the percentage of cell viability as per the following equation: V% = (Abs A/Abs C) x 100, where V% is the cell viability percentage, Abs A is the sample absorbance, and Abs C is the control absorbance.

Statistical analysis
The analyses were completely randomized. Insecticidal activity was tested using four treatments and nine repetitions, while cytotoxic activity was tested using five treatments for each sample and three repetitions containing 4 wells each. Results were submitted to analysis of variance (ANOVA) and compared using the Tukey test at 5% probability (P < 0.05). Origin 9.0 was used for statistical analysis.

Results
Both volatile oils from extraction 1 and extraction 2 presented lower density than water, yellow color, and similar, strong, and characteristic odors. Total yield, with respect to the mass of dry material, was calculated to be 0.070 ± 0,036% (v/w) for leaves and 0.081 ± 0,013% (v/w) for stems from P. meridionale.
The leaf volatile oil GC/MS data showed low concentrations of monoterpenes and triterpenes and 55.22% sesquiterpene and 41.34% diterpene concentrations. In both cases, sesquiterpene and diterpene hydrocarbons are more usual.
However, the stem volatile oil demonstrated concentrations of 53.77% and 39.60% for sesquiterpenes and diterpenes, respectively. Again, hydrocarbons were the typical compounds found. Monoterpenes and triterpenes were not observed in extraction 2. The identified volatile classes and their percentages are summarized in Table 1.  (Adams, 2007), IRc = retention index calculated, % = percentage of componente, (⎯) means non-detected. Source: The authors. Table 1 presents the volatile compounds extracted from leaf (extraction 1) and stem samples (extraction 2) of P.
meridionale. Considering the resulting chromatograms, these samples revealed 50 and 28 identified peaks, respectively.
In general, the volatile oils from leaves and stems of P. meridionale presented insecticidal activity against C. felis in both fumigant and topical trials.  Table 2. In general, P. meridionale volatile oils seem to be non-cytotoxic to human Jurkat leukemic T-cell line. The most substantial reductions in cell viability were 9.47 ± 0.06% and 11.32 ± 0.07% for leaf extraction and stem extraction, respectively. These values were observed for the highest concentration tested (400 μg.mL -1 ), while the other concentrations revealed lower values as illustrated in Table 3. Considering the low effect of P. meridionale volatile oils against Jurkat cell line, it was not possible to calculate the half maximal inhibitory concentration (IC50) since no cell death greater than 50% was achieved. The values of cell viability did not differ significantly within 0.05 (ANOVA and Tukey Test). 1 mean ± standard deviation; 2 mean value obtained from the cell growth control set at 100%. Source: The authors.

Discussion
Despite differences in some particular volatile compounds, this study confirmed the lack of remarkable changes in the percentage of sesquiterpenes and diterpenes obtained from P. meridionale leaves and stems. This information is supported by the fact that these two volatile samples share 13 of their major components (Table 1). Based on the chemical composition of P.
meridionale volatile oils, it was initially expected that both samples would provide similar biological activities. Ottobelli et al. (2011) found concentrations of 9.63 and 82.11% for monoterpenes and sesquiterpenes, respectively, when investigating an essential oil from Philodendron scabrum k. Krause stems. Castellar et al. (2013) observed concentrations of 24.3 and 72.6% for monoterpenes and sesquiterpenes in an essential oil from Philodendron fragrantissimum Kunth roots. Santiago et al. (2014) obtained 3.7 and 91.6% monoterpenes and sesquiterpenes in an essential oil from P.
bipinnatifidum roots. Therefore, the P. meridionale volatile oils followed the well-known profile reported for the genus, since they demonstrated low content of monoterpenes and higher amount of sesquiterpenes. However, the volatile composition of both volatile oils from P. meridionale presented a higher composition of diterpenes which was not observed in other species from Philodendron. This result suggests that the volatile composition of our species can be better classified as resin oil than essential oil.
Studies on the composition of volatile oils from Philodendron plants are still scarce. Limonene and copaene have been found in some other species of this genus, while caryophyllene has been observed in all of those studied taxa with some different stereochemical orientations and concentrations (Viana, Andrade-Neto & Pouliquen, 2002;Bezerra et al., 2002;Ottobelli et al., 2011;Castellar et al., 2013;Santiago et al., 2014;Bacchus et al., 2015;Joffard et al., 2017). Germacrene has also been investigated by Silva et al. (2016) for Philodendron maximum K. Krause, as well as by Joffard et al. (2017) for Philodendron melinonii Brongn and P. fragrantissimum. No other similarities were observed between the composition of P.
meridionale essential oils and those of other species from Philodendron.
Certain terpenes, mainly elemene, copaene, gurjunene, caryophyllene, cubebene, sesquisabinene, muurolene, germacrene, cadinol, and biformene, were detected in more than one stereochemical orientation (Table 1). These data demonstrate that there are compounds being produced through the same metabolic pathway. In spite of this chemical relationship, these stereoisomers can provoke different effects on the biological systems and can result in dissimilar properties from a pharmacological point of view.
This study verified a high concentration of ent-kaur-16-ene. This is the first report of this diterpene in Philodendron.
This compound is a secondary metabolite that is biosynthetically derived from the cyclization and subsequent modification of the bicyclic carbon backbone of geranylgeranyl pyrophosphate via oxidation, reduction, acetylation, methylation, and glycosylation (Toyomasu & Sassa, 2010;Riehl et al., 2015). This compound is also a precursor of steviol glycosides.
Many compounds obtained through metabolically processing of ent-kaurene present anticancer activityfor example, in SK-HEP1 human hepatocellular carcinoma cells activated by adenosine monophosphate-activated protein kinase and cytochrome p53 and inhibited by nuclear factor kB and the telomerase enzyme (Riehl et al., 2015). Ding et al. (2010) correlated  (Table 3), allowing for cellular development. According to Sylvestre et al. (2006), IC50 values of 200-300 μg/mL are indicative of very weak cytotoxic activity. Taking all these into account, one might suggest that a substance, though predominant, does not present the same effect when it is isolated.
Therefore, it can be hypothesized that other volatile compounds present in the P. meridionale essential oils provided a synergic protective effect against Jurkat cell death which supported the high results of cell viability experimentally observed (Table 3).
The KT50 results demonstrate remarkable insecticidal potential (Table 2) by the fumigant activity and the topical application, even when compared to other studies (Yang et al., 2004;Toloza et al., 2006;Toloza et al., 2008). Yang et al. (2004)  Secondary metabolites play an important role in the resistance of plants to insects. According to Ibrahim et al. (2001), they can yield contact toxicity by penetrating the cuticle of the insect, fumigant activity by entering through the respiratory system, and ingestant effects by moving through the digestive system. The chemical features of essential oils contribute to their insecticidal activity, particularly in the toxokinetic and toxicodynamic stages of penetration, distribution, metabolism, and interaction with the site of action. This is due to the lipophilicity of the molecules present, which allows easy permeability of the insect integument with respect to topical action. In addition, these compounds are characterized by their high vapor tension, enabling fumigant activity (Sfara, Zerba & Alzogaray, 2009). Ellse and Wall (2014) attested that the fumigant action is connected to a neurotoxic pathway, rather than simply a mechanical one. This was confirmed by Genovese, (2012), who pointed out the possible interference of essential oils as neuromodulators of octopamine and gamma amino butyric acid receptors. In addition, the natural hydrophobicity of the oils could have mechanical effects on the parasites by preventing them from excreting water by perspiration, thereby resulting in osmotic stress or suffocation (Ellse & Wall, 2014;Burgess, 2009). This effect could also competitively inhibit the enzyme acetylcholinesterase by occupying the hydrophobic area in its active center (Ryan & Byrne, 1988). Finally, insect feeding behavior depends on integrating the central nervous system with chemoreceptors located in the legs, parts of the mouth, and oral cavity. Insecticides might therefore act on these chemoreceptors, thereby inhibiting feeding; this is characterized as an antifeedant activity (Santiago et al., 2014). Schmelz et al. (2014) stated that the sequential activity of the enzyme ent-kaurene synthase produces diterpene phytoalexins that present antifeedant activity in insects. This information highlights both its nature as a defense mechanism of the plant as well as its mechanism of action as a bio-pesticide. In this case, in addition to its own insecticidal effect, ent-kaur-16-ene could serve as a precursor to substances that act as insecticides. Ultimately, the concentrations and synergetic or complementary actions resulting from the mixture of terpenes that are present in P. meridionale volatile oils play an extremely important role in their insecticidal activity (Ibrahim et al., 2001;Yang et al., 2004;Izumi et al., 2013;Ellse & Wall, 2014).

Mclean and Khan
In that sense, the great finding of this paper is related to the confirmation of an effective insecticidal activity against the parasite C. felis with no pronounced effects on cell viability of human Jurkat leukemic T-cells. Thus, it is possible to propose a biological selectivity to the volatile oils under study, which guarantees, at the same time, effect against this insect with no evident damage to the human biological system.
In addition, this study shows that P. meridionale volatile oils work against ectoparasites, e.g. C. felis, and that they can be used as an alternative to more traditional insecticides like organochloride, organophosphate, and pyrethroid compounds whose use has been restricted due to their harmful environmental and human health effects. Moreover, they are also feasible compounds to be used in organic farming (Ellse & Wall, 2014). However, not all plants have the ability to accumulate volatile oils. On the other hand, P. meridionale has particular anatomical structures, such as secretory cells (idioblasts), cavities and ducts focused on producing natural products (Swiech et al., 2016) that can be successfully used to obtain volatile oils. In that sense, this work paves the way for using P. meridionale volatile oils as raw materials to obtain novel insecticidal products to treat animal and human ectoparasites.

Final Considerations
Volatile oils from P. meridionale are an important source of terpenes, mainly sesquiterpenes and diterpenes, which demonstrates a remarkable potential for the development of veterinary and human medicines against fleas. It is possible that the major volatile compound found in P. meridionale leaves and stems, ent-kaur-16-ene, is partially responsible for the insecticidal effects obtained in this study; however, the synergistic effect of all other volatile constituents appears to be important.
These volatile oils could be further used as bio-pesticides, given that they showed high efficacy in combating C. felis both topically and as a fumigant agent. Furthermore, they did not change the enzymatic activity of the mitochondrial/nonmitochondrial succinate dehydrogenase of human Jurkat leukemic T-cells, a primary target that could lead to cell death. As such, they also demonstrated safety to be applied on animals and humans with no risk of cell damage.
Future studies may address other biological and toxicological actions, as well as the insecticidal action on other adult insects ora t stages of development.