Bioactive Compounds and Insecticidal Activity of Hysterionica pinifolia, a Native South American Plant

Ingrid M. Cufre^1,2*, Lucas E. Fabián^2,3, Sandra V. Clemente⁴, Arnaldo L. Bandoni^1,2, Adriana M. Broussalis^1,2

¹Facultad de Farmacia y Bioquímica, Catedra de Farmacognosia, Universidad de Buenos Aires, Buenos Aires, Argentina.
²CONICET, Instituto de la Química y Metabolismo del Fármaco (IQUIMEFA), Buenos Aires, Argentina.
³Facultad de Farmacia y Bioquímica, Catedra de Química Medicinal, Universidad de Buenos Aires, Buenos Aires, Argentina.
⁴Facultad de Agronomía, área de Producción Vegetal Orgánica, Universidad de Buenos Aires, Buenos Aires, Argentina.

DOI: 10.4236/ajps.2022.136055                

Abstract

Phytochemical investigation of the purified fractions of the active dichloromethane extract of Hysterionica pinifolia (Poir.) Baker led to the identification of five compounds. New acetylenic alcohol (E)-undec-3-en-5,7-diyne-1-ol (1) and three other compounds (3), (4), and (5) were reported for the first time in this species. Furthermore, forty-six components from the volatile fraction of H. pinifolia were identified. These compounds were elucidated using 1D and 2D NMR spectroscopy as well as MS-ESI and GC-FID-MS experiments. The dichloromethane extract, its fractions, and the methanolic extract were tested for insecticidal activity against Tribolium castaneum under laboratory conditions. The dichloromethane extract and the fraction F2 were found to be active, showing high larval mortality. The dichloromethane extract was also active against T. castaneum adults. The results have shown that H. pinifolia could be considered, in a near future, as a potential source for the development of a botanical insecticide for pest control.

Keywords

Hysterionica pinifolia, Bioactive Compounds, Polyacetylene Compounds, (E)-undec-3-en-5, 7-diyne-1-ol, Insecticidal Activity

(E)-undec-3-en-5,7-diyne-1-ol, Insecticidal Activity

1. Introduction

Hysterionicapinifolia (Poir.) Baker (Asteraceae) is a perennial subshrub, native to the Argentine flora. It grows in hilly areas of the province of Buenos Aires, between 0 and 800 meters above sea level, and in other South American countries such as Brazil and Uruguay [1]. Previous investigations on this plant have led to the identification of acetylenic compounds [2] but no reports of biological activities were found for this species.

At the moment, there is growing concerned about the negative effects on human health, and the environment due to the widespread and indiscriminate use of conventional insecticides [3] [4]. For that reason, obtaining and characterizing new molecules of plant origin with insecticidal activity is, currently, one of the main research focuses for pest control [5] [6] [7].

In this work, we report the identification of a new acetylenic alcohol and four other compounds from the active CH₂Cl₂ extract of H. pinifolia. The insecticidal activity of the CH₂Cl₂ extract, its fractions, and the CH₃OH extract were assayed against Triboliumcastaneum Herbst, the red flour beetle, a stored grain pest.

2. Materials and Methods

2.1. Plant Material

Aerial parts of Hysterionica pinifolia were collected in Tandil, Buenos Aires province (west 59.1369, south 37.3286), Argentina, in March 2016 and were authenticated by Dr. Gustavo Giberti. A voucher specimen (BAF 858) was deposited in the Pharmacobotanical Museum of the Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Argentina. The material was dried at room temperature and finely powdered.

2.2. Extraction and Bioguided Fractionation

The powered aerial parts (100 g) were extracted at room temperature by maceration using CH₂Cl₂ (1000 ml) for 1 h under continuous shaking. This procedure was repeated 5 times changing the solvent each time. These extracts were gathered. The plant residue was then extracted using the same volume of CH₃OH. After evaporation of the solvent, 4.6 and 8.1 g of each extract were obtained respectively.

A portion of the active CH₂Cl₂ extract (2.0 g) was fractionated by a preparative chromatography column (60 cm × 5 cm) using silica gel 60 MN (70 - 230 mesh, Macherey-Nagel, Germany), eluted with a gradient mixture of hexane (Hx)/ethyl acetate (EtOAc) (100:0 to 50:50, v/v), then EtOAc/CH₃OH (100:0 to 0:100, v/v). The chromatographic analyses were made by Thin Layer Chromatography (TLC) on silica gel F254 (MERCK-Germany), developed by different solvent systems and the spots were visualized under UV light at 254 and 366 nm. Four fractions (F1 to F4) were collected based on their TLC profiles. The insecticidal activity was found to be concentrated only in F2 (Table 4). Then, F2 (313 mg) was rechromatographed on an analytical column (45 cm × 3 cm) using silica gel 60 MN (70 - 230 mesh, Macherey-Nagel, Germany) and was eluted with gradient mixtures of Hx/ethyl ether (Et₂O) (100:0 to 50:50, v/v), then Et₂O/CH₃OH (100:0 to 0:100, v/v). Fifteen subfractions (F2-A to F2-O) were obtained. After the TLC analysis, the cromatographic profile of the subfractions F2-C and F2-E showed the main compounds present in the active fraction F2. For this reason, the subfractions F2-E and F2-C were selected for the identification of their bioactive compounds.

2.3. Volatile Fraction Obtention

Volatile fraction was obtained by hydrodistillation of naturally air dried aerial parts during 3 h in a Clevenger-type trap [8]. The resulting volatile fraction was dried over anhydrous sodium sulfate and stored at 2˚C prior to GC-FID-MS analysis

2.4. Compounds Identification

The identification of the compounds in the subfractions F2-E and F2-C was conducted by GC-FID-MS, ¹H and ¹³C NMR 1D and 2D and MS-ESI. The identification of unknown compounds in complex mixtures using the combination of 1D and 2D NMR and MS, without the need for prior isolation, allows rapid identification [9] [10].

2.4.1. NMR and MS Analysis

¹H and ¹³C-NMR spectra and homo- and heteronuclear correlation spectroscopy experiments were recorded in chloroform-d₃ (CDCl₃) on an Avance II 500 Bruker at 500 and 125 MHz. Chemical shifts (δ) are reported as ppm based on the tetramethylsilane signal.

Mass spectra were measured on a Bruker micrOTOF-Q II spectrometer, ionization was performed by electrospray (ESI) in positive mode.

2.4.2. GC-FID-MS Analysis

GC-FID-MS analysis was performed using a GC-FID-MS Agilent 7890A/5975C equipped with one injector (split ratio 1:100) connected by a flow splitter to two capillary columns (HPWAX and HP-1, both 60 m × 0.25 mm with 0.25 microns of fixed phase). The polar column was connected to a FID, whereas the non-polar column was connected to a quadrupolar mass detector (HP 5975C) (70 eV). Helium was used as gas carrier, at 1.8 mL/min. The injector temperature was set at 250˚C. The column temperature was programmed according to the following gradient: 100˚C, increasing at 2˚C/min to 240˚C and kept constant for 15 min. FID temperature was 260˚C, and temperatures for the transference line and the ionic source were set at 280 and 230˚C, respectively. Mass range (m/z) was 40 - 500 Da. Data acquisition, processing and instrument control was performed using the Agilent Chem Station software.

2.4.3. GC-FID-MS Identification

The identification of the compounds was achieved by analyzing the retention indexes (relative to C8-C24 n-alkanes) obtained in both columns and compared with those of reference compounds, compounds identified in chemically well-known essential oils and from bibliography [11] [12]. Additionally, each mass spectra obtained was compared to those from the literature libraries [11] [13] [14] and mass spectra obtained from reference compounds.

Relative percentage contribution of the compounds was calculated from the FID responses by a computerized integration assuming all of the responses factors were 1.

2.5. Insecticidal Activity Bioassay

2.5.1. Insect Rearing

T. castaneum Herbst. (Coleoptera: Tenebrionidae) larvae from an established laboratory colony in the Organic Plant Production Laboratory, Faculty of Agronomy, University of Buenos Aires, Argentina, were employed. T. castaneum larvae were reared with an artificial diet (wheat flour, beer yeast, cornstarch 10:1, 5:10) and environmental standard conditions (25˚C ± 1˚C, 60% ± 5% RH, in darkness). This laboratory strain is susceptible to all insecticides [15].

2.5.2. Larvicidal Activity Ingestion Bioassay

The insecticidal activity of the CH₂Cl₂ extract, its fractions (F1 to F4) and the CH₃OH extract were investigated.

The concentrations employed for the determination of larvicidal activity were: CH₂Cl₂ and CH₃OH extracts: 5.0 to 25.0 mg/ml and the fractions (F1 to F4): 25.0 mg/ml. Two grams of the artificial diet were mixed with 1.0 ml of each acetonic solution of H. pinifolia extracts and fractions. The solvent was evaporated from the diet at room temperature during 24 h. Then, ten neonate larvae of T. castaneum were placed in each glass vessel containing a treated artificial diet. All tests were performed in quadruplicate with acetone as a negative control. The bioassay was conducted for nine weeks under standardized conditions (25˚C ± 1˚C and 60% ± 5% RH). The mortality at each stage of the life cycle of the red flour beetle as well as the overall mortality was weekly assessed. The sublethal effects expressed as delays in the development of the insect produced by the extracts and fractions were also evaluated [15].

2.5.3. Study of the Survival of T. castaneum Adults

Ten adults of T. castaneum were added to each glass vessel containing two grams of artificial diet previously mixed with an acetonic solution of H. pinifolia CH₂Cl₂ extract. The concentrations employed for the study of the survival of T. castaneum adults were: 25.0 and 50.0 mg/ml. The number of surviving insects was observed and recorded for 35 days or until the death of all the individuals. Insects were considered dead when there were no responses to tactile stimuli. All tests were performed in quadruplicate with acetone as a negative control [16].

2.5.4. Statistical Analysis

Statistical differences in mortality (p ≤ 0.05) were calculated with ANOVA and Tukey’s multiple range tests [17]. Sublethal effects and EC₅₀ values were calculated by Probit analysis using a computer program [18].

3. Results and Discussion

3.1. Phytochemical Analysis

In order to identify the bioactive compounds, the subfractions F2-E and F2-C from the active CH₂Cl₂ extract of H. pinifolia were analyzed. The analysis was conducted by GC-FID-MS, ¹H and ¹³C NMR 1D and 2D experiments (COSY, TOCSY, HSQC, HMBC) and MS-ESI. The compounds were identified in the subfractions using the combination of 1D and 2D NMR and MS, without the need of prior isolation, wich allowed to a rapid identification [9] [10].

A new acetylenic alcohol was identified in subfraction F2-E. The molecular formula was assigned as C₁₁H₁₄O₁ on the basis of its MS-ESI analysis, which showed a protonated molecular ion peak at m/z 145.10 [M + H]⁺ (calcd for C₁₁H₁₄O₁, 162.23) (FigureS1). This molecular ion was obtained by dehydration of the alcohol functional group under electrospray ionization conditions [19]. In the ¹³C-NMR spectrum the signal of a carbon linked to a hydroxyl group at δ_C 72.11 ppm, the signals of alkenes carbons at δ_C 148.47 and 108.83 ppm and the signals of alkyl carbons at δ_C 75.02, 72.99, 65.19 and 84.42 ppm were observed. Aliphatic carbon signals δ_C 36.87, 21.52, 21.75 and 13.41 ppm were also observed (FigureS2). The ¹H-NMR spectrum showed signals of protons from alkenes with trans isomerism at δ_H 6.28 and 6.19 ppm (J = 15.9 Hz) and signals from protons bound to aliphatic carbons at δ_H 4.19, 2.32, 1.60, 1.56 and 1.02 ppm (FigureS3). The ¹H and ¹³C NMR spectra showed the signals corresponding to an acetylene skeleton. These compounds are characterized by the presence of two or more triple bonds [20]. With the aid of ¹H-¹H COSY, TOCSY, HSQC and HMBC spectroscopic data, all protons and carbons were fully assigned (Table 1, Figures S4-S7). Analysis of the ¹H-¹H COSY and TOCSY spectrum allowed to determine the connection between the protons and led to the establishment of two spin systems (H-1 to H2/H3/H4 and H9 to H10/H11) (Figure1). Furthermore, the HMBC correlations between C1/H3 and H4; C3/H1; C4/H1; C5/H4 and H9; C6/H3, H4 and H9; C7/H9; C8/H9 and H10; C9/H10 and H11; C10/H9 and H11 and C11/H9 and H10, confirm the structure of this new polyacetylene alcohol as (E)-undec-3-en-5,7-diyne-1-ol (1). This compound is reported for the first time in H. pinifolia and no reports have been found in other species.

The major compound, cis lachnophyllum methyl ester (2), was identified in subfraction F2-C by GC-FID-MS (63.0%, rt: 45.45 min) and ¹H and ¹³C NMR 1D and 2D (Table 2, Figures S8-S11). In the ¹³C-NMR spectrum, signals of a carbonyl carbon at δ_C 166.8 ppm and of a methyl carbon at δ_C 51.6 ppm were observed, indicating the presence of an ester group. The alkene carbon signals at δ_C 130.7 and 122.5 ppm and the alkyl carbon signals δ_C70.8, 86.6, 65.2 and 90.1 ppm indicated the presence of one double bond and two triple bonds in the molecule. Signals of aliphatic carbons δ_C 21.60, 21.79 and 13.5 ppm were also observed (FigureS8). The ¹H-NMR spectrum showed signals of protons of alkenes with cis isomerism at δ_H 6.23 and 6.19 ppm (J = 11.4 Hz), a singlet at δ_H 3.80 ppm corresponding to the methyl protons of the ester group and the signals of the protons linked to aliphatic carbons at δ_H 2.37, 1.61 and 1.03 ppm (FigureS9). Like in compound 1 the ¹H and ¹³C NMR spectra showed the signals corresponding to an acetylene skeleton. Additionally, the heteronuclear correlation spectra HSQC (FigureS10) and HMBC allowed to fully assign the protons and carbons in the molecule (Table 2, Figure1). The HMBC correlations between C1/H2, H3 and H11; C3/H2; C4/H2, H3 and H8; C5/H2 and H8; C6/H2, H3 and H8; C7/H8 and H9; C8/H9 and H10; C9/H8 and H10 and C10/H8 and H9 (FigureS11) confirm the structure of this polyacetylene ester as cis lachnophyllum methyl ester. The results of NMR analysis agreed with the data described in the literature [21]. This compound was previously described by Bohlmann et al. [2] for this species.

The compounds eucalyptol (3), benzoic aldehyde (4) and benzyl alcohol (5) were identified in fractions F2-E and F2-C by GC-FID-MS. Eucalyptol (rt: 6.51 min) 3.50% in F2-C and 4.90% in F2-E, benzoic aldehyde (rt: 14.2 min) 4.90% in F2-C and 30.0% in F2-E and benzyl alcohol (rt: 28.69 min) 2.50% in F2-C and 13.5 % in F2-E. These compounds are reported for first time in H. pinifolia.

Figure 1. Key H-¹H COSY (blue line) and HMBC (arrows) correlations for compounds 1 and 2.

Table 1. ¹³C and ¹H NMR data and 2D correlations of compound 1 in CDCl₃.

^a Data recorded at 125 MHz. ^bData recorded at 600 MHz.

Table 2. ¹³C and ¹H NMR data and 2D correlations of compound 2 in CDCl₃.

^a Data recorded at 125 MHz. ^bData recorded at 600 MHz.

Acetylenic compounds have also been found in essential oils [22]. For this reason, the volatile fraction of H. pinifolia was obtained and analyzed. Forty-six components were identified by GC-FID-MS and these constituted the 84.2% of the volatile fraction.

The principal compounds identified were cis lachnophyllum methyl ester (38.4%) as the major constituent, trans-β-ocimene (17.5%), γ-curcumene (12.5%), ar-curmumene (4.5%), β-caryophyllene (1.9%) and geranylacetone (1.3%) (Table 3).

3.2. Insecticidal Activity Results

The insecticidal activity was evaluated on T. castaneum larvae from a laboratory strain susceptible to all insecticides. The CH₂Cl₂ extract exhibited only larval mortality with EC₅₀ value of 15.7 mg/ml (CI = 14.9 - 16.6 mg/ml) and no sublethal effects were observed (Table 4). These promising results led to a bioguided fractionation in order to identify the active compounds present in the CH₂Cl₂ extract. For that reason, the activity of the four fractions (F1 to F4) of the CH₂Cl₂ extract was evaluated. Only the fraction F2 showed lethal effects with 95% of larval mortality at 25.0 mg/ml and no sublethal effects were observed (Table 4). The mortality observed in F2 fraction was comparable to that obtained with the crude CH₂Cl₂ extract (92.5%) at the same concentration of 25.0 mg/mL (Table 4). These results showed that the insecticidal activity of the crude CH₂Cl₂ extract would be concentrated in the F2 fraction. The CH₃OH extract did not present lethal or sublethal effects (Table 4).

The high larval mortality obtained with the CH₂Cl₂ extract and the fraction F2 was very interesting due to the extensive losses in stored grains caused by the voracity of T. castaneum larvae in their different instars [23]. In addition, adults of T. castaneum also cause an important damage. Therefore, the insecticidal activity of the CH₂Cl₂ extract on T. castaneum adults was evaluated. Mortality and immobility were initially observed and, at the end of the trial, 100% mortality only at 50.0 mg/ml was obtained. In the control group, no mobility alteration or mortality was exhibited.

Table 3. Relative percentage of the main constituents of the volatile fraction of H. pinifolia.

T: trace, less than 0.05%. ^aLinear retention index on non polar column, experimentally determined using homologous series of C8-C24 alkanes. ^bLinear retention index on polar column experimentally determined using homologous series of C8-C24 alkanes.

Table 4. Insecticidal activity of the CH₂Cl₂ extract, its fractions (F1 to F4) and the CH₃OH extract against T. castaneum larvae.

Different letters indicate significant differences (p < 0.05).

The insecticidal activity showed with the CH₂Cl₂ extract and its F2 fraction could be attributed to the identified compounds. The major compound cis lachnophyllum methyl ester has reported larvicidal, nematicidal, and antifeedant activities on various pests [24] [25] [26] as well as other acetylene alcohols [27]. Additionally, the compounds eucalyptol, benzoic aldehyde, and benzyl alcohol have also reported insecticidal activity [28] [29] [30]. The cis lachnophyllum methyl ester was also the major compound in the volatile fraction. Therefore it is expected that this volatile fraction presents insecticidal activity such as the CH₂Cl₂ extract and the F2 fraction.

4. Conclusion

In the present study, the acetylenic alcohol (E)-undec-3-en-5,7-diyne-1-ol (1) was reported for the first time in H. pinifolia and no reports have been found in other species. The compounds eucalyptol (3), benzoic aldehyde (4), and benzyl alcohol (5) were also reported for the first time in this species. The volatile fraction was studied and forty-six components were identified. cis lachnophyllum methyl ester (2) was the major compound identified in the fraction F2-C and in the volatile fraction. Our results showed that the CH₂Cl₂ extract of H. pinifolia and its fraction F2 have significant insecticidal activity in T. castaneum neonate larvae. The CH₂Cl₂ extract was also active on T. castaneum adults. This is the first report of bioactivity for this species. These results allow considering H. pinifolia as a potential source of extracts, purified fractions, and eventually secondary metabolites with insecticidal activity for the development, in a near future, of a botanical insecticide for controlling pests.

Acknowledgements

We thank the NMR Service of the Chemistry and Metabolism of Drugs Institute (IQUIMEFA)—CONICET, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, especially its director Dr. Albertina Moglioni.

Funding

This work was supported financially by grants UBACYT (University of Buenos Aires, Science, and Technology) 20020170100752BA and UBATCYT 20020130100705BA of the University of Buenos Aires.

Appendix

Figure S1. Massspectrumof fraction F2-E(1).

Figure S2. ¹³CNMR spectrum of fraction F2-E(1) in CDCl₃.

Figure S3. ¹H-NMR spectrum of fraction F2-E(1) in CDCl₃.

Figure S4. COSY spectrumof fraction F2-E(1) in CDCl₃.

Figure S5. TOCSY spectrumof fraction F2-E(1) in CDCl₃.

Figure S6. HSQC spectrumof fraction F2-E(1) in CDCl₃.

Figure S7. HMBC spectrumof fraction F2-E(1) in CDCl₃.

Figure S8. ¹³C-NMR spectrum of fraction F2-C(2) in CDCl₃.

Figure S9.¹H-NMR spectrum of fraction F2-C(2) in CDCl₃.

Figure S10.HSQC spectrum of fraction F2-C(2) in CDCl₃.

Figure S11.HMBC spectrum of fraction F2-C(2) in CDCl₃.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References

[1]	Cabrera, A.L. (1963) Flora de la provincia de Buenos Aires. Colección Científica del INTA. INTA Ed, Buenos Aires.
[2]	Bohlmann, F., Burkhardt, T. and Zdero, C. (1973) Naturally Occurring Acetylenes. Academic Press, London, New York.
[3]	Cufre, I., Tarcaya, V., Broussalis, A. and Miño, J. (2014) Hybanthus parviflorus and H. bigibbosus (Violaceae): Acute and Subchronic Oral Toxicity Assessment of Hydroalcoholic Extracts in Rodents. Revista Latinoamericana de Química, 42, 50-56.
[4]	Chen, Y., Luo, J., Zhang, N., Yu, W., Jiang, J. and Dai, G. (2021) Insecticidal Activities of Salvia hispanica L. Essential Oil and Combinations of Their Main Compounds against the Beet Armyworm Spodoptera exigua. Industrial Crops and Products, 162, Article ID: 113271. https://doi.org/10.1016/j.indcrop.2021.113271
[5]	Broussalis, A., Clemente, S. and Ferraro, G. (2010) Hybanthus parviflorus (Violaceae): Insecticidal Activity of a South American Plant. Crop Protection, 29, 953-956. https://doi.org/10.1016/j.cropro.2010.06.001
[6]	Céspedes, C.L., Salazar, J.R., Ariza-Castolo, A., Yamaguchi, L., ávila, J.G., Aqueveque, P., Kubo, I. and Alarcón, J. (2014) Biopesticides from plants: Calceolaria integrifolia s.l. Environmental Research, 132, 391-406. https://doi.org/10.1016/j.envres.2014.04.003
[7]	Tarcaya, V., Di Leo Lira, P., Cufre I., González, S., Clemente, S. and Broussalis, A. (2014) Ovidia andina: actividad insecticida, extracto y compuestos bioactivos. Revista Latinoamericana de Química, 42, 89-96.
[8]	IRAM (Argentine Institute for Standardization and Certification) (1996) IRAM Standard 18729.
[9]	Bingol, K., Bruschweiler-Li, L., Cao, Y., Somogyi, A., Zhang, F. and Bruschweiler F. (2015) Metabolomics beyond Spectroscopic Databases: A Combined MS/NMR. Strategy for the Rapid Identification of New Metabolites in Complex Mixtures. Analytical Chemistry, 87, 3864–3870. https://doi.org/10.1021/ac504633z
[10]	Wang, C., He, L., Li, D., Bruschweiler-Li, L., Marshall, A. and Bruschweiler R. (2017) Accurate Identification of Unknown and Known Metabolic Mixture Components by Combining 3D NMR with FT-ICR MS/MS. Journal of Proteome Research, 16, 3774-3786. https://doi.org/10.1021/acs.jproteome.7b00457
[11]	Joulain, D. and König, W. (1998) The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. E. B.-Verlag, Hamburg.
[12]	Babushok, V., Linstrom, P. and Zenkevich, I. (2011) Retention Indices for Frequently Reported Compounds of Plant Essential Oils. Journal of Physical and Chemical Reference Data, 40, Article ID: 043101. https://doi.org/10.1063/1.3653552
[13]	Adams, R. (2007) Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. 4th Edition, Allured Publishing, Illinois.
[14]	Wiley/NIST (2008) The Wiley/NBS Registry of Mass Spectral Data. 8th Edition, J. Wiley & Sons, Inc., New York.
[15]	Clemente, S., Mareggiani, G., Broussalis, A., Martino, V. and Ferraro, G. (2003) Insecticidal Effects of Lamiaceae Species against Stored Products Insects. Boletín de Sanidad Vegetal. Plagas, 29, 421-426.
[16]	Casadío, A. and Zerba, E. (1996) Desarrollo poblacional de Triboilum castaneum (Herbst) (coleoptera: Tenebrionidae) en distintas dietas y su influencia sobre la toxicidad y resistencia a Malatión. Boletín de sanidad vegetal. Plagas, 22, 511-520.
[17]	Steel, R. and Torrie, J. (1993) Bioestadística. Principios y procedimientos. McGraw-Hill, México.
[18]	Finney, D.J. (1971) Probit Analysis. Cambridge University Press, New York.
[19]	Demarque, D., Crotti, A., Vessecch, R., Lopes, J. and Lopes, N. (2016) Fragmentation Reactions Using Electrospray Ionization Mass Spectrometry: An Important Tool for the Structural Elucidation and Characterization of Synthetic and Natural Products. Natural Products Research, 33, 432-455. https://doi.org/10.1039/C5NP00073D
[20]	Konovalov, D. (2014) Polyacetylene Compounds of Plants of the Asteraceae Family (Review). Pharmaceutical Chemistry Journal, 48, 613-631. https://doi.org/10.1007/s11094-014-1159-7
[21]	Albuquerque, M., Souza, E., Lins, M., Nogueira, N., Leda Lemos, T., Silveira, E. and Pessoa, O. (2004) Composition and Antimicrobial Activity of the Essential Oil from Aerial Parts of Baccharis trinervis (Lam.) Pers. ARKIVOC, 6, 59-65. https://doi.org/10.3998/ark.5550190.0005.608
[22]	Kumar, V., Shekhar, M. C., Tewari, G., Singh, D., Tewari, A. and Bisht, K. (2014) Chemical Composition and Antifungal Activity of Essential Oils from three Hymalayan Erigeron Species. Food Science and Technology, 56, 278-283. https://doi.org/10.1016/j.lwt.2013.12.007
[23]	Sreeramoju, P., Prasad, M. and Lakshmipathi, X. (2016) Complete Study of Life Cycle of Tribolium castaneum and its Weight Variations in the Developing Stages. International Journal of Plant, Animal and Environmental Sciences, 6, 95-100.
[24]	Kumar, V., Shekhar, M.C., Tewari, G. and Kumar, P.A. (2015) Biopesticide potential of (7R)-Trans, Trans-Nepetalactone and cis Lachnophyllum Ester in Control of Mustard Aphid, Lipaphis erysimi (kalt.). Journal Teknology, 77, 19-24. https://doi.org/10.11113/jt.v77.5981
[25]	Kimura, Y., Mori, Y., Suzuki, A. and Bayashi, A. (1981) Isolation and Identification of Two Nematicidal Substances from Roots of Erigeron philadelphicus L. and Nematicidal Activities of Their Related Compounds. Agricultural and Biological Chemistry, 45, 2915-2917. https://doi.org/10.1271/bbb1961.45.2915
[26]	Faini, F., Labbe, C. and Coll, J. (1999) Seasonal Changes in Chemical Composition of Epicuticular Waxes from the Leaves of Baccharis linearis. Biochemical Systematics and Ecology, 27, 673-679. https://doi.org/10.1016/S0305-1978(98)00131-8
[27]	Eckenbach, U., Lampman, R., Seigler, D., Ebinger, J. and Novak, R. (1999) Mosquitocidal Activity of Acetylenic Compounds from Cryptotaenia canadensis. Journal of Chemical Ecology, 25, 1885-1893. https://doi.org/10.1023/A:1020938001272
[28]	Clemente, S., Mareggiani, G., Broussalis, A. and Ferraro, G. (2007) Actividad insecticida de 1,8-cineol sobre mosca de los frutos, Ceratitis capitata Wied. (Diptera: Tephritidae). Dominguezia, 23, 29-34.
[29]	Park, I.-K. (2014) Fumigant Toxicity of Oriental Sweetgum (Liquidambar orientalis) and Valerian (Valeriana wallichii) Essential Oils and Their Components, Including Their Acetylcholinesterase Inhibitory Activity, against Japanese Termites (Reticulitermes speratus). Molecules, 19, 12547-12558. https://doi.org/10.3390/molecules190812547
[30]	Silva, D., Arrigoni-Blank, M., Bacci, L., Fitzgerald Blank, A., Nunes Faro, R., Oliveira Pinto, J. and Garcia Pereira, K. (2019) Toxicity and Behavioral Alterations of Essential Oils of Eplingiella fruticosa Genotypes and Their Major Compounds to Acromyrmex balzani. Crop Protection, 116, 181-187. https://doi.org/10.1016/j.cropro.2018.11.002