Introduction

Bursera graveolens (Kunth) Triana & Planch, (Burseraceae) is a tree (3-15 m) with a bark of reddish-brown color, alternate leaves grouped at the tips of their branches and unisexual flowers (Muñoz-Acevedo, Serrano-Uribe, Parra-Navas, Olivares-Escobar, & Niño-Porras, 2013). The tree can be seen in Figure 1. It is a native species of tropical America (from south Mexico down to Peru). The different parts of the plant, e.g., leaves, stems and resin, have been used in folk medicine (Peru, Costa Rica, Nicaragua, Guatemala, Cuba, Colombia), in different forms (smoked, infusions, cataplasms, compresses), as healing, abortive, anti-inflammatory, anti-tumor, analgesic, antidiarrheal, depurative, diaphoretic, expectorant, insecticidal, for the treatment of anemia, rheumatism, dermatitis, asthma and colic as well as a mosquito repellent (Nakanishi et al., 2005; Zúñiga et al., 2005; Monzote, Hill, Cuellar, Scull, & Setzer, 2012). Essential oil obtained from B. graveolens has shown diverse activities as an anti-inflammatory (Zuñiga et al., 2005; Manzano-Santana et al., 2009), antiproliferative (Monzote et al., 2012), antioxidant and antimicrobial agent (Andrade-Santiago et al., 2016; Sotelo-Mendez, Figueroa-Cornejo, Césare-Coral, & Alegría-Arnedo, 2017).

Essential oils (EO’s) are a mixture of chemical compounds of an organic nature, which are found as end products of the secondary metabolism of aromatic plants, which are responsible for their characteristic aromas and bioactivities (Andres et al., 2017). Biological pesticides consisting mainly of essential oils have been shown to exert fungicidal, insecticidal and bactericidal effects, among others; therefore, their use in agroindustry has increased exponentially, what has been known as green alternatives in recent years (Prieto, Patiño, Delgado, Moreno, & Cuca, 2011; Benelli et al., 2017). Essential oils have been specially investigated as natural insecticides against several insects from stored products, and many studies have used the red flour beetle Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) as a biological model. It is a pest from stored products of great economic importance that is found in grain and flour at homes and stores. T. castaneum is a secondary pest of a wide variety of cereals, legumes, oilseeds, cakes, nuts, spices and products of animal origin. It abounds in warm and dry conditions (Manivannan, 2015).

There are various reports of the fumigant and repellent activities of essential oils on T. castaneum (Stefanazzi, Stadler, & Ferrero, 2011; Jaramillo-Colorado, Martelo, & Duarte, 2012; Taban, Saharkhiz, & Hooshmandi, 2017; Candy, Nicolas, Andriantsoanirina, Izri, & Durand, 2018; Hu, Wang, & Dai, 2019). However, there are no reports on B. graveolens and its components. This study aimed to determinate the volatile chemical composition of B. graveolens essential oil and evaluate its repellent and fumigant activities.

Material and methods

Vegetal material

Fresh leaves from B. graveolens, were collected at the locality of Malagana, municipality of Mahates, Bolivar, Colombia. They were collected in January of 2016. Identification taxonomy was carried out at the Institute of Biology of the Faculty of Exact and Natural Sciences of the University of Antioquia. The EO’s from B. graveolens were stored at the Agrochemical Research Laboratory according to the GIA 015 code.

Extraction of essential oils

The hydro-distillation method was used for the extraction for B. graveolens, using clevenger-type equipment, according to the procedure described by Jaramillo-Colorado, Duarte-Restrepo, and Jaimes (2016). 500 g of fresh leaves, stems, finely chopped and submerged in 2 L of water were used. The extraction time was 2 hours. Essential oil (EO) was separated from the water by decantation. The extractions were done three times. For each EO, an aliquot of 30 μL was taken and diluted in 1 mL of dichloromethane for chromatographic analyzes.

Gas chromatography analysis

Essential oils (20 µL mL^-1) were analyzed by means of Agilent Technologies 4890D (Palo Alto, California, USA) gas chromatography equipment (GC) equipped with a split/splitless (250°C, ratio split 1:30) injection port and a flame ionization detector (FID) (280°C). An Agilent 6890N GC coupled to an Agilent 5973N Mass Detector (electron ionization, 70 eV) was used for GC-MS analysis equipped with HP-5 capillary columns (phenyl-dimethylpolysiloxane, 0.2 µm film thickness) 25 m × 0.2 mm id). Working conditions were as follows: injector temperature 260°C; column temperature 70-190°C, 5°C min.^-1. The carrier gas was helium (99.995%, Linde, S.A) with a linear velocity of 35 cm s^-1. The temperature of the transfer line connected to the mass spectrometer was 280°C; Oven temperature 40 (15 min.) up to 250°C (15 min.) @ 5°C min.^-1. For identification of compounds, standard terpenes were used, by analyzing under the same instrumental conditions and by comparing the mass spectrum with those available in the Wiley Mass Spectral Database library. The Mass spectral and retention indexes obtained were compared with the literature reported (Adams, 2007).

Insects and bioassays

Adults of T. castaneum used in the experiments were collected seven days after hatching. Bioassays were carried out in the dark in incubators at 28-30ºC and 70-80% relative humidity (r.h) at the Agrochemical Research laboratory of the University of Cartagena. Oat (Avena sativa) was employed to feed T. castaneum.

Repellent activity

The repellent activity was performed according to Tapondjou, Adler, Fontem, Bouda, & Reichmuth (2005). The experimental method was evaluated using the area preference method. Whatman No. 1 filter papers (diameter 9 cm) were cut in half. The essential oil from B. graveolens was dissolved in acetone.

The concentrations chosen to evaluate the repellent activity were 0.001, 0.01, 0.1 and 1%. A volume of 0.5 mL of each essential oil solution was applied slowly and uniformly to one half of each filter paper, while the other half was treated with an equal volume of acetone alone as a control. The treated and control half discs were dried at room temperature in order to allow evaporation of the solvent. Treated and untreated halves were attached using adhesive tape and placed in Petri dishes. Twenty adults (5-7 day old) of T. castaneum were released separately at the center of each filter paper disc. The dishes were then covered and transferred to an incubator at room temperature. Five replications were used for each concentration.

Fumigant activity

The toxic effect of B. graveolens EO’s was tested on T. castaneum adults. Filter paper discs (Whatman No. 1, 2 cm diameter), deposited at the bottom of Petri dish covers (90 x 15 mm) were used. These were impregnated with oil at doses calculated in such a manner that equivalent fumigant concentrations of 500, 350, 250, 150, 50 µg of oil mL-1 air, respectively, were given. Twenty adult insects (1 to 10-d-old) were introduced and tightly capped (replicated four times for each concentration). Pirilan, a commercial insecticide containing methyl pirimiphos (organophosphorus compound, 300 μg mL^-1 air) as an active ingredient was used as positive control. The mortality percentage was determined after 24 hours from the start of exposure (Prieto et al., 2011).

Statistical analysis

The data presented in terms of repellent activity are submitted as the mean ± standard deviation through an ANOVA statistical formula and a t-test of for students; as for the fumigant activity, the mean lethal concentration (LC₅₀ = dose at which 50% mortality of the insect population is produced) was calculated by a linear regression analysis using the STATGRAPHIC Centurion XVI statistical program, version 16.1.03.

Results

The yield of essential oil from B. graveolens was 0.3%. The main components found in the oil were monoterpenes: Limonene (42.2%), pulegone (20.9%), carvone (7.5%), Caryophyllene (4.1%), and trans-carveol (3.8%); see Table 1.

Repellent activity of essential oil

The results of the repellent activity from B. graveolens EO’s are presented in Table 2. The oil from B. graveolens exhibited repellent activity against the Tribolium castaneum weevil at a concentration of 1% at 2 and 4 hours of exposure (88.1 and 88.6% respectively). Limonene (standard), the major compound present in the oil, had the highest repellent activity at a concentration of 1% at 2 and 4 hours of exposure (100%); this had a repellent capacity greater than that presented by the commercial repellent, Ethylbutylacetylaminopropionate (approx. 78%). Caryophyllene exhibited a weak repellent effect, as shown in Table 2.

Fumigant activity

EO from B graveolens exhibited 95 and 100% of mortality on T. tribolium at 350 and 500 µg EO mL-1 of air, respectively, which is similar to the commercial pesticide methyl-pirimiphos (see Figure 2). However, the essential oil from B. graveolens (LC₅₀ = 108.2 μg mL-1 of air) is not more toxic than methyl-pirimiphos (LC₅₀ = 87.4 μg m-1 of air) versus T.castaneum (as deduced by the LC50 confidence intervals in Table 3). On the other hand, limonene, the main compound in the EO of B. graveolens was more toxic (LC50 = 189.6 μL L^-1) than caryophyllene (LC₅₀ = 472.6) on T. castaneum (Table 3).

Discussion

EO from Bursera graveolens has monoterpenes as its principal compounds: Limonene (42.2%), pulegone (20.9%), carvone (7.5%), caryophyllene (4.1%), and trans-carveol (3.8%). Figure 3 shows the structure of its major components. Other investigations carried out in Havana (Cuba) reported, for the EO of B. graveolens, compounds such as limonene (30.7%), (E)-β-ocimene (20.8%) and β-elemene (3.0%) (Carmona, Quijano-Celís, & Pino, 2009). Young, Chao, Casabianca, Bertrand, and Minga (2007) found limonene (58.6%) and a-terpineol (10.9%) in the essential oil from Ecuadorian B. graveolens. Muñoz-Acevedo et al. (2013) described germacrene D (20.7%), caryophyllene (18.0%), viridiflorol (8.0%), limonene (6.6%), linalool (6.5%) and dendrolasin (5.3%) in volatile fractions from leave extracts of B. graveolens. Luján-Hidalgo et al. (2012) reported limonene (42.90%), β-ocimene (17.39%), β-elemene (11.82%), menthofuran (6.79%) as major compounds for Mexican B. graveolens.

These divergences related to the composition of essential oils are attributed to different causes, including variations in ecological conditions (climate, type of soil, season of the year, geographical location) in which the plant develops; (extraction method, time, raw material) which can produce qualitative and quantitative changes in the oil (Figueiredo, Barroso, Pedro, & Scheffer, 2008; Andrade et al., 2008). Table 4 shows the major compounds present in B. graveolens EO’s from Cuba, Ecuador, Mexico, Peru and Colombia (department of Atlántico).

This work showed that the essential oil of B. graveolens had repellent and fumigant activities. Many researchers explain the differences in repellent properties between the essential oil and the individual components, among which are the molecular structure, synergistic effect and different species reaction (Ju-Qin et al., 2018; Saad, El-Deeb, & Abdelgaleil, 2019). Malacrino, Campolo, Laudani, & Palmeri (2016) reports a higher repellent activity of R-(+)-limonene compared to the other enantiomer versus Triboliumconfusum. Monoterpenes and phenylpropenes have exhibited significant repellent effects on adults of T. castaneum, among which (-)-menthone, trans-cinnamaldehyde, and α-terpinene are notable (Saad et al., 2019).

Studies show that the fumigant properties of essential oils are associated with the presence of mono and sesquiterpene compounds. Terpenes can be toxic due to their penetration of the insect cuticle (contact effect), respiratory system (fumigant effect) and via the digestive apparatus (ingestion effect) (Ibrahim, Kainulainen, & Aflatuni, 2001).

Plant-based pesticides are considered attractive alternatives to synthetic pesticides, as they usually are safer due to their rapid biodegradation and consequent short-term persistence in the environment (Isman, 2006; 2008). The action of some terpenes is similar to that of the organophosphorus and carbamate compounds present in some conventional insecticides, inhibitors of the enzyme acetylcholinesterase, which causes rapid death due to respiratory failure in certain insects from stored grain (López & Pascual-Villalobos, 2010; Abou-Taleb, Mohamed, Shawir, & Abdelgaleil, 2016). For example, limonene is a component of citrus essential oils recommended for controlling scale insects in ornamental plants and agricultural activities in the United States (Hollingsworth, 2005). Terpinen-4-ol, 1,8-cineol, linalool, R - (+) - limonene and geraniol were tested in vapor form against different stages of Tribolium confusum (Stamopoulos, Damos, & Karagianidou, 2007). Kim, Kang, & Park (2013) reported α-pinene as the most potent inhibitor of AchE activity followed by β-pinene and limonene, while Saad et al. (2019) reported in a fumigant toxicity assay claiming that (-)-terpinen-4-ol (LC₅₀ = 20.47 μl l air^-1) and α-terpinene (LC₅₀ = 23.70 μl l air^-1) exhibited the highest toxicity without any significant differences between them. Moreover, (-)-menthone and p-cymene showed strong toxicity. Chaubey (2012) found that 1-8-cineole was most effective against Sitophilus oryzae and Tribolium castaneum. Pulegone also displayed strong fumigant toxicity against adults of S. zeamais and T. castaneum (7 day LC₍₅₀₎ = 3.47 and 11.56 mg cm^-3, respectively) Liu, Chu, & Jiang (2011).

Conclusion

The repellent and fumigant activity of B. graveolens essential oil against T. castaneum (stored-product insect) was tested in this work. EO and limonene were more active than commercial repellent (Ethylbutylacetylaminopropionate). The results demonstrate that B. graveolens EO and limonene have the potential for the development of natural fumigants and repellents.

Acknowledgments

Thanks to the Agrochemical Research Group. Thanks to Palacio-Herrera F.M for their technical support. The authors acknowledge the support provided by the University of Cartagena, the Support Research Groups Program (2014-2019), sponsored by the Vice-Presidency for Research at the University of Cartagena.

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