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INTRODUCTION
Plectranthus amboinicus (Lour.) Spreng. (synonyms: Colus amboinicus [Lour.]) is an herbaceous, succulent, aromatic plant with fleshy leaves, generally less than 1 m tall. Native to tropical Asia and Africa, it is cultivated in tropical areas of the world (Tramil, 2017), and it belongs to the Lamiaceae family (The Plant List, 2013). In Colombia, it is found in the biogeographic regions of the Caribbean, Pacific, Valle del Cauca, and Valle del Magdalena (0-1,700 m altitude) (Bernal et al., 2019).
P.amboinicus is a medicinal species, whose extracts and/or essential oils have various ethnobotanical uses, among which are the treatment of respiratory tract disorders as a bronchodilator and antitussive (Lopes et al., 2017); anti-diarrheal (Shubha and Bhatt, 2015); antiparasitic (Lima et al., 2014; Ramli et al., 2014); antihyperglycemic and antihyperlipidemic (Viswanathaswamy et al., 2011); anti-inflammatory (Chen et al, 2014); larvicide (Huang et al., 2019); antimicrobial and antioxidant (Ajitha et al., 2014; Gupta and Negi, 2016; Santos et al., 2016; Swamy et al., 2017); anticancer (Yulianto et al., 2016); among others.
The essential oils produced by plants contain a wide range of secondary metabolites, such as derivatives of monoterpenes, sesquiterpenes, diterpenes, aromatics, hydrocarbons, and fatty acids (Dehsheikh et al., 2020). Plants produce essential oils for various purposes; these have been considered a species of botanical pesticides, and some have been evaluated as insecticides against mosquitoes, flies, etc. (Cossetin et al., 2018; Luz et al., 2020). The biocidal effect of essential oils and pure compounds on insects can be manifested in various ways including toxicity, mortality, antifeedant activity, growth inhibitor, suppression of reproductive behavior, reduction of fecundity, and fertility (Jankowska et al., 2018). Given the diverse biological activities of P. ambicoides, the objective of this work was to evaluate its repellent and fumigant activity by using the Tribolium castaneum (Herbst) weevil as a biological model and to relate the results to the volatile chemical composition of the essential oil.
MATERIALS AND METHODS
Vegetal material
Plectranthus amboinicus (Lour.) Spreng. plants were collected in Condoto, Choco, Colombia, in 2019. Taxonomic identification was made at the National Herbarium of Colombia. The control leaves of each plant are archived as a permanent sample at the Herbarium (COL No-538449).
Extraction of the essential oil (EO)
EO was obtained through the hydrodistillation method by using Clevenger type distillation equipment (Jaramillo-Colorado et al., 2012). 500 g of leaves and stems from P. amboinicus were used, finely chopped, and submerged in boiling water by using conventional heating for 2 h. The EO was separated by decantation and then anhydrous Na2SO4 (Merck, Darmstadt, Germany) was added to the oil. One EO aliquot (30 µL) was diluted in 1 mL of dichloromethane (Panreac AppliChem, Darmstadt, Germany) for gas chromatography analysis (Jaramillo-Colorado et al., 2020).
Chromatography analysis
The EO was analyzed in an Agilent Technologies GC-MS system model 7890A Network GC coupled to a mass selective detector model 5975 (Palo Alto, CA) equipped with a split/split-less injection port (230°C, split ratio 20:1). The mass spectra were obtained by electron-impact ionization at 70 eV energy. GC conditions were as follows: A HP-5MS capillary column (30 m × 0.25 mm id × 0.25 μm df) with 5% phenyl-poly (methyl siloxane) stationary phase was used for the separation of mixtures. The initial oven temperature was 50°C for 2 min and then resumed at a rate of 5°C min. up to 250°C (5 min). The carrier gas was helium, with an inlet pressure at the head of the column of 12.667 psi at a rate of 1.172 mL min-1, at 50°C. The mass spectra and Kovàts retention indexes obtained were compared to those reported in the literature (Adams, 2007).
Insects rearing
Adults of T. castaneum were reared in oat (Avena sativa). Bioassays were carried out in the dark in incubators at 28-30°C and 70-80% relative humidity at the Agrochemical Research Laboratory of the Universidad de Cartagena.
Repellent activity
The repellent property of P. amboinicus EO was analyzed on adult specimens of T. castaneum. It was evaluated by using the area preference method. The oil was dissolved in acetone (Panreac AppliChem, Darmstadt, Germany) after preparing 5 solutions (1, 0.1, 0.01, 0.001, and 0.0001%). A 9-cm in diameter filter paper sheet was cut in half and 500 µL of each concentration was applied separately to one of the halves of the filter paper as evenly as possible with a micropipette. The other half (control) was treated with 500 µL of acetone. DEET (N, N-diethyl-toluamide) (Dr. Ehrenstorfer, Germany), which refers to a commercial repellent, was a positive control.
The treated and control media disks were dried at room temperature for 10 min to allow for the evaporation of the solvent. The treated and untreated halves were attached using adhesive tape and placed on Petri dishes. Twenty adult specimens (5 to 7 d old) of T. castaneum were placed, one by one, in the center of each filter paper disc with the help of tweezers. The dishes were then covered and, after approximately 5 min, transferred to an incubator at room temperature (Jaramillo-Colorado et al., 2020). Four replicates were used for each concentration. Weevil preference was measured for each Petri dish at 2 and 4 h of exposure.
To determine the percentage of repellency (PR), check the following equation following the parameters identified by Jaramillo-Colorado et al. (2012) (Eq. 1):
where, Nc number of insects in the control area (acetone) and Nt number of insects in the treated area (EO + acetone).
Fumigant activity
Fumigant activity was performed according to Jaramillo-Colorado et al. (2020). The toxic effect from P. amboinicus EO and terpenes were assayed on T. castaneum. Filter paper discs (Whatman No. 1, 2-cm in diameter), laid down at the bottom of Petri dish covers (90 × 15 mm) were used. These were impregnated with oil at doses calculated as to provide equivalent fumigant concentrations of 500, 350, 250, 150, and 50 µg mL-1 air of oil, respectively. Twenty adult insects (1 to 10 d old) were introduced and tightly capped (replicated four times for each concentration). Pirilan, a commercial pesticide containing methyl pirimiphos, (Syngenta, Colombia) (organophosphorus pesticide, 300 μg L-1 air) as an active ingredient, was used as a positive control. The mortality percentage was determined after 24 and 48 h from the start of exposure.
The percentage of mortality (% mortality) was calculated using the following Equation 2:
where, MT and MC are the number of dead insects in the treated and control areas, respectively.
Statistical analysis
The results were converted into repellent and fumigant percentages and analyzed by ANOVA (Kruskal-Wallis test). Mortality rates were calculated by using the statistical formulas of Abbott and Probit to determine the LC50, chi-square values, and related parameters. Biostat, a statistical software (Analyst Soft Robust Business Solutions, BioStat v 2009) was used, with a confidence level of 5%. Four replicates for each analysis were performed.
RESULTS AND DISCUSSION
The essential oil of P. amboinicus obtained by hydrodistillation presented a yield of 0.2% (w/w). Table 1 shows the major compounds found in the EO of P. amboinicus, extracted by hydrodistillation. Fifteen compounds with a relative area greater than 0.5% were found, where the main analytes were 3-hexen-2-ol- (z) - (0.59%), 1-octen-3-ol (1.97%), 2-carene (0.50%), p-cymene (3.48%), γ-terpinene (0.54%), 4-terpineol (2.53%), carvacrol (75.88%), isothymol (0.57%), β-Guaiene (2.1%), α-bergamotene (4.4%), humulene (2.7%), α-murolene (1.3%), β-bisabolene (0.50%), caryophyllene oxide (2.39%).
Table 1. Major compounds found in the essential oil of P. amboinicus, obtained by GC-MS.
| Peak, No. | Compound | TR (min) | Molecular ion | Ik (HP-5) | Relative area (%) |
|---|---|---|---|---|---|
| 1 | 3-Hexen-2-ol | 4.24 | 100.16 | 857 | 0.6±0.08 |
| 2 | 1-Octen-3-ol | 6.54 | 128.21 | 979 | 2.0±0.50 |
| 3 | 2-Carene | 7.36 | 136.23 | 1010 | 0.5±0.05 |
| 4 | p-Cymene | 7.57 | 134.22 | 1048 | 3.5±0.50 |
| 5 | γTerpinene | 8.38 | 136.23 | 1063 | 0.6±0.22 |
| 6 | 4-Terpineol | 11.66 | 154.25 | 1089 | 2.5±0.50 |
| 7 | Carvacrol | 16.03 | 150.22 | 1298 | 75.9±1.20 |
| 8 | Isothymol | 16.99 | 150.22 | 1299 | 0.6±0.09 |
| 9 | Caryophyllene | 18.42 | 204.35 | 1420 | 1.0±0.22 |
| 10 | -Bergamotene | 18.54 | 204.35 | 1431 | 4.4±0.20 |
| 11 | -Humulene | 18.79 | 204.35 | 1440 | 2.7±0.50 |
| 12 | -Guaiene | 19.28 | 204.35 | 1455 | 2.1±050 |
| 13 | -Muurolene | 20.13 | 204.35 | 1499 | 1.3±0.60 |
| 14 | -Bisabolene | 20.23 | 204.35 | 1509 | 0.5±0.05 |
| 15 | Caryophyllene oxide | 21.69 | 220.35 | 1582 | 2.4±0.60 |
TR: Retention time. Ik: Kováts index performed in apolar column HP-5 (5% phenyl -95% polymethyl siloxane) (30 m × 0.25 mm di × 0.25 um df).
The main compound found in this oil was carvacrol, which is a phenolic monoterpenoid that has a wide range of bioactivities, such as clinical applications (antioxidant, antimicrobial, and anticancer properties), (Sharifi-Rad et al., 2018), repellent, acaricide (Tabari et al., 2017, 2015), and insecticidal (Youssefi et al., 2019), among others.
The chemical composition in this study differs slightly from the results obtained through other essential oils of P. amboinicus from other countries, i.e., in the EO from Paraiba, Brazil, where the principal compounds found were carvacrol (33.50), p-cymene (28.20%) and γ-terpinene (14.77%). While a study in India reported the caryophyllene, caryophyllene oxide, aromadendrene oxide, and selinene as the majority components (Vishnu et al., 2021), in Malaysia the main constituents of the P. amboinicus EOs were carvacrol (43-45%), γ-terpinene (11-16%), and p-cymene (12-16%) (Arumugam et al., 2020), and in Taiwan, these were was carvacrol (61,5%), β-Caryophyllene (12.79%), γ-terpinene (8.51%) and p-cymene (9.42%) (Huang et al., 2019). The variation in the proportion and yield percentage of essential oils and their chemical composition can be due to the influence of agroecology and environmental conditions (Aguiar et al., 2015).
The results of the repellent activity of the essential oil of P. amboinicus and a commercial repellent against T. castaneum, are shown in table 2.
Table 2. Repellent activity of the essential oil of Plectranthus amboinicus, and Stay off Amazonic, a commercial repellent (DEET) against Tribolium castaneum, at different exposure times.
| Essential oil (EO) | Concentrations (%) | Repellent activity (%)a | |
|---|---|---|---|
| 2 h | 4 h | ||
| P.amboinicus | 1 | 78.67±5.51 | 84.67±5.42 |
| 0.1 | 83.33±2.70 | 92.67±2.06 | |
| 0.01 | 64.67±6.75 | 70.00±5.25 | |
| 0.001 | 48.67±5.68 | 58.00±5.36 | |
| 0.0001 | 18.67±2.74 | 34.33±4.75 | |
| DEET (N,N-diethyl-toluamide - Stay off) | 1 | 76.25±5.25 | 78.00±8.44 |
| 0.1 | 50.05±6.54 | 60.85±6.61 | |
| 0.01 | 40.62±7.24 | 54.75±2.74 | |
| 0.001 | 16.44±3.36 | 18.20±6.75 | |
| 0.0001 | 10.98±5.42 | 16.25±2.06 | |
Repellent activity value = mean ± standard deviation.
The essential oil of P. amboinicus presented the highest percentage of repellency at a concentration of 0.1% at 2 and 4 h of exposure (83.33 and 92.67%, respectively). EO results were compared to those of the commercial repellent based on DEET (N, N-diethyl-toluamide). At the concentration of 0.1% DEET, the repellency percentages obtained at 2 and 4 h of exposure against T. castaneun were 50.05 and 60.85%, respectively. Significant differences (Kruskal-Wallis test (P<0.05) were found between the concentrations for the repellent percentage.
Other studies have shown the potential of essential oils from P. amboinicus and their blend as mosquito repellents against A. aegypti, the vector of dengue, chikungunya, and yellow fever (Lalthazuali and Mathew, 2017), as well as against bites of Lutzomyia migonei, the Leishmania vector (Nieves et al., 2010), and L. (Viannia) braziliensis (Lima et al., 2014).
The results obtained for the fumigant activity of the essential oil of P. amboinicus against the flour weevil (T. castaneum) are recorded in table 3. Therein, it can be observed that at a concentration of 250 µg mL-1 of AE air it reached a mortality rate of 100%.
Table 3. Fumigant activity of essential oil from P. amboinicus against T. castaneum.
| Concentrations (µg mL-1) | Mortality (%) | |
|---|---|---|
| 24 h | 48 h | |
| 50 | 17.50±2.5 | 21.61±3.0 |
| 150 | 73.33±1.9 | 85.00±1.8 |
| 250 | 100±0 | 100±0 |
| 350 | 100±0 | 100±0 |
| 500 | 100±0 | 100±0 |
The values reflect the average of the four replicates ± the standard mean deviation.
Significant differences (Kruskal-Wallis test, (P<0.05) were found between the concentrations and the mortality percentage.
Table 4 exhibits the mean lethal concentrations (LC50) obtained for the essential oil of P. amboinicus in two exposure periods. The fumigant toxicity of the EO was evaluated on adult T. castaneum weevils.
Table 4. Toxicity of the essential oil from P. amboinicus and pirimiphos-methyl against T. castaneum.
| Treatments | Period (h) | LC50(95% FL) | Χ2 (df) | Slope ± SE |
|---|---|---|---|---|
| P. amboinicus | 24 | 182.070 [158.040; 209.753] | 0,998 (39) | 1.977± 0.031 |
| 48 | 136.937 [116.743; 160.623] | 1.000 (36) | 1.751 ± 0.035 | |
| Commercial insecticide (pirimiphos-methyl) | 24 | 188.673 [114.824; 246.333] | 1.449 (3) | 0.0198 ± 0.0015 |
| 48 | 84.2145 [77.023; 147.414] | 1.758 (3) | 0.0159 ± 0.0015 |
FL: Fiducial limits,χ2:Chi-square, df: Degrees of freedom, SE: standard error, n=5.
The interpretation for 95% FL is that, with 95% confidence, the required lethal concentration to achieve 50% mortality in the study population species will be within the lower limit and upper limit.
The results of the probit analysis showed that the pyrimiphos methyl (positive control), at 24 h of exposure obtained an LC50 = 188.673 [114.824; 246.333] µg mL-1 air, at 48 h at LC50 = 84.2145 [77.023; 147.414] µg mL-1 air, pirimiphos-methyl. The P. amboinicus EO’s had a toxicity level in the first 24 h like that of the pirimiphos-methyl, but 2.16 times more lethal in the first 48 h times than the essential oil under study, because P. amboinicus EO yielded an LC50 = 182.070 µg mL-1 air at 24 h, and LC50 = 136.937 µg mL-1 air at 48 h of exposure.
The EO from P. amboinicus evaluated in this study showed significant fumigant and repellent activity. According to the literature, the main compound found in the oil, carvacrol, had insecticidal activity and neurophysiological effects against Cimex lectularius L (Gaire et al., 2019); Culex pipiens pallens (Youssefi et al., 2019;Ma et al., 2014); and R. dominica and L. serricone (Ramadan et al., 2020), Ixodes ricinus (Tabari et al., 2017); Dermanyssus gallinae (Tabari et al., 2015); among others.
Carvacrol has delocalized electron and hydroxyl groups. Ultee et al. (2002) suggested that the hydroxyl group and delocalized electron of carvacrol is essential for biological activities.
