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INTRODUCTION
Piper gorgonillense Trel. & Yunck is an accepted name for a species in the Piper genus (Piperaceae family) (The Plant List, 2013). It is a bush native to Colombia that can reach 3 m, which has been found in the canyon and valley of Bajo Cauca, valley of Magdalena Medio, the jungle valley of Atrato and Uraba (0-500 m altitude), eastern slope of the Central Mountain Range or Cordillera Central (0-1,000 m), and the Nariño department (Idárraga and Callejas, 2011). Plants of the Piper genus have shown various biological activities, such as anti-inflammatory (Rengifo et al; 2019), phytotoxic, antifungal (Parra et al., 2019), antimicrobial (Gamboa et al., 2018), repellent and insecticidal properties (Jaramillo-Colorado et al., 2019a; Chaubey; 2011), among others. Approximately, two hundred and fifty species of insects attack grains and their products during storage, of which around twenty are of paramount importance (Opit et al., 2012); among these, one of the most damaging is the brown flour beetle, whose common name comes from its color and its habits of infesting flour, while its scientific name is Tribolium castaneum Herbst. This beetle belongs to the Insect class, Coleoptera order and Tenebrionidae family (Athanassiou et al., 2016). It is one of the most important stored product blights found in homes and grocery stores. It has an Indo-Australian origin and currently occurs worldwide in warmer climates. T. castaneum Herbst is a secondary pest that generally feeds on stored cereals and grains (Adamski et al., 2019).
To minimize these losses, chemical pesticides are normally used, where mixtures of insecticides and fungicides are utilized in order to protect the seeds during storage; however, chemicals can generate seed toxicity and lead to resistance problems, environmental contamination, and food residues (Daglish et al., 2015).Several authors have reported the use of essential oils as a non-chemical option for the management of these insect pests (Singh and Pandey, 2018; Andrés et al., 2017). EOs are obtained from aromatic plants and are made up of different volatile organic compounds, so their biological activities will rely on their composition and the interaction between their components (Kendra et al., 2017).
The objective of this study was to determine the volatile chemical composition of the essential oil from P. gorgonillense by assessing its repellent and fumigant activity against T. castaneum.
MATERIALS AND METHODS
Reagents and chemicals
Anhydrous Na2SO4 was acquired from Merck (Darmstadt, Germany). Dichloromethane, acetone, and hexane were acquired from AppliChem Panreac (Darmstadt, Germany), along with DEET (Dr. Ehrenstorfer, Germany) and pirimiphos methyl (Syngenta, Colombia). Filter paper was purchased from GE Healthcare (Hangzhou, China).
Vegetal material
Fresh Piper gorgonillense Trel. & Yunck. leaves and stems were collected in a rural area of Quibdo (Colombia) in July 2018. The collection site is characterized as a growing area and is also vulnerable to flooding. Taxonomic identification was done at the Herbarium Universidad de Antioquia. The control leaves of each plant are archived as a permanent sample at the Herbarium (No-493853).
Extraction of the essential oil
The extraction was performed using the hydrodistillation technique, according to Jaramillo-Colorado et al. (2012). 500 g of leaves and stems from P. gorgonillense were used, finely chopped and submerged in boiling water with conventional heating for 2 h. The EO was separated with decantation, and then anhydrous Na2SO4 was added to the oil. One EO aliquot (30 µL) was diluted in one mL of dichloromethane for the gas chromatography analysis (Jaramillo 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 with electron-impact ionization at 70 eV energy. The 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, increased 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 were compared to those reported in the literature (Adams, 2007).
Insects and bioassays
T. castaneum adults were collected 7 d after hatching. 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 University of Cartagena. Oat (Avena sativa) was used to feed T. castaneum.
Repellent activity
The repellent property of P. gorgonillense EO was analyzed on adult specimens of T. castaneum using the area preference method. The P. gorgonillense EO was dissolved in acetone after preparing five solutions (1, 0.1, 0.01, 0.001, and 0.0001%). A filter paper of 9-cm diameter 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), a commercial repellent, was the positive control.
The treated and control media disks were dried at room temperature for 10 min to allow the solvent to evaporate. 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., 2019b). Four replicates were used for each concentration. The weevil preference was measured for each Petri dish at 2 and 4 h of exposure.
The following equation was used to determine the percentage of repellency (PR), following the parameters identified by Jaramillo-Colorado et al. (2012) (Eq. 1):
where, Nc is the number of insects in the control area (acetone), and Nt is the number of insects in the treated area (EO + acetone).
Fumigant activity
Fumigant activity was performed according to Jaramillo-Colorado et al. (2019b). The toxic effect of P. gorgonillense EO and terpenes were assayed on T. castaneum. Filter paper discs (Whatman No. 1, 2-cm in diameter) were laid down at the bottom of Petri dish covers (90 x 15 mm), which were impregnated with oil at doses calculated to provide equivalent fumigant concentrations of 500, 350, 250, 150, 50 µg mL-1 air of oil, respectively. Twenty adult insects (1 to 10 days 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 the positive control. The mortality percentage was determined after 24 h from the start of exposure.
The percentage of mortality was calculated using 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 fumigant percentage and analyzed with ANOVA and Student t tests. The mortality rates were calculated using the statistical formulas of Abbott and Probit to determine the LC50, chi-square values and related parameters. Biostat, a statistical program (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 hydrodistillation technique isolated the essential oil from P. gorgonillense, and a yield of 0.14% was obtained, which, compared to other studies of this genus, was lower than the 0.21% seen with extraction with hydrodistillation of Piper corcovadensis EO (Miq.) (da Silva et al., 2016). Similarly, a study in Brazil obtained an essential oil yield of 0.44% with hydrodistillation extraction from Piper cubataonum (Santos et al., 2014); another study where oil was extracted with steam distillation from Piper hispidinervum saw 0.95% (Sauter et al., 2012); finally, the essential oil yield from P. hispidinervum obtained with steam distillation reached 0.632% in a study by Andrés et al. (2017). The volatile chemical composition of EO is shown in table 1. Figure 1 shows the chemical structure of the main compounds of the P. gorgonillense essential oil.
Table 1 Volatile chemical composition of essential oil from Colombian P. gorgonillense.
| No | Compoundsa | RI (HP-5)b experimental | RI (HP-5) theoretical | Relative areac (%) |
|---|---|---|---|---|
| 1 | -Pinene | 935 | 939 | 0.2 |
| 2 | Camphene | 950 | 954 | 0.3 |
| 3 | -Pinene | 968 | 979 | 0.5 |
| 4 | Limonene | 1030 | 1029 | 0.2 |
| 5 | Linalool | 1100 | 1096 | 0.6 |
| 6 | Anisole | 1225 | 1232 | 0.2 |
| 7 | -Elemene | 1332 | 1335 | 0.6 |
| 8 | 1,5,5-Trimethyl-6-methylene-cyclohexene | 1340 | 1338 | 1.8 |
| 9 | Cubebene | 1342 | 1345 | 1.3 |
| 10 | -Copaene | 1368 | 1374 | 13.5 |
| 11 | -Bourbonene | 1380 | 1387 | 2.0 |
| 12 | -Elemene | 1385 | 1389 | 0.9 |
| 13 | -Gurjunene | 1410 | 1409 | 2.7 |
| 14 | -Caryophyllene | 1425 | 1419 | 28.7 |
| 15 | -Gurjunene (-Calarene) | 1430 | 1433 | 3.2 |
| 16 | Aromadendrene | 1444 | 1441 | 2.3 |
| 17 | Humulene | 1450 | 1454 | 2.1 |
| 18 | Allo-Aromadendrene | 1462 | 1460 | 1.3 |
| 19 | -Muurolene | 1474 | 1479 | 1.6 |
| 10 | Germacrene D | 1480 | 1484 | 1.0 |
| 21 | trans-Muurola-4(14),5-diene | 1496 | 1493 | 2.1 |
| 22 | -Muurolene | 1500 | 1500 | 2.7 |
| 23 | Epi-bicyclosesquiphellandrene | 1515 | 1521 | 1.9 |
| 24 | -Cadinene | 1520 | 1523 | 7.3 |
| 25 | -Cadinene | 1530 | 1538 | 1.1 |
| 26 | -Calacorene | 1538 | 1544 | 1.7 |
| 27 | Spathulenol | 1570 | 1578 | 2.5 |
| 28 | Caryophyllene oxide | 1581 | 1583 | 0.9 |
| 29 | Globulol | 1588 | 1590 | 1.7 |
| 30 | Guaiol | 1596 | 1600 | 1.0 |
| 31 | trans-β-Elemenone | 1600 | 1601 | 0.9 |
| 32 | Cubenol <1-epi-> | 1622 | 1628 | 0.5 |
| 33 | Cadin-4-en-7-ol <cis-> | 1628 | 1636 | 0.8 |
| 34 | tau-Cadinol (epi-α-cadinol) | 1636 | 1640 | 3.6 |
| 35 | Epoxide-allo aromadendrene | 1639 | 1641 | 0.5 |
| 36 | -Cadinol (tau-Muurolol) | 1645 | 1642 | 2.1 |
| 37 | Cubenol | 1650 | 1646 | 1.4 |
| 38 | Patchouli alcohol | 1656 | 1658 | 1.0 |
| 39 | Eudesm-7(11)-en-4-ol (Juniper camphor) | 1720 | 1700 | 0.8 |
| 40 | Phytol | 1932 | 1943 | 0.5 |
a Identification made with mass spectrometry (EI: electron impact ionization, 70 eV; peak matching >90%) and LRIs. Spectral databases wiley8, NIST08. b Experimentally RI on the HP-5, averages of three independent extractions. cRI -Linear retention indices relative to C7-C30 n-alkanes. Forty compounds were identified, where sesquiterpenic type compounds predominated. The main components were -copaene (13.5%), -caryophyllene (28.7%), -cadinene (7.3%) and tau-cadinol (3.6%).
Figure 1 Chemical structure of the main compounds of the essential oil from P. gorgonillense. Source: MarvinSketch.
After an intense bibliographic search, it was confirmed that this study is the first report on the volatile chemical composition of P. gorgonillense essential oil. The main compound found in this oil was β-caryophyllene, which is a cyclic sesquiterpene; these types of compounds are characterized by their unique structure, which has a bicyclo undecane ring system in the essential oil of a large variety of plant species. This compound is characterized as being volatile and aromatic with low solubility. It is recognized for deploying effective biological activities, such as anti-inflammatory, antiviral, anticarcinogenic, and antimutagenic, among other properties (De Oliveira-Tintinoet al., 2018, Wu et al., 2018; Parisotto et al., 2020).
The chemical composition in this study differed slightly from the results obtained by other species belonging to the Piperaceae family, such as essential oil from Piper guineense with the following main components: β-sesquiphellandrene (20.9%), linalool (6.1%), limonene (5.8%), Z-β-bisabolene (5.4%) and α-pinene (5.3%) (Oyemitan et al., 2015); some of these compounds were found in the Piper gorgonillense EO but at a much lower proportion, for example, linalool (0.6%), limonene (0.2%) and α-pinene (0.2%). On the other hand, the oils extracted from Piper dilatatum had apiol (79.0%), trans-caryophyllene (8.3%), spathulenol (4.2%) and γ-cadinene (1.7%); Piper divaricatum EO had eugenol (37.5%), methyl eugenol (36.3%), γ-elemene (10.7%), asarone (4.6%) and trans-caryophyllene (3.0%); Piper aff. hispidum contained δ-3-carene (9.6%), p-cymene (10.9%), limonene (17.2%), elemol (14.1), γ-elemene (7.3%) and β-eudesmol (5.7%), whose composition markedly differs from that of Piper gorgonillense (Jaramillo-Colorado et al., 2019a).
Whereas, the Piper tuberculatum EO is composed of sesquiterpenes and their oxygenated derivatives, such as, (-)-spathulenol (11.37%), α-farnesene (6.22%), (-)-humulene epoxide II ( 6.04%), β-eudesmol (4.36%), 2-tridecanone (4.27%), 2-pentadecanone (4.06%), ledane (3.62%), (E,E) -farnesyl-acetone (3.55%), β-cedrenoxide ( 2.96%), α-cadinol (2.86%), dibutylphthalate (2.84%), (1RS, 2SR) -2-hydroxy-2,4,4-trimethyl-3- (3-methyl-3-butenylidene) cyclopentyl methyl ketone (2.68%), 4-norpyridoxol (2.26%), epiglobulol (2.14%) and 8-acetyl-3, 3-epoxymethane-6,6,7-trimethylbicyclo [5,1,0] octan-2-one (2.11 %) (Ordaz et al., 2011). In addition, the chemical identification of Piper marginatum had a higher percentage of sesquiterpenes; the main compounds included 3,4-methylenedioxyproprophenone (22.90%), δ-3-carene (10.19%), trans-caryophyllene (9.67%) and spathulenol (6.89%) (Gomes-Macêdo et al., 2020). However, the characterization of the essential oil from Piper aleyreanum saw a slight similarity in the composition of P. gorgonillense because sesquiterpenoids prevailed in P. aleyreanum, with β-elemene (16.3%), bicyclogermacrene (9.2%), δ-elemene (8.2%), germacrene D (6.9%), β-caryophyllene (6.2%) and spathulenol (5.2 %) as the main components; as for the Piper anonifolium oil, the composition was dominated by selin-11-en-4-α-ol (20.0%), β-selinene (12.7%), α-selinene (11.9%) and α-pinene (8.8%); and the Piper hispidum oil had β-caryophyllene (10.5%), α-humulene (9.5%), δ-3-carene (9.1%), α-copane (7.3%), limonene (6.9%), caryophyllene oxide (5.9%) and β-selinene (5.1%) (da Silva et al., 2014).
Essential oils from the Piperaceae family are characterized by the presence of sesquiterpenes and derivatives of such oxygenates in their chemical composition; therefore, there are some similarities in the composition of these species (Chellappandian et al., 2018).Monoterpenes and sesquiterpenes are a significant and very versatile class of secondary metabolites that are commercially used in food, cosmetic, pharmaceutical, biotechnology, and agricultural industries, among others (Koyama et al., 2019).
Table 2 shows the results achieved for the repellent activity of the essential oil from P. gorgonillense on T. castaneum. The best repellent activity was at a concentration of 1%, with a repellency percentage of 75±10% and 90±5% at 2 and 4 h of exposure, respectively. This was compared to the commercial DEET repellent (N, N-diethyl-toluamide) at the same concentration with an activity of 76±6% during the first 2 h and 78±5% after 4 h (Tab. 2). These results show that the P. gorgonillense EO exhibited a higher repellent activity than the commercial repellent.
Table 2 Repellent activity of the essential oil from P. gorgonillense against T. castaneum.
| Sample | Concentration (%) | Repellency (%) | |
|---|---|---|---|
| Exposure time 2 h | Exposure time 4 h | ||
| Piper gorgonillense EO | 0.0001 | 16±5 | 24±5 |
| 0.001 | 32±8 | 36±5 | |
| 0.01 | 56±5 | 64±5 | |
| 0.1 | 68±4 | 74±5 | |
| 1.0 | 78±8 | 90±7 | |
| N,N-diethyl-toluamide (DEET) | 0.0001 | 10±5 | 16±5 |
| 0.001 | 16±5 | 18±8 | |
| 0.01 | 40±10 | 57±13 | |
| 0.1 | 50±5 | 59±11 | |
| 1.0 | 76±6 | 78±5 | |
a Repellent values = mean ± SE of the four replicates (SE = standard error). Paired t test (P<0.05).
After a statistical analysis of repellent activity results, bilateral significance was obtained with a value greater than 0.005, which were 0.106 at 2 h of exposure and 0.109 at 4 h of exposure; the intervals of confidence were [(-2.55464) -(25.75464)] and [(-2.78010) - (26.78010)] for 2 and 4 h of exposure, respectively. Lastly, the t-test showed an equal value for the two trials: the P. gorgonillense EO and DEET, with 1,648 at 2 h and 1,632 at 4 h for both. This analysis allowed us to infer that the P. gorgonillense EO and DEET had similar behaviors; therefore, it is possible to infer that the oil has a repellent activity very similar to that of a commercial repellent.
-Caryophyllene has shown repellent activity against Aedes aegypti and Anopheles minimus (Nararak et al., 2019), as well as against Sitophilus granarius (Plata et al., 2018). In the study reported by Parisotto et al. (2020), β-caryophyllene acted as a powerful anti-inflammatory agent and demonstrated efficiency in the repair of skin tissue; β-caryophyllene provided anti-inflammatory and antiedemagnetic activity in in vivo tests of acute and chronic inflammation through a series of laboratory tests (De Oliveira-Tintinoet al., 2018). β-caryophyllene has shown an effective action against parasites such as Trypanosoma cruzi and Leishmania brasiliensis. Thanks to these results, this compound is considered an alternative against the aforementioned species (Leite et al., 2013), i.e., -cadinene deployed larvicidal activity against the malaria vector Anopheles stephensi, (Govindarajan et al., 2016). -copaene has been evaluated as an insecticide against different types of insects, including the beetle, Xyleborus glabratus Eichhoff (Kendra et al., 2016), the beetle Euwallacea fornicatus Eichhoff (Kendra et al., 2017), the fly Bactrocera oleae (Rossi) (de Alfonso et al., 2014), and Ceratitis capitata (Flat et al., 1994). Different -cadinene activities have also been reported, such as anticancer (Xu et al., 2015), antifungal (Kundu et al., 2013), and acaricidal activities (Guo et al., 2017).
The fumigant activity results for the P. gorgonillense essential oil are shown in figure 2. The P. gorgonillense EO reached a fumigant activity of 100% at a concentration of 350 µg mL-1 air. Essential oils are considered fumigants because of their physical properties of low molecular weight, low boiling point and high volatility (Zhang et al., 2017).
Table 3 presents the mean lethal concentrations (LC50) obtained for the essential oil of P. gorgonillense in three exposure periods. The fumigant toxicity of the EO was evaluated on adult T. castaneum weevils. The results of the probit analysis showed that the pyrimiphos methyl (positive control), at 24 h of exposure, obtained an LC50 = 188,673 µg mL-1 air, LC 50 = 84.2145 µg mL-1 air at 48 h, and LC50 = 68.0253 at 72 h, with pirimiphos-methyl being 2.3493 times more toxic in the first 24 h, 4.13622 times, more toxic in the first 48 h and 3.4261 times more lethal than the studied essential because the P. gorgonillense EO yielded an LC50 = 443.262 µg mL-1 air at 24 h, an LC50 = 348,328 µg mL-1 air at 48 h and an LC50 = 233,066 µg mL-1 air at 72 h of exposure.
On the other hand, several studies have reported the repellent and/or insecticidal activity of essential oils from plants of the Piper genus on Tribolium castaneum, i.e. Piper nigrum, Piper dilatatum, Piper aduncum, Piper divericatum and Piper sp. (Scott et al., 2008; Jaramillo-Colorado et al., 2019a); P. guineense (Adarkwah et al., 2018); and Piper cubeba (Chaubey, 2011); among others. The P. gorgonillense EO evaluated in this study showed significant fumigant and repellent activity. According to the literature, the major sesquiterpenes found in the oil, such as (-caryophyllene, (-copaene and (-cadinene, have exhibited insecticidal and/or repellent properties. Kim et al. (2010) reported that the fumigant and repellent activity of essential oils are related to the presence of monoterpenes, such as (-pinene, p-cymene, carvacrol thymol, and (-and myrcene,and sesquiterpene compounds, such as caryophyllene oxide and caryophyllene (Kim et al., 2010).
Table 3 Toxicity of the essential oil P. gorgonillense and pirimiphos- methyl against T. castaneum.
| Treatments, n=5 | Period (h) | LC50 (95% FL) | LC95 (95% FL) | X 2 (df) | Slope ± SE |
|---|---|---|---|---|---|
| P. gorgonillense | 24 | 443.262 [395.696; 495.943] | 938.338 [831.046; 1100.81] | 8.345 (3) | 1.79± 0.18 |
| 48 | 348.328 [315.464; 382.645] | 671.424 [608.674; 760.233] | 6.776 (3) | 1.77 ± 0.17 | |
| 72 | 233.066 [202.345; 262.862] | 515.145 [465.407; 583.398] | 4.011 (2) | 1.35 ± 0.16 | |
| Commercial insecticide (methyl pirimiphos) | 24 | 188.673 [114.824; 246.333] | 333.766 [277.547; 394.697] | 1.449 (3) | 0.0198 ± 0.0015 |
| 48 | 84.2145 [77.023; 147.414] | 216.133 [207.333; 320.308] | 1.758 (3) | 0.0159 ± 0.0015 | |
| 72 | 68.0253 [54.157; 79.856] | 153.084 [143.502; 177.335] | 0.193 (2) | 0.0136 ± 0.0012 |
