Chromatographic profile and allelopathic potential of the essential oil of Acritopappus confertus

The present study aims to test the Acritopappus confertus (Gardner) R. M. King & H. Rob. essential oil allelopathic potential on the germination and inital development of Cenchrus echinatus L. and Lactuca sativa L. seedlings, in addition to identifying and quantifying chemical constituents. The chemical composition analysis was performed by gas chromatography (GC-FID) and gas chromatography coupled to mass-spectrometry (GC-MS). For the allelopathic assays, the essential oil was emulsified with dimethylsulfoxide (DMSO) in a 1:1 ratio, and diluted in distilled water to obtain 0.001, 0.01, 0.10, 0.25, 0.50, 0.75 and 1% c. The control consisted of 1% aqueous DMSO solution.Treatments were performed in five repetition with 20 seeds each. The pH of the oils were measured and adjusted to a scale of 6 to 7. Assays were conducted in a germination chamber at 25 °C with a 12h photoperiod. Seed germination, Germination Speed Index (GSI), caulicle and radicle length were analyzed. Research, Society and Development, v. 9, n. 12, e1991210450, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10450 3 The data were subjected to analysis of variance and the means were compared by Tukey test (p<0.05), through the ASSISTAT. The constituents mycene, β-pinene and limonene stood out the most in chemical analysis. The essential oil did not influence C. echinatus and L. sativa seed germination, however it influenced GSI and seedling development. The effects observed herein may be due to chemical constituents found in the studied species, which may act in an isolated or combined manner.

Allelochemical action can affect plant growth, interfering with cell division and elongation processes, modifying the synthesis of a plant's main constituents and carbon distribution in cells, they may also affect hormones which play an important role in growth regulation, as well as interfering with enzymes, increasing or inhibiting their activities (Pires & Oliveira, 2011).
The discovery of new allelochemicals has been used an alternative to conventional herbicide usage for the control of invasive plants. Conventional herbicides have increased the risk of environmental contamination, causing changes in invasive species population and increasing resistance to these compounds. In this sense, studies carried out with allelochemicals may contribute to new bioherbicide discoveries, either for direct use or as possible precursors for the synthesis of new agrochemicals (Oliveira et al., 2012). Miranda et al. (2015a) reinforce that bioherbicide use contributes to the development of more sustainable agriculture.
According to Ootania et al. (2017), the essential oils present in plants can be used for the production of herbicides, since they are more specific and less harmful to the environment. The authors draw attention to the phytotoxic effects of essential oils such as herbitoxins, insecticides, fungitoxins, in addition to the allelopathic effects, responsible for interfering in the germination, growth and development processes of other plants.
The bioassays carried out in the laboratory have proved to be an essential tool in studies aimed at determining the allelopathic potential of different plant species, whether native, naturalized, exotic or cultivated (Costa et al., 2019).
Weeds have been used as receptor species in allelopathic assays and, several authors, like Pires and Oliveira (2011), confirm the allelopathic influences of such species, which in many cases affect native and cultivated plants, causing delay or impediment germination, reduced growth and interference in the symbiosis process of cultures.
Through allelopathic potential and constituent analysis, new alternatives for spontaneous and cultivated plant management may be observed, in addition to allowing diversification in agricultural crops (Silva et al., 2011;Gusman, Vieira & Vestena, 2012).
Species of the genus Acritopappus have been studied from the phytochemical point of view, having been observed the presence of several chemical compounds such as diterpene monoterpenes and diverse sesquiterpenes (Ferreira, Marturano, Carollo & Oliveira, 2011;Giacomini et al., 2005;Lima et al., 2005), however, few studies relate these compounds to their allelopathic potential.
Based on the aforementioned, the present study aims to test the A. confertus essential oil allelopathic potential on the germination and development of Cenchrus echinatus L. and Lactuca sativa L. seedlings, in addition to identifying and quantifying chemical constituents.

Methodology
The present study is characterized a experimental research with a quantitative approach. In the experimental research, the variables to be analyzed are selected, establishing the form of control over them, in addition to investigating the implications on the object of study in pre-established situations. As the researcher exercises control over the variables, flaws and biases can be eliminated, thus ensuring greater reliability in his results (Fontelles, Simões, Farias & Fontelles, 2009).

Botanical material collect and identification
In order to extract the essential oil, fresh A. confertus leaves were collected from a Cerrado area in the Araripe National Forest, located at 7° 28'92" S and 39º 54'11" W, 933m altitude, in the morning. For identification of the species under study, 5 branches in the reproductive phase, with flowers and/or fruits were collected. These were herborized, treated and identified using identification keys and incorporated into the collection at the Caririense Dárdano de Andrade-Lima Herbarium (HCDAL) of the Regional University of Cariri (URCA), under registration number 12.462.

Essential oil extraction
For essential oil extraction, the hydrodistillation technique using a Clevenger type apparatus was employed.
For the extraction of essential oil, the hydrodistillation technique was used, using the Clevenger type apparatus. For that, 649g of leaves were crushed and placed in 3 portions in a 3 liter glass bottle, in which 1.5 liters of distilled water were added for each extraction, totaling 4.5 liters (Matos, 1997). Two hours after the start of the extraction process, during which it was observed to be exhausted, the essential oil was collected, packed in an amber bottle, labeled and kept refrigerated until the moment of use.

Gas chromatography (GC-FID)
The analyzes were performed following the protocol described by Cunha et al.
(2015) using a gas chromatography (GC) system with an Agilent Technologies System 6890N GC-FID equipped with a DB-5 capillary column (30m x 0.25mm, film thickness 0.25mm) coupled to a FID detector. The injector and detector temperatures were set to 280°C. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The thermal programmer consisted of 50-300 °C at a rate of 5 °C/min. Two sample replicates were processed the same way. The constituents relative concentrations were calculated based on the CG peak areas without correction factors. The A. confertus essential oil injection volume was 1 μL.

Gas chromatography coupled to mass spectrometry (GC-MS)
Gas chromatography coupled to mass spectrometry (GC-MS) analysis were performed on an Agilent Technologies AutoSystem XL GC-MS system operating in EI mode at 70 eV, equipped with a split/splitless injector (250°C). The transfer line temperature was 280 °C. Helium was used as the carrier gas (1.0 mL/min) and the capillary columns used were HP 5MS (30m x 0.25mm, film thickness 0.25mm) and HP Innowax (30m x 0.32mm id, film thickness 0.50mm). The temperature program was the same as that used for GC analyzes. The Essential oil injection volume was 1 μL (Cunha et al., 2015).

Chemical constituents identification
The A. confertus essential oil chemical constituents identification was performed based on retention indices (RI), determined with n-alkanes homologous series references, C7-C30, under identical experimental conditions, comparing with mass spectra library (NIST and Wiley) and with mass spectra literature according to Adams (1995).

Allelopathic activity
The A. confertus leaf essential oil influence on seed germination, Germination Speed Index (GSI) and initial C. echinatus L. and L. sativa L. seedling development using diverse concentrations was tested.
The essential oil was emulsified with dimethyl sulfoxide (DMSO) at a 1:1 proportion and then dissolved in distilled water to obtain solutions at the concentrations of 0.001, 0.01, 0.10, 0.25, 0.50, 0.75 and 1%. The control group consisted of 1% aqueous DMSO solution.
The pH of the solutions in their various concentrations were adjusted to a range between 6.0 and 7.0 due to high acidity. The recipient species seeds were distributed in sterilized petri dishes, having three sheets of filter paper moistened with distilled water as a substratum (at a ratio of 1 gram of paper to 3 mL of distilled water). Each treatment consisted of five replicates with 20 seeds totaling 140 seeds per treatment. After sowing, 3 mL of the oil solution were distributed in two filter paper sheets at each established concentration and placed in the plate cover, adopting the indirect contact methodology (Leite et al., 2015).
The experiments were conducted in a BOD-type germination chamber at 25 °C with a 12-hour photoperiod for five days for C. echinatus seeds and seven days for L. sativa seeds.

Statistical analysis
For germination data, caulicular and radicular development, an analysis of variance (ANOVA) was performed where Tukey's test was applied at a 5% probability for comparison between the samples. All statistical analyzes were performed using the ASSISTAT version 7.7 beta program.

Essential oil chemical analysis
The fresh A. confertus leaf essential oil presented a yield of 0.34%. Chemical analysis allowed the identification and quantification of 12 compounds, representing 89.51% of the total chemical composition. Myrcene (49.16%), β-pinene (17.09%) and limonene (8.73%) were identified as the major constituents, corresponding to 74.98% of the total oil composition (Table 1). Research, Society and Development, v. 9, n. 12, e1991210450, 2020 (CC BY 4. (Adams, 1995). Source: Authors. Lima et al. (2005) performed studies on the chemical composition of A. confertus leaf volatile oils and identified the occurrence of monoterpenes composing 81.0% of the total, with myrcene (52.0%) as its main component, followed by β-pinene (16.8%) and limonene (8.2%). These results corroborate with those of the present study.
Moreover, Miranda et al. (2015b) state it is of paramount importance to perform comparative analyzes of essential oil allelopathic effects and their major constituents.

Essential oil Ph
Essential oil pH values varied between 5.0 and 5.5 for the 0.5% concentration, and in very acidic ranges between 4.8 and 4.7 for the 0.75 and 1% concentrations, respectively; these values were adjusted to the 6 range to prevent interferences with seedling growth and development (table 2). According to Oliveira, Diógenes, Coelho and Maia (2009)  Periotto, Perez and Lima (2004) state pH values at extreme conditions such as acidity or alkalinity can negatively affect seedling growth and development. In these cases a pH adjustment to 6.0, the appropriate range for germination and allelopathic effect verification, is indicated (Macias, Gallindo & Molinillo, 2000). As stated by Ferreira and Áquila (2000), pH verification is extremely important in studies addressing allelopathic action since extracts may contain sugars, amino acids and organic acids capable of masking such effect, due to pH interference. Research, Society andDevelopment, v. 9, n. 12, e1991210450, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10450

Essential oil allelopathic effects
In the bioassays carried out with the A. confertus leaf essential oil, no significant statistical differences were observed regarding to C. echinatus seed germination when compared to the control (Figure 1a), but it caused a delay in the Germination Speed Index of C. echinatus seeds at all concentrations tested, when compared to the control (Figure 1b).  Rosado et al. (2009) state that even without allelochemical interferences in the final germination percentage, it is possible that these compounds cause changes in the germination pattern through differences in seed germination speed and synchrony.
Such effect in natural environment may be important so competition from a donor species with other functionally recipient species is avoided. In this context, Gatti, Perez and Ferreira (2007) reported this condition could represent an important ecological factor since plants which germinate in a slower manner may have a reduced size, which contributes to them being more vulnerable to stress, and present few conditions for resource competition.
C. echinatus seedling stem length at all concentrations tested was significantly reduced compared to the control (Figure 2a). Significant inhibitory effects on root growth and elongation were also observed, this effect being more evident at higher concentrations (0.50, 0.75 and 1%) (Figure 2b). Research, Society and Development, v. 9, n. 12, e1991210450, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10450   Ribeiro and Lima (2012) verified inhibitory effects on E. heterophylla L. roots exposed to the C. sinensis L. bark essential oil, whose main component is limonene. In addition, lesions and malformations were also observed in the roots, some of which were serious enough to kill seedlings.
According to Duschatzky et al. (2007) studies on the Heterothalamus alienus (Spreng.) Kuntze and H. psiadioides Less. (Asteraceae) essential oil chemical analysis, indicated β-pinene as the major constituent. When evaluating the oil cytotoxicity of these two species, Schmidt-Silva, Pawlowski, Santos, Zini and Soares (2011) observed a reduction in the mitotic index of L. sativa (lettuce) and A. cepa (onion) meristematic cells, resulting in chromosomal abnormalities such as chromosomal adhesion, c-mitosis and micronucleus formation. Such effects may be associated with a reduction in root length.
Allelochemicals have the ability to interfere in primary metabolic processes and plant growth, where the actions of these compounds can directly affect cell division and elongation, which are extremely important processes to ensure plant growth and development of the plants (Pires & Oliveira, 2011).
According to Maia et al. (2011) studies have pointed out that monoterpenes are capable of causing significant damage to the membranes and respiratory processes of some plants, since these substances can be absorbed through their secretory structures. Rosado et al. (2009) reported that monoterpenes are capable of altering the structure and function of membranes, impeding cell growth and performance.
Regarding L. sativa seeds, the A. confertus essential oil did not interfere with germination at any of tested concentrations ( Figure 3a). As for GSI, no statistical differences in relation to the control were found at the lowest concentrations (0.001, 0.01, 0.10 and 0.25%). However, at the highest concentrations (0.50, 0.75% and 1%), a delay in L. sativa seed GSI with statistical differences when compared to control was observed (Figure 3b).  Regarding radicular development, the oil stimulated L. sativa radicular growth at the 0.001, 0.01, 0.10% concentrations, while the 0.25% concentration did not display an effect when compared to the control group, however, from the 0.50% concentration upwards radicular length inhibition was observed, presenting statistical differences only in the concentration of 1% (Figure 4b). Such results may be due to the fact that an allelochemical can be both inhibitory and stimulatory depending on the concentration in which they are found and their combination (Rice, 1984;An, Johnson & Lovette, 1993). Silva, Overbeck and Soares (2014) observed the H. psiadioides Less. (Asteraceae) essential oil inhibitory effect over L. sativa and A. cepa seedling aerial and root length.
According to these authors, the phytotoxic effects of this species can be attributed to the interaction of their chemical components, which reveal the presence of monoterpenes, mainly β-pinene, Δ 3 -carene e limonene.
The allelopathic effects observed in the present study can be attributed to the constituents identified in the A. confertus leaf essential oil.
Souza Filho, Guilhon, Zoghbi and Cunha (2009b) state the presence of major constituents in essential oils may explain the allelopathic effects over the germination and growth of the receptive plant.

Conclusion
Myrcene, β-Pinene and limonene were the most outstanding compounds in the A.
confertus leaf essential oil chemical analysis. The negative interference observed in the Germination Speed Index and seedling development of L. sativa and C. echinatus may be due to the combined or isolated action of these compounds. However, further studies are needed to purify and isolate these compounds, to investigate the specific constituents that cause the allelopathic effects verified in the bioassays performed.