Essential oils of Lippia gracilis and Lippia sidoides chemotypes and their major compounds carvacrol and thymol: nanoemulsions and antifungal activity against Lasiodiplodia theobromae

The aim of this work was to evaluate the antifungal activity of essential oils (EOs) of Lippia gracilis and Lippia sidoides accessions, their major compounds and nanoemulsions. Nanoemulsions with 18% of EO or major compound were produced by spontaneous emulsification method. The EO of two L. gracilis accessions (LGRA-106 and LGRA-109) and two L. sidoides accessions (LSID-102 and LSID-104) were extracted by hydrodistillation and the major compounds thymol and carvacrol were purchased commercially. Antifungal activity was tested against Lasiodiplodia theobromae by calculating the percentage of inhibition of mycelial growth caused by different concentrations (0.1, 0.5, 1.0, 5.0, and 10.0 mL.L), in relation to the control. The EOs and their respective nanoemulsions presented as major compounds Research, Society and Development, v. 11, n. 3, e36511326715, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i3.26715 2 thymol (LGRA-106: 61.84% and Nano-106: 63.43%; LSID-102: 64.07% and Nano-102: 83.03%) or carvacrol (LGRA109: 54.56% and Nano-109: 45.63%; LSID-104: 69.06% and Nano-104: 38.66%). Nano-104 presented 35.91% of an unidentified compound. The fungicidal activity of the EOs was similar to that of the major compounds, with a minimum fungicidal concentration of 1.0 mL.L for LGRA-106, LSID-102 as well as for thymol, and 0.5 mL.L for LGRA-109, LSID-104 as well as for carvacrol. The nanoemulsions were only able to reduce the mycelial growth of the fungus except for Nano-104 that exhibited fungicidal activity at the concentration of 10 ml.L. It was possible to observe that the EOs exhibited greater toxicity against L. theobromae than the nanoemulsions. These results can help in the development of products for the control of this important phytopathogen.


Introduction
The fungus Lasiodiplodia theobromae (Pat.) Griffon and Maubl. is a typical pathogen of the tropical and subtropical regions where it occurs in several species of plants of economic interest (Punithalingam, 1980;Slippers & Wingfield, 2007). Its ability to infect fruits such as mangoes and other tropical species puts it among the most efficient pathogens spread through seeds and causes of post-harvest diseases (Pereira, Martins, Michereff, Silva, & Câmara, 2012;Yang et al., 2021). This pathogen can cause different symptoms in infected plants, including dry-down (die back); cancer in branches, stems and roots; lesions in piles, leaves, fruits and seeds; besides inciting the death of seedlings and grafts.
The alternative control pathogenic fungi as L. theobromae, in a global context, has been much discussed given that the use of synthetic chemicals has been for many years, the primary means pest control (Yang et al., 2019). Despite their significant contribution to agricultural production, intensive and indiscriminate use of these products favored the emergence of secondary pests and failed to eliminate existing problems (Marques et al., 2004). Furthermore, studies revealed the emergence of strains resistant to several synthetic active principles (He et al., 2018;Yang et al., 2019). Studies suggest the use of essential oils with antifungal properties in pathogens control in post-harvest of fruits (Peixinho et al., 2017;Santos et al., 2014;Sharma & Tripathi, 2008).
Among the essential oils with significant antimicrobial activity against fungi and bacteria are those obtained from leaves of Lippia gracilis and L. sidoides (França et al., 2020;Melo et al., 2013;Veras et al., 2017). These herbs are endemic in northeastern Brazil and are found predominantly in the States of Ceará, Rio Grande do Norte, Bahia, Sergipe, and Piauí (Lorenzi & Matos, 2002). The essential oils of these species have high concentrations of the monoterpenes thymol and carvacrol (Cavalcanti et al., 2010).
However, certain limitations are observed in the direct use of essential oils, such as poor solubility in aqueous medium, high volatility, and quick degradation in the environment (Cadena, Preston, Van der Hoorn, Townley, & Thompson, 2018).
These characteristics can lead to low retention at the application site, thus diminishing the effect and effectiveness as an antimicrobial agent (Marreto et al., 2008).
The use of nanometric systems carrying these compounds have been investigated as a way to minimize the volatility of essential oils, prolonging the retention time of the substance at the site of action (Asbahani et al., 2015;Donsi, Annunziata, Sessa, &Ferrari, 2011;Sansukcharearnpon et al., 2010).
Nanoemulsions are nanometric dispersion systems of oil droplets in an external aqueous phase stabilized by a suitable surfactant (Bouchemal et al., 2004). Such systems are obtained when the size of the globules of the nanoemulsions reaches approximately 20-500 nm (Bernardi et al., 2011).
Thus, the objective of this work was to study the antifungal activity of essential oils, major compounds and nanoemulsions of L. gracilis and L. sidoides accessions, against the pathogen L. theobromae.

Plant material
Leaves of accessions LGRA-106 and LGRA-109 of L. gracilis, and accessions LSID-102 and LSID-104 of L. sidoides were collected from plants of the Active Germplasm Bank of the Federal University of Sergipe (SISGEN Register number A8CCB3B), located at the Research Farm "Campus Rural da UFS", municipality of São Cristóvão, Sergipe State, Brazil (10°55'28,35" S and 37°11'58,06 "W) (Table 1). The leaves were dried at 40 ± 1 °C in a drying oven with air flow for five days. Essential oils were extracted by hydrodistillation for 140 minutes using a Clevenger apparatus (Ehlert et al., 2006). Essential oils were stored in amber flasks at -20 ± 2 ° C until the moment of chemical analysis and bioassays. The essential oil content was calculated and expressed in % (v/w). The standards of the compounds carvacrol and thymol were obtained from Sigma-Aldrich.
The MS and FID data were simultaneously acquired employing a Detector Splitting System; the split flow ratio was 4:1 were acquired in the full scan mode (m/z of 40-350) at a scan rate of 0.3 scan/s using the electron ionization (EI) with an electron energy of 70 eV. The injector temperature was 250 °C and the ion-source temperature was 250°C. The FID temperature was set to 250 ºC, and the gas supplies for the FID were hydrogen, air, and helium at flow rates of 30, 300, and 30 mL/min, respectively.
Quantification of each constituent was estimated by FID peak-area normalization (%). Compound concentrations were calculated from the GC peak areas and they were arranged in order of GC elution.
Identification of individual components of the essential oil was performed by computerized matching of the acquired mass spectra with those stored in NIST21, NIST107 and WILEY8 mass spectral library of the GC-MS data system. A mixture of hydrocarbons (C9H20-C19H40) was injected under these same conditions and identification of constituents was then performed by comparing the spectra obtained with those of the equipment data bank and by the Kovats index, calculated for each constituent as previously described (Adams, 2007). Retention indices were obtained with equation proposed by Van Den Dool and Kratz (1963).

Preparation of nanoemulsions
The nanoemulsions were produced according to the method of spontaneous emulsification (Lovelyn & Attama, 2011).
The oil phase was composed of 1.8 ml essential oil, thymol or carvacrol. The aqueous phase was composed of 1.0 ml water and 7.2 ml of Procetyl, an emulsifying agent. Then, the two phases were mixed and the material was subjected to moderate magnetic agitation (600 rpm) with GOstirrer MS-M-S10 magnetic stirrer. After 90 minutes, nanoemulsions were obtained with a final concentration of 18% of essential oil, thymol or carvacrol (Flores, Ribeiro, Ourique, Rolim, & Silva, 2011).

Characterization of nanoemulsions
The nanoemulsions were characterized in terms of macroscopy, zeta potential, particle diameter and polydispersity.
Particle size and polydispersity index were determined by photon correlation spectroscopy after dilution of the sample with ultrapurified water in the ratio 1:500 (v:v) with Zs-ninth Nanoseries Zetasizer (Malvern Instruments, Worcestershire, UK ). The pH of the samples was determined directly using a calibrated pH meter (MD-20 Digimed) at room temperature (Danielli et al., 2013).
To determine the volatile components of the nanoemulsions samples, GC analyses, using a GC-MS/FID (QP2010 Ultra, Shimadzu Corporation, Kyoto, Japan) equipped with an autosampler AOC-20i (Shimadzu), were realized under the conditions described above.

Fungal strain and pathogenicity test
The fungal strain Lasiodiplodia theobromae was obtained from the fungi collection of the Federal University of Sergipe.
The fungus viability was assessed by pathogenicity testing. The test involved mango fruit inoculated with a fungal spore suspension (105 UFC.mL -1 ). Subsequently, the fungus was used to re-isolation from the fruit that has been damaged.

Antifungal activity
The nanoemulsions were evaluated at concentrations of 10.0; 5.0; 1.0; 0.5 and 0.1 mL.L -1 , diluted directly in the PDA culture medium (potato dextrose agar HIMEDIA), and then the solutions were poured into petri dishes of 9 cm, as described by After one week of incubation the mycelial disks of concentrations that had no visible growth were transferred to petri plates containing only culture medium. After 7 days, lower concentrations corresponding to plates that remained without visible growth were considered the fungicidal concentration.

Statistical analysis
The standard deviation values were obtained by Excel software.

Physicochemical properties of nanoemulsions
Through spontaneous nanoemulsification technique, it was possible to obtain six translucent, homogeneous formulations with different particle sizes ranging from 15-253 nm ( Table 3). The nanoemulsions produced showed pH acid (5.49-6.50) and polydispersity index ranging from 0.171 to 0.411.

Antifungal activity
The fungicidal activity exhibited by essential oils were similar to the activities exhibited by their respective major compounds. Accessions LGRA-106 and LSID-102 showed fungicide activity from the concentration of 1.0 mL.L -1 of essential oil, as well as the major compound thymol, while the accessions LGRA-109 and LSID-104 exhibited the same capacity from 0.5 mL.L -1 of essential oil, as well as the major compound carvacrol. It should be noted that the essential oil from accession LSID-104 was able to inhibit the growth of the fungus by 50% at the lowest concentration tested (0.1 mL.L -1 ), while the other essential oils were not able to cause any inhibition of mycelial growth in this concentration (Table 4). It was observed that carvacrol was more toxic as it inhibits mycelial growth from 0.1 mL.L -1 , while for thymol this capacity was observed from 0.5 mL.L -1 . Similarly, the minimum fungicidal concentration (MFC) for carvacrol was 0.5 mL.L -1 while for thymol it was 1.0 mL.L -1 ( Table 4).
The nanoemulsions obtained from the essential oil of the Lippia species were only able to slow the mycelial growth of the fungus L. theobromae, except for Nano-104 which not only inhibited the growth of the fungus, but caused its death in the highest tested concentration. Nano-106, Nano-109 and Nano-102 began to exhibit some effect on mycelial growth from the concentration of 5.0 ml.L -1 , while Nano-104 started the same effect from the concentration of 1.0 mL.L -1 (Table 5).  Nano-thymol and Nano-carvacrol emulsions inhibited 100% of mycelial growth from a concentration of 5.0 ml.L -1 .
However, for this same concentration, the first showed a fungistatic effect, while the second showed a fungicidal effect (Table   5).

Discussion
Chemical analysis of essential oils and nanoemulsions from L. gracilis and L. sidoides accessions confirmed the stability of the two major compounds, thymol and carvacrol, in nanoemulsified oil-water systems without the addition of organic solvents.
The studied species have different essential oil composition, and these differences are often used to separate them into distinct chemotypes. In addition to presenting different chemical compositions, all essential oils contained a significant proportion of the precursors of the organic phenolic compounds, ρ-cymene and γ-terpinene, precursors of the monoterpenes carvacrol and thymol (Stević et al., 2014). The compounds ρ-cymene and γ-terpinene represent important intermediates used in the pharmaceutical industry, in the production of fungicides, pesticides and flavoring agents, in addition to providing antimicrobial properties together with thymol and carvacrol to nanoemulsions of essential oils of L. gracilis and L. sidoides (Selvaraj et al., 2002).
The nanoemulsions exhibited particle sizes ranging between 15 and 253 nm. These droplet sizes are similar to those reported in the literature for nanoemulsions obtained from essential oils from other plant species (Danielli et al., 2013;Duarte et al., 2015;Ngan et al., 2015). This efficiency in the production of nanoemulsions containing essential oils from L. gracilis and L.
sidoides is possibly associated with the presence of low molecular weight molecules with amphiphilic character, which tend to act as co-surfactants. Furthermore, the small size of essential oil particles in an oil-in-water dispersion is directly related to maintaining the stability of the nanoscale system (Lovelyn & Attama 2011;Shang et al., 2014).
In the chemical composition of the nanoemulsion Nano-104 obtained from the essential oil of the accession LSID-104, a possible isomerization of carvacrol was observed. In this nanoemulsion, unidentified compounds (NI-I, II and III) that were not present in the essential oil of LSID-104 were detected. These molecules belong to the isopropylmethylphenol class, of which the main representatives are thymol and carvacrol. Therefore, it is believed that these unidentified compounds are possibly isomers produced from carvacrol.
The essential oils exhibited L. theobromae mycelial growth inhibition capacities, similar to their respective major compounds. Accessions LGRA-109 and LSID-104, whose essential oils have carvacrol as the major compound, were more efficient in inhibiting the growth of the fungus when compared to the others, which have thymol as the major compound. The fact that pure carvacrol can inhibit 100% of mycelial growth at the lowest concentration tested and exhibit CFM lower than thymol, proves that this compound is the main responsible for the greater toxicity of the essential oils of the accessions LGRA-109 and LSID-104. Although essential oils whose main compound is thymol (LGRA-106 and LSID-102), as well as pure thymol are also toxic to the studied fungus, but they have a lower capacity to inhibit and kill the fungus than the other treatments.
Nano-104 stood out in relation to other essential oil nanoemulsions for having shown a fungicidal effect at 10.0 mL.L -1 . By analyzing the chemical composition, it is observed that this emulsion has carvacrol as the major compound (38.66%). When comparing it with Nano-carvacrol, it is possible to see that it also stands out in relation to Nano-104 (Table 5). At a concentration of 10.0 mL.L -1 , Nano-104 is equivalent to a concentration of approximately 0.7 mL.L -1 of carvacrol (considering 18% LSID-104 essential oil in the composition) while Nano-carvacrol, at a concentration of 5.0 mL.L -1 , is equivalent to 0.9 mL.L -1 of carvacrol.
That means that Nano-carvacrol needed a larger amount of carvacrol to present the same fungicidal effect as Nano-104, with a smaller amount of carvacrol. It is important to mention that Nano-104 had high content of carvacrol and three unidentified compounds, one of them in large quantities (35.91%). It is possible that this compound acted synergistically with carvacrol, providing greater activity against the fungus L. theobromae.
On the other hand, when comparing the activity displayed by Nano-thymol with Nano-106 and Nano-102, it is observed that Nano-thymol is more effective. This nanoemulsion at a concentration of 5.0 mL.L -1 is equivalent to 0.9 mL.L -1 of thymol, while Nano-106 and Nano-102 at a concentration of 10.0 mL.L -1 are equivalent to 1.14 and 1.49 mL.L -1 of thymol, respectively.
Nano-106 and Nano-102 have a higher content of thymol, but were not able to inhibit the growth of the fungus. In this case, other compounds present in these nanoemulsions may have had an antagonistic effect on thymol, reducing its toxicity.
When comparing the antifungal activity displayed by essential oils and their respective nanoemulsions, it was possible to observe that the essential oils exhibited greater toxicity against L. theobromae when compared to the emulsified systems. For example, the essential oil LGRA-106 inhibited 100% of mycelial growth at a concentration of 1.0 mL.L -1 , which is equivalent to approximately 0.62 mL.L -1 of thymol (Table 4). However, when we observe Nano-106, at a concentration of 10.0 mL.L -1 , which is equivalent to 1.14 mL.L -1 of this compound (Table 5), there was still growth of the fungus. The only nanoemulsion that approached the inhibition capacity exhibited by its respective essential oil was Nano-104. One of the causes of this lower effectiveness of nanoemulsions in relation to essential oils may be due to changes that occurred in the chemical constitution of nanoemulsified systems. It was observed that the compounds ρ-cymene and γ-terpinene had their contents reduced in emulsions, when compared to essential oils. Except for Nano-109, which presented change of ρ-cymene content, but a reduction of γterpinene (from 9.36 to 5.84%) and carvacrol (from 54.56 to 45.63%) was observed. These compounds, although minor, act synergistically or antagonistically with thymol and carvacrol, increasing or reducing the antimicrobial properties of essential oils and their emulsions.
The significant biological activity of the nanoemulsions and essential oils from L. gracilis and L. sidoides can be attributed to the presence of the phenolic compounds carvacrol and thymol. Previous research found that there is synergistic activity between carvacrol and thymol and mutual interaction plays an important role in the overall activity of essential oils that contain them (Stević et al., 2014). It is suggested that carvacrol and thymol act by promoting an increase in permeability of the plasmatic membrane of the target cell favors the entry of other active compounds (Calo, Crandall, O'Bryan, & Ricke, 2015).
However, the fact that other bioactive components present in the essential oil, such as ρ-cymene, suggests the existence of synergism between the components of the essential oils.
The form of action of these terpenes is not fully understood, it is considered that is related to changes in the lipophilic properties of the plasmatic membrane of the fungus, causing increased permeability or even their break (Pina-Vaz et al., 2004;Danielli et al., 2013). Thus, carvacrol and thymol can be easier transport across the cell wall of the fungus, causing it to become more permeable to other constituents of essential oils, toxic fungal cells. This action may be associated with the cell membrane lipid oxidation (Montanari et al., 2012) and significantly reducing ergosterol, which is an essential constituent for cell membranes of fungi (Khan et al., 2010).

Conclusion
Some changes were observed in the concentration of the major compounds present in the nanoemulsions in relation to the respective essential oils. Both, L. gracilis and L. sidoides essential oils and emulsions, were able to inhibit the mycelial growth of the fungus L. theobromae. Essential oils were more effective than emulsions when comparing the equivalent concentration of the major compounds present. Nanoemulsion Nano-104, obtained from L. sidoides LSID-104 accession, stood out. These results can help in the development of products for the control of this important fungal species. Future studies should search for alternatives to obtain emulsions with a higher concentration of essential oil and greater stability of the chemical composition.