Drying Effects on Chemical Composition and Antioxidant Activity of Lippia thymoides Essential Oil, a Natural Source of Thymol

Leaves of Lippia thymoides (Verbenaceae) were dried in an oven at 40, 50 and 60 °C and the kinetic of drying and the influence of the drying process on the chemical composition, yield, and DPPH radical scavenging activity of the obtained essential oils were evaluated. The composition of the essential oils was determined with gas chromatography-mass spectrometry and gas chromatography-flame ionization detection analyses. The influence of drying on the chemical composition of the essential oils of L. thymoides was evaluated by multivariate analysis, and their antioxidant activity was investigated via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The Midilli model was the most appropriate to describe the behavior of drying kinetic data of L. thymoides leaves. Thymol was the major compound for all analyzed conditions; the maximum content was obtained from fresh leaves (62.78 ± 0.63%). The essential oils showed DPPH radical scavenging activity with an average of 73.10 ± 12.08%, and the fresh leaves showed higher inhibition (89.97 ± 0.31%). This is the first study to evaluate the influence of drying on the chemical composition and antioxidant activity of L. thymoides essential oils rich in thymol.


Introduction
Essential oils and their constituents have been described as antioxidants [1][2][3][4][5], food preservatives [6][7][8], and mosquito repellents [9][10][11]. Essential oils containing thymol as a major component have been used in the industry as flavorings and food preservatives, and even in aromatherapy [4,12,13]. This compound has been registered by the European Commission for use as flavoring in foodstuffs; it is also used as a food additive in the USA and China [12,14]. Furthermore, isolated thymol as well as essential oils with high levels of thymol demonstrated a protective action in the treatment of soybean seeds [15]; they also exhibit antifungal [16][17][18], antibacterial [19,20], antiviral [21], and antioxidant

Drying Kinetics
The initial moisture content of L. thymoides fresh leaves was 68.15 ± 0.01% on a wet basis. At the end of the drying process, the moisture content was 31.28 ± 1.51% for the 40 • C conditions, 12.34 ± 0.09% at 50 • C, and 9.29 ± 0.07% at 60 • C. During the drying of L. thymoides leaves, the moisture ratio decreased exponentially, and an increase in the air temperature led to higher water removal from plant matrices ( Figure 1). To explain a similar behavior, Perea-Flores et al. (2012) [38] reported that during the falling period, the surface of the material was no longer saturated with water, and that the phenomenon of internal diffusion controlled the drying rate [38]. The kinetic curves in Figure 1 presented a typical pattern of drying plots [39][40][41]. In the present study, the moisture ratio reached stable values in the range of 0.03 after 350 min (40 • C), 210 min (50 • C), and 160 min (60 • C), as presented in Figure 1. In this condition, we obtained a moisture content of 6.54% on a wet basis. The pattern of the kinetic drying curves described in Figure 1 is in accordance with the results previously described for Ocimum basilicum [42,43] and Warionia saharae [44]. Kucuk et al. (2014) [45] reported that different models have been applied to predict drying conditions of several products [45]. The Midilli model presented the best fit of the experimental drying data at different temperatures (Table 1, Figure 1), and presented SEE < 0.02% and MRE < 1.18%. The R 2 values were 99.91% (40 • C), 99.95% (50 • C), and 99.87% (60 • C). The Midilli model is indicated to describe the thin layer drying process [45] and it was appropriated to describe the drying kinetic of L. thymoides. Midilli was also suitable for describing the drying curves of leaves of Piper umbellatum [46], Ocimum basilicum [43], and Vernonia amygdalina [47]. According to Table 1, for the Lewis, Diffusion approach, and Henderson & Pabis models, the "k" values increased as the drying temperature increased. However, for the Page and Midilli models, the temperature variation (40, 50 and 60 • C) did not cause a defined trend in the values of "k". The parameter "k" is defined as the constant of drying (min −1 ) and according to Dorneles et al. (2019) [46], it is related to the effective diffusivity during the falling period of kinetic drying, in which liquid diffusion controls the process [46]. Molecules 2021, 26, x FOR PEER REVIEW 3 of 14  [45] reported that different models have been applied to predict drying conditions of several products [45]. The Midilli model presented the best fit of the experimental drying data at different temperatures (Table 1, Figure 1), and presented SEE < 0.02% and MRE < 1.18%. The R 2 values were 99.91% (40 °C), 99.95% (50 °C), and 99.87% (60 °C). The Midilli model is indicated to describe the thin layer drying process [45] and it was appropriated to describe the drying kinetic of L. thymoides. Midilli was also suitable for describing the drying curves of leaves of Piper umbellatum [46], Ocimum basilicum [43], and Vernonia amygdalina [47].

Drying Effects on Essential Oil Yield
The drying conditions had a significant effect on the yield of the essential oils (p < 0.05) ( Table 2). The yield obtained from fresh leaves of L. thymoides was similar to the yield obtained by drying for 390 min at 60 • C (0.50% on average). The highest yields were obtained for dried leaves at 40 • C and 50 • C (0.74% on average). Therefore, according to the experimental conditions here described, to achieve better yields, it is necessary to reduce the moisture content of L. thymoides leaves, but the samples should be dried at temperatures in the range of 40 to 50 • C. At higher temperatures such as 60 • C, there is a greater loss in the components by volatilization, resulting in lower yields during extraction.
Considering the storage structures found in different organs and the volatile constituents of the aromatic plants, the selection of the appropriate drying methods is important because drying can affect the yield and the chemical composition of the essential oil [35,48]. We did not find previous studies evaluating the effect of different drying conditions on the yield of essential oil of L. thymoides, however, Ebadi et al. (2015) [49] investigated the drying of Lippia citriodora leaves using different methodologies including oven-drying (40, 50, and 60 • C), and they found that the increase in temperature generated damage to glandular trichomes, which may have favored the loss of volatiles and the reduction in the yield [49]. Mashkani et al. (2018) [50] subjected the leaves of Thymus daenensis to different drying methods (sun and shade, oven, microwave, and vacuum drying) and observed that, in general, there was a reduction in the yield of essential oils at increased temperatures [50]. Alves et al. (2015) [51] investigated the influence of Ocimum basilicum leaf-drying on the yield and composition of the essential oil and observed that the oil yield decreased linearly over eight days at 40 • C (6.0% to 3.9%) [51]. Therefore, an increase in the temperature contributes to high evaporation rates, which can damage the cells that store the essential oil, intensifying the release of volatile compounds and resulting in lower yields after extraction. Notes: * Columns with the same letter did not differ by the Tukey test at 5% probability; RI (L) : Retention index from literature [52,53]; RI (c) : Retention index calculated using an n-alkane standard solutions (C8-C40) in column DB5-MS; ** Relative percentage areas calculated based on the peak areas.

Drying Effects on Chemical Composition
Thirty-two volatile constituents were identified, comprising a range of 90.07 ± 3.11% to 98.53 ± 0.05% of the total composition of the analyzed essential oils (Table 2). Thymol was the major compound obtained from both fresh and dried leaves. The highest content was observed in fresh leaves (62.78 ± 0.63%), while the levels in the extracted essential oils after drying at 40, 50 and 60 • C were 49.21 ± 11.46%, 53.03 ± 11.76%, and 59.29 ± 2.89%, respectively.
Here, we utilized the multivariate analysis to investigate possible similarities between the chemical classes of the constituents identified in the essential oils of the fresh and dried leaves of L. thymoides (Table 2). From the PCA data (Figure 2A), we deducted a fitting of 85.57% (PC1 + PC2) of the chemical variation in the samples. The principal components (PC1 and PC2) divided the chemical composition of the fresh and dried leaves into two groups (Figure 2A). Notes: * Columns with the same letter did not differ by the Tukey test at 5% probability; RI(L): Retention index from literature [52,53]; RI(c): Retention index calculated using an n-alkane standard solutions (C8-C40) in column DB5-MS; ** Relative percentage areas calculated based on the peak areas.
Here, we utilized the multivariate analysis to investigate possible similarities between the chemical classes of the constituents identified in the essential oils of the fresh and dried leaves of L. thymoides (Table 2). From the PCA data (Figure 2A), we deducted a fitting of 85.57% (PC1 + PC2) of the chemical variation in the samples. The principal components (PC1 and PC2) divided the chemical composition of the fresh and dried leaves into two groups (Figure 2A). These results confirmed that regardless of the applied drying temperature (40, 50 and 60 • C), the essential oils extracted from the dried leaves had a similar chemical profile and formed Group 1. The percentages of monoterpene hydrocarbons increased in essential oils extracted from leaves after the drying process when compared to fresh leaves. However, for the class of oxygenated monoterpenes, a reduction in their contents was detected when compared to oil extracted from fresh leaves (control). This variation may have favored the formation of Group 2, composed only by the chemical constituents of the fresh leaves, which was confirmed by the analysis of the dendrogram in Figure 2B.
Previously, a drying study described the effect of temperature and air velocity of drying on the composition of the Piper umbellatum leaves. They also reported a significant difference between the chemical profile of the fresh and dried leaves, but they found little difference between the samples dried at 40, 50, 60 and 70 • C (0.4 and 0.7 m.s −1 ) [46].
Other studies have also evaluated the effect of drying on monoterpene content and reported an effect similar to that described in the present study. Mashkani et al. (2018) [50] evaluated different drying methods for Thymus daenensis and observed that when compared with fresh plants, oven-drying at 35 • C after a pre-drying operation resulted in a higher content in monoterpene hydrocarbons and reduced the oxygenated monoterpenes (especially the content of thymol and carvacrol) [50]. Shahhoseini et al. (2013) [54] subjected the aerial flowering parts of Lippia citriodora to different drying methods and observed that during oven-drying, the percentage of monoterpene hydrocarbons increased and those of oxygenated monoterpenes decreased with an increase in the drying temperature [54].
The thymol content decreased after the drying process when compared to the content presented in the fresh leaves. Similarly, Calín-Sánchez et al. (2013) [55] evaluated the effects of different methodologies and temperatures in the chemical composition of Thymus vulgaris essential oils, which presented high levels of thymol. They found 732 mg·100g −1 of thymol in the fresh leaves and after oven-drying, the levels changed to 571 mg·100g −1 (40 • C), 479 mg·100g −1 (50 • C), and 451 mg·100g −1 (60 • C) [55]. In the study with the essential oil of Lippia origanoides, Queiroz et al. (2018) [56] also observed a reduction in thymol content after the drying process [56]. Therefore, the authors found that the drying process promoted a reduction in the levels of thymol, which corroborate with our results.

Antioxidant Activity
The DPPH radical scavenging activity of the essential oils of L. thymoides obtained from fresh and dried leaves (40 • C, 50 • C, and 60 • C) are listed in Table 3. The variation in drying temperature did not show a defined trend in the results of antioxidant activity, but the highest percentage of inhibition of the DPPH radical was observed for the essential oil sample from the fresh material (89.97 ± 0.31%), which presented the highest percentage of thymol (62.78%, Table 2). In our study, we observed that the samples of the essential oils obtained after the drying process showed lower antioxidant activity, which may be associated with lower levels of thymol. A previous study investigated the antioxidant activity of some pure essential oil components using lipid model systems, and the authors concluded that the phenolic compounds were efficient antioxidants and they considered thymol as one of the most active components in the class of oxygenated monoterpenes [22]. It is interesting to note that thymol has been indicated as an alternative to synthetic antioxidants in the food matrix [14]. Although the chemical composition of the essential oils extracted after drying presented high similarity ( Figure 2B), only the oils of L. thymoides extracted from dried leaves at 40 • C and 60 • C inhibited the DPPH radical in a statistically similar manner (Table 3). Natural products such as extracts and essential oils are formed by a complex mixture of organic compounds that act synergistically, increasing the biological activity or even antagonistically, thus reducing the verified activity, so identifying the compounds involved with a given biological response represents a methodological challenge [57][58][59]. Based on this, we conjecture that this variation in the results of DPPH inhibition percentage for the dried leaves could be involved with the synergist or antagonist effects (Table 3). Other studies identified synergist effects in the antioxidant [60] and antibacterial [61][62][63] activities of essential oils. For example, Ranjbaran et al. (2019) [13] reported that the high antioxidant activity of Trachyspermum ammia essential oil could be associated with the strong synergism between the monoterpenes (p-cymene and γ-terpinene), the phenolic monoterpenes (thymol), and the oxygenated monoterpenes [13].
Essential oils with high thymol contents have been evaluated for their DPPH radical scavenging activities such as the essential oil from Trachyspermum ammia [13]. This essential oil showed robust antioxidant activity, which may be associated with its high content of thymol (40.25%). Using the DPPH method, the IC 50 were 54 ± 3.2 µg·mL −1 and 90 ± 5.4 µg·mL −1 for the essential oil and thymol, respectively [13]. The essential oil of oregano (Origanum vulgare) with thymol (45%) and carvacrol (37.4%) as the major compounds was subjected to DPPH radical scavenging tests and resulted in IC 50 values between 0.76 µg·mL −1 and 0.33 µg·mL −1 , indicating that this essential oil is an interesting natural source of antioxidant compounds with applications in the chemical, pharmaceutical, cosmetics, and food industries [64].
Few studies have reported the antioxidant profile of the essential oil of the species L. thymoides. Silva et al. (2016) [29] applied scavenging of the DPPH free radical and βcarotene bleaching tests to determine the antioxidant potential of the L. thymoides essential oil, rich in E-caryophyllene (17.22-26.27%). The authors did not find a satisfactory antioxidant activity for the essential oil, with EC 50 (concentration that caused 50% of the DPPH radical scavenging) values more than 236 mg·mL −1 in the DPPH assay and a reduction of less than 11% in the β-carotene bleaching test [29]. There is a growing interest in the development of antioxidant substances, mainly from natural products [65].

Plant Material
Leaves of L. thymoides were collected in the municipality of Abaetetuba (State of Pará, Amazon region, Brazil). The botanical identification (MG 213373) was performed in the Herbarium of the Museu Paraense Emílio Goeldi (Belém, State of Pará, Brazil).

Thin-Layer Drying Kinetics of Lippia thymoides Leaves
The drying kinetics were measured at 40, 50 and 60 • C using 12.03 ± 0.05 g of fresh leaves. All assays were conducted in duplicate for 390 min using a convection oven (model SL-102, Solab, Piracicaba, São Paulo, Brazil) until reaching the equilibrium moisture content. The moisture content was determined according to the standard methods of analysis [66] at 105 • C. After the experiments, the moisture ratio (XR) was calculated according to previous studies [42,46,67].
Some mathematical models applied to describe the experimental drying kinetics of natural products were selected (Table 4): Lewis, Henderson & Pabis, Page, Diffusion approach, Midilli, and Wang & Singh, using a nonlinear regression procedure to estimate the parameters associated with each model.  [69,71] Note: k is the drying constant (min −1 ); a, b, and n are the model coefficients.

Drying Conditions Versus Yield and Chemical Composition
Samples of 90.11 ± 0.06 g of fresh leaves were dried at 40 • C, 50 • C, and 60 • C for 390 min in a convection oven. Then, the dried samples were subjected to hydrodistillation.

Essential Oil Extraction
Leaves of L. thymoides (fresh and dried) were subjected to hydrodistillation using a modified Clevenger glass extractor for 3 h, as described by Nascimento et al. (2020) [73]. We utilized 30 g of plant material for each experiment. The yield (%) of the essential oil was expressed as the percentage of oil in relation to the dry matter of the leaves.

Analysis of Chemical Composition
The chemical composition was analyzed with gas-phase chromatography coupled to mass spectrometry (GC/MS) using a Shimadzu QP 2010 Plus System (Shimadzu Corporation, Kyoto, Japan), equipped with silica capillary columns (DB-5MS, length of 30 m, inner diameter of 0.25 mm, and film thickness of 0.25 µm); carrier gas: helium, linear velocity: 36.5 cm·s −1 ; type of injection: splitless (solution of 2 µL of oil in 500 µL of hexane); injector temperature: 250 • C, temperature range: 60 • C to 250 • C, gradient of 3 • C·min −1 ; electron impact mass spectrometry: 70 eV; ion source temperature and connection parts: 220 • C. The quantification of each component was performed by peak-area normalization using a flame ionization detector (GC-FID, QP 2010 system, Shimadzu Corporation, Kyoto, Japan) under the same conditions as GC/MS, except for the carrier gas, which was hydrogen.
The components were identified based on the retention index (RI), which was calculated using the retention times of a homologous series of n-alkanes (C8-C40, Sigma-Aldrich, San Luis, Missouri, USA). The pattern of fragmentation observed in the spectra was compared with existing patterns of authentic samples in data system libraries and the literature [52].

2,2-Diphenyl-1-picrylhydrazyl (DPPH) Free Radical Scavenging Assay
The antioxidant activity of the essential oils was evaluated using the 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging method. A stock solution of DPPH (0.5 mM) was prepared in ethanol. The solution was diluted to approximately 60 µM, measuring an initial absorbance of 0.62 ± 0.02 at 517 nm at 22 • C. The absorbance was measured at the start of the reaction, every 5 min during the first 30 min, and then at 30 min intervals until a constant absorbance value (plateau of reaction, 2 h). Each essential oil sample (50.0 µL, 20 mg·mL −1 ) was mixed with a Tween 20 solution (0.5%, 50 µL, w/w), and added to DPPH (0.5 mM, 1900 µL) in ethanol. The control sample was prepared in the same conditions, replacing the essential oil sample by ethanol. The standard curves were prepared using Trolox (6-hydroxy-2

Statistical Analyses
The most appropriate models to describe the drying kinetics curves were chosen based on the mean relative error (MRE, Equation (1)), standard error of estimate (SEE, Equation (2)), and coefficient of determination (R 2 ). A MRE value of less than 10% was one of the criteria for selecting the best model [76]. Statistical analyses were conducted using Statistica ® 13.1 (TIBCO Software Inc., Palo Alto, CA, USA), and the Quasi-Newton method was applied. N represents the number of experiments, df the degrees of freedom, XR exp the experimental moisture ratio, and XR pre the predicted moisture ratio.
The yield and antioxidant activity data were subjected to variance analysis (ANOVA) and their means were compared performing the Tukey test (at a significance level of p < 0.05). Principal component analysis (PCA) was performed using a matrix correlation. In addition, the Euclidean distance and complete binding were used for the hierarchical cluster analysis (HCA) of the samples. The statistical analyses were conducted using Statistica ® 13.1 (TIBCO Software Inc., Palo Alto, CA, USA).

Conclusions
The study of drying kinetics provides data for the selection of the type of dryer as well as the best operating drying conditions. Midilli was the best model to describe the drying kinetics of L. thymoides leaves. In the present study, the drying conditions reached an appropriate moisture content for safe storage. The highest yields of essential oil were obtained from leaves that were dried at 40 • C and 50 • C after drying for 390 min. The chemical composition showed a similar quantitative profile for all the drying conditions (40, 50 and 60 • C), but it differed from the chemical composition of the fresh material.
Thymol was the major compound identified in the essential oil obtained from fresh and dried leaves. The highest thymol content (62.78 ± 0.63%) and inhibition percentage of the DPPH radical (89.97 ± 0.31%) were obtained from fresh leaves. Thus, L. thymoides can be a natural source of thymol, with levels greater than 50% of the composition of the essential oil.