Enhancing Essential Oil Extraction from Lavandin Grosso Flowers via Plasma Treatment

Citation: Molina, R.; López-Santos, C.; Balestrasse, K.; Gómez-Ramírez, A.; Sauló, J. Enhancing Essential Oil Extraction from Lavandin Grosso Flowers via Plasma Treatment. Int. J. Mol. Sci. 2024,25, 2383. https:// doi.org/10.3390/ijms25042383 Academic Editor: William N. Setzer Received: 15 January 2024 Revised: 12 February 2024 Accepted: 15 February 2024 Published: 17 February 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Enhancing Essential Oil Extraction from Lavandin Grosso Flowers via Plasma Treatment Ricardo Molina 1, * , Carmen López-Santos 2,3 , Karina Balestrasse 4,5 , Ana Gómez-Ramírez 2,6 and Jordi Sauló 7 1 Department of Biological Chemistry, Institute of Advanced Chemistry of Catalonia (IQAC), Spanish National Research Council (CSIC), 08034 Barcelona, Spain 2Nanotechnology on Surfaces and Plasma Group, Institute of Materials Science of Seville (US-CSIC), 41092 Sevilla, Spain; [email protected] (C.L.-S.); [email protected] (A.G.-R.) 3 Departamento de Física Aplicada I, Escuela Politécnica Superior, Universidad de Sevilla, 41011 Sevilla, Spain 4Instituto de Investigaciones en Biociencias Agrícolas y Ambientales (INBA), Facultad de Agronomía, Universidad de Buenos Aires (UBA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires C1417DSE, Argentina; [email protected] 5Cátedra de Bioquímica, Departamento de Biología Aplicada y Alimentos, Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires C1417DSE, Argentina 6Departamento de Física Atómica, Molecular y Nuclear, Facultad de Física, Universidad de Sevilla, 41012 Sevilla, Spain 7 Laboratory of Dioxins, Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDÆA), Spanish National Research Council (CSIC), 08034 Barcelona, Spain; [email protected] *Correspondence: ricar[email protected]; Tel.: +34-4006100 (ext. 437851) Abstract: This study explores the impact of plasma treatment on Lavandin Grosso flowers and its influence on the extraction of essential oils (EOs) via hydrodistillation. Short plasma treatment times enhance the yield of EO extraction from 3.19% in untreated samples to 3.44%, corresponding to 1 min of plasma treatment, while longer treatment times (10 min) show diminishing returns to 3.07% of yield extraction. Chemical characterization (GC/MS and ATR-FTIR) indicates that plasma treatments do not significantly alter the chemical composition of the extracted EOs, preserving their aromatic qualities. Investigations into plasma–surface interactions reveal changes at the nanometer level, with XPS confirming alterations in the surface chemistry of Lavandin Grosso flowers by reducing surface carbon and increasing oxygen content, ultimately resulting in an increased presence of hydrophilic groups. The presence of hydrophilic groups enhances the interaction between the surface membrane of the glandular trichomes on Lavandin Grosso flowers and water vapor, consequently increasing the extraction of EOs. Furthermore, microscopic SEM examinations demonstrate that plasma treatments do not affect the morphology of glandular trichomes, emphasizing that surface modifications primarily occur at the nanoscale. This study underscores the potential of plasma technology as a tool to enhance EO yields from botanical sources while maintaining their chemical integrity. Keywords: essential oil; Lavandin Grosso; hydrodistillation; plasma technology; surface modification; GC/MS; ATR-FTIR; XPS; SEM; contact angle 1. Introduction Lavandula (common name lavender) is a genus of around 30 known species and subspecies and numerous hybrids of flowering plants and belongs to the Lamiaceae plant family [ 1 , 2 ]. Lavandula species are among the most useful aromatic and medicinal plants with great economic value in perfumery, aromatherapy, cosmetics, pharmaceutical, and food industry, mainly due to the commercial significance of lavender’s essential oil [ 2 , 3 ]. The most common species in cultivation are Lavandula angustifolia,Lavandula x intermedia (lavandin, hybrid), and Lavandula latifolia. Among the main lavandin varieties (Grosso, Int. J. Mol. Sci. 2024,25, 2383. https://doi.org/10.3390/ijms25042383 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2024,25, 2383 2 of 17 Abrial, Super), the Grosso variety is the most famous for its essential oil yield and its odor [ 4 ]. Additionally, Lavandin Grosso essential oils have phytotoxic properties [ 5 ], antiplatelet/antithrombotic properties [ 6 ], antimicrobial activity [ 7 , 8 ], and antioxidant [ 9 ] and antifungal properties [ 10 , 11 ], to name a few. Lavender essential oil is located in a round shape of glandular trichomes with an average diameter of 100 µ m present on the outer surface of the flower [ 12 ]. Generally, the essential oil (EO) is isolated from lavender mostly by hydrodistillation or steam distillation of fresh or dried flowers. During the distillation process, the essential oil is heated and flows out through the cuticle of the glandular trichomes to the vapor phase [12,13]. Plasma, known as the fourth state of matter, is naturally present in the universe (e.g., aurora borealis) but can be generated artificially by an electrical discharge in a gas (e.g., neon tubes). Plasma is composed of different types of reactive species (e.g., radicals, electrons, ions, and atoms) and UV–visible radiation. The modifications produced by these chemical species, such as oxidation and the generation of hydrophilic chemical groups, are limited to a few nanometers from the surface of a material in contact with a plasma and therefore do not alter the internal properties of the material. Low-temperature or nonthermal plasmas (NPTs) are widely used for polymeric material treatments, biomaterials, sterilization, or medicine [ 14 , 15 ]. The application of NTPs in agriculture is a recent and rapidly growing field [ 16 , 17 ]. NTP technology is environmentally friendly and can be economically competitive compared to traditional chemical treatments. The application of plasma treatments in the modification of raw materials for essential oil extraction has emerged as an intriguing and promising possibility. Recent studies have successfully employed plasma technology to improve the extraction yield of essential oils from plants, including lemon peel and leaves [ 18 – 20 ], grape and tomato pomace [ 21 , 22 ], Camelina sativa and cumin seeds [ 23 , 24 ], fennel seeds and spearmint leaves [ 25 ], betel leaves [ 26 ], and thyme leaves [ 27 ]. In the field of extracting natural compounds, including essential oils, the application of plasma treatments prior to the extraction process has shown the potential to alter the chemical and physical properties of plant raw materials, which could influence the efficiency and quality of the subsequent extraction process [ 16 , 17 ]. In particular, microwaveexcited low-pressure plasma treatments, as well as atmospheric pressure dielectric barrier discharge (DBD) plasma treatments, have mainly induced small defects such as cracks in the epidermal cell structure along with a drying of the peel directly related to an improved oil extraction process [ 19 , 26 ]. In addition, the formation of hydrophilic groups on the surface has also been related to improved essential oil extraction yield [22,27]. The present study focuses on the application of low-temperature and pressure plasma treatments to Lavandin Grosso flowers as a precursor to enhance the extraction of essential oils. Via a systematic approach, possible changes in the chemical profiles of essential oils will be investigated, along with the physico-chemical changes induced by low-temperature and pressure plasma treatment on the surface of Lavandin Grosso flowers. Ultimately, this study aims to shed light on the potential of plasma technology as a promising tool to improve the extraction of essential oils from plant materials while providing valuable information on how plasma treatment parameters can influence the resulting chemical and aromatic profiles. 2. Results and Discussion 2.1. Hydrodistillation Kinetics and Characterization of Essential Oils from Lavender Flowers Hydrodistillation extraction kinetics of the cumulative essential oil (EO) compounds of untreated and plasma-treated Lavandin Grosso flowers is shown in Figure 1. Untreated Lavandin Grosso extraction yield (final percentage of essential oil extraction weight respect total flower weight) after 2 h (3.19 ± 0.06%) agrees with previously reported values in Lavandin Grosso flowers (3.2%) [ 28 ]. It can be observed that the initial rate (curve slope in the first 20 min) and the oil extraction yield after 2 h increase with 1 min of plasma treatment (3.44 ± 0.08%). A subsequent increase in plasma treatment time tends to decrease both the initial rate and oil extraction yield with values of 3.31 ± 0.05% and 3.28 ± 0.02% for 3 min Int. J. Mol. Sci. 2024,25, 2383 3 of 17 and 5 min, respectively. At 10 min plasma treatment time, the rate and the extraction yield obtained (3.07 ± 0.04%) are lower than the corresponding for untreated flowers. Therefore, while short plasma treatment times are beneficial for increasing the amount of essential oils extracted by hydrodistillation, long plasma treatment times (10 min) have a detrimental effect on extraction. Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 19 in the first 20 min) and the oil extraction yield after 2 h increase with 1 min of plasma treatment (3.44 ± 0.08%). A subsequent increase in plasma treatment time tends to decrease both the initial rate and oil extraction yield with values of 3.31 ± 0.05% and 3.28 ± 0.02% for 3 min and 5 min, respectively. At 10 min plasma treatment time, the rate and the extraction yield obtained (3.07 ± 0.04%) are lower than the corresponding for untreated flowers. Therefore, while short plasma treatment times are beneficial for increasing the amount of essential oils extracted by hydrodistillation, long plasma treatment times (10 min) have a detrimental effect on extraction. Figure 1. Kinetics of essential oil extracted by hydrodistillation from untreated (UT) and plasmatreated Lavandin Grosso flowers for increasing treatment times. The identification of essential oils composition was carried out to determine if plasma active species can chemically modify the oils present inside the glandular trichomes of Lavandin Grosso flowers and therefore alter their fragrance. Figure 2 shows the GC/MS analysis performed on the cumulative essential oils obtained after hydrodistillation (2 h) of untreated and plasma-treated Lavandin Grosso flowers. 1,8-Cineole (Eucalyptol), linalool, camphor, and linalyl acetate are the most intense compounds observed in the gas chromatogram. No significant difference in the intensity of the peaks is observed between oils extracted from untreated or plasma-treated Lavandin Grosso flowers, even for long treatment times (10 min). Figure 1. Kinetics of essential oil extracted by hydrodistillation from untreated (UT) and plasmatreated Lavandin Grosso flowers for increasing treatment times. The identification of essential oils composition was carried out to determine if plasma active species can chemically modify the oils present inside the glandular trichomes of Lavandin Grosso flowers and therefore alter their fragrance. Figure 2shows the GC/MS analysis performed on the cumulative essential oils obtained after hydrodistillation (2 h) of untreated and plasma-treated Lavandin Grosso flowers. 1,8-Cineole (Eucalyptol), linalool, camphor, and linalyl acetate are the most intense compounds observed in the gas chromatogram. No significant difference in the intensity of the peaks is observed between oils extracted from untreated or plasma-treated Lavandin Grosso flowers, even for long treatment times (10 min). Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 of 19 Figure 2. GC/MS chromatogram for essential oils extracted from untreated (UT) and plasma-treated Lavandin Grosso flowers for different treatment times. Detailed identification of the chemical compounds (Table S1) has evidenced that Lavandin Grosso EOs are composed of ~100 chemical compounds. The chemical composition of Lavandin Grosso EOs depends on the lavender species (Lavandula angustifolia, Lavandula latifolia, and Lavandula hybrid are the most employed in the perfume and cosmetics industry), the geographic region of origin, altitude, soil, and climate conditions, morphology, processing of the plant material, its nature (fresh or dried), and extraction method [3,29–36]. Although with different concentrations, which are attributed to the different parameters described above, the most representative chemical compounds found in the EOs extracted from untreated Lavandin Grosso flowers (Table 1) are in concordance with that of EOs found by other authors [3,4,37,38]. It is important to remark that no new chemical compounds appear, there is no significant variation in chemical composition concentrations, and no new chemical compounds are formed as a result of possible oxidative or reductive chemical reactions of the plasma active species with the EOs located inside the glandular trichomes. The same effect has been previously reported in essential oils extracted from plasma-treated fennel seeds and plasmas-treated spearmint leaves where no significant differences in extracted EO chemical composition are observed between untreated and plasma-treated samples [25]. These results seem to evidence that in the experimental conditions used in this work, at short plasma treatment times (<3 min), active species just modify the surface membrane of the glandular trichomes, modifying their permeability and favoring the diffusion of EOs towards the exterior of the glandular trichomes, thereby increasing the EO extraction yield in this way. Table 1. GC/MS determination of main compounds (area %) in EOs extracted from untreated (UT) and plasma-treated Lavandin Grosso flowers, comparative with ISO normative. Compound UT 1 min 3 min 5 min 10 min Lavandin Grosso, French Type [37] Linalool 35.61 35.93 35.45 35.71 36.10 24.0–37.0 Linalyl acetate 9.61 8.96 9.58 8.75 8.45 25.0–38.0 1.8-Cineole 10.83 10.61 10.14 10.62 10.03 4.0–8.0 Camphor 10.98 10.02 10.27 10.39 10.34 6.0–8.5 Limonene 0.48 0.52 0.49 0.47 0.44 0.5–1.5 Figure 2. GC/MS chromatogram for essential oils extracted from untreated (UT) and plasma-treated Lavandin Grosso flowers for different treatment times. Int. J. Mol. Sci. 2024,25, 2383 4 of 17 Detailed identification of the chemical compounds (Table S1) has evidenced that Lavandin Grosso EOs are composed of ~100 chemical compounds. The chemical composition of Lavandin Grosso EOs depends on the lavender species (Lavandula angustifolia,Lavandula latifolia, and Lavandula hybrid are the most employed in the perfume and cosmetics industry), the geographic region of origin, altitude, soil, and climate conditions, morphology, processing of the plant material, its nature (fresh or dried), and extraction method [ 3 , 29 – 36 ]. Although with different concentrations, which are attributed to the different parameters described above, the most representative chemical compounds found in the EOs extracted from untreated Lavandin Grosso flowers (Table 1) are in concordance with that of EOs found by other authors [ 3 , 4 , 37 , 38 ]. It is important to remark that no new chemical compounds appear, there is no significant variation in chemical composition concentrations, and no new chemical compounds are formed as a result of possible oxidative or reductive chemical reactions of the plasma active species with the EOs located inside the glandular trichomes. The same effect has been previously reported in essential oils extracted from plasma-treated fennel seeds and plasmas-treated spearmint leaves where no significant differences in extracted EO chemical composition are observed between untreated and plasma-treated samples [25]. Table 1. GC/MS determination of main compounds (area %) in EOs extracted from untreated (UT) and plasma-treated Lavandin Grosso flowers, comparative with ISO normative. Compound UT 1 min 3 min 5 min 10 min Lavandin Grosso, French Type [37] Linalool 35.61 35.93 35.45 35.71 36.10 24.0–37.0 Linalyl acetate 9.61 8.96 9.58 8.75 8.45 25.0–38.0 1,8-Cineole 10.83 10.61 10.14 10.62 10.03 4.0–8.0 Camphor 10.98 10.02 10.27 10.39 10.34 6.0–8.5 Limonene 0.48 0.52 0.49 0.47 0.44 0.5–1.5 Cis-β-Ocimene 0.73 1.07 1.02 1.04 0.98 0.5–1.5 Terpinen-4-ol 3.63 3.78 3.84 3.82 3.95 1.5–5.0 Lavandulyl acetate 2.69 2.82 2.96 2.95 3.05 1.5–3.5 Lavandulol 1.06 1.09 1.13 1.12 1.18 0.2–1.0 α-Terpineol 4.23 4.32 4.34 4.42 4.69 0.3–1.3 Borneol 4.29 4.03 4.16 4.12 4.25 1.5–3.5 These results seem to evidence that in the experimental conditions used in this work, at short plasma treatment times (<3 min), active species just modify the surface membrane of the glandular trichomes, modifying their permeability and favoring the diffusion of EOs towards the exterior of the glandular trichomes, thereby increasing the EO extraction yield in this way. A detailed statistical analysis of the results presented in Table 1shows a high correlation of the areas corresponding to the different EOs between the untreated and plasmatreated samples (Table 2), indicating that statistically, the plasma treatments scarcely modify the chemical composition of the extractedEOs. Table 2. Pearson correlation analysis attending to the areas of the different Eos obtained for untreated (UT) and plasma-treated samples. UT 1 min 3 min 5 min 10 min UT 1.00000 0.99919 0.99949 0.99934 0.99847 1 min 0.99919 1.00000 0.99963 0.99989 0.99950 3 min 0.99949 0.99963 1.00000 0.99952 0.99917 5 min 0.99934 0.99989 0.99952 1.00000 0.99967 10 min 0.99847 0.99950 0.99917 0.99967 1.00000 Int. J. Mol. Sci. 2024,25, 2383 5 of 17 Additionally, it was analyzed by means of linear regression fitting whether individually the area of the different EOs extracted in each sample showed any trend as a function of the plasma treatment time. Table 3shows the values corresponding to the Pearson coefficients and the adjusted R-squared of the different samples analyzed. A significant trend (r > ± 0.70) can be seen in the decrease in the concentrations of Linalyl acetate, 1,8-Cineole, and Limonene as a function of treatment time (negative values of r) and a tendency to increase the area as a function of treatment time forEOs, Terpinen-4-ol, Lavandulyl acetate, Lavandulol, and α -Terpineol (positive values of r). However, taking into account the adjusted R-squared values, it can be observed that only α -Terpineol presents a statistically significant value (0.95838) and that the variation in the concentration of this compound represents only a variation of 0.46% of the total (4.23 for UT sample to 4.69 for 10 min plasma treatment). This slight increase is not considered a significant change from the perspective of applications such as cosmetics, given the wide dispersion of concentrations already observed among essential oils obtained from Lavandin Grosso in different geographical areas [37]. Table 3. Values corresponding to the linear regression analysis of areas of the different EOs as a function of plasma treatment time. Compound Number of Points Degrees of Freedom Residual Sum of Squares Pearson’s Coefficient (r) Adjusted R-Squared Linalool 5 3 0.17464 0.5852 0.12328 Linalyl acetate 5 3 0.40244 −0.78546 0.48926 1,8-Cineole 5 3 0.21091 −0.74332 0.40336 Camphor 5 3 0.46929 −0.25306 −0.24795 Limonene 5 3 0.0011 −0.82236 0.56838 Cis-β-Ocimene 5 3 0.06641 0.33987 −0.17932 Terpinen-4-ol 5 3 0.01172 0.88417 0.70902 Lavandulyl acetate 5 3 0.0175 0.8822 0.70437 Lavandulol 5 3 0.0175 0.8822 0.70437 α-Terpineol 5 3 0.00385 0.98427 0.95838 Borneol 5 3 0.0407 0.23124 −0.26204 As shown by GC/MS Lavandin Grosso, EOs are concentrated solutions of volatile compounds with ~100 constituents. Thus, their FTIR spectra are complex due to the spectra of individual components overlapping and the mixing of various vibrational modes [ 34 , 39 ]. However, ATR-FTIR spectra obtained from the essential oil samples display characteristic spectral fingerprints that can be used to discriminate different plant species and chemotypes [ 40 – 46 ]. ATR-FTIR spectra of the cumulative essential oils extracted by hydrodistillation of untreated and plasma-treated Lavandin Grosso flowers (Figure 3) show characteristic bands corresponding to O − H stretching vibration (3443 cm −1 ), C–H stretching of vinyl groups CH 2 =CH– typical for the monoterpenes linalool and linalyl acetate (small band at 3085 cm−1), symmetric and antisymmetric C–H stretching of –CH 2 and –CH 3 groups of alkane chains (2970–2860 cm −1 ), C=O stretching of ester group corresponding to camphor and lynalyl acetate (1737 cm −1 ), methylene C–H bend (1450 cm −1 ), C–H in-plane bend of vinyl groups (1412 cm −1 ), C–O stretching (1240–1027 cm −1 ), and C–H out-of-plane bend of vinyl groups (995 cm −1 and 918 cm−1) [4,34,39,41,43,44]. ATR-FTIR spectra of the cumulative essential oils extracted by the hydrodistillation of Lavandin Grosso flowers treated with plasma show a remarkable overlapping of all the peaks with respect to the untreated flowers, confirming that no significant variation in chemical composition can be observed after hydrodistillation. Therefore, the results seem to confirm that plasma active species just modify the surface of the glandular trichomes and do not interact significantly with the essential oils located inside the glandular trichomes. Int. J. Mol. Sci. 2024,25, 2383 6 of 17 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 19 groups of alkane chains (2970–2860 cm−1), C=O stretching of ester group corresponding to camphor and lynalyl acetate (1737 cm−1), methylene C−H bend (1450 cm−1), C−H in-plane bend of vinyl groups (1412 cm−1), C−O stretching (1240–1027 cm−1), and C−H out-of-plane bend of vinyl groups (995 cm−1 and 918 cm−1) [4,34,39,41,43,44]. Figure 3. ATR-FTIR spectra of the cumulative essential oils extracted by hydrodistillation of untreated (UT) and plasma-treated Lavandin Grosso flowers for different treatment times. ATR-FTIR spectra of the cumulative essential oils extracted by the hydrodistillation of Lavandin Grosso flowers treated with plasma show a remarkable overlapping of all the peaks with respect to the untreated flowers, confirming that no significant variation in chemical composition can be observed after hydrodistillation. Therefore, the results seem to confirm that plasma active species just modify the surface of the glandular trichomes and do not interact significantly with the essential oils located inside the glandular trichomes. 2.2. Characterization of Plasma Active Species Present in the Plasma OES (Optical Emission Epectroscopy) is a very powerful method to identify active chemical species present in plasma. The identification of such species can provide information on the plasma/surface reactions that are taking place. The UV-vis emission spectra obtained during the plasma treatment with and without Lavandin Grosso flowers inside the plasma reactor are shown in Figure 4. The emission spectra recorded during the plasma treatment without Lavandin Grosso flowers inside the plasma reactor show different characteristic emission bands associated with nitroxide molecules (NOϒ) in the region 225–283 nm, OH radicals (OH•: 308.9 nm, 287 nm) [47,48], nitrogen molecules (second positive system, N2 SPS) in the region 297–430 nm, nitrogen first negative system (N+2 FNS) in the region 390–490 nm and nitrogen first positive system (N2 FPS) in the region 660–760 nm [49–52], and atomic oxygen lines at 715 and 777 nm [53–58]. The presence of OH radicals can be attributed to the water-rich atmosphere employed, whereas the remaining peaks associated with nitrogen and oxygen reactive species result from the presence of air in the reactor environment. The spectrum collected at 10 min of plasma treatment with Lavandin Grosso flowers inside the reactor shows a slight decrease in the peak’s intensity attributed to the smaller effective area of the electrode due to the presence of the Lavandin Grosso flowers on it. However, no emission bands associated with carbon Figure 3. ATR-FTIR spectra of the cumulative essential oils extracted by hydrodistillation of untreated (UT) and plasma-treated Lavandin Grosso flowers for different treatment times. 2.2. Characterization of Plasma Active Species Present in the Plasma OES (Optical Emission Epectroscopy) is a very powerful method to identify active chemical species present in plasma. The identification of such species can provide information on the plasma/surface reactions that are taking place. The UV-vis emission spectra obtained during the plasma treatment with and without Lavandin Grosso flowers inside the plasma reactor are shown in Figure 4. The emission spectra recorded during the plasma treatment without Lavandin Grosso flowers inside the plasma reactor show different characteristic emission bands associated with nitroxide molecules (NO Υ ) in the region 225–283 nm, OH radicals (OH • : 308.9 nm, 287 nm) [ 47 , 48 ], nitrogen molecules (second positive system, N 2 SPS) in the region 297–430 nm, nitrogen first negative system (N +2 FNS) in the region 390–490 nm and nitrogen first positive system (N 2 FPS) in the region 660–760 nm [ 49 – 52 ], and atomic oxygen lines at 715 and 777 nm [ 53 – 58 ]. The presence of OH radicals can be attributed to the water-rich atmosphere employed, whereas the remaining peaks associated with nitrogen and oxygen reactive species result from the presence of air in the reactor environment. The spectrum collected at 10 min of plasma treatment with Lavandin Grosso flowers inside the reactor shows a slight decrease in the peak’s intensity attributed to the smaller effective area of the electrode due to the presence of the Lavandin Grosso flowers on it. However, no emission bands associated with carbon species (CN, CH, and C 2 ) are observed [ 57 , 59 , 60 ], suggesting that EO exudation from the glandular trichomes during plasma treatment is not appreciable in the experimental conditions used in this work, even for the longest time of plasma treatment (10 min). Int. J. Mol. Sci. 2024,25, 2383 7 of 17 1 Figure 4. Optical emission spectra of the plasma generated with (red curve) and without (black curve) Lavandin Grosso flowers inside the reactor. 2.3. Weight Loss Tracking of Lavandin Grosso Flowers Treated with Plasma Biopolymeric materials exposed to plasmas or low-pressure conditions usually exhibit a measurable weight loss due to different physical or chemical processes such as water desorption and/or hydrocarbon oxidative etching [ 61 – 63 ]. Figure 5depicts the weight loss for Lavandin Grosso flowers subjected to the different plasma treatment times used in this work. Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 of 19 species (CN, CH, and C2) are observed [57,59,60], suggesting that EO exudation from the glandular trichomes during plasma treatment is not appreciable in the experimental conditions used in this work, even for the longest time of plasma treatment (10 min). Figure 4. Optical emission spectra of the plasma generated with (red curve) and without (black curve) Lavandin Grosso flowers inside the reactor. 2.3. Weight Loss Tracking of Lavandin Grosso Flowers Treated with Plasma Biopolymeric materials exposed to plasmas or low-pressure conditions usually exhibit a measurable weight loss due to different physical or chemical processes such as water desorption and/or hydrocarbon oxidative etching [61–63]. Figure 5 depicts the weight loss for Lavandin Grosso flowers subjected to the different plasma treatment times used in this work. Figure 5. Lavandin Grosso flowers’ weight loss (%) as a function of plasma treatment time. As observed, Lavandin Grosso flowers experience progressive weight loss as the treatment time increases, reaching a maximum of 1.2% at 10 min. It is worth mentioning Figure 5. Lavandin Grosso flowers’ weight loss (%) as a function of plasma treatment time. As observed, Lavandin Grosso flowers experience progressive weight loss as the treatment time increases, reaching a maximum of 1.2% at 10 min. It is worth mentioning that despite a minor weight loss (approximately 0.5%) found for 1 min treatment, the essential oil yield values depicted in Figure 1for this treatment (3.44%) exceed those of the untreated sample (3.19%). This suggests that the weight loss primarily stems from the removal of water content within the flowers. On the other hand, the slight increase in weight loss (Figure 5) and the progressive decrease in the extraction yield of the essential Int. J. Mol. Sci. 2024,25, 2383 8 of 17 oils (Figure 1) with treatment time could indicate that in addition to the water loss process, there could be other processes such as exudation of the essential oils during plasma treatment or possible chemical modification of the essential oils located within the glandular trichome of the Lavandin Grosso flower such that they are not easily evaporated during the hydrodistillation process and therefore cannot be detected in the extracted essential oil. 2.4. Wettability and Water Vapor Adsorption of Plasma-Treated Lavandin Grosso Flowers To further investigate the mechanism by which Lavandin Grosso essential oil extraction is enhanced by short plasma treatment times, the interaction of Lavandin Grosso flowers with water and water vapor occurring during hydrodistillation was evaluated by means of water contact angle and water vapor adsorption. Small droplets placed onto Lavandin Grosso flowers can be visualized in Figure 6, and the apparent water contact angle value is obtained from these images. Due to the small size of dry Lavandin Grosso flowers (~5 mm long and ~1 mm width) and the high curvature of their surface, the apparent water contact angle is strongly affected by the location where the small water drop is placed onto the flower, and therefore the results obtained can only be interpreted qualitatively. Taking this into account, untreated Lavandin Grosso flowers evidence a hydrophobic apparent water contact angle (126.5 ◦ ) that can be related to their surface chemical composition and to their high topographic surface roughness [ 64 ]. On this basis, plasma treatment progressively affects apparent water contact angle and superhydrophilic behavior can be observed if water droplet is deposited onto a flower area exposed directly to plasma active species. However, as can be observed in Figure 6b, a flower area not directly exposed to plasma active species (i.e., part of the flower leaning onto the lower electrode) preserves a more hydrophobic behavior. Nevertheless, the apparent water contact angle measured on those “hidden” regions tends to decrease as a function of plasma treatment time becoming hydrophilic after 5 min of plasma treatment. Although the apparent water contact angle measured on Lavandin Grosso flowers can just report qualitative results, it is clear that plasma treatment noticeably modifies the wetting behavior from hydrophobic to superhydrophilic. Additionally, the induced surface chemical and/ or topographic heterogeneity (differences between changes in the faced and opposite regions to the plasma discharge) of Lavandin Grosso flowers tend to decrease as a function of plasma treatment time [ 62 , 65 , 66 ]. Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 9 of 19 that despite a minor weight loss (approximately 0.5%) found for 1 min treatment, the essential oil yield values depicted in Figure 1 for this treatment (3.44%) exceed those of the untreated sample (3.19%). This suggests that the weight loss primarily stems from the removal of water content within the flowers. On the other hand, the slight increase in weight loss (Figure 5) and the progressive decrease in the extraction yield of the essential oils (Figure 1) with treatment time could indicate that in addition to the water loss process, there could be other processes such as exudation of the essential oils during plasma treatment or possible chemical modification of the essential oils located within the glandular trichome of the Lavandin Grosso flower such that they are not easily evaporated during the hydrodistillation process and therefore cannot be detected in the extracted essential oil. 2.4. Wettability and Water Vapor Adsorption of Plasma-Treated Lavandin Grosso Flowers To further investigate the mechanism by which Lavandin Grosso essential oil extraction is enhanced by short plasma treatment times, the interaction of Lavandin Grosso flowers with water and water vapor occurring during hydrodistillation was evaluated by means of water contact angle and water vapor adsorption. Small droplets placed onto Lavandin Grosso flowers can be visualized in Figure 6, and the apparent water contact angle value is obtained from these images. Due to the small size of dry Lavandin Grosso flowers (~5 mm long and ~1 mm width) and the high curvature of their surface, the apparent water contact angle is strongly affected by the location where the small water drop is placed onto the flower, and therefore the results obtained can only be interpreted qualitatively. Taking this into account, untreated Lavandin Grosso flowers evidence a hydrophobic apparent water contact angle (126.5°) that can be related to their surface chemical composition and to their high topographic surface roughness [64]. On this basis, plasma treatment progressively affects apparent water contact angle and superhydrophilic behavior can be observed if water droplet is deposited onto a flower area exposed directly to plasma active species. However, as can be observed in Figure 6b, a flower area not directly exposed to plasma active species (i.e., part of the flower leaning onto the lower electrode) preserves a more hydrophobic behavior. Nevertheless, the apparent water contact angle measured on those “hidden” regions tends to decrease as a function of plasma treatment time becoming hydrophilic after 5 min of plasma treatment. Although the apparent water contact angle measured on Lavandin Grosso flowers can just report qualitative results, it is clear that plasma treatment noticeably modifies the wetting behavior from hydrophobic to superhydrophilic. Additionally, the induced surface chemical and/ or topographic heterogeneity (differences between changes in the faced and opposite regions to the plasma discharge) of Lavandin Grosso flowers tend to decrease as a function of plasma treatment time [62,65,66]. Figure 6. (a) Images of small water droplets placed onto plasma-treated Lavandin Grosso flowers and (b) evolution of water contact angle with the plasma treatment time for two different water droplet locations (the region in direct contact with the plasma-faced and the region in contact with the electrode (opposite)). Note: Lines representing standard deviations have been intentionally truncated in some data points for visual clarity. Corresponding error bars (upper or lower) are maintained for some data points. The untreated sample is denoted by the abbreviation UT. Int. J. Mol. Sci. 2024,25, 2383 9 of 17 During the hydrodistillation process, the surface of Lavandin Grosso flowers also interacts with water vapor. Thus, the water vapor adsorption of untreated and plasma-treated Lavandin Grosso flowers at short and long treatment times (1 and 10 min, respectively) was evaluated. In order to minimize the evaporation process of the essential oil that would overlap with the water adsorption process under the conditions used in the hydrodistillation (100 ◦ C and 100% R.H.), milder humidity and temperature conditions have been chosen during the dynamic vapor sorption (DVS) measurements (25 ◦ C and 60% R.H.). Figure 7 shows that plasma treatment of Lavandin Grosso flowers increases both the water vapor adsorption rate (higher slope in the curve) and the final water vapor yield, evidencing an increase in the interaction of the Lavandin Grosso flower surface with water vapor. This behavior correlates with the increase in hydrophilicity observed by means of the contact angle and suggests that the interaction between water molecules (vapor or liquid) and glandular trichomes is favored, increasing desorption of aromatic oils in a more effective way in Lavandin Grosso flowers treated with plasma. Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 10 of 19 Figure 6. (a) Images of small water droplets placed onto plasma-treated Lavandin Grosso flowers and (b) evolution of water contact angle with the plasma treatment time for two different water droplet locations (the region in direct contact with the plasma-faced and the region in contact with the electrode (opposite)). Note: Lines representing standard deviations have been intentionally truncated in some data points for visual clarity. Corresponding error bars (upper or lower) are maintained for some data points. The untreated sample is denoted by the abbreviation UT. During the hydrodistillation process, the surface of Lavandin Grosso flowers also interacts with water vapor. Thus, the water vapor adsorption of untreated and plasmatreated Lavandin Grosso flowers at short and long treatment times (1 and 10 min, respectively) was evaluated. In order to minimize the evaporation process of the essential oil that would overlap with the water adsorption process under the conditions used in the hydrodistillation (100 °C and 100% R.H.), milder humidity and temperature conditions have been chosen during the dynamic vapor sorption (DVS) measurements (25 °C and 60% R.H.). Figure 7 shows that plasma treatment of Lavandin Grosso flowers increases both the water vapor adsorption rate (higher slope in the curve) and the final water vapor yield, evidencing an increase in the interaction of the Lavandin Grosso flower surface with water vapor. This behavior correlates with the increase in hydrophilicity observed by means of the contact angle and suggests that the interaction between water molecules (vapor or liquid) and glandular trichomes is favored, increasing desorption of aromatic oils in a more effective way in Lavandin Grosso flowers treated with plasma. Figure 7. Water vapor adsorption (at 60% R.H. and 25 °C) for untreated (UT) and plasma-treated Lavandin Grosso flowers at short and long treatment times (1 min and 10 min). 2.5. XPS Surface Chemistry Analysis of Lavandin Grosso Flowers Treated with Plasma The modification of the surface chemical composition induced on plasma-treated Lavandin Grosso flowers was determined by XPS. Table 4 shows the elemental chemical composition as a function of plasma treatment time. Untreated Lavandin Grosso flowers show a high content of surface carbon (~88%), oxygen (~10%), and traces of other elements (Ca, Si, and Al). Based on the high apparent contact angle (~125 °) of the untreated Lavandin Grosso flowers, it is suggested that the outermost part of the flower surface is mainly composed of hydrocarbon groups (C-C and C-H), which are hydrophobic in nature and combined with the high surface roughness of the Lavandin Grosso flowers give a hydrophobic behavior. Depending on the time of treatment with water vapor plasma, a progressive decrease in the carbon content and an increase in surface oxygen are observed, while the chemical composition associated with the traces (Ca, Si, and Al) remains practically constant. Figure 7. Water vapor adsorption (at 60% R.H. and 25 ◦ C) for untreated (UT) and plasma-treated Lavandin Grosso flowers at short and long treatment times (1 min and 10 min). 2.5. XPS Surface Chemistry Analysis of Lavandin Grosso Flowers Treated with Plasma The modification of the surface chemical composition induced on plasma-treated Lavandin Grosso flowers was determined by XPS. Table 4shows the elemental chemical composition as a function of plasma treatment time. Untreated Lavandin Grosso flowers show a high content of surface carbon (~88%), oxygen (~10%), and traces of other elements (Ca, Si, and Al). Based on the high apparent contact angle (~125 ◦ ) of the untreated Lavandin Grosso flowers, it is suggested that the outermost part of the flower surface is mainly composed of hydrocarbon groups (C-C and C-H), which are hydrophobic in nature and combined with the high surface roughness of the Lavandin Grosso flowers give a hydrophobic behavior. Depending on the time of treatment with water vapor plasma, a progressive decrease in the carbon content and an increase in surface oxygen are observed, while the chemical composition associated with the traces (Ca, Si, and Al) remains practically constant. 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