Post-Distillation By-Products of Aromatic Plants from Lamiaceae Family as Rich Sources of Antioxidants and Enzyme Inhibitors

There is currently no use for the vast quantities of post-distillation by-products, such as spent plant materials and residual waters, produced by the essential oil (EO) industry of aromatic herbs. In this study, the EOs of three Lamiaceae species (thyme, oregano, and basil) and their total, spent, and residual water extracts were phytochemically characterized and biologically assessed. The collected information was put through a series of analyses, including principal component analysis, heatmap analysis, and Pearson correlation analysis. Concerning the EOs, 58 volatile compounds were present in thyme (e.g., p-cymene, thymol), 44 compounds in oregano (e.g., thymol, carvacrol), and 67 compounds in basil (e.g., eucalyptol, linalool, estragole, (E)-methyl cinnamate). The LC-HRMS/MS analysis of the total, spent, and residual water extracts showed the presence of 31 compounds in thyme (e.g., quercetin-O-hexoside, pebrellin, eriodictyol), 31 compounds in oregano (e.g., rosmarinic acid, apigenin, kaempferol, salvianolic acids I, B, and E), and 25 compounds in basil (e.g., fertaric acid, cichoric acid, caftaric acid, salvianolic acid A). The EOs of the three Lamiaceae species showed the highest metal-reducing properties (up to 1792.32 mg TE/g in the CUPRAC assay), whereas the spent extracts of oregano and basil displayed very high radical-scavenging properties (up to 266.59 mg TE/g in DPPH assay). All extracts exhibited anti-acetylcholinesterase (up to 3.29 mg GALAE/g), anti-tyrosinase (up to 70.00 mg KAE/g), anti-amylase (up to 0.66 mmol ACAE/g), and anti-glucosidase (up to 1.22 mmol ACAE/g) effects. Thus, the present research demonstrated that both the raw extracts (EOs and total extracts) and the post-distillation by-products (spent material and residual water extracts) are rich in bioactive metabolites with antioxidant and enzyme inhibitory properties.


Introduction
Aromatic plants/herbs are important constituents of human nutrition, valued as agents of aroma, flavor, color, and preservation in food. Additionally, aromatic plants contain various phytochemicals endowed with potent biological activity, making them promising candidates for the treatment of a variety of chronic diseases [1]. The culinary, cosmeceutical, and medicinal uses of plants in the Lamiaceae family have made them stand out among aromatic herbs over the years [2].
Thymus L. is one of the largest genera of the Lamiaceae family, with over 350 species found in Europe, Asia, and North Africa [3]. Among them, the most significant species is antimicrobial activity of decocts and macerates from the spent materials of thyme and basil were also performed [2,8,35].

Plant Material
Dried aerial parts of thyme (Thymus vulgaris L.), oregano (Origanum vulgare L.), and basil (Ocimum basilicum L.) were purchased from local markets in Germany; their identity was confirmed by one of the authors (A.T.). Vouchers (TV/220714, OV/220714, OB/220714) were stored in the Department of Pharmacognosy and Phytotherapy, Faculty of Pharmacy, "Grigore T. Popa" University of Medicine and Pharmacy Iasi, Romania.

Essential Oil Isolation
The powdered plant material (50 g from each species) was subjected to hydrodistillation with 500 mL of deionized water for 4 h in a Clevenger-type apparatus. The obtained essential oils (EOs) were measured (with the scale of the apparatus in mL), collected, dried over anhydrous sodium sulfate, and stored in dark glass tubes at 4 • C until further analysis. Each procedure was performed in duplicate.

Preparation of Total, Spent, and Residual Water Extracts
At the end of the hydrodistillation process, the water in the flask was filtered, and aliquots (25 mL) were freeze-dried, yielding the residual water extracts (WE). The residue (spent plant material) was dried in an oven at 40 • C for 48 h, and 10 g was extracted with 3 × 100 mL of methanol/water 75/25 (v/v) by ultra-sonication (30 min). After filtration, the obtained extracts (spent extracts, SE) were evaporated to dryness under vacuum and kept at −20 • C until further analysis. For comparison purposes, unprocessed powdered plant material (10 g from each species) was extracted similarly with 3 × 100 mL of methanol/water 75/25 (v/v) by ultra-sonication (30 min). After filtration, the obtained extracts (total extracts, TE) were evaporated to dryness under vacuum and kept at −20 • C until analysis. Each procedure was performed in duplicate.
The extraction yields to obtain the EOs, WE, SE, and TE from thyme, oregano, and basil are provided in Table 1.

GC-MS Analysis
The analyses were performed on a TRACE gas chromatograph (GC) coupled to an ISQ™ mass spectrometer (MS) from Thermo Fisher (Waltham, MA, USA). The column was Zebron™ ZB-5MS (30 m × 0.25 mm i.d., 0.25 µm film thickness) from Phenomenex (Torrance, CA, USA); Carrier gas-helium; flow rate-1.43 mL/min; inlet temperature-250 • C; split ratio-50:1; injection volume-1 µL. The oven temperature was programmed as follows: 4 min held at 60 • C; then ramped up to 280 • C at a rate of 10 • C/min and held at 280 • C for 5 min; then ramped to 300 • C at 10 • C/min and held at 300 • C for 10 min. The MS was set in full scan mode from 50-350 amu, with the following parameters: ionization energy-70 eV; source temperature-230 • C; transfer line temperature-320 • C. Linear retention indices (LRI) were calculated for the individual compounds using a standard mixture of C8-C20 n-alkanes. The compounds were identified by comparing their mass spectra with those from the NIST 11 Mass Spectra Library and their LRI with literature data. Each analysis was performed in triplicate.

Statistical and Data Processing
Data are presented as mean ± standard deviation of the number of replicates. Oneway analysis of variance with Tukey's post-hoc test was conducted; p < 0.05 was considered statistically significant. The relationship between the molecules and antioxidant activities and enzyme inhibitory activities was assessed by calculating the Pearson correlation coefficient. Pearson coefficient values >0.8 were considered significant. Afterward, the principal component analysis (PCA) and Heatmap analysis were performed. Before the PCA, the LC-HRMS/MS data (peak areas from the base peak chromatograms) were log-transformed. The statistical and data processing was done using R software v. 4.1.2.
In the case of oregano EO, 44 compounds accounted for up to 99% of the total peaks observed in the GC-MS chromatograms ( Table 2). Similar to the thyme EO, 95.03% were monoterpenes, whereas 4.74% were sesquiterpenes. Carvacrol (69.93%) was the dominant constituent, followed by thymol (9.40%) and p-cymene (6.71%). These findings are consistent with previous reports. Ozdemir et al. [16] showed that O. vulgare EOs obtained from differently processed plant materials contained levels of carvacrol between 45.09-46.71% and thymol between 14.67-15.72%. Carvacrol (72.8-79.5%) was also shown as the main constituent in the EO obtained from the leaves and stems of oregano [17].

LC-HRMS/MS Analysis of Total, Spent, and Residual Water Extracts
The total (unspent plant material) extracts (TE), residual water extracts (WE), and spent plant material (solid residue) extracts (SE) were obtained from thyme, oregano, and basil, as described in Section 2.2.2. For all samples, the highest extraction yields were obtained for the WE, followed by the TE and SE (Table 1). All extracts were phytochemically profiled by LC-HRMS/MS, with the spectro-chromatographic data provided in Table 3.
A total of 25 specialized metabolites were labeled in the basil extracts ( Table 3). As mentioned above for the thyme and oregano extracts, danshensu (2), hydroxybenzoic acid (4), caffeic acid (9), salvianolic acid H (21), salvianolic acid D (25), rosmarinic acid (26), and 3,4,4'-trihydroxy-5,5'-diisopropyl-2,2'-dimethylbiphenyl (52) were also annotated as phenolic compounds in WE, SE, and TE of oregano. In addition, caftaric acid (5), fertaric acid (13), cichoric acid (29), and salvianolic acid isomer (34) were also evidenced. Most of these phenolic metabolites were previously reported in Ocimum species by LC-MS analyses [28,40]. The group of flavonoids was represented by eight derivatives, such as free aglycones: luteolin (35), cirsimaritin (38), ladanein (40), and cirsilineol (45) (22), and luteolin-O-deoxyhexoside-O-hexoside (24); and C-glycosides: luteolin-C-deoxyhexoside-C-hexoside (14). Flavonoids with similar structures were previously documented in the Ocimum genus [22,28,40]. Lastly, four fatty acids (trihydroxyoctadecadienoic acid I, 36, hydroperoxyoctadecadienoic acid, 46, hydroxyoctadecatrienoic acid, 49, and hydroxyoctadecadienoic acid 51), quinic acid (1), and roseoside (8) were also spotted in the obtained basil extracts. The differences observed between WE, SE, and TE were not significant. For instance, caffeic acid (9), a few flavonoids (19,22,24,35, and 40), and salvianolic acid D (25) were absent from the TE of oregano. In contrast, some lipophilic fatty acids (46 and 51) were not retrieved in the WE.    As can be seen from the above discussion, numerous specialized metabolites were simultaneously found in all three Lamiaceae species. The heatmap approach was used to identify the key molecules unique to thyme, oregano, and basil. Firstly, as shown in Figure 1  In conclusion, the by-products of the hydrodistillation of the three Lamiaceae herbs, residual water and spent material extracts, can be regarded as rich sources of bioactive metabolite, mainly phenolic compounds. Compared with the total (unspent material) extracts, no groundbreaking qualitative differences in the phytochemical profiles were observed for the by-product extracts. Generally, the spent extracts showed the highest abun- In conclusion, the by-products of the hydrodistillation of the three Lamiaceae herbs, residual water and spent material extracts, can be regarded as rich sources of bioactive metabolite, mainly phenolic compounds. Compared with the total (unspent material) extracts, no groundbreaking qualitative differences in the phytochemical profiles were observed for the by-product extracts. Generally, the spent extracts showed the highest abundance of phytochemicals; several possible theories can be formulated. On the one hand, compounds that might be found in small amounts in the original (unspent) plant materials and are transferred neither into the hydrodistillate nor into the water used for hydrodistillation may become more accessible to the subsequent solvent extraction of the spent plant materials. On the other hand, following the long exposure of the plant material to boiling water, a cell permeation effect can be assumed, favoring the subsequent extraction of the metabolites or even the transfer of medium or low polarity molecules into the hydrodistillation water. In addition, due to the harsh hydrodistillation conditions (high temperatures and long exposure times), the formation of phenolic artifacts in the residual water and spent extracts cannot be excluded.

Total Phenolic and Flavonoid Content of Total, Spent, and Residual Water Extracts
Phenolic compounds are one of the most popular class of phytochemicals that are gaining increasing interest due to their wide range of biological activities [46]. In this sense, estimating the phenolic content could provide the first clues about their biological potential. In this section, the total phenolic and flavonoid content of the total, spent, and residual water extracts of thyme, oregano, and basil were determined, with the results summarized in Table 4. In basil, the highest total phenolic content was found in the spent extract with 93.66 mg GAE/g, followed by the residual water (63.57 mg GAE/g) and total (58.85 mg GAE/g) extracts. Regarding thyme, the residual water extract had the highest concentration of total phenolics (88.69 mg GAE/g). However, the spent and total extracts of thyme had lower values. In oregano, the phenolic levels were reported as 115.71, 113.34, and 110.35 mg GAE/g for total, spent, and residual water extract, respectively. Concerning the total flavonoid content, the spent extracts of all three species had higher concentrations than the total and residual water extracts. The highest level of total flavonoid was determined in the spent extracts of oregano (34.88 mg RE/g), followed by thyme (28.16 mg RE/g) and basil (27.59 mg RE/g). The extraction process subsequent to hydrodistillation significantly increased the total flavonoid concentration. Based on these findings, the hydrodistillation process may destroy the cell wall, allowing the methanol to enter the cell faster and more efficiently during the extraction process. Some antioxidant compounds were not dissolved in boiling water in a previous study; thus, extraction with polar solvents such as methanol or ethanol could help extract more compounds from materials after hydrodistillation [47]. In line with our findings, several researchers have reported that post-hydrodistillation extraction increased flavonoid levels [48,49].

Antioxidant Activity of Essential Oils, Total, Spent, and Residual Water Extracts
Antioxidants are thought to protect against the onslaught of free radicals, which are the primary cause of the progression of severe health conditions, such as Alzheimer's, diabetes, and cancer. In this regard, identifying new raw materials as sources of antioxidants has become one of the most popular topics within the scientific community [50]. The EOs and extracts of thyme, oregano, and basil were tested in various antioxidant assays (Table 5). In the DPPH assay, the extracts showed a more significant activity than EOs. Concerning the thyme extracts, the highest activity was noticed in the residual water extract (DPPH: 121.00 mg TE/g; ABTS: 150.09 mg TE/g). The most active oregano samples were the spent (266.59 mg TE/g) and total extracts (347.67 mg TE/g) for DPPH and ABTS, respectively. In basil, the highest free radical scavenging ability was determined in spent extract (DPPH: 135.88 mg TE/g; ABTS: 144.57 mg TE/g). When all extracts were evaluated together, oregano extracts were more active than the corresponding basil and thyme extracts. As can be seen in Table 5, the EOs showed a more substantial CUPRAC power than the extracts; the EO of oregano was the most active sample at 1792.32 mg TE/g. In the CUPRAC assay, the spent extracts of basil and oregano displayed the highest capacity, while the residual water of thyme was the most active. Basil and thyme EOs were the most potent in the FRAP assay, whereas oregano's spent extract was the most effective. The EO of basil (31.24 mmol TE/g) was the most active in the PBD assay, followed by the EOs of thyme (10.14 mmol TE/g) and oregano (5.74 mmol TE/g). These findings are consistent with the CUPRAC and FRAP results. Overall, the results of the extracts from all samples were almost the same in the free radical scavenger and reducing power tests. Some compounds in the extracts, including caftaric acid, roseoside, fertaric acid, and cichoric acid, were strongly correlated with antioxidant activity (Table S1). Consistent with our findings, several researchers have described these compounds as powerful antioxidants [51][52][53][54]. In addition, Table S2 shows the correlation between EO compounds and antioxidant properties. For example, cis-terpineol and thymol methyl ester strongly correlated with DPPH scavenging ability, while limonene and camphene mainly contributed to the FRAP power.
In contrast to other antioxidant assays, the highest metal chelating propensities were found in the residual water extracts of the three species (10.74-17.50 mg EDTAE/g). As can be seen in Table S1, no compounds correlated with the observed metal chelating ability. In this sense, the activity can be explained by the presence of non-phenolic chelating agents, including polysaccharides or peptides. Regarding the metal chelating potential of EOs, only the EO from basil was active with 1.34 mg EDTAE/g, but others showed no chelating ability. The spent extracts from the tested Lamiaceae species (especially basil and oregano) contained significant antioxidant properties [55][56][57]. Consequently, the byproducts of post-distillation can be considered rich sources of natural antioxidants with potential pharmaceutical and nutraceutical applications. Values are reported as mean ± SD of three parallel measurements. TE: Trolox equivalent; EDTAE: EDTA equivalent; na: not active; Different letters indicate significant differences among the extracts/essential oils from each species (p < 0.05).

Enzyme Inhibitory Activity of Essential Oils, Total, Spent, and Residual Water Extracts
Inhibiting key enzymes associated with the pathologies of global health issues may benefit disease treatment. For example, amylase and glucosidase are essential enzymes for hydrolyzing carbohydrates, and when inhibited, diabetic patients' blood glucose levels can be regulated [58]. Similarly, AChE breaks down acetylcholine in the synaptic cleft under normal conditions. If AChE is inhibited in Alzheimer's patients, the level of acetylcholine in the synaptic cleft can rise, thereby enhancing the cognitive functions in the patients [59]. In this sense, several compounds have been chemically designed as inhibitor drugs used in clinical practice. Their long-term use, however, resulted in adverse health effects such as gastrointestinal disturbances and hepatotoxicity [60]. This study evaluated the enzyme inhibitory properties of thyme, oregano, and basil against cholinesterases, tyrosinase, amylase, and glucosidase ( Table 6). Basil and thyme EOs had the highest AChE inhibitory activity (basil: 3.29 mg GALAE/g; thyme: 1.80 mg GALAE/g), followed by spent, total, and residual water extracts. Regarding the AChE inhibitory effects of oregano samples, the order of potency was total extract > EO > spent extract > residual water. Surprisingly, only the EOs showed inhibitory effects on BChE. The best BChE inhibitory effect was observed with EOs of basil, with 1.28 mg GALAE/g, followed by oregano and thyme. In summary, the tested EOs had good potential as cholinesterase inhibitors. A strong correlation between several EOs constituents (e.g., α-pinene, cis-α-terpineol, and p-cymene) and their cholinesterase inhibitory properties was noticed (Table S4). In the literature, several authors pointed out that the EOs have significant cholinesterase inhibiting effects [61][62][63].
In terms of the tyrosinase inhibitory activity, the best capacity was found among the EOs; for the extracts, the following decreasing order of activity was noticed: spent > total > residual water. In the correlation analysis, some compounds in the extracts and EOs might contribute to the observed tyrosinase inhibitory properties (Tables S3 and S4). Concerning the antidiabetic-related properties, the anti-amylase activity of the EOs was succeeded by that of the spent, total, and residual water extracts. Interestingly, no EO showed any inhibitory effects on glucosidase. The highest glucosidase inhibitory values were recorded in the spent extracts, followed by the total and residual water extracts. Several previous studies were conducted on the enzyme inhibitory properties of basil, thyme, and oregano EOs or extracts. The literature indicates that these species had significant potential as natural enzyme inhibitors as well as antioxidants [64][65][66][67][68][69][70][71]. Furthermore, our findings shed light on the valuable activity of post-distillation by-products, which can be used to develop novel enzyme inhibition drugs. These findings could also aid in the recycling of plant materials following EO extraction.

Multivariate Analysis
Principal component analysis (PCA) and heatmapping are widely used to analyze complex chemical and biological data. While PCA is used to reduce the dimensionality of high-dimensional data, heatmaps represent a data matrix in which rows and/or columns of the matrix are clustered to allow the visualization of the values in the cells by using a color gradient [72].
In this study, PCA was initially used to explain the differences between the samples and obtain information on bioactivity. The results of the PCA are given in Figure 2A-C. Dimension 1 accounted for 52.4% of the total variance and was primarily associated with amylase, BChE, tyrosinase, and, to a lesser extent, PBD and glucosidase. Dimension 2 accounted for 27.3% of the total variance and was determined primarily by ABTS, DPPH, FRAP, and AChE, whereas dimension 3 accounted for 9.6% of the total variance and was defined by CUPRAC, PBD, and MCA. These results suggest that the extracts are separated along each dimension according to the bioactivities that characterize them. These three dimensions were retained because they had eigenvalues greater than 1. The next step was to explore the different scatter plots formed by the three retained dimensions. In the scatter plot Dim-1 vs. Dim-2, the EOs of the three species were separated from the other extracts. In addition, the spent and total extracts of oregano were more distant from the other extracts. In the scatter plot Dim-1 vs. Dim-3, the EO of basil stood out from the EOs of thyme and oregano. This trend was confirmed in the scatter plot Dim-2 vs. Dim-3. In this study, PCA was initially used to explain the differences between the samples and obtain information on bioactivity. The results of the PCA are given in Figure 2A-C. Dimension 1 accounted for 52.4% of the total variance and was primarily associated with amylase, BChE, tyrosinase, and, to a lesser extent, PBD and glucosidase. Dimension 2 accounted for 27.3% of the total variance and was determined primarily by ABTS, DPPH, FRAP, and AChE, whereas dimension 3 accounted for 9.6% of the total variance and was defined by CUPRAC, PBD, and MCA. These results suggest that the extracts are separated along each dimension according to the bioactivities that characterize them. These three dimensions were retained because they had eigenvalues greater than 1. The next step was to explore the different scatter plots formed by the three retained dimensions. In the scatter plot Dim-1 vs. Dim-2, the EOs of the three species were separated from the other extracts. In addition, the spent and total extracts of oregano were more distant from the other extracts. In the scatter plot Dim-1 vs. Dim-3, the EO of basil stood out from the EOs of thyme and oregano. This trend was confirmed in the scatter plot Dim-2 vs. Dim-3. Next, a Heatmap analysis was performed to better represent the various clusters and visualize the differences in bioactivities between each cluster. As shown in Figure 3, the extracts were separated into four distinct groups. Oregano EO and thyme EO were in cluster I, while basil EO represented cluster II. Cluster I showed stronger anti-tyrosinase and anti-amylase activities, as also demonstrated by cluster II. In addition, cluster I exhibited remarkable CUPRAC activity, while cluster II showed the highest anti-BChE, PBD, and FRAP activities. Next, a Heatmap analysis was performed to better represent the various clusters and visualize the differences in bioactivities between each cluster. As shown in Figure 3, the extracts were separated into four distinct groups. Oregano EO and thyme EO were in cluster I, while basil EO represented cluster II. Cluster I showed stronger anti-tyrosinase and anti-amylase activities, as also demonstrated by cluster II. In addition, cluster I exhibited remarkable CUPRAC activity, while cluster II showed the highest anti-BChE, PBD, and FRAP activities.
Furthermore, it was noticed that the EOs of clusters I and II were segregated from the other extracts, which represented clusters III and IV. Hence, spent and total extracts of oregano showed a similar bioactivity profile and were placed in cluster III. This cluster exhibited higher ABTS and DPPH activities. Lastly, the remaining extracts were grouped in cluster IV. Overall, the extracts of this cluster were characterized by low bioactivity, except for the residual water extracts of thyme and oregano, which showed significant chelating activity. The merged result showed that the heatmap conclusion agreed with the PCA observation. Accordingly, we can suggest that PCA coupled with the heatmap was suitable for discriminating the study samples. Furthermore, it was noticed that the EOs of clusters I and II were segregated from the other extracts, which represented clusters III and IV. Hence, spent and total extracts of oregano showed a similar bioactivity profile and were placed in cluster III. This cluster exhibited higher ABTS and DPPH activities. Lastly, the remaining extracts were grouped in cluster IV. Overall, the extracts of this cluster were characterized by low bioactivity, except for the residual water extracts of thyme and oregano, which showed significant chelating activity. The merged result showed that the heatmap conclusion agreed with the PCA observation. Accordingly, we can suggest that PCA coupled with the heatmap was suitable for discriminating the study samples.

Conclusions
In this work, two raw extracts (EOs obtained by hydrodistillation and total extracts) and two post-distillation by-products (spent material and residual water extracts) of thyme, oregano, and basil were phytochemically and biologically evaluated. More than 90 volatile compounds (hydrocarbon monoterpenes, oxygenated monoterpenes, hydrocarbon sesquiterpenes, oxygenated sesquiterpenes, etc.) were identified by GC-MS in the essential oils of the three Lamiaceae species. Around 50 specialized metabolites belonging to various phytochemical classes, such as phenolic and organic acids (e.g., quinic acid, hydroxybenzoic, caffeic acid, salvianolic acids, etc.), flavonoids (luteolin, kaempferol, quercetin and their O-and C-glycosides), diterpenes (e.g., carnosol), and fatty acids, were assigned with the use of LC-HRMS/MS in the solvent-based extracts. The Heatmap analysis revealed that the spent extracts showed a higher abundance of phenolic phytochemicals than the total or residual water extracts. Concerning the biological evaluations, all tested samples displayed strong antioxidant potential; the EOs were the most active metal reducing agents, whereas the spent and total extracts were the best scavengers of DPPH and ABTS. In addition, the EOs also displayed the highest cholinesterase and tyrosinase inhibitory properties, while the highest anti-glucosidase effects were recorded for the spent extracts.

Conclusions
In this work, two raw extracts (EOs obtained by hydrodistillation and total extracts) and two post-distillation by-products (spent material and residual water extracts) of thyme, oregano, and basil were phytochemically and biologically evaluated. More than 90 volatile compounds (hydrocarbon monoterpenes, oxygenated monoterpenes, hydrocarbon sesquiterpenes, oxygenated sesquiterpenes, etc.) were identified by GC-MS in the essential oils of the three Lamiaceae species. Around 50 specialized metabolites belonging to various phytochemical classes, such as phenolic and organic acids (e.g., quinic acid, hydroxybenzoic, caffeic acid, salvianolic acids, etc.), flavonoids (luteolin, kaempferol, quercetin and their Oand C-glycosides), diterpenes (e.g., carnosol), and fatty acids, were assigned with the use of LC-HRMS/MS in the solvent-based extracts. The Heatmap analysis revealed that the spent extracts showed a higher abundance of phenolic phytochemicals than the total or residual water extracts. Concerning the biological evaluations, all tested samples displayed strong antioxidant potential; the EOs were the most active metal reducing agents, whereas the spent and total extracts were the best scavengers of DPPH and ABTS. In addition, the EOs also displayed the highest cholinesterase and tyrosinase inhibitory properties, while the highest anti-glucosidase effects were recorded for the spent extracts.
Overall, the results of the current study provide new and insightful contributions to the continuously expanding body of knowledge regarding the potential valorization of post-distillation by-products as a low-cost source of high-value constituents for the food, pharmaceutical, and cosmetics industries. Furthermore, this can constitute a starting point for finding new ways of exploiting the large amounts of waste produced worldwide by the EO industry, with beneficial environmental, technological, and economic advantages.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox12010210/s1, Table S1. Correlation value between chemical compounds of extracts and antioxidant abilities (R 2 ). Table S2. Correlation value between chemical compounds of essential oils and antioxidant abilities (R 2 ). Table S3. Correlation value between chemical compounds of extracts and enzyme inhibitory abilities (R 2 ). Table S4. Correlation value between chemical compounds of essential oils and enzyme inhibitory abilities (R 2 ).