Chemical and Biochemical Characterization of Essential Oils and Their Corresponding Hydrolats from Six Species of the Lamiaceae Family

Many plants belonging to the Lamiaceae family are rich in essential oils (EOs) which are intensively used for aromatherapy, food and beverage flavoring, alternative medicine, cosmetics, and perfumery. Aerial parts of Thymus vulgaris L., Thymus pannonicus All., Lavandula angustifolia L., Lavandula x intermedia, Origanum vulgare L., and Origanum vulgare var. aureum L. were subjected to hydrodistillation, and both resulting fractions were analyzed. The purpose of this study was to determine the chemical composition, antioxidant activity, and total phenolic content of six essential oils and their corresponding hydrolats (HDs) through GC-MS and spectrophotometric analyses. Overall, 161 compounds were identified, some found exclusively in essential oils and others in hydrolats, making them individual products with specific end purposes. The total phenolic content was the highest for the Thymus vulgaris L. EOs (3022 ± mg GAE L−1), because of its high phenolic oxygenated monoterpenes content (thymol and carvacrol) and the smallest for the Lavandula angustifolia L. EOs (258.31 ± 44.29 mg GAE L−1), while hydrolats varied from 183.85 ± 0.22 mg GAE L−1 for Thymus vulgaris L. HD and 7.73 mg GAE L−1 for Thymus pannonicus All. HD. Significant antioxidant effects determined through DPPH• and ABTS•+ assays were also observed in samples with higher hydrophilic compounds. The highest antioxidant activity was determined for Thymus vulgaris L. EO and its corresponding HD. Although EOs are the principal traded economic product, HDs represent a valuable by-product that could still present intense antiseptic activities, similar to their corresponding EOs (thyme and oregano), or have multiple aromatherapy, cosmetics, and household applications (lavender and lavandin).


Introduction
Medicinal and aromatic plants have always served as sources of compounds with bioactive properties for ritual, food flavoring, medicinal, cosmetic, and hygienic purposes [1]. These compounds are secondary metabolites which serve many functions in plants, e.g., from signaling to defense molecules, improving the plant's chances of survival when faced with unfavorable environmental conditions [2]. Essential oils are mostly composed of highly complex, volatile organic compounds which are insoluble in water, mainly composed of monoterpenes and sesquiterpenes [3], representing one of the four main biological classes of natural compounds alongside polyphenols, alkaloids, and glycosides [1,4]. industry known as HDs or floral water [45]. The water-soluble EO components dissolved in the distillation water give the resulting HD its characteristic scent and flavor [36,46]. Most HDs have no further applications because of the low abundance of compounds of interest, and as such, are generally discarded [1,36]. Nonetheless, they are sometimes used in the food industry as flavoring agents (deserts and beverages), and in the cosmetic sector in skincare products [47].
This study had the following objectives: (i) to broaden the limited existing data regarding the chemical compositions of several EOs and their corresponding HDs using GC-MS analysis; and (ii) to perform biochemical analysis to determine the antioxidant activity and the total phenolic content of the EOs and their correspondent HDs. Table 1 presents the chemical composition of six EOs and their corresponding concentrated HDs resulting from the same distillation process. In total, 161 compounds were identified, of which 54 were specific to the EOs, 66 were commonly found in both EOs and HDs, and 41 were found exclusively in HDs. The major classes of compounds are presented in Figure 1. Considering that distillation is still the most used method for obtaining EOs, many HDs are generated in the process as by-products. These HDs have low concentrations of bioactive compounds, i.e., usually under 1000 mg per litter [48,49], while still presenting antioxidant and antimicrobial effects.

Essential Oils and Hydrolats Chemical Composition
Comparing the chemical profiles of the EOs and HDs resulting from the same distillation batch (Figure 2a) showed that both products had common compounds in different ratios. However, they both contained unique compounds and should be considered independent products. In Figure 2b, for lavender EO, we considered only constituents with concentrations above 0.5%. We compared 20 components, of which 14 were shared with their corresponding HD, and only five compounds with a concentration above 0.5% were exclusively found in the HD.    ------0.06  -----38  1130  2-Pinen-7-one  MO  ---0.42  --------39  1135  Limonene oxide  MO  ----0.34  0.3  ------40  1138  Limonene epoxide  MO  ---0.1  --------41  1142  cis verbenol  MO  --0.86  ---------42 1144 1147       RI: calculated retention indices relative to n-alkanes (C10-C35); Compounds identified by using comparison with standards were marked with an asterisk (*). To further compare the chemical compositions of the EOs and HDs in the analyzed samples, we determined the ratios of commonly found compounds in both samples. The results are shown in Figure 3.

Total Phenolic Content
Folin-Ciocâlteu reagent was used to determine the total phenolic content of the EO and HDs samples. For the EO, a 1:10 dilution with methanol was needed, while the HD was used without dilution. The results varied between 3022 mg GAE L −1 for TVEO and 7.73 mg GAE L −1 for TPHD.
The data are presented in Table 2.

Antioxidant Activity
For all EOs and HDs, the antioxidant activity was evaluated using DPPH • and ABTS •+ assays ( Table 3). The inhibition results ranged from 4.89% for LIHD to 94.27% for TVEO for DPPH • assay and from 10.11% for TPHD to 98.38% for LAEO for ABTS •+ assay.
In several countries, the production of EOs is one of the most important industries, with a trading market worth billions of USD annually. It has been projected that the EO market will reach 27 billion USD in 2022. The majority of HDs are wasted, and the recovery of this by-product could be economically valued.
The chemical composition of EOs and HDs were determined using the GC-MS technique, which is a gold standard in the field, allowing the determination of all major and minor compounds. The average chemical composition of EOs comprised two major compound classes: hydrocarbonated compounds or terpenes (monoterpenes, sesquiterpenes, diterpenes) and oxygenated compounds or terpenoids (derived from terpenes, alcohols, aldehydes, phenols, esters, ketones, lactones) [50].
Hidrocarbonated terpenes (limonene, β-caryophyllene, pinenes) are nonpolar and do not bond with water molecules. Therefore, they are found almost exclusively in EOs and rarely in HDs; their presence in the latter usually indicates poor separation [50].
In contrast, HDs are rich in many oxygenated compounds which are more soluble in water, making thyme, oregano, and lavender HDs rich in biologically active compounds, like terpenes, compared to citrus or coniferous HDs [50].
According to Šilha et al. [3], the abundance of compounds determined in the HDs results from the favorable conditions present during the steam distillation process instead of hydrodistillation. Some compounds can interact with the surrounding boiling water and get transformed into different compounds through oxidation, polymerization, or hydrolyzation. (e.g., β-caryophyllene to caryophyllene oxide, limonene to limonene oxide) [51].
According to Garneau et al. [48], Melissa officinalis has around 30 compounds found specifically in its EO, 24 in the HD, among which 11 are commonly found in both products.
In the wild thyme samples analyzed in this study, i.e., TVEO and TVHD, 57 and 19 compounds respectively were identified, of which three were exclusively found in the HD. The chemical profiles were represented mainly through oxygenated monoterpenes 52.4% in TVEO and 93.68% in TVHD, represented by thymol methyl ether, carvacrol methyl ether, thymol, carvacrol, followed by hydrocarbonated monoterpenes comprising 34.02% in TVEO and 2.47% in TVHD, represented by p-cymene. Other studies have indicated that thymol, p-cymene, limonene, and carvacrol are the major terpenes detected [11,52]. Sesquiterpenes were almost exclusively identified in TVEO, with 11.96% instead of 1.91% being determined in TVHD.
Usually, the chemical compositions of HDs are different from those of their corresponding EOs, being enriched in hydrophilic oxygenated terpenes, especially phenols [53].
Because of their phenolic structure, thymol 62.96% and carvacrol 21.48% were present in high quantities in TVHD, as previously reported in other studies [53,54].
The ratio of hydrolats to essential oils has been calculated for the major common compounds that occur in both sample types (Figure 3). These results confirmed, once again, the presence of oxidized compounds as major compounds in the corresponding HDs. For example, the highest ratio values for one of the compounds found in HDs compared with EOs is carvacrol 11.61 for TVHD/TVEO; elemol 7.20 for TPHD/TPEO; α-terpineol 5.13 for LAHD/LAEO; cis-geraniol 9.29 for LIHD/LIEO; terpinen-4-ol 247 for OVHD/OVEO; trans-sabinene hydrate (4-thujanol) 9.82 for OVAHD/OVAEO.
Overall, a similar chemical pattern was observed for all EOs and their corresponding HDs, polarity influencing the distribution of chemical compounds. The absence of oxygen in the nonpolar molecules like hydrocarbonated monoterpenes and sesquiterpenes (p-cymene, γ-terpinene, germacrene D, aromandendrene), make these compounds specific to the EO, and their presence in the HDs could indicate a poorly performed separation.
Instead, slightly more water-soluble compounds, like oxygenated monoterpenes and sesquiterpenes (alcohols: linalool, terpinen-4-ol, eucalyptol, geraniol; ketones: camphor, piperitone, D-carvone; aldehydes: β-citral, geranial; phenols: thymol, carvacrol) are usually present in higher quantities, and the higher the polarity, the higher the proportion of dissolved compounds in HDs. Therefore, a difference between the EO and its corresponding HD is visible, and both products need to be considered independent of each other to recommend them for different purposes.
The total phenolic content among all six EOs samples varied as follows: TVEO > OVAEO > TPEO > OVEO > LIEO > LAEO and for their six corresponding HDs TVHD > OVAHD > LIHD > LAHD > OVHD > TPHD. As previously reported by other studies [52,56], thyme plants have high amounts of thymol and carvacrol, both of which are phenolic oxygenated monoterpenes, leading to a high total phenolic content. The lowest phenolic equivalent for EOs was observed in LAEO with 258.31 mg GAE L −1 , while the highest was recorded for TVEO with 3022.36 mg GAE L −1 .
Among the HDs, TVHD has the highest phenolic content, i.e., 183.85 mg GAE L −1 in accordance with its chemical composition, thymol, and carvacrol, accounting for over 80% of its constituents. In comparison, TPHD and OVHD presented the lowest phenolic content, i.e., 7.7 mg GAE L −1 following their low oxygenated terpenes content.
The antioxidant activity of the EOs and HD samples was evaluated using DPPH • and ABTS •+ assays. The antioxidant activity for all six EOs and their corresponding HDs was influenced mainly by the presence of p-cymene, γ-terpinene, eucalyptol, linalool, thymol, carvacrol, and the synergistic role that the combination of one or more compounds played was reported previously by other studies [58].
As presented by other studies [59][60][61], the more hydrophilic compounds present in the samples (EOs and HDs) were better reproduced by the ABTS •+ assay than the DPPH • assay, which is more sensitive for samples containing phenolic compounds and derivates. The data suggested that using the ABTS •+ assay, we obtained higher values of the antioxidant activity of EOs and HDs compared to DPPH • assay. The different values of the antioxidant activity measured for the same sample (EO or HD) could be explained by the different mechanisms involved in the reactions of radical antioxidant and sample compounds [62,63].

Plant Material
Fresh herbs of Thymus vulgaris L., Thymus pannonicus All., Lavandula angustifolia L., Lavandula x intermedia L., Origanum vulgare L., and Origanum vulgare var. aureum L. were obtained from a local producer in Arad County, Romania. Voucher specimens from these plants are deposited at "Aurel Valicu" University of Arad, Romania. All plant materials were air-dried and stored in paper bags before distillation.

Essential Oil and Hydrolat Extraction
Dried aerial parts were submitted to steam distillation using small-scale copper distillation equipment. The resulting EOs and HDs were separated using a separation funnel and stored at +4 • C until further usage.

Hydrolat Liquid-Liquid Extraction (LLE)
The dispersed and dissolved compounds in the HD samples were separated by LLE using a modified method, as described by Paolini et al. [47]. Briefly, 1 mL hexane and 25 mL HD were mixed and sonicated at room temperature, at 35 kHz, for 1 h at 100% power, using the Elmasonic TI-H5 (Elma, Schimdbauer GmbH, Singen, Germany). Subsequently, the hexane-hydrolat mixture was centrifuged for 5 min at 7000 rpm using a Hettich ultracentrifuge (Rotina 380 R, Hettich GmbH, Tuttlingen, Germany), and the organic layer was filtered through a 0.24 µm PTFE syringe filter before GC-MS analysis. This process was performed three times, and the resulting organic extracts were combined.

Annotations
For all the EOs and HDs, the annotations used are presented in Table 4.

Chemical Composition of EOs and HDs Determined by GC-MS
EOs and HDs were analyzed by using a gas chromatograph (GC, Shimadzu 2010, Kyoto, Japan) coupled with a mass spectrometer (MS, TQ 8040, Shimadzu, Kyoto, Japan) using a method described earlier in Moisa et al. [7]. Briefly, the EOs and HDs constituents were determined by a gas chromatograph (Shimadzu2010, Kyoto, Japan) coupled with a triple quadrupole mass spectrometer (TQ 8040, Shimadzu, Kyoto, Japan), and an optima 1MS + WAX column (30 m × 0.25 mm i.d., 0.25 µm film thickness, Macherey-Nagel, Duren, Germany). The carrier gas used was He, with a 1 mL min −1 flow. The oven temperature was initially 70 • C that was held for 11 min, and raised to 190 • C at a rate of 5 • C min −1 and then to 240 • C at a rate of 20 • C min −1 where it was kept for 5 min. Injector and MS source temperatures were set to 250 • C and 200 • C, respectively. The injection volume was 1 µL, with a split ratio of 10:1. Before the injection, EOs samples were diluted (1:25, v:v), and the HDs samples were injected as obtained. All the samples were were filtered using 0.45 µm PTFE membrane. All chemical constituents were identified using spectra libraries NIST 14 and Wiley 09 [6,7], compared with some commercial standards (α-pinene; sabinene; β-pinene; β-myrcene; α-phellandrene; 3-carene; D-limonene; cis-β-ocimene; trans-β-ocimene; carvacrol; caryophyllene), and by comparing their retention indices (abbreviated RI), determined relative to the time of retention values of n-alkanes (C10-C35), on capillary columns with those found in the literature [64].

Total Phenolic Content
All samples were analyzed spectrophotometrically for total phenolic content using Folin-Ciocâlteu reagent (FCR) [65], and the results were calculated and expressed as GAE/L (mg gallic acid equivalents). The HDs were analyzed without dilution, adding to 1 mL of sample, 0.5 mL FCR, 2 mL Na 2 CO 3 20%, and 5 mL distilled water. The reaction time was 1.5 h in the dark at room temperature. Afterward, the absorbance was recorded at 765 nm against a blank prepared in the same conditions, using a UV-Vis spectrophotometer (Specord 200, Analytik Jena AG, Jena, Germany) [65].

Antioxidant Activity
The radical scavenging activity of the EOs and HDs was analyzed using 1,1-diphenyl-2-picrylhidrazyl radical (DPPH • ) and measured spectrophotometrically at 517 nm after 1 h reaction time in the dark, as described by Moisa et al. [65]. The inhibition was calculated with the following equation: where A control is the absorbance of the DPPH • solution, and A sample is the absorbance recorded for the mixture of extract and DPPH • solution.

Statistical Analysis
All statistical analyses were further conducted with GraphPad Prism version 9.2.0 (San Diego, CA, USA). The resulting data were further analyzed with ANOVA and Tukey's post hoc test. The significantly different means p < 0.05 were marked with different letters.

Conclusions
In this work, chemical investigations using GC-MS revealed that the same batch of EOs and their corresponding HDs obtained from six plants from Lamiaceae family (thyme, lavender, and oregano) had different compositions of bioactive compounds. HDs, as byproducts obtained in the distillation of EOs, have a pleasant smell, a natural taste, and, depending on their oxygenated terpenes content, sometimes their smell is superior to that of the corresponding EO.
In our work, DPPH • and ABTS •+ assays demonstrated that both EOs and HDs present antioxidant activities, further confirming that HDs could be considered as independent products and with high economic value as flavoring agents in soft drinks and food products, or in aromatherapy, natural cosmetics, or green synthesis applications.
Although EOs are the principal traded economic product, HDs represent a valuable by-product that could present intense antiseptic activities, similar to those of their corresponding EOs (thyme and oregano), or have multiple aromatherapy applications or cosmetic and household uses (lavender and lavandin).