4.2.1. Qualitative Analysis

The HPLC-ESI/MS analysis enabled the determination of 12 phytochemicals as shown in Table 1. Figure 1 shows a typical total ion and UV chromatogram of the aqueous *Lavandula stoechas* extract pointing out with numbers the identified phytochemicals according to retention time.


**Table 1.** Phytochemicals identified in methanolic and aqueous extracts of Ladastacho.

RT, retention time (minutes).

**Figure 1.** Total ion and UV chromatograms of *Lavadula stoechas* aqueous extract. Phytochemicals are numbered according to the retention time shown in Table 1.

Caffeic acid has been previously reported to be the main natural phenol in argan oil [17]. It has also been found in the freshwater fern *Salvinia molesta* [18], in the mushroom *Phellinus linteus* [19], in the bark of *Eucalyptus globulus* [20] and in barley grain [21]. Its antioxidant potential has been reported in vitro and also in vivo [22]. In addition, caffeic acid has been reported to possess an inhibitory effect on cancer cell proliferation in human HT-1080 fibro-sarcoma cells [23]. Despite its antioxidant potential, there are some studies that report adverse results regarding the carcinogenicity or anti-carcinogenicity of caffeic acid [24].

Rosmarinic acid is mainly found in culinary herbs, such as *Ocimum basilicum* (basil), *Ocimum tenuiflorum* (holy basil), *Melissa officinalis* (lemon balm), *Rosmarinus officinalis* (rosemary), *Origanum majorana* (marjoram) and *Salvia officinalis* (sage), thyme and peppermint or in plants with medicinal properties, such as common self-heal (*Prunella vulgaris*) or in species of the genus Stachys [25]. It is also found in other Lamiales, such as *Heliotropium foertherianum*, a plant in the family Boraginaceae. Rosmarinic acid accumulation is favored in hornworts in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms [26], which may be found also as a derivative in Anthocerotophyta (*Anthoceros agrestis*) in the form of rosmarinic acid <sup>3</sup>-*O*-β-D-glucoside [27].

Regarding some potential health effects, rosmarinic acid is a potential anxiolytic as it acts as a gamma-aminobutyric acid (GABA) transaminase inhibitor, more specifically on 4-aminobutyrate transaminase [28]. Rosmarinic acid also inhibits the expression of indoleamine 2,3-dioxygenase via its cyclooxygenase-inhibiting properties [29], whereas it was found to be effective in a mouse model of Japanese encephalitis [30].

Salvianolic acid B (Sal B) is a phenolic acid derived from the dried root and rhizome of *Salvia miltiorrhiza* (Labiatae), which has been used traditionally in most Asian countries for the clinical therapy of various vascular diseases for hundreds of years. Salvia has been also used for various diseases related to blood stasis syndrome in China for thousands of years, and now it is widely used for cardiovascular diseases (CVDs) [31]. In addition, Sal B protects diverse kinds of cells from damage caused by a variety of toxic stimuli [32].

Furthermore, flavonoids may also have numerous beneficial effects in human health. For instance, apigenin and the substituted derivatives (i.e., -apigenin-7-*O*-glucuronide) isolated from the *Marrubium deserti* de Noé plant have been reported to possess antioxidant and antigenotoxic properties [33]. The same holds for Luteolin 7-glucoside, often called cynaroside, which is a flavone. It has been identified in *Campanula persicifolia* and *Campanulla rotundifolia* [34], *Teucrium gnaphalodes* [35], *Ferula varia* and *Ferula foetida* [36], the bamboo *Phyllostachys nigra* [37], dandelion coffee, *Cynara scolymus*

(artichoke) [38] and *Phoenix hanceana* var. formosana [39]. What is remarkable is that antioxidant and neural cell protective effects have also been reported for the latter ethanolic extract, in which the cynaroside was identified [39].

#### 4.2.2. Quantitative Analysis: Total Phenolic Content

The total phenolic content of the aqueous extract of *Lavandula stoechas* recorded a much higher value (ca. 4289 mg <sup>L</sup>−1) compared to the methanolic one (ca. 217 mg <sup>L</sup>−1), indicating that the aqueous medium (higher polarity) favoured the effective release/isolation of phytochemicals, especially phenolic acids and flavonoids, among other reducing agents (i.e., ascorbic acid). In a recent work [40], the preparation of *Lavandula angustifolia* beverages with either the method of infusion or decoction resulted in a beverage with a higher phenolic content when water, among other solvents, was used for the extraction of phytochemicals, in agreemen<sup>t</sup> with the present results.

The alcoholic and water extracts (1:1, *v*:*v*) of Romanian *Lavandula angustifolia* resulted in a significantly lower total phenolic content expressed as gallic acid (GA) equivalents (50.6 ± 3.2 mg GA g<sup>−</sup>1) compared to the results of the present study [41].

#### 4.2.3. In Vitro Antioxidant Activity

Both methanolic and aqueous extracts of *Lavandula stoechas* showed in vitro antioxidant activity during the DPPH assay. However, the aqueous extract (of higher polarity) recorded a significantly (*t* = −4.196, *df* = 4, *p* = 0.014) higher antioxidant activity. In addition, higher antioxidant activity was recorded for the higher concentration of the extracts (Figure 2). The EC50 values were 7.05 mg mL−<sup>1</sup> and 1.78 mg mL−<sup>1</sup> for the methanolic and aqueous extracts, respectively.

**Figure 2.** Antioxidant activity (AA%) of *Lavavdula stoechas* with respect to extract concentration (mg <sup>L</sup>−1).

What is also of grea<sup>t</sup> importance is that there was a perfect correlation between the in vitro antioxidant activity and the total phenolic content of the methanolic and aqueous extracts using Pearson's bivariate statistics (*r* = 1, *p* = 0.01). The present results are in agreemen<sup>t</sup> with those of Hu et al. [37], who reported that solvent-extracted bamboo leaf extract (BLE) containing chlorogenic acid, caffeic acid and luteolin 7-glucoside showed in vitro antioxidant activity using the DPPH assay (among others). The authors also reported that BLE exhibited a concentration-dependent scavenging activity of the [DPPH•], in agreemen<sup>t</sup> with the present results.

*Lavandula angustifolia* extracts from the region of Southeast Romania showed a considerable in vitro antioxidant activity against the DPPH free radical, as reported by Spiridon et al. [41].

In a more recent work, *Lavandula angustifolia* (from the region of Polykarpi Pozar in Greece) aqueous extracts prepared with infusion or decoction also showed a high in vitro antioxidant activity against the DPPH free radical (>90%), in agreemen<sup>t</sup> with the present results [40].

#### 4.2.4. Volatile Compounds of *Lavandula stoechas*

Fifty volatile compounds were tentatively identified using HS-SPME/GC-MS, belonging to alcohols, aldehydes, ketones, norisoprenoids and numerous terpenoids. In Table 2 is given (among other data) the percentage contribution of each volatile compound to the total ones. Figure 3 shows a typical gas chromatogram of *Lavandula stoechas*, pointing with numbers to some indicative volatile compounds. The most abundant volatiles were a-thujone (32.14%), 1,3,3-trimethyl-2-oxabicyclo[2.2.2] octane (9.77%), (+)-myrtenyl acetate (7.75%), camphor (6.07%), 1-octen-3-yl-acetate (3.94%) and fenchyl acetate (3.91%), followed by minor proportions of numerous others. A considerable number of these compounds have been reported in the essential oil of plants with edible modified stems, roots and bulbs in the Solanaceae, Tropacolaceae, Typhaceae and Zingiberaceae families, which are grown all over the world [42].

The biosynthesis of thujone in plants starts with the formation of geranyl diphosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP), catalyzed by the enzyme geranyl diphosphate synthase [43]. In addition, Umlauf and Zapp [44], using carbon nuclear magnetic spectroscopy (13C-NMR), reported that the isoprene units used to form thujone in plants are derived from an alternative pathway, the methylerythritol phosphate pathway (MEP).

Different opinions occur regarding the use of thujone as a food or drink supplement. In particular, the maximum thujone levels in the E.U. are related to the Artemisia species used along with the matrix (food or beverage). In that sense, the respected range may be within 0.5–35 mg kg−<sup>1</sup> [45,46].

On the other hand, in the U.S., the addition of pure thujone to foods is not permitted [47]. Foods or beverages that contain Artemisia species, white cedar, oak moss, tansy or yarrow must be free of thujone [48], which practically means that these contain less than 10 mg L−<sup>1</sup> of thujone [49]. Sage and sage oil (which may contain up to 50% thujone) are on the Food and Drug Administration's list of generally recognized as safe (GRAS) substances [50]. The average lethal dose value, or LD50, of alpha-thujone, the more active of the two isomers (alpha- and beta-) found in plant-derived products, has been reported to be ca. 45 mg kg−<sup>1</sup> in mice, with a 0% mortality rate at 30 mg kg−<sup>1</sup> and a 100% mortality rate at 60 mg kg−1, respectively [51].

The biochemical pathway of camphor production involves the presence of geranyl pyrophosphate, which, via the cyclisation of linaloyl pyrophosphate, turns to bornyl pyrophosphate, followed by hydrolysis to borneol and then oxidation to camphor. Camphor has been used in traditional medicine since ancient times in countries where it was native. Its characteristic odour and its decongestant effect on the treatment of sprains, swellings and inflammation have probably led to its use in medicine [52,53]. Camphor was also used for centuries in Chinese medicine for a variety of health issues. The lethal doses in adults are in the range of 50–500 mg kg−<sup>1</sup> after oral exposure. In particular, 2 g may cause serious toxicity and 4 g is potentially lethal [54]. To the best of our knowledge, this is the first report in the literature reporting the volatile profile of Greek *Lavandula stoechas*.


**Table 2.** Volatile compounds identified in *Lavandula stoechas*



RT: retention time (minutes), KI: experimental retention indices values based on the calculations of Kovats index values (KI) using the standard mixture of alkanes. RIlit: Retention indices of the identified compounds according to literature data included in Wiley 7 NIST MS library. Qualification: Percentage accuracy of volatile compounds identified using Wiley 7 NIST MS library data. Results reported are the average ± standard deviations values of two independent replicates (*n* = 2).

**Figure 3.** A typical gas chromatogram of *Lavandula stoechas*. Indicative volatile compounds (markers) are numbered according to the retention time shown in Table 2. 1: alpha-Pinene, 2: alpha-Fenchene, 3: Camphene, 4: para-Cymene, 5: cis-Linalool oxide, 6: alpha-Thujone, 7: Camphor, 8: alpha-Terpineol, 9: Myrtenal, 10: cis-Geranyl acetate, 11: (+)-Sativene, 12: beta-Selinene, 13: Viridiflorol, 14: (+)-Aromadendrene.
