**1. Introduction**

The systematic cultivation and exploitation of the health benefits and beneficial applications of historical flowering plants/herbs is of grea<sup>t</sup> value for the modern world as new challenges arise day-by-day. The most important challenge, however, is the maintenance of the welfare and healthcare of humans in lieu of the use of synthetic drugs that may cause adverse effects.

Indeed, since ancient times, there has been all over the world a systematic use of herbs for the treatment of several health disorders. For example, in Greece, such herbs as oregano, marjoram, dill, fennel, mint, rosemary, mountain tea, sage, chamomile, thyme, parsley and basil have been used not only to flavour the cuisine or traditional dishes but also for medicinal purposes. Another important flowering plant that has a long history is Lavandula or, more commonly, lavender.

Approximately 47 species of the Lavandula genus are known, belonging to the mint family Lamiaceae. These flowering plants have a long history and have been found in numerous regions of the world, including Europe, the Mediterranean zone, Africa, Asia and India [1]. The temperate climates favor the cultivation of *Lavandula* spp. as ornamental plants or culinary herbs, and also at a commercial level for the extraction of essential oils [2]. Lavandula is grown in well-drained soils and in light places with good air circulation. It cannot grow in the shade [3,4]. The suitable soil pH may vary from acid to neutral and basic, whereas lavender can favorably grow in very alkaline soils. The optimum pH for the growth of Lavandula is between 6 and 8 [5]. In most cases, lavender is harvested by hand, depending on the usage [5].

In the Greek and Mediterranean zones, the species that grow originally are *Lavandula stoechas* [6]. Furthermore, the species of *Lavandula stoechas*, *Lavandula pedunculata* and *Lavandula dentata* were reported in Roman times [7]. The most commonly cultivated plant is the English lavender *Lavandula angustifolia* (formerly named *L. officinalis*). Some other commonly grown ornamental species are *Lavandula stoechas*, *Lavandula dentata* and *Lavandula multifida* (Egyptian lavender).

The lavender flower has been reported to have plenty of uses in practices of herbalism. The German scientific committee enhanced its use in alternative medicine, including its use for restlessness or insomnia, Roehmheld's syndrome, intestinal discomfort and cardiovascular diseases, among others [8]. The National Institutes of Health (NIH) in Maryland, U.S., stated that lavender is considered ''likely" safe in food amounts and ''possibly" safe in medicinal amounts. However, the NIH did not recommend the use of lavender in pregnan<sup>t</sup> or breast-feeding women because of a lack of knowledge of its effects. Lavender oil should not be used in young boys due to the possible hormonal effects leading to gynecomastia [9,10]. In addition, the NIH stated that lavender may cause skin irritation and could be poisonous if consumed orally [11]. In 2005, a review on lavender essential oil reported that lavender is traditionally regarded as a 'safe' oil, whereas skin problems (contact dermatitis) may occur at only a very low frequency [12]. In a study dealing with the relationship between various fragrances and photosensitivity carried out in 2007, it was reported that, even though lavender is known to favour cutaneous photo-toxic reactions, it did not induce photohaemolysis [13].

Saidona is a beautiful village in the Messiniaki Mani. Organic olive oil, table olives, sage, mountain tea, *Lavaldula stoechas*, and other flowering plants flourish in considerable amounts in Saidona. There is a long historical tradition of the local citizens using *Lavandula stoechas*, commonly referred as ''ladastacho", for the treatment of hair loss by preparing a gently boiled aqueous solution of its flowers.

Based on the above, the aim of the present work was to investigate the aqueous and methanolic extracts of *Lavandula stoechas* in terms of phenolic profile and in vitro antioxidant activity along with volatile compounds; some physico-chemical parameters were also considered. To the best of our knowledge, this is the first report in the literature that reports a combination of different parameter analyses for the characterisation of such a flowering plant grown in the Greek zone.

#### **2. Materials and Methods**

#### *2.1. Collection of Lavandula stoechas and Preparation of Samples*

Two kilograms (kg) of *Lavandula stoechas* was collected from the region of Saidona, Mani, (Messinia, Greece) (36◦5255"N 22◦1701"E) during June of 2017. *Lavandula stoechas* is shelf-grown in this region without the use of any cultivation system/procedure. The flowers were left alone to dry in a dark place and were then gently removed from the body of the plant. Finally, these were stored in aluminum foil at 4 ± 1 ◦C until further analysis.

#### *2.2. Reagents and Solutions*

Gallic acid (3,4,5-trihydrobenzoic acid) anhydrous for synthesis was purchased from Merck (Darmstadt, Germany). Methanol for analysis, Folin–Ciocalteu phenol reagent, sodium chloride (NaCl), sodium hydroxide (NaOH) and sodium carbonate (Na2CO3) were purchased from Merck. Finally, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (Darmstadt, Germany).

#### *2.3. Physicochemical Parameter Analysis*

#### 2.3.1. Determination of Acidity

For the determination of acidity, 10 g of *Lavandula stoechas* was transferred to a conical flask and 100 mL of distilled water was added. The obtained solution was titrated with a standard solution of sodium hydroxide (0.1 N) after the addition of a few drops of phenolphthalein, serving as an indicator of equivalent point. The results reported are the average values ± standard deviation values of three replicates (*n* = 3) and are expressed as g NaOH per 100 g of dried *Lavandula stoechas* [14].

#### 2.3.2. Determination of pH

pH values of *Lavandula stoechas* were measured using a Delta OHM, model HD 3456.2, pH-meter (Delta OHM, Padova, Italy) (precision of ± 0.002). Samples (10 g) were diluted with 100 mL of distilled water and the homogenate was used for the pH determination. Reported results are the average values ± standard deviation values of three replicates (*n* = 3). All measurements were carried out at 15 ± 1 ◦C after immersion of the electrode (that was firstly cleaned with distilled water) in the herb aqueous solution, until constant values were reached.

#### 2.3.3. Determination of Electrical Conductivity, Salinity and Total Dissolved Solids

Electrical conductivity, liquid resistivity, salinity and total dissolved solids of 10% (*w v*<sup>−</sup>1) aqueous herb solutions in distilled water were measured using a Delta OHM, model HD 3456.2, conductimeter (Delta OHM, Padova, Italy) with four-ring and two-ring conductivity/temperature probes. Temperature was measured by four-wire Pt100 and two-wire Pt1000 sensors by immersion. The probe was calibrated automatically using a 1413 μS cm<sup>−</sup><sup>1</sup> conductivity standard solution (Hannah Instruments, Inc., Woonsocket, RI, USA). Results are expressed as mS cm<sup>−</sup>1, Ohm (Ω), g L−<sup>1</sup> and mg <sup>L</sup>−1, respectively. The results reported are the average ± standard deviation values of three replicates (*n* = 3).

#### 2.3.4. Extraction of Phenolic Compounds

Extraction of phenolic compounds was carried out in two independent extraction systems: methanol and water. In particular, 0.34 g of dried and gently blended *Lavandula stoechas* was placed in plastic volumetric flasks containing 30 mL of methanol or water (1.13% (*w v*<sup>−</sup>1), (11,333.33 mg <sup>L</sup>−1)). The vials were then centrifuged for 2 h at 4000 rpm. The supernatant was filtered using a filtrate paper and the extracts were collected in plastic vials, having been wrapped with parafilm and aluminum foil. Finally, these were kept at −18 ◦C until analysis and used for the determination of phenolic compounds and in vitro antioxidant activity.

#### 2.3.5. Analysis of *Lavandula stoechas* Phenolic Compounds Using High-Performance Liquid Chromatography Electro Spray Ionization Mass Spectrometry (HPLC/ESI-MS)

An Agilent, model 1100 series, HPLC system (Agilent, Santa Clara, California, USA) was used for the chromatographic analysis. The respected wavelength was that of 280 ± 2 nm. Water and acetonitrile (Merck, Darmstadt, Germany) were used as the mobile phase at a flow rate of 1 mL min−1. The gradient elution program followed the sequence: 10% of acetonitrile then increasing to 30% for 20 min, further increasing to 40% at 30 min, to 50% at 35 min, and finally to 50% at 40 min. To avoid any memory effects, the column was eluted isocratically for 10 min before the next injection. A clear separation of the phenolic compounds was achieved using the Eclipse XDB C8 column (250 mm × 4.6 mm × 5 μm, Agilent, Santa Clara, USA) at 25 ◦C.

The mass spectrometer was the LC/MSD trap SL (Agilent). The MS conditions were as follows: Injection volume: 3.5 μL; Source conditions: Drying gas (nitrogen) 8 L/min at 330◦ C; Nebulizer pressure: 50 psi; Mass range: 100–1000; Scan mode: negative (−). Identification of phenolic compounds was achieved by comparing the mass to charge values [M−H]- of individual peaks shown at total

ion chromatograms with those identified previously in the literature. Analysis of *Lavandula stoechas* samples was run in duplicate (*n* = 2).

2.3.6. Determination of in Vitro Antioxidant Activity

Preparation of DPPH free radical standard solution

A standard solution of [DPPH•] equal to ca. 0.08 mM (mmol <sup>L</sup>−1) was prepared by dissolving 0.031 g of the free radical in 100 mL of methanol.

2.3.7. Preparation of the DPPH Free Radical Calibration Curve

A calibration curve of concentration versus absorbance of [DPPH•] was prepared as follows: The aforementioned standard solution of DPPH was diluted with the addition of methanol. The resulting solutions (range of 0–31 mg <sup>L</sup>−1) were vortexed, left in the dark (until measurements were made), and the absorbance was measured in a UV/VIS Spectrometer (Lambda 25, PerkinElmer, Waltham, USA) at λmax of 517 nm. The calibration curve of absorbance (*y*) versus concentration (*x*) of [DPPH•] was expressed by the following equation:

$$y = 0.0243x - 0.0001; \; R^2 = 0.998 \tag{1}$$

2.3.8. Determination of in Vitro Antioxidant Activity of *Lavandula stoechas* Aqueous and Methanolic Extracts

The antioxidant activity of *Lavandula stoechas* aqueous and methanolic extracts was estimated in vitro using the [DPPH•] assay. For the determination of antioxidant activity, a volume of 3.0 mL of [DPPH•] solution (0.08 mM) was placed in the cuvette (final volume of 3 mL) and the absorbance of the [DPPH•] radical was measured at *t* =0(*A*0 = 0.7565). Subsequently, 0.20 mL of *Lavandula stoechas* aqueous and methanolic extracts was placed in the respective cuvettes plus 2.8 mL of the [DPPH•] solution. The absorbance was measured every 15 min (regular time periods) until the value reached a plateau (steady state, *At*). The reaction between the free radical and *Lavandula stoechas* water soluble and methanolic antioxidants was accomplished at 30 min. The absorbance of the reaction mixture was measured at 517 nm.

The [DPPH•] antioxidant activity (%AA) with respect to aqueous and methanolic extract of *Lavandula stoechas* was calculated using the following equation:

$$\%AA = ((A\_0 \\_\\_A\_t)/A\_0) \times 100\tag{2}$$

where *A*0 is the initial absorbance of the [DPPH•] free radical standard solution and *At* is the absorbance of the remaining [DPPH•] free radical after reaction with *Lavandula stoechas* antioxidants, at steady state (plateau). For the estimation of *Lavandula stoechas* effective concentration EC50, termed as the concentration of antioxidants that could cause a decrease in the DPPH free radical by 50%, further dilutions from the initial extracts (11,333.33 mg <sup>L</sup>−1) were prepared for methanolic and aqueous extract from the initial mother solution in the range 500–11,333.33 mg L−<sup>1</sup> and the value of EC50 was obtained from graphs of [DPPH•] remaining versus concentration of extracts. For this antioxidant test, methanol was used as the blank. Prior to absorbance measurements, the extracts were filtered using Whatman filters (Whatman plc, Buckinghamshire, UK) with a pore size of 0.45 μm. Each sample was run in triplicate (*n* = 3).

#### 2.3.9. Determination of Total Phenolic Content

The total phenolic content of *Lavandula stoechas* was determined in its aqueous and methanolic extracts using the Folin-Ciocalteu assay according to Singleton et al. [15]. In particular, in a 5 mL volumetric flask, 0.2 mL of herb extract, followed by 2.5 mL of distilled water and 0.25 mL of Folin-Ciocalteu reagent, were added. After 3 min, 0.5 mL of saturated Na2CO3 (30%, *w v*<sup>−</sup>1) was also

added and the final volume of 5 mL was reached using distilled water. The flask was then stored in a dark place and the absorbance was measured after 2 h at 760 nm in a UV/VIS Spectrophotometer (SHIMADZU, UV-1280, Kyoto, Japan). Before any absorbance measurements, the solutions were filtered using Whatman filters (Whatman plc, Buckinghamshire, UK) with a pore size of 0.45 μm. For the quantification analysis, a calibration curve of standard gallic acid was constructed in wide range of concentrations: 0–6240 mg L−1. The equation obtained was of the following form:

$$y = 0.0004x + 0.1479,\\
R^2 = 0.9895\tag{3}$$

Total phenolic content was expressed as mg of gallic acid equivalents per mL (or g <sup>L</sup>−1) of *Lavandula stoechas* extract. Each sample was analysed in triplicate (*n* = 3).

#### **3. Headspace Solid Phase Microextraction Coupled to Gas Chromatography/Mass Spectrometry (HS-SPME/GC-MS)**

#### *3.1. Headspace Extraction of Volatile Compounds*

The extraction of volatile compounds of *Lavandula stoechas* was carried out using a divinyl benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber of 50/30 μm purchased from Supelco (Bellefonte, PA, USA). Before analysis of samples, the fiber was conditioned according to the manufacturer's recommendations and was cleaned daily using the method of ''clean" program to avoid any source of contamination. In particular, the injector and MS-transfer line were maintained at 260 ◦C and 270 ◦C, respectively, whereas during the ''cleaning" of the fiber, the oven temperature was held at 80 ◦C for 0 min, and then was increased to 270 ◦C at 10 ◦C per min (2 min hold). A split ratio of 10:1 was used.

For the sample analysis, the following conditions were followed: 15 min equilibration time, 30 min sampling time, and 45 ◦C water bath temperature. Approximately, 0.05 g of *Lavandula stoechas* was then placed in a 15 mL vial equipped with a polytetrafluoroethylene PTFE/silicone septa screw-cap, along with a magnetic stirrer. The vials were introduced into a water bath of 45 ◦C under continuous stirring (600 rpm) during the headspace extraction [16].

#### *3.2. Gas Chromatography-Mass Spectrometry Unit and Analysis Conditions*

The GC unit used in the study for the gas chromatography/mass spectrometry analysis of *Lavandula stoechas* samples was an Agilent 7890A model coupled to a MS detector (Agilent 5975, Agilent, Santa Clara, USA). The DB-5MS capillary column (Agilent, Santa Clara, USA) was used in the analysis with the following characteristics: cross-linked 5% PH ME siloxane, of 60 m × 320 μm i.d., × 1 μm film thickness. Helium of excellent purity (99.999%) was the carrier gas at a flow rate of 1.5 mL min−1. The injector and MS-transfer line were maintained at 250 ◦C and 270 ◦C, respectively, whereas, during the analysis, the oven temperature was maintained at 40 ◦C for 3 min, and then was increased to 260 ◦C at a rate of 8 ◦C min−<sup>1</sup> (6 min hold). The electron impact mass spectra were recorded at a mass range of 50–550, and the ionization energy was 70 eV, whereas a split ratio of 1:2 was used to introduce the appropriate amount of sample onto the column [16].

#### *3.3. Identification of* Lavandula stoechas *Volatile Compounds*

The identification of volatile compounds was achieved using the Wiley 7, NIST 2005 mass spectral library (John Wiley & Sons, New York, USA). The Kovats indices were determined by the use of a mixture of n-alkanes (C8–C20) supplied by Supelco (Bellefonte, PA, USA). Prior to gas chromatography-mass spectrometry (GC/MS) analysis, the mixture was dissolved in n-hexane and the retention time of standards was determined according to the aforementioned temperature-programmed run. Only volatile compounds having ≥80% similarity with the Wiley library were tentatively identified using the GC-MS spectra. The method of identification was based on the combination of MS data found in the Wiley 7 NIST 2005 mass spectral library and data of Kovats index values that were determined for each volatile compound and then compared with those included in the Wiley MS library [16]. Data were expressed as a percentage (contribution of the area of each volatile to the total peak area multiplied by 100). Volatile compounds identified only in replicated samples were used in the study.

#### *3.4. Graph Preparation and Statistical Analysis*

Graphs and average ± standard deviation values of physico-chemical parameters, in vitro antioxidant activity, total phenolic content and volatile compounds were prepared using the SPPS statistics software (v.20, 2013, IBM, New York, USA) and Microsoft Office Excel spread sheets for Windows 2007 (Microsoft, Redmond, USA). A t-test and Pearson's bivariate statistics were carried out using the SPPS statistics software. The level of significance was considered to be that of *p* ≤ 0.05.

#### **4. Results and Discussion**

#### *4.1. Physico-Chemical Parameter Analysis*

The pH of *Lavandula stoechas*, showing the active acidity of the sample, was slightly acidic in the range of 5.74 ± 0.03. The electric potential (E0), which in our case could show how the electric potential energy of any charged bio-molecule(s) at any location in the solution is divided by the charge of that/those molecule(s), was 73.4 ± 0.2 (mV). In addition, low salinity content was observed (0.29 ± 0.00 g <sup>L</sup>−1) and the total dissolved solids content was in the range of 280 ± 2 mg L−1. The electrical conductivity, a measure that defines the charged molecules in an aqueous medium, was 579 ± 1 μS cm<sup>−</sup><sup>1</sup> and the liquid resistivity, a measure that defines how strongly a material opposes the flow of electric current, was 1780 ± 20 Ω (Ohm). Finally, free acidity was in the range of 33 ± 3 g NaOH per 100 g of dried *Lavandula stoechas*. To our knowledge, the data are scarce on the physicochemical parameters of *Lavandula stoechas* aqueous extracts. Therefore, comparisons with literature data cannot be provided. In the work of Murthy et al. [14], it was reported that *Lavandula bipinnata* seed oil showed a much lower acidity: ca. 5.76 mg NaOH per g.

#### *4.2. Phenolic Profile: Qualitative and Quantitative Determinations*
