The Influence of Cryogrinding on Essential Oil, Phenolic Compounds and Pigments Extraction from Myrtle (Myrtus communis L.) Leaves

Abstract

The aim of this study was to investigate the influence of cryogrinding pretreatment on the recovery of essential oil, phenolics and pigments from myrtle leaves. The duration of cryogrinding (3, 6 and 9 min) in combination with the duration of hydrodistillation (30, 60 and 90 min) for the isolation of essential oils and the duration of hydroethanolic extraction (5, 10 and 15 min) for the isolation of phenols and pigments were studied as independent factors in a full factorial design. The major volatile components detected in myrtle leaf essential oil were myrtenyl acetate, 1,8-cineole, α-pinene and linalool. The most abundant phenols detected were myricetin derivatives (myricetin 3-O-galactoside and myricetin 3-O-rhamnoside), galloylquinic acid, myricetin and digalloylquinic acid, while the major pigments were chlorophyll b, pheophytin a and lutein. A 3 min cryogrinding pretreatment significantly increased the yield and concentrations of essential oil volatile compounds and reduced the distillation time to 30 min. A 9 min cryogrinding pretreatment and 15 min extraction resulted in at least 40% higher concentrations of phenolic compounds and pigments in the extracts when compared to the untreated control. According to the results obtained, cryogrinding can significantly increase the yield of myrtle EO and extracts and also modulate their composition.

1. Introduction

Myrtle (Myrtus communis L., Myrtaceae) is an evergreen shrub common in the Mediterranean region, growing in groups as a wild plant characterised by pleasantly fragrant leaves and attractively coloured (dark indigo blue) berries [1]. In Croatia, it grows along the coast and on the islands of the Adriatic Sea [2]. The plant is traditionally used as a spice, for medicinal purposes and for food preparation [3]. The most commonly used parts of myrtle are the leaves for essential oil (EO) production in the fragrance and flavour industry [4] and the berries for liqueur production (Mirto Rosso and Mirto Bianco), especially in Sardinia [5].

EO and polyphenolic compounds are the main secondary metabolites in myrtle leaves [3]. EOs are oily aromatic liquids with many compounds, mainly terpenes and terpenoids, responsible for the pleasant odour and used mainly as food flavourings [6], in food preservation [7] and aromatherapy [8,9]. In general, the yield of EOs in plants is very low and rarely exceeds 1% [10]. Due to their hydrophobic nature, EOs are soluble in nonpolar or weakly polar solvents and are colourless or pale yellow, with the exception of chamomile oil, which is blue. 1,8-cineole, myrtenyl acetate and α-pinene, the main terpenoids from myrtle leaves [11], exhibit antimicrobial [12], cytotoxic [13] and anti-inflammatory [14] effects, among others. Climatic, genetic and edaphic factors [15] as well as season [2] can influence the composition of EO. The occurrence of EO chemotypes is the result of different chemical composition within the same plant species, i.e., different genetic compositions [16]. There are two main EO chemotypes of myrtle leaves based on myrtenyl acetate content: (i) the cineoliferum type (rich in terpenes and terpenoid oxides); and (ii) the myrtenilacetatiferum type (rich in terpene esters and terpenoid oxides) [17]. Further subdivision is made into subgroups according to the relative ratio of α-pinene to myrtenyl acetate or α-pinene to cineole [18,19]. Distillation is a crucial step in obtaining the EO from a plant, and the duration of distillation has a great influence on the quality and yield of the EO, which is about 0.4–0.5% for myrtle leaves [1]. The most common conventional methods of distillation are hydrodistillation, steam distillation and steam/water distillation [16], but there are also innovative methods that use green technologies, such as solvent-free microwave extraction and supercritical fluids [20]. The main drawback of conventional methods is that they are long-lasting and un-economical, so the distillation time and energy consumption should be reduced [21,22].

Furthermore, the main phenolic compounds of myrtle leaves are flavonols (myricetin 3-O-rhamnoside/galactoside, myricetin), phenolic acids (hydroxybenzoic acids) and flavan-3-ols (epigallocatechin gallate) [9,23,24]. These compounds have shown a broader spectrum of therapeutic effects, e.g., anti-inflammatory, antimutagenic, antigenotoxic, antihyperglycemic and antioxidant [25]. In addition to phenolic compounds, myrtle leaf extracts are rich in pigments, i.e., carotenoids such as lutein and β-carotene, as well as chlorophylls such as chlorophyll b and pheophytin a, whose properties were discussed in a previous study by Cvitković et al. [26].

The amount of these secondary metabolites in plant species can be affected by various factors such as harvest time, genotype, abiotic stress, climatic conditions and salt stress as environmental factors [27,28,29]. Moreover, in order to optimise the extraction yield during the processing of the plant material, it is necessary to pay attention to each step in the processing of the plant, including the drying/grinding procedure, distillation/extraction parameters and the solvent/method selection. For example, different chemical structures affect the polarity of bioactive molecules, so the solvent type should be suitable. The amount of extracted phenolic compounds increases with increasing solvent polarity [30], while medium and nonpolar solvents are suitable for the extraction of the carotenoid β-carotene [26].

Since EOs contain highly volatile and thermolabile compounds that can be lost at classical grinding temperatures (42–93 °C) due to evaporation or oxidation [31], grinding at −196 °C using nitrogen as a cryogen, i.e., cryogrinding, can avoid such undesirable losses and changes. In this way, it is possible to preserve the flavour, colour and nutritional value of ground herbs [22], having a finer and more uniform size [32], which in turn also increases the yield of distillation/extraction. Compared to grinding at room temperature, the EO content of black pepper was 26% higher after cryogrinding [31,33]. The yields of cumin [22] and coriander seed [34] EOs were also significantly increased by cryogrinding as a pretreatment when compared to conventional grinding. In addition to the EO, cryogrinding had a positive effect and increased the total phenolic, flavonoid and antioxidant content in cumin [35] and ajwain seed extracts [36]. Balbino et al. [37] showed that in pumpkin seed cake pretreated with cryogrinding, the amount of total phenolic compounds and protochlorophyll (precursor of chlorophylls) as well as antioxidant capacity increased significantly. The additional advantage of using liquid nitrogen is its safety and chemical and biological inertness [38].

The aim of this study was to evaluate the effect of cryogrinding as a pretreatment used to increase the yields of bioactive compounds of myrtle leaves and its effect on the components of the (i) EO obtained by hydrodistillation and (ii) the phenolic compounds and pigments obtained by agitation-assisted extraction.

2. Materials and Methods

2.1. Chemicals

Commercial standards of (+)-α-pinene, camphene, (−)-β-pinene, (R)-(−)-α-phellandrene, 3-carene, p-cymene, o-cymene, γ-terpinene, a mixture of α-fenchyl acetate, (+)-carvone, 1,8-cineole, myrtenyl acetate, myrtenol, geraniol, carvacrol and alkane standard solution C₇–C₃₀ were procured from Sigma Aldrich (St. Louis, MO, USA); myrcene, butyl butyrate, linalool, α-terpineol and eugenol from Merck (Darmstadt, Germany); R-(+)-limonene and nerol from Fluka^® Analytical (Munich, Germany); and α-terpinene from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The standards of caffeic, gallic, chlorogenic and p-coumaric acids; quercetin-3-glucoside; and myricetin were purchased from Sigma-Aldrich (Steinheim, Germany). Epicatechin, catechin, epigallocatechin gallate, apigenin and luteolin were from Extrasynthese (Genay, France). Carotenoids (β-carotene, lutein, zeaxanthin) and chlorophylls (chlorophyll a, chlorophyll b) were obtained from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Acetonitrile was procured from J.T. Baker Chemicals (Deventer, Netherlands), and formic acid (98–100%) was from T.T.T. d.o.o. (Sveta Nedjelja, Croatia). Purified water was of Milli-Q quality (Millipore, Bedford, MA, USA). Ethanol 96% and n-hexane 95% were purchased from J. T. Baker (Phillipsburg, NJ, USA). All chemicals and solvents were of HPLC grade.

2.2. Plant Material

Myrtle leaves (Myrtus communis L.) were collected in February 2021 from plants growing in the wild and an ecologically pure environment on the island of Mljet (Babino Polje, 42°43′54.8″ N; 17°35′02.9″ E) during the fruit ripening phenophase and dried at room temperature until constant weight. Prior to analysis, the dried leaves were gently ground using an electric laboratory knife mill (WSG30, Waring Commercial, Torrington, CT, USA) and passed through a 5 mm sieve. This plant material was also used as a control in experiments without cryogrinding (labelled as 0 min).

2.3. Cryogrinding (CG)

CG of dried myrtle leaves was performed on a Spex 6875D freezer/mill (Metuchen, NJ, USA) with pre-cooling time set to 2 min and total grinding time of 3, 6 and 9 min applied during 2 cycles with intermediary cooling of 2 min. The particle size distribution of cryogenically ground leaves was determined using a Mastersizer laser diffraction particle size analyser equipped with a Scirocco 2000 dispersion unit and Mastersizer 2000 software (Malvern Instruments, Worcestershire, UK). Mean particle size (D₅₀) was used to evaluate the effect of cryogrinding on particle size reduction.

2.4. Hydrodistillation (HD)

The EO was obtained by HD of 20 g of cryogenically ground or untreated (control) myrtle leaves with 200 mL of water in a Clevenger-type apparatus according to the European Pharmacopoeia [39]. The distillation times were set to 30, 60 and 90 min. After volume measurement in the graduated part of the Clevenger-type apparatus, the EO was released and collected in a vial, dried from possible water residues with anhydrous sodium sulphate, transferred to another vial and stored at –18 °C until GC analysis. Distillations were performed twice for each set of parameters.

2.5. Volatile Compounds Analysis

To prepare the sample for gas chromatographic (GC) analysis, 10 µL of EO, 100 µL of internal standard (nerol, c = 10.6500 mg mL⁻¹) and 890 µL of n-hexane (HPLC purity, 95%) were added to the vial. The oil sample thus prepared was analysed on an Agilent Technologies 6890N Network GC System gas chromatograph (Santa Clara, CA, USA) using an Agilent Technologies 5973 inert Mass Selective Detector. The analysis was performed on an Agilent HP-5MS capillary column (5% phenylmethylsiloxane; 30 m × 0.25 mm × 0.25 μm) under the following conditions: injected sample volume 1 μL, split ratio 100:1 and helium carrier gas at a constant flow rate of 1 mL min⁻¹. During the analysis, the following column temperature program was used: initial temperature 60 °C, then 60–145 °C (3 °C min⁻¹), 145–250 °C (30 °C min⁻¹) and retention for 3 min at maximum temperature (250 °C). The transfer-line temperature was 280 °C, and the injector temperature was 250 °C, while MS source and quadrupole temperatures were 230 and 150 °C, respectively. The volatile compounds were identified by comparing the retention times and mass spectra (m/z) of the tested compounds with the retention times and mass spectra of commercial standards and comparing the obtained mass spectra with those in the NIST17, NIST14 and WILEY9 databases using MSD ChemStation software Data Analysis. In addition, to confirm the identified compounds, their retention index was calculated and compared with the data in the available literature. Quantitative values for each volatile component were calculated from the calibration equations of the standard compounds. The results were expressed in mg per 100 g of dry weight (dw).

2.6. Agitation Assisted Extraction (AAE)

One gram amounts of cryogenically ground or untreated (control) myrtle leaves were exhaustively extracted with 20 mL of solvent (twice with 96% and twice with 30% EtOH), as this solvent combination was shown to give the highest content of pigments and phenols in the preliminary experiments (data not shown). AAE was performed using an agitation water bath (SBS40, Cole-Parmer, Stone, UK) at 60 °C and 120 rpm. The extraction times were set at 5, 10 and 15 min. Then the mixture was centrifuged (ROTOFIX 32 A, Hettich, Westphalia, Germany) at 5000 rpm for 10 min and filtered. The extractions were performed twice for each set of parameters.

2.7. Pigment Analysis

The content of carotenoids and chlorophylls was determined in myrtle ethanolic extracts by high performance liquid chromatography (HPLC) using an Agilent 1260 Infinity quaternary LC system (Agilent Technologies, Santa Clara, CA, USA) and a diode array detector (DAD). A Phenomenex Develosil RP-Aqueous (C30) reversed phase column (250 mm × 4.6 mm i.d., 5 µm particle size) (Phenomenex, Torrance, CA, USA) and a mixture of MeOH:methyl tert-butyl ether (MTBE):water (90:7:3, v/v/v) as mobile phase A and MeOH:MTBE (10:90, v/v) as mobile phase B were used. The flow rate was 0.8 mL min⁻¹, and 10 µL was the injection volume. As previously described by Castro-Puyana et al. [40], the elution gradient of the mobile phase was 0 min, 0% B; 20 min, 30% B; 35 min, 50% B; 45 min, 80% B; 50 min, 100% B; and 52 min, 0% B. The signal intensities used for the quantification of carotenoids and chlorophyll were detected at 450 and 660 nm. Chlorophyll derivatives were identified by their DAD absorption spectra and relative retention times [41,42], while their quantification was based on the calibration curve of their original form (chlorophyll a or b) [43]. The results were expressed in mg per 100 g of dry weight (dw).

2.8. Phenolic Compounds Analysis

Ultra-performance liquid chromatography–tandem mass spectrometer (UPLC–MS/MS) phenol identification and quantification was performed using an Agilent series 1290 RRLC instrument (Agilent, Santa Clara, CA, USA) equipped with an Agilent 6430 triple quadrupole mass spectrometer (QQQ) and Agilent MassHunter Workstation Software (ver. B.04.01). A 100 × 2.1 mm Fortis C18 column with a 1.7 µm particle size was used for reversed phase separation. Ionization of analytes was performed by ESI with nitrogen as the desolvation and collision gas (temperature 300 °C, flow rate 11 L h⁻¹) in negative and positive mode (m/z 100–1000). The column temperature was kept at 35 °C, the injection volume was 1 µL, and the flow rate was 0.3 mL min⁻¹. The composition and gradient of the solvents were described by Elez Garofulić et al. [44]. Quantitative determinations were performed using the calibration curves of the standards, where myricetin-3-O-galactoside and myricetin-3-O-rhamnoside were calculated according to the myricetin, quercitrin according to the quercetin-3-glucoside, apigenin according to the luteolin and epicatechin according to the catechin calibration curve. The 5-galloylquinic and digalloylquinic acids were calculated according to the gallic acid calibration curve. Myricetin 3-O-galactoside and myricetin 3-O-rhamnoside were not used for quantification and qualification because it was not possible to obtain the specified standards from the supplier at the time the analyses were performed due to the COVID crisis. According to various authors, if standards are not available, compounds with similar structure or representatives of a family of phenolic compounds can be used for quantification of the phenolic compounds and the results expressed as equivalents [45,46]. The results were expressed in mg per 100 g of dry weight (dw).

2.9. Statistical Analysis

The experiment was based on a full factorial experimental design with two independent variables: cryogrinding time (0, 3, 6 and 9 min) and hydrodistillation time (30, 60 and 90 min) or hydroethanolic extraction time (5, 10 and 15 min), with a total of 12 experimental conditions, each performed in two replicates (N = 24). XLSTAT 2020.3.1 statistical software (Addinsoft, Paris, France) was used for data processing. Results were analysed using multifactorial analysis of variance (ANOVA) in combination with Tukey’s multiple comparison test at a significance level of p ≤ 0.05 (95% confidence interval). All analytical determinations were performed in two replicates, and the mean values were calculated, which were then used for statistical evaluation. Data on pigments and phenols in the extracts and on EO composition (total and individual) were presented as least squares means (LS) ± standard error (SE) of n = 8 for the cryogrinding time and n = 6 for the extraction or distillation time.

3. Results and Discussion

3.1. The Influence of CG on the Composition of Myrtle EO

To the best of our knowledge, this is the first study that examines the influence of cryogrinding and distillation time on the yield of myrtle EO. In total, 22 volatile components of myrtle EO were detected and quantified by GC–MS analysis, and

Table 1. Profile of myrtle leaf EO (mg/100 g dw) obtained by different CG and extraction times.

Time (min)	α-Pinene	Myrcene	p-Cymene	Limonene	1,8-Cineole	Linalool	α-Terpineol	Myrtenol	Geraniol	Myrtenyl Acetate	Eugenol
Cryogrinding	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *
0	71.85 ± 0.35 d	3.06 ± 0.02 c	2.52 ± 0.01 c	12.19 ± 0.07 d	135.12 ± 0.63 b	49.67 ± 0.26 c	16.64 ± 0.08 c	5.05 ± 0.02 a	4.60 ± 0.02 d	136.15 ± 0.44 b	3.16 ± 0.01 d
3	97.60 ± 0.35 a	4.26 ± 0.02 a	2.90 ± 0.01 a	16.00 ± 0.07 a	158.30 ± 0.63 a	60.66 ± 0.26 a	20.91 ± 0.08 a	4.85 ± 0.02 b	5.64 ± 0.02 a	153.62 ± 0.44 a	3.41 ± 0.01 a
6	85.50 ± 0.35 b	3.55 ± 0.02 b	2.57 ± 0.01 b	13.71 ± 0.07 b	134.48 ± 0.63 b	50.09 ± 0.26 c	16.25 ± 0.08 d	4.06 ± 0.02 d	4.89 ± 0.02 c	135.15 ± 0.44 b	3.29 ± 0.01 c
9	81.30 ± 0.35 c	3.59 ± 0.02 b	2.53 ± 0.01 c	12.92 ± 0.07 c	132.70 ± 0.63 b	52.31 ± 0.26 b	17.54 ± 0.08 b	4.24 ± 0.02 c	5.22 ± 0.02 b	131.29 ± 0.44 c	3.38 ± 0.01 b
Distillation	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p = 0.096	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *
30	78.20 ± 0.30 c	3.54 ± 0.01 b	2.53 ± 0.01 c	12.90 ± 0.06 c	141.89 ± 0.54 a	54.09 ± 0.22 a	17.90 ± 0.07 a	4.32 ± 0.02 c	4.81 ± 0.02 b	132.03 ± 0.38 c	3.14 ± 0.00 c
60	86.19 ± 0.30 b	3.80 ± 0.01 a	2.74 ± 0.01 a	14.25 ± 0.06 a	141.43 ± 0.54 a	53.67 ± 0.22 a	17.90 ± 0.07 a	4.59 ± 0.02 b	5.24 ± 0.02 a	143.56 ± 0.38 a	3.38 ± 0.00 b
90	87.79 ± 0.30 a	3.51 ± 0.01 b	2.62 ± 0.01 b	13.97 ± 0.06 b	137.13 ± 0.54 b	51.78 ± 0.22 b	17.70 ± 0.07 a	4.74 ± 0.02 a	5.22 ± 0.02 a	141.57 ± 0.38 b	3.41 ± 0.00 a
Interaction	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p = 0.003 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p = 0.01 *
0 × 30	49.06 ± 0.60 g	2.41 ± 0.03 f	2.27 ± 0.01 f	9.47 ± 0.13 f	138.17 ± 1.08 b,c	48.16 ± 0.45 e	15.94 ± 0.14 e	4.75 ± 0.04 b,c	3.82 ± 0.04 g	117.47 ± 0.76 g	2.87 ± 0.01 e
0 × 60	77.73 ± 0.60 e	3.33 ± 0.03 e	2.79 ± 0.01 b	13.47 ± 0.13 c,d	135.50 ± 1.08 b,c,d,e	52.11 ± 0.45 d	17.49 ± 0.14 c	5.27 ± 0.04 a	4.93 ± 0.04 e	150.57 ± 0.76 b	3.27 ± 0.01 c
0 × 90	88.77 ± 0.60 c	3.44 ± 0.03 d,e	2.49 ± 0.01 d	13.63 ± 0.13 c d	131.69 ± 1.08 d,e	48.74 ± 0.45 e	16.50 ± 0.14 d,e	5.13 ± 0.04 a	5.05 ± 0.04 d,e	140.42 ± 0.76 c,d	3.35 ± 0.01 b
3 × 30	105.98 ± 0.60 a	4.77 ± 0.03 a	3.00 ± 0.01 a	16.72 ± 0.13 a	160.43 ± 1.08 a	64.83 ± 0.45 a	21.99 ± 0.14 a	4.81 ± 0.04 b	5.76 ± 0.04 a	157.82 ± 0.76 a	3.31 ± 0.01 b,c
3 × 60	94.40 ± 0.60 b	4.56 ± 0.03 b	2.95 ± 0.01 a	16.30 ± 0.13 a	159.93 ± 1.08 a	60.38 ± 0.45 b	20.57 ± 0.14 b	4.62 ± 0.04 b,c	5.82 ± 0.04 a	150.81 ± 0.76 b	3.45 ± 0.01 a
3 × 90	92.42 ± 0.60 b	3.45 ± 0.03 d,e	2.76 ± 0.01 b	14.98 ± 0.13 b	154.53 ± 1.08 a	56.76 ± 0.45 c	20.16 ± 0.14 b	5.12 ± 0.04 a	5.35 ± 0.04 b,c	152.21 ± 0.76 b	3.48 ± 0.01 a
6 × 30	83.51 ± 0.60 d	3.44 ± 0.03 d,e	2.47 ± 0.01 d	13.21 ± 0.13 d	139.09 ± 1.08 b	50.63 ± 0.45 d,e	15.89 ± 0.14 e	3.75 ± 0.04 f	4.48 ± 0.04 f	127.44 ± 0.76 f	3.19 ± 0.01 d
6 × 60	84.18 ± 0.60 d	3.65 ± 0.03 c	2.63 ± 0.01 c	14.05 ± 0.13 c	134.26 ± 1.08 b,c,d,e	50.44 ± 0.45 d,e	16.40 ± 0.14 d,e	4.26 ± 0.04 d	5.08 ± 0.04 d,e	143.75 ± 0.76 c	3.34 ± 0.01 b
6 × 90	88.80 ± 0.60 c	3.57 ± 0.03 c,d	2.61 ± 0.01 c	13.86 ± 0.13 c,d	130.09 ± 1.08 d,e	49.19 ± 0.45 e	16.46 ± 0.14 d,e	4.17 ± 0.04 d,e	5.1 ± 0.04 d,e	134.27 ± 0.76 e	3.34 ± 0.01 b
9 × 30	74.26 ± 0.60 f	3.55 ± 0.03 c,d	2.37 ± 0.01 e	12.19 ± 0.13 e	129.86 ± 1.08 e	52.73 ± 0.45 d	17.78 ± 0.14 c	3.97 ± 0.04 e,f	5.16 ± 0.04 c,d	125.39 ± 0.76 f	3.2 ± 0.01 d
9 × 60	88.44 ± 0.60 c	3.65 ± 0.03 c	2.60 ± 0.01 c	13.16 ± 0.13 d	136.02 ± 1.08 b,c,d	51.76 ± 0.45 d	17.15 ± 0.14 c,d	4.20 ± 0.04 d	5.12 ± 0.04 d,e	129.11 ± 0.76 f	3.44 ± 0.01 a
9 × 90	81.19 ± 0.60 d	3.58 ± 0.03 c,d	2.61 ± 0.01 c	13.40 ± 0.13 d	132.22 ± 1.08 c,d,e	52.43 ± 0.45 d	17.69 ± 0.14 c	4.54 ± 0.04 c	5.38 ± 0.04 b	139.38 ± 0.76 d	3.49 ± 0.01 a

Results are expressed as mean (n = 8 for cryogrinding time, and n = 6 for distillation time) ± SE. Different letters indicate significant differences between the means (* p ≤ 0.05).

shows only those obtained in concentrations >2.5 mg/100 g dw. In addition, camphene, β-pinene, butyl butyrate, α-phellandrene, 3-carene, α-terpinene, o-cymene, γ-terpinene, fenchyl acetate, carvone and carvacrol were also identified. The EO was characterised by a high percentage of oxygenated monoterpenes (77%) followed by monoterpene hydrocarbons (23%). Oxygenated compounds are more valuable than monoterpene hydrocarbons in the fragrance industry [47]. Predominant EO compounds were 1,8-cineole, myrtenyl acetate, α-pinene and linalool, which is in accordance with the studies of Jerkovic et al. [2], Pereira et al. [48], Bradesi et al. [19] and Mulas and Melis [17]. According to the presence of notable amounts of myrtenyl acetate in the examined myrtle EO, myrtle used in this study was characterised as a myrtenyl acetate chemotype, which is characteristic for countries such as Morocco, Portugal, France, Albania and countries of the former Yugoslavia [19]. These oils have considerable amounts of myrtenyl acetate and α-pinene, while in this study myrtenyl acetate was present in an amount almost twice that as α-pinene. The same chemotype of myrtle EO from Vis island (Croatia) was confirmed in the study by Jerkovic et. al. [2]. In contrast, EO from eastern and central–northern Algeria belong to the “α-pinene-cineole” chemotype [49] and in Italy to the cineoliferum type due to the dominance of α-pinene in relation to myrtenyl acetate [17].

Considering the effect of pretreatment, a 3 min CG yielded 16.6% more of the total volatiles when compared to the control, and the concentrations of individual compounds, especially low boiling point volatiles such as α-pinene, myrcene, limonene and 1,8-cineole, increased by 26.4, 28.2, 23.8 and 14.6%, respectively. On the other hand, prolonged grinding time (6 and 9 min) caused a decrease in total volatile content as well as individual ones in comparison with the control. Similar results were reported by Tischer et al. [50] on Baccharis articulata, who found that smaller material particles obtained by CG can cause the loss of EO and flavouring constituents due to glandular trichome rupture. They assumed that glandular trichomes and secretory ducts containing EO could break during prolonged CG and be lost by adsorption to the walls of the grinding vessel or by volatilization at room temperature. In our case, 3 min of CG did not cause such a reduction in particle size that would lead to this phenomenon, but with 6 and 9 min of grinding, i.e., further reduction, lower distillation yields were achieved. CG time is proportional to size reduction, which means higher energy requirements for longer grinding [51]; therefore, optimisation is crucial to avoid unnecessary losses. Measurement of particle size in our study confirmed this rule, as approximately a double size reduction was achieved for every 3 min of CG; i.e., for 3, 6 and 9 min of CG, D₅₀ was 106.94, 46.80 and 28.65 μm, respectively. Due to lower yields and higher costs, 6 and 9 min of grinding were not necessary; therefore, for maximum retention of myrtle leaf EO, a shorter CG time (3 min) is the best choice. Čukelj Mustač et al. [52] also reported that longer CG, i.e., further reduction of particle size, may lead to loss of antioxidant activity.

In terms of distillation time, 60 min gave the highest yield of total volatiles and most of the individual compounds (Figure 1). However, considering the interaction between grinding and distillation time, 3 min of CG and 30 min of distillation were sufficient to obtain the highest yield of total volatiles, which therefore significantly reduced the distillation time. Standard distillation usually lasts 180 min according to the European Pharmacopeia [53], since longer distillation times give higher yields [54]. Thus, CG pretreatment enables rapid exhaustion of the plant material i.e., accelerates the extraction/distillation phenomena [47], since the volatile compounds are immediately available, and all evaporate and condense in the first 30 min of HD. The study of Akloul et al. [21] examined the effect of CG compared to ambient grinding as a pretreatment for microwave-assisted HD and steam distillation of Curcuma longa L. rhizome and Carum carvi L. fruit and also concluded that a sufficient amount of EO is obtained in a shorter time, most components are obtained in the first minutes of extraction and the composition of major components varies during the extraction time. Additionally, more monoterpene and sesquiterpene hydrocarbons were obtained with CG when compared to ambient grinding. Furthermore, Berka-Zougali et al. [55] also successfully reduced the distillation time of myrtle leaf EO from 180 min (conventional HD) to 30 min with solvent-free-microwave-extraction (SFME), which was richer in oxygenated compounds. In comparison with our results, myrtle from Algeria generally contained a lower EO content.

3.2. The Influence of CG on the Phenolic and Pigment Compositions of Myrtle

Table 2. Phenolic profile (mg/100 g dw) of myrtle leaf extract obtained by different cryogrinding and extraction times.

Time (min)	Myricetin 3-O-G	Myricetin 3-O-R	Q-3-G	EGKG	Quercitrin	Myricetin	Digalloylquinic A	Galloylquinic A	Caffeic A	Gallic A
Cryogrinding	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *
0	1287.19 ± 2.89 d	1081.19 ± 2.89 d	16.14 ± 0.43 b	21.68 ± 0.29 d	12.32 ± 0.26 c	186.97 ± 0.87 b	95.50 ± 0.87 c	280.26 ± 0.58 b	15.61 ± 0.29 c	59.93 ± 0.23 b
3	1364.81 ± 2.89 c	1179.41 ± 2.89 c	17.84 ± 0.43 b	24.55 ± 0.29 c	13.52 ± 0.26 b	190.26 ± 0.87 b	121.55 ± 0.87 b	244.73 ± 0.58 d	18.51 ± 0.29 b	66.36 ± 0.23 a
6	1506.90 ± 2.89 b	1256.85 ± 2.89 b	21.18 ± 0.43 a	26.57 ± 0.29 b	16.94 ± 0.26 a	229.06 ± 0.87 a	124.94 ± 0.87 b	248.91 ± 0.58 c	21.80 ± 0.29 a	52.94 ± 0.23 c
9	1698.98 ± 2.89 a	1396.04 ± 2.89 a	21.15 ± 0.43 a	28.09 ± 0.29 a	16.40 ± 0.26 a	228.43 ± 0.87 a	139.76 ± 0.87 a	334.84 ± 0.58 a	18.22 ± 0.29 b	66.32 ± 0.23 a
Extraction	p < 0.0001 *	p < 0.0001 *	p = 0.137	p < 0.0001 *	p = 0.025 *	p = 0.176	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *
5	1383.56 ± 2.50 c	1174.73 ± 2.50 c	19.48 ± 0.37 a	22.74 ± 0.25 c	15.36 ± 0.23 a	207.52 ± 0.75 a	115.23 ± 0.75 b	258.12 ± 0.50 c	18.99 ± 0.25 b	46.04 ± 0.20 c
10	1532.05 ± 2.50 a	1268.50 ± 2.50 a	19.33 ± 0.37 a	27.50 ± 0.25 a	14.67 ± 0.23 a,b	208.90 ± 0.75 a	130.79 ± 0.75 a	295.19 ± 0.50 a	20.59 ± 0.25 a	62.69 ± 0.20 b
15	1477.80 ± 2.50 b	1241.88 ± 2.50 b	18.42 ± 0.37 a	25.43 ± 0.25 b	14.37 ± 0.23 b	209.62 ± 0.75 a	115.29 ± 0.75 b	278.24 ± 0.50 b	18.10 ± 0.25 b	75.44 ± 0.20 a
Interaction	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *
0 × 5	1323.18 ± 5.00 f	1093.95 ± 5.00 h	16.52 ± 0.75 c,d	20.10 ± 0.50 e	11.64 ± 0.45 e	179.30 ± 1.50 g	103.63 ± 1.50 g	272.09 ± 1.00 e	24.10 ± 0.50 d	48.04 ± 0.40 g
0 × 10	1475.54 ± 5.00 d	1233.61 ± 5.00 e,f	18.36 ± 0.75 b,c	25.76 ± 0.50 c,d	13.43 ± 0.45 d,e	201.35 ± 1.50 f	115.13 ± 1.50 e,f	384.11 ± 1.00 b	13.88 ± 0.50 f	67.17 ± 0.40 d
0 × 15	1062.86 ± 5.00 h	916.01 ± 5.00 j	13.56 ± 0.75 d	19.17 ± 0.50 e	11.88 ± 0.45 e	180.27 ± 1.50 g	67.75 ± 1.50 h	184.59 ± 1.00 i	61.16 ± 0.50 a	64.59 ± 0.40 e
3 × 5	1111.21 ± 5.00 g	1051.48 ± 5.00 i	16.18 ± 0.75 c,d	20.42 ± 0.50 e	12.84 ± 0.45 d,e	181.74 ± 1.50 g	109.99 ± 1.50 f,g	335.02 ± 1.00 c	45.89 ± 0.50 b	50.69 ± 0.40 f
3 × 10	1500.58 ± 5.00 d	1258.00 ± 5.00 e	18.80 ± 0.75 b,c	25.77 ± 0.50 c,d	13.65 ± 0.45 c,d,e	181.63 ± 1.50 g	127.40 ± 1.50 b,c,d	165.71 ± 1.00 j	13.49 ± 0.50 f	67.70 ± 0.40 d
3 × 15	1482.63 ± 5.00 d	1228.76 ± 5.00 f	18.54 ± 0.75 b,c	27.45 ± 0.50 a,b,c,d	14.08 ± 0.45 b,c,d,e	207.41 ± 1.50 e,f	127.27 ± 1.50 b,c,d	233.45 ± 1.00 g	7.29 ± 0.50 g	80.69 ± 0.40 b
6 × 5	1567.01 ± 5.00 b	1259.66 ± 5.00 e	25.76 ± 0.75 a	25.22 ± 0.50 d	20.79 ± 0.45 a	249.94 ± 1.50 a	119.07 ± 1.50 d,e	191.45 ± 1.00 h	33.01 ± 0.50 c	45.01 ± 0.40 h
6 × 10	1379.56 ± 5.00 e	1165.40 ± 5.00 g	18.37 ± 0.75 b,c	28.22 ± 0.50 a,b,c	15.04 ± 0.45 b,c,d	222.37 ± 1.50 c,d	132.90 ± 1.50 b	251.82 ± 1.00 f	7.44 ± 0.50 g	45.42 ± 0.40 h
6 × 15	1574.14 ± 5.00 b	1345.50 ± 5.00 c	19.42 ± 0.75 b,c	26.28 ± 0.50 b,c,d	15.00 ± 0.45 b,c,d	214.88 ± 1.50 d,e	122.86 ± 1.50 c,d,e	303.46 ± 1.00 d	24.94 ± 0.50 d	68.40 ± 0.40 c,d
9 × 5	1532.84 ± 5.00 c	1293.84 ± 5.00 d	19.48 ± 0.75 b,c	25.20 ± 0.50 d	16.15 ± 0.45 b,c	219.12 ± 1.50 d	128.25 ± 1.50 b,c	233.93 ± 1.00 g	12.95 ± 0.50 f	40.42 ± 0.40 i
9 × 10	1772.53 ± 5.00 a	1417.01 ± 5.00 b	21.79 ± 0.75 a,b	30.23 ± 0.50 a	16.54 ± 0.45 b	230.26 ± 1.50 b,c	147.73 ± 1.50 a	379.12 ± 1.00 b	23.56 ± 0.50 d	70.47 ± 0.40 c
9 × 15	1791.57 ± 5.00 a	1477.26 ± 5.00 a	22.17 ± 0.75 a,b	28.84 ± 0.50 a,b	16.51 ± 0.45 b	235.91 ± 1.50 b	143.29 ± 1.50 a	391.46 ± 1.00 a	19.01 ± 0.50 e	88.07 ± 0.40 a

Myricetin 3-O-G = myricetin 3-O-galactoside, Myricetin 3-O-R = myricetin 3-O-rhamnoside, Q-3-G = quercetin-3-glucoside, EGKG = epigallocatechin gallate, A = acid. Results are expressed as mean (n = 8 for cryogrinding time, and n = 6 for extraction time) ± SE. Different letters indicate significant differences between the means (* p ≤ 0.05).

shows the most significant phenolic compounds identified in myrtle leaf ethanol extracts. Other polyphenols (epicatechin, catechin, luteolin, apigenin, chlorogenic and p-coumaric acid) that were detected in very low quantities or in traces (below 5 mg/100 g dw) are not shown, but their values were added up to the total.

Myricetin derivatives (myricetin 3-O-galactoside and myricetin 3-O-rhamnoside) were the most abundant compounds, followed by galloylquinic acid, myricetin and digalloylquinic acid. Phenolic acids (gallic and caffeic) and the flavonoids epigallocatechin gallate, quercetin-3-glucoside and quercitrin were present in lower amounts in all extracts, which was in accordance with a study by Taamalli et al. [56]. Total phenolic compounds ranged from 2531.83 to 4224.07 mg/100 g dw (Figure 2), which was also similar to the values reported by Amensour et al. [30]. In their study, the total phenolic content of myrtle leaves ranged from 29.0 (ethanol) to 35.6 mg/g (methanol) depending on the solvent used. On the other hand, results of the present study showed an almost three-fold higher content of total phenols in control extracts after 10 min of extraction (3560.63 mg/100 g dw) when compared to the values of Čulina et al. [24] (1293.55 mg/100 g dw), probably as the choice of solvent favoured higher yields.

Significant variation in total phenolic and flavonoid content in cryogenically ground Trigonella foenum-graecum seed extract compared to conventionally ground seeds were observed by Saxena et al. [57] as well as in Canavalia gladiata bean extract [58]. In the present study, total phenolic content showed a cascading increase in yield with increasing CG time when compared to control, with a 22% increase at 9 min. The same behaviour applied to the individual compounds with the highest increases for digalloylquinic acid (95.50–139.76 mg/100 g dw), quercitrin (12.32–16.40 mg/100 g dw) and myricetin 3-O-galactoside (1287.19–1698.98 mg/100 g dw). Grinding time had a significant effect on all individual compounds. This is another example of how reducing the particle size, i.e., increasing the specific surface area exposed to the extraction solvent, leads to a positive increase in the yield of bioactive molecules and ultimately justifies the use of this technique [34,59,60]. Regarding total phenolics content, extraction time had a significant effect on the content of all phenolic compounds (Table 2), with 10 min of extraction being sufficient for maximum yield, except for quercetin-3-glucoside and myricetin, for which the effect of extraction time was not significant. The reason could be that prolonged exposure of certain phenolic compounds to temperatures, usually above 60 °C, leads to their hydrolysis and oxidation [61]. Therefore, a slight decrease in yield was observed after 15 min. Only for quercitrin and gallic acid were the highest yields achieved with 5 (14.37–15.36 mg/100 g dw) and 15 (46.04–75.44 mg/100 g dw) min of extraction, respectively. The proportion of total phenolic compounds was also significantly affected by the interaction of CG and extraction time; as much as 40% more phenolic compounds were obtained at 9 min of CG and 15 min of extraction when compared to conventional grinding at the same extraction time.

From the data shown in

Table 3. Pigment profile (mg/100 g dw) of myrtle leaf extract obtained by different cryogrinding and extraction times.

Time (min)	Chl b	Chl b’	Chl a	Pheo a	Lutein	9-cis Lutein	β-Carotene
Cryogrinding	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *
0	36.04 ± 0.23 d	3.68 ± 0.09 d	11.15 ± 0.17 b	57.77 ± 1.44 c	35.88 ± 0.12 d	6.06 ± 0.13 c	14.39 ± 0.20 d
3	65.67 ± 0.23 c	5.29 ± 0.09 c	13.92 ± 0.17 a	108.67 ± 1.44 b	68.99 ± 0.12 c	9.86 ± 0.13 b	27.14 ± 0.20 c
6	69.46 ± 0.23 a	6.48 ± 0.09 b	8.84 ± 0.17 c	121.99 ± 1.44 a	75.93 ± 0.12 a	10.19 ± 0.13 b	29.99 ± 0.20 a
9	67.13 ± 0.23 b	7.24 ± 0.09 a	8.15 ± 0.17 c	120.36 ± 1.44 a	72.77 ± 0.12 b	10.85 ± 0.13 a	28.28 ± 0.20 b
Extraction	p = 0.012 *	p < 0.0001 *	p < 0.0001 *	p = 0.006 *	p < 0.0001 *	p = 0.048 *	p = 0.454
5	59.19 ± 0.20 b	5.35 ± 0.08 b	12.43 ± 0.15 b	99.61 ± 1.25 b	62.00 ± 0.10 c	9.13 ± 0.11 b	24.78 ± 0.17 a
10	60.16 ± 0.20 a	5.37 ± 0.07 b	13.41 ± 0.15 a	100.78 ± 1.25 b	63.14 ± 0.10 b	9.09 ± 0.11 b	24.96 ± 0.17 a
15	59.37 ± 0.20 b	6.30 ± 0.08 a	5.71 ± 0.15 c	106.20 ± 1.25 a	65.04 ± 0.10 a	9.50 ± 0.11 a	25.10 ± 0.17 a
Interaction	p < 0.0001 *	p < 0.0001 *	p < 0.0001 *	p = 0.000 *	p < 0.0001 *	p = 0.000 *	p < 0.0001 *
0 × 5	41.77 ± 0.40 f	4.427 ± 0.15 d	12.01 ± 0.30 c	69.68 ± 2.50 f	42.08 ± 0.20 h	6.94 ± 0.23 d	16.40 ± 0.35 f
0 × 10	32.94 ± 0.40 g	3.159 ± 0.15 e	14.03 ± 0.30 b	48.38 ± 2.50 g	31.96 ± 0.20 j	5.39 ± 0.23 e	13.01 ± 0.35 g
0 × 15	33.41 ± 0.40 g	3.468 ± 0.15 e	7.42 ± 0.30 f	55.26 ± 2.50 g	33.61 ± 0.20 i	5.84 ± 0.23 d,e	13.74 ± 0.35 g
3 × 5	65.19 ± 0.40 d	4.671 ± 0.15 d	18.74 ± 0.30 a	102.48 ± 2.50 e	66.98 ± 0.20 g	9.84 ± 0.23 b,c	27.04 ± 0.35 d,e
3 × 10	66.98 ± 0.40 c,d	5.094 ± 0.15 d	17.71 ± 0.30 a	109.33 ± 2.50 c,d,e	69.25 ± 0.20 f	9.62 ± 0.23 c	27.24 ± 0.35 c,d,e
3 × 15	64.85 ± 0.40 d	6.102 ± 0.15 c	5.30 ± 0.30 g	114.19 ± 2.50 b,c,d,e	70.74 ± 0.20 e	10.11 ± 0.23 b,c	27.14 ± 0.35 c,d,e
6 × 5	67.96 ± 0.40 b,c	6.281 ± 0.15 c	10.31 ± 0.30 d,e	117.62 ± 2.50 a,b,c,d	72.28 ± 0.20 d	10.01 ± 0.23 b,c	29.02 ± 0.35 a,b,c
6 × 10	71.31 ± 0.40 a	5.988 ± 0.15 c	11.02 ± 0.30 c,d	122.91 ± 2.50 a,b,c	77.10 ± 0.20 b	10.36 ± 0.23 b,c	30.93 ± 0.35 a
6 × 15	69.11 ± 0.40 a,b,c	7.176 ± 0.15 b	5.20 ± 0.30 g	125.46 ± 2.50 a,b	78.40 ± 0.20 a	10.22 ± 0.23 b,c	30.01 ± 0.35 a,b
9 × 5	61.86 ± 0.40 e	6.021 ± 0.15 c	8.67 ± 0.30 e,f	108.68 ± 2.50 d,e	66.66 ± 0.20 g	9.72 ± 0.23 c	26.67 ± 0.35 e
9 × 10	69.40 ± 0.40 a,b	7.241 ± 0.15 b	10.86 ± 0.30 c,d	122.51 ± 2.50 a,b,c,d	74.25 ± 0.20 c	11.01 ± 0.23 a,b	28.67 ± 0.35 b,c,d
9 × 15	70.12 ± 0.40 a,b	8.460 ± 0.15 a	4.93 ± 0.30 g	129.90 ± 2.50 a	77.41 ± 0.20 a,b	11.82 ± 0.23 a	29.51 ± 0.35 a,b

Chl = chlorophyll, Pheo = pheophytin. Results are expressed as mean (n = 8 for cryogrinding time, and n = 6 for extraction time) ± SE. Different letters indicate significant differences between the means (* p ≤ 0.05).

, it is obvious that chlorophyll b and pheophytin a were chlorophylls present in significant concentrations in the extracts, while lutein and β-carotene were the most abundant carotenoids. Noted differences in the composition of pigments and the higher content of pheophytin in myrtle plant extracts were caused by extraction temperatures and were already explained in the study by Cvitković et al. [26]. The pigment contents of the cryogenically ground samples were also significantly higher than those of the control myrtle leaf samples. In general, 6 min of CG was sufficient to obtain the highest yields of total pigments as well as most of the individual ones (Table 3 and Figure 3). When compared to the control, 6 min of CG yielded almost 50% more total pigments. The increase in the content of individual pigments ranged from a minimum of 41% for 9-cis lutein to 53% for pheophytin a and lutein. Only chlorophyll a showed a decrease after 3 min of grinding, so 3 min of grinding could be sufficient for the extraction of this compound. Castro-Puyana et al. [62] proved that among four different pretreatments, CG for 3 min was the best pretreatment for pressurized liquid extraction to obtain the highest contents of total carotenoids from the microalgae sample. The study by Balbino et al. [37] also demonstrated a positive effect of CG time on protochlorophyll yield from pumpkin seed cake. Furthermore, the highest amounts of chlorophyll b’, pheophytin a, lutein and 9-cis lutein were obtained at an extraction time of 15 min, while 10 min were sufficient for chlorophyll a and b. The reason for this is probably the higher stability of carotenoids as well as chlorophyll b when compared to chlorophyll a at the temperature used [63]. Pheophytin a, as mentioned earlier, is a form of the chlorophyll a structure, and its higher values were expected. It is obvious that with CG there were minimal differences in the proportion of extracted individual and total pigments with increases of extraction time, as shown by the interactions. A 9 min CG and 15 min extraction proved to be the best interaction to obtain the highest yield of total pigments, even 55% more than the lowest values obtained when CG was not applied at 10 and 15 min of extraction.

Except for higher retention of volatiles and flavouring components, colour and particle size distribution are better with CG than with conventional grinding [64,65]. CG of raw plant materials could therefore improve the biological activity and sensory properties of the product such as antioxidant, colour, taste and odour in comparison with non-ground material [66].

4. Conclusions

The four predominant terpenoid compounds of myrtle EO were myrtenyl acetate, 1,8-cineole, α-pinene and linalool. Myricetin derivatives (myricetin 3-O-galactoside and myricetin 3-O-rhamnoside), galloylquinic acid, myricetin and digalloylquinic acid were the most abundant phenolics, while chlorophyll b, pheophytin a and lutein were the most abundant pigments detected in the extracts. In general, CG enabled higher yields and more rapid extraction of volatiles, phenolic and pigment compounds. The content of EO and the extracts was significantly affected by grinding and distillation/extraction time, as well as by their interaction. Three minutes of CG gave the highest yield of volatiles, while their yield decreased with prolonged CG due to their loss by adsorption to the walls of the grinding vessel or by volatilization at room temperature. On the other hand, 9 and 6 min gave the highest yields of phenols and pigments, respectively. The highest extractability of the compounds was obtained at 60 min distillation and 10 min extraction (only for total phenols). The best interaction of grinding and distillation time was 3 × 30 min, while grinding and extraction times for both phenols and pigments was 9 × 15 min. The longer extraction time had a double effect: on the one hand, there was an increased yield due to the longer contact of the plant material with the solvent; and on the other hand, there was degradation due to the effect of prolonged exposure to increased temperatures. These results suggest that CG can significantly increase the yield of HD and extraction and also modulate the composition of myrtle EO and extracts.