Essential Oil Variability of Azorean Cryptomeriajaponica Leaves under Different Distillation Methods, Part 1: Color, Yield and Chemical Composition Analysis

Abstract

This study mainly deals with the effect of hydrodistillation (HD) and water-steam distillation (WSD) methods on the color, yield, and chemical profile of the essential oil (EO) from Cryptomeria japonica fresh leaves from São Miguel Island (Azores Archipelago, Portugal). The yields of EO–HD (pale-yellowish) and EO–WSD (colorless) samples were 1.21% and 0.45% (v/w), respectively. The GC–FID, GC–MS, and ¹³C-NMR analyses of EO–HD vs. EO–WSD revealed (i) a high-content of monoterpenes (72.8% vs. 86.7%), mainly α-pinene (34.5% vs. 46.4%) and sabinene (20.2% vs. 11.6%), and oxygenated mono- and sesquiterpenes (20.2% vs. 9.6%); (ii) similar sesquiterpene (1.6% vs. 1.6%), β-myrcene (5.9% vs. 5.8%), and camphene (3.5% vs. 3.8%) contents; and (iii) significant differences in other classes/components: EO–HD is richer in oxygenated sesquiterpenes (17.1%, mainly elemol (10.4%) and α-eudesmol (3.4%)) and diterpenes (3%; mostly phyllocladene), while EO–WSD is richer in oxygenated monoterpenes (7.2%, mainly terpinen-4-ol (5.4%)), p-cymene (4.4%), and limonene (3.2%). Overall, the color, yield, and quantitative composition of the EO samples studied are strongly influenced by the distillation method. Nonetheless, this C. japonica leaf EO displayed a consistent α-pinene- and sabinene-rich composition. The same chemotype was found in a commercial Azorean C. japonica leaf EO sample, obtained by industrial steam distillation (SD), as well as in Corsica C. japonica leaf EO–HD. Furthermore, the bioactive composition of our EO samples revealed the potential to be used in green plant protection and in the medical, food, cosmetic, and household industries.

1. Introduction

Essential oils (EOs) are generally complex mixtures of volatile, lipophilic, and odiferous secondary metabolites synthesized and emitted by several plants to facilitate their growth and survival. Thus, they can be valuable sources of bioactive components with application, for example, in the fight against human and animal infectious diseases as well as in green plant protection. At present, about 3000 EOs are known, but only 10% of them are commercially important [1,2]. EOs are mainly composed of two types of components: terpenes (including mono-, sesqui-, and diterpenes and their oxygenated derivatives), and the aromatic and aliphatic components, derived from phenylpropane. They can be composed of only a few to up to more than 100 components in different concentrations. Nevertheless, they are generally characterized by few major components being present at high concentrations compared with other components that are found at low or trace amounts. In general, the main components determine the characteristic odor and the functional and biological properties of the EOs. However, the overall bioactivity of botanical extracts can result from mixtures of components with synergistic, additive, or antagonistic effects. Thus, to investigate the viability of a specific EO application, knowledge about their total chemical composition is required [1,3,4].

Plant-derived EOs have been widely used all over the world since ancient times, and their production has increased tremendously over the last five decades due the strong demand for natural products, which are less hazardous to the public health and environment than their synthetic equivalents. In fact, numerous EOs, as natural products with GRAS (Generally Recognized as Safe) statuses, have commercial applications in many important industries such as food, beverage, fragrances, cosmetics, cosmeceuticals, perfumery, household products, pharmaceutical, aromatherapy, phytomedicine, and agronomic (e.g., pesticides) industries [2,3,4,5,6]. However, as is well established, the yield (usually 1 to 3% of the plant weight) and chemical composition of EOs obtained from aromatic plants and, consequently, their functional bioactivity value, commercial uses, and price, depend on many factors, such as species, EO chemotypes, varieties or subspecies, plant organ, age and vegetative cycle stage, biotic interactions, spatial and temporal changes in environmental parameters (including light intensity, temperature, rainfall, relative humidity, and soil conditions), management practices (e.g., daily harvest period) and post-processing of plant material (e.g., drying), extraction protocols (e.g., method/apparatus used and extraction time), storage of EO, and analytical methods [1,2,7,8,9]. It should be noted that several reports have highlighted that the extraction method of EOs could lead to damage or alteration of their chemical profile with impacts on their quality [5,9,10]. Several processes can be used to isolate EOs from plants, such as the traditional distillation that is still used in many parts of the world in view of its simplicity and environmentally friendly practice due to the use of water compared with solvent extraction. Steam distillation (SD), water-steam distillation (WSD), and hydrodistillation (HD) using a Clevenger-type apparatus are among the most traditional and commonly used methods [5]. The SD method has been widely used, especially for industrial-scale production. In small-scale industries, WSD is an alternative method. The system consists of a packed bed of the plant material, which sits above a steam source (boiling water). On the other hand, the HD method involves the complete immersion of plant materials in water, followed by boiling [5], and is widely used at the laboratory scale. Since EO components are volatile and non-polar, the most widely used method for EO analysis is gas chromatography–mass spectrometry (GC–MS), while gas chromatography with flame ionization detection (GC–FID) is used to determine the percentage of EO components [8].

Terpenes, in particular monoterpenes, are the main components in the EOs of conifer families, such as Cupressaceae, the most widely distributed of all gymnosperm families [11,12]. In view of their odoriferous properties, their lipophilicity, and their broad spectrum of valuable biological properties (e.g., antioxidant properties; inhibitory activity towards acetyl- and butyrylcholinesterase, and α-amylase and α-glucosidase; antimicrobial, hepatoprotective, and sedative properties; and skin permeation enhancement) [13], monoterpenes find extensive commercial applications especially in the fragrance, flavor, pharmaceutical, and medical fields [14].

Species of the Cupressaceae family, such as Cryptomeria japonica (Thunberg ex Linnaeus f.) D., are among economically important EO producers with interest in various industries and folk medicine [6,15]. C. japonica, an evergreen ornamental tree, is one of the four species belonging to the subfamily Taxodioideae [16]. It is an endemic species of Japan, commonly called Japanese cedar or sugi, and is one of the most commercially important plantation trees in Japan and other Asian countries, such as Korea, Taiwan, India, and China, due to its valuable wood characteristics, which include excellent durability for exterior construction, pleasant aroma, insect resistant properties, and a beautiful yellowish-red heartwood color. Moreover, the tree itself is valued in landscaping [17,18,19]. Owing to its industrial importance, the chemical composition and biological activities of the extracts and the EOs from different parts of C. japonica as well as the bioactive properties of its isolated components, including flavonoids, terpenes, and terpenoids, have been investigated in numerous studies ([1,20,21] and the references therein). In particular, there have been several reports over the last eight decades [22] on the bioactivity and chemical composition of the EOs from C. japonica leaves, since they are usually discarded by the wood industry. The leaf EO of this plant from various countries has demonstrated a broad range of bioactivities, including analgesic [23], antibacterial [24,25,26,27], antifungal [20,26,27], anti-inflammatory [25], antimelanogenesis [28], antioxidant [15], antitussive [29], antiulcer [30], anxiolytic [23], cancer chemopreventive [31], neuropharmacological [32], whitening [15], antitermite [33], insecticidal [34], mosquito larvicidal [35,36,37], mosquito repellent [38], and silverfish repellent [34] properties. Therefore, the interest in leaf EO from C. japonica as an eco-resource of valuable bioactive components is rapidly increasing. Concerning the leaf EO composition of this plant from various countries, great differences not only in the identity of the EO components but also in their percentage were found in several studies ([1,17] and the references therein), as expected. For the extraction of EO from C. japonica leaves, the majority of the referenced studies have utilized the standard HD method. Moreover, few studies [33] have reported the effects of different distillation methods on the properties of C. japonica leaf EO (e.g., color, odor, yield, chemical composition, and bioactivity).

In Azores Archipelago (Portugal), located in the northern Mid-Atlantic Ridge and composed of nine volcanic islands, C. japonica, known in Portugal as “criptoméria”, was introduced from Japan in the mid-19th century and was extensively cultivated for economic uses. Moreover, C. japonica is a determinant element of the Azorean landscape. At present, about 12,500 hectares of plantations is established in mountainous regions, representing 60% of the total wood producing forest area, 85% of which is in São Miguel Island [39]. Thus, the commercialization of EO isolated from wastes produced by the C. japonica wood industry in Azores has social, environmental, and economic importance. Previous investigations on the chemical composition and biological activity of EOs from different parts of C. japonica from Faial Island (Azores) revealed their potential as natural biocides, namely bactericide and fungicide [11].

As a part of our continuing phytochemical investigation of wastes from abundant Azorean resources and knowing that the quality of EOs depends mainly on its chemical composition that, as reported previously, can be influenced by the extraction method used, the purposes of the present study were (i) to compare, for the first time, the color, yield, and chemical composition of the EO obtained by HD and WSD from autumn fresh leaves of C. japonica growing in São Miguel Island (Azores), and (ii) to compare the results with those from published literature on C. japonica leaf OE from other origins. Furthermore, the chemical composition of a commercial Azorean C. japonica leaf EO sample, obtained by industrial steam distillation (SD), was also presented for comparative purposes. Overall, this study can contribute to predicting the potential of the investigated EOs as eventual raw materials (crude EO, fractions, or individual components) for specific commercial applications, thus contributing to the potential valorization of unused Azorean C. japonica biomass and, consequently, to the local circular economy.

2. Materials and Methods

2.1. Plant Material

The fresh aerial parts of red heartwood-type C. japonica, provided by Marques, S.A. (the largest local producer of EOs), were collected in November 2016 (autumn) in Cryptomeria forest (ca. 30 to 40-year-old, average height of 20 m, and andosol-type soil) at São Miguel Island (Azores Archipelago, Portugal). The aerial parts were randomly picked from the healthy plants of two locations in the northeast region of São Miguel: Achadinha (altitude 733 m, latitude 37°48′28.59″ N, longitude 25°16′16.14″ W, code sample “Lote B”), and Algarvia (altitude 663 m, latitude 37°49′00.45″ N, longitude 25°13′39.22″ W, code sample “Lote 3 talhão 3”). The aerial parts were placed in plastic bags and immediately brought to the laboratory at the University of Azores, where their leaves were separated immediately prior to the distillation process. A voucher specimen (UA-DCQFE 183) was deposited at the Department of Physics, Chemistry, and Engineering, University of Azores.

For comparative purposes concerning the chemical composition of the studied OE samples (EO–HD and EO–WSD), the EO composition data from industrial SD of fresh leaves of the red heartwood-type Azorean C. japonica sample collected in Achadinha (altitude 733 m, latitude 37°48′28.59″ N, longitude 25°16′16.14″ W, code sample “UGF1 T2”) in June 2017, were provided by Marques, S.A.

2.2. EO Isolation by Distillation Methods

Fresh leaves of an Azorean C. japonica sample (code “Lote B” plus “Lote 3 talhão 3”, as referenced in Section 2.1) were subjected to HD and WSD over 3 h after the first drop of the distillate fell, followed by a determination of their EO yields (%, v/w) on the basis of the leaf fresh weight. In both distillations, the ratio of the plant material to water was 1:10 g/mL. HD was performed in a Clevenger-type apparatus according to the European Pharmacopoeia [40], and WSD was also conducted in a laboratory glass apparatus that consisted of a column where plant material was packed, which sat above the steam source (boiling water) [41]. Each distillation was performed in triplicate, and the isolated EO was dehydrated with anhydrous sodium sulphate and stored in a sealed amber vial at 4 °C for further analysis.

Fresh leaves of Azorean C. japonica sample (code “UGF1 T2”, as referenced in Section 2.1) were subjected to SD by Marques, S.A., using stainless-steel distillers (1100 L, Vieirinox^®, Henrique Vieira & Filhos, Aveiro, Portugal) over 2 h at 0.4 bar.

2.3. EO Composition Analysis

2.3.1. Gas Chromatography (GC)

Gas chromatographic analyses were performed on a Perkin–Elmer Clarus 400 gas chromatograph (Shelton, CT, USA) equipped with two flame ionization detectors (FID), a data-handling system, and a vaporizing injector port into which two fused-silica capillary columns with different polarities were installed: a DB-1 (polydimethylsiloxane, 30 m × 0.25 mm i.d., 0.25 μm film thickness) and a DB-17HT (50% phenyl-methyl-polysiloxane, 30 m × 0.25 mm i.d., 0.15 μm film thickness), both from J & W Scientific Inc. (Rancho Cordova, CA, USA). The analytical conditions were an oven temperature from 45 to 175 °C at a rate of 3 °C/min followed by a temperature increase at 15 °C/min until 300 °C and hold during 10 min; injector and detector temperatures, 280 °C and 300 °C, respectively; carrier gas (hydrogen) at a linear velocity of 30 cm/s; and a split ratio of 1:50. The volume injected was 0.1 μL of a n-pentane–EO solution (1:1). The percentage composition of the EOs was computed by the normalization method from the GC peak areas and calculated as mean values of two injections from each EO, without using correction factors, in accordance with ISO 7609 [42].

2.3.2. Gas Chromatography–Mass Spectrometry (GC–MS)

GC–MS analyses were carried out with a Perkin–Elmer Clarus 600 gas chromatograph hyphenated with a Perkin–Elmer Turbomass Clarus 600T mass spectrometer (software version 5.4, PerkinElmer, Shelton, CT, USA), using a DB-1 column as described above. The GC–MS settings were as follows: injector and oven temperatures were as above; transfer line and ion source temperatures, 280 °C and 220 °C, respectively; carrier gas (helium) at a linear velocity of 30 cm/s; split ratio, 1:40; electron impact (EI) mode at 70 eV; ionization current of 60 μA; mass scan range of 40–300 amu; and scan time of 1 s. The identity of the EO components was assigned by comparison of their retention indices, calculated in accordance with ISO 7609 [42], relative to C₉–C₂₁ n-alkane indices, and GC–MS spectra with corresponding data of (1) components of reference EOs—Thymus caespititius [43], L. azorica [44], Coriandrum sativum, Satureja montana, Santolina chamaecyparissus, Thymus vulgaris [45], and Monizia edulis [46]; (2) laboratory-synthesized components following the methods described by Grosso et al. [45]; and (3) commercially available standards from a home-made library.

2.3.3. Carbon-13 Nuclear Magnetic Resonance (¹³C NMR)

The identity of the EO components was also confirmed by ¹³C NMR without previous separation of the components, and according to an experimental procedure and computerized method, as detailed in Cavaleiro et al. [47]. ¹³C NMR spectra were recorded on a Bruker Avance 400 spectrometer from Bruker Optik GmbH (Ettlingen, Germany), operating at 100 MHz, equipped with a 5 mm probe in deuterated chloroform (CDCl₃), using tetramethylsilane (TMS) as an internal standard.

3. Results and Discussion

3.1. Extraction, Color, and Yield of C. japonica Leaf EO

3.1.1. Effects of the Distillation Method on the Color and Yield of the Investigated Leaf EO Samples (EO–HD and EO–WSD)

Table 1. Yield and color of the essential oil of Azorean Cryptomeria japonica fresh leaves, isolated by two distillation methods (HD and WST).

Distillation Method	Essential Oil
	Yield (%, v/w) 1	Color
Hydrodistillation (HD)	1.21 ± 0.03 a	Pale yellow
Water-steam distillation (WSD)	0.45 ± 0.01 b	Colorless

¹ Values are mean ± standard deviations (n = 3). Statistical comparison was performed using Student’s T-test. Different superscript letters are significantly different (p < 0.05).

shows the color and yield of the EO–HD and EO–WSD samples. The yield results were expressed as the mean ± standard deviation of three replicates. Comparing the leaf EO yield (%, v/w), on a fresh weight (FW) basis, a significantly higher value was obtained by HD (1.21 ± 0.03%) than that produced by WSD (0.45 ± 0.01%). Concerning the EO color, the EO–HD sample showed a pale-yellowish, while the EO–WSD sample was colorless. Based on the results obtained in this study, it was proven that the distillation method significantly affects the C. japonica leaf EO color and yield, with HD being the most efficient EO extraction method compared with the WSD. The higher EO yield in the HD method, using a Clevenger-type apparatus, may be due to the conditions prevailed during distillation such as the recycling of hydrosol that allowed it to recover the dissolved EO, since it can be re-boiled.

The same yield pattern was also observed in similar studies with other aromatic plants, such as the case of rose-scented geranium (Pelargonium sp.) EO, which presented yield values of 0.16–0.22% and 0.09–0.12% for EO–HD and EO–WSD, respectively [9].

3.1.2. Comparison to Previous Studies on C. japonica Leaf EO Isolated by HD

The HD method, using a Clevenger-type apparatus, is the mostly commonly used to obtain C. japonica leaf EO at a laboratory scale due to its simplicity and efficient EO extraction, as demonstrated in this study.

Table 2. Data from the literature on the yield and color of the essential oil (EO) isolated by hydrodistillation (HD) from Cryptomeria japonica leaves from different origins and on information about the HD protocol and the plant material characteristics compared with those of the leaf EO–HD sample investigated.

C. japonica Origin	Pant Age (years)	Harvested Time	Leaves Status	EO Isolated by HD 2 Using a Clevenger-Type Apparatus			Ref.
				DT	Yield	Color
São Miguel Island (Azores) 1	30–40	Nov. 2016	fresh	3 h	1.21% (v/w)	pale yellow	-
Faial Island (Azores)	–	July 2007	dried	4 h	0.5–0.8% (w/w; FW)	–	[11]
Corsica Island (France)	–	–	–	–	0.61%	–	[48]
Yakushima Island (Japan)	30	Nov. 2013	fresh	2 h	2.2 mL/kg	–	[28]
Hefei (China)	–	May 2009	fresh	4 h	1.15% (w/w)	pale yellow	[1]
Bagmati Zone (Nepal)	–	May 2011	dried	4 h	0.5% (w/w)	pale yellow	[17]
Jeju Island (South Korea)	–	2010	fresh	24 h	4.7% (w/w; DW)	–	[15]
		–		–	0.6% (v/w)		[25]
		June 2007		6 h	0.84% (v/w)		[27]
Nantou County (Central Taiwan)	58	June 2001	–	6 h	2.37% (w/w; DW)	–	[36]
	42				1.91% (w/w; DW)
	26				2.19% (w/w; DW)
Nantou County (Central Taiwan)	28	August 2009	fresh	6 h	1.60% (w/w; DW)	–	[33]

¹ This study. ² Data on solute-to-solvent ratio were only reported in our study and that by Nakagawa et al. [28], being 1:10 and 1:1 g/mL, respectively. DT—distillation time; DW—dry weight; FW—fresh weight; Nov.—November; (–)—not reported.

shows data from the literature on the yield and color of EO–HD samples from leaves of red heartwood-type C. japonica according to geographic region, for comparative purposes. Other relevant data are summarized in Table 2, namely plant age; harvest period; status of the plant material (fresh or dry) before the distillation process; and HD conditions, such as solute-to-solvent ratio and distillation time (DT).

The results on the leaf EO-HD yield of C. japonica samples from different origins revealed that the yield obtained in the present study (1.21%, v/w, FW) is within the range reported in the literature (Table 2), being higher than that observed in two Korean samples (0.6% and 0.84%, v/w, FW) [25,27], respectively. As also observed in Table 2, Kim et al. [15] reported a significantly higher yield (4.7%, w/w, dry weight) for a Korean sample that, however, was distillated over 24 h [15] (an unusual DT). In fact, the majority of the referenced studies used a DT that ranged between 2 and 6 h, with 4 h or 6 h being the predominant distillation period (Table 2).

The differences observed on the leaf EO yield from C. japonica plant can be due to the impact of different factors, namely genetic background of the plant, plant age, vegetative cycle stage, region and altitude where it is grown, environmental parameters variations, sampling time, processing and storage practices, and HD conditions (e.g., solute-to-solvent ratio and DT). Furthermore, it should be highlighted that a comparison of the yield data between different studies is very difficult when information about the plant material and the extraction protocol is incomplete and/or different units of measurement are used, as demonstrated in the data from literature presented in Table 2.

3.2. Chemical Composition of EOs

3.2.1. Effects of the Distillation Method on the Chemical Composition of the Investigated Leaf EO Samples (EO–HD and EO–WSD)

Figure 1A,B show the total ion current (TIC) chromatogram recorded by GC–MS of the studied leaf EO samples, and their chemical compositions are shown in

Table 3. Percentage of components of leaf essential oil (EO) from Cryptomeria japonica growing in the northeast region of São Miguel Island (Azores), isolated by hydrodistillation (HD), water-steam distillation (WSD), and industrial steam distillation (SD).

Components	RI	Relative Content (%)			IP 3
		OE–HD 1	OE–WSD 1	OE–SD 2
Monoterpene Hydrocarbons (MH)
Tricyclene	921	0.5	0.6	0.6	c, d
α-Thujene	924	2.1	2.8	1.6	c, d
α-Pinene	930	34.5	46.4	39.3	a, c, d
Camphene	938	3.5	3.8	3.7	c, d, e
Sabinene	958	20.2	11.6	19.3	a, c, d, e
β-Pinene	963	t	1.7	2.1	a, c, d, e
β-Myrcene	975	5.9	5.8	5.7	a, c, d, e
α-Phellandrene	995	t	0.1	0.1	c, d, e
δ-3-Carene	1000	1.1	1.1	2.6	a, e
α-Terpinene	1002	1.0	1.1	1.2	a, c, d, e
p-Cymene	1003	0.7	4.4	0.3	a, c, d, e
β-Phellandrene	1005	0.4	0.7	0.8	c, e
Limonene	1009	1.4	3.2	8.3	a, c, d, e
γ-Terpinene	1035	1.1	2.6	1.9	a, c, d, e
Terpinolene	1064	0.4	0.8	1.0	a, c, e
Oxygenated Monoterpenes (OCM)
trans-2-p-Menthen-1-ol	1099	-	-	0.1	e
Borneol	1134	-	-	0.1	a, c
Terpinen-4-ol	1148	1.8	5.4	2.0	a, c, d, e
α-Terpineol	1186	-	0.2	-	a, c, d, e
Linalyl acetate	1245	-	-	0.1	b
Bornyl acetate	1265	1.3	1.6	1.7	b, d
α-Terpenyl acetate	1334	-	-	0.1	b, c, d
Sesquiterpene Hydrocarbons (SH)
β-Elemene	1388	-	-	0.2	c, d
β-Caryophyllene	1414	-	-	0.2	c, f
γ- Muurolene	1469	-	-	0.7	c
α-Muurolene	1494	-	0.2	0.3	c
γ-Cadinene	1500	-	0.2	0.4	c, d
trans-Calamenene	1505	-	-	t	c
δ-Cadinene	1505	1.6	1.2	1.1	c, d
Oxygenated Sesquiterpenes (OCS)
Elemol	1530	10.4	1.5	0.8	c
γ-Eudesmol	1609	1.2	0.2	-	g
τ-Cadinol	1616	0.2	0.1	0.2	c, d
τ-Muurolol	1616	0.3	0.1	-	g
β-Eudesmol	1620	1.6	0.2	-	c
α-Eudesmol	1634	3.4	0.3	-	c
Diterpene Hydrocarbons (DH)
Sandaracopimara-8(14),15-diene	1956	0.1	-	t	g
Phyllocladene	2006	2.9	0.2	0.9	a
Identified components (%)		97.6	98.1	97.4

¹ Data from our study. ² Data provided by Marques, S.A. ³ Identification procedure (IP): a = commercial standards (Sigma-Aldrich-Fluka); b = laboratory-synthesized components following the methods described by Grosso et al. [45]; c = reference EO (Thymus caespititius) [43]; d = reference EO (Laurus azorica) [44]; e = reference EO (Coriandrum sativum, Satureja montana, Santolina chamaecyparissus, and Thymus vulgaris) [45]; f = reference EO (Monizia edulis) [46]; g = MS, RI, ¹³C-NMR. RI–linear retention indices relative to C₉–C₂₁ n-alkanes on a DB-1 column; t–trace (<0.05%).

and Figure 2.

Concerning the chemical composition of the leaf EO samples studied, analyzed using the GC–FID and GC–MS methods and confirmed by ¹³C NMR, 26 and 28 components were identified in the EO–HD and EO–WSD samples, respectively, accounting for 97.6% and 98.1% of the total content of identified EO components (Table 3) and grouped into five classes: monoterpene hydrocarbons (MH), oxygen-containing monoterpenes (OCM), sesquiterpene hydrocarbons (SH), oxygen-containing sesquiterpenes (OCS), and diterpene hydrocarbons (DH), as illustrated in Figure 2. The leaf EO–HD sample was characterized by a high content of MH (72.8%) followed by OCS (17.1%), OCM (3.1%), and DH (3.0%), but a low content of SH (1.6%). In general, a similar pattern was reported for C. japonica leaves collected in summer in Faial Island, Azores [11]. On the other hand, the leaf EO–WSD sample was also characterized by a high content of MH (86.7%), followed by OCM (7.2%), OCS (2.4%), SH (1.6%), and DH (0.2%). The main components (percentages above ca 3%) in leaf EO–HD were α-pinene (34.5%), sabinene (20.2%), elemol (10.4%), β-myrcene (5.9%), camphene (3.5%), α-eudesmol (3.4%), and phyllocladene (2.9%), while in leaf EO–WSD, they were α-pinene (46.4%), sabinene (11.6%), β-myrcene (5.8%), terpinen-4-ol (5.4%), p-cymene (4.4%), camphene (3.8%), limonene (3.2%), α-thujene (2.8%), and γ-terpinene (2.6%). Moreover, sandaracopimara-8(14),15-diene was detected only in EO–HD, whereas α-terpineol, α-muurolene, and γ-cadinene were present only in EO–WSD, with all of these EO components being found in low amounts (0.1 to 0.2%).

The results show that MH α-pinene and sabinene were the main components of the leaf EO extracted using both methods and that MH β-myrcene and camphene presented similar levels in both EOs. Furthermore, in both the EO–HD and EO–WSD samples, the content of grouped components decreased as follows: MH + SH (74.4 vs. 88.3%) >> OCM + OCS (20.2% vs. 9.6%) >> DH (3.0% vs. 0.2%), with SH having the same value (1.6%).

The results also show that the EO–HD sample was richer in OCS (mainly elemol and α-eudesmol) and DH (mostly phyllocladene), while the EO–WSD sample was richer in OCM (mainly terpinen-4-ol) and in MH p-cymene, limonene, α-thujene, and γ-terpinene.

The above results indicate that the qualitative composition of the São Miguel C. japonica leaf EO extracted using two different distillation methods was similar, whereas the relative contents of certain components varied. In fact, of the twenty-nine components identified in our C. japonica leaf EO, only thirteen (namely tricyclene, α-thujene, camphene, β-myrcene, α-phellandrene, δ-3-carene, α-terpinene, β-phellandrene, terpinolene, bornyl acetate, δ-cadinene, τ-cadinol, and τ-muurolol) presented similar relative contents in both methods with concentrations that ranged between 0.1% and 5.9% of the total EO composition.

The results obtained in similar studies with other aromatic plants, such as the case of Pelargonium sp. EO [9], also proved that the distillation methods (HD and WSD) had a strong effect on the percentage of EO components. It should also be pointed out that, to the best of our knowledge, the distillation methods mentioned above have not previously been compared in C. japonica EO.

3.2.2. Comparison to Previous Reports on C. japonica Leaf EO Isolated by SD

Table 3 and Figure 2 also show the chemical composition of one commercial leaf EO sample obtained by industrial SD from C. japonica also growing in the northeast region of São Miguel Island, Azores, but collected in a different season (June 2017) compared with our investigated samples (November 2016). In the EO–SD sample, 32 components were identified, accounting for 97.4% of the total content of identified EO components (Table 3), revealing a higher complexity compared with that in the EO–HD and EO–WSD samples (26 and 28 components, respectively). The study by Řebíčková et al. [8] in bay leaf EO also reports that the EO–SD sample contains more components than the EO–HD sample. According to the authors, it can be due to thermal modification or degradation of certain EO components during contact with boiling water.

As can also be seen, the leaf EO chemical composition of the three samples (EO–HD, EO–WSD, and EO–SD) determined using the same analytical conditions revealed that α-pinene was the main component (34.5%, 46.4%, and 39.3%, respectively), followed by sabinene (20.2%, 11.6%, and 19.3%, respectively) (Table 3). Thus, our C. japonica leaf EO displayed a consistent α-pinene- and sabinene-rich composition, despite different distillation methods and sampling times, suggesting that the synthesis of these components is deeply dependent on the genetic background of the plant. Furthermore, in the three samples referenced, the content of grouped components decreased as follows: MH + SH (74.4%, 88.3%, and 91.4%, respectively) >> OCM + OCS (20.2%, 9.6%, and 5.1%, respectively) >> DH (3.0%, 0.2%, and 0.9%, respectively) (Figure 2).

However, concerning other major components (percentages above 4%), the following results were observed: elemol (10.4%) and β-myrcene (5.9%) in EO–HD; β-myrcene (5.8%), terpinen-4-ol (5.4%) and p-cymene (4.4%) in EO–WSD, and limonene (8.3%) and β-myrcene (5.7%) in EO–SD (Table 3). Moreover, eight components were detected only in EO–SD: OCM trans-2-p-menthen-1-ol, borneol, linalyl acetate, and α-terpenyl acetate (0.1% each); SH β-elemene (0.2%), β-caryophyllene (0.2%), γ-muurolene (0.7%), and trans-calamenene (traces). Nonetheless, in general, a more similar pattern was observed between the EO–WSD and EO–SD compositions, except in the OCM group (Table 3 and Figure 2).

The study by Kusumoto and Shibutani [49] on leaf EO isolated by industrial SD from Japanese C. japonica (collected in Akita in October 2010) revealed that the major group was MH (58.0%), with α-pinene (17.0%) and sabinene (12.0%) being the main components, followed by the OCS group (22.0%), which was dominated by elemol (9.6%). Interestingly, this pattern is similar to the one found in our EO–HD sample. However, a different pattern was found regarding the DH group that consisted of ent-kaurene (6.66%) and phyllocladene (1.73%) in Japanese OE, and of phyllocladene (2.9%) and sandaracopimara-8(14),15-diene (0.1%) in Azorean EO-HD.

The effect of different distillation methods (HD and SD) on the composition of C. japonica leaf EO from Taiwan has been studied by Cheng et al. [33]. The authors found that the EO sample is dominated by two components (kaur-16-ene and elemol), in both distillation methods and proved that the distillation methods had an effect on the percentage of EO components. These findings are similar to that of our study.

Furthermore, the results obtained in similar studies with other aromatic plants, such as the case of lavender EO [50], also revealed that the highest MH and SH contents were observed in the EO isolated using the SD method followed by the WSD and HD methods. According to the authors, this may be due to the non-polar nature of the components possessing strong lipophillic bondages with the fatty (oil) components, which require higher energy to break the bondage. This energy can be met by the higher enthalpy of the steam, which was generated in a separate boiler and delivered at a higher pressure in the distillation tank during the SD method.

On the other hand, the higher content of oxygenated and DH components in the EO–HD sample may be due to the practice of returning the distilled water to the still after the EO was separated from it so that it can be re-boiled and, thus, to minimize the losses of these components.

3.2.3. Comparison to Previous Studies on C. japonica Leaf EO Isolated by HD

Table 4. Data from the literature on the main components (≥2%) of the essential oils (EOs) isolated by hydrodistillation (HD) from Cryptomeria japonica leaves from different origins and compared with those of the leaf EO–HD sample investigated ¹.

Main Components	Cryptomeria japonica Origin
	São Miguel Island, Azores 2	Corsica Island [48]	Reunion Island [51]	Japan [28]	China [1] 3	Nepal [17]	Korea			Taiwan
							[15]	[25]	[27]	[36]	[33]	[34]
MH group
α-Thujene	2.1	1.4	-	0.9	0.5	0.3	1.2	-	0.6	0.9–1.3	0.5	0.8
α-Pinene	34.5	19.1	16.7	13.1	8.0	4.2	9.8	3.5	3.0	4.4–4.9	8.5	5.6
Camphene	3.5	3.1	2.4	3.6	0.8	0.03	-	0.5	0.6	0.5	3.4	1.0
Sabinene	20.2	19.6	8.5	2.7	-	4.3	5.5	8.9	5.1	6.8–10.8	3.8	9.4
β-Pinene	t	1.2	0.7	8.2	0.7	0.1	-	-	-	-	-	0.2
β-Myrcene	5.9	4.3	2.9	3.9	-	0.6	1.6	1.2	1.3	1.0–1.2	1.6	2.6
δ-3-Carene	1.1	0.4	2.5	0.7	0.5	0.1	3.1	1.7	0.6	0.8–1.2	1.5	9.7
α-Terpinene	1.0	2.0	2.0	0.8	0.1	0.5	3.0	1.0	1.4	0.8–1.2	0.9	1.9
p-Cymene	0.7	0.2	1.3	-	0.1	0.3	-	-	-	1.9–2.7	-	0.2
β-Phellandrene	0.4	0.6	0.7	-	6.0	-	-	-	-	-	-	-
Limonene	1.4	9.0	2.2	5.1	1.6	1.1	2.0	1.2	1.9	2.0–2.4	6.8	5.3
γ-Terpinene	1.1	3.2	3.5	1.5	0.6	1.2	2.0	1.9	2.2	1.8–2.4	1.4	3.1
Terpinolene	0.4	1.3	1.0	0.8	0.3	0.5	1.1	1.1	0.9	0.8–1.0	0.6	1.6
OCM group
Terpinen-4-ol	1.8	6.4	5.9	1.4	0.7	0.9	5.7	4.1	4.6	6.2–8.3	2.0	9.1
α-Terpineol	-	0.3	0.5	0.1	-	-	13.4	-	0.3	0.3–0.5	0.2	0.5
Bornyl acetate	1.3	2.2	1.6	2.2	0.5	0.1	0.6	-	0.7	0.9–1.3	3.8	0.8
SH group
β-Elemene	-	0.1	0.1	0.5	5.9	0.03	-	-	0.2	-	-	-
Widdrene	-	-	-	-		-	-	2.6	-	-	-	-
β-Caryophyllene	-	tr	0.2	2.5	0.3	-	-	-	0.1	-	-	-
Thujopsene	-	-	-	8.8		-	0.7	-	-	-	-	-
δ-Cadinene	1.6	0.5	0.5	1.9	1.7	1.5	0.6	3.3	1.8	0.8–1.1	0.4	0.5
OCS group
Elemol	10.4	10.7	11.8	2.9	0.5	20.4	10.9	10.9	6.9	18.3–19.1	18.3	18.2
α-Elemol	-	-	-	-	20.1	-	-	-	-	-	-	-
Cedrol	-	-	0.2	3.3		-	-	1.0	-	0.0–0.6		-
γ-Eudesmol	1.2	1.3	0.1	1.2	4.1	7.0	10.6	9.4	19.0	6.3–7.2	8.2	-
β-Eudesmol	1.6	1.5	3.4	-	5.0	5.0	-	5.1	6.0	11.5–11.8	4.8	5.7
α-Eudesmol	3.4	1.6	2.9	2.3	5.6	4.7	12.2	5.3	7.9		6.5	-
α-Cadinol	-	-	0.4	-	-	2.3	-	2.2	-	-	-	-
Liriodenine	-	-	-	-	2.7	-	-	-	-	-	-	-
Lendene	-	-	-	-	-	-	-	3.8	-	-	-	-
DH group
Phyllocladene	2.9	-	-	-	-	-	-	-	-	-	-	-
Kaur-16-ene	-	-	-	-	14.8	42.1	-	-	-	19.5–20.7	23.3	11.6
Kaurene	-	6.5	10.0	9.2	1.5	-	19.4	17.2	26.3	-	-	-
Comp. ident.	26	45	80	73	57	32	30	24	38	29–30	22	22
GC column	DB1	BP1	WAX52	DB5	HP5	HP5	HP5	DB1	HP5	DB5

¹ Data on the total number of components identified (comp. ident.) and GC column used are also reported. ² This study. ³ Wild-growing C. japonica. t–trace (<0.05%); MH—monoterpene hydrocarbons; OCM—oxygen-containing monoterpenes; SH—sesquiterpene hydrocarbons; OCS—oxygen-containing sesquiterpene; DH—diterpene hydrocarbons..

shows data from the literature on the main components of EO–HD samples from leaves of red heartwood-type C. japonica according to geographic region, for comparative purpose. As can be observed, deep differences were found, not only in the identity of the EO components but also in their percentage. Nonetheless, on a basis of the component groups (MH, OCM, SH, OCS, and DH), a similar pattern was found in all C. japonica OE samples (Table 4), as summarized below.

Concerning the MH group, the OE samples were characterized by the association of α-pinene and sabinene as major MH or by the dominance of α-pinene, except for one Taiwan EO sample [34] that presented the following decreasing order of abundance in their major MH: δ-3-carene ≈ sabinene > α-pinene. In the OCM group, terpinen-4-ol was the main component, except (i) for one Korean EO sample [15], in which α-terpineol presented the highest content followed by terpinen-4-ol, and (ii) for the Japanese [28] and one Taiwan [33] EO samples, in which the main OCM was bornyl acetate followed by terpinen-4-ol. Relative to the SH group, δ-cadinene was the only SH that was found in all samples, presenting the highest or similar content compared with the other SH, except for Japanese [28] and Chinese [1] EO samples, which were dominated by thujopsene and β-elemene, respectively. In the OCS group, elemol presented the highest content, except (i) for two Korean EO samples, in which α-eudesmol [15] and γ-eudesmol [27] presented the highest content, and (ii) for the Chinese EO sample [28], which was dominated by α-elemol, which was reported for the first time in C. japonica EO composition [28]. In relation to the DH group, kaurene was dominant in the Corsica Island, Reunion Island, Japan, and Korea EO samples, while kaur-16-ene (ent-kaurene) was dominant in the China, Nepal, and Taiwan EO samples. The two DHs mentioned above are absent in our EO sample that, in contrast, was the only one that presented the DH phyllocladene. In fact, the alternative occurrence of (−) kaurene or (+) phyllocladene in the leaves of C. japonica was discussed in previous studies [51].

Similar to São Miguel Island (Azores, Portugal), C. japonica from Japan was introduced during the last century in other European countries, such as in Corsica [48] and Reunion [51] Islands (France). As can be observed in Table 4, the EOs from Corsica [48] and the São Miguel Islands were characterized by the association of α-pinene, sabinene, and elemol as major components, decreasing in the following order of abundance: α-pinene >> sabinene >> elemol in São Miguel, and sabinene ≈ α-pinene >> elemol in Corsica. On the other hand, the Corsica EO sample was richer in limonene and a good source of the DH kaurene, which was absent in our EO sample (Table 4), as mentioned above. The Reunion EO sample [51] was also characterized by the association of α-pinene, sabinene, β-elemol, and kaurene, with α-pinene being its major component. Compared with the European EO samples, the Japanese EO [28] is also a good source of MH fraction and kaurene, with its major component also being α-pinene. In addition, all of these samples are poor in γ-eudesmol. On the contrary, other studies (Table 4) demonstrated the occurrence of other chemotypes in C. japonica, such as the elemol chemotype from Taiwan [34], the α-elemol chemotype from China [1], the kaur-16-ene chemotype from Nepal [17] and Taiwan [33,36], and the kaurene chemotype from Korea [15,25,27].

Overall, this comparative study revealed the occurrence of the α-pinene chemotype in C. japonica leaf OE from São Miguel Island, Reunion Island [51], and Japan and that C. japonica had considerable qualitative and quantitative variations in its leaf EO composition. This variation can be due to the effect of genetics, plant age, vegetative cycle stage, region, environmental conditions, season, and the analytical methods used, among other factors, as already referenced in Section 3.1.2. The same finding has been reported in many studies, such as the one from Pavela et al. [52], which compared the chemical profiles identified in nine conifer EOs from Italy with those from different geographic origins. Thus, variations in the EO chemical profile of conifer species indicate the importance of optimizing protocols for the collection, processing, and extraction of plant material.

3.3. Previous Studies on the Bioactivity of the Main Components of Azorean C. japonica Leaf EO

As can be seen in Table 3, 37 components were identified in the Azorean C. japonica leaf EO, with 13 being major components: (i) MH α-pinene (34.5–46.4%), sabinene (11.6–20.2%), limonene (1.4–8.3%), β-myrcene (5.7–5.9%), p-cymene (0.3–4.4%), camphene (3.5–3.8%), α-thujene (1.6–2.8%), δ-3-carene (1.1–2.6%), and γ-terpinene (1.1–2.6%); (ii) OCM terpinen-4-ol (1.8–5.4%); (iii) OCS elemol (0.8–10.4%) and α-eudesmol (0.3–3.4%); and (iv) DH phyllocladene (0.2–2.9%). All of the terpene/terpenoid components referenced have already demonstrated valuable biological properties [13,53,54,55,56], as shown in

Table 5. Data from the literature on the bioactivity of the main components (≥3% of total content of identified EO components) of Azorean C. japonica leaf EO.

Component	Biological Activity	Ref.
α-Pinene	Flavoring and Fragrance; Antiseptic and Herbicidal Properties	[53]
	Analgesic, Anti-AChE, Antiapoptotic, Antibacterial, Anticancer, Anticoagulant, Anticonvulsant, Antidepressant, Antifungal, Anti-Inflammatory, Anti-Leishmania, Antimalarial, Antimetastatic, Antioxidant, Anxiolytic, Bronchodilating, Cytogenetic, Cytoprotective, Gastroprotective, Neuroprotective, Insecticidal, Larvicidal, and Nematicidal Properties; Cognitive Impairment and Insomnia Effects	[13,57,58,60]
	Substrate for Chemical Reactions	[61,62]
	Mosquito Repellency Effect; Anti-Quorum-Sensing Properties	[38,59]
Sabinene	Flavoring and Fragrance; Antiseptic and Anti-Quorum-Sensing Properties	[53,59]
	Anticancer, Anti-Inflammatory, and Antioxidant Properties	[31]
	Antibacterial Properties (e.g., against several oral bacteria and Helicobacter pylori)	[24,53]
	Antifungal, Anti-AChE, and Anti-BChE Properties; Skin Penetration Enhancer	[13]
Limonene	Flavoring and Fragrance; Antiseptic, Anti-AChE, Anti-Asthmatic, Antibacterial, Anticancer, Antidepressant, Antifungal, Anti-Inflammatory, Antimalarial, Antimutagenic, Antiproliferative, Antispasmodic, Antiviral, and Anxiolytic Properties; Detoxicant; Expectorant; Gastroprotective and Immunomodulatory Properties; Muscle Relaxant; Sedative	[53,54,55]
	Neuropharmacological Properties; Skin Penetration Enhancer	[13,23]
	Insecticidal Properties; Mosquito Repellency Effect	[38,52]
β-Myrcene	Fragrance; Analgesic, Anti-Inflammatory, and Antipsychotic Properties; Sedative; Muscle Relaxant	[54]
	Antibacterial, Anticancer, Antifungal, and Antimalarial Properties	[55]
	Anti-AChE and Antioxidant Properties	[13]
	Larvicidal against Aedes aegypti and Aedes albopictus	[36]
p-Cymene	Anticancer, Antifungal, and Antioxidant Properties	[13,55]
	Neuroprotective Properties; Skin Penetration Enhancer	[13]
	Larvicidal against A. aegypti and A. albopictus	[36]
Camphene	Anticancer, Antifungal, and Antioxidant Properties	[13,55]
	Anti-AChE and Cardioprotective Properties	[54]
	Larvicidal against Helicoverpa armigera (cotton bollworm)	[52]
α-Thujene	Antifungal Properties	[13]
δ-3-Carene	Antifungal Properties	[13]
	Larvicidal against A. aegypti and A. albopictus	[36]
	Mosquito Repellency Effect	[38]
γ-Terpinene	Analgesic, Anticancer, Anti-Inflammatory, and Antimicrobial Properties	[54]
	Anti-AChE, Anti-BChE, and Antioxidant Properties	[13]
	Larvicidal against A. aegypti and A. albopictus	[36]
	Mosquito Repellency Effect	[38]
Terpinen-4-ol	Antiseptic, Antiallergic, Anti-Asthmatic, Antibacterial (e.g., against several oral bacteria), Anti-Inflammatory, Antitussive, and Expectorant Properties	[24,53]
	Anticancer and Antiulcer Properties	[30,31]
	Antioxidant Properties; Relaxing Effects	[54]
	Antifungal Properties; Skin Penetration Enhancer	[13]
	Mosquito Repellency Effect	[38]
Elemol	Anticancer Properties	[54]
	Acaricidal against Tetranychus urticae and Tetranychus kanzawai	[63]
	Antitermite Properties	[33]
	Silverfish (Lepisma saccharina) Repellency Effect	[34]
α-Eudesmol	Antiangiogenic, Anticancer, Antifungal, and Antimicrobial Properties	[56]
Phyllocladene	Antimicrobial Properties	[11]

AChE—acetylcholinesterase; BChE—butyrylcholinesterase.

, with α-pinene being one of the most extensively studied [57,58,59,60]. It should be highlighted that α-pinene plus sabinene account for about 60% of the total content of identified EO components in all studied samples (Table 3). A recent study [59] revealed that Juniperus communis EO and its major components (α-pinene and sabinene) exhibit anti-quorum-sensing properties and are capable of repressing the spoilage phenotype of pseudomonads. Thus, this juniper OE can be used for the biopreservation of cold-stored fishery products [59]. Interestingly, the yields of α-pinene (48.3%) and sabinene (8.4%) in juniper EO–HD sample [59] are similar to those found in our EO–WSD sample (46.4% and 11.6%, respectively). Furthermore, in the latter sample, the yield of α-pinene accounts for about 50% of the total content of identified EO components (Table 3). This MH can be found in many higher plant species (e.g., conifers and Cannabis ssp.) and has been studied for decades. Nonetheless, the number of publication on α-pinene-related research is steadily increasing. α-Pinene has been used to treat respiratory tract infections for centuries and plays a crucial role in the fragrance and flavor industry [60]. Furthermore, it is a major component of aromatherapy EOs [58]. Moreover, due to their high natural abundance and their broad spectrum of valuable pharmacological and biocidal activities (Table 5), α-pinene is considered “a miracle gift of nature” [57] and a “molecule of interest” for future drug and agrochemical discovery [60] as well for further bioactivity studies (in vitro and in vivo). Concerning their pharmacological importance, Weston-Green et al. [58] have highlighted that α-pinene is a promising candidate for further investigation as a novel medicine for illnesses, including stroke, ischemia, inflammatory and neuropathic pain (including migraine), cognitive impairment (relevant to Alzheimer’s disease and aging), insomnia, anxiety, and depression. Additionally, α-pinene exhibited activity against methicillin-resistant Staphylococcus aureus (MRSA) ([60] and the references therein), which also highlights its value in one of the most significant health concerns worldwide: antibiotic resistance. Several studies related to biocidal activity revealed that α-pinene-rich EOs show a broad range of activity against different organisms ([60] and the references therein), such as Sitophilus zeamais, a key insect pest attacking stored maize, and pine wood nematode (Bursaphelenchus xylophilus). However, synergistic interactions between EO components have to be considered. For example, α-pinene exhibits a synergistic relationship with limonene (a known ingredient of commercial insecticidal) responsible for the high insecticidal efficacy of some conifer EOs [52]. In this context, it should be noted that the EO–WSD and EO–SD samples investigated are richer in α-pinene and limonene compared with the EO–HD sample.

Overall, the biological activities of the OE components mentioned (Table 5), specifically fragrance; flavor; skin permeation enhancement; and antiseptic, antimicrobial, antioxidant, anti-inflammatory, anticancer, antidepressant, neuroprotective, and pesticidal properties, could open new approaches to utilizing C. japonica leaf OE as raw materials (crude EO or its fractions) for potential commercial applications in the food (e.g., biopreservatives), phytomedicine (e.g., aromatherapy), pharmaceutical (e.g., natural antimicrobial products), cosmetic, cosmeceutical, perfumery, and household fields or in the agrochemical industry (as acaricidal, herbicidal, insecticidal, termicidal, or nematicidal products) with impacts in future integrated pest management (IPM) systems if formulated through emulsion or encapsulation.

4. Conclusions

The interest in EO from C. japonica wastes by the scientific community and EO markets is rapidly increasing. In fact, due to their chemical variability and high abundance, these EOs can find applications in several industries with many approaches and, therefore, are of great economic, environmental, and social importance.

Several studies show that the spatial and temporal changes in environmental factors and the genetic background of the plant can influence not only the yield but also the qualitative and quantitative variations in components of C. japonica leaf EO. On the contrary, few studies have reported the effect of different distillation methods on these parameters. Furthermore, it should be highlighted that, to the best of our knowledge, the HD and WSD methods have not previously been compared in C. japonica EO. Thus, our study presents important information in this issue.

Based on our results, it was clearly demonstrated that the different distillation methods used significantly affect the yield, color, and chemical composition of the Azorean C. japonica fresh leaf EO, as reported in similar studies with other aromatic plants. In fact, the EO–HD sample revealed a yield that was about three times higher than that of EO–WSD and higher than that with several C. japonica leaf EO–HD samples from other origins. Furthermore, the EO–HD sample showed a characteristic pale-yellow color, while the EO–WSD sample was colorless. Concerning the EO composition, the major components were MH α-pinene and sabinene, regardless of the distillation method used (HD, WSD, or SD), together accounting for about 60% of the total content of identified EO components. A similar chemotype pattern was found in the literature for C. japonica leaf EO–HD samples from Corsica Island, Reunion Island, and Japan. However, the presence and/or relative contents of other biologically important EO components varied significantly. The EO–HD sample was richer in elemol, isomers of eudesmol (mainly α-eudesmol), and phyllocladane; the EO–WSD sample was richer in terpinen-4-ol, p-cymene, and γ-terpinene; and the EO–SD sample was richer in limonene and δ-3-carene. Nonetheless, in general, a more similar pattern was observed between the EO–WSD and EO–SD compositions, except in the OCM group. Moreover, both samples, when compared with EO–HD, were richer in α-pinene and limonene. Furthermore, the EO–WSD and EO–SD samples contained more components than the EO–HD sample, in decreasing order as follows: EO–SD > EO–WSD > EO–HD.

Future research on the bioactivity of these chemically different EO samples is needed in order to improve the industrial applications of Azorean C. japonica leaf EO by creating new high value-added products, thus contributing to the local circular economy.