Variations in Essential Oil Chemical Composition and Biological Activities of Cryptomeria japonica (Thunb. ex L.f.) D. Don from Different Geographical Origins—A Critical Review

: The scientiﬁc community is paying increasing attention to plant waste valorization, and also to “greener” practices in the agriculture, food and cosmetic sectors. In this context, unused forest biomass (e.g., leaves, seed cones, branches/twigs, bark and sapwood) of Cryptomeria japonica , a commercially important tree throughout Asia and the Azores Archipelago (Portugal), is currently waste/by-products of wood processing that can be converted into eco-friendly and high added-value products, such as essential oils (EOs), with social, environmental and economic impacts. Plant-derived EOs are complex mixtures of metabolites, mostly terpenes and terpenoids, with valuable bioactivities (e.g., antioxidant, anti-inﬂammatory, anticancer, neuroprotective, antidepressant, antimicrobial, antiviral and pesticide), which can ﬁnd applications in several industries, such as pharmaceutical, medical, aromatherapy, food, cosmetic, perfumery, household and agrochemical (e.g., biopesticides), with manifold approaches. The EOs components are also of value for taxonomic investigations. It is known that the variation in EOs chemical composition and, consequently, in their biological activities and commercial use, is due to different exogenous and endogenous factors that can lead to ecotypes or chemotypes in the same plant species. The present paper aims to provide an overview of the chemical composition, biological properties and proposals of valorization of C. japonica EO from several countries, and also to indicate gaps in the current knowledge.


Introduction
Numerous plant-derived essential oils (EOs), due to their valuable odoriferous and bioactivity properties, and GRAS (Generally Recognized as Safe) status, have applications in many fields, such as aromatherapy, cosmetic, cosmeceutical, food, beverage, household, pharmaceutical, phytomedicine and pest control. However, the bioactivity and potential commercial use of EOs depends on their complex mixture of organic compounds, produced through the secondary metabolic pathways of aromatic plants [1][2][3][4], such as the conifer Cupressaceae family [4]. its chemotypes, main reported biological activities and consequent industrial/commercial applications.

Data Analysis
The principal component analysis (PCA) and hierarchical cluster analysis (HCA) were performed by using SPSS version 27.0 software (SPSS Inc., Chicago, IL, USA) to show the relationship among the C. japonica EO from different regions based on their percentage chemical composition. The PCA was achieved selecting the three highest principal components (PCs) obtained by the linear regressions operated on scaled data. For the statistical evaluation of the complete EO composition, the covariance data was a 19 × 14 matrix (19 EO samples × 14 chemical compounds = 266 data). The first three PCs (PC1, PC2 and PC3) explained 46.8%, 14.7% and 10.0% of the total variance, respectively, allowing the visualization of more than 70% of the information contained within the dataset. HCA was performed using Ward's method and squared Euclidean distance.

Yield and Chemical Composition of C. japonica EO
Plant-derived EOs are generally complex mixtures of low-molecular-weight metabolites, particularly monoterpenes (C10) and sesquiterpene (C15) hydrocarbons, and their oxygenated derivatives, usually extracted from non-woody parts of the plant, such as foliage. However, to a lesser extent, it could also be extracted from other plant tissues (e.g., wood, seeds and bark). Hydrodistillation and steam distillation are the traditional extraction methods used to obtain EO from plants. Another less-used technique is the classic extraction with organic solvents [23], which is a non-eco-friendly process. In order to improve conventional extraction methods, other innovative and green techniques have been developed, such as supercritical fluid extraction [24][25][26].
Leaves of C. japonica are by far the plant organ most used to obtain EO. C. japonica leaf EO, typically obtained by hydrodistillation and chemotyped by gas chromatography and mass spectrometry analysis, is pale yellow in color and has a fresh aromatic odor [27][28][29]. Terpenes and terpenoids are the major components found in C. japonica leaf EO, as illustrated in Figure 1.
limitations, such as inconsistency in EO chemical profile between different regions, volatility, lipophilicity and effectivity of the EO under field conditions, must be reviewed. Therefore, the present review aims to update the current knowledge on the C. japonica EO, its chemotypes, main reported biological activities and consequent industrial/commercial applications.

Data Analysis
The principal component analysis (PCA) and hierarchical cluster analysis (HCA) were performed by using SPSS version 27.0 software (SPSS Inc., Chicago, IL, USA) to show the relationship among the C. japonica EO from different regions based on their percentage chemical composition. The PCA was achieved selecting the three highest principal components (PCs) obtained by the linear regressions operated on scaled data. For the statistical evaluation of the complete EO composition, the covariance data was a 19 × 14 matrix (19 EO samples x 14 chemical compounds = 266 data). The first three PCs (PC1, PC2 and PC3) explained 46.8%, 14.7% and 10.0% of the total variance, respectively, allowing the visualization of more than 70% of the information contained within the dataset. HCA was performed using Ward's method and squared Euclidean distance.

Yield and Chemical Composition of C. japonica EO
Plant-derived EOs are generally complex mixtures of low-molecular-weight metabolites, particularly monoterpenes (C10) and sesquiterpene (C15) hydrocarbons, and their oxygenated derivatives, usually extracted from non-woody parts of the plant, such as foliage. However, to a lesser extent, it could also be extracted from other plant tissues (e.g., wood, seeds and bark). Hydrodistillation and steam distillation are the traditional extraction methods used to obtain EO from plants. Another less-used technique is the classic extraction with organic solvents [23], which is a non-eco-friendly process. In order to improve conventional extraction methods, other innovative and green techniques have been developed, such as supercritical fluid extraction [24][25][26].
Alternatively, EO of C. japonica could be classified by the diterpene chemotype of the leaves, which contained ent-kaurene, phyllocladene or ent-sclarene as a main diterpene hydrocarbon or mixtures of these compounds [42]. It is believed that these diterpenes chemotypes are mainly genetically controlled [27,33,42]. Moreover, Yamashita et al. [31] had reported that the diterpene chemotype of the leaves has an impact on the biological activity of the EO, i.e., the ent-kaurene chemotype exhibited higher acaricidal activity than the other diterpene hydrocarbon chemotypes (phyllocladene or ent-sclarene), where the difference between ent-kaurene and phyllocladene was a diastereomer with the same planar structure. This study [31] highlights the importance of the stereostructure/stereochemistry of the compounds on their biological activities. In addition, Suzuki et al. [43] have achieved the same conclusions, regarding sesquiterpenoids from C. japonica extracts.  Alternatively, EO of C. japonica could be classified by the diterpene chemotype of the leaves, which contained ent-kaurene, phyllocladene or ent-sclarene as a main diterpene hydrocarbon or mixtures of these compounds [42]. It is believed that these diterpenes chemotypes are mainly genetically controlled [27,33,42]. Moreover, Yamashita et al. [31] had reported that the diterpene chemotype of the leaves has an impact on the biological activity of the EO, i.e., the ent-kaurene chemotype exhibited higher acaricidal activity than the other diterpene hydrocarbon chemotypes (phyllocladene or ent-sclarene), where the difference between ent-kaurene and phyllocladene was a diastereomer with the same planar structure. This study [31] highlights the importance of the stereostructure/stereochemistry of the compounds on their biological activities. In addition, Suzuki et al. [43] have achieved the same conclusions, regarding sesquiterpenoids from C. japonica extracts.
Ent-kaurene is a diterpene that, apart from its function in plant defense, is the precursor of gibberellins, which represent an important group of plant hormones involved in various physiological plant processes [44]. Although this diterpene is generally the major component of C. japonica leaf EO in East Asia, the oxygenated sesquiterpenes made up the higher contribution in this EO [12,45]. However, in α-pinene chemotype, the major EO fraction is normally composed by monoterpene hydrocarbons [18].
In Azores Archipelago, Moitero et al. [32] reported that the C. japonica leaf EO chemotype is α-pinene type, and regarding diterpene hydrocarbon is an ent-kaurene-phyllocladene type. These authors found that ent-kaurene compound is higher in leaf EO of red heartwood-type than on black-type C. japonica populations [32]. Nevertheless, in our PCA (Figure 2), α-pinene chemotype was positively associated with phyllocladene but negatively correlated with ent-kaurene. Ent-kaurene is a diterpene that, apart from its function in plant defense, is the precursor of gibberellins, which represent an important group of plant hormones involved in various physiological plant processes [44]. Although this diterpene is generally the major component of C. japonica leaf EO in East Asia, the oxygenated sesquiterpenes made up the higher contribution in this EO [12,45]. However, in α-pinene chemotype, the major EO fraction is normally composed by monoterpene hydrocarbons [18].
In Azores Archipelago, Moitero et al. [32] reported that the C. japonica leaf EO chemotype is α-pinene type, and regarding diterpene hydrocarbon is an ent-kaurenephyllocladene type. These authors found that ent-kaurene compound is higher in leaf EO of red heartwood-type than on black-type C. japonica populations [32]. Nevertheless, in our PCA (Figure 2), α-pinene chemotype was positively associated with phyllocladene but negatively correlated with ent-kaurene.
In addition, in China, Xie et al. [46] reported that elemol-rich leaf EO (elemol plus ent-kaurene chemotype) is usually poor in phyllocladene content. Moreover, this last chemotype is associated with high biological activities (see Sections 4 and 5).
Concerning the EO yield from C. japonica, leaves cultivated in different regions varied from 0.5% to 4.7% (w/w) of dry weight, with the highest value found in South Korea ( Table 1). The yield of different parts of C. japonica in decreasing order is leaf > bark > heartwood > sapwood [10]. However, Garcia et al. [33] have reported that the EO yield from C. japonica cones growing in Corsica (France) is 2.7 fold higher than that of leaf EO. Moreover, these authors reported that the cone chemical profile is similar to that of the leaf EO. Further studies should explore this plant organ.
Oppositely to cones, the EO of other tissues of C. japonica (bark, heartwood and sapwood) exhibits a dissimilar chemical profile as compared to that of the leaf EO. Briefly, the major fraction in bark EO is usually composed by monoterpenes and monoterpenoids, where ferruginol is a main component [10,12,22,40]. In heartwood EO, the major fraction is composed by sesquiterpene hydrocarbons, mainly cadinene isomers [10,12,40]. Lastly, sapwood EO contains sesquiterpenes and sesquiterpenoids as main fractions, where ferruginol is also a major compound [12,40].
Overall, based on the data described above, a large chemical variability is observed among C. japonica EO. Such variation can be attributed to several factors, including exogenous (such as light, precipitation, growing region, nature of the soil and season) or/and endogenous factors (e.g., plant age, plant organ, developmental stage and genotype). Also, the EO yield can be influenced by these same parameters [27,[46][47][48] along with the extraction method used. However, there are conflicting reports in the literature about the influence of environmental conditions on C. japonica EO chemotypes, with some studies reporting no effects [33] and others [27] indicating that environmental factors strongly influenced the EO chemical profile. Apart from these studies, it is reasonable to envisage that chemotype variation (within the same plant species) appearing as a plant adaptive process to the local ecologic conditions.

Antimicrobial Activity of C. japonica EO in Food Industry and Human Diseases
EOs are generally accepted as natural antimicrobials and antioxidants that can be used in the food industry as bio-preservatives to increase shelf life and quality of food products [49]. In addition, infections caused by fungi and bacteria represent a key issue due to the development of resistant species to current fungicides and antibiotics. Therefore, EOs could be an ecological and effective alternative to synthetic antimicrobial agents [49]. Table 3 shows the antimicrobial activity of C. japonica EO from different tissues and geographical origins, against several gram-positive and gram-negative bacterial strains, as well as against various fungal species. The minimal inhibitory concentrations (MICs) were determined by the broth dilution method.
Cha et al. [37] reported that EO from leaves of South Korean C. japonica has excellent antimicrobial activity against several oral bacteria, except Escherichia coli, with MICs ranging from 0.025 to 12.8 mg/mL (Table 3). According to the authors, this activity is mainly due to the presence of α-pinene, sabinene, α-terpineol and 4-terpineol compounds. In fact, the major chemical group of the studied EO is the monoterpene hydrocarbons (highly lipophilic), which possibly possess cytotoxicity by the disruption of bacteria membrane integrity. On the other hand, Lee et al. [50] showed that EO from needles and twigs of South Korean C. japonica had weak antibacterial activity, namely against gram-negative bacteria, with MICs greater than 10 mg/mL (Table 3). Similar findings have already been reported for heartwood components of C. japonica [51]. The observed discrepancy between the two referred studies can be attributed to the influence of the plant organ on the EO chemical composition. However, it is known that gram-negative bacteria have an outer membrane, rich in lipopolysaccharides, which acts as a permeability barrier against hydrophobic molecules, hence it is expected that EOs are less effective against gram-negative than gram-positive bacteria. Nevertheless, it is worth noting that EOs could also be cytotoxic to eukaryotic cell membranes [52]. Therefore, further studies are needed with respect to both target and non-target organisms.
Oppositely, in the Lee et al. [50] study, C. japonica EO (which is ent-kaurene type) exhibited antifungal activity against fungal strains that cause foot rot and other human diseases, namely against Cryptococcus neoformans (Table 3). Contrary to the leaves, twigs or heartwood are rich in sesquiterpene hydrocarbons, namely δ-cadinene, which is associated with superior antifungal activities [10,53]. In fact, Cheng et al. [10] showed that the heartwood EO of C. japonica from Taiwan, which is rich in sesquiterpene hydrocarbons, has the strongest antifungal activity against wood decay and tree pathogenic fungi, followed by leaf EO. This activity was associated mainly with δ-cadinene, the major compound in the EO. Therefore, EO from C. japonica could also have applications in timber industry as natural wood preservatives. Moreover, in Takao et al. [53] study, the EO from the heartwood of Japanese C. japonica inhibited the growth of Trichophyton rubrum, while no antibacterial activity was observed with respect to Staphylococcus epidermidis (Table 3), possibly due to the lack of monoterpene hydrocarbons, as the authors stated. Contrary to heartwood EO, leaf EO of C. japonica, which is rich in monoterpene hydrocarbons, is highly active against this gram-positive strain [37,38] (Table 3). Furthermore, diterpenoids [54] and diterpenes in EO from C. japonica leaves can also affect microbes that cause skin [38] and human diseases, such as tuberculosis [32]. However, these last authors were more restrictive as considering EOs with MIC > 0.250 mg/mL as inactive (Table 3). Yet, C. japonica EO had shown weak antibacterial activity against Legionella pneumophila, with a minimal bactericidal concentration (MBC) value higher than 2 mg/mL [55]. Overall, components from C. japonica EO have strong antimicrobial effects that can inhibit food decay-related microbial growth and can be promising antibiotic therapeutic agents. Antimicrobial compounds are usually highly correlated with antioxidant effects [56]. In fact, besides microbial contamination, lipid peroxidation is a real problem related to food deterioration and the addition of antioxidants is an attractive strategy to retard or even stop oxidation processes. C. japonica extracts from different tissues are rich in phenolics, which are well-known natural antioxidants and antibacterial agents [43,57]. Contrary to C. japonica extracts, EO from various tissues of this plant exhibited weak antioxidant activities (in 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assays), presenting the sapwood EO with the best value (IC 50 = 113 µg/mL), followed by twigs, heartwood, bark and lastly leaf EOs [12]. The effect of sapwood EO on the free radical scavenging assay is attributed to its hydrogen-donating ability, possibly due to the high content of ferruginol (an oxygenated diterpene). Thus, sapwood EO or ferruginol can also be used as natural food preservatives in food industry. In fact, it has already been reported that diterpenes show higher antioxidant and antimicrobial effects than monoterpenes [39].
As discussed earlier, it is noteworthy that enrichment of C. japonica EO fractions with specific compounds can be a useful strategy to pest/microbes management, where deterpenation with vacuum fractional distillation can be effective. Kusumoto and Shibutani [18] had submitted EO from C. japonica leaves to an open system mild heat treatment, which decreased the content of monoterpene hydrocarbons and increased the content of oxygenated sesquiterpenes and diterpenes. They found that the evaporation residue had higher antifungal activity than the crude EO. Also, Salha et al. [20] reported the same findings for Origanum majorana EO. Further studies should explore this issue.

Acaricidal and Insecticidal Activities of C. japonica EO
The EOs play a functional role in the plant chemical defense against phytopathogens and pests [2,15]. For that reason, they are a potentially good source of environmentally friendly pesticides or pest-control agents. Table 4 shows the insecticidal and acaricidal effects of C. japonica EO from different tissues.
Spider mites, such as Tetranychus urticae and Tetranychus kanzawai, are known as world pests of agricultural crops. However, since spider mites easily build up a tolerance to pesticides, EOs have been studied as alternative eco-pesticides. Yamashita et al. [31] found that EO from the leaves of C. japonica is a fruitful option to control these mites (Table 4). They mainly attributed this acaricidal activity to the ent-kaurene compound, followed by elemol.
Silverfish (Lepisma saccharina), another pest common in libraries and museums where paper books and labels are abundant, are insects that owe their survival to their secretive life in damp, cool places. As observed in a Wang et al. [34] study, this pest is also sensitive to C. japonica leaf EO, which highly repelled silverfish (>80% of repellency at 0.01 mg/cm 3 after 4 h) and killed it on contact assay (LD 50 value of 0.087 mg/cm 3 after 10 h) ( Table 4). These authors assigned these toxic effects to volatiles (monoterpenes) and non-volatiles compounds (such as ent-kaurene and elemol), respectively. Biodegradation of wood caused by termites is one of the most serious problems for wood utilization, and extractives from wood tissues can provide natural protection against harmful pests. Therefore, and as shown in Table 4, EO from C. japonica (namely from leaf and heartwood) can be a natural termicide. In particular, elemol plus ent-kaurene chemotype is associated with high antitermitic activity (by contact assay), which in the Cheng et al. [36] study was correlated with elemol and α-terpineol compounds.
In addition to pest control, EOs have been used since ancient times to repel insects, especially insect vectors of human diseases, such as yellow fever, dengue and malaria. Investigations on the insecticidal and repellent effects of C. japonica EO (elemol plus entkaurene chemotype) on Aedes aegypti and A. albopictus larvae and adults were carried out by Cheng's research team [21,35,58]. When compared to other plant tissues, leaf EO showed superior larvicidal [58] and repellent activities against those mosquitoes (Table 4), and when the authors [35] assessed individual EO components, they found that 4-terpineol had the best repellent activity. Other monoterpenes, such as 3-carene, were also important in larvicidal effects [21]. Apart from bark, leaves are the main plant tissue that have high concentrations of monoterpenoids (volatile compounds), which are usually associated with the best insect repellency [34,35].
It is interesting to note that EO from C. japonica leaves is more effective in larvicidal and repellent activities against A. aegypti and A. albopictus than their methanolic extracts [21,45]. However, methanolic extracts from other C. japonica tissues (such as sapwood) have also been stated as excellent mosquito larvicidal agents [45,59].
A further study [17] reported the larvicidal effects of C. japonica leaf EO on Anopheles gambiae (the main malaria vector), and revealed promising results in the laboratory (Table 4) and semi-field conditions.

Other Biocidal Activities of C. japonica EO
Although studies regarding elemol plus ent-kaurene chemotype or ent-kaurene chemotype are the most reported in the literature, EO from leaves of C. japonica from Azores (α-pinene chemotype) has recently been reported to have high molluscicidal activity against Radix peregra (Lymnaeidae), a freshwater snail that hosts a number of significant parasites, such as Fasciola hepatica [61]. This parasite is the causing agent of fascioliasis, a well-known veterinary problem of vertebrate domestic livestock, which causes animal production losses and consequent economic costs [62]. The lethal concentrations (LC 50 ) of the EO varied between 33.3 and 61.8 ppm on an aqueous suspension assay [61]. Although the author has not assessed individual EO components for molluscicidal activity, it is worth pointing out that this EO has α-pinene as the major component. Moreover, this same EO chemotype exhibited inhibitory activities against Pseudaletia unipuncta (Lepidoptera: Noctuidae), an important pest of agricultural crops. Particularly, this leaf EO inhibited eggs hatching and thereafter exhibited lethal and sub-lethal effects. Additionally, it showed high repellency properties against adults [63].
More recently, some other C. japonica bioactivities have been described. For instance, Tanaka et al. [64] reported that chemical components of C. japonica leaves suppress the growth of invasive plants (Robinia pseudoacacia) and weeds (Trifolium repens), which are the largest competitor of agricultural crops, with negative impact on productivity.
In addition, there are some studies on the growth inhibition activities of C. japonica chemical components (mainly from bark) against harmful marine [22] and freshwater [43] algae. The strong growth-inhibitory activity of the C. japonica bark EO against the bacillariophyceae Skeletonema costatum (commonly known as red tide plankton) was correlated with the presence of ferruginol [22], which has a strong antioxidant activity, as already mentioned. The results of this study [22] indicate the potential of a new use for components of C. japonica bark EO to control red tide plankton growth, a serious environmental problem in the world's oceans, which can have an adverse impact on aquaculture. Further studies should explore this item, as the C. japonica bark is a promising waste that is available in large quantities.

Pharmacological Properties of C. japonica EO
Aromatic plants, such as C. japonica, synthesized and emitted complex mixtures of volatile organic compounds (VOCs) in order to facilitate their growth and survival [13,65].
The VOCs from C. japonica have already been reported to provide relaxing and stressrelieving effects on mice [66] and on humans [67]. In this way, C. japonica EO could be a useful tool in mental health management.
In addition, the most abundant VOCs emitted from C. japonica wood during the drying process, in the wood industry, are sesquiterpenes, such as δ-cadinene, α-muurolene and β-cadinene. These VOCs were assessed as having soothing effects in a human sensory evaluation [68]. Moreover, the main constituent of C. japonica heartwood EO is δ-cadinene [10,13]. It is believed that this compound and other VOCs of C. japonica suppress the sympathetic nervous system activity in humans, especially in women, stimulating relaxing and pleasurable emotions [69]. Furthermore, VOCs from C. japonica leaves display antitussive effects in guinea pigs [70] and increases the output of fluid in the respiratory tract [71]. However, further studies regarding the long-term effects of the inhalation of these volatiles are required.
On the other hand, different tissues of C. japonica also have bioactive compounds with benefits to the skin, which can have several skin health applications (inhibiting melanin production, skin ageing, antiseptic, etc.) [39,72,73]. In particular, phytocompounds from this heartwood have been reported to possess antifungal properties in vitro against Trichophyton rubrum, a major cause of Tinea pedis [53], as already seen in Section 4. Moreover, EO from C. japonica leaves also showed excellent anti-inflammatory and antibacterial activities in vitro, being an attractive acne-mitigating candidate [38]. Another recent study [54] also stated that phytochemicals compounds from C. japonica by-products, namely diterpenoids, can be useful in skin health.
In addition, the EO from C. japonica leaves, namely its diterpenes, is a strong inhibitor of acetylcholinesterase activity, which could be an effective therapeutic agent for Alzheimer's disease [74]. Alternatively, this EO can be explored in the preventive dentistry field, as an effective inhibitor of oral bacteria [37,75], or as an anticancer chemopreventive [76] and antiulcer [77] agent. The results of this last study, suggested that 4-terpineol could have gastroprotective activity.
Overall and according to the literature [76], various parts of C. japonica have been used in Asian folk medicine for a variety of indications, including liver ailments, and an antitussive, and for its antiulcer activities. Hence, more studies are warranted with respect to these health benefits, namely, the toxic effects.

Conclusions
In the last two decades, several investigations have been conducted on components of C. japonica EO, mainly on Asian chemotypes. However, all C. japonica EO chemotypes exhibited antimicrobial properties and hence can have applications as food preservatives and/or as antibiotic alternatives. Moreover, this EO can have applications in agrochemical (as repellent, larvidical, insecticidal, acaricidal or termicidal) and cosmeceutical industries (as skin whitening agents or oral bacteria inhibitors). Nevertheless, future research should establish EO safe concentration prior any of these biological applications. Additionally, other C. japonica by-products (such as seed cones, bark and sawdust), as well as the enrichment of C. japonica EO fractions with specific compounds, should be exploited.
Thus, the interest in the C. japonica EO by the scientific community and the EOs market, quickly increases.