Abstract
Hydrodistillation (HD), organic solvent extraction (OSE), and supercritical fluid extraction (SFE) were compared in terms of the chemical composition and antifungal activity of leaf essential oils from Chamaecyparis formosensis and C. obtusa var. formosana. Gas chromatography–mass spectrometry revealed notable differences among extraction methods. In C. formosensis, HD-derived oil was dominated by α-pinene (83.4%), SFE-derived oil by kaur-16-ene (51.1%), and OSE-derived oil by phytol (44.4%). In C. obtusa var. formosana, HD oil was rich in sabinene (36.2%) and thujopsene (22.5%), SFE oil in totarol (50.9%), and OSE oil in thujopsene (27.6%) and cedrol (24.8%). Bioassays demonstrated that OSE oil of C. formosensis exhibited the strongest inhibitory effects against Trichoderma sp., Trametes versicolor, Laetiporus sulphureus, and Gloeophyllum trabeum. For C. obtusa var. formosana, HD oil was most effective against Trichoderma sp. and L. sulphureus, whereas SFE oil was most active against G. trabeum. These results highlight the strong influence of the extraction method on both chemical composition and antifungal efficacy of leaf essential oils.
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Comparative Evaluation of Hydrodistillation, Supercritical Fluid Extraction, and Organic Solvent Extraction on Leaf Essential Oils of Chamaecyparis formosensis and C. obtusa var. formosana and Their Potential as Wood-Protective Agents
Ying-Ju Chen 
,a Fu-Lan Hsu ,b and Sen-Sung Cheng c,*
Hydrodistillation (HD), organic solvent extraction (OSE), and supercritical fluid extraction (SFE) were compared in terms of the chemical composition and antifungal activity of leaf essential oils from Chamaecyparis formosensis and C. obtusa var. formosana. Gas chromatography–mass spectrometry revealed notable differences among extraction methods. In C. formosensis, HD-derived oil was dominated by α-pinene (83.4%), SFE-derived oil by kaur-16-ene (51.1%), and OSE-derived oil by phytol (44.4%). In C. obtusa var. formosana, HD oil was rich in sabinene (36.2%) and thujopsene (22.5%), SFE oil in totarol (50.9%), and OSE oil in thujopsene (27.6%) and cedrol (24.8%). Bioassays demonstrated that OSE oil of C. formosensis exhibited the strongest inhibitory effects against Trichoderma sp., Trametes versicolor, Laetiporus sulphureus, and Gloeophyllum trabeum. For C. obtusa var. formosana, HD oil was most effective against Trichoderma sp. and L. sulphureus, whereas SFE oil was most active against G. trabeum. These results highlight the strong influence of the extraction method on both chemical composition and antifungal efficacy of leaf essential oils.
DOI: 10.15376/biores.20.4.9332-9347
Keywords: Chamaecyparis formosensis; Chamaecyparis obtusa var. formosana; Hydrodistillation; Organic solvent extraction; Supercritical fluid extraction; Antifungal activity
Contact information: a: Forest Products Utilization Division, Taiwan Forestry Research Institute, Ministry of Agriculture, Taipei 100051, Taiwan; b: Taimalee Research Center, Taiwan Forestry Research Institute, Ministry of Agriculture, Taitung 963001, Taiwan; c: The Experimental Forest, College of Bio-Resources and Agriculture, National Taiwan University, Nantou 55750, Taiwan;
* Corresponding author: sscheng@ntu.edu.tw
INTRODUCTION
Chamaecyparis formosensis Matsum. and Chamaecyparis obtusa var. formosana (Hayata) are emblematic cypresses of Taiwan’s montane forests. They are prized for both their durable timber and culturally valued aroma. The chemical composition of their essential oils was first delineated in 1931 (Kafuku and Ichikawa 1931; Kafuku et al. 1931). Later studies showed that hydrodistilled leaf oils of C. formosensis are dominated by α-pinene, while reports on C. obtusa var. formosana have yielded differing results. Su et al. (2006) found α-pinene (76.7%) to be the predominant constituent, whereas Chen et al. (2011), based on samples collected across multiple regions in Taiwan, demonstrated that the leaf oils of this taxon could be classified into three distinct chemotypes—β-elemol type, (–)-thujopsene type, and cis-thujopsenal type—highlighting pronounced geographic variation. Leaf essential oils of C. obtusa var. formosana have demonstrated strong antitermitic potential (Cheng et al. 2007). Nevertheless, compared with leaf essential oils, wood-derived oils and their constituents have been investigated more extensively, with numerous studies confirming their antimicrobial, antifungal, and antitermitic activities (Wang et al. 1987; Wu and Wang 1990; Wang et al. 2005; Chen and Chang 2017). Although previous analyses reported that leaf essential oils are rich in monoterpenes and sesquiterpenes with pesticidal properties (Su et al. 2006; Chen et al. 2011), systematic evaluations of the influence of different extraction methods—such as hydrodistillation (HD), organic solvent extraction (OSE), and supercritical fluid extraction (SFE)—on their chemical composition and bioactivity remain limited (Zhou et al. 2023; Karimnejad and Ghavam 2024). Moreover, valorizing leaf biomass, typically regarded as a low-value forestry byproduct, for essential oil production represents a more environmentally sustainable strategy, supporting circular bioeconomy objectives and alleviating pressure on slow-growing, economically valuable heartwood resources. As recent research has primarily focused on wood-derived oils or isolated compounds (Hsu et al. 2016; Lin et al. 2022; Chen et al. 2023), the biological potential and practical applications of leaf essential oils, as well as the effects of extraction techniques, have yet to be fully elucidated.
Extraction technology plays a decisive role in shaping essential oil profiles and downstream efficacy. Conventional HD is simple and inexpensive, but it may degrade heat-labile constituents and often delivers limited yields (Zhou et al. 2023; Wang et al. 2025). OSE can increase recovery of high molecular weight or less volatile compounds. Hexane is among the most commonly used solvents for OSE because its strong hydrophobicity matches the lipophilic nature of most essential oil constituents, and its relatively low boiling point (63 to 69 °C) facilitates efficient solvent removal after extraction (Ghahramanloo et al. 2017; Bourgou et al. 2021). Nevertheless, residual solvents and energy demands raise safety and environmental concerns (Ghahramanloo et al. 2017; Park et al. 2022; Wang et al. 2025). SFE offers tunable selectivity and solvent-free products but requires costly high-pressure equipment (Ha et al. 2008; Donelian et al. 2009; Huo et al. 2024). Comparative studies on other taxa (e.g., Aquilaria spp., Mentha longifolia; Satureja bachtiarica Bunge.) demonstrate that switching extraction methods can shift major constituents, thereby modulating antioxidant, antibacterial, and antifungal activities (Memarzadeh et al. 2015; Yousefi et al. 2019; Karimnejad et al. 2024; Wang et al. 2025). However, no systematic evaluation of HD, OSE, and SFE has been reported for Taiwanese cypress leaves, leaving a critical knowledge gap for both science and industry.
Essential oils from conifers, including Taiwanese cypresses, have been reported to exhibit antifungal activity against several wood-decaying fungi such as Trametes versicolor, Laetiporus sulphureus, and Gloeophyllum trabeum (Wang et al. 2005; Chen and Chang 2017; Chen et al. 2023). In particular, essential oils from C. formosensis and C. obtusa var. formosana have shown good inhibitory effects against a range of decay fungi in both liquid and vapor phases, underscoring their potential as natural antifungal agents. However, the extent to which extraction methods influence their antifungal performance against wood-decaying fungi has not yet been systematically investigated.
In this work it was hypothesized that extraction technique would significantly influence (i) the yield and chemical composition of C. formosensis and C. obtusa var. formosana leaf essential oils and (ii) their antifungal performance against wood decay fungi. Accordingly, this study employed HD, OSE, and SFE under optimized, reproducible conditions to obtain oils, which were then profiled by GC–MS and evaluated for antifungal activity. By identifying the method that maximizes bioactive constituents while minimizing environmental burdens, the aim was to provide a sustainable pathway for converting forestry residues into natural wood protective agents, thereby adding economic value to leaf litter and advancing sustainable forest management.
EXPERIMENTAL
Materials
Plant materials
Leaves of C. formosensis and C. obtusa var. formosana were collected from the Experimental Forest of National Taiwan University during the winter season. Specifically, leaves of C. formosensis were collected from Compartment 29 of Plantation No. 79-2 in the Duigaoyue Tract (longitude: 120°51′10.19″, latitude: 23°29′12.67″) at an elevation of 2,457 m. Similarly, leaves of C. obtusa var. formosana were collected from Compartment 21 in Plantation No. 62-1A in the Neimaopu Tract (longitude: 120°48′14.35″, latitude: 23°38′43.51″) at an elevation of 1,908 m. Once on-site collection was complete, the samples were immediately transported to the laboratory at a low temperature. The sample trees were identified by Mr. Yen-Ray Hsui (Taiwan Forestry Research Institute, TFRI). Voucher specimens were deposited at the department of Forest Products Utilization, TFRI, Taiwan. After the branches were separated from the leaves, the leaf samples were subjected to essential oil extraction procedures with HD, SFE, and OSE.
Chemicals and reagents
Ethanol (99.5%, analytical grade) and anhydrous sodium sulfate (Na₂SO₄, ≥99%) were purchased from Sigma-Aldrich (USA). Positive control reagents included didecyldimethylammonium chloride (≥98%, Sigma-Aldrich) and triadimefon (a commercially available fungicide, Bayer CropScience). Potato dextrose agar (PDA) medium was obtained from Difco Laboratories (USA). All reagents were of analytical grade and used without further purification.
Essential Oil Extraction
Hydrodistillation (HD)
Fresh leaves (approximately 1 kg per species) were transported to the TFRI and subjected to hydrodistillation using a Clevenger-type apparatus. The leaves were placed in a large round-bottom flask containing 3 L of distilled water and heated for 6 to 8 h to obtain the essential oil fraction separated from the recondensed water. Residual moisture was removed by treatment with anhydrous sodium sulfate, and the oils were subsequently stored in airtight vials until further analysis. Essential oil yields were determined gravimetrically, and all reported values represent the mean of triplicate extractions.
Supercritical fluid extraction (SFE)
Essential oils were extracted using SFE with CO₂ as the supercritical solvent in a Speed SFE system equipped with a 50 mL extraction vessel (Applied Separations, Allentown, PA, USA). Briefly, approximately 7 g of leaves was loaded into the vessel, and the vessel was packed with propylene wool. After preliminary tests were conducted, the optimal extraction conditions were determined to be a temperature of 40° C, a pressure of 150 bar, and a flow rate of approximately 4 to 6 L/min. Following a 20 min static extraction phase, a 30 min dynamic extraction phase was performed, and this cycle was repeated twice. Finally, the supercritical fluid extract was collected, and the oil yield was calculated.
Organic solvent extraction (OSE)
Essential oils were extracted using OSE following a method outlined by Ahmadian et al. (2018) with some modifications. Briefly, 300 g of fresh leaves was soaked in 1 L of n-hexane (high-performance liquid chromatography grade) and agitated at 150 rpm for 24 h. Subsequently, the extract was filtered, and n-hexane was removed using a rotary evaporator at 40 °C, yielding concrete. To eliminate pigment and wax, the concrete was redissolved in 60 mL of absolute ethanol and stirred for 2 h in a water bath at 50 °C. Next, the solution was cooled to −10 °C, and the waxes were completely removed by filtration. Finally, the mixture was concentrated using a rotary evaporator, and the extraction yield was calculated on the basis of the initial dry weight. All extraction procedures were performed in triplicate.
Essential Oil Composition Analysis
The chemical compositions of the essential oils were analyzed using a PerkinElmer Clarus 600 gas chromatography mass spectrometry system (PerkinElmer Instruments, Waltham, MA, USA). Briefly, a 1-μL aliquot of diluted essential oil was injected into the system. Subsequently, separation was performed using a DB-5ms column (Crossbond 5% phenyl methylpolysiloxane, 30 m × 0.25 mm × 0.25 μm), with helium used as the carrier gas at a flow rate of 1 mL/min. Next, the injection port temperature was set to 250 °C, with an ionization voltage of 70 eV, and the mass range was scanned from m/z 40 to 450 amu. The oven temperature was programmed to be isothermal at 60 °C for 5 min, raised to 150 °C at the rate of 5 °C/min, raised to 180 °C at the rate of 2 °C/min, then to 250 °C at a rate of 25 °C/min and held for 2 min. Helium was employed as the carrier gas at a 1 mL/min flow rate. The relative content of each essential oil component was calculated based on the peak area in the chromatogram (not shown here). Component identification was performed by comparing their mass spectra with those in the National Institute of Standards and Technology (NIST) 2.0 and Wiley 8 databases. Terpenoids were further identified using the arithmetic index (AI), in conjunction with the mass spectra library and reference AI (rAI) (Adams 2007). The AI was calculated using the Eq.1,
Arithmetic index = 100 × [n + (RT(x) − RT(n))/(RT(n+1) − RT(n))] (1)
where RT(n) and RT(n+1) are the retention times of n-alkanes and (n + 1)-alkanes, respectively, and RT(x) is the retention time of the unknown compound, where RT(n) < RT(x) < RT(n+1).
Antifungal Assay
The antifungal activity of essential oils was evaluated using an agar dilution method following Cheng et al. (2006) with minor modifications. Samples of Trametes versicolor (BCRC 35253), Laetiporus sulphureus (BCRC 35305), Gloeophyllum trabeum (BCRC 31614), and Trichoderma sp. (BCRC 35296) were obtained from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute, Hsinchu City, Taiwan. After essential oils were extracted, they were dissolved in 150 μL of 99.5% ethanol and added to 15 mL of sterilized potato dextrose agar in 9-cm Petri dishes. Ethanol was used as a negative control, and didecyldimethylammonium chloride and triadimefon (a commercially available fungicide) were used as positive controls. A fungal mycelial plug was placed at the center of the medium and incubated at 27 ± 2 °C at 70% relative humidity. Fungal growth was monitored on a daily basis. Once the mycelia in the control group reached the edges of the Petri dishes, the antifungal index was calculated by Eq. 2,
Antifungal index (%) = (1 − Da/Db) × 100 (2)
where Da and Db are the diameters (in centimeters) of the fungal growth zone in the oil-treated dish and control dish, respectively. All experiments were conducted in triplicate, and the results are presented as mean ± standard deviation.
Statistical Analysis
All statistical analyses were conducted using SPSS version 17.0 (SPSS, Chicago, IL, USA). One-way analysis of variance was conducted to compare the antifungal activity of various essential oils obtained using different extraction methods. Scheffé’s post hoc test was used to identify significant differences, with the confidence interval set at 95%.
RESULTS AND DISCUSSION
Essential Oil Yield Obtained from Different Extraction Methods
Table 1 presents the average essential oil yields of C. formosensis and C. obtusa var. formosana obtained using different extraction methods. For C. formosensis, OSE (7.2 ± 0.1%) was associated with the highest essential oil yield, followed by SFE (1.0 ± 0.2%) and HD (0.3 ± 0.0%). For C. obtusa var. formosana, OSE (7.5 ± 0.1%) was associated with the highest essential oil yield, followed by SFE (2.2 ± 1.2%) and HD (1.4 ± 0.4%). Among all methods, OSE was associated with the highest essential oil yield, consistent with the findings of previous studies. In a study on Ocimum basilicum L. essential oils, HD, OSE, and SFE were reported to yield 0.26, 2.39, and 0.43% essential oils, respectively (de Barros et al. 2014). Similarly, in a study on Artemisia annua essential oils, HD was reported to yield 0.49% essential oils, OSE was reported to yield 7.28% essential oils, and SFE was reported to yield 5.27 and 5.73% essential oils at 30 °C/150 bar and 50 °C/300 bar, respectively (Zhou et al. 2023).
Each extraction method has a unique effect on the yield of essential oils. This variability is primarily attributable to differences in the solubility, diffusion rates, and extraction efficiency of volatile compounds, which affect their final yield. Additionally, different extraction methods exhibit varying degrees of effectiveness depending on plant material and essential oil composition. Therefore, selecting an appropriate extraction method is crucial and should be guided by the target essential oil composition and quality requirements.
Among the traditional extraction methods, HD is typically associated with the lowest yield of essential oils, particularly because essential oils contain both nonpolar and high-molecular-weight compounds, and the high polarity of water facilitates the extraction of polar compounds, making it less effective for nonpolar compounds. HD is regarded as the most suitable method for extracting essential oils with boiling points exceeding 100 °C, which are insoluble or only slightly soluble in water (de Barros et al. 2014; Zhou et al. 2023). Prolonged heating during distillation may cause the loss of volatile aromatic compounds and may change the composition of essential oils instead of improving their yield. High temperatures may also cause the degradation of heat-sensitive compounds (Huo et al. 2024) or the loss of volatile compounds (Manzoor et al. 2019), further reducing their yield. Despite these limitations, HD remains one of the most commonly used extraction methods because of its relatively low equipment cost, operational simplicity, and high reproducibility (Zhou et al. 2023). Compared with HD, OSE is associated with a higher essential oil yield and enables the extraction of a wider range of compounds (Fekri et al. 2021). Previous studies have shown that hexane can be efficiently recovered and reused through conventional evaporation or distillation (Burger et al. 2019; Bourgou et al. 2021). Nevertheless, the final extracts may still contain residual solvents or suffer from purity issues, which pose potential risks to human health (Fekri et al. 2021; Zhou et al. 2023).
Compared with HD and OSE, SFE has been reported to have a higher extraction rate and extraction efficiency and is more capable of mitigating the loss of active ingredients (Zhang et al. 2016; Zhou et al. 2023). Because of its relatively low critical temperature and pressure requirements, SFE can effectively extract heat-resistant compounds under mild conditions, which in turn reduces the risk of thermal degradation. In addition, SFE involves carbon dioxide as the extraction medium. Carbon dioxide is a colorless, odorless, inert, nonflammable, and inexpensive gas that can be rapidly removed after extraction, resulting in a pure and pollutant-free essential oil. Therefore, SFE is particularly suitable for the extraction of heat-sensitive compounds and can facilitate high-purity, high-efficiency extraction while addressing the problem of solvent residues. The main limitations of SFE are its high equipment cost and its precise pressure and temperature control requirements. In summary, each extraction method is suitable for different types of plant materials and target components. Therefore, when selecting an extraction method, factors such as plant material characteristics, target component stability, extraction efficiency, and operational cost should be carefully considered.
Table 1. Average Essential Oil Yields of C. formosensis and C. obtusa var. formosana Obtained Using Different Extraction Methods
Chemical Composition of Leaf Essential Oils
C. formosensis
As shown in Table 2, the chemical compositions of the C. formosensis leaf essential oils significantly varied depending on the extraction method used. For instance, the essential oils extracted by HD were dominated by α-pinene (83.4%) followed by β-myrcene (4.4%) and β-pinene (3.0%). The essential oils extracted by SFE were dominated by kaur-16-ene (51.1%) followed by germacrene D (23.0%). The essential oils extracted by OSE were dominated by phytol (44.4%), terpinen-4-ol (11.2%), and an unidentified component (19.7%).
The leaf essential oils of C. formosensis were categorized into four major classes: monoterpene hydrocarbons, sesquiterpene hydrocarbons, diterpene hydrocarbons, and oxygenated diterpenes (Table 2). The predominant compound types varied depending on the extraction method used. HD primarily yielded monoterpene hydrocarbons (92.4%), SFE produced a mixture dominated by diterpene hydrocarbons (51.1%) and sesquiterpene hydrocarbons (34.2%), while OSE resulted mainly in oxygenated diterpenes (44.4%).
Table 2. Chemical Compositions of C. formosensis Leaf Essential Oils Obtained Using Different Extraction Methods
Research into the leaf essential oils of C. formosensis dates back to 1931, when Kafuku and Ichikawa (1931) first analyzed the chemical composition of these essential oils and confirmed that α-pinene was the main compound. Similarly, Su et al. (2006) identified α-pinene (71.6%) as the primary compound in C. formosensis leaf essential oils, followed by δ-2-carene (4.6%), β-myrcene (4.1%), γ-muurolene (3.1%), β-pinene (2.7%), and α-caryophyllene (2.0%). Chen et al. (2011) reported that the leaf essential oils extracted from C. formosensis in different regions of Taiwan primarily consist of monoterpene hydrocarbons (87.1 to 93.7%), with α-pinene (71.6 to 86.0%) as the primary compound, followed by β-pinene (2.7 to 3.3%), β-myrcene (2.5 to 3.7%), and Δ3-carene (0.0 to 6.7%).
Table 3. Chemical Compositions of C. obtusa var. formosana Leaf Essential Oils Obtained Using Different Extraction Methods
The compositions of HD-extracted essential oils observed in this study were consistent with those reported previously. Few studies have examined the biological activity of C. formosensis leaf essential oils, and their chemical compositions obtained by SFE or OSE remain unexplored. While comparative studies on extraction methods and the antimicrobial activity of essential oils have been reported for other taxa, to our knowledge this is the first study to compare the leaf essential oils of C. formosensis and C. obtusa var. formosana across different extraction methods.
C. obtusa var. formosana
As shown in Table 3, the chemical compositions of C. obtusa var. formosana leaf essential oils varied depending on the extraction method used. For instance, the essential oils extracted by HD were dominated by sabinene (36.2%) and thujopsene (22.5%); the essential oils extracted by SFE were dominated by totarol (50.9%), elemol (15.7%), and an unidentified compound (17.7%). The essential oils extracted by OSE were dominated by thujopsene (27.6%), cedrol (24.8%), and elemol (17.7%).
The leaf essential oils of C. obtusa var. formosana were divided into four categories based on their structural characteristics (Table 3). HD primarily yielded monoterpene hydrocarbons (59.6%), SFE produced mainly oxygenated diterpenes (50.9%), and OSE resulted in a high proportion of oxygenated sesquiterpenes (45.8%) and sesquiterpene hydrocarbons (31.8%).
Multiple studies have examined the chemical compositions of C. obtusa var. formosana essential oils. Su et al. (2006) compared the leaf essential oil compositions of five coniferous species in Taiwan and discovered that α-pinene was the main compound in C. obtusa var. formosana, C. formosensis, and Calocedrus formosana. They also reported that the leaf essential oils of C. obtusa var. formosana contained α-pinene (76.7%), β-myrcene (5.7%), β-pinene (3.2%), γ-muurolene (2.8%), δ-2-carene (2.1%), and β-phellandrene (2.1%). Cheng et al. (2007) extracted various essential oils from the heartwood, bark, and leaves of C. obtusa var. formosana and examined their insecticidal activity against termites (Coptotermes formosanus). However, they did not provide details on the chemical composition of each essential oil.
Chen et al. (2011) compared the chemical compositions of C. obtusa var. formosana leaf essential oils from different regions and reported that the concentration of each component varied depending on the region. Using cluster analysis, they classified these essential oils into three chemotypes depending on their chemical composition: β-elemol, thujopsene, and cis-thujopsene. According to this classification, the essential oil composition obtained in the present study belongs to the category of thujopsene.
According to the aforementioned results, for both C. formosensis and C. obtusa var. formosana, HD is primarily associated with monoterpene compounds, whereas SFE and OSE are primarily associated with high-molecular-weight oxygenated sesquiterpenes and diterpenes. Research into the extraction of essential oils from Satureja bachtiarica has indicated a significant effect of the extraction method used on the chemical composition of the essential oils extracted from aerial plant parts (p < 0.01) (Memarzadeh et al. 2015). This effect may be attributable to the loss or enhancement of certain compounds, potentially as a result of oxidation, glycoside hydrolysis, esterification, or other chemical processes (Ghasemi Pirbalouti et al. 2013; Memarzadeh et al. 2015).
The bioactivity of essential oils primarily depends on their chemical composition, specifically on whether they are alcohols, aldehydes, acids, phenols, esters, or terpenes (Zhou et al. 2023). In addition, the extraction conditions significantly influence the chemical composition of essential oils, which in turn affects their bioactivity. Many studies have indicated that the essential oils extracted by SFE have excellent antimicrobial activity. For example, Glišić et al. (2007) reported that the fruit essential oils of Daucus carota L. extracted by SFE were significantly more effective in killing Bacillus cereus compared with those extracted by HD, indicating the ability of SFE to retain compounds with antimicrobial activity.
Although SFE can enhance the antimicrobial and antioxidant activity of essential oils in many cases, few studies have confirmed the superiority of SFE to HD (Glišić et al. 2007). Yousefi et al. (2019) reported that the Aquilaria crassna essential oils extracted by HD had lower minimum inhibitory concentrations against Staphylococcus aureus and Candida albicans compared with those extracted by SFE. This discrepancy in findings may be attributable to the different concentrations of volatile compounds in essential oils, because many compounds with antioxidant and antibacterial activity are often volatile, and HD-extracted essential oils tend to contain a high concentration of volatile compounds (Huo et al. 2024). In summary, the extraction method used directly affects the composition and antibacterial and antioxidant capacity of the essential oils extracted.
Antifungal Activity of C. formosensis and C. obtusa var. formosana Leaf Essential Oils
Poisoned food assay
Figure 1 depicts the antifungal effects of essential oils (800 μg/mL) extracted from C. formosensis and C. obtusa var. formosana leaves against Trichoderma sp., T. versicolor, and two brown-rot fungi, namely L. sulphureus and G. trabeum. Among all essential oils, the essential oils extracted from C. formosensis leaves by OSE exhibited the strongest antifungal activity against Trichoderma sp., T. versicolor, L. sulphureus, and G. trabeum, with inhibition indices of 76.1, 81.8, 79.7, and 67.4%,respectively (Fig. 1). The essential oils extracted by HD achieved an inhibition index of 71.5% against L. sulphureus, but they did not achieve an inhibition index greater than 70%against the other fungal strains. As shown in Table 2, the C. formosensis leaf essential oils extracted by OSE were dominated by (E)-phytol (44.4%),which is a common antifungal compound. Multiple studies have indicated that essential oils rich in (E)-phytol have high antifungal activity (Rajab et al. 1998; Kobaisy et al. 2001). These findings suggest that the strong antifungal effect of OSE-extracted essential oils is attributable to their high concentration of (E)-phytol.
Fig. 1. Antifungal activity of C. formosensis leaf essential oils obtained using different extraction methods. Data are presented as mean ± SD of five replicates. Abbreviations: L.s., Laetiporus sulphureus; G.t., Gloeophyllum trabeum; T.v., Trametes versicolor.
Studies on the bioactivity of C. formosensis essential oils have primarily focused on wood-derived instead of leaf-derived essential oils. Wang et al. (2005) analyzed the chemical composition of C. formosensis heartwood essential oils and identified α-eudesmol as the main compound. They reported that α-eudesmol had a strong antifungal effect against L. sulphureus and T. versicolor, achieving complete inhibition at concentrations of 50 and 100 μg/mL, respectively. Similarly, Kuo et al. (2007) analyzed the chemical composition of C. formosensis heartwood essential oils and identified myrtenal as the main compound. They reported that myrtenal had an insecticidal effect against Lepisma saccharina (silverfish). Although the antifungal activity of C. formosensis leaf essential oils are lower than that of wood-derived oils, leaf essential oils remain a promising alternative from a sustainability perspective. Utilizing these essential oils ensures high resource efficiency while maintaining bioactivity. OSE not only produces large quantities of essential oils but also enhances their antifungal activity by enriching bioactive compounds.
In this study, C. obtusa var. formosana essential oils exhibited stronger antifungal effects than those of C. formosensis essential oils, regardless of the extraction method. At an essential oil concentration of 800 μg/mL, the OSE-extracted essential oils had inhibition indices of 65.7, 84.3, 86.5, and 68.6% against Trichoderma sp., T. versicolor, L. sulphureus, and G. trabeum, respectively (Fig. 2). At the same concentration, the SFE-extracted essential oils had inhibition indices of 81.4, 78.9, 88.4, and 82.1% against Trichoderma sp., T. versicolor, L. sulphureus, and G. trabeum, respectively, and the HD-extracted essential oils had inhibition indices of 100.0, 77.9, 100.0, and 75.2% against Trichoderma sp., T. versicolor, L. sulphureus, and G. trabeum, respectively (Fig. 2). At an essential oil concentration of 400 μg/mL, the inhibition indices remained at 77.8, 74.8, 97.4, and 34.8%, respectively (data not shown).
These results indicated that the essential oils extracted in this study exhibited varying inhibitory effects on each fungus. At a concentration of 800 μg/mL, the HD-extracted essential oils exhibited the strongest inhibitory effect against Trichoderma sp. and L. sulphureus, achieving 100% inhibition. At the same concentration, the SFE-extracted essential oils exhibited the strongest inhibitory effect against G. trabeum, achieving 82.1% inhibition, and the OSE-extracted essential oils exhibited the strongest inhibitory effect against T. versicolor, achieving 84.3% inhibition.
The fungi tested in this study (T. versicolor, L. sulphureus, G. trabeum, and Trichoderma sp.) are all important wood-decay species; therefore, the inhibitory effects of the leaf essential oils indicate their potential as natural wood-protective agents. Similar studies have also demonstrated that leaf essential oils from other tree species, such as Litsea coreana (Ho et al. 2010), Metasequoia glyptostroboides, and Melaleuca leucadendron (Bajpai and Kang 2010; Rini et al. 2012), exhibit pronounced antifungal activities against both white-rot and brown-rot fungi. These findings are consistent with the present results and further highlight the potential of leaf essential oils as natural wood protectants, particularly in substituting for chemical preservatives, promoting the sustainable use of wood products, and reducing reliance on heartwood resources, thereby aligning with the principles of environmental sustainability.
In summary, the results of this work demonstrate that the method selected for extraction plays a decisive role in shaping both the chemical composition and antifungal efficacy of cypress leaf essential oils. While wood-derived oils of cypresses have long been studied for their bioactivities, the present findings provide the first systematic comparison of C. formosensis and C. obtusa var. formosana leaf essential oils obtained by HD, OSE, and SFE. Beyond antifungal activity, it is worth noting that leaf powders and methanolic extracts of C. obtusa have also been reported to exhibit weed-suppression potential through allelochemicals such as (−)-hinokiic acid and (+)-dihydrosesamin, which were not detected in the present essential oil fractions (Kato-Noguchi et al. 2024). This underscores that extraction approaches targeting different chemical classes can lead to distinct biological applications, highlighting the broader value of leaf biomass as a renewable resource.
Fig. 2. Antifungal activity of C. obtusa var. formosana leaf essential oils obtained using different extraction methods. Data are presented as mean ± SD of five replicates. Abbreviations: L.s., Laetiporus sulphureus; G.t., Gloeophyllum trabeum; T.v., Trametes versicolor.
CONCLUSIONS
- The essential oil yields of both C. formosensis and C. obtusa var. formosana varied markedly depending on the extraction method, with organic solvent extraction (OSE) consistently producing the highest yields, followed by supercritical fluid extraction (SFE) and hydrodistillation (HD). In addition to these yield differences, the chemical composition also depended strongly on extraction method: HD predominantly yielded volatile monoterpenes, whereas SFE and OSE produced higher-molecular-weight sesquiterpenes and diterpenes.
- Essential oils obtained by OSE exhibited the strongest antifungal activity against Trichoderma sp., T. versicolor, L. sulphureus, and G. trabeum, which may be attributable to their high (E)-phytol content.
- Similarly, the essential oils extracted by HD from C. obtusa var. formosana had the strongest inhibitory effect against Trichoderma sp. and L. sulphureus, which may be attributable to their high sabinene and thujopsene content.
- The findings of this study provide valuable insights into the potential applications of C. formosensis and C. obtusa var. formosana leaf essential oils, which may serve as eco-friendly wood preservatives.
ACKNOWLEDGMENTS
The authors express special thanks to the Experimental Forest of National Taiwan University for providing the materials, to Mr. Yen-Ray Hsui of Taiwan Forestry Research Institute for his technical assistance.
Conflicts of Interest
The authors have no conflicts of interest to declare.
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Article submitted: July 24, 2025; Peer review completed: August 16, 2025; Revised version received and accepted: August 28, 2025; Published: September 4, 2025.
DOI: 10.15376/biores.20.4.9332-9347