Phytochemical composition, biological activities, and toxicity of the leaf essential oils obtained from Eucalyptus grandis × Eucalyptus urophylla cultivated in Malaysia

Abstract

This study aimed to evaluate the phytochemical composition, antimicrobial properties, mosquito larvicidal effects, and brine shrimp toxicity of essential oils obtained using hydrodistillation from the fresh and dried leaves of Eucalyptus grandis × Eucalyptus urophylla at two age groups. Leaves from trees aged 17 to 31 months old yielded more essential oils than those aged 40 to 50 months. Gas chromatography-mass spectrometric analysis revealed that 1,8-cineol (13.1% to 26.7%) and α-terpinyl acetate (18.3% to 26.1%) were the dominant components across all essential oils. All tested essential oils inhibited Gram-positive bacteria, yeasts, and the dermatophyte Trichophyton rubrum, but failed to exhibit activity against most of the tested Gram-negative bacteria and Aspergillus fumigatus. The minimum inhibitory concentrations ranged from 0.16 to 2.50 mg/mL for bacteria and 0.04 to 1.25 mg/mL for fungi, highlighting the greater antifungal efficacy of the essential oils. All tested essential oil samples were also active against third instar larvae of Aedes aegypti and Aedes albopictus, with median lethal concentrations of 52.3 to 134 µg/mL after 24 h, lower than that of against Artemia franciscana nauplii (209 and 222 µg/mL). Therefore, Eucalyptus grandis × Eucalyptus urophylla essential oils are potential larvicidal agents for mosquito control with low toxicity to aquatic organisms.

Full Article

Phytochemical Composition, Biological Activities, and Toxicity of the Leaf Essential Oils Obtained from Eucalyptus grandis × Eucalyptus urophylla Cultivated in Malaysia

See Cheng Yip  , Lai Yee Ho  , and Nam Weng Sit  ,*

This study aimed to evaluate the phytochemical composition, antimicrobial properties, mosquito larvicidal effects, and brine shrimp toxicity of essential oils obtained using hydrodistillation from the fresh and dried leaves of Eucalyptus grandis × Eucalyptus urophylla at two age groups. Leaves from trees aged 17 to 31 months old yielded more essential oils than those aged 40 to 50 months. Gas chromatography-mass spectrometric analysis revealed that 1,8-cineol (13.1% to 26.7%) and α-terpinyl acetate (18.3% to 26.1%) were the dominant components across all essential oils. All tested essential oils inhibited Gram-positive bacteria, yeasts, and the dermatophyte Trichophyton rubrum, but failed to exhibit activity against most of the tested Gram-negative bacteria and Aspergillus fumigatus. The minimum inhibitory concentrations ranged from 0.16 to 2.50 mg/mL for bacteria and 0.04 to 1.25 mg/mL for fungi, highlighting the greater antifungal efficacy of the essential oils. All tested essential oil samples were also active against third instar larvae of Aedes aegypti and Aedes albopictus, with median lethal concentrations of 52.3 to 134 µg/mL after 24 h, lower than that of against Artemia franciscana nauplii (209 and 222 µg/mL). Therefore, Eucalyptus grandis × Eucalyptus urophylla essential oils are potential larvicidal agents for mosquito control with low toxicity to aquatic organisms.

DOI: 10.15376/biores.21.1.237-266

Keywords: Eucalyptus grandis × Eucalyptus urophylla; Aedes aegypti; Aedes albopictus; Antibacterial; Antifungal; Larvicidal

Contact information: Department of Allied Health Sciences, Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat, 31900 Kampar, Perak, Malaysia; *Corresponding author: sitnw@utar.edu.my

Graphical Abstract

INTRODUCTION

In Malaysia, afforestation or reforestation through forest plantations has been identified as a crucial strategy to address the declining timber supply resulting from the depletion of natural forest resources. The Forest Plantation Development Program, initiated by the Malaysian Timber Industry Board (MTIB), provides financial incentives to encourage the development of commercial forest plantations of four hectares or more (MTIB 2021). Among the species listed under this program, Eucalyptus species stands out as a promising candidate for large-scale planting due to its fast growth, limited space occupancy, high phenotypic plasticity, and adaptability to diverse climates and soil types (Yahya et al. 2020). Eucalyptus is a genus of evergreen hardwood (angiosperm) species in the family Myrtaceae, which is commonly cultivated in subtropical regions for plantation purposes (Mieres-Castro et al. 2021). Globally, the top three Eucalyptus-cultivating countries are Brazil, India, and China. The main planted species in Brazil are E. camaldulensis, E. citriodora, E. globulus, E. grandis, E. saligna, E. urophylla, and their hybrids such as E. grandis × E. camaldulensis,  E. urophylla × E. camaldulensis,  E. urophylla × E. globulus,  and E. urophylla × E. grandis (Florêncio et al. 2022). In India, the commercially important species are E. camaldulensis, E. citriodora, E. globulus, E. grandis, and E. tereticornis (Shikha et al. 2025), while China primarily cultivates E. camaldulensis,  E. dunnii,  E. globulus,  E. grandis,  E. maidenii, E. saligna,  E. tereticornis,  E. urophylla, as well as E. urophylla × E. grandis  and E. camaldulensis × E. grandis  hybrids (Zhou and Wingfield 2011).

Malaysia is ideal for growing Eucalyptus, as its low cold tolerance and high-water requirement are well-suited to the country’s hot and humid climate conditions (Zhang and Wang 2021).  Eucalyptus  pellita  and E. grandis × E. urophylla  are the commonly cultivated species. However, the hybrid type has gained popularity using clones sourced from Southern China, as its growth outperformed E. pellita in terms of tree height and diameter (Yahya et al. 2020). This hybrid was developed through controlled pollination, a technique used to select and combine the desirable traits of both parent species. Eucalyptus grandis contributes to fast growth and ease of vegetative propagation, while Eucalyptus urophylla provides beneficial traits such as enhanced disease resistance, improved adaptability to diverse environmental conditions, and higher wood density (Kullan et al. 2012; Van den Berg et al. 2015).

Eucalyptus trunks are valued for various applications, including paper pulp, plywood, furniture, poles, and sawn timber (Lu et al. 2014), while the remaining parts, including fruits, flowers, and leaves, could be harvested for essential oil extraction. The leaves are particularly noteworthy, as more than 300 Eucalyptus species have been reported to contain volatile oils (Mieres-Castro et al. 2021). Essential oils are colorless or pale-yellow, aromatic, oily products that are typically soluble in organic solvents and less dense than water (Haro-González et al. 2021). According to the International Organization for Standardization (2021), essential oils are natural products derived from botanical sources obtained via hydrodistillation, dry distillation, steam distillation, or mechanical compression in the case of citrus fruit, after separation of aqueous phase using physical methods. This definition highlights that essential oils are commonly obtained through hydrodistillation, a method that involves the use of boiling water to release volatile compounds from plant material, which are subsequently condensed and collected. Hydrodistillation is widely used due to its efficiency, simplicity, and the absence of organic solvents (Salehi et al. 2019).

The essential oils of Eucalyptus species are recognized for their abundance of bioactive compounds, primarily monoterpenes, such as α-pinene, 1,8-cineol (eucalyptol), and limonene, as well as sesquiterpenes, such as β-eudesmol, α-humulene, and globulol (Yip et al. 2024). However, their phytochemical composition can vary significantly depending on the extraction techniques, tree age, species, geographical location, and leaf condition (Zhang et al. 2010; Achmad et al. 2018; Shiferaw et al. 2019). Furthermore, existing literature has documented a wide range of biological activities associated with Eucalyptus essential oils, including antioxidative, antimicrobial, antiviral,  antidiabetic, anti-inflammatory, analgesic, mucolytic, and bronchodilatory effects (Barbosa et al. 2016; Salehi et al. 2019; Chandorkar et al. 2021). For example, Zhou et al. (2021) reported minimum inhibitory concentrations of between 0.023 to 0.091 mg/mL for  E.  grandis × E.  urophylla  leaf essential oils, obtained using steam distillation, against human pathogenic bacteria, i.e., Bacillus cereus, B. subtilis, Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Escherichia coli. Moreover, Lucia et al. (2008) demonstrated the larvicidal activity of essential oils derived from  Eucalyptus  hybrids:  E. grandis × E. camaldulensis  and E. grandis × E. tereticornis, against Aedes aegypti, the yellow fever mosquito.

Given that the biological activities of essential oils derived from Eucalyptus species are closely associated with their phytochemical composition, this study aimed to explore the phytochemical composition of essential oils obtained from the leaves of Eucalyptus grandis × Eucalyptus urophylla  cultivated in Malaysia at different tree ages using hydrodistillation. In addition, this study assessed the toxicity of the isolated essential oils and evaluated their antibacterial activity against bacterial pathogens, antifungal efficacy against human pathogenic fungi, and larvicidal potential against Aedes mosquitoes, which are the vectors of arboviral diseases such as dengue, chikungunya, and Zika. Through these assessments, this study seeks to explore natural alternatives  to synthetic disinfectants and mosquito larvicides.

EXPERIMENTAL

Materials

The iodonitrotetrazolium chloride, itraconazole, along with the standards for alkanes (C7-C40), α-pinene, α-terpineol, borneol, β-caryophyllene, 1,8-cineol, and limonene were purchased from Sigma-Aldrich (St. Louis, USA), whereas the aromadendrene standard was purchased from ChemFaces (Wuhan, China). Chloramphenicol was sourced from Duchefa Biochemie (Haarlem, The Netherlands), 3-morpholinopropanesulfonic acid from Bio Basic (Markham, Canada), 5-fluorocytosine from Acros Organics (Hong Kong, China), temephos from Dr. Ehrenstorfer GmbH (Augsburg, Germany), potassium dichromate from Systerm Chemical (Shah Alam, Malaysia), anhydrous sodium sulphate and acetone (spectroscopy grade) from Merck (Darmstadt, Germany), and methanol and ethanol (analytical grade) from Rank Synergy (Kuala Lumpur, Malaysia). The culture media used included Mueller-Hinton broth and Mueller-Hinton agar (HiMedia, Thane, India), potato dextrose agar (Liofilchem, Roseto degli Abruzzi, Italy), oatmeal agar (Laboratorios Conda, Madrid, Spain), and Roswell Park Memorial Institute (RPMI)-1640 medium (Biowest, Nuaillé, France).

Ten bacterial species comprising six reference strains from the American Type Culture Collection (ATCC), Enterococcus hirae ATCC^® 10541^™, Staphylococcus aureus  ATCC^® 6538^™,  Pseudomonas aeruginosa ATCC^® 15442^™,  Escherichia coli ATCC^® 35218^™, Klebsiella pneumoniae  ATCC^® 13883^™, and Bacillus cereus ATCC^® 14579^™, as well as four clinical isolates, Staphylococcus aureus SA-LWE23#1, Staphylococcus aureus SA-LWE23#2, Klebsiella pneumoniae KP-LWE23#1, and Escherichia coli EC-LWE23#1, were used in this study. Eight fungal species, including six yeasts, namely Candida tropicalis ATCC^® 750^™, Candida albicans  ATCC^® 90028^™, Candida auris derived from CDC B11903, Candida parapsilosis  ATCC^® 22019^™,  Cryptococcus neoformans ATCC^® 13690^™, and Nakaseomyces glabratus  ATCC^® MYA-2950^™, along with two filamentous fungi, Trichophyton rubrum  ATCC^® 28188^™  and Aspergillus fumigatus ATCC^® 204305^™, were also tested in the study.

Preparation of Plant Materials

Fresh leaves from the Eucalyptus grandis × Eucalyptus urophylla were used for essential oil extraction. The leaf samples were harvested from trees cultivated at two different estates in Gua Musang, Kelantan, Malaysia. Trees aged 17 to 31 months from the first estate (4° 39′ 48″ N 101° 36′ 58″ E) were designated as Eucalyptus A, while those aged 40 to 50 months from the second estate (4° 48′ 192” N 101° 55′ 15″ E) were designated as Eucalyptus B. Leaf vouchers were prepared (code: UTAR/FSC/23/001) and deposited at the Faculty of Science, Universiti Tunku Abdul Rahman, Kampar, Malaysia. The collected leaves were rinsed and divided into two batches. One batch was kept in a 4 °C fridge to maintain freshness, while another batch was dried in an oven (Memmert GmbH, Schwabach, Germany) at 40 °C for 5 to 7 days. The average mass loss (n=3) of the leaves following drying was 32.0% ± 3.46% for Eucalyptus A and 59.1% ± 3.59% for Eucalyptus B. Both fresh and dried leaf samples were then cut into smaller pieces and blended prior to hydrodistillation.

Hydrodistillation

Hydrodistillation of the plant sample was carried out using a stainless-steel distiller (Laboratory & Scientific Enterprise, Klang, Malaysia) to obtain the essential oils. The distiller consisted of a 10 L distilling pot connected to a condensation tower filled with cold water, as shown in Fig. 1. The cold water was maintained using a chiller (Buchi Labortechnik AG, Flawil, Switzerland). One kg of blended leaf material was placed in the distilling pot and submerged in 4 L of deionized water. The mixture was heated using an induction cooker (Philips, China) operating at 800 W. The pot was equipped with a thermometer for continuous temperature monitoring. The entire extraction was run at 100 °C for 8 h, and the distillate was collected in a glass collecting flask, where the essential oil layer was separated from the hydrosol. The resulting essential oils were collected and dried using anhydrous sodium sulphate, then stored in glass vials at 4 °C prior to analysis. Each extraction was performed in triplicate. The extraction yield (w/w) was calculated based on the fresh leaf weight for fresh leaf essential oils (FLEO) and the dried leaf weight for dried leaf essential oils (DLEO). For clarity, the FLEO samples from Eucalyptus A and Eucalyptus B were designated as HfA and HfB, respectively, while the corresponding DLEO samples were labeled HdA and HdB, respectively.

Fig. 1. Instrumental setup for hydrodistillation

Phytochemical Composition

A Shimadzu model, QP2010 Plus (Tokyo, Japan) GC-MS was used to identify the phytochemical composition of essential oil samples. The component separation was performed using a 30.0 m × 0.25 mm × 0.25 µm capillary column of 5% diphenyl-95% dimethyl polysiloxane (SH-I-5Sil MS, Shimadzu, Tokyo, Japan). The temperature of the capillary column was initially set at 50 °C and held for 5 min, then the temperature rose at a rate of 5 °C/min until reaching 200 °C and remained for 5 min, giving a total duration of 40 min for the analysis of each sample. The mobile phase, helium gas, flowed at a linear velocity rate of 36.3 cm/s. The sample was prepared in acetone at 1.0 mg/mL and filtered using a 0.45 µm nylon syringe filter. One µL of sample was injected per run, with a split ratio of 20. The injector port temperature was maintained at 200 °C. For the mass spectrometer settings, both interface temperature and ion source temperature were 200 °C. Electron impact ionization at 70 eV was used. The fragment ions produced by each component were scanned at m/z 35 to 600. The identification of compounds was carried out by matching their mass spectra with those recorded in the NIST 23 Mass Spectral Library (National Institute of Standards and Technology, Gaithersburg, USA). Only components with matching similarity ≥86% were reported (Lim et al. 2023). Further confirmation of selected identified compounds was accomplished through comparison of their retention times with the authentic standards (aromadendrene, β-caryophyllene, α-terpineol, α-pinene, limonene, borneol, and 1,8-cineol) analyzed under the same settings. The retention indices were determined with respect to C7 to C40 alkanes. The relative percentage of compounds in the total ion chromatogram was calculated using a peak area normalization method. The analysis was performed in duplicate.

Antibacterial Assay

The colorimetric broth microdilution antibacterial assay was adapted from the method of Sit et al. (2017) with slight modifications. Bacteria were first cultured on Mueller-Hinton agar prior to the assay. A 10 mg/mL stock solution of essential oil was prepared in an ethanol-water mixture (2:1, v/v) and sterilized using a 0.45 µm syringe filter. This stock solution was subjected to two-fold serial dilutions in a 96-well microtiter plate with Mueller-Hinton broth to obtain final concentrations of 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, and 2.50 mg/mL. After that, 50 µL of bacterial suspension (1×10⁶ colony-forming unit/mL) was inoculated into each well, bringing the final volume to 100 µL per well. Four controls were included in each assay to validate the results: positive control (chloramphenicol ranging from 1 to 128 µg/mL), medium control (broth), growth control (bacterial inoculum without essential oil), and negative control (essential oil without bacteria). The microtiter plates were incubated at 37 °C for 24 h. After incubation, 20 µL of iodonitrotetrazolium chloride solution at 0.4 mg/mL was added to each well. Bacterial growth was assessed based on the formation of a purple formazan precipitate. The minimum inhibitory concentration (MIC) was recorded as the lowest concentration that inhibited bacterial growth. Subsequently, the minimum bactericidal concentration (MBC), which was the lowest concentration capable of killing 99.9% bacteria, was determined by inoculating 20 µL from wells that showed no bacterial growth onto Mueller-Hinton agar plates. The plates were then incubated at 37 °C for 24 h, and the formation of bacterial colonies was observed to determine the MBC.

Antifungal Assay

The colorimetric broth microdilution method was also used to determine the antifungal properties of the essential oils. All yeasts and A. fumigatus were maintained on potato dextrose agar, while T. rubrum was cultured on oatmeal agar prior to testing. The 10 mg/mL stock solution of each essential oil, prepared in an ethanol-water mixture and filter-sterilized through a 0.45 µm syringe filter, was two-fold serially diluted in a 96-well microtiter plate to obtain final concentrations ranging from 0.02 to 2.50 mg/mL. The fungal inoculums were prepared according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2008a; 2008b), and 50 μL of each inoculum was added to the wells. Itraconazole and 5-fluorocytosine served as the positive controls for filamentous fungi and yeasts, respectively. Wells containing only RPMI-1640 medium, only essential oil, and only fungal inoculum served as sterility control, negative control, and growth control, respectively. The microtiter plates were incubated at 35 °C for 48 h for Candida spp. and N. glabratus, 72 h for C. neoformans and A. fumigatus, and at 30 °C for 5 d for T. rubrum. After incubation, 20 µL of iodonitrotetrazolium chloride solution (0.4 mg/mL) was pipetted into each well to determine the MIC. The spread plate technique was performed by swabbing 20 µL from wells that showed no visible purple precipitate on potato dextrose agar. The lowest essential oil concentration that completely inhibited fungal growth on the agar was determined as the minimum fungicidal concentration (MFC).

Mosquito Larvicidal Testing

The mosquito larvicidal properties of the essential oils were evaluated against Aedes aegypti and Aedes albopictus based on the World Health Organization guidelines (WHO 2005), with slight modifications. The assay began with the hatching of mosquito eggs, sourced from the Vector Control Research Unit (VCRU) of Universiti Sains Malaysia (USM), Penang, Malaysia, in dechlorinated water and maintained until they reached the third instar larval stage. A stock solution of the essential oil, which was prepared at 40 mg/mL in methanol, was then diluted with deionized water to obtain five concentration levels (25, 50, 100, 200, and 400 µg/mL), using round plastic containers. The final methanol concentration in the assay was maintained at ≤1% to avoid methanol-induced larval mortality. For each test, 20 third instar larvae were transferred into plastic containers containing 100 mL of the diluted essential oil solutions, following a 1.0 h holding period. The containers were maintained at room temperature (24 °C) and relative humidity (70%). Larval mortality was recorded at 24 h and 48 h post-treatments. The negative control was 1% methanol, while the positive control was 1000 µg/mL temephos. Larvae were considered dead if they failed to move when gently probed with a pipette tip in the cervical region and were unable to reach the water surface. Dead larvae were examined for morphological abnormalities under a stereo microscope (SMZ-161, Motic Asia, Hong Kong, China). The percentage of larval mortality was calculated as (number of dead larvae in treatment/total number of larvae) × 100.

Brine Shrimp Lethality Testing

The brine shrimp lethality assay was employed to assess the toxicity of the essential oils. Artemia franciscana cysts, obtained from Universiti Malaysia Terengganu (UMT), Terengganu, Malaysia, were hatched in artificial seawater under constant aeration at room temperature for 48 h, and the nauplii were then fed every 24 h with PKC-Nutri+® (Tiong et al. 2024). A stock solution of essential oil at 10,000 µg/mL was prepared using 10% ethanol and subsequently diluted using artificial seawater to obtain final concentrations of 1, 10, 100, 500, and 1000 µg/mL in separate plastic containers. Each container had a final volume of 5 mL. Ten brine shrimp nauplii were introduced into each container and incubated at room temperature for 24 h. Artificial seawater containing 1% ethanol served as the negative control, while potassium dichromate prepared at the same concentrations (1, 10, 100, 500, and 1000 µg/mL) served as the positive control. After incubation, the number of surviving nauplii in each container was counted, and the percentage of mortality (%) was calculated as [(number of survivors in control – number of survivors in treatment)/number of survivors in control] × 100.

Statistical Analysis

Antimicrobial assays and toxicity assessment were triplicated, while the mosquito larvicidal assay was performed in four independent experiments. Data from the extraction yields, mosquito larvicidal assays, and brine shrimp lethality assays were presented as means ± standard deviations. Results for antibacterial and antifungal assays were expressed as either mean values or ranges. Microsoft Excel 2019 was used for the calculation of the means and standard deviations, while IBM Statistical Package for the Social Sciences (SPSS) version 27 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. The median lethal concentration (LC₅₀) and 95% lethal concentration (LC₉₅) of the essential oils against brine shrimp nauplii and mosquito larvae were determined using Probit analysis. Student’s t-test was applied to compare the extraction yields between two tree age groups. Two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was applied to analyze brine shrimp mortality rates with essential oil type and concentration as independent factors. Four-way ANOVA followed by Tukey’s post-hoc test was employed to assess mosquito larval mortality rates with essential oil type, concentration, exposure time, and mosquito species being independent variables. The significance level of P < 0.05 was adopted throughout the study.

RESULTS AND DISCUSSION

Extraction Yield and Phytochemical Composition

After 8 h of hydrodistillation, essential oil yields from fresh Eucalyptus hybrid leaves were 0.118% ± 0.012% (w/w) for HfA and 0.026% ± 0.002% (w/w) for HfB, calculated on a fresh weight basis. On the other hand, dried leaves yielded 0.191% ± 0.029% (w/w) for HdA and 0.046% ± 0.004% (w/w) for HdB, based on dry weight. Correspondingly, for 1 kg of plant material, the extraction volumes obtained were 1.225, 1.977, 0.273, and 0.477 mL for HfA, HdA, HfB, and HdB, respectively. Notably, the younger Eucalyptus hybrid trees (HfA, HdA) consistently produced significantly higher essential oil yields than the older Eucalyptus hybrid trees (HfB, HdB) under both fresh and dried leaf conditions (P < 0.05).

Thirty-eight components were successfully identified in the essential oil samples, accounting for 95.6% to 99.7% of the total peak area (Table 1), with corresponding total ion chromatograms shown in Tables S1-S4. 1,8-cineol and α-terpinyl acetate were the dominant components across all samples. 1,8-cineol constituted 22.9% in HfA, 26.7% in HdA, 13.0% in HfB, and 21.9% in HdB, while α-terpinyl acetate accounted for 26.1% in HfA, 19.7% in HdA, 24.6% in HfB, and 18.3% in HdB. In addition to these two major constituents, HfA and HdA also exhibited high abundance of α-pinene (13.9%; 15.7%), α-terpineol (7.7%; 6.3%), and limonene (5.9%; 8.2%), respectively. In contrast, HfB and HdB displayed more variations in their phytochemical profiles, with HfB being dominated by α-terpineol (9.2%), borneol (6.6%), and globulol (5.1%), while HdB was characterized by high levels of α-terpineol (9.3%), α-pinene (7.8%), and borneol (7.5%).

A comparison of phytochemical profiles between the two age groups revealed that essential oils obtained from younger Eucalyptus trees uniquely contained seven chemical constituents, namely aromadendrene (HfA: 1.95%; HdA: 1.79%), epiglobulol (HfA: 0.41%; HdA: 0.53%), isoamyl isobutyrate (HfA: 0.35%), terpinen-4-ol (HfA: 0.63%), bornyl acetate (HfA: 0.50%), isocarveol (HfA: 0.39%), and α-cubebene (HfA: 0.36%). Conversely, five components were found exclusively in essential oils from older Eucalyptus trees: butyl isobutyl phthalate (HfB: 1.02%), humulene (HfB: 0.81%), τ-muurolol (HfB: 0.72%), δ-cadinene (HfB: 1.12%; HdB: 0.68%), and caryophyllene oxide (HfB: 1.64%; HdB: 0.93%).

Additionally, some compounds were detected exclusively in either fresh or dried leaf essential oils, regardless of tree age. FLEO contained eight unique compounds that were not present in DLEO: namely, terpinen-4-ol (HfA: 0.63%), isocarveol (HfA: 0.39%), bornyl acetate (HfA: 0.50%), α-cubebene (HfA: 0.36%), isoamyl isobutyrate (HfA: 0.35%), humulene (HfB: 0.81%), τ-muurolol (HfB: 0.72%), and butyl isobutyl phthalate (HfB: 1.02%). Conversely, DLEO exclusively contained fenchene (HdA: 0.51%; HdB: 0.48%), isoterpinolene (HdA: 0.79%; HdB: 0.48%), and cubebin-11-ol (HdA: 0.47%; HdB: 0.71%).

The essential oil yields obtained in the present study were compared to those reported by da Silva et al. (2020), who hydrodistilled fresh leaves of Eucalyptus grandis × Eucalyptus urophylla cultivated in Brazil for 4 h and achieved an essential oil yield of 1.03% (w/w). In contrast, despite using a longer hydrodistillation period of 8 h, the yields obtained in the present study were much lower, with 0.118% (w/w) for HfA and 0.026% (w/w) for HfB. Essential oil yield in aromatic plants is affected by a complex group of factors, and the observed variations in the yield may be attributed to differences in cultivation practices and environmental conditions such as geographical location, soil type, rainfall, climate, and air temperature (Gilles et al. 2010; Malaka et al. 2022). Prolonged extraction at high temperatures may cause thermal degradation of essential oils. For example, the yields of Piper nigrum (green pepper) essential oil increased as the hydrodistillation time extended from 30 min to 180 min, but they decreased when further prolonged to 300 min (Dao et al. 2020).

This study also highlighted the influence of tree age on essential oil yield. Leaves harvested from older trees yielded significantly less essential oil. Essential oils accumulate in the specialized glands during leaf development but may diminish after full leaf expansion via evaporation and leakages (Fikremariam et al. 2019). In addition, the branches become wider and denser as trees grow, which may reduce sunlight exposure to the leaves, impairing photosynthesis and reducing carbon flux for secondary metabolite production (Fajar et al. 2019). These findings align with the research of Shiferaw et al. (2019), who reported a decline in essential oil yield from 1.32% to 1.10%, obtained from Eucalyptus globulus as the age of the tree increased from 3 years to 8 years.

Table 1. Phytochemical Composition of Eucalyptus grandis × Eucalyptus urophylla Essential Oils Identified Using NIST 23 Mass Spectral Library

Table 2. Phytochemical Composition of Essential Oils Obtained from Eucalyptus grandis × Eucalyptus urophylla and Its Parental Species via Hydrodistillation

A comparative summary of essential oil compositions with previous studies is presented in Table 2. Despite the differences in yield, the predominant constituents identified in this study were generally consistent with earlier studies. 1,8-cineole was consistently the major component across all essential oil samples. α-Pinene was also found in considerable abundance in most samples, except in HfB from the current study, as well as the FLEOs reported by Insuan et al. (2021) and Borges et al. (2024). In addition, the high percentage of α-terpinyl acetate reported by da Silva et al. (2020) and Borges et al. (2024) in their FLEOs from the same Eucalyptus hybrid was similarly observed as one of the dominant constituents in all our samples. In contrast, p-cymene, detected in high proportions in some E. grandis and E. urophylla essential oils, was absent in the current study. These findings suggest that variations in chemical composition may result from differences in species, extraction parameters, and cultivation environments. Moreover, phytochemical analysis revealed that essential oil composition varied according to the plant age and leaf condition, indicated by the presence of unique compounds detected in younger and older Eucalyptus hybrids, as well as between fresh and dried leaf samples (Table 1). Disparities in the phytochemical profiles of essential oils between fresh and dried plant samples have been recorded in earlier studies on basil and thyme essential oils (Ghasemi Pirbalouti et al. 2013; Rahimmalek and Goli 2013). Such variations may arise due to the degradation or transformation of compounds via oxidation, glycoside hydrolysis, dehydration, and esterification during the drying process, as well as the rupture of plant cells that release the volatile compounds (Díaz-Maroto et al. 2004; Sewanu et al. 2015).

Bioactivities and Toxicity of Essential Oils

Table 3 summarizes the antibacterial activity of the essential oils against ten tested bacterial species. Generally, Gram-positive bacteria were more susceptible to the essential oils than Gram-negative bacteria. Among the essential oil samples, HdB displayed slightly stronger antibacterial activity against Gram-positive ATCC strains. It inhibited the growth of Bacillus cereus and Staphylococcus aureus ATCC strains at concentrations between 0.16 to 0.31 mg/mL, and Enterococcus hirae at 0.63 mg/mL, representing the lowest MIC values recorded among all tested essential oils. Although all essential oils, except HfA, inhibited the two clinically isolated S. aureus strains at 2.50 mg/mL, only HdB exhibited bactericidal activity against S. aureus SA-LWE23#1, a methicillin-resistant clinical isolate, at the same concentration. On the other hand, the Gram-negative bacteria were all generally less sensitive. Out of the five Gram-negative bacterial strains tested, only Klebsiella pneumoniae ATCC strain showed susceptibility to all four essential oils, with MIC values ranging from 1.25 to 2.50 mg/mL, and with bactericidal effect observed only for HfB at 2.50 mg/mL.

The antibacterial results demonstrated that Gram-positive bacteria were generally more sensitive to the essential oils than Gram-negative bacteria. This difference can be explained by the presence of a lipopolysaccharide outer membrane in Gram-negative bacteria, which serves as a hydrophilic barrier against the penetration of hydrophobic compounds (Simpson et al. 2015). In contrast, the more permeable cell wall in Gram-positive bacteria allows hydrophobic compounds in essential oils to penetrate them and interact with the phospholipid bilayer, leading to increased ion permeability, intracellular components leakage, and bacterial enzyme disruption (Silva et al. 2011; Barbosa et al. 2016). Furthermore, in a related study, Zhou et al. (2021) demonstrated stronger antibacterial activity from E. grandis × E. urophylla leaf essential oils obtained using steam distillation, which inhibited Escherichia coli and Pseudomonas aeruginosa at concentrations of 0.091 mg/mL and 0.023 mg/mL, respectively; unlike the current study, where no inhibition was observed against both bacterial species. Additionally, their reported MIC against Bacillus cereus was 0.045 mg/mL, which is substantially lower than the MIC values (0.16-0.63 mg/mL) found in this study (Table 3).

Table 3. Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations of Eucalyptus grandis × Eucalyptus urophylla Essential Oils Against Bacterial Species

The antifungal activity of essential oil samples was assessed against eight fungal species. As shown in Table 4, the antifungal efficacies of the four essential oils did not differ noticeably, as the variations in MIC and MFC values for each fungal strain did not exceed a two-fold difference. The tested yeasts demonstrated susceptibility to all four essential oils, with MIC and MFC values ranging from 0.16 to 1.25 mg/mL and 0.31 to 2.50 mg/mL, respectively. Candida tropicalis was the most susceptible among the Candida species, with the lowest MIC values of 0.16 to 0.31 mg/mL recorded for HdA and HfB. Notably, all four essential oils exhibited fungicidal activity against Cryptococcus neoformans, with an MFC of 0.31 mg/mL. Strong antifungal activity was also observed against the ringworm-causing dermatophyte Trichophyton rubrum, where the HfB and HdB achieved the lowest MFC value of 0.04 mg/mL, followed by 0.08 mg/mL for HfA and HdA, as their MFC values were <0.10 mg/mL (Saraiva et al. 2011). In contrast, Aspergillus fumigatus exhibited high resistance to the essential oils, with no inhibitory effects observed for any of the samples. To the best of our knowledge, this is the first study of the essential oils from E. grandis × E. urophylla leaves using hydrodistillation against human pathogens. Previous antimicrobial studies on essential oils of this hybrid all deployed the steam distillation technique, as summarized in Table 5.

Table 4. Minimum Inhibitory Concentrations and Minimum Fungicidal Concentrations of Eucalyptus grandis × Eucalyptus urophylla Essential Oils Against Fungal Species

This study assessed the larvicidal activity of essential oils obtained from Eucalyptus hybrid leaves by exposing third instar larvae of Ae. aegypti and Ae. albopictus to five concentrations ranging from 25 to 400 µg/mL for 24 h and 48 h. No mortality was observed in the negative control (1% methanol), while temephos (positive control) caused 100% mortality to the larvae of both species within 24 h of treatment. In the assay against Ae. aegypti larvae (Fig. 2), essential oils derived from younger Eucalyptus hybrid trees (HfA and HdA) consistently induced higher larval mortality after 48 h compared to those obtained from older Eucalyptus hybrid trees (HfB and HdB).

Table 5. Reported Bioactivities of Essential Oils Obtained from Eucalyptus grandis × Eucalyptus urophylla or Its Parental Species

Specifically, HfA and HdA achieved over 60% mortality at the lowest concentration of 25 µg/mL (HfA: 62.8% ± 24.0%; HdA: 69.0% ± 17.4%) and reached complete mortality at 200 and 400 µg/mL after 48 h. In contrast, HfB and HdB exhibited less than 15% mortality (HfB: 12.5% ± 9.57%; HdB: 7.50% ± 6.45%) at the lowest concentration and only achieved complete mortality at the highest concentration. Comparatively, Ae. albopictus larvae were more susceptible to the essential oils. HfA, HdA, and HfB induced 100% mortality at 200 and 400 µg/mL within 24 h, while HdB required 48 h to reach the same effect (Fig. 3). Although HdB consistently demonstrated lower larval mortality than the essential oils from younger Eucalyptus hybrid trees (HfA and HdA), HfB unexpectedly exhibited higher efficacy at lower concentrations, producing 77.5% ± 11.9% mortality at 50 µg/mL and 95.0% ± 0% at 100 µg/mL after 48 h.

Fig. 2. Larval mortality rate of Aedes aegypti at five concentration levels of Eucalyptus grandis × Eucalyptus urophylla essential oils after 24 h (A) and 48 h (B) post-exposure. Bars with different letters (a, b, c) represent significant differences (P < 0.05) between concentrations for each sample. HfA: Fresh leaf essential oil from Eucalyptus hybrid aged 17 to 31 months; HdA: Dried leaf essential oil from Eucalyptus hybrid aged 17 to 31 months; HfB: Fresh leaf essential oil from Eucalyptus hybrid aged 40 to 50 months; HdB: Dried leaf essential oil from Eucalyptus hybrid aged 40 to 50 months

Fig. 3. Larval mortality rate of Aedes albopictus at five concentration levels of Eucalyptus grandis × Eucalyptus urophylla essential oils after 24 h (A) and 48 h (B) post-exposure. Bars with different letters (a, b, c) represent significant differences (P < 0.05) between concentrations for each sample. HfA: Fresh leaf essential oil from Eucalyptus hybrid aged 17 to 31 months; HdA: Dried leaf essential oil from Eucalyptus hybrid aged 17 to 31 months; HfB: Fresh leaf essential oil from Eucalyptus hybrid aged 40 to 50 months; HdB: Dried leaf essential oil from Eucalyptus hybrid aged 40 to 50 months

Four-way ANOVA analysis demonstrated that larval mortality rates differed significantly (P < 0.001) across essential oils, exposure times, mosquito species, and concentration levels. Furthermore, interactions among these variables were also statistically significant (P < 0.05), except for the interaction between exposure time and essential oil (P = 0.057), as shown in Table 6. Probit regression analysis was performed to determine the median lethal concentration (LC₅₀) and 95% lethal concentration (LC₉₅) of the essential oils. Among the four samples, HfA exhibited the strongest larvicidal activity against Ae. aegypti, consistently demonstrating the lowest LC₅₀ values across both 24 h (52.3 µg/mL) and 48 h (14.3 µg/mL), along with corresponding LC₉₅ values of 130 µg/mL and 71.9 µg/mL, respectively (Table 7). For Ae. albopictus, HfB was most effective in killing the larvae, demonstrating the lowest LC₅₀ of 67.3 µg/mL after 24 h and the corresponding LC₉₅ of 131 µg/mL. Its efficacy further increased after 48 h, with the LC₅₀ being reduced to 42.1 µg/mL and the LC₉₅ to 81.2 µg/mL (Table 7). These findings indicate that the larval susceptibility to essential oils was influenced by the mosquito species.

Table 6. Four-way ANOVA Analysis for Mosquito Larvicidal Activity of Eucalyptus grandis × Eucalyptus urophylla Essential Oils

Table 7. Lethal Concentrations and Probit Analysis of Eucalyptus grandis × Eucalyptus urophylla Essential Oils against Third-Instar Larvae of Aedes Mosquitoes

Moreover, morphological deformities were observed in the treated larvae compared to the untreated controls (Fig. 4). Larvae of both Ae. aegypti and Ae. albopictus exposed to the essential oil samples showed notable elongation in the neck region and blackening of the midgut.

Fig. 4. Elongated necks (circle) and blackened midgut (arrow) were observed in the third instar larvae treated with essential oils of Eucalyptus grandis × Eucalyptus urophylla at 400 µg/mL (30× magnification under stereo microscope). (a) Control, untreated Ae. aegypti larvae; (b) HfA, Aedes aegypti; (c) HdA, Ae. aegypti; (d) HfB, Ae. aegypti; (e) HdB, Ae. aegypti; (f) Control, untreated Ae. albopictus larvae; (g) HfA, Ae. albopictus; (h) HdA, Ae. albopictus; (i) HfB, Ae. albopictus; (j) HdB, Ae. albopictus; HfA: Fresh leaf essential oil from Eucalyptus hybrid aged 17 to 31 months; HdA: Dried leaf essential oil from Eucalyptus hybrid aged 17 to 31 months; HfB: Fresh leaf essential oil from Eucalyptus hybrid aged 40 to 50 months; HdB: Dried leaf essential oil from Eucalyptus hybrid aged 40 to 50 months

The mosquito larvicidal activity of E. grandis × E. urophylla essential oils observed in this study was also compared to those reported in the literature (Table 5). The hybrid essential oils in the study of Gallon et al. (2020) showed higher Ae. aegypti larval mortality at 100 μg/mL than in the present study. However, the LC₅₀ values in the present study were lower than those described by Cheng et al. (2009a) against Ae. albopictus after 24 h post-treatment. Moreover, morphological deformities observed in the treated Aedes larvae, which revealed elongated necks and blackened midguts, are consistent with previous studies and may reflect underlying physiological damage. For instance, Seye et al. (2021) reportedly found intestinal tissue degradation, muscular disruption, and damaged microvilli in Ae. aegypti larvae that were treated with Cymbopogon citratus (lemongrass) essential oil. Similarly, Soonwera and Phasomkusolsil (2016) reported multiple deformities in Ae. aegypti larvae treated with essential oils of C. citratus and Syzygium aromaticum (clove), including elongated necks, enlarged thorax, and degraded respiratory tracheae and digestive tract, which suggested possible disruptions in hormonal regulation and chitin synthesis during the molting process.

Figure 5 illustrates the concentration-dependent mortality rate of brine shrimp nauplii following 24-h exposure to various concentrations of essential oil samples. A similar mortality trend was observed across all four essential oils, with 100% survival of exposed nauplii at the lower concentrations of 1 and 10 µg/mL. At 100 µg/mL, HfA, HdA, and HfB caused no mortality, whereas HdB resulted in a slight mortality rate of 3.33% ± 5.77%. Complete mortality was observed for all essential oils at the highest concentrations of 500 µg/mL and 1000 µg/mL.

Fig. 5. Mortality rate of Artemia franciscana shrimp nauplii at different concentration levels of Eucalyptus grandis × Eucalyptus urophylla essential oils after 24 h post-exposure. No nauplii mortality was observed at 1 and 10 µg/mL. Bars with different letters (a, b) represent significant differences (P < 0.05) between concentrations for each sample. HfA: Fresh leaf essential oil from Eucalyptus hybrid aged 17 to 31 months; HdA: Dried leaf essential oil from Eucalyptus hybrid aged 17 to 31 months; HfB: Fresh leaf essential oil from Eucalyptus hybrid aged 40 to 50 months; HdB: Dried leaf essential oil from Eucalyptus hybrid aged 40 to 50 months.

Significant differences in nauplii mortality rates were observed only between concentration levels (P < 0.001) in the two-way ANOVA analysis (Table 8), with no significant effects among the essential oil samples (P = 0.403) or their interaction (P = 0.446). Probit regression analysis revealed LC₅₀ and LC₉₅ values of 222 and 468 µg/mL, respectively, for all HfA, HdA, and HfB. In contrast, a lower LC₅₀ of 209 µg/mL and LC₉₅ of 438 µg/mL were determined for HdB (Table 9). The positive control, potassium dichromate, revealed a much lower LC₅₀ of 20.2 µg/mL and LC₉₅ of 172 µg/mL, indicating that the essential oils possess substantially lower toxicity relative to the control.

Table 8. Two-way ANOVA Analysis for Artemia franciscana Nauplii Mortality of Eucalyptus grandis × Eucalyptus urophylla Essential Oils

The essential oil’s bioactivity is highly related to its phytochemical composition. In this study, the essential oils contained a relatively higher proportion of terpenes and terpenoids, which are known for their bioactivities. Specifically, 1,8-cineol, α-terpinyl acetate, and α-pinene have demonstrated antimicrobial properties against various bacteria and fungi (Ložienė et al. 2018; Marei et al. 2019; Ivanov et al. 2021).

Table 9. Lethal Concentrations and Probit Analysis of Eucalyptus grandis × Eucalyptus urophylla Essential Oils against Artemia franciscana Nauplii

Moreover, Perumalsamy et al. (2009) reported the larvicidal activities of 1,8-cineol, limonene, α-pinene, and α-terpineol against third instar Ae. aegypti larvae, with LC₅₀ values ranging from 24.5 to 112 µg/mL.

Cheng et al. (2009b) also documented an LC₅₀ of 74.0 µg/mL for α-pinene against Ae. albopictus larvae. However, the overall bioactivity of the essential oils was likely attributable to the synergistic or additive interactions between components, rather than from the individual components alone. Badr et al. (2021) revealed that pure α-terpinyl acetate exhibited greater antifungal and antibacterial activities on Candida albicans (half maximal effective concentration, EC₅₀ = 0.3 mg/mL) and S. aureus (MIC = 0.8 mg/mL) than the α-terpinyl acetate-rich lavender essential oil (EC₅₀ = 0.6 mg/mL and MIC = 3.0 mg/mL). Additionally, Mulyaningsih et al. (2010) reported synergistic antibacterial effects between aromadendrene and 1,8-cineol in inhibiting multidrug-resistant bacteria, while Sarma et al. (2019) found that the binary combinations of 1,8-cineol and α-pinene at a 1:1 ratio demonstrated synergistic larvicidal activity against Ae. aegypti, which achieved >90% larval mortality after 24 h. Notably, in the present study, no distinguishable differences in bioactivities were observed between the fresh and dried leaf essential oils or between the younger and older trees. Therefore, constituents such as 1,8-cineole, α-terpinyl acetate, and α-pinene, which were persistently detected in all tested samples, as well as their possible synergistic interactions, are proposed to contribute to the observed bioactivities. Their relatively stable presence across both tree age groups and leaf conditions also indicates that these major constituents are not susceptible to loss during leaf drying or tree maturation.

Importantly, comparison of the LC₅₀ values for brine shrimp lethality and Aedes mosquito larvicidal  assays revealed relatively lower concentrations were required to achieve larval mortality. Given that the brine shrimp lethality assay is widely used to assess the toxicity of substances towards aquatic organisms, the current findings suggest that essential oils of  E. grandis × E. urophylla  are relatively safe for non-target aquatic ecosystems while retaining their potency as natural mosquito larvicide.

CONCLUSIONS

1. Hydrodistillation of fresh and dried leaves from Eucalyptus grandis × Eucalyptus urophylla at two different tree age groups successfully yielded essential oils, with younger trees consistently producing significantly higher yields.
2. Gas chromatography-mass spectrometry (GC-MS) analysis identified 1,8-cineol and α-terpinyl acetate as the dominant chemical components across all essential oil samples.
3. Although there was no notable variation in bioactivity observed between fresh leaf essential oils (FLEO) and dried leaf essential oils (DLEO), as well as between younger and older Eucalyptus hybrid trees, the essential oils exhibited stronger antifungal activity, particularly against the dermatophyte Trichophyton rubrum, than antibacterial effects.
4. The essential oils demonstrated concentration-dependent mosquito larvicidal activity against Aedes aegypti and Aedes albopictus, with effective larval mortality achieved at concentrations lower than those required to produce toxicity in the brine shrimp nauplii, suggesting a favorably safe level for non-target aquatic organisms.
5. The bioactivity of the essential oils could be attributed to the known antimicrobial and larvicidal properties of major components of the essential oils, as well as the possible synergistic interactions between the components.
6. The findings of this study are indicative of the potential of essential oils from E. grandis × E. urophylla as natural disinfectants and mosquito larvicides, offering a low-risk alternative to synthetic chemicals for combating infectious and vector-borne diseases. This aligns with the Sustainable Development Goal (SDG) 3: Good Health and Well-being. Furthermore, by exploring the valorization of agro-industrial wastes such as Eucalyptus hybrid leaves, the study contributes to SDG 12: Responsible Consumption and Production, promoting sustainable applications of natural resources.

ACKNOWLEDGMENTS

The work was supported by the funding from Edubest Plantation Holdings Sdn. Bhd. (Number: 8168/0001). The funders had no role in the study design, collection, analysis and interpretation of data, in the writing of the manuscript and in the decision to submit the article for publication.

Conflict of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Article submitted: September 6, 2025; Peer review completed: October 25, 2025; Revised version received and accepted: November 9, 2025; Published: November 17, 2025.

DOI: 10.15376/biores.21.1.237-266

APPENDIX

Table S1. Chemical Composition of the Fresh Leaf Essential Oils Obtained from Eucalyptus grandis × Eucalyptus urophylla with Tree Ages of 17 to 31 months (HfA) Using Gas Chromatography-Mass Spectrometry

Table S2. Chemical Composition of the Dried Leaf Essential Oils Obtained from Eucalyptus grandis × Eucalyptus urophylla with Tree Ages of 17 to 31 months (HdA) Using Gas Chromatography-Mass Spectrometry

Table S3. Chemical Composition of the Fresh Leaf Essential Oils Obtained from Eucalyptus grandis × Eucalyptus urophylla with Tree Ages of 40 to 50 months (HfB) Using Gas Chromatography-Mass Spectrometry

Table S4. Chemical Composition of the Dried Leaf Essential Oils Obtained from Eucalyptus grandis × Eucalyptus urophylla with Tree Ages of 40 to 50 months (HdB) Using Gas Chromatography-Mass Spectrometry