Materials

The leaves of M. alternifolia were harvested after about 9 months of age in Lam Dong province, Vietnam (Coordinates: 11°40'1.20"N, 107°19'58.80"E). The plant specimen (coded MA151022VST) has been archived at the Plant Biotechnology Laboratory of the Institute of Biotechnology and Food Technology, Ho Chi Minh City University of Industry. On average, the per-batch yield was approximately 50 kg leaves/batch and MaEO was extracted using steam distillation for 3 h at 100 °C, with the yield obtained at about 1.5% (v/w), and the EO was stored in a dark sealed bottle at 4 °C before analysis.

Bacterial strains used in this study include Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778), Escherichia coli (ATCC 25922), and Salmonella enterica (ATCC 10376).

Determination of physicochemical properties of MaEO

The freezing point (FP), relative density (RD), absolute density (AD), acid value (AV), esters value (EV), and saponification value (SV) were evaluated according to International Organization for Standardization including ^{ISO 1041 (1973)}, ^{279 (1998)}, ^{1242 (2023)}, and ^{7660 (1983)}, respectively.

Determination of fragrance retention (FR) of MaEO

According to the procedure described by ^{Mahajan (2022)}, fragrance retention (FR) is determined by the concentration of the flavoring ingredient and its retention time, with some minor corrections. EO was mixed into concentrations (5, 10, 15, 20, and 25%, v/v) in 96% ethanol. Next, three drops of EO were placed on the odor test paper, and a few seconds were allotted for the EO to penetrate the paper. Finally, the time until the smell of EOs disappeared was calculated and the results recorded.

Gas chromatography-mass spectrometry (GC-MS)

The chemical volatile composition of EO was analyzed using the GC-MS method. 1 μL of EO was injected into a gas chromatograph (Shimadzu Nexis GC-2030, Japan) with a versatile capillary column (Rtx-5sil-MS, 30 m×0.25 mm×0.25 μm, Restek Technologies, USA) equipped with a quadrupole mass analyzer (Shimadzu GC-MS-QP2020 NX, Japan). Helium was used as a carrier gas at a constant flow rate of 3 mL min^-1, and a split ratio of 10:1. The injection temperature was 250 °C and the temperature program was set as follows: initial temperature of 50 °C, held for 2 min, increased until 250 °C at a rate of 10 °C min^-1, and held for 5 min; finally, increased to 280 °C at a rate of 10 °C min^-1, and held for 3 min. Mass spectra were recorded at the ionization energy of 70 eV in EI mode (^{Hao and Quoc 2024}).

Determination of antioxidant capacity (AC) of MaEO

The AC of MaEO was determined using DPPH assay according to the procedure described by ^{Rahman et al. (2015)} with some minor corrections. EOs were diluted into concentrations (100, 200, 300, 400, 500, and 600 mg mL^-1) in 96% ethanol. 0.3 mL of the obtained EO and 3.7 mL DPPH 0.1 M in 96% ethanol solution were mixed. The mixture was kept in dark conditions for 30 min at room temperature. DPPH radical scavenging capacity (DPPH_RSC) of MaEO was expressed by the degree of color reduction of the DPPH solution, as determined by measuring the absorbance at a wavelength of 517 nm, and antioxidant capacity was calculated following equation 1, while vitamin C was used as a control. Percent inhibition plotted against EO concentration to estimate concentration gives 50% inhibition (IC₅₀).

Determination of the antibacterial activity (AA) of MaEO

The AA was determined by susceptibility testing by the agar plate diffusion method described by ^{Quyen and Quoc (2023)} with minor adjustments. First, a sterile spreader spread 100 μL of bacterial suspension (0.5 McFarland standard, approximately 1.5×10⁸ CFU mL^-1) onto Muller-Hinton agar medium (MHA). Then, paper plates with a diameter of 6 mm were impregnated with essential oils (5 μL) put on MHA media, while ampicillin (10 μg disc^-1) and 5% dimethyl sulfoxide (DMSO) solution (5 μL disc^-1) were used as a positive and negative control. Petri dishes were incubated for 24 h at 37 °C, and the diameter of the inhibition zone was measured in mm.

Statistical data analysis

All the experimental results were analyzed using Statgraphics Centurion software (Version 15.1.02). Every assay was done in triplicates. Analysis of variance (ANOVA) with Fisher's least significant difference procedure was used to determine the significant differences (P<0.05) between means.

Determination of physicochemical properties of Melaleuca alternifolia leaf essential oils

MaEO is a light-yellow liquid with a characteristic odor and a bitter taste. The physicochemical properties of EO are shown in Table 1. The pH value of MaEO is about 4.94 and this result is higher than that in some EOs from other materials, such as Melaleuca cajuputi leaves (pH=4.46) (^{Quoc 2021}) and Ceratonia siliqua pulp (pH=4.3) (^{Ouis and Hariri 2018}). The chemical component of EO affects pH strongly, leading to a significant difference in pH in materials.

Table 1 Physicochemical properties of Melaleuca alternifolia leave essential oils. 

The FP of EO remains undetermined at -40 °C; the oil still exists in liquid form, with no freezing phenomenon. The results show that the FP of MaEO must be below -40 °C; compared to other materials, FPs of the EO of M. cajuputi leaves and Ocimum gratissimum were -45 and -38 °C (^{Quoc 2021}; ^{Hao and Quoc 2024}). This indicated that FP depends on the chemical composition of the EO significantly. In addition, the RD and AD of the oil were also analyzed; both values are lower than one, 0.9064 and 0.9048 g mL^-1, respectively. These findings agree with the ^{ISO 4730 (2017)}, and they did not differ significantly from those of M. cajuputi (RD: 0.9102 and AD: 0.9086 g mL^-1) (^{Quoc 2021}) or M. arvensis (RD: 0.8987 and AD: 0.8959 g mL^-1) leaves' EO (^{Quoc 2022}). The obtained values also revealed that the MaEO belongs to the EO group lighter than water.

The AV, SV, and EV of MaEO were determined to be 2.10, 7.34, and 5.24 mg KOH g^-1 EO, respectively. In general, the AV is agreement with that of previous studies for other materials, such as C. siliqua pulp and seeds (AV: 3.82 and 2.2 mg g-1 EO) (^{Ouis and Hariri 2018}), basil EO (AV: 3.95 mg KOH g^-1 EO), and lemongrass (AV: 4.09 mg KOH g^-1 EO) (^{Mustapha 2018}), while the obtained SV is significantly lower than that of M. cajuputi leaves EO (SV: 28.05 mg KOH g^-1 EO) (^{Quoc 2021}), basil EO (SV: 198 mg KOH g^-1 EO), and lemongrass (SV: 143 mg KOH g^-1 EO) (^{Mustapha 2018}). The AV, SV, and EV are indicators used to assess the quality of the EO and they are influenced by distillation technique, cultivar, climatic conditions, harvest period, and chemical composition of the initial materials. The staying power is also the critical index to evaluate the quality of EO. In this study, FR of MaEO can reach a range of 6-10 h at 20-25%, and this shows that FR is quite long, similar to perfume at a fragrance concentration of 20-30%, and it can last for more than 6-8 h (^{Mahajan 2022}), while pure MaEO can last more than 24 h. This finding proves that MaEO has potential applications in cosmetics and the food industry.

Chemical composition of Melaleuca alternifolia leaves essential oils

The volatile compounds of MaEO were analyzed by GC-MS. The results are displayed in Table 2. A total of 45 compounds were identified and quantified in the EO isolated from the M. alternifolia leaves; they account for 99.35% of the oil and are tested by retention times ranging from 7 to 22 min. Compounds comprising the highest content in MaEO, include Terpinen-4-ol (44.55%), γ-Terpinene (19.42%), p-Cymene (8.75%), α-Terpinene (6.73%), and Terpineol (3.33%). These components play an important role in the quality of MaEO and there is variation in the chemical composition of EO from tea tree leaves collected in different regions. In previous studies, authors also used the distillation method to extract EOs from the initial material. For example, MaEO isolated from Tien Giang province (Vietnam) possess 19 volatile components: Terpinen-4-ol (36%), γ-Terpinene (17.8%), 1,8-Cineole (10%), etc. (^{Hòa et al. 2016}), while MaEO from Slovakia has 47 identified components, including Terpinen-4-ol (40.3%), γ-Terpinene (11.7%), 1,8-Cineole (7%), p-Cymene (6.2%), etc. (^{Borotová et al. 2022}), and while MaEO collected in Thailand has 24 components, including Terpinen-4-ol (30.42-34.76%), γ-Terpinene (25.08-26.23%), α-Terpinene (12.31-12.43%) and 1,8-Cineole (5.99-9.08%), etc. (^{Sukatta et al. 2011}). In this study, the main component of MaEO is Terpinen-4-ol and its content is higher than that in MaEO from other places. The MaEO from the Lam Dong province complied with the ^{ISO 4730 (2017)} standard analysis with few variations in the contents of major constituents. This difference in the chemical composition of MaEO can be explained due to different extraction methods, climatic conditions, harvesting periods, genes, etc.

Table 2 Chemical composition of M. alternifolia leaves essential oils. 

Antioxidant capacity (AC) of Melaleuca alternifolia leaves essential oils

Figures 1 and 2 show the efficiency of the antioxidant process of EO and control (vitamin C). An increase in MaEO concentration leads to an increase in AC. Based on the obtained curve, the IC₅₀ of MaEO was determined at 360 mg mL^-1 (Figure 1), while the IC₅₀ of vitamin C was only about 26 µg mL^-1 (Figure 2). The results show that the IC₅₀ of MaEO is much higher than that of vitamin C, showing that the AC of EO is very weak. These results are consistent with those of MaEO from Thailand (IC₅₀: 29.34-38.68 mg mL^-1) (^{Sukatta et al. 2011}) (Their AC is extremely low compared to the control). Besides, compared to some EOs from other materials, such as C. siliqua pulp and seeds (IC₅₀: 7.8 and 31.25 µg mL-1) (^{Ouis and Hariri 2018}), M. arvensis (IC₅₀: 330 mg mL^-1) (^{Quoc 2022}), the AC of MaEO is also weaker. This is a disadvantage when applying this EO in the cosmetic and food industry. Differences in AC of various EOs may be due to differences in their chemical composition.

Figure 1 Antioxidant capacity of the Melaleuca alternifornia leaves EO. 

Figure 2 Antioxidant capacity of vitamin C. 

Antibacterial activity (AA) of Melaleuca alternifornia leaves essential oils

Table 3 shows that MaEO exhibited to AA against four bacterial strains, and ampicillin was used as an inhibitory control against the bacteria used in the study. AA of ampicillin are arranged in order of susceptibility: E. coli > S. enterica>S.aureus/B. cereus. Similarly, AA of MaEO is arranged in order of susceptibility: E. coli >S. enterica/S. aureus/B. cereus. The AA of MaEO was 1.5 times stronger than that of ampicillin for S. aureus and B. cereus, while the AA of MaEO was much lower than that of ampicillin for S. enterica and E. coli. These obtained results are also different from those of MaEO collected from Thailand (^{Sukatta et al. 2011}) and other materials (C. siliqua pulp and seeds, M. cajuputi leaves, and O. gratissimum EO) (^{Ouis and Hariri 2018}; ^{Quoc 2021}; ^{Hao and Quoc 2024}). The AA of MaEO was observed in the presence of a high content of Terpinen-4-ol; according to ^{Halcón and Milkus (2004)}, Terpinen-4-ol strongly influences the bacterial cell wall and compromises the cytoplasmic membrane of bacteria (S. aureus), giving it a bacteriostatic and bactericidal effect. In addition, combining other bioactive ingredients, such as γ-Terpinene, 1,8-Cineole, α-Terpineol, etc., can also increase the ability to inhibit bacteria.

Table 3 Antibacterial zones of M. alternifornia leaves essential oils.