The leaves of M. cajuputi and M. quinquenervia were collected in Phong My, Phong Dien, Thua Thien Hue, Vietnam [(16˚30'56''N, 107˚18'08''E) and (16˚30'41''N, 107˚16'56''E)] in May, 2023, while the leaves of M. leucadendra were collected in An Minh Bac, U Minh Thuong, Kien Giang, Vietnam (9˚37'06''N, 105˚05'50''E) in June, 2023. These plant materials were identified by Dr Le Tuan Anh (Mientrung Institute for Scientific Research, Vietnam National Museum of Nature, Thua Thien Hue, Vietnam) and the voucher specimens (MISR2023-7, MISR2023-8 and MISR2023-11) were kept in the herbarium of the Mientrung Institute for Scientific Research, Vietnam National Museum of Nature.

Fresh leaves of M. cajuputi, M. quinquenervia and M. leucadendra (200 g) were cut into small sections and then subjected to steam distillation using a glass apparatus to extract the EOs for 2 h at normal pressure. The EOs were then collected, dried with 0.5 g Na₂SO₄ (Merck, KGaA), stored in the dark and sealed in vials at 4˚C until further chemical analysis and biological activity testing.

The EOs extracted from the leaves of M cajuputi, M. quinquenervia and M. leucadendra were analyzed via GC-MS using a Shimadzu GCMS-QP2010 Plus system (Shimadzu Corporation). This system was equipped with a flamer ionization detector (FID) and an Equity-5 capillary column (30 m x 0.25 mm, 0.25 µm film thickness). The EOs were diluted to a concentration of 1% in n-hexane and 1.0 µl of the solutions were injected into the instrument for analysis. The GC was operated with helium as carrier gas at a flow rate of 1.5 ml/min. The GC oven temperature was initiated at 60˚C for 2 min, then increased to 240˚C at a rate of 4˚C/min and maintained for 10 min before further programming to 280˚C at a rate of 5˚C/min. The injector temperature was set at 260˚C. Mass spectrometry was performed at 70 eV in a mass range of 40-500 amu with a sampling rate of 0.5 scan/sec.

The chemical components of the EOs were identified by comparing their relative retention indices (RI) with those of a series of reference n-alkanes C7-C40 (Merck, KGaA). Additionally, the identification relied on computer matching against commercial libraries (WILEY7 Library and NIST11 Library), as well as MS and RI data of known compounds from the literature (21,22). The chemical structures of the compounds were drawn using ChemDraw Ultra 8.0 (CambridgeSoft Corporation).

The antimicrobial activity of the EOs was evaluated against a panel of five reference microorganisms, including Staphylococcus aureus (ATCC 25923; S. aureus), Enterococcus faecalis (ATCC 29212; E. faecalis), Escherichia coli (ATCC 25922; E. coli), Pseudomonas aegurinosa (ATCC 27853; P. aegurinosa) and Candida albicans (ATCC 10231; C. albicans). The minimum inhibitory concentrations (MICs) of the EOs against these microorganisms were determined using the broth microdilution method as previously reported by Dat et al (23). Briefly, the bacterial inoculum (100 µl at a concentration of 1x10⁶ CFU/ml) was introduced into the wells of 96-well plates containing various concentrations of the EOs (100 µl) ranging from 1.0 to 2,560 µg/ml. The plate was incubated at 37˚C for 24 h, followed by the measurement of absorbance at 630 nm using an ELx800 absorbance microplate reader (BioTek Instruments, Inc.; Agilent Technologies). The MICs of the antibacterial EOs were defined at the lowest concentration where no bacterial growth was observed through absorbance records at 630 nm. Similarly, for yeast, the yeast inoculum (100 µl at a concentration of 2-5x10⁵ CFU/ml) was added to wells containing the EOs (100 µl) at various concentrations ranging from 1.0 to 2,560 µg/ml in 96-well plates, which were then incubated at 28˚C for 48 h. The MICs of the anti-yeast EOs were determined at the lowest concentration where no yeast growth was observed through absorbance records at 530 nm using an ELx800 absorbance microplate reader. The antibiotics, ciprofloxacin and fluconazole (MilliporeSigma) ranging from 0.5 to 8.0 µg/ml, served as the positive controls for bacteria and yeast, respectively. The experiments were performed in triplicate.

The inhibitory effect of the EOs on α-amylase (MilliporeSigma) was assessed according to the method previously described by Nguyen et al (24). In brief, the starch azure solution supplemented with 0.01 M CaCl₂ in 0.05 M Tris-HCl buffer (pH 6.9) (MilliporeSigma) was boiled for 5 min and pre-incubated at 37˚C for 5 min. The reaction containing 50 µl of the EO, 50 µl of the substrate solution, and 25 µl of α-amylase solution (2 U/ml) was incubated in 96-well plates at 37˚C for 10 min. The reaction was terminated by the addition of 75 µl of 50% acetic acid, followed by the measurement of absorbance at 650 nm using an ELx800 absorbance microplate reader (BioTek Instruments, Inc.). The inhibitory activity was calculated as follows: Inhibition (%)=100 x [1-(A_s-A_bs)/(A_c-A_cb)], where: A_s is the absorbance of the sample, A_sb is the absorbance of the sample blank, A_c is the absorbance of the control, and A_cb is the absorbance of the control blank. Acarbose (MilliporeSigma) was used as a positive control with tested concentrations ranging from 10 to 200 µl/ml. The IC₅₀ value was calculated using GraphPad Prism v8.0 (Dotmatics). The experiments were performed in triplicate and data are expressed as the mean ± standard deviation.

ii) Glucosidase inhibitory activity. The inhibitory effect of the EOs on α-glucosidase (MilliporeSigma) was determined according to the method previously described by Nguyen et al (24). Briefly, the reaction containing 50 µl of the EO and 100 µl of α-glucosidase solution (0.5 U/ml) in 0.1 M potassium phosphate buffer (pH 6.8) (MilliporeSigma) was incubated in 96-well plates at 37˚C for 10 min. The reaction was initiated by the addition of 50 µl of 5 mM 4-Nitrophenyl β-D-glucopyranoside (MilliporeSigma), followed by incubation at 37˚C for 30 min. The reaction was then terminated by the addition of 75 µl of 0.2 M Na₂CO₃ (MilliporeSigma) and absorbance was recorded at 405 nm using an ELx800 absorbance microplate reader. The inhibitory activity was calculated as follows: Inhibition (%)=100 x [1-(A_s-A_bs)/(A_c-A_cb)], where: A_s is the absorbance of the sample, A_sb is the absorbance of the sample blank, A_c is the absorbance of the control, and A_cb is the absorbance of the control blank. Acarbose (MilliporeSigma) was used as a positive control with tested concentrations ranging from 10 to 200 µl/ml. The IC₅₀ value was calculated using GraphPad Prism v8.0 (Dotmatics). The experiments were performed in triplicate and data are expressed as the mean ± standard deviation.

iii) XO inhibitory activity. The inhibitory effect of the EOs on XO (MilliporeSigma) was determined according to the method described in the study by Dat et al (23). In summary, the reaction mixture containing 50 µl of the EO, 35 µl of 70 mM phosphate buffer (pH 7.5) and 30 µl of enzyme solution (0.01 U/ml) was pre-incubated at 25˚C for 15 min, followed by the addition of 60 µl of 150 mM xanthine (MilliporeSigma). Subsequently, the reaction was incubated at 25˚C for 30 min, followed by the addition of 25 µl of 1.0 N HCl (MilliporeSigma). The absorbance of the reaction was then measured at 290 nm using an ELx800 absorbance microplate reader. The inhibitory activity was calculated as follows: Inhibition (%)=100 x [1-(A_s-A_bs)/(A_c-A_cb)], where: A_s is the absorbance of the sample, A_sb is the absorbance of the sample blank, A_c is the absorbance of the control and A_cb is the absorbance of the control blank. Allopurinol (MilliporeSigma) was used as a positive control with tested concentrations ranging from 1.0 to 50 µl/ml. The IC₅₀ was calculated using GraphPad Prism v8.0. The experiments were performed in triplicate and the data are expressed as the mean ± standard deviation.

iv) AChE inhibitory activity. The inhibitory activity of the EOs on AChE (MilliporeSigma) was determined according to the method previously described by Thai et al (25). The reaction containing 100 µl of 3 mM 5,5-dithiobis-2-nitrobenzoate (MilliporeSigma), 20 µl of the EO and 20 µl of AChE (0.2 U/ml; MilliporeSigma) was pre-incubated at 25˚C for 15 min, and then initiated by the addition of 20 µl of 15 mM acetylthiocholine iodide (MilliporeSigma). Following incubation at 25˚C for 20 min, the reaction was terminated by the addition of 20 µl of 4% SDS (MilliporeSigma). Subsequently, the absorbance of the reaction was measured at 415 nm using an ELx800 absorbance microplate reader. The inhibitory activity was calculated as follows: Inhibition (%)=100 x [1-(A_s-A_bs)/(A_c-A_cb)], where: A_s is the absorbance of the sample, A_sb is the absorbance of the sample blank, A_c is the absorbance of the control and A_cb is the absorbance of the control blank. Galantamine (MilliporeSigma) was used as a positive control with tested concentrations ranging from 1.0 to 20 µl/ml. The IC₅₀ value was calculated using GraphPad Prism v8.0 software (Dotmatics). The experiments were performed in triplicate and the data are expressed as the mean ± standard deviation.

The antimicrobial activity of the EOs presented in Table II demonstrated that the EOs of the Melaleuca species examined in the present study exhibited antimicrobial activity against all tested microorganisms (S. aureus, E. faecalis, E. coli, P. aegurinosa, C. albicans) with MICs in the range of 640-2560 µg/ml. The leaf EO of M. cajuputi exhibited the highest antimicrobial activity against E. coli and P. aegurinosa with MICs of 640 µg/ml, whereas the leaf EOs of M. quinquenervia and M. leucadendra exhibited the highest antimicrobial activity againts S. aureus with MIC of 640 µg/ml. The difference in the antimicrobial activity of the EOs derived from the Melaleuca species may be attributed to the varying chemical composition and content of bioactive compounds present in the EOs.

Previous investigations have revealed that the EOs of Melaleuca species exhibit antimicrobial activity against a broad spectrum of pathogenic microbes. The EO of M. cajuputi has been shown to inhibit the growth of a range of Gram-positive bacteria, including Bacillus cereus, Bacillus subtilis, Corynebacterium diphtheriae, Corynebacterium minutissimum, Enterococcus faecium, Listeria monocytogenes, Micrococcus luteus, S. aureus, Staphylococcus capitis, Staphylococcus epidermidis, E. faecalis and Klebsiella spp. at concentrations ranging of 0.4-0.6% (32,33), Gram-negative bacteria such as Alcaligenes faecalis, Enterobacter cloacae, E. coli and Proteus vulgaris, as well as the fungi such as C. albicans, Gardnerella. vaginalis, Candida glabrata, Aspergillus niger, Penicillium notatum at concentrations ranging of 0.4-0.6% (15,34,35). The EO of M. quinquenervia has been reported to have effective antimicrobial activity against bacteria, including E. coli, S. aureus, P. aeruginosa, Staphylococcus epidermidis, Propionibacterium acnes, Streptococcus peroris, Klebsiella pneumonia, Acinetobacter baumannii and Proteus vulgaris with MICs of 0.5-16 mg/ml and fungi C. albicans, Candida tropicalis, Aspergillus niger with MICs of 0.2-4 mg/ml (17). The EO from M. leucadendra leaf has been shown to exhibit antibacterial activity against S. aureus and E. coli, Salmonella thiphymurium, Proteus mirabilis, Klebsiella pneumonie, E. coli, Enterobacter aerogenes, Providencia rettgeri, Shigella fexnerii, P. aeruginosa, E. faecalis, Staphylococcus saprophyticus and S. aureus with MICs of 7.8-62.5 mg/ml (10,36).

In vitro and in silico investigations have demonstrated that terpenes and terpenoids are the main active compounds in antimicrobial EOs (17,37). Furthermore, other biomolecules, such as phenols, alcohols and aldehydes found in the EOs, induce antimicrobial activity with varying specificity and effectiveness. These variations are often attributed to the functional groups within the EO and the hydrogen bonding dynamics during their interactions (38). Terpenes are recognized for their antimicrobial properties, mainly attributed to their ability to disrupt cell membranes, inhibit cell growth, and interfere with protein and DNA synthesis (39). Specific terpenes, such as carvone, carvacrol, eugenol, thymol and geraniol have demonstrated antibacterial effects (40), whereas compounds such as menthol, azadirachtin, ascaridol, toosendanin, methyl eugenol and volkensin have exhibited anti-fungal effects (41,42). These compounds have exhibited antimicrobial effects by compromising cellular membrane integrity (43). Monoterpenoids also exhibit antibacterial effects by disrupting microbial multiplication and development, as well as by interfering with their metabolic and physiological functions (44). Monoterpene terpineol isomers, such as terpinen-4-ol, α-terpineol and δ-terpineol have demonstrated effective inhibition against Gram-negative bacteria, particularly Shigella flexneri, by inducing permeability changes in bacterial membranes, leading to the release of nucleic acids and proteins, alongside a decrease in membrane potential (45). Eugenol exhibits potent bactericidal activity against Salmonella enterica serovar Typhimurium and S. aureus (46).

Previous investigations have also demonstrated the mechanisms of action of the main antimicrobial compounds in the EOs of Melaleuca. For instance, limonene and 1,8-cineole enhance the permeability of the bacterial membrane, whereas 1,8-cineole and viridiforol inhibit enzyme peptidoglycan glycosyltransferase (36). Limonene affects cell membrane permeability of in Gram-positive bacteria by attacking cell integrity and cell wall structure (47). Other compounds, such as lupene, guaiol and 1,8-cineole exhibit antifungal effects by inhibiting the target enzymes cellobiohydrolase, laccase and lignin peroxidase (48).

Enzyme inhibition activity. The enzyme inhibitory activities of the EOs presented in Table III revealed that the EOs of Melaleuca species exerted inhibitory effects on the enzymes α-amylase, α-glucosidase, AChE and XO. The leaf EO of M. cajuputi exhibited inhibitory activities against the enzymes α-amylase, α-glucosidase, AChE and XO with IC₅₀ values of 862.6±65.74, 1212±73.49, 765.7±26.14 and 331.9±20.64 µg/ml, respectively; the leaf EO of M. quinquenervia exhibited enzyme inhibitory effects with IC₅₀ of 786.3±58.42, 1453±93.79, 815.5±20.72, and 380.1±17.85 µg/ml, respectively; and the leaf EO of M. leucadendra exhibited enzyme inhibitory effects IC₅₀ of 714.3±38.09, 1066±45.01, 535.5±19.84 and 433.8±18.02 µg/ml, respectively. The variations in the composition and content of bioactive compounds presented in the EOs of Melaleuca species may contribute to the differences in their enzyme inhibitory properties. Therefore, further research is needed to identify key compounds in the EOs that inhibit the enzymes.

Although the antimicrobial activity of the EOs of Melaleuca species has been reported in recent investigations, only a limited number studies on the enzyme inhibitory activity of Melaleuca species against α-amylase, α-glucosidase, AChE, XO have been identified to date. As regards the AChE and BChE enzymes, the EOs of M. cajuputi leaf and M. citrina leaf exhibited AChE inhibitory effects with 21.18±0.54 and 71.77±2.11% inhibition, respectively at a concentration of 100 µg/ml (49). The methanol extract from M. cajuputi leaf also exhibited potent AChE inhibitory effects with IC₅₀ of 282 µg/ml (50). The EO from M. alternifolia leaf showed strong AChE and BChE inhibitory effects with IC₅₀ of 153.7±1.25 and 85.6±0.7 µg/ml, respectively (51). In the case of the XO enzyme, the methanol and methanol-water extracts from M. leucadendra have been reported to have a XO inhibitory activity with IC₅₀ values of 76.7 and 78.9 µg/ml, respectively (52). Involving α-amylase and α-glucosidase enzymes, the EOs from M. alternifolia leaf and M. viridiflora leaf demonstrated α-amylase inhibitory activity with 14 and 28% inhibition at a concentration of 0.67 mg/ml (53). To the best of our knowledge, the findings of the present study provide additional insight into the enzyme inhibitory properties of the leaf EO from Melaleuca species that have not been reported in previous research. However, a limitation of the present study is that the potential activity of compounds in the EOs was not investigated. Therefore, further studies are required to determine the most active compounds in the EO, such as molecular docking, in silico methods for drug design and discovery, from which the most potential.

The present study determined the chemical composition of the leaf EOs of M. cajuputi, M. quinquenervia and M. leucadendra with the dominance of oxygenated monoterpenes and sesquiterpenes. The present study also highlighted the chemical composition variation of EOs of Melaleuca species over geographical regions, in agreement with previous observations (7-12,14,26). The subsequent bioassays revealed that the leaf EOs of M. cajuputi, M. quinquenervia and M. leucadendra exhibited antimicrobial, as well as enzyme inhibitory activities against α-amylase, α-glucosidase, AChE and XO. The findings presented herein provide additional insight into the enzyme inhibitory properties of the leaf EO from Melaleuca species not reported in previous research.