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Introduction

Vietnam is a Southeast Asian country characterized by a tropical monsoon climate with a rich vegetation system. Out of >12,000 plant species in Vietnam, 5,117 of these have medicinal value (1,2), representing a key source of active compounds for research or applications in medicine, pharmacy and the pharmaceutical industry. The objective of the present study was to investigate the chemical constituents and biological activities of the essential oils (EOs) of three Melaleuca species growing in Vietnam.

The genus Melaleuca (Myrtaceae) includes 280 species, of which four species have been found in Vietnam, including Melaleuca alternifolia, Melaleuca cajuputi, Melaleuca leucadendra and Melaleuca quinquenervia (1). According to traditional Vietnamese medicine, the species M. cajuputi, M. leucadendra and M. quinquenervia are widely used in the treatment of diseases, such as cold, flu, fever, malaria, indigestion, bone pain, diarrhea, inflammatory skin diseases, allergies and eczema (2). Previous studies on the chemical constituents of the plant EOs have demonstrated that the EOs of M. cajuputi are mainly composed of 1,8-cineol, α-pinene, γ-terpinene, p-cymene, α-terpineol, caryophyllene, α-humulene and α-gurjunene (3-6). The EOs of M. leucadendra L. have been found to be mainly composed of p-cymene, α-terpinene, γ-terpinene, 4-terpineol, caryophyllene, methyleugenol and E-nerolidol (7-10). The main components of the EOs of M. quinquenervia have been shown to be monoterpenes (1,8-cineole, α-pinene, α-terpineol, limonene) and sesquiterpene (viridiflorol, E-nerolidol) (11,12). Furthermore, pharmacological studies on the EOs from M. cajuputi, M. quinquenervia and M. leucadendra have demonstrated their antibacterial, antimycrobacterial, antivirus, antifungal, antiinsecticidal and antioxidant effects (4,6,9,10,13-18); however, other investigations have revealed that the chemical compositions of EOs of M. cajuputi, M. quinquenervia and M. leucadendra vary extensively in different geographic and ecological conditions, even from different regions of the same country, resulting in several chemotypes (7,9,10,12,18-20). In addition, studies on the biological activities of EOs from M. cajuputi, M. quinquenervia and M. leucadendra species grown in Vietnam are still limited and primarily focus on the EOs from M. cajuputi (4,6,14,18).

The present study evaluated the antimicrobial activity and, for the first time, to the best of our knowledge the enzyme inhibitory effects against α-amylase, α-glucosidase, acetylcholinesterase (AChE) and xanthine oxidase (XO) of EOs extracted from the leaves of three Melaleuca species widely distributed in Vietnam (Fig. 1).

Figure 1 Melaleuca species examined in the present study.

Materials and methods

Plant materials

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.

Extraction of EOs

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.

Gas chromatography-mass spectrometry (GC-MS) analysis

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.

Identification of the compounds

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).

Determination of biological activities of EOs: Antimicrobial activity

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.

Determination of biological activities of EOs: Enzyme inhibition activity. i) Amylase inhibitory activity

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-(As-Abs)/(Ac-Acb)], where: As is the absorbance of the sample, Asb is the absorbance of the sample blank, Ac is the absorbance of the control, and Acb 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-(As-Abs)/(Ac-Acb)], where: As is the absorbance of the sample, Asb is the absorbance of the sample blank, Ac is the absorbance of the control, and Acb 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-(As-Abs)/(Ac-Acb)], where: As is the absorbance of the sample, Asb is the absorbance of the sample blank, Ac is the absorbance of the control and Acb 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-(As-Abs)/(Ac-Acb)], where: As is the absorbance of the sample, Asb is the absorbance of the sample blank, Ac is the absorbance of the control and Acb 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.

Results and Discussion

For each of the three steam distillations of the fresh leaves of M. cajuputi, M. quinquenervia, and M. leucadendra (200 g), the average yield of the EOs was 1.40 g (0.70%, wt/wt), 2.35 g (1.18%, wt/wt) and 0.62 g (0.31%, wt/wt), respectively. The GC chromatograms of the EOs of M. cajuputi, M. leucadendra, and M. quinquenervia are presented in the Fig. 2, Fig. 3 and Fig. 4. The chemical constituents of the EOs are listed in Table I and the chemical structures of the main compounds are illustrated in Fig. 5.

Figure 2 Gas chromatogram of the leaf EO of M. cajuputi.

Figure 3 Gas chromatogram of the leaf EO of M. quinquenervia.

Figure 4 Gas chromatogram of the leaf EO of M. leucadendra.

Figure 5 Chemical structure of the major compounds of the leaf EOs of Melaleuca species in Vietnam. The numbers in bold font in parentheses next to name of each compound correspond to their order of presentation in Table I.

Table I Chemical composition of the leaf EOs of M. cajuputi, M. quinquenervia and M. leucadendra.

Table I

Chemical composition of the leaf EOs of M. cajuputi, M. quinquenervia and M. leucadendra.

Compound no.	Compounds	RIa	RIb	M. cajuputi (%)	M. quinquenervia (%)	M. leucadendra (%)
1	α-Thujene	925	924	-	-	1.76
2	α-Pipene	932	932	0.44	0.84	7.69
3	Benzaldehyde	957	960	0.32	-	-
4	β-Pinene	975	974	0.42	0.79	0.44
5	Myrcene	989	990	0.58	0.58	-
6	α-Phellandrene	1,004	1,002	-	-	0.73
7	p-Cymene	1,023	1,024	-	0.62	5.38
8	(-)-Limonene	1,027	1,029	2.16	2.66	1.43
9	1,8-Cineole	1,030	1,031	30.87	42.51	-
10	γ-Terpinene	1,057	1,059	0.35	0.79	12.94
11	Terpinolene	1,087	1,088	-	0.83	11.77
12	Linalool	1,099	1,096	3.86	3.38	0.55
13	Terpinen-4-ol	1,177	1,177	0.52	1.02	4.65
14	α-Terpineol	1,190	1,188	8.31	12.00	1.25
15	β-Caryophyllene	1,420	1,419	1.61	0.95	14.11
16	α-Humulene	1,454	1,454	1.12	0.75	8.54
17	β-Selinene	1,487	1,490	1.17	0.50	-
18	δ-Selinene	1,492	1,492	0.52	-	-
19	α-Selinene	1,496	1,498	1.26	0.63	0.65
20	Palustrol	1,569	1,568	-	-	1.06
21	Caryophyllene oxide	1,584	1,583	-	-	7.22
22	Viridiflorol	1,592	1,592	-	-	2.48
23	Guaiol	1,598	1,600	9.71	6.86	-
24	Khusimone	1,604	1604	-	-	9.87
25	Isolongifolanone	1,610	1613	-	-	3.42
26	1-epi-cubenol	1,627	1,628	1.59	1.01	-
27	γ-Eudesmol	1,633	1,632	6.16	3.90	-
28	Alloaromadendrene epoxide	1,641	1,641	1.02	-	-
29	β-Eudesmol	1,652	1,650	9.23	6.53	-
30	α-Cadinol	1,655	1,654	11.29	7.81	-
Total identified				92.50	95.95	95.93
- Non-terpenoids				0.32	0.62	5.38
- Monoterpene hydrocarbons				3.94	6.48	36.76
- Oxygenated monoterpenes				43.56	58.92	6.45
- Sesquiterpene hydrocarbons				5.68	2.82	23.30
- Oxygenated sesquiterpenes				39.00	26.11	24.04
Total unidentified				7.50	4.05	4.07

[i] RI^a, retention indices calculated from retention time in relation to those of a series of C7-C40 n-alkanes on an Equity-5 capillary column;

[ii] RI^b, literature retention indices (17,18,23). EO, essential oil; M. cajuputi, Melaleuca cajuputi; M. quinquenervia, Melaleuca quinquenervia; M. leucadendra, Melaleuca leucadendra. Values in bold indicate main compounds in essential oils.

The GC-MS analysis of the leaf EO of M. cajuputi and the comparisons of relative RIs of reference n-alkanes and the spectral databases of known compounds revealed the presence of 21 compounds, accounting for 92.50% of the total amount of the EO. Of these, monoterpene hydrocarbons accounted for 3.94%, oxygenated monoterpenes for 43.56%, sesquiterpene hydrocarbons for 5.68%, oxygenated sesquiterpenes for 39.00% and non-terpenoids for 0.32%. These results indicated that oxygenated monoterpenes and sesquiterpenes were the main components of the EO of M. cajuputi. Among these, 1,8-cineole and α-terpineol were the main compounds in the group of oxygenated monoterpenes, accounting for 30.87 and 8.31%, respectively. Moreover, guaiol (9.71%), γ-eudesmol (6.16%), β-eudesmol (9.23%) and α-cadinol (11.29%) were the main compounds in the oxygenated sesquiterpene component of the EO of M. cajuputi. Previous studies have indicated that 1,8-cineole is one of the main compounds in the leaf EO of M. cajuputi, with widely varying concentrations ranging of 27.78-59.90% (3-6,14).

In addition, the presence of 20 compounds was identified in the EO of M. quinquenervia. Among these, oxygenated monoterpenes (58.92%) accounted for the highest proportion, whereas other compound groups, including oxygenated sesquiterpenes, monoterpene hydrocarbons, sesquiterpene hydrocarbons and non-terpenoids accounted for 26.11, 6.48, 2.82 and 0.62%, respectively. The compounds 1,8-cineole (42.51%), α-terpineol (12.00%), guaiol (6.68%), β-eudesmol (6.53%) and α-cadinol (7.81%) were the main constituents of the EO extracted from M. quinquenervia species grown in Thua Thien Hue province, Vietnam. However, the main compounds in the EO of M. quinquenervia vary widely among different geographical regions. For example, a report on the chemical composition of the EO of M. quinquenervia in Australia and Papua New Guinea regions indicated that EOs extracted from M. quinquenervia species growing from Sydney, North along the East coast of Australia to Selection Flat, New South Wales and Maryborough, Queensland, typically contain linalool (14.0-30.0%) and E-nerolidol (74.0-95.0%) as major components. Moreover, oils from M. quinquenervia species in areas ranging from Sydney to Papua New Guinea and New Caledonia often contain main constituents, such as α-terpineol (0.5-14.0%), β-caryophyllene (0.5-28.0%), viridiflorol (13.0-66.0%) and 1,8-cineole (10.0-75.0%) (12). The EO extracted from M. quinquenervia species harvested in Taiwan contains main chemical components, including α-terpineol (13.73%), viridiflorol (14.55%), α-pinene (15.93%) and 1,8-cineole (21.60%) (11). On the other hand, the EO of M. quinquenervia collected in Costa Rica exhibits different compositions, including α-terpineol (6.5%), α-pinene (17.9%), viridiflorol (21.7%) and 1,8-cineole (31.5%) (26).

The results of the chemical composition analysis of the leaf EO of M. leucadendra identified 19 compounds, accounting for 95.93% of the EO. Compounds, such as α-pinene (7.69%), p-cymene (5.38%), γ-terpinene (12.94%), terpinolene (11.77%), β-caryophyllene (14.11%), α-humulene (8.54%), caryophyllene oxide (7.22%) and khusimone (9.87%) were the main compounds found in the EO of M. leucadendra. Previous investigations on the chemical composition of the EO have revealed marked differences among M. leucadendra. In particular, the EO extracted from M. leucadendra species harvested in Fujian, China, has been reported to be rich in compounds, such as α-pinene (4.96%), α-terpinene (7.82%), p-cymene (5.74%), γ-terpinene (18.4%), α-terpineol (4.92%) and 4-terpineol (36.85%) (8). Furthermore, compounds such as limonene (4.8%), 1,8-cineole (61.0%), α-terpineol (15.6%) and viridiflorol (7.9%) dominate in the composition of the EO extracted from M. leucadendra species harvested in Havana, Cuba (9). Moreover, the EO extracted from M. leucadendra leaves in India has been shown to contain (E)-nerolidol (90.85%) as the absolute predominant compound among the 28 identified compounds present in the oil (7). Additionally, the EO of M. leucadendra in Senegal has been shown to contain methyleugenol (98.4-99.5%) as the main component (10). Furthermore, a report on the composition of the EO of M. leucadendra from Danang, Vietnam, indicated that α-humulene (4.4%), β-selinene (3.7%), α-selinene (3.7%), guaiol (10.9%) and α-eudesmol (17.6%) are the major compounds in this oil (18). The findings of the present study, as well as those of previous reports (7-10,18), indicate that the chemical composition of the leaf EO of M. leucadendra varies significantly due to geographical differences.

Although numerous studies have indicated that the EO of Melaleuca species includes several chemotypes, the environmental factors responsible for essential oil chemotype distribution of these species remain unclear. Therefore, further studies on environmental factors influencing the EO chemotype of Melaleuca species are required. Several investigations of EO chemotypes from other plants have revealed that the environment factors responsible for EO chemotype distribution include soil properties and nutrients (pH, Ca2+, K+, organic matter, aridity and texture), bioclimatic regions (temperature, precipitation, altitude and seasonal variation), cultivating conditions, maturation of harvested plants, plant storage, plant preparation and methods of extraction (27-31).

Biological activities of essential oils. Antimicrobial activity

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.

Table II Antimicrobial activity of the EOs (MIC, µg/ml).

Table II

Antimicrobial activity of the EOs (MIC, µg/ml).

EOs	S. aureus	E. faecalis	E. coli	P. aegurinosa	C. albicans
M. cajuputi	1,280	1,280	640	640	2,560
M. quinquenervia	640	2,560	1,280	1,280	2,560
M. leucadendra	640	1,280	1,280	2,560	1,280
Ciprofloxacin	2	4	2	2	-
Fluconazole	-	-	-	-	4

[i] EO, essential oil; M. cajuputi, Melaleuca cajuputi; M. quinquenervia, Melaleuca quinquenervia; M. leucadendra, Melaleuca leucadendra; S. aureus, Staphylococcus aureus; E. faecalis, Enterococcus faecalis; E. coli, Escherichia coli; P. aegurinosa, Pseudomonas aegurinosa; C. albicans, Candida albicans.

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.

Table III α-Amylase, α-glucosidase, acetylcholinesterase, and xanthine oxidase inhibitory activities of the EOs (IC50, µg/ml).

Table III

α-Amylase, α-glucosidase, acetylcholinesterase, and xanthine oxidase inhibitory activities of the EOs (IC50, µg/ml).

	Inhibitory activity
EOs	α-Amylase	α-Glucosidase	Acetylcholinesterase	Xanthine oxidase
M. cajuputi	862.6±65.74	1212±73.49	765.7±26.14	331.9±20.64
M. quinquenervia	786.3±58.42	1453±93.79	815.5±20.72	380.1±17.85
M. leucadendra	714.3±38.09	1066±45.01	535.5±19.84	433.8±18.02
Acarbose	60.84±2.23	111.5±2.96	-	-
Galantamine	-	-	5.95±0.14	-
Allopurinol	-	-	-	7.44±0.37

[i] EO, essential oil; M. cajuputi, Melaleuca cajuputi; M. quinquenervia, Melaleuca quinquenervia; M. leucadendra, Melaleuca leucadendra.

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.

Acknowledgements

Not applicable.

Funding

Funding: The present study was funded by the Vietnam Academy of Science and Technology (grant no. CSCL.01/23-24).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

THDT and CVCL were involved in the conceptualization and methodology of the study, as well as in the formal analysis, writing of the original draft, and in the writing, review and editing of the manuscript. PHT and TTTV were involved in the methodology of the study, and in data investigation and formal analysis. TDTP, VMN and TNMN were involved in the formal analysis. PHT and TTTV confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References