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In the last decade, research into essential (volatile) oils has received increasing attention from both industrial and academic sectors because of the growing interest in green consumerism and the need for alternative techniques to assure the quality and safety of perishable foods [[
Several studies conducted on natural plant essential oils have indicated that these oils may be used as antimicrobial agents and have potential use in industrial applications [[
Melaleuca alternifolia (M. alternifolia) samples were collected in four locations of Zhaoqing City of Guangdong Province at altitudes between 300 and 600 m. Samples were dried in well-ventilated spaces away from sunlight. Samples were then placed at 50°C to dry the samples to a constant, dry weight and disintegrated through a 90-mesh sieve. Air-dried plant materials were hydrodistilled using a Clevenger-type apparatus. The composition of M. alternifolia essential oil was analyzed in the laboratory using Hewlett-Packard 6890 gas chromatograph equipped with a cross-linked 5% PH ME siloxane Hewlett-Packard-5MS capillary column (25 m × 0.25 mm ID, 0.25 μm film thickness), coupled to a Hewlett-Packard 5972A mass spectrometer (Hewlett-Packard Ltd., Bracknell, UK). The GC operating conditions were as follows: helium as carrier gas with a flow rate 2.0 ml/min; column temperature programming from 60°C to 275°C at 4°C/min; injector and FID detector temperatures of 215 and 275°C, respectively. The MS operating parameters were as follows: ionization potential, 70 ev; resolution, 1000; ion source temperature, 250°C. Identification of components was based on GC retention indices and the fragmentation patterns of the mass spectra with those of authentic samples, as well as the NIST 98 and HPCH 2205 GC–MS libraries. Relative percentage amounts were obtained directly from GC peak areas. The composition of the essential oil from M. alternifolia is presented in Table 1.
Chemical composition of Melaleuca alternifolia essential oil.
Componentsa Composition% Terpinene-4-ol 31.11 γ-Terpinene 25.30 α-Terpinene 12.70 1,8-Cineole 6.83 ρ-Cymene 4.23 Terpinolene 4.03 Limonene 2.50 α-Terpineol 2.35 Aromadendrene 1.75 δ-Cadinene 1.41 Sabinene 0.28 Globulol 0.24 Viridiflorol 0.14 Total 92.87
Thea dates were provided by Meriden Animal Health Lt.
This spectrophotometric assay uses the stable 2,2-diphenylpicrylhydrazyl (DPPH) radical as a reagent [[
The thiobarbituric acid reactive substances (TBARS) method was used as described by [[
The hydroxyl radical scavenging activity was measured by the method described in [[
Escherichia coli ATCC25922 (E. coli), Staphylococcus aureus ATCC25923 (S. aureus), Pseudomonas aeruginosa ATCC27853 (P. aeruginosa), Penicillium italicum Wehmer (P. italicum Wehmer), and Penicillium digitatum Sacc. (P. digitatum Sacc.) were obtained from the China Center for Type Culture Collection (Wuhan University, China).
Paper disks with 6 mm diameter were soaked with 0.1 mL of the essential oil from M. alternifolia and placed on the surface of solid media plates previously inoculated with the different microorganisms (6.0 logCFU/mL) tested in this study. P. italicum Wehmer and P. digitatum Sacc. were cultured at 28°C and 120 rpm for 48 h, whereas E. coli, S. aureus, and P. aeruginosa were cultured at 37°C and 120 rpm for 24 h. The size of the halo for each microorganism was recorded by measuring the zones of growth inhibition surrounding the disks. Individual samples were examined in triplicate. The size of the halos is presented as means ± standard deviation.
A broth microdilution method was used to determine the MIC and MBC. The potato dextrose broth and Mueller Hinton broth were prepared at twice the final concentration. M. alternifolia essential oil was added to glass tubes to yield final sample media concentrations at 0.2 to 48 mg/mL for the potato dextrose and Mueller Hinton broths, respectively. The control sets were run simultaneously without the addition of an antimicrobial agent. An appropriate volume of inoculum (6.0 logCFU/mL) was added to the media to give an approximate final cell concentration of 4.0 logCFU/mL. P. italicum Wehmer and P. digitatum Sacc. cultures in potato dextrose broth were incubated at 28°C and 120 rpm for 48 h, whereas E. coli and S. aureus cultures in Mueller Hinton broth were incubated at 37°C and 120 rpm for 24 h. The essential oil concentration that yielded no visible growth for a tested species was considered the MIC for that particular species. Then, 0.1 mL suspension obtained from the above transparent tubes was spread onto either potato dextrose agar (PDA) or Mueller Hinton agar (MHA). After 48 h culturing at 28°C (fungi) or 48 h culturing at 37°C (bacteria), the concentration of inoculated suspension containing the lowest level of antimicrobial agent that showed no colonies on the plate was determined as the MBC.
The essential oil from M. alternifolia can be obtained easily from the hydrodistillation of M. alternifolia leaves, and the chemical composition is dependent on the extraction method used and crop region the samples are taken from [[
As previously described, the use of different methods is required when assessing antioxidant activity [[
Antioxidative activity of the essential oil of Melaleuca alternifolia.
Sample (μg/mL) Test system DPPH assay (EC50) Hydroxyl radical scavenging activity (EC50) TBARS method (IC50) Vitamin C 7.79 7.43 185.7 Vitamin E 34.59 113.1 14.19 Quercetin 4.46 9.64 7.82 Alpha lipoic acid 305.6 1877 3414 Melaleuca alternifolia essential oil 48.35 43.71 135.9
E. coli, S. aureus, P. aeruginosa, P. italicum Wehmer, and P. digitatum Sacc. are different types of microorganisms. E. coli, S. aureus, and P. aeruginosa are pathogenic bacteria [[
Antimicrobial activity of the essential oil of Melaleuca alternifolia.
Microorganisms Melaleuca alternifolia essential oil DD (mm) MIC (mg/mL) MBC (mg/mL) Escherichia coli 12±1.63 8 8 Staphylococcus aureus 26±2.80 2 2 Pseudomonas aeruginosa 10±0.94 12 12 Penicillium italicum Wehmer 9±0.41 12 12 Penicillium digitatum Sacc. 8±0.47 24 24
DD, diameter of zone of inhibition (mm) including disc diameter of 6 mm.
In conclusion, our study is the first report describing the in vitro antioxidant properties of the essential oil from M. alternifolia. Because of its strong antibacterial and excellent protective features exhibited in antioxidant activity tests, this essential oil and extracts from the herbal parts of M. alternifolia represent a potential natural source that can be used freely in food, agriculture, and pharmaceutical industries as a culinary herb.
The authors declare that they have no conflicts of interest.
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PHOTO (COLOR): Antioxidant activity of different antioxidants by DPPH assay. (a) Vitamin C; (b) vitamin E; (c) quercetin; (d) alpha lipoic acid; and (e) Melaleuca alternifolia essential oil. Values represent means ± SEM, n=3.
PHOTO (COLOR): Antioxidant activity of different antioxidants by hydroxyl radical scavenging activity. (a) Vitamin C; (b) vitamin E; (c) quercetin; (d) alpha lipoic acid; and (e) Melaleuca alternifolia essential oil. Values represent means ± SEM, n=3.
PHOTO (COLOR): Antioxidant activity of different antioxidants by TBARS method. (a) Vitamin C; (b) vitamin E; (c) quercetin; (d) alpha lipoic acid; and (e) Melaleuca alternifolia essential oil. Values represent means ± SEM, n=3.
By Xiaofeng Zhang; Yanjun Guo; Liying Guo; Hui Jiang and Qianhua Ji