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Chemical composition, antibacterial and antioxidant activities of essential oils from leaves of three Melaleuca species of Pakistani flora
⁎Corresponding author. saimesiddique@gmail.com (Saima Siddique)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
This study presents essential oil composition of three Melaleuca species namely, Melaleuca bracteata F. Muell, Melaleuca fulgens R. Br. subsp. steedmanii and Melaleuca leucadendron (L.) L. collected from different regions of Pakistan. The chemical composition of essential oils was analyzed by GC-FID and GC–MS. Eugenol methyl ether was identified as a principal component in M. bracteata (82.3%), M. fulgens (87.8%) and M. leucadendron (95.4%) oils. In vitro antibacterial studies were done by agar well diffusion and microdilution method and the tested essential oils exhibited bacteriostatic and bactericidal effects against the tested foodborne pathogens at 4–8 µg/ml. Time kill assay showed significant bactericidal effect of oils for four weeks. The antioxidant potential was assessed by free radical scavenging activity and reducing power assay. The oils showed strong antioxidant activity with approximately 89.0–89.5% inhibition of 2,2-diphenyl-1-picrylhydrazyl radical and ferric reducing power in the range of 1.94 ± 0.007–2.04 ± 0.04% at 100 µg/ml.
Keywords
Melaleuca bracteata
M. fulgens
M. leucadendron
Essential oils
Antibacterial activity
Antioxidant potential
1 Introduction
Food preservation involves control of oxidative damage to the food products and growth of foodborne pathogens. Different chemicals are used to inhibit oxidation and microbial growth in foods (Russell and Gould, 2003). However, the negative consumers’ perception of chemical preservatives due to their harmful effects and increased microbial resistance to the majority of currently used antibacterial drugs (Shan et al., 2007), has directed the attention of scientists toward the use of natural alternatives for the extension of products’ shelf-life. Among the emerging natural preservatives, essential oils have been widely investigated and have gained momentum in the recent years due to the presence of antioxidants and their antibacterial, anti-mutagenic and anti-carcinogenic properties (Gutierrez et al., 2009).
Myrtaceae is one of the most diverse and widespread plant families, rich in essential oils. The main genera of Myrtaceae include Eucalyptus, Eugenia, Leptospermum, Melaleuca, Myrtus, Pimenta, Plinia, Psidium, Pseuocaryophyllus and Syzygium. The genus Melaleuca L. consists of around 260 species and occurs predominantly in Australia but is also domesticated in South-East Asia, the Southern United States and the Caribbean (Tran et al., 2013). They are shrubs or trees, generally found in open forests, woodlands or shrublands, particularly along the watercourses and the edges of swamps. Melaleuca genus shows significant phenotypic diversity in a variety of ecosystems. They can adapt to climate change through Wright’s ‘migrational adaptation’, and can be managed to attain sustainable benefits (Tran et al., 2013). The essential oils from Melaleuca have demonstrated antibacterial (Hussein et al., 2007), anti-inflammatory (Caldefie-Chezet et al., 2006), fungicidal (Bagg et al., 2006), acaricidal (Iori et al., 2005), antioxidant (Pino et al., 2010) and antiviral properties (Minami et al., 2003).
Studies on chemical composition of essential oils of various Melaleuca species from Benin, Brazil, Egypt, India, Thailand and Tunisia have been previously reported. Eugenol methyl ether, 1,8-cineole, terpinen-4-ol, terpinolene, α-terpinene, caryophyllene and caryophyllene oxide were identified as major constituents in the essential oils of most of the Melaleuca species (Lohakachornpan and Rangsipanuratn, 2001; Farag et al., 2004; Kumar et al., 2005; Silva et al., 2007; Chabir et al., 2011; Rini et al., 2012a, 2012b).
Melaleuca genus was introduced in the Punjab, Pakistan, long ago (Parker, 1956). Literature survey revealed that essential oil composition of Melaleuca species from Pakistan has not yet been investigated. The screening of these indigenous essential oil resources is needed for their use in the industry. Three species of Melaleuca were selected for identification of chemical constituents present in their oils and for investigation of their biological properties. This research was also aimed at identification of volatile constituents of the selected Melaleuca species and their evaluation for use in the food industry as alternate to chemical preservatives.
2 Material and Methods
2.1 Plant material
Fresh leaves of M. bracteata F. Muell were collected from Government College University Botanical Garden, Lahore (Longitude 74.31°E, Latitude 31.57°N), M. fulgens R. Br. subsp. steedmanii from Qarshi Botanical Garden, Hattar, Abottabad (Longitude 72.85°E, Latitude 33.85°N) and M. leucadendron (L.) L. from Jinnah Garden, Lahore (Longitude 74.26°E, Latitude 31.50°N) in the Early Summer in 2014. Plant herbaria were authenticated by Prof. Dr. A.N. Khalid at the Herbarium, Department of Botany, University of Punjab, Lahore, Pakistan and voucher specimens (BDSS # 4039, BDSS # 4040, BDSS # 4041) were also deposited in the same herbarium.
2.2 Chemicals
Homologous series of C8–C25 n-alkanes used in this study were obtained from Sigma Chemical Co. (St. Louis, MO, USA) while 2,2-diphenyl-1-picrylhydrazyl (99.0%) was purchased from ACE, Germany. Butylated hydroxytoluene (BHT, 99.0%) was obtained from ACROS-Organics, Belgium. Anhydrous sodium sulphate, ferrous chloride, potassium ferricyanide, trichloroacetic acid, ethanol and methanol used in this study were purchased from Merck (Darmstadt, Germany). Culture media (Nutrient broth, Nutrient agar, Plate count agar) were purchased from OXOID Ltd. Hampshire, UK.
2.3 Isolation of oils
Fresh leaves (4.30 kg, 0.97 kg and 1.25 kg) of M. bracteata, M. fulgens and M. leucadendron respectively were subjected to hydro-distillation for 3 h using Clevenger-type apparatus in triplicate, according to the method recommended in the European Pharmacopoeia (EDQM, 2005). The oils obtained were dried over anhydrous sodium sulphate, filtered and stored at −4 °C further analyses. The essential oil contents (%) were expressed as volume of essential oil vs. weight of fresh leaves (v/w).
2.4 Chemical analysis of essential oils
2.4.1 GC-FID
GC analysis of the essential oils was carried out on Shimadzu GC 2010 equipped with the flame ionization detector (FID) and AOC-20i auto-sampler using a DB-5 MS (30 m × 0.25 mm id, 0.25 µm film thickness) capillary column. The column oven temperature was programmed initially at 40–90 °C at the rate of 2 °C/min and then raised to 90–240 °C at the rate of 3 °C/min. The final temperature was held constant for 5 min. Injector and detector temperatures were maintained at 240 and 280 °C, respectively. Essential oil (0.5 µl) was injected in a split mode ratio of 1:5. Helium was used as a carrier gas at the flow rate of 1 ml/min. Quantification of constituents was carried out by integration of peak areas without using the correction factors. The essential oils samples were ran in triplicate.
2.4.2 GC–MS
The identification of components was carried out on GCMS-QP 2010 Plus, Shimadzu, Japan operating in electron ionization mode at 70 eV. Mass units were monitored from 35 to 500 AMU. A DB-5 MS (30 m × 0.25 mm id, 0.25 µm film thickness) capillary column was used. Column conditions and temperatures of injector and detector were the same as in GC analysis.
Linear retention indices were calculated using a homologous series of n-alkanes (C8-C25) under the same temperature-programmed conditions. The components were identified by comparison with linear retention indices (RI) from literature Adams (2001); mass spectra with those of NIST mass spectral library (Mass spectral library, 2001) or co-injection with standards.
2.5 Evaluation of antibacterial activities of essential oils
2.5.1 Tested Microorganisms
Seven bacterial strains from American Type Culture Collection (ATCC, Rockville) were selected for in vitro antibacterial activity of the essential oils. Of the seven bacterial strains Bacillus spizizenii (ATCC 6633) and Staphylococcus aureus (ATCC 25923) were Gram positive while Enterobacter aerogenes (ATCC 13048), Escherichia coli (ATCC 8739), Klebsiella pneumoniae (ATCC 13882), Pseudomonas aeruginosa (ATCC 27853) and Salmonella enterica (ATCC 14028) were Gram negative strains. All the bacterial strains were sub-cultured at 35 °C for 24 h on nutrient agar slants prior growing them in nutrient broth overnight.
2.5.2 Agar well diffusion method
Antibacterial activity of the selected essential oils was checked by agar well diffusion method (Zaika, 1988). Molten agar medium (20 ml) was inoculated with microbial suspension containing the indicator strain having 106 cfu/ml concentration. The inoculated medium was poured into Petri plates and allowed to solidify. Wells were made on solidified agar with a sterilized cork borer and 90 μl of the tested oil was added to each. Ampicillin was used as a positive control. The plates were incubated at 35 °C for 24 h. The diameters of inhibition zones were measured in millimeters and results were recorded in triplicate.
2.5.3 Minimum Inhibitory Concentration (MIC) assay
Serial dilutions of 4 μg/ml, 8 μg/ml, 15 μg/ml, 65 μg/ml and 250 μg/ml were used in triplicate to determine MIC levels by agar well method (Zaika, 1988). The lowest concentration of oil inhibiting visible growth of each microbe after incubation was taken as the MIC.
2.5.4 Minimal Bactericidal Concentration (MBC) assay
Minimum Bactericidal Concentration (MBC) was determined by broth microdilution method (Rabe et al., 2002). Bacterial load (106 cfu/ml) was poured in tubes containing respective culture broth and oil with concentration of MIC. Broth tubes with and without bacterial load were used as controls. The tubes were incubated for 24 h at 35 °C. After incubation, 100 μl from tubes having no visible growth was removed and poured in plates along with agar to enumerate total viable count. The lowest concentration with no visible growth after 24 h of incubation at 35 °C was defined as the MBC, indicating 99.9% killing of the original inoculum.
2.5.5 Time kill assay
A time kill study was carried out with the MIC values found previously by the agar well method to discern whether the tested oils had bacteriostatic or bactericidal effect over a period of time for use as a food preservative (White et al., 1996). Microorganisms (bacterial suspension) with 106 cfu/ml and oil having concentration equal to MIC were added respectively in the tube of corresponding culture medium. Broth tubes with and without microbial suspension were used as controls. The cultures were incubated for one month at 35 °C. An inoculant of 100 μl, removed after 2, 5, 8, 11, 14 and 30 d was poured in agar plates in triplicate to determine the total reduction in viable counts. The mean number of the colonies (cfu/ml) was counted and compared with that in the control culture at the end of the incubation period. The test tubes with turbidity after a certain incubation period depicted bacteriostatic effect of the tested essential oil at the applied concentration. To determine the bactericidal concentration of essential oils against that particular strain, higher concentrations (15 μg/ml, 65 μg/ml and 250 μg/ml) were applied and lethal effect of essential oils was observed as mentioned above.
2.6 Antioxidant activity
2.6.1 DPPH assay
The antioxidant activities of essential oils were evaluated by measurement of their ability to scavenge 2,2′-diphenyl-1-picrylhydrazyl (DPPH) stable radical. The assay was carried out spectrophotometrically as described by Shimada et al. (1992).
Various solutions at different concentrations in the range of 20 to 100 μg/ml of essential oils were prepared in methanol. To 0.1 ml of each test concentration, 3 ml of methanolic solution of DPPH (0.004%) were added. The resulting mixtures were incubated in the dark for 30 min at room temperature and absorbance was recorded as Asample at 517 nm using spectrophotometer (Cecil CE 7200). A blank experiment was also carried out applying the same procedure to a solution without essential oil, and absorbance was recorded as Ablank. Scavenging (%) of DPPH free radical by essential oils was calculated as follows:
Antioxidant activity of essential oils or standard was expressed as IC50 which is defined as the concentration of test material required to cause a 50% decrease in initial DPPH concentration. All determinations were performed in tripilcate. Butylated hydroxytoluene (BHT) was used as a standard.
2.6.2 Total reduction ability by Fe3+-Fe2+ transformation
The total reduction ability of essential oils was determined by the method of Oyaizu (1986). The capacity of essential oils to reduce the ferric ion (Fe3+) to the ferrous ion (Fe2+) was evaluated by measuring the absorbance at 700 nm. To the different concentrations of the essential oils 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide (1%) were added. The mixture was incubated at 50 °C for 20 min. Then 2.5 ml of trichloroacetic acid (10%) were added. The mixtures were revolved at 3000 rpm for 10 min. The supernatant (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of ferric chloride. Absorbance was measured at 700 nm on UV spectrophotometer after allowing the solution to stand for 30 min. Butylated hydroxytoluene (BHT) was used as a standard.
2.7 Statistical analysis
The mean values, ± standard deviations were calculated using MS Excel 2007. Data was analysed by using analysis of variance (ANOVA) and differences among the means were determined for significance at P < 0.05 using Duncan’s multiple range test by SPSS (version 16.0).
3 Results & discussion
3.1 Essential oil yield
Hydrodistillation of fresh leaves of M. bracteata, M. fulgens and M. leucadendron yielded 0.14 ± 0.01%, 2.10 ± 0.10% and 0.42 ± 0.03% of essential oils respectively. The essential oil yield of M. bracteata and M. leucadendron was lower than that reported by previous researchers (Zhong et al., 2009; Oyedeji et al., 2014; Almarie et al., 2016). Essential oil yield from M. fulgens leaves has not been reported previously.
3.2 Chemical composition
The chemical compositions of essential oils from Melaleuca species is given in Table 1. The essential oils of M. bracteata, M. fulgens and M. leucadendron were characterized by high percentage of aromatic compounds (95.1–96.4%). Eugenol methyl ether was identified as a principal component in M. bracteata (82.3%), M. fulgens (87.8%) and M. leucadendron (95.4%) oils. Melaleuca bracteata essential oil contained methyl cinnamate (11.4%) in significant amounts along with eugenol methyl ether. Our findings on M. bracteata essential oil are in agreement with the previous reports (Aboutabl et al., 1991; Ye et al., 2014; Almarie et al., 2016). Ye et al. (2014) reported comparable methyl eugenol content (83.55%) but a higher methyl cinnamate content (4.55%) in M. bracteata essential oil, extracted using petroleum ether. Zhong et al. (2009) however, reported a higher concentration of eugenol methyl ether (>95%) in M. bracteata essential oil extracted from branches and leaves.
| Compounds | RIexp | RIlit | Area (%) | ||
|---|---|---|---|---|---|
| M. bracteata | M. fulgens | M. leucodendron | |||
| α-Pinene | 930 | 932 | 0.2 | 0.1 | 0.1 |
| α-Phellandrene | 1002 | 1002 | 0.1 | 0.1 | – |
| δ-3-Carene | 1004 | 1008 | tr | tr | – |
| p-Cymene | 1026 | 1020 | 1.7 | 1.2 | 0.1 |
| Limonene | 1029 | 1024 | 0.2 | 0.2 | 0.1 |
| 1,8-Cineole | 1028 | 1026 | 0.3 | 0.2 | 0.1 |
| cis-β-Ocimene | 1043 | 1032 | tr | 0.1 | – |
| γ-Terpinene | 1055 | 1054 | tr | 0.1 | – |
| Terpinolene | 1083 | 1086 | 0.3 | 0.5 | 0.1 |
| Linalool | 1095 | 1095 | 1.0 | 1.0 | 0.4 |
| Citronellal | 1148 | 1148 | tr | tr | – |
| p-Menthan-3-ol | 1167 | 1167 | 0.1 | – | – |
| p-Cymen-8-ol | 1183 | 1179 | 0.4 | tr | 0.1 |
| α-Terpineol | 1186 | 1186 | 1.0 | 0.9 | 0.7 |
| Citronellol | 1223 | 1223 | 0.4 | tr | 0.1 |
| 2-Isopropenyl-5-methylhex-4-enal | 1198 | – | 0.1 | tr | – |
| α-Cubebene | 1345 | 1345 | tr | tr | tr |
| 3-Allyl-2-methoxyphenol | – | – | 0.3 | 0.3 | 0.8 |
| Nerol acetate | 1365 | 1359 | tr | tr | tr |
| α-Copaene | 1374 | 1374 | tr | 0.1 | tr |
| (E)-methyl cinnamate | 1379 | 1376 | 11.4 | 5.8 | 0.8 |
| Eugenol methyl ether | 1402 | 1403 | 82.3 | 87.8 | 95.4 |
| β-Caryophyllene | 1417 | 1417 | – | 0.1 | 0.1 |
| Germacrene D | 1484 | 1484 | 0.2 | 0.6 | 0.4 |
| Germacrene B | 1559 | 1559 | tr | 0.2 | 0.1 |
| Caryophyllene oxide | 1582 | 1582 | tr | tr | 0.1 |
| Epiglobulol | 1585 | – | tr | 0.1 | tr |
| Total identified | 99.6 | 99.4 | 98.7 | ||
| Monoterpene hydrocarbons | 0.8 | 1.1 | 0.3 | ||
| Oxygenated monoterpenes | 2.6 | 2.1 | 1.3 | ||
| Sesquiterpene hydrocarbons | 0.2 | 1.0 | 0.6 | ||
| Oxygenated sesquiterpenes | tr | 0.1 | 0.1 | ||
| Aromatic compounds | 95.9 | 95.1 | 96.4 | ||
| Others | 0.1 | tr | – | ||
RIexp = Retention Indices relative to C9-C25 n-alkanes on the DB-5 column; RIlit = Retention Indices from literature (2); tr = trace < 0.05%.
Essential oil from M. fulgens and M. leucadendron leaves showed qualitative and quantitative variability in chemical composition when compared with the earlier reports. Previously, 1,8-cineole was reported as the principal component in the essential oil of M. leucadendron species grown in Cuba, India, Indonesia, Ivory Coast and Egypt (Farag et al., 2004; Kumar et al., 2005; Rini et al., 2012a, 2012b; Tia et al., 2013). Terpinolene (29.21%), α-terpinene (22.55%), 2-γ-carene (8.53%) and α-phellandrene (7.61%) were reported as main components in the essential oil of M. leucadendron grown in Thailand (Lohakachornpan and Rangsipanuratn, 2001). Our results are similar to the Brazilian M. leucadendron essential oil (Silva et al., 2007) wherein eugenol methyl ether (96.6%) was identified as the principal component. However, Pakistan has a dry sub-tropical climate as compared to Brazil having wet tropical climatic situation. Contrasting the previous reports, 1,8-cineole (0.1%) and terpinolene (0.1%) were found in traces in Pakistani variety of M. leucadendron.
Lee et al. (2004a, 2004b) reported 1,8-cineole (77.5%) as a major component in M. fulgens essential oil from Australia along with an appreciable amount of limonene (6.14%), α-pinene (3.21%), myrcene (1.43%) and methyl geranate (1.0%). On the contrary, eugenol methyl ether (87.78%) was found as the major component alongwith minor quantities (0.1–0.2%) of limonene, α-pinene and 1,8-cineole in essential oil from M. fulgens leaves in Pakistan. These differences in essential oils composition might have arisen due to environmental and climatic conditions, geographic variations and genetic differences.
3.3 Antibacterial activity
The evaluated essential oils manifested good antibacterial properties against all tested microbes but the level of bacterial growth inhibition was found to be dependent on essential oils concentration and the bacterial strain (Table 2).
| Essential oil | Conc. (µg/ml) | Zones of inhibition (mm) on tested microbial strains | ||||||
|---|---|---|---|---|---|---|---|---|
| B. spizizenii | S. aureus | E. aerogenes | E. coli | K. pneumoniae | P. aeruginosa | S. enterica | ||
| M. bracteata | 250 | 44.0 ± 3.5f | 12.8 ± 0.3a | 15.7 ± 0.6ab | 16.7 ± 0.3b | 18.9 ± 0.4bc | 12.5 ± 0.0a | 17.8 ± 0.8b |
| 100 | 20.8 ± 0.8c | 12.3 ± 0.3a | 15.0 ± 0.0ab | 15.3 ± 0.3ab | 17.2 ± 0.3b | 12.0 ± 0.0a | 15.2 ± 0.3ab | |
| 65 | 14.8 ± 0.3ab | 12.2 ± 0.3a | 14.3 ± 0.6ab | 14.8 ± 0.3ab | 16.3 ± 0.3b | 11.8 ± 0.3a | 13.7 ± 0.3ab | |
| 15 | 13.2 ± 0.3ab | 11.7 ± 0.3a | 13.2 ± 0.3ab | 12.8 ± 0.3a | 14.0 ± 0.0ab | 11.7 ± 0.3a | 12.0 ± 0.3a | |
| 8 | 12.7 ± 0.3a | 11.5 ± 0.0a | 13.0 ± 0.0ab | 12.5 ± 0.0a | 13.3 ± 0.3ab | 11.2 ± 0.3a | 11.8 ± 0.3a | |
| 4 | 12.2 ± 0.3a | 11.3 ± 0.3a | 11.5 ± 0.3a | 11.8 ± 0.3a | 12.2 ± 0.3a | 11.0 ± 0.0a | 11.7 ± 0.3a | |
| M. fulgens | 250 | 20.8 ± 0.8c | 12.7 ± 0.6a | 17.0 ± 1.0b | 17.2 ± 0.3b | 17.8 ± 0.8b | 16.3 ± 0.5b | 17.2 ± 0.3b |
| 100 | 19.2 ± 0.3bc | 12.5 ± 0.0a | 16.3 ± 0.3b | 16.3 ± 0.3b | 17.3 ± 0.3b | 15.7 ± 0.3ab | 16.3 ± 0.3b | |
| 65 | 18.8 ± 2.0bc | 12.3 ± 0.3a | 16.0 ± 0.5b | 16.0 ± 0.9b | 17.2 ± 1.0b | 15.0 ± 1.0ab | 15.8 ± 0.8ab | |
| 15 | 17.5 ± 1.0b | 11.7 ± 0.3a | 14.8 ± 0.2ab | 15.5 ± 0.0ab | 16.8 ± 2.1b | 11.3 ± 0.6a | 14.8 ± 0.6ab | |
| 8 | 16.7 ± 1.5b | 11.2 ± 0.3a | 14.2 ± 0.2ab | 14.8 ± 0.3ab | 16.2 ± 0.0b | 11.0 ± 0.0a | 14.2 ± 0.3ab | |
| 4 | 14.3 ± 0.6ab | 10.8 ± 0.3a | 13.2 ± 0.3ab | 13.8 ± 0.8ab | 14.5 ± 0.5ab | 10.8 ± 0.3a | 12.8 ± 0.3a | |
| M. leucodendron | 250 | 16.3 ± 0.6b | 13.2 ± 0.3ab | 16.2 ± 0.6b | 17.3 ± 0.8b | 16.7 ± 0.6b | 12.2 ± 0.3a | 15.8 ± 0.3ab |
| 100 | 16.2 ± 0.3b | 12.5 ± 0.0a | 15.7 ± 0.3ab | 16.7 ± 0.3b | 16.5 ± 0.0b | 12.0 ± 0.0a | 15.7 ± 0.3ab | |
| 65 | 16.0 ± 0.0b | 12.3 ± 0.3a | 15.5 ± 2.6ab | 15.3 ± 0.8ab | 16.3 ± 0.6b | 11.8 ± 0.3a | 15.5 ± 0.5ab | |
| 15 | 15.0 ± 0.0ab | 12.3 ± 0.3a | 15.3 ± 0.3ab | 14.7 ± 0.6ab | 15.7 ± 1.5ab | 11.7 ± 0.6a | 14.5 ± 0.0ab | |
| 8 | 13.8 ± 0.3ab | 11.8 ± 0.3a | 14.7 ± 0.3ab | 13.8 ± 0.3ab | 15.3 ± 1.2ab | 11.7 ± 0.6a | 13.5 ± 0.0ab | |
| 4 | 13.2 ± 0.3ab | 11.0 ± 0.0a | 13.7 ± 0.3ab | 13.3 ± 0.6ab | 14.8 ± 0.3ab | 11.0 ± 0.0a | 11.8 ± 0.3a | |
| Ampicillin | 1000 | 32.0 ± 1.0d | 16.3 ± 0.5b | 17.6 ± 0.5b | 39.3 ± 0.5e | 12.5 ± 0.5a | ND | 33.3 ± 0.5d |
The diameter of the inhibition zones (mm), including the well diameter (6 mm), are given as mean ± SD of triplicate experiments. ND: Not detected.
The values with the same lower case letters are not statistically significant at P = 0.05% according to Duncan’s Multiple Range Test.
Melaleuca bracteata exhibited excellent activity against B. spizizenii with inhibition zones (IZ) of 12.2–44.0 mm followed by M. fulgens (14.3–20.8 mm) and M. leucadendron (13.2–16.3 mm). The tested essential oils showed moderate activity against S. aureus with inhibition zones between (11.0–13.2 mm). The evaluated essential oils showed good activity against tested Gram negative bacterial strains with zones of inhibition ranging from 11.0 to 18.9 mm at concentrations of 4–250 μg/ml. Melaleuca leucadendron essential oil demonstrated appreciable activity against E. aerogenes (IZ = 13.7–16.2 mm) while M. bracteata and M. fulgens showed comparable zones of inhibition too i-e between 11.5 and 17.0 mm. Melaleuca bracteata showed moderate zones of inhibition against K. pneumoniae (12.2–18.9 mm) while it ranged from 14.5 to 17.8 mm for M. fulgens and M. leucadendron essential oils. Melaleuca essential oils showed similar inhibitory effect against E. coli, P. aeruginosa, and S. enterica as marked by zones of inhibition ranging from 11.8 to 17.3 mm, 11.0–16.3 mm and 11.7–17.8 mm in the three species respectively. Melaleuca fulgens exhibited a slightly larger zone of inhibition as compared to M. bracteata and M. leucadenderon i-e. 13.8–17.2 mm and 10.8–16.3 mm for E. coli and P. aeruginosa while M. bracteata was found more inhibitory to S. enterica with inhibition zone 11.7–17.8 mm. The variation in the antibacterial activities of Melaleuca essential oils with respect to all species was statistically significant (P < 0.05).
The MIC and MBC values of Melaleuca essential oils ranged from 4 to 8 μg/ml for the tested bacterial strains (Table 3). There are a few reports on antibacterial activity of the selected Melaleuca species for comparison. Higher MIC values were reported against E. coli, P. aeruginosa and S. aureus (25–100 μl/ml) for M. leucadendron essential oil (Lohakachornpan and Rangsipanuratn, 2001; Dehghan and Bawazir, 2012). Antibacterial activity of M. fulgens has not been reported previously. Our findings on antibacterial activity of M. bracteata essential oils showed discrepancies with the previously published data. Oyedeji et al. (2014) reported larger zones of inhibition (10.3–15.3 mm) against S. aureus for different varieties of M. bracteata from South Africa while smaller IZ were observed against Gram negative bacteria viz. E. coli (11.8–14.5 mm), P. aeruginosa (8.5–9.0 mm) and K. pneumoniae (11.5–14.5 mm). The MICs were found 0.62–1.25 μg/ml for S. aureus and 0.62–1.25 μg/ml and 1.25–2.5 μg/ml for K. pneumoniae and P. aeruginosa respectively.
| Tested microbial strains | M. bracteata | M. fulgens | M. leucodendron | ||||||
|---|---|---|---|---|---|---|---|---|---|
| MIC | MBC, 24 h | MBC, 4 w | MIC | MBC, 24 h | MBC, 4 w | MIC | MBC, 24 h | MBC, 4 w | |
| B. spizizenii | 4 | 4 | 8 | 4 | 4 | 250 | 4 | 4 | 65 |
| S. aureus | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| E. aerogenes | 4 | 4 | 65 | 4 | 4 | 250 | 4 | 4 | 15 |
| E. coli | 4 | 4 | 8 | 4 | 4 | 8 | 4 | 4 | 15 |
| K. pneumoniae | 4 | 4 | 8 | 4 | 4 | 8 | 4 | 4 | 8 |
| P. aeruginosa | 8 | 8 | 65 | 8 | 8 | 250 | 8 | 8 | 250 |
| S. enterica | 4 | 4 | 15 | 4 | 4 | 15 | 8 | 8 | 250 |
*Abbreviation: w = week.
The differences in the antibacterial activity of the selected essential oils and subsequently the MICs and MBCs may result from different chemical compositions and percentage content of active constituents in essential oils. Factors such as the choice of bacterial strains and their sensitivity, volume of inoculum, cultivation conditions (incubation time, temperature, oxygen etc), concentration of test substance, the culture medium, the solvents used to dilute the essential oils and different methods used for in vitro antibacterial activity could also be related to the variation in the experimental results.
The time kill assay was carried out to check the potency of Melaleuca essential oils for use as food preservative. The study was carried out to evaluate whether the tested essential oils have bacteriostatic or bactericidal effects at 4–8 μg/ml for four weeks. Our data showed that the response of bacteria to the tested essential oils varied among the strains and was concentration and time dependent. Melaleuca bracteata essential oil proved highly lethal to B. spizizenii with MBC value of 8 μg/ml whilst the highest bactericidal concentration (250 μg/ml) was observed for M. fulgens essential oil against B. spizizenii. Melaleuca leucodenderon essential oil showed moderate bactericidal effects against B. spizizenii with MBC of 65 μg/ml. Evaluated essential oils showed bactericidal effect against S. aureus at 8 μg/ml for a month.
Klebsiella pneumoniae was found to be sensitive toward all tested oils with lowest MBC i-e. 8 μg/ml among Gram negative strains whilst P. aeruginosa was found to be most resistant among the tested strains with MBC values of 250 μg/ml for M. fulgens and M. leucadendron essential oils. Melaleuca bracteata, however, showed relatively lower MBC value against P. aeruginosa of 65 μg/ml.
Melaleuca leucadendron essential oil was resistant toward S. enterica with MBC value of 250 μg/ml, but showed lower MBC value against E. aerogenes and E. coli i-e 15 μg/ml. The MBC values for M. bracteata ranged from 8 to 65 μg/ml against E. aerogenes, E. coli and S. enterica. For M. fulgens essential oil the MBC values were 8 μg/ml, 15 μg/ml and 250 μg/ml against E. coli, S. enterica and E. aerogenes respectively.
The significant antibacterial activity of assayed essential oils could be related to eugenol methyl ether; the major compound with known antibacterial potential (Lawal et al., 2014). The enhanced inhibitory effect of M. fulgens and M. bracteata essential oils could be related to their higher methyl cinnamate content which has also been reported to possess antibacterial property (Stefanović et al., 2015).
3.4 Essential oils antioxidant activities
DPPH assay and reducing power assay were used to assess antioxidant potential of Melaleuca essential oils. The synthetic antioxidant BHT was used as an equivalence parameter for the antioxidant activity of the essential oils. The Melaleuca essential oils showed DPPH scavenging activity (89.0–89.5%). The DPPH scavenging activity of the tested oils was found higher than butylated hydroxytoluene (BHT) as is evident from lower IC50 value of essential oils. In reducing power assay, Melaleuca essential oils showed comparable ferric reducing power to BHT at the tested concentrations of 20–100 µg/ml (Table 4). Previously, Hou et al. (2016) reported good ferric reducing power (FRP) (2.37 ± 0.01 mM Fe2+/g DW) and DPPH radical scavenging activity (86.0 ± 0.3%) of M. bracteata ethanolic extract with eugenol methyl ether (86.86%) and trans-cinnamic acid methyl ester (6.41%) as major components. No data is available for comparison of antioxidant activity of M. fulgens essential oil. Rini et al. (2012a, 2012b) reported IC50 values of M. leucadendron essential oil ranging from 4240 to 9460 µg/ml in a DPPH scavenging assay. The IC50 value of evaluated M. leucadendron essential oil was quite low (39.1 ± 0.3 µg/ml) showing its strong antioxidant potential.
| Test system | Conc. (μg/ml) | M. bracteata | M. fulgens | M. leucodendron | BHT | IC50 value, μg/ml |
|---|---|---|---|---|---|---|
| DPPH radical scavenging capacity (%) | 20 | 35.3 ± 0.4 | 34.9 ± 1.6 | 34.1 ± 1.1 | 30.8 ± 0.6 | 37.3 ± 0.9 M. bracteata 37.8 ± 1.6 M. fulgens 39.1 ± 0.3 M. leucodendron 41.5 ± 0.50 (BHT) |
| 40 | 54.3 ± 1.0 | 54.1 ± 0.5 | 52.6 ± 0.9 | 52.4 ± 1.0 | ||
| 60 | 69.1 ± 0.3 | 67.7 ± 0.8 | 66.8 ± 0.4 | 66.5 ± 0.7 | ||
| 80 | 77.2 ± 0.3 | 77.3 ± 1.2 | 75.6 ± 0.8 | 76.8 ± 0.6 | ||
| 100 | 89.2 ± 0.4 | 89.0 ± 0.6 | 89.5 ± 0.8 | 85.8 ± 0.8 | ||
| Reducing power (absorbance at 700 nm) | 20 | 1.20 ± 0.02 | 1.25 ± 0.03 | 1.22 ± 0.08 | 1.16 ± 0.18 | – |
| 40 | 1.38 ± 0.01 | 1.44 ± 0.05 | 1.40 ± 0.05 | 1.36 ± 0.03 | ||
| 60 | 1.60 ± 0.01 | 1.68 ± 0.05 | 1.63 ± 0.03 | 1.57 ± 0.05 | ||
| 80 | 1.88 ± 0.02 | 1.92 ± 0.03 | 1.89 ± 0.03 | 1.82 ± 0.07 | ||
| 100 | 2.02 ± 0.02 | 2.11 ± 0.04 | 2.06 ± 0.05 | 1.95 ± 0.04 | ||
4 Conclusion
Our results show that the essential oils from leaves of Melaleuca species are rich in eugenol methyl ether. The remarkable antibacterial activity of Melaleuca essential oils based on MIC, MBC, and kill-time study against common food-borne pathogens and strong antioxidant activity suggests their possible use in the food industry as a potential new source of natural antibacterial and antioxidant agents. However, in vivo studies are recommended to determine the toxicity profile of essential oils.
Authors contributions
Saima Siddique and Sania Mazhar designed and performed the bioassays; Zahida Parveen collected the species and extracted the essential oils; Saima Siddique and Firdaus-e-Bareen prepared the manuscript and analyzed the data statistically.
Acknowledgement
The authors are thankful to Prof. Dr. A.N. Khalid in the herbarium, University of the Punjab, Lahore for identification of plants and the authentication of specimens. Authors are also very thankful to Mr. Muhammad Akram (Senior Scientific Officer), Medicinal Botanic Centre, Peshawar for his help in the GC-MS analysis of essential oils.
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