The essential oil of Melaleuca armillaris (Sol. ex Gaertn.) Sm. leaves from Pakistan: A potential source of eugenol methyl ether

Industrial Crops & Products 109 (2017) 912–917

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Research paper

The essential oil of Melaleuca armillaris (Sol. ex Gaertn.) Sm. leaves from Pakistan: A potential source of eugenol methyl ether

MARK

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Saima Siddiquea, , Zahida Parveenb, Firdaus-e-Bareenc, Muhammad Nawaz Chaudharya, Sania Mazhard, Shaista Nawazd a

College of Earth & Environmental Sciences, University of the Punjab, Lahore 54890, Pakistan Applied Chemistry Research Centre, PCSIR Laboratories Complex, Lahore 54600, Pakistan c Department of Botany, University of Punjab, Lahore 54890, Pakistan d Food & Biotechnology Research Centre, PCSIR Laboratories Complex, Lahore 54600, Pakistan b

A R T I C L E I N F O

A B S T R A C T

Keywords: Melaleuca armillaris Eugenol methyl ether Antimicrobial studies Microdilution method Antioxidant activity

The importance of Melaleuca armillaris (Sol. ex Gaertn.) Sm. leaves as a commercially important source of aroma chemicals is known. This study aims at analyzing the chemical composition of M. armillaris essential oil and its antioxidant and antimicrobial activities for evaluating its potential use in the industry. The chemical composition was analysed by gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC–MS). Twenty-eight components were identified representing 90.6% of the total oil components. Melaleuca armillaris essential oil mainly consisted of aromatic compounds (84.5%). Eugenol methyl ether was identified as a major component (89.57%) followed by p-cymene (2.8%). The antioxidant potential was assessed by free radical scavenging activity, metal chelating activity and reducing power assay. It showed strong antioxidant activity with approximately 86.6% inhibition of 2,2-diphenyl-1-picrylhydrazyl radical and ferric reducing power (1.91 ± 0.02%) at 100 μg/ml while it did not show any metal chelating activity. In vitro antimicrobial studies were done by agar well diffusion and microdilution methods Melaleuca armillaris showed moderate activity against tested bacterial strains with inhibition zones (IZ) ranging from 11.0–19.3 mm. It exhibited excellent activity against fungal strains with (IZ) 24.3–46.0 mm. The minimum inhibitory concentration (MIC), minimum bactericidal concentrations (MBC) and minimum fungicidal concentrations (MFC) values of M. armillaris essential oil ranged from 2 to 8 μg/ml for the tested microbial strains. Time kill assay showed a significant microbiocidal effect of essential oil for four weeks.

1. Introduction The family Myrtaceae is one of the most diverse and widespread in the plant kingdom. 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 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. The genus 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). Plants of Melaleuca genus are rich in essential oils and volatile constituents (1,8-cineole, eugenol methyl ether, (E)-nerolidol, linalool,

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α-terpineol, terpinen-4-ol) of commercial importance. The essential oils from Melaleuca genus are also well known for their antibacterial (Siddique et al., 2015), anti-inflammatory (Caldefie-Chézet et al., 2006), fungicidal (Terzi et al., 2007), insecticidal (Liao et al., 2016), antioxidant (Pino et al., 2010) and antiviral properties (Garozzo et al., 2009). Melaleuca armillaris (Sol. ex Gaertn.) Sm. is the most widely cultivated species of the genus Melaleuca. It is commonly known as the Bracelet Honey Myrtle and grows into a large spreading shrub or small tree (Hayouni et al., 2008). Based on essential oil composition, two types of chemotypes have been described for M. armillaris growing in Brazil, Egypt and Tunisia. One was characterized by high monoterpenoid content with 1,8-cineole as the major component along with terpinen-4-ol, α-terpineol (Aboutabl et al., 1991; Chabir et al., 2011; Farag et al., 2004; Silva et al., 2007; Yvon et al., 2012). Another chemotype contained sesquiterpenes (cis-calamenene) and

Corresponding author. E-mail address: [email protected] (S. Siddique).

http://dx.doi.org/10.1016/j.indcrop.2017.09.048 Received 4 July 2017; Received in revised form 21 August 2017; Accepted 21 September 2017 Available online 05 October 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.

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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 oil samples were ran in triplicate.

sesquiterpenoids (torreyol, dihydrocarveol) as prominent components (Amri et al., 2012). The chemotype, 1,8-cineole has demonstrated strong virucidal effects against Herpes simplex virus type 1, exhibited low antifungal effects against A. niger while strong anti-candidal effect were shown against C. albicans. It has also exhibited antioxidant, anticancer, antimalarial and antihypertensive activities. In addition, aforementioned chemotype has proven a potent fumigant against three stored products insects namely Sitophilus oryzae, Tribolium castaneum and Ryzopertha dominica (Chabir et al., 2011; Farag et al., 2004; Lee et al., 2004a; Yvon et al., 2012). On the other hand, sesquiterpenic fraction rich M. armillaris essential oil from Tunisia has demonstrated phytotoxic activity against germination and initial radicle growth of Lepidium sativum L., Phalaris canariensis L., Raphanus sativus L., Sinapis arvensis L. and Triticum durum L. seeds and low antibacterial activity (Amri et al., 2012). Thus, geographical location is an important factor affecting chemical variability of essential oil and biological activities. A review of literature revealed that essential oil composition and biological properties of M. armillaris from Pakistan have not yet been investigated. The screening of these indigenous essential oil resources is needed for their use in the industry. Thus, this research is aimed at identification of volatile constituents of M. armillaris, evaluation of its antimicrobial, antioxidant properties and comparison of data with the species growing in other parts of the world.

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 (Liao et al., 2016) or co-injection with standards. 2.5. Evaluation of antibacterial activities of essential oils

2. Material and methods

2.5.1. Tested microorganisms Seven bacterial strains from American Type Culture Collection (ATCC, Rockville) and six local fungal strains (fungal bank, Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan) were selected for in vitro antimicrobial activity of essential oil. Of 7 bacterial strains Bacillus spizizenii (ATCC 6633) and Staphylococcus aureus (ATCC 25923) were Gram positive while Enterobacter aerogenes (ATCC 13048), Escherichia coli (ATCC 8739), Salmonella enterica (ATCC 14028), Klebsiella pneumoniae (ATCC 13882) and Pseudomonas aeruginosa (ATCC 27853) were Gram negative strains. Aspergillus niger (AC 1109), Aspergillus flavus (AC 1110), Fusarium oxysporum (AC 1175), Fusarium solani (AC 1199) and Penicillium digitatum (AC 1160) were selected for anti-fungal activity. All the bacterial strains were sub-cultured at 35 °C for 24 h on nutrient agar slants prior to being grown in nutrient broth overnight whereas the fungal strain was sub-cultured at 25 °C for 120 h on potato dextrose agar (PDA) slants to prepare spore suspension before testing.

2.1. 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 ACROSOrganics, 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, Potato dextrose agar, Plate count agar) were purchased from OXOID Ltd. Hampshire, UK and HiMEDIA, Mumbai. India. 2.2. Plant material Fresh leaves of M. armillaris (Sol. ex Gaertn.) Sm. were collected from plant grown at Government College University Botanical Garden, Lahore (Longitude 74.31°E, Latitude 31.57°N) in April 2013 during daytime (noon) and authenticated by Prof. Dr. A. N. Khalid at the Herbarium, Department of Botany, University of Punjab, Lahore, Pakistan. A voucher specimen (BDSS # 4030) was also deposited in the same herbarium.

2.5.2. Agar well diffusion method Antimicrobial activity of M. armillaris essential oil 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 and nystatin were used as positive control. The plates with bacterial strains were incubated at 35 °C for 24 h and at 25 °C for 48 h for the fungal strains. The diameters of inhibition zones were measured in millimeters and results were recorded in triplicate.

2.3. Isolation of oils The fresh leaves (285 g) of M. armillaris were subjected to hydrodistillation a day after collection for 3 h using Clevenger-type apparatus (EDQM, 2005). The obtained oil was subsequently dried over anhydrous Na2SO4 and stored under refrigeration until analyzed and tested. The essential oil content (%) was expressed as volume of essential oil vs. weight of fresh leaves (v/w). 2.4. Essential oil analysis

2.5.3. Minimum inhibitory concentration (MIC) assay Serial dilutions of 4 μg/ml, 8 μg/ml, 15 μg/ml, 65 μg/ml, 100 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.

2.4.1. GC-FID GC analysis of the essential oil was carried out on Shimadzu GC 2010, Japan 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

2.5.4. Minimal Bactericidal/Fungicidal concentration (MBC) assay Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) was determined by broth microdilution method (Rabe et al., 2002). Bacterial/fungal 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 913

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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.

incubated for 24 h at 35 °C for bacterial and at 25 °C for a period of 48 h for fungal growth. 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 killing 99.9% of the original inoculum with no visible growth after 24 h of incubation at 35 °C was defined as the MBC while the minimum oil concentration that entirely seized the fungal growth and didn’t allow slightest growth revival even after 48 h of incubation was considered as MFC − minimum fungicidal concentration.

2.6.3. Metal chelating activity Metal chelating activity of essential oil was estimated by the Ferrozine assay as described previously by (Gülçin et al., 2012). Essential oil (15 μg/ml) in 0.4 ml was added to 0.05 ml of 2 mM FeCl2 solution followed by addition of 5 mM ferrozine (0.2 ml) solution. The final volume was made up to 4 ml with ethanol. This mixture was shaken well and left for ten minutes at room temperature. Absorbance of the reaction mixture was determined by spectrophotometer at 562 nm. The percentage of inhibition of ferrozine-Fe2+ complex formation was calculated as:

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 M. armillaris oil had microbiostatic or microbiocidal effect over a period of time for use as a food preservative (White et al., 1996). Microorganisms (bacterial/ fungal spore 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 microstatic effect of the tested essential oil at the applied concentration. To determine the bactericidal and fungicidal concentration of M. armillaris essential oil against that particular strain, higher concentrations (8 μg/ml, 15 μg/ml, 65 μg/ml, 100 μg/ml and 250 μg/ml) were applied and lethal effect of essential oil was observed as mentioned above.

Ferrous ion chelating effect (%) = [(AControl − ASample)/AControl] × 100 The control contains only FeCl2 and ferrozine, complex formation molecules. 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 and chemical composition

2.6. Antioxidant activity

Hydrodistillation of fresh leaves of M. armillaris yielded (1.5 ± 0.04)% essential oil. (Farag et al., 2004) reported (0.39–0.92%) yield from fresh leaves of M. armillaris collected during February to November from Egypt. (Amri et al., 2012) reported essential oil yield of (0.65%) from dried hydrodistilled leaves of M. armillaris collected in April (Tunisia). (Aboutabl et al., 1991) reported comparable oil content (1.3–1.5%) in fresh leaves of M. armillaris from Egypt. The variability in essential oil yield can be attributed to the season of harvest, age of the leaves (fresh, mature, senescent) and geographical variation (Verma et al., 2015; Zutic et al., 2016). Pakistan is included geographically and climatically in the semi-tropical zone. Although it lies just above the tropic of Cancer, it is climatically hot and dry due to east-west mountain ranges of Himalyasin the north. It represents a unique climatic situation from others where M. armillaris grows, so it can be expected that its essential oil will also have different constituents. The chemical composition of M. armillaris essential oil is given in Table 1. Twenty eight components were identified from M. armillaris essential oil by GC–MS representing 90.6% of the total oil components. Melaleuca armillaris essential oil mainly consisted of aromatic compounds (84.5%). Monoterpenoids were found in appreciable amount (4.01%) followed by monoterpene hydrocarbons (1.2%). Sesquiterpene hydrocarbons and sesqueterpenoids constituted a minor portion of essential oil i-e. 0.21% and 0.23% respectively. Eugenol methyl ether (80.6%) was the principal component among aromatic compounds followed by p-cymene (2.8%). Linalool (1.4%) and α-terpineol (1.4%) were prominent among monoterpenoids. Previous reports on chemical composition of Melaleuca essential oils showed monoterpenes hydrocarbons (terpinolene), monoterpenoids (1,8-cineole, terpinen-4-ol), sesquiterpenes hydrocarbons (longifolene, allo-aromadendrene) sesquiterpenoids (E-nerolidol, viridifloral) and phenylpropanoids (eugenol methyl ether) as the principal component (Almariea et al., 2016a,b; Farag et al., 2004; Kumar et al., 2005; Lee et al., 2004b; Lohakachornpan et al., 2001; Noumi et al., 2011; Pino et al., 2011; Pujiarti et al., 2012; Siddique et al., 2017; Silva et al., 2007;

2.6.1. DPPH assay The antioxidant activities of M. armillaris essential oil was evaluated by measurement of its 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 20–100 μg/ml of essential oil 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: Scavenging (%) = (Ablank − Asample/Ablank) × 100 Antioxidant activity of essential oil 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 oil was determined by the method of (Oyaizu, 1986). The capacity of essential oil 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 oil 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 914

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contradictory to our results where eugenol methyl ether was the major component and 1,8-cineole was found in insignificant amount (0.29%). These dissimilarities in the essential oil composition extracted from any plant material could arise by geographical location and environmental conditions (Ghasemi et al., 2013; Verma et al., 2015; Zutic et al., 2016). Pakistan has a dry sub-tropical climate which seems to endorse shikimic acid pathway for the synthesis of (eugenol methyl ether), a phenylpropanoid derivative as compared with Brazil, Egypt and Tunisia have wet tropical climatic situation leading to mevalonic acid pathway producing terpenoids (1,8-cineole). Although M. linarrifolia essential oil has been characterized in latitudinally equivalent city of Pantnagar, Uttarakhand in India with Lahore, the Lahore city is climatically dry and tropical with topography of plains while Pantnagar is on the foothills of Himaliyas and is sub-temperate (Pino et al., 2010). This appears to be the reason for (77.40%) 1,8-cineole in M. linarrifolia as compared with M. armillaris from Lahore having eugenol methyl ether (89.%) as the major components.

Table 1 Chemical composition of essential oil from M. armillaris leaves. S. No.

RI

Components

Area (%)

Mode of identification

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

926 930 978 988 1002 1026 1029 1032 1083 1095 1178 1183 1148 1186 1354 1402 1417 1446 1457 – 1484 1547 1559 1576 1588 1592 1601 –

α-Thujene α-Pinene β-Pinene β-Myrcene α-Phellandrene p-Cymene Limonene 1,8-Cineole Terpinolene Linalool Terpinen-4-ol p-Cymen-8-ol Citronellol α-Terpineol Eugenol Eugenol methyl ether β-Caryophyllene (E)-Isoeugenol Alloaromadendrene Humulen-(V1) Germacrene D Elemol Germacrene B Spathulenol Globulol Viridifloral Ledol +-(Selin-7(11)-en-4-alpha-ol Total Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Aromatic compounds

0.07 0.06 0.06 0.25 0.53 2.80 0.26 0.29 0.41 1.40 0.21 0.80 0.28 1.42 0.39 80.57 0.11 0.39 0.02 0.02 0.04 0.01 0.02 0.13 0.07 0.03 0.02 0.08 90.6 1.23 4.01 0.23 0.21 84.95

RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT,

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS MS

3.2. Antimicrobial activity Melaleuca armillaris essential oil showed moderate activity against Gram positive bacterial strains with IZ ranging from 11.0–12.3 mm. The IZ against Gram negative bacterial strains were 10.5–19.3 mm Klebsiella pneumoniae was moderately sensitive to M. armillaris essential oil (IZ = 19.3 mm) whilst E. aerogenes, E. coli, P. aeruginosa and S. enterica were resistant to M. armillaris oil with IZ = 10.5–12.0 mm Melaleuca armillaris exhibited excellent activity against fungal strains with IZ 24.3–46.0 mm (Table 2). The MIC, MBC and MFC values of M. armillaris essential oil ranged from 2 to 8 μg/ml for tested microbial strains (Table 3). Antimicrobial activities of various Melaleuca species have been explored earlier against a range of bacterial and fungal strains (Dehghan and Bawazir, 2012; Kim and Park, 2012; Lee et al., 2008; Lohakachornpan et al., 2001; Oyedeji et al., 2014; Sfeir et al., 2013; Siddique et al., 2017; Touqeer et al., 2014). The essential oil components, 1,8-Cineole, terpinen-4-ol and eugenol methyl ether have been reported as main antimicrobial agents. There are few reports on antimicrobial activity of M. armillaris essential oil. Lower antimicrobial effect of M. armillaris essential oil (Egypt) in the form of smaller IZ against B. spizizenii (9.7 mm), E. coli (9.0 mm) and A. niger (11.7 mm) has been reported by Farag et al. (2004). (Amri et al., 2012) reported higher MIC and MBC values of M. armillaris essential oil against S. aureus (ATCC 25923), (MIC 100 μg/ml; MBC > 100 μg/ml), E. coli (ATCC 25922) (MIC 50 μg/ml; MBC not active), K. pneumoniae (ATCC 10031) (MIC 100 μg/ml; MBC > 100 μg/

RT: Retention time; MS: Mass spectra; RI: Retention Index; tr: traces ≤ 0.1%; -: not detected.

Tia et al., 2013; Wheeler et al., 2007; Ye et al., 2014). Earlier studies on chemical composition M. armillaris essential oil have highlighted the prominence of 1,8-cineole (33.93−85.8%) as a principal component in M. armillaris leaves oil from Brazil and Egypt (Aboutabl et al., 1991; Chabir et al., 2011; Farag et al., 2004; Silva et al., 2007; Yvon et al., 2012) whereas this study reveals eugenol methyl ether as the main component (89.57%). (Amri et al., 2012) reported cis-calamenene (19.0%), torreyol (15.1%), dihydrocarveol (9.0%), α-terpineol (7.7%), myrtenol (6.4%) and spathulenol (4.4%) as prominent components in M. armillaris essential oil from Tunisia. This is Table 2 Antimicrobial activity of M. armillaris essential oil by Agar well diffusion method. Tested Microbial Strains

Zones of Inhibition (mm) on tested microbial strainsa Pure oil

B. spizizenii S. aureus E. aerogenes E. coli K. pneumoniae P. aeruginosa S. enterica A. flavus A. niger F. oxysporum F. solani P. digitatum

12.3 11.0 11.2 10.5 19.3 12.0 11.5 46.0 26.7 24.3 24.7 25.3

± ± ± ± ± ± ± ± ± ± ± ±

0.3a 0.0a 0.3a 0.0a 1.2e 0.0bc 0.5 0.0a 1.2c 0.6d 0.6c 1.2c

250 μg/ml

100 μg/ml

65 μg/ml

15 μg/ml

8 μg/ml

18.7 15.2 15.7 16.7 19.3 15.8 15.7 – – – – –

16.3 15.2 15.5 15.7 18.7 15.7 15.5 – – 46.0 41.0 44.8

15.8 14.7 15.2 15.2 18.3 15.3 15.2 – 44.3 34.0 38.0 39.0

14.0 12.2 14.7 14.8 17.3 12.3 14.7 – 30.3 14.7 14.3 13.0

13.5 12.0 14.0 14.5 16.7 12.2 14.0 46.0 21.3 13.7 13.2 12.2

± ± ± ± ± ± ±

1.4c 0.3d 0.3 cd 0.3d 0.6e 0.3c 0.3d

± ± ± ± ± ± ±

0.3b 0.3d 0.0 cd 0.3 cd 0.3d 0.3c 0.0 cd

± 0.0f ± 1.0e ± 1.0e

± ± ± ± ± ± ±

0.3b 0.6c 0.3c 0.3c 0.6 cd 0.6c 0.3c

± ± ± ±

1.2e 1.0e 1.0d 1.0d

± ± ± ± ± ± ±

0.0ab 0.3b 0.3c 0.3c 0.6c 0.3bc 0.3c

± ± ± ±

1.5d 0.6b 0.6b 1.0b

± ± ± ± ± ± ± ± ± ± ± ±

4 μg/ml

0.0a 0.0b 0.0c 0.5c 0.6c 0.3bc 0.0c 0.0c 1.2b 1.5a 0.3b 0.3a

12.7 11.3 12.7 13.2 14.2 11.5 12.2 20.8 12.3 12.5 12.0 11.8

± ± ± ± ± ± ± ± ± ± ± ±

0.3a 0.3a 0.3b 0.3b 0.3b 0.0a 0.3b 0.8a 0.3a 0.5a 0.0a 0.3a

Ampicillin (1000 μg/ml)

Nystatin (8 μg/ml)

32.0 16.3 17.6 39.3 12.5 ND 33.3 – – – – –

– – – – – – – 25.7 ± 0.4b 25.3 ± 0.4bc 17.3 ± 0.4c 22. ± 0.4c 26.3 ± 0.4c

± ± ± ± ±

1.0d 0.5e 0.5d 0.5e 0.5a

± 0.5e

a 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.

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Table 3 Minimum Inhibitory Concentration (MIC), minimum bactericidal concentration (MBC) and fungicidal concentration (MFC) of M. armillaris essential oil. Tested Microbial Strains

MIC (μg/ml)

MBC (μg/ml) 24 h

MBC (μg/ml) 4w

MFC (μg/ml) 48 h

MFC (μg/ ml) 4w

B. spizizenii (ATCC 6633) S. aureus (ATCC 25923) E. aerogenes (ATCC 13048) E. coli (ATCC 8739) K. pneumoniae (ATCC 13882) P. aeruginosa (ATCC 27853) S. enterica (ATCC 14028) A. flavus (AC 1110) A. niger (AC 1109) F. oxysporum (AC 1175) F. solani (AC 1199) P. digitatum (AC 1160)

4

4

8

–

–

4 2

4 2

8 65

– –

– –

4 2

4 2

15 8

– –

– –

2

2

250

–

–

4

4

15

–

–

Fig. 2. Ferric reducing power of M. armillaris essential oil.

2 4 4 4 4

– – – – –

– – – – –

8 8 8 8 8

8 8 8 8 8

The good antimicrobial activity of M. armillaris essential oil could be related to eugenol methyl ether; the major compound with known antimicrobial potential (Farag et al., 2004; Lawal et al., 2014). 3.3. Antioxidant activity

h = hours; w = week.

DPPH assay and reducing power assay were used to assess antioxidant potential of M. armillaris essential oil. The synthetic antioxidant BHT was used as an equivalence parameter for the antioxidant activity of the essential oil. Melaleuca armillaris essential oil showed DPPH 41.1−86.6% scavenging effect at the concentrations (20−100 μg/ml) (Fig. 1). The IC50 value (32.2 ± 1.3) was lower than BHT (41.5 ± 0.50) indicating strong antioxidant potential of M. armillaris essential oil. In reducing power assay, M. armillaris essential oil showed comparable ferric reducing power (1.91 ± 0.02) to BHT (1.95 ± 0.04) at the tested concentrations 20–100 μg/ml (Fig. 2). The essential oil from M. armillaris leaves did not show metal chelating activity. Previously, (Farag et al., 2004) studied antioxidant effects of M. armillaris essential oil in carbon teterachloride (CCl4) treated rats, as a free radical inducer. An increase in vitamin C, vitamin E, superoxide dismutase (SOD) and catalase (CAT) levels by M. armillaris essential oil was also observed. (Chabir et al., 2011) and (Yvon et al., 2012) reported IC50 value of 2183.6 ± 44.3 mg/L in DPPH assay by M. armillaris essential oil from Tunisia. The IC50 value (32.2 ± 1.3 μg/ml) of evaluated M. armillaris essential oil was lower than previous reports showing its strong antioxidant potential. Eugenol methyl ether; the major compound could be considered responsible for good antioxidant activity.

Fig. 1. DPPH radical scavenging activity of M. armillaris essential oil.

ml), P. aeruginosa (ATCC 27853) (MIC 50 μg/ml; MBC not active) using the broth dilution method. The MIC and MBC values of M. armillaris essential oil (Pakistan) were quite low when compared with (Amri et al., 2012) reflecting it efficacy as a bactericidal agent. The differences in the antimicrobial activity of the evaluated essential oil and subsequently the MICs, MBCs, MFCs may result from different chemical compositions and percentage content of active constituents in essential oil. Factors such as the choice of microbial 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 oil and different methods used for in vitro antimicrobial activity could also be related to the variation in the experimental results. The time kill assay was carried out to check the potency of M. armillaris essential oil for use as food preservative. The study was carried out to evaluate whether the essential oil has microbiostatic or microbiocidal effects at 4–250 μg/ml for four weeks. The oil showed fungicidal effect against all tested fungal strains at 8 μg/ml for a month. Melaleuca armillaris essential oil showed bactericidal effect at 8 μg/ml against B. spizizenii, S. aureus and K. pneumoniae; 15 μg/ml for E. coli and S. enterica and 250 μg/ml for P. aeruginosa in time kill assay. The higher MBC against E. coli and S. enterica and P. aeruginosa observed during four weeks of time kill assay suggest that essential oil of M. armillaris has bacteriostatic effect upto 24 h at low concentration but is bactericidal at high concentration for these bacterial strains.

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