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Chemical Characterization, Antioxidant and Antimicrobial Activities Of Indian Journal of Experimental Biology Vol. 56, September 2018, pp. 686-693 Chemical characterization, antioxidant and food can have a deteriorating effect on food colour, 1 antimicrobial activities of essential oil from flavour, texture, quality, wholesomeness and safety . Melaleuca quinquenervia leaves Synthetic preservatives are usually preferred in food industry to retard discoloration, spoilage and Saima Siddique*1, Sania Mazhar2, Firdaus-e-Bareen1 & microbial contamination due to their effectiveness 2 Zahida Parveen3 and low price . However, concerns for the safety 1College of Earth & Environmental Sciences, University of of some chemical preservatives and negative the Punjab, Ferozpur Road, Lahore, Pakistan customers feedbacks over their use have encouraged 2Food & Biotechnology Research Centre; 3Applied Chemistry growing interest in natural green alternatives Research Centre, PCSIR Laboratories Complex, Lahore, Pakistan for the extension of product shelf-life3. Among emerging natural preservatives, essential oils Received 20 August 2016; revised 18 May 2018 have been widely investigated and have gained Niaouli oil is an essential oil known for its applications in momentum in recent years due to their antimicrobial aromatherapy and pharmaceutical preparations for coughs, colds, (antifungal, antibacterial and antiviral), antioxidants, rheumatism and neuralgia. It also serves as a sedative, possesses antimutagenic and anticarcinogenic properties4. antifungal activity, and used in perfume industry. Melaleuca quinquenervia (Cav.) S.T. Blake, commonly called, paper bark tea Myrtaceae family comprising of at least 133 genera tree or punk tree, is a potential source of niaouli oil. Here, and approximately 3800 is the rich source of essential we analyzed the chemical composition of essential oil from oils. It is found abundantly in Australia, Southeast M. quinquenervia leaves and evaluated its antioxidant and Asia and tropical to southern temperate America antimicrobial potential. Chemical analysis of the oil by GC-FID 5 and GC-MS revealed 1,8-cineole (31.0%) as a major component while few are domesticated in Africa . Essential oils followed by p-cymen-8-ol (19.7%), p-cymene (16.5%), α-terpineol belonging to Myrtaceae have been reported to have (9.9%), limonene (6.8%), α-pinene (4.2%) and terpinolene (4.2%). diverse bioactivity, such as insecticidal, antimicrobial M. quinquenervia essential oil demonstrated good antioxidant and nematicidal activity6-8. diphenyl-1-picrylhydrazyl-׳activity by inhibiting 84.3 % of 2,2 radical and ferric reducing power (1.94±0.007) at 100 µg/mL. Melaleuca quinquenervia (Cave) S. T. Blake Further, it was highly effective against tested food borne bacterial (Fam. Myrtaceae) is a source of 1,8-cineole-rich as well as fungal pathogens inducing 11.0-46.0 mm and 11.8-46.0 mm essential oil called Niaouli oil, which is used in zones of inhibition, respectively at concentration of 8-250 μg/mL. aromatherapy and pharmaceutical preparations The high degrees of antibacterial and antifungal activities were further confirmed at 8 μg/mL minimum bactericidal concentrations for the relief of coughs, colds, rheumatism and 9 and minimum fungicidal concentrations, respectively. Time kill neuralgia . The compounds E-nerolidol and linalool assay showed significant bactericidal and fungicidal effects of present in niaouli oil have widespread use in essential oil for four weeks. The high antimicrobial and the perfume industry10. Besides, linalool has antioxidant activities of M. quinquenervia essential oil substantiate demonstrated acaricidal11, bactericidal and fungicidal its potential use as alternative to chemical preservatives in food 12 industry. properties . Pioneer reports have elucidated chemical ׳ Keywords: 1,8-Cineole, 2-Diphenyl-1-picrylhydrazyl radical, Food composition13-16 and antifungal activity17,18 of borne pathogens, Limonene, Minimum fungicidal concentration, Niaouli oil, 2Paper bark tea tree, Preservatives, Punk tree, Time essential oil of M. quinquenervia from different kill assay parts of the world. To the best of our knowledge, the chemical composition of Pakistani variety of Microorganisms and oxidation are the major causes M. quinquenervia has not been described previously. of food deterioration. Microbial growth is a major The present work, thus, aims to study the chemical concern as some microorganisms can potentially composition of locally growing M. quinquenervia cause food-borne illnesses. Auto-oxidation of lipids in species and to evaluate its antioxidant potential and ——————— antimicrobial activity against food borne pathogens * Correspondence: and spoilage microorganisms, for a potential use as a Phone: +92 992 30688 E-mail: [email protected] natural preservative in food industry. SIDDIQUE et al.: ESSENTIAL OIL FROM MELALEUCA QUINQUENERVIA LEAVES 687 Material and Methods library (Mass spectral library 2001) and Adam (2001) as well as by comparing their retention indices Collection of Materials either with those of authentic compounds or with Plant materials literature values20,21. Fresh leaves (1.16 Kg) of M. quinquenervia were collected during 2013 from the botanical garden, Evaluation of antimicrobial activities of essential oil Hattar, Pakistan and authenticated at the Herbarium, Test microorganisms Department of Botany, University of Punjab, Lahore, Seven bacterial strains from American Type Pakistan. The voucher specimen (code 4057) was also Culture Collection (ATCC, Rockville) and six local deposited in the same herbarium. fungal strains (fungal bank, Institute of Agricultural Chemicals Sciences, University of the Punjab, Lahore, Pakistan) Homologous series of C8–C25 n-alkanes, were selected for in vitro antimicrobial activity of diphenyl-1-picrylhydrazyl (90.0%), butylated essential oil. Of 7 bacterial strains, Bacillus spizizenii-׳2,2 hydroxytoluene (BHT, 99.0%), and various reference (ATCC 6633) and Staphylococcus aureus (ATCC chemicals used in this study were obtained from 25923) were Gram positive while Enterobacter Sigma Chemical Co. (St. Louis, MO, USA). All other aerogenes (ATCC 13048), Escherichia coli (ATCC chemicals (analytical grade), i.e., anhydrous sodium 8739), Salmonella enterica (ATCC 14028), Klebsiella sulphate, ferrous chloride, potassium ferricyanide, pneumonie (ATCC 13882) and Pseudomonas trichloroacetic acid, ethanol and methanol used in this aeruginosa (ATCC 27853) were Gram negative study were purchased from Merck (Darmstadt, strains. Aspergillus niger (AC 1109), A. flavus (AC Germany). All culture media (Nutrient broth, Nutrient 1110), Fusarium oxysporum (AC 1175), F. solani agar, Potato dextrose agar, Plate count agar) were (AC 1199), Alternaria alternata (AC 1200) and purchased from OXOID Ltd. Hampshire, UK and Penicillium digitatum (AC 1160) were selected for HiMEDIA, Mumbai. India. antifungal activity. All the bacterial strains were subcultured at 35°C for 24 h on nutrient agar slants Isolation and Chemical analysis of essential oil prior to being grown in nutrient broth overnight The air-dried and finely ground plant material whereas the fungal strain was sub-cultured at 25°C (1.16 kg) was subjected to hydrodistillation for 3 h using for 120 h on potato dextrose agar (PDA) slants to Clevenger-type apparatus according to the method 19 prepare spore suspension before testing. recommended in the European Pharmacopoeia . Distillate of essential oil was dried over anhydrous Antimicrobial activity sodium sulfate, filtered and stored at 4°C until analyzed. Antimicrobial activity of M. quinquenervia GC analysis of the essential oil was carried out on essential oil was evaluated by agar well diffusion Shimadzu GC 2010 using DB-5 MS (30 m×0.25 mm method22. Twenty millilitres of molten agar medium id, 0.25 µm film thickness) capillary column. The were inoculated with microbial suspension column oven temperature was programmed initially at containing indicator strain at 106 cfu/mL. The 40-90°C at the rate of 2°C/min and then 90-240°C at inoculated medium was poured into a Petri plate the rate of 3°C/min. The final temperature was held and allowed to solidify. Wells were made on constant for 5 min. Injector and detector temperatures solidified agar and 90 μg of M. quinquenervia were maintained at 240 and 280°C, respectively. essential oil was added to each. The plates with Essential oil (0.5 μL) was injected in a split mode bacterial strains were incubated at 35ºC for 24 h and ratio of 1:5. Helium was used as a carrier gas at the at 25°C for 48 h for the fungal strain. The diameters flow rate of 1 mL/min. GC-MS analysis was carried out of inhibition zones were measured in millimeters on GCMS-QP 2010 Plus, Shimadzu, Japan operating in and results were recorded in triplicate. electron ionization mode at 70 eV. Column conditions were same as in GC analysis. The mass spectrometer Minimum inhibitory concentration (MIC) assay was capable of scanning from 35 to 500 AMU Four different concentrations of oil (8, 25, 65 and every second or less. The data acquisition system 250 μg/mL) were used in triplicate to determine MIC continuously acquires and stores all data analyses. levels by agar well method22. The lowest concentration The components were identified by comparing of oil inhibiting visible growth of each microbe was their mass spectra with those of NIST mass spectral taken as the MIC. 688 INDIAN J EXP BIOL, SEPTEMBER 2018 Minimal bactericidal concentration (MBC) and fungicidal DPPH assay concentration (MFC) The antioxidant activity of the essential oil from
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