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Journal of Oleo ScienceCopyright ©2016 by Japan Oil Chemists’ Societydoi : 10.5650/jos.ess16042J. Oleo Sci. 65, (8) 629-640 (2016)

Bioactive Compounds of Cold-pressed Thyme (Thymus vulgaris) Oil with Antioxidant and Antimicrobial PropertiesAdel M. A. Assiri1, Khaled Elbanna2, 3, Hussein H. Abulreesh3 and Mohamed Fawzy Ramadan4, 5,＊

1 Biochemistry Department, Faculty of Medicine, Umm Al-Qura University, Makkah, Kingdom of Saudi ARABIA2 Deptartment of Agricultural Microbiology, Faculty of Agriculture, Fayoum University, EGYPT3 Department of Biology, Faculty of Applied Science, Umm Al-Qura University, Makkah, Kingdom of Saudi ARABIA4 Institute of Scientific Research and Revival of Islamic Heritage, Umm Al-Qura University, Makkah, Kingdom of Saudi ARABIA5 Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, 44519 Zagazig, EGYPT

1 IntroductionAromatic plants rich in bioactive phytochemicals are in-

creasingly used in foods, pharmaceuticals and cosmetics. Medicinal plants have been also used as spices and condi-ments to confer aroma and flavor to food and beverages1－3）. Phytochemicals have become attractive to scientists as natural bioactive agents that could be safer than synthetic compounds4, 5）. Scientific research on aromatic plants has focused on their oils and extracts because of their potential

＊Correspondence to: Mohamed Fawzy Ramadan, Institute of Scientific Research and Revival of Islamic Heritage, Umm Al-Qura University, Makkah, Kingdom of Saudi ARABIAE-mail: [email protected] Accepted March 29, 2016 (received for review February 23, 2016)Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 onlinehttp://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs

use in as natural antioxidants and antimicrobial agents6, 7）.Thyme（Thymus vulgaris L., family Lamiaceae）is a sub-

shrub native to the western Mediterranean region. Thyme（fresh or dried）is widely used as a spice to add a distinc-tive flavor to food. Thyme essential oil（TEO）extracted from fresh leaves can be used as aroma additives in food, pharmaceuticals, and cosmetics8）. Thyme acts as an expec-torant and spasmolytic agent for the bronchia, and in folk medicine it is part of herbal teas and infusions9, 10）. Various

Abstract: Herbs rich in bioactive phytochemicals were recognized to have biological activities and possess many health-promoting effects. In this work, cold-pressed thyme (Thymus vulgaris L.) oil (TO) was studied for its lipid classes, fatty acid profile, tocols and phenolics contents. Antioxidant activity and radical scavenging potential of TO against free radicals (DPPH・ and galvinoxyl) was determined. Antimicrobial activity (AA) of TO against food borne bacteria, food spoilage fungi and dermatophyte fungi were also evaluated. Neutral lipids accounted for the main lipid fraction in TO, followed by glycolipids and phospholipids. The major fatty acids in TO were linoleic, oleic, stearic, and palmitic. γ-Tocopherol (60.2% of total tocols) followed by α-tocotrienol (26.9%) and α-tocopherol (9.01% of total tocols) were the main tocols. TO contained high amounts of phenolic compounds (7.3 mg/g as GAE). TO had strong antiradical action wherein 65% of DPPH・ radicals and 55% of galvinoxyl radical were quenched after 60 min of incubation. Rancimat assay showed that induction time (IT) for TO: sunflower oil blend (1:9, w/w) was 6.5 h, while TO: sunflower oil blend (2:8, w/w) recorded higher IT (9 h). TO inhibited the growth of all tested microorganisms. TO exhibited various degrees of AA against different food borne bacteria, food spoilage fungi and dermatophyte fungi, wherein the highest AA was recorded against dermatophyte fungi and yeasts including T. mentagrophytes (62 mm), T. rubrum (40 mm), and C. albicans (20 mm) followed by food spoilage fungi including A. flavus (32 mm) with minimal lethal concentrations (MLC) ranging between 80 to 320 μg/mL. Furthermore, TO exhibited broad-spectra activity against food borne bacteria including S. aureus (30 mm), E. coli (25 mm) and L. Monocytogenes (20 mm) with MLC ranging between 160 to 320 μg/mL. The results suggest that TO could be used economically as a valuable natural product with novel functional properties in food, cosmetics and pharmaceutical industries.

Key words: lipid classes, tocols, antiradical, food spoilage fungi, dermatophyte fungi

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bioactive compounds with antioxidant traits were de-scribed in thyme including TEO constituents, flavonoids and phenolic acids11, 12）. In TEO, 25 compounds were iden-tified7, 13, 14）. Thymol（48.6％）, and p-cymene（22.9％）were the main compounds present in the TEO followed by α-terpinolene（6.49％）7）. Recently, Karabagias et al.15） re-ported a reduction of meat oxidation and bacterial counts when TEO was used to extend the shelf-life of lamb meat. Solomakos et al.16） tested the antimicrobial impact of TEO on Escherichia coli in minced beef meat. during cold storage.

Microbial spoilage affects the shelf life and the cost of the product17）. It was estimated that 48 million people ex-perience food borne illness in the United States every year18）. Food borne pathogens are responsible for 325.000 hospitalizations and 5000 deaths in the US annually19）. Yeasts spoil foods resulting in changes in physical and chemicals properties. Candida, Pichia, Rhodotorula, Torulopsis, Saccharomyces, Zygossacharomyces, Han-senula and Trichosporon are important food spoiling yeasts17）. On the other hand, candidiasis caused by Candida species has increased dramatically in the past years. Candida species are opportunistic pathogen that causes local and systemic infections in predisposed persons, commonly affecting immunologically compro-mised patients and those undergoing prolonged antibiotic treatment20）. Candida infections created a great burden on the public healthcare sector and this situation has gone worse by various epidemiological changes. The difficulties associated with the management of Candida infections necessitate the discovery of new antifungal agents21, 22）. Plant-derived bioactive compounds and plant extracts may offer potential lead to new antimicrobial and antioxidant compounds23）.

Cold-pressed oils have been internationally marketed, wherein bio-information on their chemical composition, antioxidant potential and antimicrobial properties have not been fully reported. Increased interest on cold-pressed oils has been recognized as these oils have high levels of lipid soluble bioactives with nutritive properties. The cold press-ing technology is becoming an interesting substitute for traditional practices because of consumers’ desire for natural products. Cold pressing involves no chemical or heat or refining treatments. Therefore, cold pressed oils usually contain a high level of lipophilic phytochemicals in-cluding natural antioxidants24）.

It is hard to find any data in the literature on cold-pressed thyme（Thymus vulgaris）oil（TO）. This study was performed to: 1）determine lipid classes, fatty acids and tocols of TO, 2）measure the total phenolics content, in vitro antiradical activity and antioxidant potential in food model（Rancimat assay）of TO; and 3）estimate the antimi-crobial activity（AA）of TO.

2 Material and methods2.1 Oils and chemicals

Three different samples of TO and extra virgin olive oil were obtained from a local market in Mekkah（KSA）. Total phenolics content（TPC）in olive oil as determined by the Folin-Ciocalteu was 3.6 mg/g as gallic acid equivalents（GAE）. Neutral lipid（NL）standards were obtained from Sigma（St. Louis, MO, USA）. Standards used for glycolipids（GL）including monogalactosyldiacylglycerol（MGD）, diga-lactosyldiacylglycerol（DGD）, cerebrosides（CER）, steryl glucoside（SG）and esterified steryl glucoside（ESG）were purchased from Biotrend（Köln, Germany）. Standards used for phospholipids（PL）including phosphatidylserine（PS）, phosphatidylethanolamine（PE）, phosphatidylinositol（PI）from bovine liver and phosphatidylcholine（PC）from soybean were purchased from Sigma（St. Louis, MO, USA）. Standards used for tocols were purchased from Merck（Darmstadt, Germany）.

2.2 Methods2.2.1 Column chromatography（CC）and thin layer chroma-

tography（TLC）of lipid classes2.2.1.1 Fractionation of lipid classes and subclasses

TO was separated into neutral and polar lipid classes by elution with solvents over a glass column（20 mm×30 cm）packed with a slurry of activated silicic acid（70 to 230 mesh; Merck, Darmstadt, Germany）in chloroform（1: 5, w/v）. NL was eluted with 3-times the column volume of chlo-roform25）. The major portion of GL was eluted with 5-times the column volume of acetone and that of PL with 4-times the column volume of methanol. The amount of the lipid classes obtained was determined by gravimetry. By means of TLC on Silica gel F254 plates（thickness 0.25 mm; Merck, Darmstadt, Germany）a further characterisation of the GL and PL subclasses was carried out with chloroform/metha-nol/25％ ammonia solution（65: 25: 4, v/v/v）. For the char-acterisation of NL subclasses, TLC plates were developed with n-hexane/diethyl ether/acetic acid（60: 40: 1, v/v/v）. For detection of the lipids, TLC plates were sprayed with the following agents: for the marking of all lipids with sul-phuric acid（40％）, for the marking of GL with α-naphthol/sulphuric acid and for the marking of PL with the molyb-date-blue reagent26）. Each spot was identified with lipid standards as well as their reported retention factor（Rf）values. Individual bands were visualized under ultraviolet light, scraped from the plate and recovered by extraction with chloroform: methanol（2: 1, v/v）. Fatty acid composi-tion of TO as well as NL, GL and PL was determined by GLC/FID as described below.2.2.1.2 Quantitative determination of lipid subclasses

For the quantitative determination of NL classes, indi-vidual bands were scraped from the plate and recovered by extraction with 10％ methanol in diethyl ether, followed by diethyl ether. For the quantitative estimation of GL sub-

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classes, the acetone fraction obtained by CC was separated by TLC in the above given solvent system. The silica gel regions with the corresponding GL classes were scraped out followed by hexose measurement photometrically at 485 nm using the phenol: sulphuric acid in acid-hydrolysed lipids27）. The percent distribution of each component was obtained from the hexose values. From the extinction values the quantitative amount was determined and related to their portion of the GL fraction. The determined portion was set into relation with the amount of oil, which had been separated by CC into the main lipid fractions. For the determination of the PL, the methanol fraction from CC was also separated by TLC in the above given solvent system and after scraping out of the individual PL sub-classes brought to reaction with the hydrazine sulphate: sodium molybdate reagent at 100℃ for 10 min and photo-metrically analyzed at 650 nm. From the obtained extinc-tion values via a calibration chart for phosphorus the amount of PL was calculated. The individual values were put into relation to the PL fraction（methanol fraction from CC）and to the amount of oil.2.2.2 Gas chromatography（GC）analysis of fatty acid meth-

yl esters（FAME）Fatty acids of TO and lipid classes were transesterified

into FAME using N-trimethylsulfoniumhydroxide（Mach-erey-Nagel, Düren, Germany）according to Arens et al.28）. FAME were identified on a Shimadzu GC-14A equipped with flame ionization detector（FID）and C-R4AX chroma-topac integrator（Kyoto, Japan）. The flow rate of the carrier gas helium was 0.6 mL/min and the split value with a ratio of 1: 40. A sample of 1 μL was injected on a 30 m×0.25 mm×0.2 μm film thickness Supelco SPTM-2380（Bellefonte, PA, USA）capillary column. The injector and FID temperature was set at 250℃. The initial column tem-perature was 100℃ programmed by 5℃/min until 175℃ and kept 10 min at 175℃, then 8℃/min until 220℃ and kept 10 min at 220℃. A comparison between the retention times of the samples with those of an authentic standard mixture（Sigma, St. Louis, MO, USA; 99％ purity specific for GC）, run on the same column under the same condi-tions, was made to facilitate identification.2.2.3 HPLC analysis of tocols

For tocols analysis, a solution of 250 mg of TO in 25 mL n-heptane was directly used for the HPLC24）. The HPLC analysis was conducted using a Merck Hitachi low-pressure gradient system, fitted with an L-6000 pump, a Merck-Hita-chi F-1000 Fluorescence Spectrophotometer（The detector wavelength was set at 295 nm for excitation, and at 330 nm for emission）and a D-2500 integration system; 20 μL of the samples were injected by a Merck 655-A40 Autosampler onto a Diol phase HPLC column 25 cm 94.6 mm ID（Merck, Darmstadt, Germany）using a flow rate of 1.3 mL/min. The mobile phase used was n-heptane: tert-butyl methyl ether（99: 1, v/v）.

2.2.4 Extraction, quantification and characterization of phenolic compounds

Aliquots of TO and extra virgin olive oil（1 g）were dis-solved in n-hexane（5 mL）and mixed with 10 mL methanol: water（80: 20, v/v）in a glass tube for two min in a vortex29）. After centrifugation at 3000 rpm for 10 min, the hydroalco-holic extracts were separated from the lipid phase by using a Pasteur pipette then combined and concentrated in vacuo at 30℃ until a syrup consistency was reached. The oily residue was re-dissolved in 10 mL methanol: water（80: 20, v/v）and the extraction was repeated twice. Hydroalco-holic extracts were re-dissolved in acetonitrile（15 mL）and the mixture was washed three times with n-hexane（15 mL each）. Purified phenols in acetonitrile were concentrated in vacuo at 30℃ then dissolved in methanol for further analysis. Aliquots of phenolic extracts were evaporated to dryness under nitrogen. The residue was re-dissolved in 0.2 mL water and diluted（1: 30）Folin-Ciocalteu’s phenol reagent（1 mL）was added. After 3 min, 7.5％ sodium car-bonate（0.8 mL）was added. After 30 min, the absorbance was measured at 765 nm using a UV-260 visible recording spectrophotometer（Shimadzu, Kyoto, Japan）. Gallic acid was used for the calibration and the results of triplicate analyses are expressed as parts per million of gallic acid.

UV spectrum（200-400 nm）of diluted extracts（1 mg/mL）was recorded by spectrophotometry（Jenway 4605-UV–VIS Spectrophotometer）.2.2.5 Radical scavenging activity（RSA）of TO and olive oil

toward DPPH・

RSA of TO and extra virgin olive oil was assayed with DPPH・ radical dissolved in toluene30）. Toluene solution of DPPH・ radicals was freshly prepared at a concentration of 10－4 M. The radical, in the absence of antioxidant com-pounds, was stable for more than 2 h of normal kinetic assay. For evaluation, 10 mg of TO or olive oil（in 100 μL toluene）was mixed with 390 μL toluene solution of DPPH・ radicals and the mixture was vortexed for 20 s at ambient temperature. Against a blank of pure toluene without DPPH・, the decrease in absorption at 515 nm was mea-sured in 1-cm quartz cells after 1, 30 and 60 min of mixing using a UV-260 visible recording spectrophotometer（Shi-madzu, Kyoto, Japan）. RSA toward DPPH・ radicals was es-timated from the differences in absorbance of toluene DPPH・ solution with or without sample（control）and the inhibition percent was calculated from the following equa-tion:

％ Inhibition＝［（absorbance of control－absorbance of test sample）/absorbance of control］×100.

2.2.6 RSA of TO and olive oil toward galvinoxyl radicalA miniscope MS 100 ESR spectrometer（Magnettech

GmbH; Berlin, Germany）was used in this analysis29）. Ex-perimental conditions were as follows: measurement at


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room temperature; microwave power, 6 db; centerfield, 3397 G, sweep width 83 G, receiver gain 10 and modulation amplitude 2000 mG. Ten mg of oil（in 100 μL toluene）were allowed to react with 100 μL of toluenic solution of galviox-yl（0.125 mM）. The mixture was stirred on a vortex stirrer for 20 s then transferred into a 50 μL micro pipette（Hirschmann Laborgeräte GmbH, Ederstadt, Germany）and the amount of galvinoxyl radical inhibited was measured exactly after total incubation time of 60 min after the addi-tion of the galvinoxyl radical solution. The galvinoxyl signal intensities were evaluated by the peak height of signals against a control. A quantitative estimation of the radical concentration was obtained by evaluating the decrease of the ESR signals in arbitrary units after 60 min incubation using the KinetikShow 1.06 Software program（Magnettech GmbH; Berlin, Germany）. The reproducibility of the mea-surements was 5％ as usual for kinetic parameters.2.2.7 Rancimat assay

The antioxidant potential of TO blends with sunflower oil（1: 10 and 2: 8 w/w, TO: sunflower oil）was tested with Rancimat method according to Ranalli et al.31）. The Ranci-mat apparatus was operated at 120℃. A dry air flow of 20 L/h was passed through the oil sample（5 g）. The volatile oxides coming from the oxidation of the oil or blend dis-solved in cold milli-Q water（60 mL）, causing an increase in the electrical conductivity. The time（h）taken to reach a specific conductivity value, corresponding to the flex point of the peroxidation curve, was considered as the induction time（IT）. The higher the induction time the higher the an-tioxidant potency of the compounds. Tests were performed in triplicate.2.2.8 Antimicrobial activity（AA）2.2.8.1 Bacteria and fungi strains

The AA of TO were assessed against pathogenic bacteria and fungi including Staphylococcus aureus（ATCC8095）, Salmonella enteritidis（ATCC 13076）, Escherichia coli（ATCC 25922）, Listeria monocytogenes（ATCC 15313）, Candida albicans（ATCC 10231）, Aspergillus flavus, Trichophyton mantigrophytes, and Trichophyton rubrum. Stock cultures of bacteria were maintained on nutrient agar slants at 4℃, while fungi and Candida yeast were maintained on potato dextrose agar slants at 4℃.2.2.8.2 Determination of AA

AA of TO was determined by agar well diffusion method32－34）. For each bacterial or fungal strain, sterilized Mueller Hinton and potato dextrose agar（PDA）medium containing 0.1％ tween 80 were poured into sterilized Petri dishes, left to solidify at room temperature（22℃）, then swabbed from fresh bacterial or fungal strain culture. Wells in the centre of agar plate were created using a sterile cork borer（9 mm）and different concentrations of TO samples were transferred to the wells. Plates with pathogenic bac-teria and Candida albicans were incubated at 37℃ and 30℃ for 24 h, respectively. Other pathogenic fungi were

incubated at 30℃ for 48-72 h. The antimicrobial activity was determined by measuring the clear zones diameter（CZD）around each well in mm. Distilled water without test compounds was used as a control. The antibacterial activity of bacterial antibiotics was assessed by the agar disk diffu-sion method35） by measuring CZD around each disk in mm.2.2.8.3 Determination of minimum lethal concentrations

（MLC）The MLC of TO was determined according to the dilution

method36）. For pathogenic bacteria and Candida albicans, serial of two-fold concentrations of TO（20, 40, 80, 160, 320 and 640 μL）were pipetted into tubes containing 4 mL of L broth（LB）or potato dextrose broth medium（containing 0.1％ tween 80）, respectively. Each tube was inoculated with 0.4 mL（0.5 McFarland medium）of a standardized sus-pension of bacterial test species containing 1×106 cell/mL. For fungi, liquid media was inoculated with fungus and in-cubated for approximately 48 h at 30℃. Subsequently, the culture was filtered through a thin layer of sterile sintered Glass G2 to remove mycelia fragments. The titer of spores of each fungus was determined microscopically using a he-mocytometer. A suspension containing the spores was used for inoculation of PDA medium. Serial of two-fold concen-trations of TO were pipetted into tubes containing 4 mL of PD（containing 0.1％ tween 80）. Each tube was inoculated with 1×106 of prepared spores.

All inoculated tubes were incubated at appropriate tem-perature and time for each microorganism. After the incu-bation period, 0.1 mL from each tube was subcultured on LB agar or PDA plates and incubated at appropriate tem-perature and time for each microorganism. The lowest concentration of tested TO which gave a viable count less than 0.1％ of the original inocula（1×106 cell/mL）was assumed as the MLC.

Antibiotics such as Augmentin（30 μg）, Chloramphenicol（30 μg）, Flucoral（fluconazole, 100 μg/mL）and Mycosat（nystatin BP, 100 μg/mL）were used as standards for com-parison in antibacterial and antifungal tests, respectively. All work was carried out under subdued light conditions. All experimental procedures were performed in triplets if the variation was routinely less than 5％ and the mean values（±standard deviation）were determined.

3 Results and discussion3.1 Lipid classes of TO

To the best of our knowledge, we report for the first time on the composition and biological properties of TO. Levels of lipid classes and subclasses presented in TO are shown in Table 1. The level of NL was the highest（ca. 90％）, fol-lowed by GL（0.78％）and PL（0.53％）. In decreasing order, subclasses of NL contained triacylglycerol（TAG）, free fatty acids（FFA）, diacylglycerol（DG）, esterified sterols（STE）


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and monoacylglycerol（MG）. Significant amount of TAG was found（ca. 95.5％ of total NL）in TO followed by a lower level of FFA（ca. 1.9％ of total NL）, while DG and STE were recovered in lower levels. GL subclasses in TO（Table 1）were SQD, DGD, CER, SG, MGD and ESG. CER, SG and ESG were the main components and made up about 85％ of the total GL. TLC fractions of PL revealed that the major PL subclasses were PC followed by PE, PI and PS, respec-tively（Table 1）. About a half of total amount of PL in TO was PC and a quarter was PE, while PI and PS were mea-sured in lower quantities.

3.2 Fatty acid pro�le of TO and lipid classesFatty acid profiles of TO and lipid classes are presented

in Table 2. Nine fatty acids were identified in TO, wherein linoleic and oleic acids were the main fatty acids. Both fatty acids accounted for 82％ of the total FAME. TO con-tained high amounts of monounsaturated fatty acids（MUFA, 40.1 g/100 g total FAME）which is comparable to the hemp, cranberry, blueberry, onion and milk thistle cold pressed seed oils but was much lower than that of 81 and 82％ in the carrot and parsley cold pressed oils37, 38）. TO had a polyunsaturated fatty acids（PUFA）content of 43.7 g/100 g of total fatty acids（Table 2）. This PUFA content was lower than that in the cranberry（67.6 g/100 g）, onion（64-65 g/100 g）, milk thistle（61 g/100 g）and blueberry（69 g/100 g）cold-pressed oils39）. Palmitic and stearic were the major saturated fatty acids（SFA）, comprising together

Table 1　Lipid classes（g/kg TL）composition of TO.

Neutral lipid Subclass

Rf values×100a g/kg TL

Glycolipid Subclass

Rf values× 100b g/kg TL

Phospholipid Subclass

Rf values×100b g/kg TL

MG 14 6.55 SQD 6 0.40 PS 4.7 0.67

DG 39 9.69 DGD 17 0.60 PI 11 0.78

FFA 56 17.60 CER 29-35 2.55 PC 20 2.67

TG 79 869.00 SG 41 2.78 PE 30 1.19

STE 95 6.82 MGD 64 0.18

ESG 76 1.36a Solvent system used in TLC development: n-hexane/diethyl ether/acetic acid（60: 40: 1, v/v/v）.b Solvent system used in TLC development: chloroform/methanol/ammonia solution 25％（65: 25: 4, v/v/v）.Results are given as the average of triplicate determinations±standard deviation.Abbreviations: MAG, monoacylglycerols; DAG, diacylglycerols; TAG, triacylglycerols; FFA, free fatty acids; STE, sterol esters. SQD, sulphoquinovosyldiacylglycerol; DGD, digalactosyldiacylglycerol; CER, cerebrosides; SG, steryl glucoside; MGD, monogalactosyldiacylglycerol; ESG, esterified steryl glucoside; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine.

Table 2　‌‌Fatty acid composition（relative content, ％）of TO and its lipid classes.

OO Neutral lipids Glycolipids PhospholipidsC 10: 0 0.02 0.02 0.03 0.04C 12: 0 0.04 0.05 0.06 0.07C 14: 0 0.04 0.05 0.06 0.06C 16: 0 9.80 9.65 9.80 9.92C 16: 1 0.47 0.46 0.45 0.44C 18: 0 6.23 6.22 6.49 6.50C 18: 1n-9 39.70 39.85 39.60 39.37C 18: 2n-6 42.50 42.45 42.30 42.39C 18: 3 1.20 1.25 1.21 1.21ΣSFA 16.13 15.99 16.44 16.59

ΣMUFA 40.17 40.31 40.05 39.81

ΣPUFA 43.70 43.70 43.51 43.60

Results are given as the average of triplicate determinations±standard deviation.


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about 16％ of total FAME. TO contained about 16.1 g of SFA per 100 g of total FAME, which is lower than that of 30.8 g/100 g of total FAME in the cardamom cold pressed oil and comparable to that of 13.8 and 15.9 g/100 g of total FAME found in the cold pressed milk thistle and roasted pumpkin cold-pressed seed oils, respectively39）. The SFA levels were higher than those of 7.4-9.7 g/100 g of total FAME in the parsley, onion, hemp, mullein and cranberry cold-pressed seed oils37）.

Fatty acids of NL and polar lipids（GL and PL）were not differed significantly from each other（Table 2）, wherein linoleic and oleic acids were the main fatty acids. The ratio of unsaturated fatty acids to SFA, however, was not signifi-cantly higher in NL than in the corresponding polar frac-tions（GL and PL）. For the obtained results it could be said that TO contains high level of MUFA and PUFA. MUFA and MUFA have been shown to reduce LDL（low density lipo-proteins）cholesterol and retain HDL（high density lipopro-teins）cholesterol29）. Literature illustrates the health pro-moting benefits of PUFA, in alleviating many diseases including inflammatory, heart diseases, cardiovascular, ath-erosclerosis and diabetes. Fatty acid profile of TO evince the lipids as a good source of the nutritionally essential fatty acids. The fatty acid profile and high levels of MUFA and PUFA make the TO a special material for nutritional applications.

3.3 Tocols compositionFrom our results, TO was characterized by high amounts

of unsaponifiables（26.8 g/kg）. Levels of α-, β-, γ- and δ- to-copherols in TO were 718, 23, 4800 and 26 mg/kg oil, re-spectively. In addition, amounts of α-, β-, γ- and δ- tocotri-enols were 2146, 63, 62.1 and 134 mg/kg oil, respectively. γ-Tocopherol constituted ca. 60.2％ of total tocols followed by α-tocotrienol（26.9％ of total tocols）and α-tocopherol（9.01％ of total tocols）. Other tocols were measured in lower levels. α- and γ-Tocopherols proved to be the major tocopherols in edible oils and fats. γ-Tocopherol found in high levels in camelina, linseed, cold pressed rapeseed and corn oil40）. Levels of tocols detected in TO may contribute to the stability of the oil toward oxidation.

3.4 RSA of TO in comparison with extra virgin olive oilThe model of scavenging stable free radicals is widely

used to evaluate the antioxidant properties in a relatively short time, as compared to other methods. Two or more radical systems are needed to better study the antiradical potential of antioxidants or bioactive extracts. Ramadan and Moersel41） developed a simple experiment using toluene to dissolve fat or oil samples as well as the free radicals.

Antiradical properties of the TO and extra virgin olive oil（as a standard edible oil that contain high levels of antioxi-dants and bioactives）were compared using stable DPPH・

and galvinoxyl radicals. Figure 1 shows that TO had stron-ger antiradical action than olive oil. After 60 min of incuba-tion with DPPH・ radicals, 65％ of DPPH・ radicals were quenched by TO, while olive oil was able to quench only 45％（Fig. 1A）. ESR results showed also the same pattern（Fig. 1B）, wherein TO quenched 55％ of galvinoxyl radical and olive oil deactivated about 38％ after 60 min of reac-tion. Regarding the composition of TO and olive oil, they have different patterns of fatty acid and lipid-soluble bioac-tives.

Our results confirmed that TO was characterized by higher amounts of phenolic compounds（7.3 mg/g, respec-tively as GAE）than extra virgin olive oil（3.6 mg/g as GAE）. To detect bioactive phenolic compounds in TO, absorptive spectra between 200 and 400 nm were screened. Chemical structure of phenolic compounds, specifically the aromatic ring, produces strong absorbance in the UV region associ-ated with electronic transitions of the molecule which pro-vides a unique spectrum42）. Phenolic compounds in TO exhibit two major absorption bands in the UV/visible region: a first band in the range between 320 and 380 nm and a second band in 255-280 nm range. Absorption at 220

Fig. 1　 Scavenging effect at different incubation times of cold-pressed TO and olive oil on（A）DPPH・ radical as measured by changes in absorbance values at 515 nm and on（B）galvinoxyl radical as recorded by ESR.


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nm may be due to the presence of flavones or flavonol de-rivatives. The UV spectra of phenolic acids showed differ-ences over the scan range of 200-400 nm. Ferulic acid dis-plays a maximum absorbance at 215 nm, 287 nm and 312 nm. Moreover, p-coumaric acid exhibits a maximum absor-bance at 286 nm, 209 nm and 220 nm42）.

Phenolic compounds have been reported to be present in edible oils, which is very important for the oxidative sta-bility of the PUFA of these oils. The antioxidant effect of phenolic compounds is mainly due to their redox proper-ties and is the result of various mechanisms: radical scav-enging activity, transition metal chelating activity, and/or singlet oxygen quenching capacity43）. Total phenolic amount of TO was higher than that of 1.73-2.0 mg GAE/g oil for the red raspberry, blueberry and boysenberry cold pressed seed oils, and that of 1.8-3.4 mg GAE/g oil for the parsley, onion, cardamom, mullein and milk thistle cold pressed seed oils38, 39）. On the other hand, tocols levels in oils may have a great effect on their RSA. Increasing ring methyl substitution led to an increase of scavenging activi-ty against the DPPH・ radical, and also to a decrease in oxygen radical absorbance capacity44）.

The stronger RSA of TO compared to olive oil may be due to（i）the differences in content and profiel of unsaponi-fiable materials（ii）the diversity in structural characteristics of potential antioxidants present,（iii）a synergism of anti-oxidants with other bioactive components, and（iv）differ-ent kinetic behaviors of antioxidants. From the results we can suggest that TO may be used in different food or phar-maceutical applications to provide nutrition and health benefits.

3.5 Rancimat assayIt was important to assess the antioxidant potential of

TO in a in a food system such as edible oils. In our study, induction time（IT）of sunflower oil was 3 h. The IT for TO: sunflower oil blend（1: 9, w/w）was 6.5 h, while TO: sun-flower oil blend（2: 8, w/w）recorded the highest value（9 h）. Considering the results of Rancimat assay, the antioxidant activity of TO in sunflower oil blend was superior wherein high levels of tocols and phenolic compounds in TO can explain high antioxidant capacity.

3.6 Antimicrobial activity of TOO. vulgare have presented prominent results as antimi-

crobial agents to be applied in food bioconservation systems. Studies reported antimicrobial activity in Origa-num species, while O. vulgare extracts and essential oil had shown positive results in inhibiting the growth of pathogenic microorganisms and in the synthesis of micro-bial metabolites45, 46）.

In our study, AA of TO was examined against 4 bacterial and 4 yeast strains selected on the basis of their relevance as food spoilage or human pathogenic agents. The exami-

nation of AA of the TO by the agar diffusion method re-vealed that TO inhibited the growth of all microorganisms tested. The AA of TO was screened against eight patho-genic microorganisms using agar diffusion assay and the minimal lethal concentrations was further determined（Fig. 2 and Table 3）. TO exhibited various degrees of AA against different pathogens fungi and bacteria, wherein the highest AA was recorded against the dermatophyte fungi and yeasts including T. mentagrophytes（62 mm）, T. rubrum（40 mm）, and C. albicans（20 mm）followed by food spoil-age fungi including A. flavus（32 mm）with MLC ranging between 80 to 320 μg/mL. Dermatophytic fungi including Trichophyton rubrum are anthropophilic fungi which fre-quently cause acute or chronic inflammatory tinea corporis which is a superficial fungal infection（dermatophytosis）of the arms and legs, especially on glabrous skin. The data in Table 3 and Fig. 2 showed that TO had a significant anti-fungal potential against the tested dermatophytic fungi（T. mentagrophytes and T. rubrum）, whereas the CZD values were 62 and 40 mm, respectively. In addition, TO exhibited very low MLC values（ca. 80 and 160 μg/mL, respectively）. Furthermore, TO exhibited broad-spectra activity against mycotoxin-producing food spoilage fungi and food borne pathogen bacteria, wherein the AA of TO against A. flavus, S. aureus, E. coli and L. Monocytogenes were 32, 30, 25 and 20 mm, respectively. The MLC of TO against these mi-croorganisms was ranging between 160 to 320 μg/mL（Table 3 and Fig. 2）. The lowest AA of TO was recorded for S. enteritidis（13 mm）. In a recent study conducted by Ghabraie et al.46）, red thyme showed substantial antimicro-bial activity against S. aureus, E. coli and L. monocyoto-genes, particularly when it was combined with other es-sential oils. Similarly, Ivanovic et al.47） reported a strong AA of thyme against food-associated bacteria（e.g. E. coli, Sal-monella and Bacillus）and concluded that thyme is a valu-able source of antibacterial agents. Microbial spoilage is an important factor influencing food availability. A. flavus causes different clinical manifestations of human aspergil-losis such as cutaneous aspergillosis, aspergillar otomy-cosis, aspergillar onychomycosis, invasive lung aspergil-losis and aspergillar sinusitis46）. Yeasts are widely able to spoil many foods products, wherein Candida and Saccha-romyces are important food spoiling yeasts17）. Candida species are now at fourth ranks among microbes20）. Candida genus is known as the yeasts most frequently in-volved in the etiology of mycotic infections48）. Among Candida species, C. albicans is the organism most often associated with serious fungal infection and it is showing increased resistance to traditional antifungal agents49）. The difficulties associated with the management of Candida infections necessitate the discovery of novel antifungal agents23, 50）.

Worth mentioning that the AA of TO against tested mi-croorganisms recorded the same CZD without any chang-


J. Oleo Sci. 65, (8) 629-640 (2016)

636

Fig. 2　Antimicrobial activity of TO against studied microorganisms indicated by clear zone diameter.


J. Oleo Sci. 65, (8) 629-640 (2016)

637

ing when left for more than ten days as an additional incu-bation period. Furthermore, no growth was observed when new agar plates or broth media were inoculated with loop from the clear zone area, indicating that the TO have a lethal effect.

In relation to TO composition, it appears that the AA of TO is mainly linked to the presence of significant propor-tion of tocols and phenolic constituents with high antioxi-dant activity. The variation in the effectiveness of TO against different bacterial strains may depend on the dif-ferences in the permeability of cell of those microbes. In addition, the AA of TO might be due to the presence of outer membrane surrounding the cell wall in bacteria, which limits diffusion of hydrophobic substances through its lipopolysaccharide covering. Nazzaro et al.51） reported that AA of the oils might be targets variety of microbial cell components, particularly the cell membrane, cytoplasm, and in some cases, they completely change the morpholo-gy.

4 ConclusionsComposition and properties of bioactive phytochemicals

in herbs and medicinal plants are required to improve quality and nutritional value of the human diet. These are also important to improve utilization of food and pharma-ceutical products. The present study was designed to in-vestigate for the first time the composition and biological properties of cold pressed TO. This report might serve as a milestone toward development of healthy oils with high nutritional value. It could be concluded that the TO is a good source of essential fatty acids and tocols. The high levels of linoleic and oleic acids make TO nutritionally valu-able. The present study demonstrated that TO contained significant levels of natural antioxidants. Tocols and pheno-lics at the level estimated may be of nutritional importance as natural antioxidants and might directly react with and quench free radicals and prevent lipid peroxidation. To best of our knowledge, this is the first report on the bioac-tive lipids, antioxidant potential and antimicrobial proper-ties of TO. The results confirmed that TO have potential antimicrobial activity and could be used in food, cosmetics and pharmaceutical products to inhibit microbial growth. Since tested dermatophytic fungi strains showed a very low MLC, TO could be used as a novel anti-fungal agent. In addition, TO could be used in the development of novel foods and drug formulations for the prevention of chronic diseases such as Alzheimer and Type II diabetes, which are linked to oxidative stress.

Tabl

e 3 　

In v

itro

ant

ibac

teri

al a

nd a

ntif

unga

l act

ivit

ies

of T

O r

ecor

ded

by c

lear

zon

e di

amet

er（C

ZD

, mm）a

nd m

inim

al le

thal

con

cent

rati

on（

ML

C, μ

g/m

L）a,

b.

Food

born

e pa

thog

en b

acte

riaFo

od sp

oila

ge fu

ngi

Der

mat

ophy

tic fu

ngi

S. e

nter

itidi

sL.

mon

ocyt

ogen

esE.

coli

S. a

ureu

sC

. alb

ican

sA.

flav

usT.

men

tagr

ophy

tes

T. ru

brum

CZD

MLC

CZD

MLC

CZD

MLC

CZD

MLC

CZD

MLC

CZD

MLC

CZD

MLC

CZD

MLC

TO（

200 μL）

1364

020

160

2516

030

160

2032

032

160

6280

4080

Aug

men

tin（30

μg）

28 n

dc28

nd30

nd40

ndnd

ndnd

ndnd

ndnd

nd

Chl

oram

phen

icol（

30 μ

g）20

nd22

nd27

nd25

ndnd

ndnd

ndnd

ndnd

nd

Fluc

oral（

100 μg

/mL）

ndnd

ndnd

ndnd

ndnd

35nd

38nd

35nd

34nd

Myc

osat（

100 μg

/mL）

ndnd

ndnd

ndnd

ndnd

30nd

40nd

40nd

38nd

a Eac

h va

lue

repr

esen

ts m

ean

of sa

mpl

e±SD

for n

=3,

b Dia

met

er o

f inh

ibiti

on z

one

was

mea

sure

d as

the

clea

r are

a ce

nter

ed o

n th

e ag

ar w

ell c

onta

inin

g th

e sa

mpl

e,c n

d; n

ot d

eter

min

ed


J. Oleo Sci. 65, (8) 629-640 (2016)

638

Conflict of InterestsThe authors declare that they have no conflict of inter-

ests.

AcknowledgmentThis study was supported by a grant from Institute of

Scientific Research and Revival of Islamic Heritage at Umm Al-Qura University, Makkah, KSA（Project no. 43405062）.

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bioactive compounds of cold-pressed thyme

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