Journal of Oleo Science

Copyright ©2016 by Japan Oil Chemists’ Society

doi : 10.5650/jos.ess16042
J. Oleo Sci. 65, (8) 629-640 (2016)

Bioactive Compounds of Cold-pressed Thyme

(Thymus vulgaris) Oil with Antioxidant and
Antimicrobial Properties
Adel 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 ARABIA
2
Deptartment of Agricultural Microbiology, Faculty of Agriculture, Fayoum University, EGYPT
3
Department of Biology, Faculty of Applied Science, Umm Al-Qura University, Makkah, Kingdom of Saudi ARABIA
4
Institute of Scientific Research and Revival of Islamic Heritage, Umm Al-Qura University, Makkah, Kingdom of Saudi ARABIA
5
Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, 44519 Zagazig, EGYPT

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

1 Introduction use in as natural antioxidants and antimicrobial agents6, 7）.

Aromatic plants rich in bioactive phytochemicals are in- Thyme（Thymus vulgaris L., family Lamiaceae）is a sub-
creasingly used in foods, pharmaceuticals and cosmetics. shrub native to the western Mediterranean region. Thyme
Medicinal plants have been also used as spices and condi- （fresh or dried） is widely used as a spice to add a distinc-
ments to confer aroma and flavor to food and beverages1−3）. tive flavor to food. Thyme essential oil（TEO）extracted
Phytochemicals have become attractive to scientists as from fresh leaves can be used as aroma additives in food,
natural bioactive agents that could be safer than synthetic pharmaceuticals, and cosmetics8）. Thyme acts as an expec-
compounds4, 5）. Scientific research on aromatic plants has torant and spasmolytic agent for the bronchia, and in folk
focused on their oils and extracts because of their potential medicine it is part of herbal teas and infusions9, 10）. Various

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

629
 A. M. A. Assiri, K. Elbanna and H. H. Abulreesh et al.

bioactive compounds with antioxidant traits were de- 2 Material and methods
scribed in thyme including TEO constituents, flavonoids 2.1 Oils and chemicals
and phenolic acids11, 12）. In TEO, 25 compounds were iden- Three different samples of TO and extra virgin olive oil
tified7, 13, 14）. Thymol（48.6％）, and p-cymene（22.9％）were were obtained from a local market in Mekkah（KSA）. Total
the main compounds present in the TEO followed by phenolics content（TPC）in olive oil as determined by the
α-terpinolene（6.49％）7）. Recently, Karabagias et al.15） re- Folin-Ciocalteu was 3.6 mg/g as gallic acid equivalents
ported a reduction of meat oxidation and bacterial counts （GAE）. Neutral lipid（NL）standards were obtained from
when TEO was used to extend the shelf-life of lamb meat. Sigma（St. Louis, MO, USA）. Standards used for glycolipids
Solomakos et al.16）tested the antimicrobial impact of TEO （GL）including monogalactosyldiacylglycerol（MGD）, diga-
on Escherichia coli in minced beef meat. during cold lactosyldiacylglycerol（DGD）, cerebrosides（CER）, steryl
storage. glucoside（SG）and esterified steryl glucoside（ESG）were
Microbial spoilage affects the shelf life and the cost of purchased from Biotrend（Köln, Germany）. Standards used
the product17）. It was estimated that 48 million people ex- for phospholipids（PL）including phosphatidylserine（PS）,
perience food borne illness in the United States every phosphatidylethanolamine（PE）, phosphatidylinositol（PI）
year18）. Food borne pathogens are responsible for 325.000 from bovine liver and phosphatidylcholine（ PC）from
hospitalizations and 5000 deaths in the US annually19）. soybean were purchased from Sigma（St. Louis, MO, USA） .
Yeasts spoil foods resulting in changes in physical and Standards used for tocols were purchased from Merck
chemicals properties. Candida, Pichia, Rhodotorula, （Darmstadt, Germany）.
Torulopsis, Saccharomyces, Zygossacharomyces, Han-
senula and Trichosporon are important food spoiling 2.2 Methods
yeasts 17）. On the other hand, candidiasis caused by 2.2.1 Column chromatography（CC）and thin layer chroma-
Candida species has increased dramatically in the past tography（TLC） of lipid classes
years. Candida species are opportunistic pathogen that 2.2.1.1 Fractionation of lipid classes and subclasses
causes local and systemic infections in predisposed TO was separated into neutral and polar lipid classes by
persons, commonly affecting immunologically compro- elution with solvents over a glass column（20 mm×30 cm）
mised patients and those undergoing prolonged antibiotic packed with a slurry of activated silicic acid（70 to 230
treatment20）. Candida infections created a great burden mesh; Merck, Darmstadt, Germany） in chloroform（1: 5, w/
on the public healthcare sector and this situation has gone v）. NL was eluted with 3-times the column volume of chlo-
worse by various epidemiological changes. The difficulties roform25）. The major portion of GL was eluted with 5-times
associated with the management of Candida infections the column volume of acetone and that of PL with 4-times
necessitate the discovery of new antifungal agents 21, 22）. the column volume of methanol. The amount of the lipid
Plant-derived bioactive compounds and plant extracts may classes obtained was determined by gravimetry. By means
offer potential lead to new antimicrobial and antioxidant of TLC on Silica gel F254 plates（thickness 0.25 mm; Merck,
compounds23）. Darmstadt, Germany）a further characterisation of the GL
Cold-pressed oils have been internationally marketed, and PL subclasses was carried out with chloroform/metha-
wherein bio-information on their chemical composition, nol/25％ ammonia solution（65: 25: 4, v/v/v）. For the char-
antioxidant potential and antimicrobial properties have not acterisation of NL subclasses, TLC plates were developed
been fully reported. Increased interest on cold-pressed oils with n-hexane/diethyl ether/acetic acid（60: 40: 1, v/v/v）.
has been recognized as these oils have high levels of lipid For detection of the lipids, TLC plates were sprayed with
soluble bioactives with nutritive properties. The cold press- the following agents: for the marking of all lipids with sul-
ing technology is becoming an interesting substitute for phuric acid（40％）, for the marking of GL with α-naphthol/
traditional practices because of consumers’desire for sulphuric acid and for the marking of PL with the molyb-
natural products. Cold pressing involves no chemical or date-blue reagent26）. Each spot was identified with lipid
heat or refining treatments. Therefore, cold pressed oils standards as well as their reported retention factor（Rf）
usually contain a high level of lipophilic phytochemicals in- values. Individual bands were visualized under ultraviolet
cluding natural antioxidants24）. light, scraped from the plate and recovered by extraction
It is hard to find any data in the literature on cold- with chloroform: methanol （2: 1, v/v）. Fatty acid composi-
pressed thyme （Thymus vulgaris）oil
（TO） . This study was tion of TO as well as NL, GL and PL was determined by
performed to: 1）determine lipid classes, fatty acids and GLC/FID as described below.
tocols of TO, 2）measure the total phenolics content, in 2.2.1.2 Quantitative determination of lipid subclasses
vitro antiradical activity and antioxidant potential in food For the quantitative determination of NL classes, indi-
model（Rancimat assay）of TO; and 3）estimate the antimi- vidual bands were scraped from the plate and recovered by
crobial activity （AA）of TO. extraction with 10％ methanol in diethyl ether, followed by
diethyl ether. For the quantitative estimation of GL sub-
630
J. Oleo Sci. 65, (8) 629-640 (2016)
 cold-pressed thyme oil with novel antioxidant and antimicrobial properties

classes, the acetone fraction obtained by CC was separated 2.2.4 Extraction, quantification and characterization of
by TLC in the above given solvent system. The silica gel phenolic compounds
regions with the corresponding GL classes were scraped Aliquots of TO and extra virgin olive oil（1 g）were dis-
out followed by hexose measurement photometrically at solved in n-hexane（5 mL）and mixed with 10 mL methanol:
485 nm using the phenol: sulphuric acid in acid-hydrolysed water（80: 20, v/v） in a glass tube for two min in a vortex29）.
lipids27）. The percent distribution of each component was After centrifugation at 3000 rpm for 10 min, the hydroalco-
obtained from the hexose values. From the extinction holic extracts were separated from the lipid phase by using
values the quantitative amount was determined and related a Pasteur pipette then combined and concentrated in
to their portion of the GL fraction. The determined portion vacuo at 30℃ until a syrup consistency was reached. The
was set into relation with the amount of oil, which had oily residue was re-dissolved in 10 mL methanol: water（80:
been separated by CC into the main lipid fractions. For the 20, v/v）and the extraction was repeated twice. Hydroalco-
determination of the PL, the methanol fraction from CC holic extracts were re-dissolved in acetonitrile（15 mL）and
was also separated by TLC in the above given solvent the mixture was washed three times with n-hexane （15 mL
system and after scraping out of the individual PL sub- each）. Purified phenols in acetonitrile were concentrated
classes brought to reaction with the hydrazine sulphate: in vacuo at 30℃ then dissolved in methanol for further
sodium molybdate reagent at 100℃ for 10 min and photo- analysis. Aliquots of phenolic extracts were evaporated to
metrically analyzed at 650 nm. From the obtained extinc- dryness under nitrogen. The residue was re-dissolved in 0.2
tion values via a calibration chart for phosphorus the mL water and diluted（1: 30）Folin-Ciocalteu’ s phenol
amount of PL was calculated. The individual values were reagent（1 mL）was added. After 3 min, 7.5％ sodium car-
put into relation to the PL fraction（methanol fraction from bonate（0.8 mL）was added. After 30 min, the absorbance
CC）and to the amount of oil. was measured at 765 nm using a UV-260 visible recording
2.2.2 Gas chromatography（GC） analysis of fatty acid meth- spectrophotometer（Shimadzu, Kyoto, Japan）. Gallic acid
（FAME）
yl esters was used for the calibration and the results of triplicate
Fatty acids of TO and lipid classes were transesterified analyses are expressed as parts per million of gallic acid.
into FAME using N-trimethylsulfoniumhydroxide（Mach- UV spectrum（200-400 nm）of diluted extracts（1 mg/mL）
erey-Nagel, Düren, Germany）according to Arens et al.28）. was recorded by spectrophotometry（Jenway 4605-UV–VIS
FAME were identified on a Shimadzu GC-14A equipped Spectrophotometer）.
with flame ionization detector（FID）and C-R4AX chroma- 2.2.5 Radical scavenging activity（RSA）of TO and olive oil
topac integrator（ Kyoto, Japan）. The flow rate of the toward DPPH・
carrier gas helium was 0.6 mL/min and the split value with RSA of TO and extra virgin olive oil was assayed with
a ratio of 1: 40. A sample of 1 μL was injected on a 30 m× DPPH・ radical dissolved in toluene30）. Toluene solution of
0.25 mm×0.2 μm film thickness Supelco SPTM-2380 DPPH・ radicals was freshly prepared at a concentration of
（Bellefonte, PA, USA）capillary column. The injector and 10−4 M. The radical, in the absence of antioxidant com-
FID temperature was set at 250℃. The initial column tem- pounds, was stable for more than 2 h of normal kinetic
perature was 100℃ programmed by 5℃/min until 175℃ assay. For evaluation, 10 mg of TO or olive oil（in 100 μL
and kept 10 min at 175℃, then 8℃/min until 220℃ and toluene）was mixed with 390 μL toluene solution of DPPH・
kept 10 min at 220℃. A comparison between the retention radicals and the mixture was vortexed for 20 s at ambient
times of the samples with those of an authentic standard temperature. Against a blank of pure toluene without
mixture（Sigma, St. Louis, MO, USA; 99％ purity specific DPPH・, the decrease in absorption at 515 nm was mea-
for GC）, run on the same column under the same condi- sured in 1-cm quartz cells after 1, 30 and 60 min of mixing
tions, was made to facilitate identification. using a UV-260 visible recording spectrophotometer（Shi-
2.2.3 HPLC analysis of tocols madzu, Kyoto, Japan） . RSA toward DPPH・ radicals was es-
For tocols analysis, a solution of 250 mg of TO in 25 mL timated from the differences in absorbance of toluene
n-heptane was directly used for the HPLC24）. The HPLC DPPH・ solution with or without sample（control）and the
analysis was conducted using a Merck Hitachi low-pressure inhibition percent was calculated from the following equa-
gradient system, fitted with an L-6000 pump, a Merck-Hita- tion:
chi F-1000 Fluorescence Spectrophotometer （The detector
wavelength was set at 295 nm for excitation, and at 330 nm ［
％ Inhibition＝（absorbance of control−absorbance of
for emission）and a D-2500 integration system; 20 μL of the test sample） ×100.
/absorbance of control］
samples were injected by a Merck 655-A40 Autosampler
onto a Diol phase HPLC column 25 cm 94.6 mm ID （Merck, 2.2.6 RSA of TO and olive oil toward galvinoxyl radical
Darmstadt, Germany） using a flow rate of 1.3 mL/min. The A miniscope MS 100 ESR spectrometer（ Magnettech
mobile phase used was n-heptane: tert-butyl methyl ether GmbH; Berlin, Germany）was used in this analysis29）. Ex-
（99: 1, v/v）. perimental conditions were as follows: measurement at
631
J. Oleo Sci. 65, (8) 629-640 (2016)
 A. M. A. Assiri, K. Elbanna and H. H. Abulreesh et al.

room temperature; microwave power, 6 db; centerfield, incubated at 30℃ for 48-72 h. The antimicrobial activity
3397 G, sweep width 83 G, receiver gain 10 and modulation was determined by measuring the clear zones diameter
amplitude 2000 mG. Ten mg of oil （in 100 μL toluene） were （CZD） around each well in mm. Distilled water without test
allowed to react with 100 μL of toluenic solution of galviox- compounds was used as a control. The antibacterial activity
yl（0.125 mM）. The mixture was stirred on a vortex stirrer of bacterial antibiotics was assessed by the agar disk diffu-
for 20 s then transferred into a 50 μL micro pipette sion method35）by measuring CZD around each disk in mm.
（Hirschmann Laborgeräte GmbH, Ederstadt, Germany） and 2.2.8.3 Determination of minimum lethal concentrations
the amount of galvinoxyl radical inhibited was measured （MLC）
exactly after total incubation time of 60 min after the addi- The MLC of TO was determined according to the dilution
tion of the galvinoxyl radical solution. The galvinoxyl signal method36）. For pathogenic bacteria and Candida albicans,
intensities were evaluated by the peak height of signals serial of two-fold concentrations of TO（20, 40, 80, 160, 320
against a control. A quantitative estimation of the radical and 640 μL）were pipetted into tubes containing 4 mL of L
concentration was obtained by evaluating the decrease of broth（LB）or potato dextrose broth medium（containing
the ESR signals in arbitrary units after 60 min incubation 0.1％ tween 80）, respectively. Each tube was inoculated
using the KinetikShow 1.06 Software program （Magnettech with 0.4 mL （0.5 McFarland medium） of a standardized sus-
GmbH; Berlin, Germany） . The reproducibility of the mea- pension of bacterial test species containing 1×106 cell/mL.
surements was 5％ as usual for kinetic parameters. For fungi, liquid media was inoculated with fungus and in-
2.2.7 Rancimat assay cubated for approximately 48 h at 30℃. Subsequently, the
The antioxidant potential of TO blends with sunflower culture was filtered through a thin layer of sterile sintered
oil（1: 10 and 2: 8 w/w, TO: sunflower oil）was tested with Glass G2 to remove mycelia fragments. The titer of spores
Rancimat method according to Ranalli et al.31）. The Ranci- of each fungus was determined microscopically using a he-
mat apparatus was operated at 120℃. A dry air flow of 20 mocytometer. A suspension containing the spores was used
L/h was passed through the oil sample（5 g）. The volatile for inoculation of PDA medium. Serial of two-fold concen-
oxides coming from the oxidation of the oil or blend dis- trations of TO were pipetted into tubes containing 4 mL of
solved in cold milli-Q water（60 mL） , causing an increase in PD（containing 0.1％ tween 80） . Each tube was inoculated
the electrical conductivity. The time（h）taken to reach a with 1×106 of prepared spores.
specific conductivity value, corresponding to the flex point All inoculated tubes were incubated at appropriate tem-
of the peroxidation curve, was considered as the induction perature and time for each microorganism. After the incu-
time（IT） . The higher the induction time the higher the an- bation period, 0.1 mL from each tube was subcultured on
tioxidant potency of the compounds. Tests were performed LB agar or PDA plates and incubated at appropriate tem-
in triplicate. perature and time for each microorganism. The lowest
2.2.8 Antimicrobial activity（AA） concentration of tested TO which gave a viable count less
2.2.8.1 Bacteria and fungi strains than 0.1％ of the original inocula（ 1×10 6 cell/mL）was
The AA of TO were assessed against pathogenic bacteria assumed as the MLC.
and fungi including Staphylococcus aureus（ATCC8095）, Antibiotics such as Augmentin （30 μg）, Chloramphenicol
Salmonella enteritidis（ATCC 13076）, Escherichia coli （30 μg）, Flucoral（fluconazole, 100 μg/mL）and Mycosat
（ATCC 25922）, Listeria monocytogenes（ATCC 15313）, （nystatin BP, 100 μg/mL） were used as standards for com-
Candida albicans（ ATCC 10231）, Aspergillus flavus, parison in antibacterial and antifungal tests, respectively.
Trichophyton mantigrophytes, and Trichophyton All work was carried out under subdued light conditions.
rubrum. Stock cultures of bacteria were maintained on All experimental procedures were performed in triplets if
nutrient agar slants at 4℃, while fungi and Candida yeast the variation was routinely less than 5％ and the mean
were maintained on potato dextrose agar slants at 4℃. values（±standard deviation）were determined.
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 3 Results and discussion
containing 0.1％ tween 80 were poured into sterilized Petri 3.1 Lipid classes of TO
dishes, left to solidify at room temperature（22℃）, then To the best of our knowledge, we report for the first time
swabbed from fresh bacterial or fungal strain culture. Wells on the composition and biological properties of TO. Levels
in the centre of agar plate were created using a sterile cork of lipid classes and subclasses presented in TO are shown
borer（9 mm）and different concentrations of TO samples in Table 1. The level of NL was the highest（ca. 90％）, fol-
were transferred to the wells. Plates with pathogenic bac- lowed by GL （0.78％）and PL（0.53％）. In decreasing order,
teria and Candida albicans were incubated at 37℃ and （TAG）
subclasses of NL contained triacylglycerol , free fatty
30℃ for 24 h, respectively. Other pathogenic fungi were acids（FFA）, diacylglycerol（DG）, esterified sterols（STE）
632
J. Oleo Sci. 65, (8) 629-640 (2016)
 cold-pressed thyme oil with novel antioxidant and antimicrobial properties

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

Neutral lipid Rf values× Glycolipid Rf values× Phospholipid Rf values×
g/kg TL g/kg TL g/kg TL
Subclass 100a Subclass 100b Subclass 100b
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 1.36
a
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 Phospholipids
C 10: 0 0.02 0.02 0.03 0.04
C 12: 0 0.04 0.05 0.06 0.07
C 14: 0 0.04 0.05 0.06 0.06
C 16: 0 9.80 9.65 9.80 9.92
C 16: 1 0.47 0.46 0.45 0.44
C 18: 0 6.23 6.22 6.49 6.50
C 18: 1n-9 39.70 39.85 39.60 39.37
C 18: 2n-6 42.50 42.45 42.30 42.39
C 18: 3 1.20 1.25 1.21 1.21
ΣSFA 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.

and monoacylglycerol（MG）. Significant amount of TAG in Table 2. Nine fatty acids were identified in TO, wherein
was found （ca. 95.5％ of total NL） in TO followed by a lower linoleic and oleic acids were the main fatty acids. Both
level of FFA（ca. 1.9％ of total NL）, while DG and STE fatty acids accounted for 82％ of the total FAME. TO con-
were recovered in lower levels. GL subclasses in TO （Table tained high amounts of monounsaturated fatty acids
1）were SQD, DGD, CER, SG, MGD and ESG. CER, SG and （MUFA, 40.1 g/100 g total FAME）which is comparable to
ESG were the main components and made up about 85％ the hemp, cranberry, blueberry, onion and milk thistle cold
of the total GL. TLC fractions of PL revealed that the major pressed seed oils but was much lower than that of 81 and
PL subclasses were PC followed by PE, PI and PS, respec- 82％ in the carrot and parsley cold pressed oils37, 38）. TO
tively（Table 1）. About a half of total amount of PL in TO had a polyunsaturated fatty acids（PUFA）content of 43.7
was PC and a quarter was PE, while PI and PS were mea- g/100 g of total fatty acids（Table 2）. This PUFA content
sured in lower quantities. 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
3.2 Fatty acid profile of TO and lipid classes g/100 g）cold-pressed oils39）. Palmitic and stearic were the
Fatty acid profiles of TO and lipid classes are presented major saturated fatty acids（SFA）, comprising together
633
J. Oleo Sci. 65, (8) 629-640 (2016)
 A. M. A. Assiri, K. Elbanna and H. H. Abulreesh et al.

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.
Fig. 1　Scavenging effect at different incubation times of
3.3 Tocols composition cold-pressed TO and olive oil on （A）DPPH・ radical
From our results, TO was characterized by high amounts as measured by changes in absorbance values at
of unsaponifiables （26.8 g/kg） . Levels of α-, β-, γ- and δ- to- 515 nm and on（B） galvinoxyl radical as recorded by
copherols in TO were 718, 23, 4800 and 26 mg/kg oil, re- ESR.
spectively. In addition, amounts of α-, β-, γ- and δ- tocotri-
enols were 2146, 63, 62.1 and 134 mg/kg oil, respectively. and galvinoxyl radicals. Figure 1 shows that TO had stron-
γ-Tocopherol constituted ca. 60.2％ of total tocols followed ger antiradical action than olive oil. After 60 min of incuba-
by α-tocotrienol（26.9％ of total tocols）and α-tocopherol tion with DPPH・ radicals, 65％ of DPPH・ radicals were
（9.01％ of total tocols）. Other tocols were measured in quenched by TO, while olive oil was able to quench only
lower levels. α- and γ-Tocopherols proved to be the major 45％（Fig. 1A）. ESR results showed also the same pattern
tocopherols in edible oils and fats. γ-Tocopherol found in （Fig. 1B） , wherein TO quenched 55％ of galvinoxyl radical
high levels in camelina, linseed, cold pressed rapeseed and and olive oil deactivated about 38％ after 60 min of reac-
corn oil40）. Levels of tocols detected in TO may contribute tion. Regarding the composition of TO and olive oil, they
to the stability of the oil toward oxidation. have different patterns of fatty acid and lipid-soluble bioac-
tives.
3.4 RSA of TO in comparison with extra virgin olive oil Our results confirmed that TO was characterized by
The model of scavenging stable free radicals is widely higher amounts of phenolic compounds（7.3 mg/g, respec-
used to evaluate the antioxidant properties in a relatively tively as GAE）than extra virgin olive oil（3.6 mg/g as GAE）.
short time, as compared to other methods. Two or more To detect bioactive phenolic compounds in TO, absorptive
radical systems are needed to better study the antiradical spectra between 200 and 400 nm were screened. Chemical
potential of antioxidants or bioactive extracts. Ramadan structure of phenolic compounds, specifically the aromatic
and Moersel 41） developed a simple experiment using ring, produces strong absorbance in the UV region associ-
toluene to dissolve fat or oil samples as well as the free ated with electronic transitions of the molecule which pro-
radicals. vides a unique spectrum 42）. Phenolic compounds in TO
Antiradical properties of the TO and extra virgin olive oil exhibit two major absorption bands in the UV/visible
（as a standard edible oil that contain high levels of antioxi- region: a first band in the range between 320 and 380 nm
dants and bioactives）were compared using stable DPPH・ and a second band in 255-280 nm range. Absorption at 220
634
J. Oleo Sci. 65, (8) 629-640 (2016)
 cold-pressed thyme oil with novel antioxidant and antimicrobial properties

nm may be due to the presence of flavones or flavonol de- nation of AA of the TO by the agar diffusion method re-
rivatives. The UV spectra of phenolic acids showed differ- vealed that TO inhibited the growth of all microorganisms
ences over the scan range of 200-400 nm. Ferulic acid dis- tested. The AA of TO was screened against eight patho-
plays a maximum absorbance at 215 nm, 287 nm and 312 genic microorganisms using agar diffusion assay and the
nm. Moreover, p-coumaric acid exhibits a maximum absor- minimal lethal concentrations was further determined （Fig.
bance at 286 nm, 209 nm and 220 nm42）. 2 and Table 3） . TO exhibited various degrees of AA against
Phenolic compounds have been reported to be present different pathogens fungi and bacteria, wherein the highest
in edible oils, which is very important for the oxidative sta- AA was recorded against the dermatophyte fungi and
bility of the PUFA of these oils. The antioxidant effect of yeasts including T. mentagrophytes （62 mm）, T. rubrum
phenolic compounds is mainly due to their redox proper- （40 mm）, and C. albicans （20 mm）followed by food spoil-
ties and is the result of various mechanisms: radical scav- age fungi including A. flavus（32 mm）with MLC ranging
enging activity, transition metal chelating activity, and/or between 80 to 320 μg/mL. Dermatophytic fungi including
singlet oxygen quenching capacity 43）. Total phenolic Trichophyton rubrum are anthropophilic fungi which fre-
amount of TO was higher than that of 1.73-2.0 mg GAE/g quently cause acute or chronic inflammatory tinea corporis
oil for the red raspberry, blueberry and boysenberry cold which is a superficial fungal infection （dermatophytosis） of
pressed seed oils, and that of 1.8-3.4 mg GAE/g oil for the the arms and legs, especially on glabrous skin. The data in
parsley, onion, cardamom, mullein and milk thistle cold Table 3 and Fig. 2 showed that TO had a significant anti-
pressed seed oils38, 39）. On the other hand, tocols levels in fungal potential against the tested dermatophytic fungi（T.
oils may have a great effect on their RSA. Increasing ring mentagrophytes and T. rubrum） , whereas the CZD values
methyl substitution led to an increase of scavenging activi- were 62 and 40 mm, respectively. In addition, TO exhibited
ty against the DPPH・ radical, and also to a decrease in very low MLC values（ca. 80 and 160 μg/mL, respectively）.
oxygen radical absorbance capacity44）. Furthermore, TO exhibited broad-spectra activity against
The stronger RSA of TO compared to olive oil may be mycotoxin-producing food spoilage fungi and food borne
due to（i） the differences in content and profiel of unsaponi- pathogen bacteria, wherein the AA of TO against A. flavus,
fiable materials（ii） the diversity in structural characteristics S. aureus, E. coli and L. Monocytogenes were 32, 30, 25
of potential antioxidants present,（iii）a synergism of anti- and 20 mm, respectively. The MLC of TO against these mi-
oxidants with other bioactive components, and（iv）differ- croorganisms was ranging between 160 to 320 μg/mL
ent kinetic behaviors of antioxidants. From the results we （Table 3 and Fig. 2）. The lowest AA of TO was recorded
can suggest that TO may be used in different food or phar- for S. enteritidis（13 mm）. In a recent study conducted by
maceutical applications to provide nutrition and health Ghabraie et al.46）, red thyme showed substantial antimicro-
benefits. bial activity against S. aureus, E. coli and L. monocyoto-
genes, particularly when it was combined with other es-
3.5 Rancimat assay sential oils. Similarly, Ivanovic et al.47）reported a strong AA
It was important to assess the antioxidant potential of of thyme against food-associated bacteria （e.g. E. coli, Sal-
TO in a in a food system such as edible oils. In our study, monella and Bacillus） and concluded that thyme is a valu-
induction time（IT） of sunflower oil was 3 h. The IT for TO: able source of antibacterial agents. Microbial spoilage is an
sunflower oil blend（1: 9, w/w）was 6.5 h, while TO: sun- important factor influencing food availability. A. flavus
（2: 8, w/w）
flower oil blend （9 h）
recorded the highest value . causes different clinical manifestations of human aspergil-
Considering the results of Rancimat assay, the antioxidant losis such as cutaneous aspergillosis, aspergillar otomy-
activity of TO in sunflower oil blend was superior wherein cosis, aspergillar onychomycosis, invasive lung aspergil-
high levels of tocols and phenolic compounds in TO can losis and aspergillar sinusitis46）. Yeasts are widely able to
explain high antioxidant capacity. spoil many foods products, wherein Candida and Saccha-
romyces are important food spoiling yeasts17）. Candida
3.6 Antimicrobial activity of TO species are now at fourth ranks among microbes 20）.
O. vulgare have presented prominent results as antimi- Candida genus is known as the yeasts most frequently in-
crobial agents to be applied in food bioconservation volved in the etiology of mycotic infections 48）. Among
systems. Studies reported antimicrobial activity in Origa- Candida species, C. albicans is the organism most often
num species, while O. vulgare extracts and essential oil associated with serious fungal infection and it is showing
had shown positive results in inhibiting the growth of increased resistance to traditional antifungal agents49）. The
pathogenic microorganisms and in the synthesis of micro- difficulties associated with the management of Candida
bial metabolites45, 46）. infections necessitate the discovery of novel antifungal
In our study, AA of TO was examined against 4 bacterial agents23, 50）.
and 4 yeast strains selected on the basis of their relevance Worth mentioning that the AA of TO against tested mi-
as food spoilage or human pathogenic agents. The exami- croorganisms recorded the same CZD without any chang-
635
J. Oleo Sci. 65, (8) 629-640 (2016)
 A. M. A. Assiri, K. Elbanna and H. H. Abulreesh et al.

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

636
J. Oleo Sci. 65, (8) 629-640 (2016)
 cold-pressed thyme oil with novel antioxidant and antimicrobial properties

ing when left for more than ten days as an additional incu-

MLC
bation period. Furthermore, no growth was observed when
.

80
nd
nd
nd
nd
a, b

T. rubrum
（MLC, μg/mL）
Dermatophytic fungi new agar plates or broth media were inoculated with loop
from the clear zone area, indicating that the TO have a
CZD
40
nd
nd
34
38
lethal effect.
In relation to TO composition, it appears that the AA of
TO is mainly linked to the presence of significant propor-
T. mentagrophytes
MLC
80
nd
nd
nd
nd
and minimal lethal concentration

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-
CZD
62
nd
nd
35
40 ferences in the permeability of cell of those microbes. In
addition, the AA of TO might be due to the presence of
MLC

outer membrane surrounding the cell wall in bacteria,

160
nd
nd
nd
nd
A. flavus

which limits diffusion of hydrophobic substances through

its lipopolysaccharide covering. Nazzaro et al.51） reported
Food spoilage fungi

CZD

that AA of the oils might be targets variety of microbial cell

32
nd
nd
38
40

components, particularly the cell membrane, cytoplasm,

and in some cases, they completely change the morpholo-
（CZD, mm）

MLC
320

gy.
nd
nd
nd
nd
C. albicans
CZD
20
nd
nd
35
30
Table 3　In vitro antibacterial and antifungal activities of TO recorded by clear zone diameter

4 Conclusions
Diameter of inhibition zone was measured as the clear area centered on the agar well containing the sample,

Composition and properties of bioactive phytochemicals

MLC
160
nd
nd
nd
nd

in herbs and medicinal plants are required to improve

S. aureus

quality and nutritional value of the human diet. These are

also important to improve utilization of food and pharma-
CZD
30
40
25
nd
nd

ceutical products. The present study was designed to in-

vestigate for the first time the composition and biological
MLC

properties of cold pressed TO. This report might serve as a

160
nd
nd
nd
nd
Foodborne pathogen bacteria

milestone toward development of healthy oils with high

E.coli

nutritional value. It could be concluded that the TO is a

CZD

good source of essential fatty acids and tocols. The high

25
30
27
nd
nd

levels of linoleic and oleic acids make TO nutritionally valu-

able. The present study demonstrated that TO contained
L. monocytogenes
MLC
160
nd
nd
nd
nd

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
CZD
20
28
22
nd
nd

quench free radicals and prevent lipid peroxidation. To

Each value represents mean of sample±SD for n=3,

best of our knowledge, this is the first report on the bioac-

tive lipids, antioxidant potential and antimicrobial proper-
MLC

c
640
S. enteritidis

nd
nd
nd
nd

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.
CZD
13
28
20
nd
nd

Since tested dermatophytic fungi strains showed a very

low MLC, TO could be used as a novel anti-fungal agent. In
（30 μg）

addition, TO could be used in the development of novel

Mycosat（100 μg/mL）
Flucoral（100 μg/mL）

foods and drug formulations for the prevention of chronic

Augmentin（30 μg）

nd; not determined

diseases such as Alzheimer and Type II diabetes, which are

Chloramphenicol

linked to oxidative stress.

（200 μL）
TO

b
a

637
J. Oleo Sci. 65, (8) 629-640 (2016)
 A. M. A. Assiri, K. Elbanna and H. H. Abulreesh et al.

Conflict of Interests 10）Chizzola, R.; Michitsch, H.; Franz, C. Antioxidative

The authors declare that they have no conflict of inter- properties of Thymus vulgaris leaves: comparison of
ests. different extracts and essential oil chemotypes. J. Agr.
Food Chem. 56, 6897-6904（2008）.
11）Vila, R. Flavonoids and further polyphenols in the ge-
nus Thymus. Thyme. The Genus Thymus. Medici-
Acknowledgment nal and Aromatic Plantss Industrial Profiles; Stahl-
This study was supported by a grant from Institute of Biskup, E., Saez, F., Eds.; Taylor and Francis: New
Scientific Research and Revival of Islamic Heritage at Umm York, Vol. 24（2002）.
Al-Qura University, Makkah, KSA （Project no. 43405062） . 12）Miura, K.; Kikuzaki, H.; Nakatani, N. Antioxidant activ-
ity of chemical compounds of sage（Salvia officinalis）
and thyme（Thymus vulgaris）measured by the oil
stability index method. J. Agr. Food Chem. 50, 1850-
References 1855（2002）.
1）Salgueiro, L.; Martins, A. P.; Correia, H. Raw materials: 13）Tomaino, A.; Cimino, F.; Zimbalatti, V.; Venuti, V.;
the importance of quality and safety. A review. Fla- Sulfaro, V.; De Pasquale, A.; Saija, A. Influence of heat-
vour Frag. J. 25, 253-271 （2010） . ing on antioxidant activity and the chemical composi-
2）Köse, L. P.; Gülçin I.; Gören A. C.; Namiesnik J.; Marti- tion of some spice essential oils. Food Chem. 89, 549-
nez-Ayala A. L.; Gorinstein S. LC–MS/MS analysis, an- 554（2005）.
tioxidant and anticholinergic properties of galanga （Al- 14）Viuda-Martos, M.; El Gendy, N. G. S.; Sendra, E.; Fer-
pinia officinarum Hance）rhizomes. Ind. Crop. nandez-Lopez, J.; ElRazik, K. A. A.; El-Sayed, A.; Per-
Prod. 74, 712-721 （2015） . ez-Alvarez, J. A. Chemical composition and antioxi-
3）Tohma, H. S.; Gülçin I. Antioxidant and radical scav- dant and anti-listeria activities of essential oils
enging activity of aerial parts and roots of Turkish Li- obtained from some Egyptian plants. J. Agr. Food
quorice（Glycyrrhiza glabra L.） . Inter. J. Food Prop. Chem. 58, 9063-9070（2010）.
13, 657-671 （2010） . 15）Karabagias, I.; Badeka, A.; Kontominas, M. G. Shelf life
4）Albano, S. M.; Miguel, M. G. Biological activities of ex- extension of lamb meat using thyme or oregano essen-
tracts of plants grown in Portugal. Ind. Crop. Prod. tial oils and modified atmosphere packaging. Meat Sci.
33, 338-343 （2011） . 88, 109-116（2010）.
5）El-Maati, M. F. A.; Mahgoub, S. A.; Labib, S. M.; Al-Ga- 16）Solomakos, N.; Govaris, A.; Koidis, P.; Botsoglou, N.
by, A. M. A.; Ramadan, M. F. Phenolic extracts of clove The antimicrobial effect of thyme essential oil, nisin
（Syzygium aromaticum）with novel antioxidant and and their combination against Escherichia coli O157:
antibacterial activities. Eur. J. Integr. Med. in press H7 in minced beef during refrigerated storage. Meat
http: //dx.doi.org/10.1016/j.eujim.2016.02.006 （2016）. Sci. 80, 159-166（2008）.
6）El-Ghorab, A. H.; Nauman, M.; Anjum, F. M.; Hussin, S.; 17）Souza, E. L.; Stamford, T. L. M.; Lima, E. O.; Trajano, V.
Nadeem, M. A comparative study on chemical compo- N. Effectiveness of Origanum vulgare L. essential oil
sition and antioxidant activity of ginger（Zingiber offi- to inhibit the growth of food spoiling yeasts. Food
cinale）and cumin（ Cuminum cyminum）. J. Agr. Cont. 18, 409-413（2007）.
Food Chem. 58, 8231-8237 （2010） . 18）Nsoesie, E. O.; Kulberg, S. A.; Brownstien, J. S. Online
7）Viuda-Martos M.; Mohamady, M. A.; Fernández-López, reports of foodborne illness capture food implicated in
J.; Abd ElRazik, K. A.; Omer, E. A.; Pérez-Alvarez, J. official foodborne outbreak reports. Prevent. Med. 67,
A.; Sendra, E. In vitro antioxidant and antibacterial ac- 264-269（2014）.
tivities of essentials oils obtained from Egyptian aro- 19）Luther, M.; Parry, J.; Moore, J.; Meng, J.; Zhang, Y.;
matic plants. Food Cont. 22, 1715-1722 （2011） . Cheng, Z.; Yu, L. Inhibitory effect of Chardonnay and
8）Lee, S.-J.; Umano, K.; Shibamoto, T.; Lee, K.-G. Identi- black raspberry seed extracts on lipid oxidation in fish
fication of volatile components in basil（Ocimum basi- oil and their radical scavenging and antimicrobial
licum L.） and thyme leaves （Thymus vulgaris L.） and properties. Food Chem. 104, 1065-1073（2007）.
their antioxidant properties. Food Chem. 91, 131-137 20）Zhang, Z.; Elsohly, H. N.; Jacob, M. R.; Pasco, D. S.;
（2005） . Walker, L. A.; Clark, A. M. Natural products inhibiting
9）Zarzuelo, A.; Crespo, E. The medicinal and non-medic- Candida albicans secreted aspartic proteases from
inal uses of thyme. In Thyme. The Genus Thymus. Tovomita krukovii. Planta Medica 68, 49-54 （2002）.
Medicinal and Aromatic PlantssIndustrial Profiles; 21）Wong, S. S. W.; Samaranayake, L. P.; Seneviratne, C. J.
Stahl-Biskup, E., Saez, F., Eds.; Taylor and Francis: In pursuit for the ideal antifungal agent for Candida
New York, Vol. 24（2002） . infections: high-throughout screening for small mole-
638
J. Oleo Sci. 65, (8) 629-640 (2016)
 cold-pressed thyme oil with novel antioxidant and antimicrobial properties

cules. Drug Disc. Today 19, 1721-1730 （2014） . 34）Gülçin, I.; Tel, A. Z.; Kirecci, E. Antioxidant, antimicro-
22）Assiri A. M. A.; Elbanna K.; Al-Thubiani A.; Ramadan M. bial, antifungal and antiradical activities of Cyclotrich-
F. Cold-pressed oregano（Origanum vulgare）oil: a ium niveum（Boiss.）Manden and Scheng. Inter. J.
rich source of bioactive lipids with novel antioxidant Food Prop. 11, 450-471（2008）.
and antimicrobial properties. Eur. Food Res. Tech- 35）Patton, T.; Barrett, J.; Brennan, J.; Moran, N. Use of a
nol., in press. DOI 10.1007/s00217-015-2607-7 （2016）. spectrophotometric bioassay for determination of mi-
23）Khosravi, A. R.; Shokri, H.; Kermani, S.; Dakhili, M.; crobial sensitivity to manuka honey. J. Microbiol.
Madani, M.; Parsa, S. Antifungal properties of Arte- Methods 64, 84-95（2006）.
misia sieberi and Origanum vulgare essential oils 36）Jobran, E.; Finegold, S. M. Diagonative Microbiology.
against Candida glabrata isolates obtained from pa- 9th ed. part 2, pp.168-188. Mosby Saint Louis, USA
tients with vulvovaginal candidiasis. J. Mycol. Med. （1994）.
21, 93-99 （2011） . 37）Parker, T. D.; Adams, D. A.; Zhou, K.; Harris, M.; Yu, L.
24）Ramadan, M. F. Healthy blends of high linoleic sun- Fatty acid composition and oxidative stability of cold-
flower oil with selected cold pressed oils: Functional- pressed edible seed oils. J. Food Sci. 68, 1240-1243
ity, stability and antioxidative characteristics. Ind. （2003）.
Crop. Prod. 43, 65-72 （2013） . 38）Parry, J.; Su, L.; Luther, M.; Zhou, K.; Yurawecz, M. P.;
25）Ramadan, M. F.; Elsanhoty, R. M. Lipid classes, fatty Whittaker, P.; Yu, L. Fatty acid composition and anti-
acids and bioactive lipids of genetically modified pota- oxidant properties of cold-pressed marionberry, boy-
to Spunta with Cry V gene. Food Chem. 133, 1169- senberry, red raspberry, and blueberry seed oils. J.
1176 （2012） . Agr. Food Chem. 53, 566-573（2005）.
26）Ramadan, M. F.; Sharanabasappa, G.; Seetharam, Y. N.; 39）Parry, J.; Su, L.; Moore, J.; Cheng, Z.; Luther, M.; Rao, J.
Seshagiri, M.; Moersel, J.-T. Characterisation of fatty N.; Wang, J.-Y.; Yu, L. Chemical compositions, antioxi-
acids and bioactive compounds of kachnar（Bauhinia dant capacities, and antiproliferative activities of se-
purpurea L.）seed oil. Food Chem. 98, 359-365 lected fruit seed flours. J. Agr. Food Chem. 54, 3773-
（2006） . 3778（2006）.
27）Ramadan, M. F.; Asker, M. M. S.; Tadros, M. Antiradical 40）Schwartz, H.; Ollilainen, V.; Piironen, V.; Lampi, A.-M.
and antimicrobial properties of cold-pressed black Tocopherol, tocotrienol and plant sterol contents of
cumin and cumin oils. Eur. Food Res. Technol. 234, vegetable oils and industrial fats. J. Food Comp.
833-844 （2012） . Anal. 21, 152-161（2008）.
28）Arens, M.; Schulte, E.; Weber, K. Fettsäuremethyles- 41）Ramadan, M. F.; Moersel, J.-T. Screening of the anti-
ter, Umesterung mit Trimethylsulfoniumhydroxid radical action of vegetable oils. J. Food Comp. Anal.
（Schnellverfahren）. Fat Sci. Technol. 96, 67-68 19, 838-842（2006）.
（1994） . 42）Holser, R. A. Principal Component Analysis of Pheno-
29）Ramadan, M. F.; Kinni, S. G.; Seshagiri, M.; Mörsel, J.-T. lic Acid Spectra. ISRN Spectroscopy http: //dx.doi.
Fat-soluble bioactives, fatty acid profile and radical org/10.5402/2012/493203（2012）.
scavenging activity of Semecarpus anacardium seed 43）Bettaieb, I.; Bourgou, S.; Wannes, W. A.; Hamrouni, I.;
oil. J. Amer. Oil Chem. Soc. 87, 885-894 （2010） . Limam, F.; Marzouk, B. Essential oils, phenolics, and
30）Ramadan, M. F. Antioxidant characteristics of pheno- antioxidant activities of different parts of cumin
lipids（quercetin-enriched lecithin）in lipid matrices. （Cuminum cyminum L.）. J. Agr. Food Chem. 58,
Ind. Crop. Prod. 36, 363-369 （2012） . 10410-10418（2010）.
31）Ranalli, L.; Pollastri, S.; Contento, E.; Iannucci, L. Ef- 44）Müller, L.; Theile, K.; Böhm, V. In vitro antioxidant ac-
fect of olive paste kneading process time on the over- tivity of tocopherols and tocotrienols and comparison
all quality of virgin olive oil. Eur. J. Lipid Sci. Tech- of vitamin E concentration and lipophilic antioxidant
nol. 105, 57-67 （2003） . capacity in human plasma. Mol. Nutr. Food Res. 54,
32）Torres, A.; Garedew, A.; Schmolz, E.; Lamprecht, I. 731-742（2010）.
Calorimetric investigation of the antimicrobial action 45）Baydar, H.; Sagdic, O.; Ozkan, G.; Karadgan, T. Anti-
and insight into the chemical properties of“angelita” bacterial activity and composition of essential oils
honey- a product of the stingless bee Tetragonisca an- from Origanum, Thymbra and Sartureja species
gustula from Colombia. Thermochim. Acta 415, 107- with commercial importance in Turkey. Food Cont.
113（2004） . 15, 69-172（2004）.
33）Gülçin, I.; Kirecci, E.; Akkemik, E.; Topal, F.; Hisar, O. 46）Ghabraie, M.; Vu, K. D.; Tata, L.; Salmieri, S.; Lacroix,
Antioxidant and antimicrobial activities of an aquatic M. Antimicrobial effect of essential oils in combina-
plant: Duckweed （Lemna minor L.） . Turkish J. Biol. tions against five bacteria and their effect on sensorial
34, 175-188 （2010） . quality of ground meat. LWT-Food Sci. Technol. 66,
639
J. Oleo Sci. 65, (8) 629-640 (2016)
 A. M. A. Assiri, K. Elbanna and H. H. Abulreesh et al.

332-339（2016） . bicans biofilms to antifungal agents in vitro. Antimi-

47）Ivanovic, J.; Misic, D.; Zizovic, I.; Ristic, M. In vitro crob. Agents Chemother. 39, 2128-2131（1995）.
control of multiplication of some food-associated bac- 50）Zhang, X. L.; Guo, Y. S.; Wang, C.-H.; Li, G.-Q.; Xu, J.-J.;
teria by thyme, rosemary and sage isolates. Food Chung, H.-Y.; Ye, W.-C.; Li, Y.-L.; Wang, G.-C. Phenolic
Cont. 25, 110-116 （2012） . compounds from Origanum vulgare and their anti-
48）Lima, I. O.; Oliveira, R. A. G.; Lima, E. O.; Farias, N. M. oxidant and antiviral activities. Food Chem. 152, 300-
P.; Souza, E. L. Atividade antifúngica de óleos essenci- 306（2014）.
ais sobre espécies de Candida. Rev. Bras. Farma- 51）Nazzaro, F.; Fratianni, F.; Laura De Martino, L.; Cop-
cogn. 16, 197-201 （2006） . pola, R.; De Feo, V. Effect of essential oils on patho-
49）Hawser, S. P.; Douglas, L. J. Resistance of Candida al- genic bacteria. Pharmaceuticals 6, 1451-1474（2013）.

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