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This study was designed to examine the in vitro antimicrobial and antioxidant activities of the essential oil and various extracts (prepared by using solvents of varying polarity) of Salvia tomentosa (Miller). The essential oil was particularly found to possess strong antimicrobial activity while other non-polar extracts and subfractions showed moderate activities while polar extracts remained almost inactive. GC and GC/MS analyses of the oil resulted in the identification of 44 compounds, representing 97.7% of the oil; β-pinene (39.7%), α-pinene (10.9%) and camphor (9.7%) were the main components. The samples were also subjected to screening for their possible antioxidant activity by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene-linoleic acid assays. In the first case, the free radical scavenging activity of aqueous methanol extract (MW) was superior to all other extracts (IC50=18.7 μg/ml). Polar extracts exhibited stronger activities than non-polar extracts. In the case of the linoleic acid system, oxidation of the linoleic acid was effectively inhibited by the polar subfraction of the MW extract, while the oil was less effective. The MW extract showed 90.6% inhibition, that is close to the synthetic antioxidant BHT.
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Antimicrobial and antioxidant activities of the essential oil
and various extracts of Salvia tomentosa Miller (Lamiaceae)
Bektas Tepe
a,*
, Dimitra Daferera
b
, Atalay Sokmen
a
, Munevver Sokmen
c
,
Moschos Polissiou
b
a
Department of Biology, Faculty of Science and Literature, Cumhuriyet University, 58140 Sivas, Turkey
b
Laboratory of General Chemistry, Agricultural University of Athens, Iera Odos 75, Athens 118 55, Greece
c
Department of Chemistry, Faculty of Science and Literature, Cumhuriyet University, 58140 Sivas, Turkey
Received 17 June 2003; accepted 11 September 2003
Abstract
This study was designed to examine the in vitro antimicrobial and antioxidant activities of the essential oil and various extracts
(prepared by using solvents of varying polarity) of Salvia tomentosa (Miller). The essential oil was particularly found to possess
strong antimicrobial activity while other non-polar extracts and subfractions showed moderate activities while polar extracts re-
mained almost inactive. GC and GC/MS analyses of the oil resulted in the identification of 44 compounds, representing 97.7% of the
oil; b-pinene (39.7%), a-pinene (10.9%) and camphor (9.7%) were the main components. The samples were also subjected to
screening for their possible antioxidant activity by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and b-carotene-linoleic acid assays.
In the first case, the free radical scavenging activity of aqueous methanol extract (MW) was superior to all other extracts (IC50 ¼18.7
lg/ml). Polar extracts exhibited stronger activities than non-polar extracts. In the case of the linoleic acid system, oxidation of the
linoleic acid was effectively inhibited by the polar subfraction of the MW extract, while the oil was less effective. The MW extract
showed 90.6% inhibition, that is close to the synthetic antioxidant BHT.
Ó2003 Elsevier Ltd. All rights reserved.
Keywords: Salvia tomentosa; Essential oil; Antimicrobial activity; Antioxidant activity; GC-MS
1. Introduction
Essential oils and extracts obtained from many plants
have recently gained popularity and scientific interest.
Many plants have been used for different purposes, such
as food, drugs and perfumery (Heath, 1981). Research-
ers have been interested in biologically active com-
pounds isolated from plant species for the elimination of
pathogenic microorganisms because of the resistance
that microorganisms have built against antibiotics (Es-
sawi & Srour, 2000).
Plant products are also known to possess potential
for food preservation (Baratta et al., 1998a; Baratta,
Dorman, Deans, Biondi, & Ruberto, 1998b; Deans,
1991; Deans & Ritchie, 1987; Halendar et al., 1998).
Oxidation of lipids, which occurs during raw material
storage, processing, heat treatment and further storage
of final products, is one of the basic processes causing
rancidity of food products, leading to their deteriora-
tion. Due to undesirable influences of oxidized lipids on
the human organism, it seems to be essential to decrease
contact with products of lipid oxidation in food (Kar-
pinska, Borowski, & Danowska-Oziewicz, 2001). In
order to prolong the storage stability of foods, synthetic
antioxidants are used for industrial processing. But,
according to toxicologists and nutritionists, the side ef-
fects of some synthetic antioxidants used in food pro-
cessing such as, butylated hydroxytoluene (BHT) and
butylated hydroxyanisole (BHA), have already been
documented. For example, these substances can show
carcinogenic effects in living organisms (Ames, 1983;
Baardseth, 1989). From this point of view, governmen-
tal authorities and consumers are concerned about the
*
Corresponding author. Tel.: +90-346219-1010x2907; fax: +90-346-
219-1186.
E-mail address: btepe@cumhuriyet.edu.tr (B. Tepe).
0308-8146/$ - see front matter Ó2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2003.09.013
Food Chemistry 90 (2005) 333–340
www.elsevier.com/locate/foodchem
Food
Chemistry
safety of their food and about the potential effects of
synthetic additives on health (Reische, Lillard, & Ein-
tenmiller, 1998).
Salvia, the largest genus of Lamiaceae, includes about
900 species, widespread throughout the world. This ge-
nus is represented, in Turkish flora, by 88 species and 93
taxa, 45 of which are endemic (Guner, Ozhatay, Ekim, &
Baser, 2000). Some members of this genus are of eco-
nomic importance since they have been used as flavour-
ing agents in perfumery and cosmetics. Sage (S.
officinalis) has been credited with a long list of medicinal
uses: e.g. spasmolytic, antiseptic, astringent (Newall,
Anderson, & Philipson, 1996). Some of the phenolic
compounds of plants belonging to this genus have also
shown excellent antimicrobial activity, as well as scav-
enging activity of active oxygen, as in superoxide anion
radicals, hydroxyl radicals and singlet oxygen (Masaki,
Sakaki, Atsumi, & Sakurai, 1995), inhibiting lipid per-
oxidation (Hohmann et al., 1999), consequently, the
corresponding extracts have been widely used to stabilize
fat and fat-containing foods (Ternes & Schwarz, 1995).
Despite the medicinal potential of plants in Turkey
being considerable, knowledge of this area and studies
on these plants are scarce (Digrak, Alma, & Ilcim, 2001).
As far as our literature survey could ascertain, antimi-
crobial activities of S. tomentosa have been previously
published (Haznedaroglu, Karabay, & Zeybek, 2001) on
limited number of microorganisms, but no information
is available on the antioxidative nature of this plant. The
aim of the present study was to investigate the antimi-
crobial and antioxidant potential of the essential oil and
various extracts of S. tomentosa, as well as to establish
the best procedure to obtain extracts containing active
principles.
2. Materials and methods
2.1. Collection of plant material
S. tomentosa was collected from S
o
g
utl
ug
ol plateau
(1000 m), D
uzic
ßi-Osmaniye, Turkey, when flowering (late
July, 2001). The voucher specimen was identified by Dr.
Erol Donmez at the Department of Biology, Cumhuriyet
University, Sivas-Turkey and deposited at the Herbarium
of the Department of Biology, Cumhuriyet University,
Sivas-Turkey (CUFH-Voucher no: ED 6363).
2.2. Isolation of the essential oil
A portion (100 g) of the aerial parts of S. tomentosa
was submitted for 3 h to water-distillation, using a
Clevenger-type apparatus (British type) (yield 0.51% v/
w). The obtained essential oil (EO) was dried over an-
hydrous sodium sulphate and, after filtration, stored at
+4 °C until tested and analysed.
2.3. Preparation of the extracts
2.3.1. General
Extracts of air-dried and ground plant materials were
prepared by using solvents of varying polarity and the
extraction protocol of each is given below:
2.3.2. Preparation of the deodorized hot water extract
After completion of hydro-distillation, the liquid re-
tentate was collected and lyophilized. This procedure
gave deodorized hot water extract (DeHW) in a yield of
21.09% (w/w) (Dapkevicius, Venskutonis, Van Beek, &
Linssen, 1998).
2.3.3. Preparation of the deodorized methanol extract
The solid retentate of the hydrodistillation was dried
and re-extracted with methanol. The resulting extract
(DeMeOH) (11.57%, w/w) was fractionated with water
and chloroform (CHCl3) to give deodorized water-
soluble (DeMW) (2.62%, w/w) and water-insoluble
(deodorized chloroformic) (DeMC) (7.47%, w/w) sub-
fractions (Dapkevicius et al., 1998).
2.3.4. Preparation of the hexane, dichloromethane and
methanol extracts
A portion (100 g) of dried plant material was ex-
tracted with hexane (HE) (5.36%, w/w), followed by
dichloromethane (DCM) (1.67%, w/w) and methanol
(MeOH) (7.83%, w/w) in a Soxhlet apparatus (6 h for
each solvent). The latter extract was suspended in water
and partitioned with chloroform (CHCl3) to obtain
water soluble (MW) (6.01%, w/w) and water insoluble
(MC) (1.42%, w/w) subtractions. (Sokmen, Jones, &
Erturk, 1999).
All extracts obtained were lyophilized and kept in the
dark at +4 °C until used.
2.4. Gas chromatography/mass spectrometry analysis
conditions
2.4.1. Gas chromatography analysis
The essential oil was analysed using a Hewlett
Packard 5890 II GC equipped with a FID detector and
HP-5 MS capillary column (30 m 0.25 mm, film
thickness 0.25 lm). Injector and detector temperatures
were set at 220 and 290 °C, respectively. Oven temper-
ature was kept at 50 °C for 3 min, then gradually raised
to 160 °Cat3°C/min, held for 10 min and finally raised
to 240 °Cat3°C/min. Helium was the carrier gas, at a
flow rate of 1 ml/min. Diluted samples (1/100 in acetone,
v/v) of 1.0 ll were injected manually and in the splitless
mode. Quantitative data were obtained electronically
from FID area percent data without the use of correc-
tion factors.
334 B. Tepe et al. / Food Chemistry 90 (2005) 333–340
2.4.2. Gas chromatography/mass spectrometry analysis
Analysis of the oils was performed using a Hewlett
Packard 5890 II GC, equipped with a HP 5972 mass
selective detector and a HP-5 MS capillary column (30
m0.25 mm, film thickness 0.25 lm). For GC/MS de-
tection, an electron ionization system, with ionization
energy of 70 eV, was used. Helium was the carrier gas, at
a flow rate of 1 ml/min. Injector and MS transfer line
temperatures were set at 220 and 290 °C, respectively.
Oven programme temperature was the same with GC
analysis. Diluted samples (1/100 in acetone, v/v) of 1.0 ll
were injected manually and in the splitless mode. The
components were identified by comparison of their rel-
ative retention times and mass spectra with those of
standards (for the main components), NBS75K library
data of the GC/MS system and literature data, as de-
scribed by Adams (2001). The results were also con-
firmed by comparison of the compounds elution order
with their relative retention indices on non-polar phases
as reported by Adams (2001).
2.5. Antimicrobial activity
2.5.1. Microbial strains
The essential oil and extracts were individually
tested against a panel of microorganisms, including
Staphylococcus aureus ATCC 25923 and ATCC 29213
(for minimum inhibitory concentration (MIC) test),
Streptococcus pneumoniae ATCC 49619, Moraxella
catarrhalis ATCC 49143, Bacillus cereus ATCC 11778,
Acinetobacter lwoffii ATCC 19002, Enterobacter aer-
ogenes ATCC 13043, Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Proteus mirabilis
ATCC 7002, Pseudomonas aeruginosa ATCC 27853,
Clostridium perfringens K
UKENS-Turkey, mycob-
acter, Mycobacterium smegmatis CMM 2067, Candida
albicans ATCC 10239 and C. krusei ATCC 6258.
Bacterial strains were cultured overnight at 37 °Cin
Mueller Hinton agar (MHA), with the exception of S.
pneumoniae (MHA containing 50 ml citrate blood/l)
and C. perfringens (in anaerobic conditions). Yeasts
were cultured overnight at 30 °C in Sabouraud dex-
trose agar.
2.5.2. Antimicrobial screening
Two different methods were employed for the deter-
mination of antimicrobial activities: agar well-diffusion
method for the extracts and disc diffusion method for
the essential oil (NCCLS, 1999). The MICs of the es-
sential oil against the test microorganisms were deter-
mined by the broth microdilution method (NCCLS,
1997). The MICs of netilmicin and amphotericin B were
also determined in parallel experiments in order to
control the sensitivity of the test microorganisms. All
tests were performed in duplicate.
2.5.3. Agar-well diffusion method
In the agar-well diffusion method, all extracts were
weighed and dissolved in phosphate buffer saline (PBS;
pH 7.0–7.2) and dimethylsulphoxide (DMSO), respec-
tively, 10 mg/ml, followed by sterilization using a 0.45
lm membrane filter. Each microorganism was sus-
pended in sterile saline and diluted at 106colony
forming unit (cfu) per ml. They were ‘‘flood-inoculated’’
onto the surface of MHA. The wells (8 mm in diameter)
were cut from the agar and 0.06 ml of extract solution
was delivered into them. After incubation for 24 h at 37
°C, all plates were examined for any zones of growth
inhibition, and the diameters of these zones were
measured in millimetres. All tests were performed in
duplicate.
2.5.4. Disc diffusion method
The agar disc diffusion method was employed for the
determination of antimicrobial activities of the essential
oil in question (NCCLS, 1997). Briefly, a suspension of
the tested microorganism (0.1 ml of 108cells per ml) was
spread on the solid media plates. Filter paper discs (6
mm in diameter) were impregnated with 15 ll of the oil
and placed on the inoculated plates. These plates, after
remaining at 4 °C for 2 h, were incubated at 37 °C for 24
h for bacteria and, at 30 °C for 48 h, for yeasts. The
diameters of the inhibition zones were measured in
millimetres. All tests were performed in duplicate.
2.5.5. Determination of minimum inhibitory concentration
A broth microdilution broth susceptibility assay was
used, as recommended by NCCLS, for the determina-
tion of the MIC (NCCLS, 1999). All tests were per-
formed in Mueller Hinton Broth (MHB; BBL)
supplemented with Tween 80 detergent (final concen-
tration of 0.5% (v/v), with the exception of the yeasts
(Sabouraud dextrose broth-SDB + Tween 80). Bacterial
strains were cultured overnight at 37 °C in MHA and
the yeasts were cultured overnight at 30 °C in SDB. Test
strains were suspended in MHB to give a final density of
5105cfu/ml and these were confirmed by viable
counts. Geometric dilutions, ranging from 0.036 to 72.0
mg/ml of the essential oil, were prepared in a 96-well
microtitre plate, including one growth control
(MHB + Tween 80) and one sterility control
(MHB + Tween 80 + test oil). Plates were incubated un-
der normal atmospheric conditions, at 37 °C for 24 h for
bacteria, and at 30 °C for 48 h for yeasts. The bacterial
growth was indicated by the presence of a white ‘‘pellet’’
on the well bottom.
2.6. Antioxidant activity
2.6.1. DPPH assay
The hydrogen atom or electron donation abilities of
the corresponding extracts and some pure compounds
B. Tepe et al. / Food Chemistry 90 (2005) 333–340 335
were measured from the bleaching of the purple-col-
oured methanol solution of 2,20-diphenylpicrylhydrazyl
(DPPH). This spectrophotometric assay uses the stable
radical DPPH as a reagent (Burits & Bucar, 2000;
Cuendet, Hostettmann, & Potterat, 1997). Fifty mi-
crolitre of various concentrations of the extracts in
methanol were added to 5 ml of a 0.004% methanol
solution of DPPH. After a 30 min incubation period
at room temperature, the absorbance was read against
a blank at 517 nm. Inhibition of free radical by
DPPH in percent (I%) was calculated in following
way:
I%¼ðAblank Asample=Ablank Þ100;
where Ablank is the absorbance of the control reaction
(containing all reagents except the test compound), and
Asample is the absorbance of the test compound. Extract
concentration providing 50% inhibition (IC50 ) was
calculated form the plot of inhibition percentage
against extract concentration. Tests were carried out in
triplicate.
2.6.2. DPPH assay on TLC
This procedure was applied for all extracts and the
essential oil. Five microlitre of a 1:10 dilution of the
extracts in methanol were applied to the TLC plate and
methanol–ethyl acetate (1:1) mixture was used as de-
veloper. The plate was sprayed with a 0.2% DPPH re-
agent in methanol and left at room temperature for 30
min. As explained above, yellow spots formed from
bleaching of the purple colour of DPPH reagent, were
evaluated as positive antioxidant activity.
2.6.3. b-Carotene-linoleic acid assay
In this assay, antioxidant capacity is determined by
measuring the inhibition of the volatile organic com-
pounds and the conjugated diene hydroperoxides arising
from linoleic acid oxidation (Dapkevicius et al., 1998). A
stock solution of b-carotene-linoleic acid mixture was
prepared as follows: 0.5 mg b-carotene was dissolved in
1 ml of chloroform (HPLC grade) and 25 ll linoleic acid
and 200 mg Tween 40 were added. Chloroform was
completely evaporated using a vacuum evaporator.
Then, 100 ml distilled water saturated with oxygen (30
min 100 ml/min) were added with vigorous shaking.
Two thousand five hundred microlitres of this reaction
mixture were dispensed into test tubes and 350 ll por-
tions of the extracts, prepared at 2 g/l concentrations,
were added and the emulsion system was incubated for
48 h at room temperature. The same procedure was
repeated with synthetic antioxidant, BHT, as positive
control, and a blank. After this incubation period, ab-
sorbances of the mixtures were measured at 490 nm.
Antioxidative capacities of the extracts were compared
with those of BHT and blank.
2.7. Assay for total phenolics
Total phenolic constituents of the aforesaid extracts
of S. tomentosa were determined by the literature
methods involving the Folin–Ciocalteu reagent and
gallic acid as standard (Chandler & Dodds, 1983;
Slinkard & Singleton, 1977). 0.1 ml of extract solu-
tion, containing 1000 lg extract, was taken in a
volumetric flask, 46 ml distilled water and 1 ml Folin-
Ciocalteu reagent were added, and flask was shaken
thoroughly. After 3 min, 3 ml of a solution of 2%
Na2CO3were added and the mixture was allowed to
stand for 2 h with intermittent shaking. Absorbance
was measured at 760 nm. The same procedure was
repeated for all standard gallic acid solutions and a
standard curve was obtained by the equation given
below:
Absorbance :0:0012 Gallic acid ðlgÞþ0:0033:
3. Results and discussion
3.1. Chemical composition of the essential oils
About 44 compounds, representing 97.7% of the
oil, were identified. GC and GC/MS analyses revealed
that the major constituents of the oil were b-pinene
(39.7%), a-pinene (10.9%) and camphor (9.7%) as lis-
ted in Table 1.
To the best of our knowledge, there are many re-
ports on the chemical composition of the oils isolated
from the plants belonging to the genus Salvia (Ahmadi
& Mirza, 1999; Baser, Ozek, Kirimer, & Tumen, 1993;
Baser, Beis, & Ozek, 1995a; Baser, Kurkcuoglu, Ozek,
& Sarikardasoglu, 1995b; Baser, Demircakmak, & Er-
min, 1996; Baser, Duman, Vural, Adiguzel, & Aytac,
1997; Baser, Kurkcuoglu, & Aytac, 1998; Couladis,
Tzakou, Stojanovic, Mimica-Dukic, & Jancic, 2001;
Perry et al., 1999; Rustaiyan, Masoudi, Monfared, &
Komeilizadeh, 1999; Sefidkon & Khajavi, 1999; Torres,
Velasco-Negueruela, Perez-Alonso, & Pinilla, 1997;
Tumen, Baser, Kurkcuoglu, & Duman, 1998). Most of
these reports indicate that 1,8-cineole (eucalyptol) and
borneol are the main and/or characteristic constituents
of Salvia oils.
According to a study carried out by Haznedaroglu
et al. (2001), cyclofenchene, 1,8-cineole, borneol and
b-caryophyllene were the major constituents of S.
tomentosa oil. These findings are not in agreement
with the results presented here, except for borneol,
which was found to be 4.3% in our study. The
changes in the essential oil compositions might have
arisen from several differences (climatical, seasonal,
geographical, geological), as mentioned by Perry et al.
(1999).
336 B. Tepe et al. / Food Chemistry 90 (2005) 333–340
3.2. Antimicrobial activity
As can be seen in Table 2, water-soluble extracts
(DeHW and DeMW) did not exhibit antimicrobial ac-
tivity, but water-insoluble extracts were found to have
moderate activity against S. aureus,S. pneumoniae,M.
catarrhalis,B. cereus,A. lwoffii,C. perfringens,M.
smegmatis and C. albicans. Except for four test micro-
organisms (M. catarrhalis,B. cereus,A. lwoffii and C.
albicans), the essential oil of S. tomentosa exhibited
stronger antimicrobial activity against the microorgan-
isms than those of extracts. In general, weaker activity
was observed against gram-negative ones.
Results obtained from disc diffusion method, fol-
lowed by measurements of MIC, indicate that C. per-
fringens, an anaerobic microorganism, is the most
sensitive microorganism tested, with the lowest MIC
values (0.54 mg/ml) in the presence of the oil isolated
from S. tomentosa (Table 3). S. pneumoniae and M.
smegmatis were other sensitive ones against the oil
with an MIC value at 2.25 mg/ml. No activity was ob-
served against three gram-negative microorganisms
(E. coli,P. mirabilis and P. aeruginosa).
As far as our literature survey could ascertain, there
was only one report on the antibacterial activity of the
essential oil of S. tomentosa. As far as this report is con-
cerned, weak antibacterial activity was observed against
microorganisms including E. coli,S. aureus and P. aeru-
ginosa (Haznedaroglu et al., 2001). The antimicrobial
activity of the major compounds of the oil studied here
was previously well defined by several researchers (Dor-
man & Deans, 2000; Knobloch, Pauli, Iberi, Wegand, &
Weis, 1989; Tabanca, Kirimer, Demirci, Demirci, &
Baser, 2001; Vardar-Unlu et al., 2003). Based on one re-
port, pinene-type monoterpene hydrocarbons (a-pinene
and b-pinene) and borneol (oxygenated monoterpene)
had slight activity against a panel of microorganisms
(Dorman & Deans, 2000). Antimicrobial activity of
borneol was also reported by other investigators
(Knobloch et al., 1989; Tabanca et al., 2001; Vardar-
Unlu et al., 2003). On the other hand, camphor is also
known to possess slight antifungal (Alvarez-Castellanos,
Bishop, & Pascual-Villalobos, 2001) and antibacterial
activity (Demetzos, Angelopoulou, & Perdetzoglou,
2002). Despite slight activity capacities, pinene-type
monoterpenes, camphor and borneol, could be respon-
sible for the total activity spectrum. The mechanism of
action of terpenes is not fully understood but is specu-
lated to involve membrane disruption by the lipophilic
compounds (Cowan, 1999). Moreover, moderate activi-
ties of water-insoluble extracts could also be attributed to
the presence of several types of compounds belonging to
different classes, such as oleoresins in hexane extract (HE)
(Dapkevicius et al., 1998), sterols and their derivatives,
flavones and flavonoids in dichloromethane extract
(Guillen & Manzanos, 1998), and more polar thermo-
labile and/or thermo-stable phenolics in the hydrophobic
subfractions of methanol extract (Sokmen et al., 1999).
3.3. Antioxidant activity
The essential oil and various extracts were subjected
to screening for their possible antioxidant activity. Two
Table 1
Chemical composition of S. tomentosa essential oil (%)
CompoundsaRt.b(min) KIc%
1a-Thujene 9.491 930 0.7
2a-Pinene 9.781 939 10.9
3 Camphene 10.407 954 2.4
4b-Pinene 11.931 979 39.7
5b-Myrcene 12.475 991 0.7
6a-Terpinene 13.645 1017 0.4
7 p-Cymene 14.071 1025 1.1
8 Limonene 14.262 1029 2.2
9 Eucalyptol 14.344 1031 1.1
10 (Z)-b-Ocimene 14.761 1037 0.7
11 (E)-b-Ocimene 15.269 1050 0.2
12 c-Terpinene 15.758 1060 1.4
13 cis-Sabinene
hydrate
16.221 1070 0.4
14 Terpinolene 17.228 1089 0.5
15 Linalool 17.917 1097 0.9
16 trans-Sabinene
hydrate
18.960 1098 0.2
17 Camphor 20.185 1146 9.7
18 Borneol 21.291 1169 4.3
19 Terpinen-4-ol 21.763 1177 0.7
20 a-Terpineol 22.434 1189 0.3
21 Linalyl acetate 25.518 1257 0.3
22 Bornyl acetate 26.951 1289 0.5
23 Thymol 27.486 1290 0.7
24 Carvacrol 27.930 1299 0.9
25 a-Cubebene 29.935 1351 0.5
26 a-Ylangene 30.942 1375 0.3
27 a-Copaene 31.159 1377 0.4
28 b-Bourbonene 31.577 1388 0.8
29 b-Cubebene 31.794 1388 0.2
30 a-Gurjunene 32.674 1410 0.3
31 Caryophyllene 33.155 1419 2.3
32 b-Copaene 33.545 1432 0.6
33 Aromadendrene 33.980 1441 0.3
34 a-Caryophyllene 34.660 1455 1.9
35 Allo-
Aromadendrene
34.933 1460 0.2
36 c-Muurolene 35.649 1480 1.5
37 Germacrene D 35.821 1485 0.6
38 b-Selinene 36.066 1490 0.4
39 c-Amorphene 36.420 1496 1.2
40 c-Cadinene 37.245 1514 0.5
41 d-Cadinene 37.626 1523 1.4
42 Ledol 39.495 1569 0.7
43 (-)-Spathulenol 39.912 1578 0.4
44 Viridiflorol 40.583 1593 2.3
Total 97.7
a
Compounds listed in order of elution from a HP-5 MS column.
b
Retention time (as minutes).
c
Kovats Index on DB-5 column in reference to n-alkanes (Adams,
2001).
B. Tepe et al. / Food Chemistry 90 (2005) 333–340 337
complementary test systems, namely DPPH free radical
scavenging and beta carotene/linoleic acid systems. Free
radical scavenging capacities of the extracts, measured
by DPPH assay, are shown in Fig. 1. Since the reaction
followed a concentration-dependent pattern, only con-
centrations of active extracts providing 50% inhibition
were included in the table. The free radical scavenging
activity of aqueous methanol extract (MW) was superior
to all other extracts (IC50 ¼18.7 lg/ml). Polar extracts
exhibited stronger activity than non-polar extracts.
When compared to BHT, the MW is the most effective
radical scavenger. Activity should be related to its phe-
nolic content since gallic acid equivalent of total phen-
olics was estimated as 200 ± 4.00 lg/mg dry weight
extract (20%, w/w, see Table 4).
Table 3
Antimicrobial activity of the essential oil of S. tomentosa using agar disc diffusion and MIC methods
Microorganisms Essential oil The MIC’s of the antibioticsc
DDaMICbNET AMP B
Staphylococcus aureus 14.75 18.00 8 103NT
Streptococcus pneumoniae 18.00 2.25 NTdNT
Moraxella catarrhalis 7.25 72.00 NT NT
Bacillus cereus 11.00 9.00 NT NT
Acinetobacter lwoffii 10.25 18.00 NT NT
Enterobacter aerogenes 7.00 72.00 NT NT
Escherichia coli NAeNA 1 102NT
Klebsiella pneumoniae 6.50 72.00 1 102NT
Proteus mirabilis NA NA NT NT
Pseudomonas aeruginosa NA NA 1 102NT
Clostridium perfringens 15.50 0.54 NT NT
Mycobacterium smegmatis 18.75 2.25 NT NT
Candida albicans 12.50 18.00 NT 1 103
Candida krusei 12.50 36.00 NT 1 103
a
DD, agar disc diffusion method. Diameter of inhibition zone (mm) including disk diameter of 6 mm.
b
MIC, minimum inhibitory concentration; values given as mg/ml for the essential oils and as lg/ml for antibiotics.
c
NET, netilmycine; AMP B, amphotericin B.
d
NT, not tested.
e
NA, not active.
Fig. 1. Free radical scavenging capacities of the extracts measured in
DPPH assay.
Table 2
Antimicrobial activity of the various extracts of S. tomentosa using agar well diffusion methoda
Microorganisms Extracts
MeOH DeMeOH
HE DCM MC MW DeHW DeMC DeMW
Staphylococcus aureus 12.50 NA NA NA NA 11.50 NA
Streptococcus pneumoniae 17.00 13.00 12.00 NA NA 19.50 NA
Moraxella catarrhalis 11.00 NA NA NA NA NA NA
Bacillus cereus 14.50 10.50 NA NA NA 13.00 NA
Acinetobacter lwoffii 9.50 15.00 10.50 NA NA 14.00 NA
Enterobacter aerogenes NA NA NA NA NA NA NA
Escherichia coli NA NA NA NA NA NA NA
Klebsiella pneumoniae NA NA NA NA NA NA NA
Proteus mirabilis NA NA NA NA NA NA NA
Pseudomonas aeruginosa NA NA NA NA NA NA NA
Clostridium perfringens 13.00 12.50 9.00 NA NA 14.50 NA
Mycobacterium smegmatis 14.00 NA NA NA NA 14.50 NA
Candida albicans 14.50 13.50 11.50 NA NA 12.00 NA
Candida krusei NA NA NA NA NA NA NA
NA, not active.
a
Diameter of inhibition zone (mm) including well diameter of 8 mm.
338 B. Tepe et al. / Food Chemistry 90 (2005) 333–340
The extracts applied on silica gel TLC plates and main
zones were determined for each extract. With DPPH re-
agent, at least three spots appeared immediately after
spraying the TLC trace of MW extract. Although isola-
tion and the structural determination have not yet been
completed, preliminary results show that these compo-
nents are polar phenolic acids or polyphenols.
In the linoleic acid system, oxidation of linoleic acid
was effectively inhibited by the polar subfraction of MW
extract (Fig. 2) while the oil was less effective. MW ex-
tract shows 90.6% inhibition, that is close to the syn-
thetic antioxidant reagent BHT. Antioxidants minimize
the oxidation of lipid components in cell membranes or
inhibit the volatile organic compounds and the conju-
gated diene hydroperoxides, arising from linoleic acid
oxidation, that are known to be carcinogenic. Polar
extracts exhibited stronger activity than non-polar ex-
tracts, indicating that polyphenols or flavanones and
flavonoids may also play important roles in the activity.
Therefore, any extraction procedure is suitable for ob-
taining S. tomentosa active components inhibiting lipid
oxidation.
3.4. Amount of total phenolics
Based on the absorbance values of the various extract
solutions, reacting with Folin–Ciocalteu reagent and
compared with the standard solutions of gallic acid
equivalents, as described above, results of the colori-
metric analysis of total phenolics are given in Table 4.
Total phenolics was highest in the HE (27.5%), followed
by polar subfraction of methanol extract (20.0 %), polar
subfraction of deodorized methanol extract (DeMW)
and deodorized hot water extract (15.0% and 14.9%,
respectively). The lowest amount of total phenolics was
recorded in the non-polar subfraction of the deodorized
methanol extract (1.0%).
Acknowledgements
The results presented here are basically originated
from Mr Bektas TEPE’s MSc thesis, which is supported
by the research council of Cumhuriyet University, Sivas-
Turkey (Project No. F-123. Authors wish to thank Mr
Alattin S
ßIHO
GLU, Mrs Arzuhon S
ßIHO
GLU-TEPE
and people of S
o
g
utl
ug
ol plateau for their kind hospi-
tality during the collection of plant material and Dr Erol
DONMEZ for the identification of the plant material
collected.
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The chemical composition of the essential oils of Salvia verticillata L. and Salvia santolinifolia Boiss. growing wild in Iran were examined by GC and GC–MS. Twenty-seven components were characterized for S. verticillata with β-caryophyllene (24.7%), γ-muurolene (22.8%), limonene (8.9%) and α-humulene (7.8%) as the major constituents, whereas for S. santolinifolia, 17 components were identified with α-pinene (59.4%), β-pinene (12.4%) and limonene (3.8%) as the major constituents. Copyright © 1999 John Wiley & Sons, Ltd.