Available online at www.sciencedirect.

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Bioresource Technology 99 (2008) 40964104

Chemical composition, antioxidant and antimicrobial properties of

the essential oils of three Salvia species from Turkish ora
Mustafa Kelen a, Bektas Tepe b,*

a
Suleyman Demirel University, Faculty of Agriculture, Department of Horticulture, 32260 Isparta, Turkey
b
Cumhuriyet University, Faculty of Science and Literature, Department of Molecular Biology and Genetics, 58140 Sivas, Turkey

Received 13 February 2007; received in revised form 29 August 2007; accepted 4 September 2007
Available online 23 October 2007

Abstract

Essential oils of three dierent Salvia species [Salvia aucheri var. aucheri (endemic), Salvia aramiensis and Salvia pilifera (endemic)]
were screened for their possible antioxidant and antimicrobial properties as well as their chemical compositions. According to the gas
chromatography (GC)/EIMS (gas chromatography/electron impact mass spectrum) analysis results; 41 (97.2%), 51 (98.5%) and 83 com-
pounds (98.2%) were identied, respectively. While 1,8-cineole (30.5%), camphor (21.3%) and borneol (8.50%) are the major compounds
for S. aucheri var. aucheri oil, b-pinene (10.3%), was the main constituent for S. aramienesis together with 1,8-cineole (46.0%) and cam-
phor (8.7%). In the case of S. pilifera oil, a-thujene (36.1%) and a-pinene (13.8%) determined as the major compounds. Antioxidant activ-
ity was employed by two complementary test systems namely 2,2 0 -diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and
b-carotene/linoleic acid systems. Antioxidant activity of S. aramiensis was found to be higher than those of the others for the both sys-
tems (12.26 1.09 and 92.46% 1.64 lg mg1, respectively). Additionally, antioxidant activities of BHT, curcumin, ascorbic acid and
a-tocopherol were determined in parallel experiments. In the case of antimicrobial activity, similar activity pattern was obtained (both in
disc diusion and MIC tests). Antimicrobial activity of S. aramiensis was followed by S. aucheri var. aucheri and S. pilifera, respectively.
In these experiments, the most sensitive microorganism Acinetobacter lwoi was followed by Candida albicans.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Salvia aucheri var. aucheri; Salvia aramiensis; Salvia pilifera; Antimicrobial activity; Antioxidant activity; Essential oil

1. Introduction alternative medicine and natural therapies (Reynolds,

In the last decades, the essential oils and various extracts
of plants have been of great interest as they have been the
sources of natural products. They have been screened for
their potential uses as alternative remedies for the treat-
ment of many infectious diseases and the preservation of
the foods from the toxic eects of the oxidants. Particu-
larly, the antimicrobial activities of plant oils and extracts
have formed the basis of many applications, including
raw and processed food preservation, pharmaceuticals,
*
Corresponding author. Tel.: +90 346 219 10 10x2907; fax: +90 346 219
11 86.
E-mail address: bektastepe@yahoo.com (B. Tepe).
1996; Lis-Balchin and Deans, 1997).
Furthermore, plant products are also known to possess
potential as natural agents for food preservation (Baratta
et al., 1998a,b; Deans, 1991; Deans and Ritchie, 1987; Hal-
endar et al., 1998). In order to prolong the storage stability
of foods, synthetic antioxidants are mainly used in indus-
trial processing. But according to the toxicologists and
nutritionists, the side eects of some synthetic antioxidants
used in food processing such as, butylated hydroxytoluene
(BHT) and butylated hydroxyanisole (BHA) have been
documented. For example, these substances can show car-
cinogenic eects in living organisms (Ames, 1983; Baards-
eth, 1989). From this point of view, governmental
authorities and consumers are concerned about the safety

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.09.002
 M. Kelen, B. Tepe / Bioresource Technology 99 (2008) 40964104
of their food and about the potential eect of synthetic
additives on their health (Reische et al., 1998).
The genus Salvia, with about 900 species throughout the
world, as one of the most widespread members of the Lam-
iaceae family. Although Salvia species are abundant in the
Turkish ora, as being represented by 88 species and 93
taxa and 45 of which are endemic (Guner et al., 2000), only
biological activities and bioactive properties of a few are
available in the literature while most being untapped.
Among them, the species evaluated here are aromatic
plants widely distributed in Anatolia and Mediterranean
region where they commonly known as adacayi, salba 3610 and AA 3612, respectively).
and/or dadirak by the local people. They are commonly
used in local folk medical practices and in cosmetics. Plant
evaluated here together with the other members of the
genus Salvia used in diarrhea, gonorrhea and hemorrhoids,
eye diseases and as an antiseptic, antispasmodic and sto-
machic (Rizk and El-Ghazaly, 1995).
Members of this genus produce many useful secondary
metabolites including terpenes and phenolics and their
derivatives that have been in the center of pharmacopoeias
of many countries (Tepe et al., 2007). Actually, the name of
this genus means safe or well as a Latin word sal- 2.3. Gas chromatography (GC)/EIMS analysis
vus. Healing reputation of Salvia species can be traced
back to Roman times and a well-known Salvia species, S.
ocinalis (sage), is credited for this (Konemann, 1999).
Antioxidant activities of the many members of the genus
Salvia were reported elsewhere. Additionally, previous
reports concerning the biological activities of Salvia species
native to the Turkish ora conrm that this genus has great
potential, especially in antioxidant systems, for the food
and cosmetic industries (Tepe et al., 2004, 2005, 2006,
2007).
Antimicrobial activity of the genus Salvia has been well
established in the literature. But, as far as our literature
survey could as certain, biological properties of the plant
species evaluated here had not previously been reported.
From this point of view, this study could be assumed as
the rst report on the antioxidant and antimicrobial activ-
ities of Salvia aucheri var. aucheri (endemic), Salvia arami-
ensis and Salvia pilifera (endemic).
The aim of this study was to identify the chemical com-
positions of the oils of S. aucheri var. aucheri, S. aramiensis
and S. pilifera and to compare the antioxidant and antimi-
crobial activities of them, in an attempt to contribute to the
use of these as alternative products for microbial control
and food preservation.
2. Methods
2.1. Collection of plant material
Collection information for the plant species studied is
given below:
S. aucheri Bentham var. aucheri Bentham: Pozanti,
Adana Turkey, 12.07.2006
4097
S. aramiensis Rech. l.: Kuzuculu, Dortyol, Hatay
Turkey, 10.07.2006
S. pilifera Montbret et Aucher ex Bentham: Haruniye
(Duzici), Osmaniye Turkey, 14.07.2006
Plants were collected during their owering season.
They were identied by a senior taxonomist Dr. H. Askin
AKPULAT from Cumhuriyet University, Department of
Biology. The voucher specimens have been deposited at
the Herbarium of the Department of Biology, Cumhuriyet
University, Sivas, Turkey (voucher nos.: AA 3611, AA
2.2. Extraction of the essential oil
The air-dried and ground aerial parts of the plants were
submitted for 3 h to water-distillation using a Clevenger-
type apparatus. The obtained essential oil was dried over
anhydrous sodium sulphate and after ltration, stored at
+4 C until tested and analyzed (yields 0.59%, 0.76% and
0.83% v/w, respectively).
GC/EIMS analyses were performed with a Varian CP-
3800 gas-chromatograph equipped with a DB-5 capillary
column (30 m 0.25 mm; coating thickness 0.25 lm) and
a Varian Saturn 2000 ion trap mass detector. Analytical
conditions were: injector and transfer line temperatures
220 and 240 C, respectively; oven temperature pro-
grammed from 60 to 240 C at 3 C/min; carrier gas helium
at 1 ml/min; injection of 0.2 ll (10% hexane solution); split
ratio 1:30. Identication of the constituents was based on
comparison of the retention times with those of authentic
samples, comparing their linear retention indices relative
to the series of n-hydrocarbons, and on computer matching
against commercial (NIST 98 and ADAMS) and home-
made library mass spectra built up from pure substances
and components of known oils and MS literature data
(Adams, 1995; Davies, 1990; Jennings and Shibamoto,
1980; Massada, 1976; Stenhagen et al., 1974; Swigar and
Silverstein, 1981). Moreover, the molecular weights of all
the identied substances were conrmed by GC/CIMS,
using MeOH as CI ionizing.
2.4. Antioxidant activity
2.4.1. 2,2 0 -diphenyl-1-picrylhydrazyl assay
Hydrogen atoms or electrons donation ability of the
corresponding oils was measured from the bleaching of
purple coloured methanol solution of DPPH. This spectro-
photometric assay uses stable radical 2,2 0 -diphenyl-1-pic-
rylhydrazyl (DPPH) as a reagent (Cuendet et al., 1997;
Burits and Bucar, 2000). Fifty microliter of various concen-
trations of the oils in methanol was added to 5 ml of a
0.004% methanol solution of DPPH. After a 30 min incu-
bation period at 20 C the absorbance was read against a
4098 M. Kelen, B. Tepe / Bioresource Technology 99 (2008) 40964104
blank at 517 nm. Inhibition free radical DPPH in percent
(I%) was calculated in following way:
I% Ablank  Asample =Ablank  100
where Ablank is the absorbance of the control reaction (con-
taining all reagents except the test compound), and Asample
is the absorbance of the test compound. Extract concentra-
tion providing 50% inhibition (IC50) was calculated form
the linear regression algorithm of the graph plotted inhibi-
tion percentage against extract concentration. For the cal-
culation of these values, Microsoft Excel software was
used. Tests were carried out in triplicate. Values are pre-
sented as means SD of three parallel measurements.
2.4.2. b-Carotene-linoleic acid assay
In this assay antioxidant capacity is determined by indi-
rectly 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 pre-
pared as following: 0.5 mg b-carotene was dissolved in 1 ml
of chloroform (HPLC grade), 25 ll linoleic acid and
200 mg Tween 40 was added. Chloroform was completely
evaporated using a vacuum evaporator. Then 100 ml dis-
tilled water saturated with oxygen (30 min 100 ml/min.)
was added with a vigorous shaking. 2.5 ml of this reaction
mixture was dispersed to test tubes and 350 ll portions of
the oils prepared at 2 g l1 concentrations were added
and emulsion system was incubated up to 48 h at room
temperature. Same procedure was repeated with synthetic
antioxidant, buthylated hydroxytoluene (BHT), curcumine
and ascorbic acid as positive controls, and a blank. After
this incubation period absorbance of the mixtures were
measured at 490 nm. Antioxidative capacities of the oils
were compared with those BHT and blank (contains EtOH
instead of essential oil). Values are presented as
means SD of three parallel measurements.
2.5. Antimicrobial activity
2.5.1. Microbial strains
The essential oils were individually tested against a panel
of microorganisms including Streptococcus pneumoniae
IK3, Bacillus cereus RK75, Acinetobacter lwoi ATCC
19002, Escherichia coli Hak59, Klebsiella pneumoniae
A137, Clostridium perfringens Kukens-Turkey, Candida
albicans A117 and Candida krusei ATCC 6258. Bacterial
strains were cultured overnight at 37 C in 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 dextrose agar.
2.5.2. Antimicrobial screening
Disc diusion method was employed for the determina-
tion of antimicrobial activities of the essential oils
(NCCLS, 1999). The MICs of the essential oils against
the test microorganisms were determined by broth microdi-
lution method (NCCLS, 1997). Tests were carried out in
triplicate. Values are presented as means SD of three
parallel measurements.
2.5.3. Disc diusion method
Agar disc diusion method was employed for the deter-
mination of antimicrobial activities of the essential oils in
question (NCCLS, 1997). Briey, a suspension of the tested
microorganism (0.1 ml of 108 cells per ml) was spread on
the solid media plates. Filter paper discs (6 mm in diame-
ter) were impregnated with 15 ll of the oil and placed on
the inoculated plates. These plates, after staying at 4 C
for 2 h, were incubated at 37 C for 24 h for bacteria and
at 30 C for 48 h for the yeasts. The diameters of the inhi-
bition zones were measured in millimetres. Tests were car-
ried out in triplicate. Values are presented as means SD
of three parallel measurements.
2.5.4. Determination of minimum inhibitory
concentration (MIC)
A broth microdilution broth susceptibility assay was
used, as recommended by NCCLS, for the determination
of the MIC (NCCLS, 1999). All tests were performed in
Mueller Hinton Broth (MHB; BBL) supplemented with
Tween 80 detergent (nal concentration of 0.5% (v/v), with
the exception of the yeasts (Sabouraud dextrose broth
SDB + Tween 80). Bacterial strains were cultured over-
night at 37 C in MHA and the yeasts were cultured over-
night at 30 C in SDB. Test strains were suspended in
MHB to give a nal density of 5 105 cfu/ml and these
were conrmed by viable counts. Geometric dilutions rang-
ing from 0.036 mg/ml to 72.00 mg/ml of the essential oils
were prepared in a 96-well microtiter plate, including one
growth control (MHB + Tween 80) and one sterility con-
trol (MHB + Tween 80 + test oil). Plates were incubated
under normal atmospheric conditions at 37 C for 24 h
for bacteria and at 30 C for 48 h for the yeasts. The bac-
terial growth was indicated by the presence of a white pel-
let on the well bottom.
3. Results and discussion
3.1. Chemical composition of the essential oil
Gas chromatography (GC)/EIMS analysis revealed
that; 41 (97.2%), 51 (98.5%) and 83 compounds (98.2%)
were identied, respectively. While 1,8-cineole (30.5%),
camphor (21.3%) and borneol (8.50%) are the major com-
pounds for S. aucheri var. aucheri oil, b-pinene (10.3%),
was the main constituent for S. aramienesis together with
1,8-cineole (46.0%) and camphor (8.7%) also. In the case
of S. pilifera oil, a-thujene (36.1%) and a-pinene (13.8%)
determined as the major compounds (Table 1).
As can be seen from Table 1, oil samples were found to
be rich in 1,8-cineole (eucalyptol) except S. pilifera (9.2%).
Pinene type monoterpenes (a-pinene and b-pinene) consti-
 M. Kelen, B. Tepe / Bioresource Technology 99 (2008) 40964104 4099

Table 1
Chemical composition of the essential oils of Salvia species
No. LRIa Compound Composition (%)
Salvia aucheri var. aucheri Salvia aramiensis Salvia pilifera
1 802 Hexanalc trb
2 927 Tricyclenec 0.2 2.5 tr
3 930 a-Thujened 0.5 0.3 36.1
4 939 a-Pinened 7.6 4.9 13.8
5 953 a-Fenchenec tr
6 954 Camphened 7.8 4.2 0.1
7 975 Sabinenec tr 1.9 0.9
8 978 b-Pinened 5.7 10.3 0.8
9 979 1-Octen-3-olc 0.2
10 990 3-Octanolc 0.1
11 991 Myrcened 1.2 2.0 2.2
12 995 2-Octanolc 0.1
13 1004 p-Mentha-1,7(8)-diene (= Pseudolimonene)c tr
14 1017 a-Terpinened 0.2 0.3
15 1025 p-Cymened 0.2 1.4
16 1029 Limonenec 1.6 1.4
17 1030 b-Phellandrenec 1.5
18 1031 1,8-Cineoled 30.5 46.0 9.2
19 1036 (E)-b-Ocimenec 0.1
20 1037 (Z)-b-Ocimenec 0.1
21 1060 c-Terpinened 0.4 0.4 tr
22 1070 cis-Sabinene hydratec 0.3 0.2 0.1
23 1073 trans-Linalool oxide (furanoid)c 0.3
24 1085 (Z)-2-Hexenalc 0.1 0.3
25 1087 cis-Linalool oxide (furanoid)c 0.2
26 1089 Terpinolenec 0.1 0.1 tr
27 1097 Linaloold tr 0.2 1.9
28 1098 trans-Sabinene hydratec tr 0.6 tr
29 1113 3-Octyl acetatec 0.1
30 1114 trans-Thujonec tr 3.6
31 1122 cis-p-Menth-2-en-1-olc 0.1
32 1123 trans-p-Mentha-2,8-dien-1-olc 0.1
33 1139 trans-Pinocarveolc 0.2 tr
34 1141 trans-p-Menth-2-en-1-olc tr 0.1
35 1146 Camphord 21.3 8.7
36 1165 Pinocarvonec 0.1 0.1 tr
37 1166 d-Terpineold 0.1
38 1169 Borneold 8.5 3.6 0.1
39 1177 Terpinen-4-olc 1.4 0.9 3.2
40 1181 Naphthalenec 0.1
41 1183 p-Cymen-8-olc tr tr
42 1189 a-Terpineold 0.6 0.6 2.3
43 1194 Myrtenalc 0.2 tr
44 1195 Myrtenolc tr 0.3 0.1
45 1196 cis-Piperitolc 0.6
46 1199 c-Terpineold 0.5
47 1217 trans-Carveolc tr 0.1
48 1231 cis-p-Mentha-1(7),8-diene-2-olc tr
49 1253 Piperitenonec tr 4.7
50 1261 trans-Myrtanolc tr
51 1289 Bornyl acetatec 1.3 0.8 0.3
52 1290 Thymold 0.1 tr
53 1291 trans-Sabinyl acetatec 0.3
54 1299 Carvacrold 0.2 0.3
55 1343 Piperitenonec tr 0.1
56 1349 a-Terpinyl acetatec 2.6
57 1351 a-Cubebenec 0.1
58 1359 Eugenold tr
59 1375 a-Ylangenec tr 0.1
60 1377 a-Copaenec tr 0.3
61 1381 Geranyl acetatec 0.1
(continued on next page)
4100 M. Kelen, B. Tepe / Bioresource Technology 99 (2008) 40964104

Table 1 (continued)
No. LRIa Compound Composition (%)
Salvia aucheri var. aucheri Salvia aramiensis Salvia pilifera
c
62 1388 b-Bourbonene 0.1 0.4
63 1393 Z-Jasmonec tr
64 1419 (E)-Caryophyllenec 1.1 1.0
65 1421 b-Ylangenec tr 0.3
66 1441 Aromadendrenec 0.4
67 1443 (Z)-b-Farnesenec 0.2
68 1455 a-Humulenec 0.2 0.6 0.5
69 1456 Geranylacetonec tr
70 1480 c-Muurolenec 2.6
71 1481 ar-Curcumenec 0.1
72 1485 Germacrene Dd 0.5 0.1
73 1489 (E)-b-Iononec tr
74 1490 b-Selinenec 0.4
75 1494 epi-Cubenolc 0.3
76 1496 Valencenec 0.1
77 1498 a-Selinenec 0.3
78 1500 Bicyclogermacrenec 0.2
79 1502 a-Muurolenec 0.2
80 1506 cMuurolenec 0.2
81 1514 c-Cadinenec tr 1.0
82 1523 d-Cadinenec 0.1 0.1 0.4
83 1540 cis-Calamenenec 0.1
84 1546 a -Calacorenec 0.1
85 1547 Selina-3,7(11)-dienec 0.8
86 1550 Elemolc 0.1
87 1566 b-Calacorenec tr
88 1578 Spathulenold 2.6 0.8 tr
89 1583 Caryophyllene oxided 1.2 1.3 0.5
90 1587 Gleenol c tr
91 1595 Salvial-4(14)-en-1-onec 0.1
92 1601 Guaiolc 0.6 0.2
93 1608 Humulene epoxide-IIc 0.4 0.1
94 1628 Benzophenonec tr
95 1632 c-Eudesmolc 0.4
96 1646 a-Muurololc tr
97 1647 Cubenolc 0.1
98 1651 b-Eudesmolc 0.1 0.1
99 1654 a-Cadinolc 0.2 1.2
100 1656 a-Eudesmolc 0.1 0.4
101 1672 Bulnesolc 0.5 0.5
Total 97.2 98.5 98.2

Chemical classes of the constituents

Monoterpene hydrocarbones 23.8 29.6 58.8
Oxygenated monoterpenes 66.4 62.2 27.7
Sesquiterpene hydrocarbones 4.2 3.4 1.3
Diterpenes 1.4 1.0 8.7
Not iso-prenoid compounds 0.2 1.0 1.2
Oxygenated sesquiterpenes 1.2 1.3 0.5
a
LRI, linear retention indices (HP-5 column).
b
tr, trace (60.1%).
c
Tentative identication.
d
Identication of components based on standard compounds.

tutes the great percentage of the oils. On the other hand,
borneol, camphene and camphor are the other major
constituents.
To the best of our knowledge, there are many reports on
the chemical composition of the oils isolated from the
plants belonging to the genus Salvia (Torres et al., 1997;
Baser et al., 1993, 1995a,b, 1996, 1997, 1998; Tumen
et al., 1998; Ahmadi and Mirza, 1999; Perry et al., 1999;
Rustaiyan et al., 1999; Sedkon and Khajavi, 1999;
Couladis et al., 2001). Most of these reports indicate that
 M. Kelen, B. Tepe / Bioresource Technology 99 (2008) 40964104 4101

1,8-cineole (eucalyptol) and borneol are the main and/or oil of S. aramienesis (92.46% 1.64). This activity was fol-
characteristic constituents of Salvia oils. Chemical compo- lowed again by S. aucheri var. aucheri (87.64% 1.64) and
sitions of the essential oils of the plant species studied had S. pilifera (81.13 1.13), respectively. As can be seen from
been reported before (Demirci et al., 2002, 2003; Ozcan Table 2, antioxidant activity of the essential oil of S. aram-
et al., 2003). Our results show a strong similarity with these iensis was found to be higher than that of curcumine, which
reports. is an ecient antioxidative substance for the food systems
containing lipids.
As can be seen from Table 2, the essential oils improved
3.2. Antioxidant activity
50% inhibition, indicating stronger antioxidant capacity
than the positive control (BHT), except S. pilifera. On
In the light of the dierences among the wide number of
the other hand, hydrogen atoms or electron donation
test systems available, the results of a single-assay can give
capacity of essential oil was not superior, but also close,
only a reductive suggestion of the antioxidant properties of
to BHT and higher than that of curcumine.
extracts toward food matrices and must be interpreted with
In order to determine the antioxidant nature of the oils,
some caution. Moreover, the chemical complexity of
its main components, e.g. 1,8-cineole, camphor, borneol,
extracts, often a mixture of dozens of compounds with dif-
terpinen-4-ol, a-pinene, b-pinene, p-cymene were all tested
ferent functional groups, polarity and chemical behavior,
individually and none exhibited strong antioxidative activ-
could lead to scattered results, depending on the test
ity in all methods employed in this study. As reported else-
employed. Therefore, an approach with multiple assays in
where, the components mentioned above were found not to
screening work is highly advisable. Among the plethora
possess strong reducing eects using the same procedure
of methods that can be used for the evaluation of the anti-
like DPPH assay (Burits et al., 2001). Rapid TLCDPPH
oxidant activity, very few of them are useful for determin-
screening assay of the essential oils of Salvia species studied
ing the activity of both hydrophilic and liphophilic species,
here gave only one spot that might be responsible from the
thus ensuring a better comparison of the results and cover-
activity but not corresponding pure components like 1,8-
ing a wide range of possible applications (Sacchetti et al.,
cineole, camphor and borneol. Our ndings were in accor-
2005). Taking this into account, essential oils were individ-
dance with reports in the literature, indicating that activi-
ually assessed for their possible antioxidative activities by
ties observed in the TLC screening assay were explained
employing two complementary tests; DPPH free radical
by synergistic eect since each spot on TLC represents
scavenging and b-carotene/linoleic acid assays.
more than one individual compound. Moreover, commer-
Free radical scavenging capacities of the corresponding
cial chemicals, such as borneol and 1,8-cineole did not
oils were measured by DPPH assay and the results are
show any activity with other methods like hydroxyl radical
shown in Table 2. According to the results obtained, S. ara-
scavencing and lipid peroxidation when tested individually.
mienesis oil was found the most active one with an IC50
Antioxidative activity of 1,8-cineol and terpinen-4-ol were
value of 12.26 1.09 lg ml1. This activity was followed
previously tested using two dierent assays; aldehyde/car-
by S. aucheri var. aucheri (18.82 1.27 lg ml1) and S.
boxylic acid assay and lipid peroxidation (Lee and Shibam-
pilifera (24.19 0.76 lg ml1), respectively. IC50 values of
oto, 2001) with prolonged incubation periods (30 days and
the synthetic antioxidant BHT and the others (curcumine,
18 h, respectively), whereas the activity was assessed in only
ascorbic acid and a-tocopherol) were also determined in
3060 min.
parallel experiments. None of the samples showed activity
as strong as the standards.
3.3. Antimicrobial activity
In the latter case, similar activity pattern was observed
when compared to the results of DPPH system. Oxidation
Antimicrobial activities of the oils evaluated here were
of the linoleic acid was eectively inhibited by the essential
determined by the application of agar disc diusion and
MIC tests against a panel of food born microorganisms
Table 2 and pathogenic yeasts. As can be seen from Table 3, essen-
Antioxidant capacities of Salvia speciesa
tial oils showed moderate activity against the test
Samples Test system microorganisms.
DPPH IC50 value b-Carotene/linoleic acid In the case of S. aramiensis, results from disc diusion
(lg ml1) inhibition (%) method, followed by measurements of minimal inhibition
Salvia aucheri var. 18.8 1.2 87.6 1.0 concentration (MIC) indicate that, A. lwoi was the most
aucheri sensitive microorganism with the highest inhibition zone
Salvia aramiensis 12.2 1.0 92.4 1.6
(20.00 mm 1.42) and lowest MIC value (4.50 lg ml1).
Salvia pilifera 24.1 0.7 81.1 1.1
BHT 18.0 0.4 96.6 1.2 Other sensitive microorganisms were C. krusei and C. albi-
Curcumine 7.8 0.3 89.3 2.1 cans of which MIC values are 9.00 and 18.00 lg ml1,
Ascorbic acid 3.8 0.1 94.5 1.8 respectively.
a-Tocopherol 6.5 0.7 96.6 1.7 Antimicrobial activity of the essential oil of S. aramiene-
a
Values expressed are means SD of three parallel measurements. sis was followed by S. aucheri var. aucheri and S. pilifera,
4102 M. Kelen, B. Tepe / Bioresource Technology 99 (2008) 40964104

Table 3
Antimicrobial capacities of Salvia speciesa
Microorganisms S. aucheri var. aucheri S. aramiensis S. pilifera
b c
DD MIC DD MIC DD MIC
Escherichia coli Hak 59 11.0 1.6 36.0 12.0 1.2 36.0 8.0 1.1 >72.0
Streptococcus pneumoniae IK3 10.0 1.1 36.0 13.0 0.6 18.0 9.0 0.2 72.0
Bacillus cereus RK75 9.0 0.6 72.0 11.0 0.5 36.0 8.0 0.6 >72.0
Clostridium perfringens Kukens-Turkey 11.0 0.2 36.0 13.0 0.7 18.0 9.0 0.7 72.0
Candida albicans A 117 13.0 0.9 18.0 14.0 1.2 18.0 11.0 1.5 36.0
Candida krusei ATCC 6258 16.0 0.6 18.0 19.0 1.7 9.0 13.0 1.4 18.0
Acinetobacter lwoi ATCC 19002 18.0 0.2 9.0 20.0 1.4 4.5 15.0 1.2 18.0
Klebsiella pneumoniae A 137 9.0 1.2 72.0 11.0 0.7 36.0 7.0 0.6 >72.0
a
Values expressed are means SD of three parallel measurements.
b
Diameter of inhibition zone including disc diameter of 6.00 mm.
c
MIC, minimal inhibitory concentration (as mg ml1).

ous results reporting that a-pinene and p-cymene were

Table 4 both found to possess no activity against reference strains
Antimicrobial activity of the control antibioticsa (Cosentino et al., 1999) or weak activity with narrower
Microorganisms NETb AMP Bc spectrum (Dorman and Deans, 2000). Preliminary screen-
Escherichia coli 1 10 2
NAd ing of both standard and isolated compounds on the same
Streptococcus pneumoniae NA NA microorganisms under identical conditions showed similar
Bacillus cereus NA NA results mentioned above. It is also noteworthy that syner-
Clostridium perfringens NA NA
Candida albicans NA 1 103
gistic and/or antagonistic eects might be taken into
Candida krusei NA 1 103 account for the activity observed in complex systems, such
Acinetobacter lwoi NA NA as essential oils.
Klebsiella pneumoniae 1 102 NA
a
Values expressed are means SD of three parallel measurements. 4. Conclusion
b
NET, netilmicin.
c
AMP B, amphotericin B.
d
NA, not active. Antioxidant and antimicrobial properties of the essen-
tial oils and various extracts from many plants are of great
interest in both academia and the food, cosmetic and phar-
respectively. These ndings showed a correlation with the maceutical industries, since their possible use as natural
antioxidant activities of the oil samples as given above. additives emerged from a growing tendency to replace syn-
For the both plants, the most sensitive microorganisms thetic preservatives by natural ones. In this respect, study-
were A. lwoi and the yeasts. Sensitivity of the microor- ing with the endemic species maybe of great interest since
ganisms against Netilmicin (NET) and Amphotericin B their bioactive properties and secrets could be lost forever
(AMP B) was presented in Table 4. without being tapped. Owing to their excellent protective
The overall activity test results from our experiments features exhibited in antioxidant and antimicrobial tests,
indicated the resistance of B. cereus, K. pneumoniae and the essential oil of Salvia species, of which two are endemic,
E. coli against the oils. could be concluded as a natural source that can be freely
Table 3 indicates a positive correlation between the used in the food industry as a culinary herb, but, rstly,
activity mode and the presence of main constituents, par- immediate and necessary measurements should be taken
ticularly, 1,8-cineole (eucalyptol), camphor, and borneol. for the protection of these plant species. In conclusion,
Oils containing these substances possessed strong activity our study can be considered as the rst detailed document
against yeasts and A. lwoi, in particular, and therefore on the in vitro antimicrobial and antioxidant features of
1,8-cineole and camphor were concluded as the most Salvia aucheri var. aucheri, S. aramiensis and S. pilifera.
potent agents against this pathogenic yeast. Camphor
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