Chemical Composition, Antimicrobial Activities and Olfactive Evaluation of a Salvia officinalis L. (Sage) Essential Oil from Egypt

Abstract

The chemical composition of a Salvia officinalis L. (sage) essential oil from Egypt has been analyzed by GC and GC/MS. Twenty-eight volatiles were identified, and camphor (25.1%), α-thujone (22.2%) and β-thujone (17.7%) were found as main compounds. These analytical results were correlated with olfactoric evaluations for quality control of this sage oil. Furthermore, the oil, some of its terpene components, a reference compound and two commercial antibiotics were screened against several microorganisms to determine the antimicrobial activity of S. officinals from Egypt.

Full text

Edris et al.
186/Journal of Essential Oil Research Vol. 19, March/April 2007
Received: February 2006
Revised: May 2006
Accepted: May 2006
Chemical Composition, Antimicrobial Activities and
Olfactive Evaluation of a Salvia officinalis L. (Sage)
Essential Oil from Egypt
Amr E. Edris*
Aroma & Flavor Chemistry Department, National Research Center, El Behose Street, El Dokki 12622, Cairo, Egypt
Leopold Jirovetz and Gerhard Buchbauer
University of Vienna, Department of Clinical Pharmacy and Diagnostics, Althanstrasse 14, A-1090 Vienna, Austria
Zapriana Denkova, Albena Stoyanova and Alexander Slavchev
University of Food Technologies, Department of Essential Oils, 26 Maritza Boulevard, 4002 Plovdiv, Bulgaria
Abstract
The chemical composition of a Salvia officinalis L. (sage) essential oil from Egypt has been analyzed by GC
and GC/MS. Twenty-eight volatiles were identified, and camphor (25.1%), α-thujone (22.2%) and β-thujone (17.7%)
were found as main compounds. These analytical results were correlated with olfactoric evaluations for quality
control of this sage oil. Furthermore, the oil, some of its terpene components, a reference compound and two
commercial antibiotics were screened against several microorganisms to determine the antimicrobial activity of
S. officinals from Egypt.
Key Word Index
Salvia officinalis, Lamiaceae, essential oil composition, camphor, α-thujone, β-thujone, antimicrobial activity,
olfactoric evaluation.
1041-2905/07/0002-0186$14.00/0—© 2007 Allured Publishing Corp.
J. Essent. Oil Res., 19, 186–189 (March/April 2007)
*Address for correspondence
Introduction
In continuation of an international project in the field of
combined data interpretation of composition analysis, aroma
evaluation and antimicrobial activity testings (1-3), the essential
oil of Salvia officinalis L. from Egypt was investigated. Sage
(Salvia officimalis L., Lamiaceae), is a perennial subshruby
aromatic plant native to the Mediterranean regions. It is also
abundant on the Dalmation coast, areas of Adriatic Sea, Croatia
and Albania (4). Sage oil possesses a sharp camphoraceous,
thujone-like aroma (5) and is therefore used in the fragrance
industry. The oil of S. officinalis is also known for its medici-
nal-biological activities, such as antimicrobial and fungicidal
effects (6-8).
The chemical composition of sage oil from different
geo-
graphical origins has been the subject of many studies (9-14).
The major constituents of different sage oils include α-thu-
jone, β-thujone, camphor and 1,8-cineole, which determine
also their chemotypes (15). In addition, more than 300 volatiles
were also identified as components of essential oils of Salvia
officinals (16).
To the best of our knowledge, no combined interpretation
of data from GC and GC/MS analysis, olfactoric evaluation
and antimicrobial testings (using agar diffusion and agar dilu-
tion method, both) have been done on sage oil of Egyptian
origin.
Experimental
Reference compounds: Camphor (W52,660-6), β-caryo-
phyllene (W22,520-7), 1,8-cineole (W24,650-6), limonene
(W26,330-3), linalool (W26,350-8), linalyl acetate (W26,350-
5), α-terpineol (W30,452-2), α-thujone (89231) and eugenol
(W24,670-0) obtained from Sigma-Aldrich Austria Co., Vienna,
terpinen-4-ol (800760) from Kurt Kitzing Co., Wallerstein,
β-thujone (no product-number given) from Symrise Co. (for-
mer Dragoco Co.), Holzminden, CiproxinR 500mg-tabletes
(1 tablete = 582 mg Ciprofloxacin hydrochloride.water) from
Bayer Austria Co., Vienna and LidaprimR-infusion-bottle (250
mg contain 0.8 g sulfametrol and 0.16 g trimethoprim) from
Nycomed Austria Co., Vienna.
Plant material: Seeds of sage, (Salvia officinalis L.),
were sowed in the nursery in October, then the seedlings
were transplanted in the open field at the end of February; 20
m3/acer of mature organic compost was used for fertilization.
The plants were harvested in the full flowering stage (first of

S. officinalis
Vol. 19, March/April 2007 Journal of Essential Oil Research/187
Table I. Composition and olfactoric evaluation of the Salvia officinalis oil from Egypt
Component RI a/ba Percentageb Odorc
α-pinene 942/1036 0.2 pine-like, warm, herbal
camphene 952/1069 3.5 camphoraceous, fresh
β-pinene 979/1122 1.6 resinous-piney, dry
myrcene 988/1158 1.1 weak citrus- and lime-like
α-phellandrene 1002/1173 0.1 citrus-like, weak peppery
δ-3-carene 1009/1147 0.1 sweet, lemon-note
limonene 1022/1206 1.9 lemon-like, fresh
1,8-cineole 1025/1226 7.5 eucalyptus-like, fresh
(Z)-β-ocimene 1027/1233 0.1 warm-herbal, sweet
(E)-β-ocimene 1042/1253 1.3 warm-herbal, weak floral
γ-terpinene 1057/1250 0.1 citrus-like, herbal
terpinolene 1073/1282 0.3 sweet-piney
fenchone 1081/1410 0.2 camphoraceous, herbal
linalool 1093/1515 0.5 fresh-floral
α-thujone 1100/1440 22.2 warm-herbal, minty
α-fenchol 1110/1576 0.2 camphoraceous, fresh
β-thujone 1119/1460 17.7 camphoraceous-herbal
camphor 1131/1518 25.1 camphoraceous, fresh
borneol 1154/1684 1.6 camphoraceous, earthy
α-fenchyl acetate 1221/1473 2.2 camphoraceous, herbal
linalyl acetate 1243/1542 0.1 floral, weak fruity
bornyl acetate 1277/1608 0.4 piney, balsamic, sweet
β-elemene 1401/1604 3.7 woody, weak sweet-floral
β-caryophyllene 1432/1617 2.7 dry, woody-spicy
α-humulene 1437/1681 4.5 woody, spicy
(E,E)-α-farnesene 1502/1739 0.3 weak floral-fruity
caryophyllene oxide 1576/1989 0.6 warm-spicy, woody
viridiflorol 1588/2098 0.2 herbal, floral, woody
a apolar/polar column; b in relative %-peak area using GC with an apolar column (mean value of 3 analyses); c in accordance to published data elsewhere (22-27)
June). A voucher specimen of S. officinalis was deposited in the
Herbarium of National Research Center, Cairo, Egypt.
Oil isolation: A given weight of the fresh aerial part of
sage (stems, and leaves except for the flower), was hydrodis-
tilled in a Clevenger-type apparatus for 3 h. The oil was col-
lected, cooled and dried with an anhydrous sodium sulfate and
kept in the freezer until analyzed the same day. The yield of
S. officinalis oil with a pale-yellow color was 0.238%. This amount
is equivalent to 1.2% based on the leaf weight only, taking in
consideration that the stem and branches constituted 81.6%
of the distilled biomass, while the leaf weight constituted only
18.4%. The stem and branches yield of oil was considered to
be negligible (0.04%), which is in accordance with the findings
of Perry et al. (11). Thus the oil yield obtained in this study
could be considered as a leaf oil with a very small contribu-
tion of stem oil.
GC analysis: GC/FID analyses were carried out using a
GC-14A, FID and C-R6A-Chromatopac integrator (Shimadzu,
Japan), a GC-3700 with FID (Varian, Germany) and C-R1B-
Chromatopac integrator (Shimadzu). The amount injected was
1 μl of a 1% solution of the oil in dichloromethane. The carrier
gas was H; injector temperature, 250°C; detector temperature,
320°C. The temperature program was: 40°C/5 min to 280°C/5
min, with a heating rate of 6°C/min. The columns were 30 m
x 0.32 mm bonded FSOT-RSL-200 fused silica, with a film
thickness of 0.25 µm (Biorad, Germany) and 30 m x 0.32 mm
bonded Stabilwax, with a film thickness of 0.50 µm (Restek,
USA). Quantification was achieved using peak area calculations,
and compound identification was carried out partly using cor-
relations between retention times (17-21) .
GC/MS analysis: For GC/MS measurements a GC-17A
with QP5000 (Shimadzu), and Compaq-ProLinea data sys-
tem (class5k-software), a GC-HP5890 with HP5970-MSD
(Hewlett-Packard, USA) and ChemStation software on a
Pentium PC (Böhm, Austria), a GCQ (Finnigan-Spectronex,
Germany-USA) and Gateway-2000-PS75 data system (Siemens-
Nixdorf, Germany, GCQ-software), were used. The carrier
gas was He; injector temperature, 250°C; interface-heating
at 300°C, ion-source-heating at 200°C, EI-mode was 70 eV,
and the scan-range was 41-450 amu. For other parameters, see
description of GC/FID, above. Mass spectra correlations were
done using Wiley, NBS, NIST and our own library as well as
published data (17,19,20).
Antimicrobial testings: As test microorganisms, Gram-
(+) bacteria Staphylococcus aureus ATCC 6538P, Gram-(-)
bacteria Escherichia coli ATCC 8739, Pseudomonas aeruginosa
G 28 and Klebsiella pneumoniae as well as the yeast Candida
albicans ATCC 10231 – all products from the National Bank of
Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria
– were used. The antimicrobial activity of S. officinalis oil and
two of its main components, α-thujone and β-thujone, were
studied using agar diffusion disc and agar serial tube dilution
methods, in accordance to Jirovetz et al., (1,2) and Schmidt
et al. (3). Agar diffusion disc method was conducted using 6

Edris et al.
188/Journal of Essential Oil Research Vol. 19, March/April 2007
Table II. Antimicrobial activities of Salvia officinalis oil and some of the main as well as reference compounds.
Inhibition Zones (IZ) in mm and Minimum Inhibition Concentrations (MIC) in ppm of test-microorganisms
Staphylococcus Escherichia Pseudomonas Klebsiella Candida
aureus coli aeruginosa pneumoniae albicans
Compounds & 1.1013cfu/cm3 2.1012cfu/cm3 1,2.109cfu/cm3 1.1013cfu/cm3 1.1011cfu/cm3
essential Oil IZ MIC IZ MIC IZ MIC IZ MIC IZ MIC
camphora,b,c 13 60 - - 8 600 - - 20 6
β-caryophylleneb 20 60 15 60 - - 10 60 27 0.6
1,8-cineolea,b 10 600 - - 10 60 12 6 14 60
limonenec 8 60 - - - - - - 14 6
linaloola,c 19 60 9 6 8 600 9 60 16 60
linalyl acetatea,c 11 60 - - 8 600 - - 20 6
terpinen-4-olb 25 6 25 6 8 60 27 6 25 0.6
α-terpineolb 25 60 21 60 11 600 23 6 25 6
α-thujone 20 60 10 60 8 600 12 <600 14 60
β-thujone 22 60 8 600 8 600 13 600 13 60
Salvia officinalis 30 60 13 60 - - 15 <600 20 60
eugenol 15 6 12 6 10 600 8 600 27 6.0
Ciproxin 35 <3.0 22 3.0 32 3.0 25 3.0 - -
Lidaprim 27 <60 11 60 - - - - - -
-:no inhibition observed; apublished elsewhere (1); bpublished elsewhere (2); cpublished elsewhere (3)
mm paper discs and quantities of 6 μL of the sample. After
cultivation of the bacteria and the yeast at 37°C for 24 h the
diameter of the inhibition zone (IZ) was measured. The agar
serial tube dilution method with results expressed as minimum
inhibitory concentration (MIC) was performed as follows:
The pure oil and reference compounds were added to brine,
containing 1.0% (v/v) Tween 80 at the appropriate volumes
to produce final concentrations of the samples in the range
of 1-1000 ppm; the petri dishes were inoculated by pipetting
0.1cm3 of the desired culture and 6 μL of the samples as well
as the reference compounds (the tabletes of CiproxinR were
added as solution in saline at a quantity of 300 μg and 30 μg)
on paper discs (6 mm) and then incubated at 37°C for 24h.
Olfactoric evaluations: All investigated samples were
olfactorically evaluated by three professional perfumers using
one drop of the oil on a commercial odor-strip. The aroma
described are mentioned in Table I as well as correlated with
odor impressions published elsewhere (22-27).
Results and discussions
Olfactoric evaluation of the Egyptian sage oil showed that
it possessed a characteristic sage-like odor with a fresh-cam-
phoraceous and rosemary-pine-like top-note and herbal-minty
thujone-base-notes.
Chromatographic analysis (GC/FID and GC/MS) of sage
oil indicated that camphor (25.1%), α-thujone (22.2%) and
β-thujone (17.7%) were found to be the main compounds of
this oil from Egypt (Table I). This indicates that the oil belongs
to the camphor chemotype. Further constituents in a higher
concentration (higher than 3.0%, calculated as %-peak area
using an apolar GC column), were also identified, including
1,8-cineole (7.5%), α-humulene (4.5%), β-elemene (3.7%) and
camphene (3.5%). Using olfactoric data from elsewhere (refer-
ences cited in Table I) the characteristic odor of the sample can
be correlated to camphene, α-fenchol, camphor, borneol and
α-fenchyl acetate (camphoraceous), limonene and 1,8-cineole
(fresh), α-pinene, β-pinene and terpinolene (pine-like) as well
as α-thujone and β-thujone (thujone-like).
Antimicrobial testing of S. officinalis oil against the
selected bacteria and yeast, showed high effects against K.
pneumoniae and medium effects against St. aureus, E. coli
and C. albicans, while no effect was found against P. aerugi-
nosa, compared with the well known phenolic antimicrobial
active compound eugenol (Table II). On the other hand, the
synthetic antibiotics CiproxinR (no effects against the yeast C.
albicans) and LidaprimR (no effects against P. aeruginosa, K.
pneumoniae and C. albicans) were not found to possess the
expected antimicrobial activity.
Antimicrobial testings with the two main compounds
α-thujone and β-thujone show weak effects against the
Gram-(-)-bacteria P. aeruginosa, E. coli and K. pneumoniae
and strong effects against St. aureus, and C. albicans, all
compared with each other. A correlation of antimicrobial
data of further constituents of the oil of sage from Egypt were
published elsewhere (1-3), such as camphor, β-caryophyllene,
1,8-cineole, limonene, linalool, linalyl acetate, terpinen-4-ol
and α-terpineol, (Table II). The results obtained confirms our
experience from previous studies in the field that not only one
or two compounds are responsible for antimicrobial activities
of essential oils, but the whole composition with alternating
synergistic and antagonistic effects of each component.
In conclusion, we can report that the oil of S. officinalis
from Egypt was rich in camphor, α-thujone and β-thujone
as well as some further monoterpenic compounds. Also the
antimicrobial activity of Egyptian sage oil against Gram-(+)-,
Gram-(-)-bacteria and yeast is determined by many constitu-
ents with synergistic and antagonistic effects and cannot be
attributed to a single compound.

S. officinalis
Vol. 19, March/April 2007 Journal of Essential Oil Research/189
Acknowledgment
The authors acknowledge the help of Ahmad Shalaby, Medicinal
& Aromatic Plant Department, National Research Center Giza, for
the identification and characterization of the plant material, the refer-
ence compounds from Kurt Kitzing Co. and Symrise Co. as well as the
valuable discussion about the used antimicrobial testing methods with
Heinz Schilcher, emeritus of the “Freie Universität Berlin”.
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