Alimpić et al., 2015

Industrial
Crops and Products 76 (2015) 702–709
Contents lists available at ScienceDirect
Industrial Crops and Products
journal homepage: www.elsevier.com/locate/indcrop
Composition and
biological effects of Salvia ringens (Lamiaceae)
essential
oil and
extracts
Ana Alimpi´ ca,∗, Dejan Pljevljakuˇ si´ cb, Katarina ˇ Savikinb, Aleksandar Kneˇ zevi´ ca,
Milena ´ Curˇ ci´ cc,
Dragan Veliˇ ckovi´ cd,
Tatjana
Stevi´ cb, Goran Petrovi´ ce, Vlado Matevskif,
Jelena
Vukojevi´ ca, Sneˇ zana Markovi´ cc, Petar D. Marina, Sonja Duleti´ c-Lauˇ sevi´ ca
aInstitute
of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia
bInstitute
for Medicinal Plant Research “Dr. Josif Panˇ ci´ c”, Tadeuˇ sa Koˇ s´ cuˇ ska 1, 11000 Belgrade, Serbia
cDepartment
of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovi´ ca 12, 34 000 Kragujevac, Serbia
dCollege
of
Agriculture
and Food
Technology, ´ Cirila and Metodija 1, 18400 Prokuplje, Serbia
eDepartment
of Chemistry, Faculty of Natural Sciences and Mathematics, University of Niˇ s, Viˇ segradska 33, 18000 Niˇ s, Serbia
fInstitute
of Biology, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius” and Macedonian Academy of Sciences and Arts,
Blvd.
Goce Delcev 9, 1000 Skopje, Macedonia
a r t i c l e i n f o
Article history:
Received
6
March 2015
Received
in revised form 25 June 2015
Accepted
26 July 2015
Keywords:
Salvia
ringens
Essential
oil
Extracts
Phenolics
Flavonoids
Biological
activities
a b s t r a c t
This comprehensive study was carried out in order to investigate composition and biological activities
of
essential oil and extracts of Salvia ringens Sibth. & Sm. (Lamiaceae) originating from Macedonia. Major
components
of the oil, analyzed using GC-FID and GC–MS, were monoterpenes 1.8-cineole (31.99%), cam-
phene
(17.06%), borneol (11.94%) and ␣-pinene (11.52%). HPLC analysis showed presence of 17 phenolic
components,
mainly in methanol and ethyl acetate, followed by ethanol, water and dichloromethane
extracts.
Total phenolics and flavonoids as well as DPPH, ABTS, and FRAP activities were measured
spectrophotometrically.
Essential oil, ethanol, and water extracts showed antimicrobial activity using
microdilution
method. Ethanol and water extracts performed cytotoxic activity against colon carcinoma
HCT-116
cell line using MTT assay. According to the obtained results, S. ringens herb can be considered
as
the potential source of the essential oil and/or raw material for the extraction and isolation of natural
compounds
with a range of biological activities.
©
2015 Elsevier B.V. All rights reserved.
1. Introduction
The Lamiaceae family comprises aromatic plants widely used as
spices
and medicinal plants, such as rosemary, basil, sage, lavender,
thyme,
mint, and oregano. The flavor of herbs and spices derives
from
essential oil components which make food more pleasant and,
at
the same time, show a wide spectrum of biological activities
(Miguel,
2010). Some of the Lamiaceae species were reported as a
rich
source of phenolic compounds possessing strong antioxidant
activity,
and therefore, can be applied in prevention and therapy
of
free-radical associated diseases such as atherosclerosis, cancer,
cardio-vascular
disease, immune-system decline, brain dysfunc-
tion,
cataracts, skin diseases (Asadi et al., 2010; Kamatou et al.,
2010;
Li et al., 2008) and may also serve as natural food preser-
vatives
(Miguel, 2010).
∗Corresponding
author. Fax: [phone].
E-mail
address: [email protected] (A. Alimpi´ c).
The
genus Salvia is the largest member of the family Lamiaceae
which
comprises about 1000 worldwide distributed species. In
Flora
of Europe, the genus is represented by 36 species grouped
into
7 sections (Hedge, 1972). In vitro pharmacological investi-
gations
showed its antioxidant, antibacterial, antifungal, antiviral,
cytotoxic,
neuroprotective, antiinflammatory, and tumorigenesis-
preventing
as well as ecological significance such as pest-toxic
and
repellent and other activities (Asadi et al., 2010; Bariˇ cevi´ c and
Bartol,
2000; Ben Farhat et al., 2009; Kamatou et al., 2010; Orhan
et
al., 2012; Veliˇ ckovi´ c et al., 2002). Aerial parts of these plants
usually
contain flavonoids and triterpenoids as well as essential
oils
with volatile compounds such as monoterpenoids, while diter-
penoids
are the main compounds in roots (Bariˇ cevi´ c and Bartol,
2000).
It is a rich source of polyphenols, with an excess of 160
polyphenols
having been identified, some of which are unique to
the
genus (Lu and Foo, 2002).
Salvia
ringens Sibth. & Sm. is a hardy herbaceous perennial herb,
heights
of up to 60 cm. Specific epithet, ringens, refers to the wide
open
two-lipped flowers. It inhabits dry stony and grass-covered
http://dx.doi.org/10.1016/j.indcrop.2015.07.053
0926-6690/©
2015 Elsevier B.V. All rights reserved.

A. Alimpi´ c et al. / Industrial Crops and Products 76 (2015) 702–709 703
places of South and Eastern parts of Balkan Peninsula, just extend-
ing
to southeast Romania (Hedge, 1972). This is drought tolerant
and
long lived and highly valued as ornamental and melliferous
plant
species due to ponderous leaf rosette, attractive purple flow-
ers,
and pleasant intense fragrance.
Previous
researchers have partially investigated composition
and
biological activities of S. ringens essential oil and/or extracts.
Monoterpenes
1.8-cineole and ␣-pinene have been recognized as
the
major constituents of S. ringens essential oil from Greece and
Macedonia
(ˇ
Savikin
et al., 2008; Tzakou et al., 2001) and camphor
and
borneol in Bulgarian S. ringens (Georgiev et al., 2013). The
oil
and isolated main compounds showed significant antimicro-
bial
activity (ˇ
Savikin
et al., 2008; Tzakou et al., 2001). Among 27
Macedonian
medicinal plants chosen from different plant families,
Origanum
vulgare, Melissa officinalis, and Salvia ringens showed the
strongest
antioxidant activity and highest amount of total pheno-
lics,
flavonoids, and phenylpropanoids (Tusevski et al., 2014). Many
researchers
pointed out
that strong antioxidant activity of S. ringens
extracts
probably was correlated to high amount of polyphenols
(Coisin
et al., 2012; Nikolova, 2011; Tusevski et al., 2014). Extracts
and
some isolated compounds from S. ringens root performed sig-
nificant
cytotoxic
activity against several human carcinoma cell
lines
(Janicsák et al., 2007, 2011), while literature data on antimi-
crobial
activity
of extracts were not available till now.
Taking
into account the lack of comprehensive research data on
of
S. ringens herb, especially those growing wild in Macedonia, the
aim
of the present study was to investigate chemical composition
and
biological activities of its essential oil and extracts.
2.
Material and methods
2.1. Standards and reagents
Methanol, ethanol, distilled water, glacial acetic acid, hydrochlo-
ric
acid, hexane, dichlormethane, and ethyl acetate were purchased
from
Zorka Pharma, ˇ Sabac (Serbia). Gallic acid, quercetin, ascor-
bic
acid, 2(3)-t-butyl-4-hydroxyanisole (BHA), 3,5-di-tert-butyl-
4-hydroxytoluene
(BHT) 2,2-dyphenyl-1-picrylhydrazyl
(DPPH),
2,2-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid diammo-
nium
salt) (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ), potassium
acetate
(C2H3KO2), potassium-persulfate (K2S2O8), sodium car-
bonate
anhydrous (Na2CO3), alluminium nitrate nonahydrate
(Al(NO3)3×
9H2O), sodium acetate (C2H3NaO2), iron(III) chlo-
ride
(FeCl3), iron(II)-sulfate heptahydrate (FeSO4× 7H2O) and
Folin–Ciocalteu
phenol reagent were purchased from Sigma Chem-
icals
Co. (USA). The phenolic compounds standards were from
Merck
(Germany). All chemicals used in experimental procedure
were
of analytical grade purity.
2.2. Plant material
Aerial parts of the Salvia ringens Sibth. & Sm. are collected during
the
flowering period in July of 2012 at Krivolak locality (Macedo-
nia).
Voucher samples are stored in the Herbarium of the Institute
of
Botany and Botanical Garden “Jevremovac”, Faculty of Biology,
University
of Belgrade BEOU; voucher No. (16671).
2.3.
Essential oil isolation
Air-dried aerial parts of S. ringens were grounded. Essential oil
was
isolated by hydrodistillation using a Clevenger type appara-
tus,
according to the procedure I of the Yugoslavian Pharmacopoeia
(1984).
2.4. Preparation of the extracts
Extracts were prepared from whole aerial plant parts using two
parallel
extraction procedures. Dry plant material was grounded
into
small pieces (2–6 mm) in the cylindrical crusher. First, portion
of
10 g of plant material was successively extracted by 100 mL of
dichloromethane,
ethyl acetate, and methanol, according to proce-
dure
of S¸ enol et al. (2010) and Orhan et al. (2013). Second, portion of
10
g of plant material was individually extracted by 100 mL of sol-
vent
(ethanol and hot distilled water). In both cases, extraction was
performed
by classic maceration during 24 h at room temperature
(10%
w/v). The mixture was exposed to ultrasound 1 h before and
after
24 h of maceration to improve extraction process (Veliˇ ckovi´ c
et
al., 2007; Gliˇ si´ c et al., 2011). Subsequently, extracts were fil-
tered
through a filter paper (Whatman No. 1) and evaporated under
reduced
pressure by the rotary evaporator (Buchi rotavapor R-114).
After
evaporation of the solvent, the obtained crude extracts were
stored
in the
fridge at +4 ◦C for further experiments.
2.5. Essential
oil analysis
Qualitative and
quantitative analysis was carried out using GC-
FID
and GC–MS. In the first instance model HP-5890 Series II gas
chromatograph
equipped
with a split-splitless injector, HP-5 capil-
lary
column (25 m × 0.32 mm, film thickness 0.52 ␮m) and a flame
ionization
detector (FID), was employed. Hydrogen was used as
carrier
gas (1 mL min−1). The injector was heated at 250 ◦C, the
detector
at 300 ◦C, while the column temperature was linearly
programmed
from 40 to 260 ◦C (4 ◦C/min). GC–MS analyses were
carried
out under almost the same analytical conditions, using HP G
1800C
Series II GCD analytical system, equipped with HP-5MS col-
umn
(30 m × 0.25 mm × 0.25 ␮m). Helium was used as carrier gas.
The
transfer line (MSD) was heated at 260 ◦C. The EI mass spectra
(70
eV) were acquired in the scan mode in the m/z range 40–400.
In
each case, 1 ␮L of sample solution in ethanol (10 ␮L/mL) was
injected
in split mode (1:30). The identification of constituents was
performed
by matching their mass spectra and retention indices
with
those obtained from authentic samples and/or NIST/Wiley
spectra
libraries, using different types of search (PBM/NIST/AMDIS)
and
available literature data (Adams, 2001; Hochmuth, 2006). The
percentage
compositions were obtained from electronic integra-
tion
measurements using flame ionization detection (FID; 250 ◦C).
2.6. HPLC analysis of extracts
The HPLC analyses of phenolic components were performed
using
the Agilent 1100 Series and UV-DAD (UV-diode array detec-
tor)
according to procedure Veit et al. (1995). The column was
an
Agilent Eclipse XDB-C18, 5 ␮m, 150 × 4.6 mm, 80 Å. Injection
volume
was 15 ␮L of extracts in concentration of 10 mg/mL. Peak
detection
in UV region at 350 nm was used. The mobile phase was
composed
of solvent (A) 0.15% (w/v) phosphoric acid in water:
methanol
mixture (77:23, v/v, pH 2) and solvent (B) methanol as
follows:
isocratic 0–3.6 min 100% A; 3.6–24 min 80.5% A; 24–30 min
isocratic;
linear 30–60 min 51.8% A; 60–67.2 min 100% B. The flow
rate
of mobile phase was set to the 1 cm3/min and temperature to
15 ◦C.
Phenolic compounds in the samples were identified by com-
paring
their retention times and spectra with retention time and
spectrum
of standards for each component. Identification of the
glycoside
components was based on Rf values in the HPLC chro-
matogram.
2.7.
Determination of total phenolic content
The
total phenolic content of was measured using spectropho-
tometric
method (Singleton and Rossi, 1965). The reaction mixture

704 A. Alimpi´ c et al. / Industrial Crops and Products 76 (2015) 702–709
was prepared by mixing 0.2 mL of extract solution in concentra-
tion
of 1 mg/mL and 1 mL of 10% Folin–Ciocalteu reagent and after
6
min was added 0.8 mL of 7.5% Na2CO3. Blank was prepared to con-
tain
distillated water instead of extract. Absorbance was recorded
at
740 nm after 2 h incubation at room temperature using JENWAY
6305UV–vis
spectrophotometer. The same procedure was repeated
for
standard solution of water solution of gallic acid in order to
construct
calibration curve. Phenolic content in samples was cal-
culated
from standard curve equation and expressed as gallic acid
equivalents
(mg GAE/g dry extract).
2.8.
Determination of flavonoid content
Flavonoid concentrations of samples were measured spec-
trophotometrically
according to procedure of Park et al. (1997).
The
reaction mixture was prepared by mixing 1 mL of extract solu-
tion
in concentration 1 mg/mL, 4.1 mL of 80% ethanol, 0.1 mL of
10%
Al(NO3)3×
9H2O and 0.1 mL 1 M dilution CH3COOK. Blank
was
prepared to contain 96% ethanol instead of extract. After
40
min of
incubation
at room temperature, absorbance was mea-
sured
at 415 nm using JENWAY 6305UV–vis spectrophotometer.
The
same
procedure was repeated for 96% ethanol solution of stan-
dard
antioxidant quercetin in order to construct calibration curve.
Concentration
of flavonoids
in samples was calculated from stan-
dard
curve equation and expressed as quercetin equivalents (mg
QE/g
dry
extract).
2.9. Evaluation
of antioxidant
activity
For
testing of
antioxidant activity, crude extracts were dissolved
in
methanol.
(a)
DPPH assay
For
evaluation of antioxidant activity of extracts, 2,2-dyphenyl-
1-picrylhydrazyl
(DPPH) free radical scavenging method (Blois,
1958)
with slight modifications was used. This assay is spectropho-
tometric
and uses stable DPPH radical as reagent. Stock solutions
of
dry extracts were prepared in concentration of 1000 ␮g/mL
(w/v)
and then were diluted with methanolic solution of DPPH
(40
␮g/mL) to adjust the final volume of reaction mixture (2000 ␮L)
of
the test tube (extract concentrations 10–300 ␮g/mL (v/v)).
Methanol
was used as a blank, while methanol with DPPH solu-
tion
was used as a control. BHA, BHT, and ascorbic acid were used
as
positive controls (standards). Each blank, samples and stan-
dards’
absorbances were measured in triplicate. Absorbance of the
reaction
mixture was measured after 30 min in the dark at room
temperature
at 517 nm using the JENWAY 6305UV–vis spectropho-
tometer.
The decrease of absorption of DPPH radical at 517 nm was
calculated
using equation:
Inhibition of DPPH radical (%) =(Ac−
As)
Ac×
100%
where Acis
the absorbance of control (without test sample), and As
is the
absorbance of the test samples at different concentrations.
IC50 values
(␮g/mL) (concentrations of the test samples and stan-
dard
antioxidants providing 50% inhibition of DPPH radicals) were
calculated
from
DPPH
absorption curve at 517 nm.
•ABTS
assay
In
this
test, antioxidant activity of samples was tracked spec-
trophotometrically,
using change of ABTS solution colour in
presence
of antioxidants. ABTS assay is performed according to pro-
cedure
Miller et al. (1993) with some modifications. Fresh ABTS+
solution was prepared 12–16 h before use by dissolving of ABTS
in 5 mL of 2.46 mM potassium-persulfate and stored in the dark at
room
temperature. The ABTS+solution was dissolved by distilled
water
to obtain an absorbance of working solution 0.700 ± 0.020
at
734 nm. 50 ␮L of test samples (1 mg/mL) were mixed with
2
mL of diluted ABTS+solution and incubated for 30 min at 30 ◦C.
Absorbance
was recorded at 734 nm using JENWAY 6305UV–vis
spectrophotometer.
Distilled water was used as blank. BHA and
BHT
dissolved in methanol in concentration 0.1 mg/mL were used
as
standards. ABTS activity was calculated from ascorbic acid cali-
bration
curve (0–2 mg/L) and expressed as ascorbic acid equivalents
per
gram of dry extract (mg AAE/g).
•FRAP
assay
FRAP
assay evaluates total antioxidant power of the sample
using
reduction of ferric tripyridyltriazine (Fe(III)-TPTZ) complex to
the
ferrous tripyridyltriazine (Fe(II)-TPTZ) by a test sample at low
pH.
The FRAP
assay was performed according to Benzie and Strain
(1996)
procedure with slight modifications. FRAP reagent was pre-
pared
freshly
to contain sodium acetate buffer (300 mmol/L, pH
3.6),
10 mmol/L TPTZ in 40 mmol/L HCl and FeCl3× 6H2O solution
(20
mmol/L)
in proportion 10:1:1 (v/v/v), respectively. Working
FRAP
solution was warmed to 37 ◦C prior to use. 100 ␮L of test sam-
ple
(500
␮g/mL) were added to 3 mL of working FRAP reagent and
absorbance
was recorded at 593 nm after 4 min using the JENWAY
6305UV–vis
spectrophotometer. Blank was prepared to contain
methanol
instead of extract. BHA, BHT, and ascorbic acid dissolved
at
concentration of 0.1 mg/mL were used as standards. The same
procedure
was repeated for standard solution of FeSO4× 7H2O
(0.2–1.6
mmol/L) in order to construct calibration curve. FRAP val-
ues
of sample was calculated from standard curve equation and
expressed
as ␮mol (FeSO4× 7H2O/g dry extract).
2.10. Antimicrobial assays
(a) Antibacterial assay
The
antibacterial activity of essential oil and ethanol/water
extracts
was tested against six Gram-negative: Esherichia coli (ATCC
25922),
Salmonella typhimurium (ATCC 14028), Salmonella enteri-
tidis
(ATCC 13076), Pseudomonas tolaasii (NCTC 387), Pseudomonas
aeruginosa
(ATCC 27853), Proteus mirabilis (ATCC 14273) and five
Gram-positive
bacteria: Staphylococcus aureus (ATCC 25923), Bacil-
lus
cereus (ATCC 10876), Micrococcus flavus (ATCC 14452), Sarcina
lutea
(ATCC 10054) and Listeria monocytogenes (ATCC 15313). In
order
to investigate the antimicrobial activity of extracts, a mod-
ified
version of the microdilution technique was used (Daouk
et
al., 1995; Hanel and Raether, 1988). Determination of MIC
(minimum
inhibitory concentrations) was performed by a microdi-
lution
technique using 96-well microtiter plates. Serial dilutions
of
stock solutions of extracts in broth medium (Muller–Hinton
broth
for bacteria) were prepared in a 96-wells microtiter plate.
The
microbial suspensions were adjusted with sterile saline to
a
concentration of 1 × 105CFU/mL. The microplates plates were
incubated
at 37 ◦C during 48 h. The lowest concentrations with-
out
visible growth were defined as concentrations that completely
inhibited
bacterial growth (MICs). The standard antibiotic strepto-
mycin
(1 mg/mL DMSO) was used to control the sensitivity of the
tested
bacteria.
•Antifungal
assay
Antifungal
activity of extracts was tested against pathogenic
micromycetes
(human isolates): Candida krusei (Castell.) Berkhout,
Candida
albicans
(C.P. Robin) Berkhout, Candida parapsilosis (Ash-
ford)
Langeron & Talice, Aspergillus glaucus (L.) Link, Aspergillus

A. Alimpi´ c et al. / Industrial Crops and Products 76 (2015) 702–709 705
fumigatus Fresen., Aspergillus flavus Link and Trichophyton men-
tagrophytes
(C.P. Robin) Sabour. Cultures were maintained on
Sabouraud
Dextrose Agar (SDA) at 4◦C in the culture collection of
the
Institute of Botany, Faculty of Biology, University of Belgrade
(BEOFB).
Antifungal activity of extracts was studied by microdi-
lution
method using 96-well plates (Sarker et al., 2007). Spore
suspensions
were prepared by washing of SDA surface using ster-
ile
0.9% saline containing 0.1% Tween 20 (v/v). Turbidity was
determined
spectrophotometrically at 530 nm and spore number
was
adjusted to 106CFU/mL (NCCLS, 1998). Ethanol and water
extracts
were dissolved in 5% DMSO in stock concentration. Series
of
double dilutions of extract and essential oil (64–0.25 mg/mL and
4–0.125
mg/mL, respectively) in Sabouraud liquid medium were
analyzed.
Each well contained Sabouraud liquid medium, spore
suspension,
resazurine, and extract or essential oil of defined con-
centration.
The mixture without extract was used as the negative
control,
while the positive control contained commercial antimy-
cotic,
ketoconazole, instead
of extract. Incubation was continued
for
another 48 h, and results were recorded using binocular micro-
scope.
The
lowest concentration of extract or essential oil without
visible
fungal growth was defined as minimal inhibitory concen-
tration
(MIC).
The lowest concentration of extract or essential oil
which
inhibited fungal growth after re-inoculation on SDA was
defined
as
minimal fungicidal concentration (MFC).
2.11.
Cytotoxic
activity
HCT-116
cells were seeded in a 96-well plate (104cells per well).
After
24 h of cells incubation, the medium was replaced with 100 ␮L
medium
containing various doses of ethanol and water extracts
of
S. ringens at different concentrations (1, 10, 50, 100, 250, and
500
␮g/mL). Untreated cells were used as the control. After 24 and
72
h of treatment the cell viability was determined by MTT assay
(Mosmann,
1985). Solution of MTT (final concentration 5 mg/mL
in
PBS) was added to each well and incubated at 37 ◦C in 5% CO2
for
2–4 h. The colored crystals of produced formazan were dis-
solved
in 150 ␮L of DMSO. The absorbance was measured at 570 nm
on
Microplate Reader (ELISA 2100C). Cell proliferation was calcu-
lated
as the ratio of absorbance of treated group divided by the
absorbance
of control group, multiplied by 100 to give a percentage
proliferation.
2.12. Statistical analysis
All experimental measurements were carried out in triplicate
and
are expressed as average of three measurements ± standard
deviation.
Pearson’s correlation coefficients were calculated
between
on one hand total phenolics and flavonoids and on the
other
hand antioxidant assays and interpreted according to Taylor
(1990).
Calculations and constructing of the charts were performed
using
the MS Office Excel, 2007.
3. Results and discussion
3.1.
Essential oil analysis
The aerial parts of S. ringens yielded 0.19% of the yellowish essen-
tial
oil. Chemical composition of the essential oil is presented in
Table 1. Of 39 detected compounds, representing 99.62% of the total
oil,
36 were identified. The most abundant classes of terpenes were
monoterpenes
(93.60%) including oxygenated monoterpenes and
monoterpene
hydrocarbons represented with 56.89% and 36.74%,
respectively.
The main components of the oil were 1.8-cineole
(31.99%),
camphene (17.06%), borneol (11.94%), ␣-pinene (11.52%),
camphor
(5.16%) and bornyl acetate (4.52%). Monoterpenes were
Table 1
Chemical
constituents in the Salvia ringens essential oil.
Peak Compound RIam/m (%)
1.
Tricyclene 918.8 0.82
2.
␣-Thujene 925.3 0.03
3.
␣-Tinene 931.0 11.52
4.
Camphene 945.3 17.06
5.
Thuja-2,4(10)-diene
951.1 0.11
6.
ß-Pinene 973.2 3.69
7.
1-Octen-3-ol 986.0 0.73
8.
Myrcene 991.6 3.16
9.
␣-Phellandrene 1003.7 0.11
10.
␣-Terpinene 1015.8 0.05
11.
p-Cymene 1024.5 2.96
12.
1,8-Cineole 1030.4 31.99
13.
cis-Thujone 1105.4 0.35
14.
1-Octen-3-yl acetate 1110.4 0.20
15.
endo-Fenchol 1114.5 0.12
16.
ß-Phellandrene 1122.9 0.19
17.
␣-Campholenal 1126.6 0.32
18.
trans-Pinocarveol 1138.9 0.39
19.
Camphor 1142.3 5.16
20.
Camphene
hydrate 1145.4 0.16
21.
Borneol 1167.8 11.94
22.
Terpinen-4-ol 1177.7 1.15
23.
␣-Terpineol 1194.0 0.39
24.
trans-Piperitol 1208.6 0.20
25.
Bornyl acetate 1285.3 4.52
26.
␣-Cubebene 1354.0 0.17
27.
␣-Copaene
1367.0 0.31
28.
ß-Bourbonene 1375.2 0.16
29.
cis-Caryophyllene 1410.0 0.35
30.
2-epi-Beta-funebrene 1415.5 0.21
31.
␣-Humulene 1444.8 0.10
32.
9-epi-trans-Caryophyllene
1450.2 0.13
33.
␣-Muurolene 1491.2 0.14
34.
n.i. 1496.9 0.11
35.
n.i. 1500.1 0.16
36.
␥-Cadinene 1512.8 0.26
37.
n.i. 1509.1
0.11
38.
trans-Calamenene 1523.2 0.32
39.
Humulene
epoxide II 1608.5 0.16
Aliphatic
hydrocarbons 0.73
Aromatic
hydrocarbons 2.96
Total
hydrocarbons 3.69
Monoterpene
hydrocarbons 36.74
Oxygenated
monoterpens 56.89
Total
monoterpenes 93.60
Sesquiterpene
hydrocarbons 2.16
Oxygenated
sesquiterpenes 0.16
Total
sesquiterpenes 2.32
Total
identified 99.62
aRetention
index relative to n-alkanes on HP-5 capillary column; n.i., not identi-
fied.
previously
recognized as dominant class of S. ringens oil from dif-
ferent
localities. Dominant components in the oil of S. ringens from
Greece
were 1.8-cineol, ␣-pinene, bornyl acetate, and ␤-pinene
(Tzakou
et al., 2001), 1.8-cineole, ␣-pinene and myrcene in S. rin-
gens
var. baldacciana from Dautica Mt. (Macedonia) (ˇ
Savikin
et al.,
2008)
and camphor and borneol in leaves and flowers of S. ringens
from
Bulgaria (Georgiev et al., 2013). Our findings are in agreement
with
these studies with exception of particularly high percent of
camphene
(17.06%).
Differences in the chemical composition could
be
derived from several factors such as plant age, plant part, devel-
opment
phase, growing place, harvesting period, chemotype (Ben
Farhat
et al., 2009; Miguel, 2010).
3.2. The yield of extracts, total phenolic and flavonoid content
S. ringens extracts were obtained using individual and succes-
sive
extraction procedure and yields of extracts are presented in
Table
2. The yields of ethanol and methanol extracts were the high-

706
A. Alimpi´ c et al. / Industrial Crops and Products 76 (2015) 702–709
Table
2
The
yield, total phenolic content (TPC), flavonoid content (FC) and antioxidant activities evaluated by DPPH, ABTS, and FRAP assays of S. ringens.
Sample YieldaTPCbFCcDPPHdABTSeFRAPf
Methanol
6.94 185.05 ± 1.471 27.31 ± 0.588 20.29 ± 0.263 1.19 ± 0.026 274.85 ± 13.192
Dichloromethane
2.39 58.10
± 0.510 32.31 ± 0.428 266.22 ± 4.208 0.58 ± 0.021 191.13 ± 11.020
Ethyl
acetate 1.37 248.38 ± 0.455 66.67 ± 1.464 22.25 ± 0.571 2.36 ± 0.030 969.80 ± 25.238
Ethanol
7.20 208.27 ± 1.113 30.41 ± 0.640 17.26 ± 0.412 2.44 ± 0,028 1088.30 ± 17.655 ± 17.655
Essential
oil 0.19
– – 654.33 ± 6.522 nt nt
BHA
– – – 17.94 ± 0.168 2.75 ± 0.021 445.34 ± 5.772
BHT
– – – 13.37 ± 0.430 2.82 ± 0.011 583.72 ± 5.255
Ascorbic
acid – – – 5.11 ± 0.143 – 180.81 ± 8.607
nt-not tested.
aPercentage
of yield (%).
bmg
GAE/g dry
extract.
cmg
QE/g dry extract.
dIC50,
␮g/ml.
emg
AAE/g.
f␮mol
Fe(II)/g.
est (7.70 and 6.94%, respectively), while dichloromethane and ethyl
acetate
extracts showed lower yield. Previous researchers pointed
out
that polar alcoholic extracts, such as ethanol and methanol,
showed
higher yield than less and/or non-polar solvents extracts
(Akkol
et al., 2008; Orhan et al., 2012).
Total
phenolic content (TPC) and flavonoid content (FC) were
measured
using spectrophotometric assays and results are pre-
sented
in Table 2. Ethyl acetate extract showed the largest total
phenolic
content (248.38 mg GAE/g), whereas dichloromethane
extract
was the poorest in total phenolics (58.10 mg GAE/g).
Flavonoid
contents of extracts ranged from 27.31 mg QE/g for
methanol
to 66.67 mg QE/g for ethyl acetate extract. Total pheno-
lic
and flavonoid content were previously reported for many Salvia
species
from Turkey (Akkol et al., 2008; Orhan et al., 2012), South
Africa
(Kamatou et al., 2010), Iran (Asadi et al., 2010) and Greece
(Stagos
et al., 2012). Our findings were congruent with above men-
tioned
studies, as well as with polyphenolic content of S. ringens
herb
collected in Bulgaria (Nikolova, 2011) and Romania (Coisin
et
al., 2012).
3.3. Phenolic composition
of
the extracts
Phenolic
composition
of S.
ringens
extracts was determined
using
HPLC and components were classified according to Neveu
et
al. (2010)
(Table
3). Methanol and ethyl acetate extracted most of
the
components, followed by ethanol, water and dichloromethane.
As
previously
reported, the
efficiency of extraction of phenolic
components
was rising with increasing polarity of the extrac-
tion
solvent
(Akkol et al., 2008; Orhan et al., 2012). Among
phenolic
acids, gallic, caffeic and rosmarinic acids were present.
Caffeic
acid
was present in all extracts (0.18–8.27 %), excluding
dichloromethane.
Rosmarinic acid was present only in methanol
extract
(3.59%),
although it was reported as the most common
derivate
of caffeic acid in Lamiaceae family (Lu and Foo, 2002)
and
the most abundant phenolic acid in Salvia genus with strong
antioxidant
activity (Akkol et al., 2008; Ben Farhat et al., 2009;
Coisin
et al., 2012; Kamatou et al., 2010; Orhan et al., 2012).
The
absence of rosmarinic acid in the ethanol extract could be
attributed
to the extraction procedure applied. Flavonoids, includ-
ing
flavones and flavonols, were identified in examined extracts
whereby
the flavonols were present in a higher percentage. In
Table 3
Phenolic
constituents of S. ringens extracts (%).
Extractsa
Constituents DCM ETAC MEOH ETOH W
Phenolic
acids
Gallic
acid – – 0.05 – –
Caffeic
acid – 0.18 0.51 1.28 8.27
Caffeic
acid
methyl ether
–
– 0.30 – –
Rosmarinic
acid – – 3.59 – –
Flavonoids
Flavones
Apigenin
– 1.64 0.13 1.32 –
Apigenin
5-O-glucoside – – 3.31 – –
Apigenin
4-O-glucoside – 0.03 1.71 – 1.40
Genkwanin
5-O-glucoside – 1.39 – – –
Luteolin
– 2.68 0.60 2.21 –
Flavonols
Kaempferol
3-O-7-O-diglucoside – 1.18 – – –
Kaempferol
3-O-glucoside-7-O-rhamnoside – – 1.67 – –
Kaempferol
3-O-(6 -O-acetilglucoside)-7-O-rhamnoside – 12.00 28.71 46.46 48.19
Kaempferol
3-O-rhamnoside – 0.71 0.20 – –
Rutin
– 0.25 17.31 7.74 8.54
Quercetin
– 1.44 – – –
Hyperoside
12.81 2.64 5.99 2.83 –
Other
polyphenols
Coumarin
– 1.35 2.90 1.72 –
Total
of identified constituents 12.81 25.49 66.98 63.56 66.39
Number
of identified constituents 1 12 14 7 4
Number
of non-identified constituents 8 25 22 8 11
aDCM
(dichloromethane), ETAC (ethyl acetate), MEOH (methanol), ETOH (ethanol), W (water); – not identified.