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Alimpić et al., 2015

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Industrial Crops and Products 76 (2015) 702–709
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Industrial Crops and Products
journal homepage: www.elsevier.com/locate/indcrop
Composition and biological effects of Salvia ringens (Lamiaceae)
essential oil and extracts
Ana Alimpić a,∗ , Dejan Pljevljakušić b , Katarina Šavikin b , Aleksandar Knežević a ,
Milena Ćurčić c , Dragan Veličković d , Tatjana Stević b , Goran Petrović e , Vlado Matevski f ,
Jelena Vukojević a , Snežana Marković c , Petar D. Marin a , Sonja Duletić-Laušević a
a
Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia
Institute for Medicinal Plant Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia
Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34 000 Kragujevac, Serbia
d
College of Agriculture and Food Technology, Ćirila and Metodija 1, 18400 Prokuplje, Serbia
e
Department of Chemistry, Faculty of Natural Sciences and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia
f
Institute 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
b
c
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%), camphene (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 dysfunction, cataracts, skin diseases (Asadi et al., 2010; Kamatou et al.,
2010; Li et al., 2008) and may also serve as natural food preservatives (Miguel, 2010).
∗ Corresponding author. Fax: +381 113246655.
E-mail address: [email protected] (A. Alimpić).
http://dx.doi.org/10.1016/j.indcrop.2015.07.053
0926-6690/© 2015 Elsevier B.V. All rights reserved.
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 investigations showed its antioxidant, antibacterial, antifungal, antiviral,
cytotoxic, neuroprotective, antiinflammatory, and tumorigenesispreventing as well as ecological significance such as pest-toxic
and repellent and other activities (Asadi et al., 2010; Baričević and
Bartol, 2000; Ben Farhat et al., 2009; Kamatou et al., 2010; Orhan
et al., 2012; Veličković 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 diterpenoids are the main compounds in roots (Baričević 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
A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709
places of South and Eastern parts of Balkan Peninsula, just extending 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 flowers, 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 (Šavikin 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 antimicrobial activity (Šavikin 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 phenolics, 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 significant cytotoxic activity against several human carcinoma cell
lines (Janicsák et al., 2007, 2011), while literature data on antimicrobial 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, hydrochloric acid, hexane, dichlormethane, and ethyl acetate were purchased
from Zorka Pharma, Šabac (Serbia). Gallic acid, quercetin, ascorbic acid, 2(3)-t-butyl-4-hydroxyanisole (BHA), 3,5-di-tert-butyl4-hydroxytoluene (BHT) 2,2-dyphenyl-1-picrylhydrazyl (DPPH),
2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ), potassium
acetate (C2 H3 KO2 ), potassium-persulfate (K2 S2 O8 ), sodium carbonate anhydrous (Na2 CO3 ), alluminium nitrate nonahydrate
(Al(NO3 )3 × 9H2 O), sodium acetate (C2 H3 NaO2 ), iron(III) chloride (FeCl3 ), iron(II)-sulfate heptahydrate (FeSO4 × 7H2 O) and
Folin–Ciocalteu phenol reagent were purchased from Sigma Chemicals 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 (Macedonia). 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 apparatus, according to the procedure I of the Yugoslavian Pharmacopoeia
(1984).
703
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 procedure of Şenol et al. (2010) and Orhan et al. (2013). Second, portion of
10 g of plant material was individually extracted by 100 mL of solvent (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 (Veličković
et al., 2007; Glišić et al., 2011). Subsequently, extracts were filtered 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 GCFID and GC–MS. In the first instance model HP-5890 Series II gas
chromatograph equipped with a split-splitless injector, HP-5 capillary 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 column (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 integration 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 detector) 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 comparing 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 chromatogram.
2.7. Determination of total phenolic content
The total phenolic content of was measured using spectrophotometric method (Singleton and Rossi, 1965). The reaction mixture
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A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709
was prepared by mixing 0.2 mL of extract solution in concentration of 1 mg/mL and 1 mL of 10% Folin–Ciocalteu reagent and after
6 min was added 0.8 mL of 7.5% Na2 CO3 . Blank was prepared to contain 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 calculated from standard curve equation and expressed as gallic acid
equivalents (mg GAE/g dry extract).
2.8. Determination of flavonoid content
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 calibration curve (0–2 mg/L) and expressed as ascorbic acid equivalents
per gram of dry extract (mg AAE/g).
• FRAP assay
Flavonoid concentrations of samples were measured spectrophotometrically according to procedure of Park et al. (1997).
The reaction mixture was prepared by mixing 1 mL of extract solution in concentration 1 mg/mL, 4.1 mL of 80% ethanol, 0.1 mL of
10% Al(NO3 )3 × 9H2 O and 0.1 mL 1 M dilution CH3 COOK. Blank
was prepared to contain 96% ethanol instead of extract. After
40 min of incubation at room temperature, absorbance was measured at 415 nm using JENWAY 6305UV–vis spectrophotometer.
The same procedure was repeated for 96% ethanol solution of standard antioxidant quercetin in order to construct calibration curve.
Concentration of flavonoids in samples was calculated from standard 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.
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 prepared freshly to contain sodium acetate buffer (300 mmol/L, pH
3.6), 10 mmol/L TPTZ in 40 mmol/L HCl and FeCl3 × 6H2 O 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 sample (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 × 7H2 O
(0.2–1.6 mmol/L) in order to construct calibration curve. FRAP values of sample was calculated from standard curve equation and
expressed as ␮mol (FeSO4 × 7H2 O/g dry extract).
(a) DPPH assay
2.10. Antimicrobial assays
For evaluation of antioxidant activity of extracts, 2,2-dyphenyl1-picrylhydrazyl (DPPH) free radical scavenging method (Blois,
1958) with slight modifications was used. This assay is spectrophotometric 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 solution was used as a control. BHA, BHT, and ascorbic acid were used
as positive controls (standards). Each blank, samples and standards’ 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 spectrophotometer. The decrease of absorption of DPPH radical at 517 nm was
calculated using equation:
Inhibition of DPPH radical (%) =
(Ac − As )
× 100%
Ac
where Ac is 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 standard antioxidants providing 50% inhibition of DPPH radicals) were
calculated from DPPH absorption curve at 517 nm.
• ABTS assay
(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 enteritidis (ATCC 13076), Pseudomonas tolaasii (NCTC 387), Pseudomonas
aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 14273) and five
Gram-positive bacteria: Staphylococcus aureus (ATCC 25923), Bacillus 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 modified 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 microdilution 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 × 105 CFU/mL. The microplates plates were
incubated at 37 ◦ C during 48 h. The lowest concentrations without visible growth were defined as concentrations that completely
inhibited bacterial growth (MICs). The standard antibiotic streptomycin (1 mg/mL DMSO) was used to control the sensitivity of the
tested bacteria.
• Antifungal assay
In this test, antioxidant activity of samples was tracked spectrophotometrically, using change of ABTS solution colour in
presence of antioxidants. ABTS assay is performed according to procedure Miller et al. (1993) with some modifications. Fresh ABTS+
solution was prepared 12–16 h before use by dissolving of ABTS
Antifungal activity of extracts was tested against pathogenic
micromycetes (human isolates): Candida krusei (Castell.) Berkhout,
Candida albicans (C.P. Robin) Berkhout, Candida parapsilosis (Ashford) Langeron & Talice, Aspergillus glaucus (L.) Link, Aspergillus
A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709
fumigatus Fresen., Aspergillus flavus Link and Trichophyton mentagrophytes (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 microdilution method using 96-well plates (Sarker et al., 2007). Spore
suspensions were prepared by washing of SDA surface using sterile 0.9% saline containing 0.1% Tween 20 (v/v). Turbidity was
determined spectrophotometrically at 530 nm and spore number
was adjusted to 106 CFU/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 concentration. The mixture without extract was used as the negative
control, while the positive control contained commercial antimycotic, ketoconazole, instead of extract. Incubation was continued
for another 48 h, and results were recorded using binocular microscope. The lowest concentration of extract or essential oil without
visible fungal growth was defined as minimal inhibitory concentration (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 (104 cells 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 dissolved in 150 ␮L of DMSO. The absorbance was measured at 570 nm
on Microplate Reader (ELISA 2100C). Cell proliferation was calculated 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 essential 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
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Table 1
Chemical constituents in the Salvia ringens essential oil.
Peak
Compound
RIa
m/m (%)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
Tricyclene
␣-Thujene
␣-Tinene
Camphene
Thuja-2,4(10)-diene
ß-Pinene
1-Octen-3-ol
Myrcene
␣-Phellandrene
␣-Terpinene
p-Cymene
1,8-Cineole
cis-Thujone
1-Octen-3-yl acetate
endo-Fenchol
ß-Phellandrene
␣-Campholenal
trans-Pinocarveol
Camphor
Camphene hydrate
Borneol
Terpinen-4-ol
␣-Terpineol
trans-Piperitol
Bornyl acetate
␣-Cubebene
␣-Copaene
ß-Bourbonene
cis-Caryophyllene
2-epi-Beta-funebrene
␣-Humulene
9-epi-trans-Caryophyllene
␣-Muurolene
n.i.
n.i.
␥-Cadinene
n.i.
trans-Calamenene
Humulene epoxide II
918.8
925.3
931.0
945.3
951.1
973.2
986.0
991.6
1003.7
1015.8
1024.5
1030.4
1105.4
1110.4
1114.5
1122.9
1126.6
1138.9
1142.3
1145.4
1167.8
1177.7
1194.0
1208.6
1285.3
1354.0
1367.0
1375.2
1410.0
1415.5
1444.8
1450.2
1491.2
1496.9
1500.1
1512.8
1509.1
1523.2
1608.5
0.82
0.03
11.52
17.06
0.11
3.69
0.73
3.16
0.11
0.05
2.96
31.99
0.35
0.20
0.12
0.19
0.32
0.39
5.16
0.16
11.94
1.15
0.39
0.20
4.52
0.17
0.31
0.16
0.35
0.21
0.10
0.13
0.14
0.11
0.16
0.26
0.11
0.32
0.16
Aliphatic hydrocarbons
Aromatic hydrocarbons
Total hydrocarbons
Monoterpene hydrocarbons
Oxygenated monoterpens
Total monoterpenes
Sesquiterpene hydrocarbons
Oxygenated sesquiterpenes
Total sesquiterpenes
Total identified
0.73
2.96
3.69
36.74
56.89
93.60
2.16
0.16
2.32
99.62
a
Retention index relative to n-alkanes on HP-5 capillary column; n.i., not identified.
previously recognized as dominant class of S. ringens oil from different 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. ringens var. baldacciana from Dautica Mt. (Macedonia) (Šavikin 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, development 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 successive extraction procedure and yields of extracts are presented in
Table 2. The yields of ethanol and methanol extracts were the high-
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A. Alimpić 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
Yielda
TPCb
FCc
DPPHd
ABTSe
FRAPf
Methanol
Dichloromethane
Ethyl acetate
Ethanol
Essential oil
BHA
BHT
Ascorbic acid
6.94
2.39
1.37
7.20
0.19
–
–
–
185.05 ± 1.471
58.10 ± 0.510
248.38 ± 0.455
208.27 ± 1.113
–
–
–
–
27.31 ± 0.588
32.31 ± 0.428
66.67 ± 1.464
30.41 ± 0.640
–
–
–
–
20.29 ± 0.263
266.22 ± 4.208
22.25 ± 0.571
17.26 ± 0.412
654.33 ± 6.522
17.94 ± 0.168
13.37 ± 0.430
5.11 ± 0.143
1.19 ± 0.026
0.58 ± 0.021
2.36 ± 0.030
2.44 ± 0,028
nt
2.75 ± 0.021
2.82 ± 0.011
–
274.85 ± 13.192
191.13 ± 11.020
969.80 ± 25.238
1088.30 ± 17.655 ± 17.655
nt
445.34 ± 5.772
583.72 ± 5.255
180.81 ± 8.607
nt-not tested.
a
Percentage of yield (%).
b
mg GAE/g dry extract.
c
mg QE/g dry extract.
d
IC50 , ␮g/ml.
e
mg 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 presented 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 phenolic 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 mentioned 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 extraction 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, including 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
Phenolic acids
Gallic acid
Caffeic acid
Caffeic acid methyl ether
Rosmarinic acid
Flavonoids
Flavones
Apigenin
Apigenin 5-O-glucoside
Apigenin 4 -O-glucoside
Genkwanin 5-O-glucoside
Luteolin
Flavonols
Kaempferol 3-O-7-O-diglucoside
Kaempferol 3-O-glucoside-7-O-rhamnoside
Kaempferol 3-O-(6 -O-acetilglucoside)-7-O-rhamnoside
Kaempferol 3-O-rhamnoside
Rutin
Quercetin
Hyperoside
Other polyphenols
Coumarin
Total of identified constituents
Number of identified constituents
Number of non-identified constituents
a
DCM
ETAC
–
–
–
–
–
ETOH
W
0.05
0.51
0.30
3.59
–
1.28
–
–
–
8.27
–
–
–
–
–
–
–
1.64
–
0.03
1.39
2.68
0.13
3.31
1.71
–
0.60
1.32
–
–
–
2.21
–
–
1.40
–
–
–
–
–
–
–
–
12.81
1.18
–
12.00
0.71
0.25
1.44
2.64
–
1.67
28.71
0.20
17.31
–
5.99
–
–
46.46
–
7.74
–
2.83
–
–
48.19
–
8.54
–
–
–
12.81
1
8
1.35
25.49
12
25
2.90
66.98
14
22
1.72
63.56
7
8
–
66.39
4
11
0.18
–
–
DCM (dichloromethane), ETAC (ethyl acetate), MEOH (methanol), ETOH (ethanol), W (water); – not identified.
MEOH
A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709
Table 4
Pearson’s correlation coefficients (r) of antioxidant activities versus total phenolic
content (TPC) and flavonoid content (FC) of S. ringens extracts.
TPC
FC
DPPH vs. ABTS
DPPH vs. FRAP
ABTS vs. FRAP
TPC vs. FC
DPPH
ABTS
FRAP
−0.945c
−0.236a
0.886c
0.504b
−0.780c
−0.636b
0.977c
0.530b
0.770c
0.490b
707
pound is extracted in the greatest extent by the non-polar solvent
(dichloromethane), which could be explained by applying of successive extraction, also reported by Askun et al. (2012) and Orhan
et al. (2013).
3.4. Evaluation of antioxidant activity
According to Taylor (1990):
a
r ≤ 0.35 weak correlation.
b
0.36 < r < 0.67 moderate correlation.
c
0.68 < r < 1 strong correlation.
our study, flavonol kaempferol 3-O-(6”-O-acetilglucoside)-7-Orhamnoside was present in the highest amounts (12.00–48.19 %),
especially in water and ethanol extracts. Recent data reported on
powerful antioxidant activity of some flavonols such as kaempferol
rhamnoside derivatives (Tatsimo et al., 2012). The majority of
flavonoids identified in the present study, such as luteolin, apigenin,
kaempferol, rutin, quercetin and its glycosides, were recognized
previously in methanol extract of S. ringens from Romania (Coisin
et al., 2012) as well as in the extracts of other Salvia species (Akkol
et al., 2008; Ben Farhat et al., 2009; Kamatou et al., 2010; Lu and
Foo, 2002; Orhan et al., 2012). Hyperoside–glycoside, polar com-
Antioxidant activity of S. ringens extracts was measured
using three parallel test assays, i.e., DPPH, ABTS for the evaluation of free-radical scavenging activity of extracts, and FRAP
assay for measuring the total ferric-reducing power of extracts
(Table 2). DPPH scavenging activity of extracts, presented by IC50
value, ranged from 17.26 ␮g/mL for ethanol to 266.22. ␮g/mL for
dichloromethane extract. Due to the very small amount of the
essential oil obtained, it was tested only by DPPH assay, and
performed extremely weaker activity than extracts (IC50 value
of 654.33 ␮g/mL). Against ABTS radicals the most powerful was
ethanol extract (2.44 mg AAE/g, respectively) on the contrary to
the dichloromethane extract (0.58 mg AAE/g). Similarly, ethanol
extract showed the most expressive ability to reduce Fe(III) to
Fe(II) ion (1088.30 ␮mol Fe(II)/g), unlike dichloromethane with
191.13 ␮mol Fe(II)/g. In all three assays, ethanol extract exhibited
activity close to references antioxidants BHA and BHT. Our findings are congruent with previous studies on antioxidant activity of
Table 5
Antimicrobial activity of the S. ringens ethanol (ETOH), water (W), essential oil (EO) and reference substances.
Extracts
Bacteria
Gram-negative bacteria
Esherichia coli ATCC 25922
Salmonella typhimurium ATCC 14028
Salmonella enteritidis ATCC 13076
Pseudomonas tolasii NCTC 387
Pseudomonas aeruginosa ATCC 27853
Proteus mirabilis ATCC 14273
Gram-positive bacteria
Staphylococcus aureus ATCC 25932
Bacillus cereus ATCC 10876
Micrococcus flavus ATCC 14452
Sarcina lutea ATCC 10054
Listeria monocytogenes ATCC 15313
Micromycetes
Candida krusei
Candida albicans
Candida parapsilosis
Aspergillus glaucus
Aspergillus fumigatus
Aspergillus flavus
Trichophyton mentagrophytes
EO
Streptomycin
Ketoconazole
25
–
20
–
20
–
30
–
30
–
25
–
14.25
–
14.25
–
11.40
0.012
–
0.010
–
0.010
14.25
0.016
17.10
0.016
17.10
0.005
–
–
–
–
–
–
–
–
–
–
–
5
–
10
–
10
–
15
–
5
–
15
–
20
–
20
–
25
–
15
–
9.50
–
9.50
–
9.50
–
11.40
–
9.50
–
0.016
–
0.005
–
0.010
64
NA
64
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
16
NA
NA
NA
NA
NA
NA
NA
0.125
3.000
0.125
3.000
0.125
3.000
0.125
3.000
3.000
NA
0.25
NA
0.75
1.50
–
–
–
–
–
–
–
–
–
–
–
–
–
–
ETOH
W
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
15
–
10
–
15
–
20
–
20
–
15
–
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
0.012
–
0.010
–
–
–
–
–
–
–
–
–
0.0078
0.0156
0.0078
0.0156
0.0078
0.0156
0.0078
0.0078
0.0078
0.0156
0.0078
0.0078
0.0019
0.0039
708
A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709
methanol extracts of S. ringens herb from Bulgaria (Nikolova, 2011)
and Galičica Mt., Macedonia (Tusevski et al., 2014).
Table 6
Cytotoxic activity presented as IC50 values (␮g/mL) of S. ringens extracts against
HCT-116 and SW480 cell lines.
3.5. Correlation between antioxidant assays, total phenolic and
flavonoid contents
Cell line
Type of extract
24 h
72 h
HCT-116
Antioxidant activity measured by DPPH, ABTS, and FRAP assays
on the one hand and content of total phenolic (TPC) and flavonoid
content (FC) on the other hand, were correlated in different ways
(Table 4). Antioxidant assays were moderate to strongly correlate
between each other. Antioxidant assays were more strongly correlated to total phenolic than to flavonoid content and these findings
are in agreement with previous studies (Asadi et al., 2010; Ben
Farhat et al., 2009; Li et al., 2008; Stagos et al., 2012).
SW480
ETOH
W
ETOH
W
31.83 ± 1.89
9.83 ± 0.28
>500
>500
179.30 ± 4.12
406.71 ± 4.61
>500
412.36 ± 2.31
3.6. Antimicrobial activity
Antimicrobial (antibacterial and antifungal) activities of S. ringens essential oil and ethanol and water extracts were investigated
using microdilution method and data were presented in Table 5.
These extracts were selected for testing of the antimicrobial activity
and later for cytotoxic activity since the mixtures of ethanol/water
and water extracts are mostly used in phytotherapy (Miguel, 2010;
Stagos et al., 2012).
Antibacterial activity was tested against six Gram-negative and
five Gram-positive bacteria. Results of the present study showed
that the essential oil possess the strongest antibacterial activity
(MICs 9.50–17.10 mg/mL), followed by ethanol (MICs 5–20 mg/mL)
and water extract (MICs from 15 to 30 mg/mL), which is in accordance with findings previously reported by Tepe et al. (2004).
Similar to the previous study published by Šavikin et al. (2008)
dealing with S. ringens var. baldachiana from Macedonia, Grampositive strains were more sensitive. Unlike the aforementioned,
Tzakou et al. (2001) found that inhibitory effects of S. ringens oil
was stronger against Gram-negative bacteria and mainly attributed
to presence of 1,8-cineole as dominant component. Comparing
to streptomycin (MICs ranged as 0.005–0.016 mg/mL), our samples exhibited weaker activity. The most sensitive bacteria were
S. aureus and L. monocytogenes while the most resistant bacteria
were P. tolasii and P. aeruginosa, as reported before (Šavikin et al.,
2008; Tepe et al., 2004; Veličković et al., 2002).
S. ringens essential oil showed stronger antifungal activity
against seven tested mycromicetes (MICs 0.125–3 mg/mL) compared to ethanol and water extracts. Fungicidal effects were not
observed only for A. fumigatus and A. flavus, which were previously reported as very resistant micromycetes (Veličković et al.,
2002). Ethanol extract inhibited the growth of C. krusei and C. albicans at the highest applied concentration of 64 mg/mL. Similar as
in previous studies (Šavikin et al., 2008; Tepe et al., 2004; Tzakou
et al., 2001; Veličković et al., 2002), Candida species were generally
sensitive to Salvia essential oil and extracts.
3.7. Cytotoxic activity
The ethanol and water extracts of S. ringens were tested for
their cytotoxic activity against human colon carcinoma HCT-116
and SW480 cell lines using the MTT assay. Results were recorded
after 24 and 72 h of treatment and presented as IC50 values (␮g/mL)
in Table 6. Our results showed that S. ringens extracts exhibited
more significant cytotoxic activity after 24 h than after 72 h on
HCT-116 cells. The water extract showed IC50 values lower than
30 ␮g/mL, which were considered as very good cytotoxic activity according to National Cancer Institute criteria for cytotoxic
activity of crude extracts (Suffness and Pezzuto, 1990). HCT-116
cell line was more sensitive than SW480 cell line (IC50 values
above 500 ␮g/mL). Cytotoxic effects of S. ringens methanol extract
on skin cancer cell lines (A431) were evaluated as strong, while
some isolated abietane diterpenes from S. ringens root displayed
marked concentration-dependent effects on human cervix adenocarcinoma (HeLa) cells (Janicsák et al., 2007, 2011). In current study,
as the main phenolic constituent was found kaempferol 3-O-(6”-Oacetilglucoside)-7-O-rhamnoside, especially in water and ethanol
extracts. Some kaempferol glycosides were previously reported as
strong cytotoxic agents against lung and melanoma cancer cell lines
(Moon et al., 2010).
4. Conclusions
According to results of this study, it can be concluded that
essential oil and extracts of Salvia ringens showed strong antioxidant and cytotoxic activity and promising antimicrobial effects.
Essential oil was composed mainly from monoterpenes 1.8-cineole,
camphene, borneol, and ␣-pinene. Kaempferol glycosides were
dominant among 17 phenolic components, mainly in ethanol and
water extracts, while methanol and ethyl acetate extracts were
quantitatively the richest. Methanol and ethanol extracts showed
the strongest antioxidant activity. Essential oil, ethanol, and water
extract showed antimicrobial activity against selected bacteria and
micromycetes while ehanol and water extracts performed cytotoxic activity against HCT-116 colon carcinoma cell line. Obtained
results indicate that S. ringens herb can be suggested as the possible
source of natural components with a range of biological activities.
Acknowledgements
Authors are grateful to the Ministry of Education, Science, and
Technological Development of Serbia for financial support (Projects
No. 173029, 173032, 172047, 46013, 41010).
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