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Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016
KEYWORDS: Salvia officinalis, sage, antioxidant activity, phenolics, flavonoids.
Abstract Salvia officinalis L. plants originated from continental (Mt. Pleš, Serbia) and Mediterranean part of Central
Balkans (Luštica peninsula, Montenegro) grew under the same conditions in Belgrade. Various extracts of
plant material, collected during summer and winter season, were analyzed for the antioxidant activity and phenolic and flavonoid
contents. DPPH, ABTS, and FRAP assays for antioxidant activity, as well as total phenol and flavonoid content, were measured
spectrophotometrically. Phenolic and flavonoid content and antioxidant activity of the extracts mostly depended on extraction
solvent and harvesting season. The extracts of plants originated from Serbia showed stronger antioxidant activity. Generally, plants
collected in summer season performed higher activity. Among tested extracts, ethanol extract showed better antioxidant activities
compared to other analyzed extracts.
Salvia officinalis of different origins
Antioxidant activity, phenolic and flavonoid content of extracts
INTRODUCTION
Salvia (sage), one of the largest and the most important
aromatic and medicinal genera of the Lamiaceae
family, comprises about 1000 worldwide distributed
species (1). Salvia species are reported to have antioxidant,
antibacterial, antifungal, antiviral, cytotoxic, neuroprotective,
antiin ammatory and other biological activities (2-8). Salvia
of cinalis, known as Dalmatian sage, common sage or
garden sage, is a perennial subshrub native to the northern
coastal region of the Mediterranean, but widely cultivated
in many countries (9) due to its culinary and medicinal
signi cance. It is used for food preservation, as a spice for
avouring, and for treatment of many diseases (2).
Free radicals and reactive oxygen species (ROS) are well
known inducers of cellular and tissue pathogenesis leading
to several human diseases such as cancer, in ammatory
disorders, atherosclerosis and cardiovascular diseases (10). S.
of cinalis is proven to be biologically active, and promising as
antioxidant agent of natural origin (5, 11-18).
The aim of this study was to determine and compare the
antioxidant potential, phenolic and avonoid contents of
different extracts obtained from plant material of Salvia
of cinalis L. originated from continental part of Eastern Serbia
and Mediterranean part of Montenegro, cultivated under the
same conditions in Belgrade and collected during summer
and winter season.
MATERIAL AND METHODS
Plant material
Salvia of cinalis plants from their natural habitats at Mt.
Pleš (Eastern Serbia) and Luštica peninsula (Montenegro)
were transplanted in Belgrade and cultivated under the
same conditions. Plants from Pleš were transplanted in
December 2008, and plants from Luštica in August 2009. After
ve seasons living under the same environmental conditions,
aerial parts were harvested in vegetative stage during winter
(December 2013) and owering stage in summer (June 2014).
Preparation of plant extracts
Whole aerial plant parts (10 g) were grounded in small
pieces (2-6 mm) in the cylindrical crusher. The extraction was
performed successively, in a series of consecutive extraction
and ltration of plant material, by increasing polarity solvents
(100 ml of dichloromethane (DCM), chloroform (CHL), ethyl
acetate (ETAC) and ethanol (ETOH)). The plant-solvent
mixture was exposed to the ultrasound 1h before and after
24h-maceration at 30°C. The liquid extracts were ltered and
evaporated under reduced pressure by the rotary evaporator
(Buchi rotavapor R-114). The obtained crude extracts were
stored in the fridge at +4°C for further experiments.
Experimental measurements
Crude extracts of S. of cinalis were dissolved by methanol
to obtain stock extract solution at concentration 500 μg/
mL (w/v). As standard antioxidants, BHA (2(3)-t-Butyl-4-
hydroxyanisole) and BHT (3,5-di-tert-butyl-4 hydroxytoluene),
were dissolved in methanol in concentrations of 100 μg//
mL and tested for antioxidant activity. All of applied
spectrophotometric measurements were performed using
JENWAY 6305UV/Vis spectrophotometer.
Determination of total phenolic content
The total phenolic content (TPC) was measured using
specrophotometric procedure (19). 0.2 mL of extract solution
and 1 mL of 10% Folin–Ciocalteu reagent were mixed and after
PLANT EXTRACTS
SONJA DULETIĆ-LAUŠEVIĆ1, ANA ALIMPIĆ1*, DANICA PAVLOVIĆ2,
PETAR D. MARIN1, DMITAR LAKUŠIĆ1
*Corresponding author
1. Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology,
University of Belgrade, Takovska 43, Belgrade, Serbia
2. Institute of Physics, University of Belgrade, Pregrevica 118,
11080 Zemun, Belgrade, Serbia
Ana Alimpi
ć
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Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016
assay was performed (22) with some modications. Fresh
ABTS+ solution was prepared 12-16 hours before use by
dissolving ABTS in the potassium-persulfate solution (2.46
mM). The ABTS+ solution was dissolved by distilled water to
obtain an absorbance of working solution 0.700 ± 0.020 at
734 nm. 50 μL of test samples and/or standard antioxidants
were mixed with 2 mL of working ABTS+ solution and after
incubation of 30 min at 30ºC, absorbance was recorded at
734 nm. Distilled water was used as blank. 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).
Ferric-reducing ability of plasma (FRAP) assay
The FRAP assay was performed (23) with slight modications.
FRAP reagent, prepared 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, was warmed to 37°C prior to use. 100
μL of test sample were added to 3 mL of FRAP reagent and
absorbance was recorded at 593 nm after 4 minutes. Blank
was prepared to contain distilled water instead of extract.
The same procedure was repeated for standard solution
of FeSO4 * 7H2O (0.2-1.6 mmol/L) in order to construct
calibration curve. FRAP values of sample were calculated
from standard curve equation and expressed as μmol FeSO4
* 7H2O /g dry extract).
Statistical analysis
All experimental measurements were carried out in triplicate
and are expressed as average of three measurements
± standard deviation. Pearson’s correlation coefcients,
calculated between TPC, TFC and antioxidant assays,
were interpreted according to Taylor (24). Calculations
and constructing of the charts were performed using the
MS Ofce Excel, 2007. Analysis of variance (ANOVA) was
used in order to calculate critical value from F-test (F)
and p-statistical signicance (p) for analyzed characters.
Statistical analyses were performed using the package
Statistica 5.1 (25).
six minutes was added 0.8 mL of 7.5% Na2CO3. Absorbance
was recorded at 740 nm after two hours incubation. The same
procedure was repeated for standard gallic acid in order to
construct calibration curve. Phenolic content of samples was
calculated from standard curve equation and expressed as
gallic acid equivalents (mg GAE/g dry extract).
Determination of total avonoid content
Total avonoid content (TFC) was measured using
specrophotometric procedure (20). The reaction mixture was
prepared to contain 1 mL of extract solution, 4.1 mL of 80%
ethanol, 0.1 mL of 10% Al(NO3)3 * 9 H2O, and 0.1 mL 1M solution
of CH3COOK. After 40 min of incubation at room temperature,
absorbance was measured at 415 nm. The same procedure
was repeated for standard (avonol quercetin) in order to
construct calibration curve. Concentration of avonoids in
samples was calculated from standard curve equation and
expressed as quercetin equivalents (mg QE/g dry extract).
DPPH assay
DPPH (2,2-dyphenyl-1-picrylhydrazyl) free radical
scavenging method (21) with slight modications was
performed. Stock solutions of extracts were mixed
with methanolic solution of DPPH (40 μg/mL) to adjust
concentrations of 10-100 μg/mL (v/v) of reaction mixture
(2000 μL). Methanol was used as a blank, while methanol
with DPPH solution was used as a control. Absorbance
of the reaction mixture was measured after 30 minutes in
the dark at room temperature at 517 nm. The decrease of
absorption of DPPH radical at 517 nm was calculated using
equation:
Inhibition of DPPH radical (%) = [(AC-AS)/AC] * 100%
where Ac-absorbance of control and As-absorbance of the
test samples at different concentrations. IC50 values (μg/mL)
were calculated from DPPH absorption curve at 517 nm.
ABTS assay
ABTS (2,2’-azino-bis-(3-ethylbenzothiazoline–6-sulfonic acid)
Table 1. Yield of extracts, total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activities of S. officinalis extracts.
Results are expressed as mean ± standard deviation; N is the number of samples
* Values within column are signicantly different (p<0.05)
a BHA (IC50 13.37 μg/ml) and BHT (IC50 17.94 μg/ml)
b BHA (2.82 mg AAE/g) and BHT (2.75 mg AAE/g)
c BHA (583.72 μmol Fe (II)/g) and BHT (445.34 μmol Fe (II)/g)
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Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016
obtained in this study are consistent with previous ndings on
S. ofcinalis, with high efciency of ethanolic extract (16,27,28).
The ethanol extracts showed very strong DPPH activity, close
to synthetic antioxidants BHA and BHT (13.37 and 17.94 μg/ml,
respectively).
The measured ABTS activity proved to be higher in the extracts
originating from the locality Pleš than those from Luštica (1.11 mg
AAE/g and 0.99 mg AAE/g, respectively). Stronger activity was
obtained in the summer season (1.18 mg AAE/g), comparing
to winter season (0.91 mg AAE/g). Among examined extracts,
ethanol extracts performed the strongest ABTS activity (1.77
mg AAE/g). In the ABTS assay (15), IC50 values of antioxidant
capacity showed a similar range of activities as well as in the
DPPH assay (12-95 mg/ml and 8-94 mg/ml, respectively).
FRAP activities of S. ofcinalis plant extracts originating from
the locality Pleš (613.81 μmol Fe(II)/g) was stronger than those
obtained for Luštica (593.79 μmol Fe(II)/g). Extracts showed
higher FRAP capacity in the summer season (662.22 μmol
Fe(II)/g). Ethanol extracts of plants showed the strongest activity
(1157.72 μmol Fe(II)/g). The results obtained in this study are
consistent with results of other authors who have measured the
FRAP activity of extracts of S. ofcinalis (29). It was reported on
the strongest activity of ethanol extract in the analysis of the
antioxidant activity of dichloromethane, ethyl acetate and
ethanol extracts of the aerial parts and roots of 14 Turkish Salvia
species, using a FRAP method (6). The extracts obtained using
higher polarity solvents were more effective radical scavengers
comparing to those obtained using less polar ones (16).
Previously reported data on antioxidant activity of S. ofcinalis
methanol extract tested using DPPH, ABTS and FRAP assays
showed that activity varied depending on collection site and
harvesting time (7, 8). Comparing the antioxidant activity in the
vegetative and fruiting stage of S. ofcinalis from two localities in
northern Tunisia, DPPH and ABTS assays showed stronger activity
in the vegetative, while FRAP assay showed stronger activity in
the fruiting stage (8). In the present study, differences in the DPPH
activities of extracts were caused by plant origin, harvesting
season and extraction solvent, while those in the ABTS and FRAP
assays could be attributed only to extraction solvent (Table
1). Similarly to phenolic and avonoid contents, antioxidant
activity of extracts varied depending on plant part, solvent and
techniques chosen for extraction as was indicated for other
Salvia species (6,12,18). Our results are in agreement with above-
mentioned data.
The correlation between antioxidant activity and total phenolic
and avonoid contents
Based on the values of correlation coefcients (r) shown in
Table 2, it can be concluded that the antioxidant capacities
of the extracts (measured using DPPH, ABTS and FRAP assays)
were strongly correlated to total phenol (r ranging from
±0.695 to ±0.975) and weakly to total avonoid content (r
from ± 0.065 to ± 0.255). The results indicated that the total
phenols are more responsible for the antioxidant activity than
total avonoids as it was previously reported (12). The strong
correlation between antioxidant activity and total phenolic
content in plant extracts was obtained in previous studies (4,
5, 8, 16).
Antioxidant tests were strongly correlated between each
other, especially FRAP and ABTS tests (r=0.962). According to
previous reports, it was established a statistically signicant
strong correlation (r ≥0.669) between DPPH and ABTS
antioxidant activity in all tested genera of Lamiaceae family,
whether they are examined as a group or separately (16).
RESULTS AND DISCUSSION
Extract yields, total phenolic and avonoid content
The yields of extracts were expressed in percentage (%) of the
dry weight extract compared to the initial mass of dry plant
material used for successive extraction (Table 1).
Yields of extracts varied depending on the origin, season and the
solvent used for extraction. The yields of plant extracts originating
from Pleš (2.50%) were higher than those originating from Luštica
(2.03%). The yields in the summer and winter season were very
similar (2.29 and 2.24%, respectively). The highest extract yield
was obtained by dichloromethane extract (3.23%), while the
ethyl acetate extract showed the lowest yield (1.47%). Extracts
yields were mostly inuenced by extraction solvent. Similar results
were obtained before using successive extraction (6). Yield
of methanolic extract of Salvia species varied depending on
collection locality and harvesting time (7, 8).
Results of measurements of total phenolic content (TPC) and
total avonoid content (TFC) are presented in the Table 1. The
total phenolic content was generally higher in the plant extracts
originating from Pleš (98.84 mg GAE/g) comparing to results
obtained for Luštica (96.36 mg GAE/g). Extracts of the summer
season showed higher phenolic content (110.26 mg GAE/g).
Type of extraction solvent affects the efciency of extraction of
phenols as indicated before (6). It was found that S. ofcinalis
from different collection sites showed TPC ranging from 207.48 to
251.73 mg GAE/g (7), with the highest yield in the fruiting stage
comparing to vegetative and owering ones (8).
The values of total avonoids content in the extracts were
measured as 31.75 mg QE/g for Pleš locality and 29.30 mg QE/g
for Luštica locality (Table 1). Higher avonoid content was found
in extracts from plants collected in summer (34.64 mg QE/g )
than in winter (26.41 mg QE/g), which is consistent with previous
reports (8). The highest content of total avonoids showed the
ethyl acetate extract (37.74 mg QE/g), while the lowest content
of total avonoids was obtained in ethanolic extracts (27.30 mg
QE/g), which is consistent to previous reports on Salvia species
successively obtained extracts (6). Several authors (3,6,7,18,26)
concluded that differences in results of yield of total phenolics
and avonoids could be caused by genetic and environmental
(climate, location, temperature, fertility, pests and diseases)
factors, the plant part used for extraction, time of sampling,
choice of the extraction solvent, extraction techniques, etc.
Considering the plants in our study were cultivated under
the same environmental conditions for ve years, statistically
signicant differences in total phenolic and avonoids contents
of extracts should be attributed only to harvesting season and
extraction solvent.
Evaluation of antioxidant activity
Antioxidant activity of extracts was evaluated using three
parallel test assays (DPPH, ABTS, and FRAP assay) and results are
presented in Table 1.
DPPH activity of S. ofcinalis extracts, originating from Pleš
and Luštica, was measured as 28.28 μg/ml and 48.62 μg/ml,
respectively. Summer season extracts were more successful
against DPPH radical, compared to extracts of winter season
(30.02 μg/ml and 46.89 μg/ml, respectively). Ethanol extracts
showed the strongest activity (16.93 μg/ml) while chloroform
extract was the weakest (57.35 μg/ml). It was reported that
successively obtained ethanol extracts of fourteen Salvia
species showed the strongest DPPH activity comparing to their
dichloromethane and ethyl acetate extracts (6). The results
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Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016
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CONCLUSIONS
Determined variations in phenolic contents and antioxidant
capacity of S. ofcinalis extracts revealed the association of the
plant origin, harvesting time and applied solvent. Comparing to
plant origin, extraction solvent and harvesting season showed
statistically higher impact on obtained differences in measured
parameters. Strong correlation coefcients between total
phenolics and the antioxidant tests were veried. Aerial parts
extracts of S. ofcinalis are proved to be valuable as an effective
and safe source of natural antioxidants, but observed variations
in yield and antioxidant properties could serve as basis for the
selection of plants with high level of polyphenolic compounds
and good antioxidant properties for future commercial
exploration in public health.
ACKNOWLEDGEMENTS
Authors are grateful to the Ministry of Education, Science and
Technological Development of Serbia for the nancial support
(Projects No. 173029 and 173030).
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Table 2. Linear correlation coefcients (r) between antioxidant activity and total
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According to literature data (24): ar≤0.35 weak correlation; b 0.36<r<0.67 moderate correlation; c
0.68<r<1 strong correlation
