Industrial Crops and Products 94 (2016) 240250

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Industrial Crops and Products

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Metabolite proling and antioxidative activity of Sage (Salvia fruticosa

Mill.) under the inuence of genotype and harvesting period
Eirini Sarrou a, , Stefan Martens b , Paschalina Chatzopoulou a
a
Hellenic Agricultural Organization DEMETER, Institute of Plant Breeding and Genetic Resourses- IPB&GR, Department of Medicinal and Aromatic Plants,
Thermi, 57001 Thessaloniki, Greece
b
Department of Food Quality and Nutrition Department, IASMA Research and Innovation Centre, Fondazione Edmund Mach (FEM), Via E. Mach 1, 38010
San Michele allAdige, (TN), Italy

a r t i c l e

i n f o

Article history:
Received 4 May 2016
Received in revised form 11 August 2016
Accepted 12 August 2016
Keywords:
Salvia fruticosa
Antioxidant activity
Essential oils
Harvest
Phenolics

a b s t r a c t
Two cultivated accessions of Salvia fruticosa Mill. were investigated and evaluated for their essential
oil, phenolic composition and antioxidant activity, during different harvesting time. The essential oil
and its major compound 1.8 cineole, presented their higher yields during the early summer harvesting.
The advanced analytical LCMS/MS method applied in this work led to the identication of thirty ve
compounds with rosmarinic acid, the diterpene artefact carnosol and several avones and avonols as
the main phenolic constituents, the concentration of which varied largely from spring to autumn. The
antioxidant activity of respective methanolic extracts was determined using the Ferric Reducing Ability
of Plasma (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (ABTS) assays, as a quality control tool. High positive correlations were observed between
FRAP and ABTS antioxidant activities and total phenolic/avonoid content, and particular phenolic constituents.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Salvia fruticosa Miller, Lamiaceae (syn. S. triloba L.) is an endemic
species of the east Mediterranean basin, occurring in Greece in
coastal areas of the mainland and in Ionian and Aegean islands,
commonly known as Greek, or Mediterranean wild sage (Karousou
et al., 1998; Skoula et al., 1999). Its common Greek name is
faskomilo or alisfakia and in the international trade it is known
as sage. In folk medicine aerial parts of the plant are used in
the form of herbal teas against cold symptoms as antiphlogistic of the mouth and throat, cough, abdominal pains etc. (Skoula
et al., 1999; Pitarokili et al., 2003). Furthermore, several studies
have shown that S. fruticosa essential oil and alcoholic or water
extracts exhibited pharmacological properties, such as antibac-

Abbreviations: DPPH, 2.2-diphenyl-1-picrylhydrazyl; ABTS, 2, 2-azinobis 3ethylbenzothiazoline 6-sulfonic acid; FRAP, Ferric Reducing Antioxidant Power;
GCMS, gas chromatography-mass spectrometry; TPTZ, 2,4,6- tripyridyl-s-triazine;
LCMS, liquid chromatography-mass spectrometry; UPLC, ultraperformance liquid chromatography; BA, benzoic acid; CA, caffeic acid; RA, rosmarinic acid;
Ap, apigenin; CHLA, chlorogenic acid; Qu3glc, querqetin 3-O-glucoside; Kmf3glc,
kaempferol 3-O-glucoside.
Corresponding author.
E-mail address: esarroy@gmail.com (E. Sarrou).
http://dx.doi.org/10.1016/j.indcrop.2016.08.022
0926-6690/ 2016 Elsevier B.V. All rights reserved.

terial, antimicrobial, antifungal, antioxidant, anti-cholinesterase,

antiproliferative etc. (Pitarokili et al., 2003; Fu et al., 2013; Hani and
Bayachou, 2014). Due to these properties, which are related to the
manifold presence of active compounds, originated from different
secondary metabolite pathways, S. fruticosa became an important
aromatic/medicinal and commercial species.
1.8 cineole, camphor, - and -pinene and borneol are the major
compounds of S. fruticosa leaf essential oil, which was shown to
have a high variability in its chemical composition and is responsible for the characteristic aroma and avor (Catsiotis and Iconomou,
1984; Skoula et al., 1999). Rosmarinic acid, carnosic acid, carnosol
and methyl carnosate are the major phenolic compounds and effective antioxidants detected in S. fruticosa and in the close relative
and well known herbal medicine S. ofcinalis (Pizzale et al., 2002).
Skoula et al. (2000) reported that the total phenolic content and particularly those of rosmarinic acid varied signicantly within three S.
fruticosa populations from Crete. Previous studies have shown that
the essential oil and polyphenolic composition is largely affected
by the origin, environmental and growing conditions, the ontogenetic stage of the plant, the season of harvesting, genetic factors
and others (Figueiredo et al., 2008; Cheynier et al., 2013). Similarly,
the amounts of secondary metabolites, responsible for the antioxidant activity of S. fruticosa, are beside others associated to the plant
growth stage and the harvesting time (Papageorgiou et al., 2008).

E. Sarrou et al. / Industrial Crops and Products 94 (2016) 240250

The antioxidant properties of plant extracts or even specic

compounds are very important, since they may cope with the
harmful role of free radicals, the formation of which accelerate the
oxidation of lipids in biological systems and foods, and decrease
food quality and the consumers acceptance. Provided that the
bioactivity and the pharmacological properties of medicinal plants
are related to the presence and the content of specic constituents,
which are affected by intrinsic and external parameters, the standardization of the starting plant material is essential, since it is
the most important factor in manufacturing herbal products (Garg
et al., 2012). Cultivation, instead of collecting wild grown plants and
the determination of the optimum harvesting season, which ensure
high yields of the desired constituents, might contribute to obtain
uniform plant material of constant high quality, and in accordance
to different markets demands and specication.
The purpose of the present study was to investigate two cultivated S. fruticosa populations in the same pedo-climatic conditions
and acquire comprehensive data of compounds present in the
essential oil and their phenolic extracts. Additionally, we aim to
assess the seasonal uctuation of these compounds and the effect
on antioxidant activity during the whole vegetative period. The
combination of efcient genotype, well adapted to cultivation conditions and the determination of the proper harvesting period,
by using state of the art analytical tools, will pave the way standardized, high quality plant material, for multiple uses in food,
phytoherapy, cosmetic industry, etc.

2. Materials and methods

241

2.3. Essential oil isolation

The essential oil content was determined using the European
Pharmacopoeia apparatus (Clevenger-type). Fifty grams of S. fruticosa dried leaves were subjected to hydrodistillation for 2.30 h,
with a distillation rate 33.5 mL/min. Three distillations for each
harvesting sample were done. The oil content was estimated on the
basis of dry weight plant material (mL/100 g of dried leaves). The
essential oils obtained were dried over anhydrous sodium sulphate
and stored at 46 C.
2.4. Analysis of essential oil
The essential oils were analyzed by GCMS on a fused silica DB5 column, using a Gas chromatograph 17A Ver. 3 interfaced with a
mass spectrometer Shimadzu QP-5050A supported by the GC/MS
Solution Ver1.21 software, using the method described previously
(Sarrou et al., 2013). The conditions of analysis were: injection
temperature: 260 C, interface heating: 300 C, ion source heating: 200 C, EI mode: 70 eV, scan range: 41450 amu, and scan
time 0.50 s. Oven temperature programs: (a) 55120 C (3 C/min),
120200 C (4 C/min), 200220 C (6 C/min) and 220 C for 5 min
and (b) 60240 C at 3 C/min, carrier gas He, 54.8 kPa, split ratio
1:30. The relative content of each compound was calculated as percent of the total chromatographic area and the results are expressed
as means of three replicates.
The identication of the compounds was based on comparison
of their retention indices (RI) relative to n-alkanes (C7 -C22 ), with
corresponding literature data and by matching their spectra with
those of MS libraries (NIST 98, Willey) (Adams, 1995).

2.1. Plant material and harvesting season

2.5. Sample preparation and extraction of phenolic compounds
Leaves from upper parts of 3 years old plants, from two cultivated populations of S. fruticosa populations, were collected in
different phenophases, from April to October in 2014. Each sample
was constituted from 10 plants, per accession. Germplasm from
the initial populations was originated from different districts of
Greece, (1) from S. Aegean islands and (2) from W. Peloponnese.
The species was authenticated as S. fruticosa (syn. S. triloba) and
voucher specimens were deposited at the Herbarium of the Department of Medicinal and Aromatic Plants of IPB&GR. The populations
were cultivated at the experimental eld of IPB&GR (40 34 35 N
22 57 19 E), and the plants were equally and in regular basis drip
irrigated and hand weeded as well, during cultivation period. The
soil properties at the experimental site were as follows: soil type:
red loam, pH 7.73%, clay: 39.0, organic matter: 1.43%, P2 O5 (ppm):
45, K2 O (ppm): 520. The climate at the cultivation region was characterized from mild winter, warm spring and summer with high
relative humidity. The fresh plant material was dried in a shady
and dry place at ambient temperature for fteen days and then
leaves were separated from the stem. Only leaves were used for the
essential oil isolation and the extraction of phenolic compounds.

2.2. Chemicals
The n-alkanes (C7-C22) were purchased from Supelco (Bellefonte, PA, USA). The highest purity pentane used for the GCMS
was purchased from Panreac Quimica S.L.U (Barcelona, Spain). All
reagents for LCMS analysis (with LCMS grade) and assays for the
determination of antioxidant activity were purchased from SigmaAldrich (Steinheim, Germany). Water used in sample preparation
and analysis was puried by a Milli-Q water purication system.
The specic standards carnosol and carnosic acid were purchased
from TransMIT PlantMetaChem (Giessen, Germany) and inserted
in the analytical method as described below.

The samples were randomized and a part from each leaf sample was milled to a ne powder with a mill equipped with cooling
system. 100 mg of the above sample (powdered leaf tissue) was
weighed and transferred into 15 mL falcon tube. A volume of 4 mL
80% methanol was added to each sample. The samples and solvent were mixed by orbital shaker for 3 h at room temperature and
the extraction proceeded overnight at 4 C in the dark. The resulting solutions where ltered on a 0.22 m PFTE membrane into a
glass vial and analyzed as described below. Three replicates for each
harvesting sample were done.
2.6. Phenolic metabolite analysis
The analysis of phenolic compounds was performed using the
method described previously by Vrhovsek et al. (2012). Samples
were directly injected after extraction.
Ultraperformance liquid chromatography was performed on a
Waters Acquity UPLC system (Milford, MA) consisting of a binary
pump, an online vacuum degasser, an autosampler, and a column
compartment. Separation of the phenolic compounds was achieved
on a Waters Acquity HSS T3 column 1.8 m, 100 mm 2.1 mm (Milford, MA, USA), kept at 40 C. Mobile phase A was water containing
0.1% formic acid; mobile phase B was acetonitrile containing 0.1%
formic acid. The ow was 0.4 mL/min, and the gradient prole was
0 min, 5% B; from 0 to 3 min, linear gradient to 20% B; from 3 to
4.3 min, isocratic 20% B; from 4.3 to 9 min, linear gradient to 45%
B; from 9 to 11 min, linear gradient to 100% B; from 11 to 13 min,
wash at 100% B; from 13.01 to 15 min, back to the initial conditions
of 5% B. The injection volume of both the standard solutions and
the samples was 2 L. After each injection, the needle was rinsed
with 600 L of weak wash solution (water/methanol, 90:10) and
200 L of strong wash solution (methanol/water, 90:10). Samples
were kept at 6 C during the analysis.

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E. Sarrou et al. / Industrial Crops and Products 94 (2016) 240250

Mass spectrometry detection was performed on a Waters Xevo

TQMS (Milford, MA, USA) instrument equipped with an electrospray (ESI) source. Capillary voltage was 3.5 kV in positive mode and
2.5 kV in negative mode; the source was kept at 150 C; desolvation temperature was 500 C; cone gas ow, 50 L/h; and desolvation
gas ow, 800 L/h. Unit resolution was applied to each quadrupole.
Flow injections of each individual metabolite were used to optimize
the MRM conditions. For the majority of the metabolites, this was
done automatically by the Waters Intellistart software, whereas for
some compounds the optimal cone voltages and collision energies
were identied during collision-induced dissociation (CID) experiments and manually set. A dwell time of at least 25 ms was applied
to each MRM transition. Data processing was performed using the
Mass Lynx Target Lynx Application Manager (Waters).
2.7. Total phenol and avonoid contect
Total phenol content of methanolic extracts was determined
with Folin-Ciocalteu reagent using gallic acid as a standard (Scalbert
et al., 1989). Total phenolic content was expressed in mg gallic acid/g of leaf dry weight (DW). Total avonoid content was
determined colorimetrically as described by Zhisen (1999). Rutin
was used as the standard compound for quantication of total
avonoids and values were expressed in mg of rutin/g of leaf dry
weight (DW). All measurements were repeated three times.
2.8. FRAP antioxidant power assay
The Ferric Reducing Antioxidant Power was determined using
a freshly prepared solution (0.3 M acetate buffer, pH 3.6), 10 mM
TPTZ, 20 mM FeCl3* 6H2 O and 0.05 mL of methanolic extract, as
previously described by Benzie and Strain (1996).
2.9. DPPH radical scavenging activity assay
Radical scavenging activity of essential oils and methanolic
extracts was determined following the method described by Su and
Silva (2006), by preparing a 0.1 mM solution of (DPPH) in absolute
ethanol.
2.10. ABTS radical scavenging activity assay
Scavenging activity of the methanolic extracts was determined
using ABTS (7 mM) reacting with K2 S2 O8 (2.45 mM) to a water
solution as described by Re et al. (1999). All samples were measured in triplicate and the antioxidant activity was expressed in
M Trolox/g DW.
2.11. Statistical analysis
All samples were analyzed in triplicate and the results are
expressed as the means. The data were analyzed with Analysis
of Variance (ANOVA), using the statistical package SPSS 11 17.0
(SPSS Inc. Chicago, Illinois, USA). For mean comparison, the Duncans multiple range tests and standard error (S.E) were used at
p 0.05 to establish signicant differences. Correlation coefcients
to determine the relationship between variables were calculated
using Pearson Product Moment.
3. Results and discussion
3.1. Essential oil analysis
The variation in essential oil yield and composition of both
S. fruticosa populations over the time of harvesting is presented

in Table 1. The qualitative prole of two populations was similar, while signicant differences were observed concerning the
quantitative composition under the different time of harvesting.
An increase in the essential oil content from spring to summer (AprilAugust) was observed with a decrease in autumn
(SeptemberOctober). For all the harvest points (except from April),
the essential oil content of population 1 was higher (difference of
0.673%) compared to population 2.
Twenty eight and twenty seven constituents (linalyl acetate
was not detected in population 2) were identied in the essential oils of the two populations, accounting for 95.9899.10%
and 95.8498.55% of the total oil, respectively. The major compounds from all harvesting points were monoterpenes 1.8-cineole
(45.4158.41% and 44.7050.84% respectively) and -pinene
(7.4114.07% and 5.0911.51%) and in less amount camphor
(1.324.37% and 4.2614.93%). Additionally, -pinene, camphene,
myrcene, - and -thujone, - and -terpineol, terpinyl acetate
and -caryophyllene were also detected (>1%), in both accessions. These data are in agreement with previous studies, where
similar essential oil composition was reported for various S. fruticosa populations collected from the wild (Mller-Riebau et al.,
1997; Skoula et al., 1999; Papageorgiou et al., 2008; Topcu et al.,
2013). In contrast, several authors reported S. fruticosa essential oil with major compounds -thujone, camphor, borneol and
-caryophyllene, indicating the existence of more distinct chemotypes within the species (Delamare et al., 2007; Pierozan et al.,
2009; Koliopoulos et al., 2010). It was furthermore observed, that
monoterpene hydrocarbons, with major constituents -pinene,
-pinene and myrcene, were decreased from spring to summer
and increased later in autumn. The same trend was also observed
for sesquiterpene hydrocarbons, with -caryophyllene being the
most abundant in both populations (1.777.17% and 1.396.54%
respectively) and the oxygenated sesquiterpenes viridiorol and
manool accounting to 0.392.41% and 0.783.99% for populations
1 and 2, respectively. On the contrary, the content of oxygenated
monoterpenes with predominant compound 1.8-cineole, and in
lower amounts camphor, -terpineol and - and -thujone, was
augmented from spring to summer, accumulating in higher concentration in the period from June to August and diminishing
from summer to autumn. More specically, - and -thujones,
presented their maximum concentration in autumn harvesting.
Similar changes on the oxygenated monoterpenes, sesquiterpene
hydrocarbons and oxygenated sesquiterpenes were also observed
on cultivated S. ofcinalis plants (Verma et al., 2015). In addition, in
the same study it was shown that under the effect of rainy harvest
period the amounts of these groups decreased, indicating a possible
positive role of water stress on the essential oil yield and composition. Furthermore, Papageorgiou et al. (2008) observed an increase
in total essential oil yield and of thujones and -caryophyllene
in particular, in wild collected S. fruticosa leaves, from winter to
summer.
It is known that the biosynthesis of terpene volatiles is a complex process, as some of them are direct products of various terpene
synthases, while others are formed through further transformation of the initial products by oxidation, dehydrogenation, acylation
and other reactions (Croteau et al., 2000; Dudareva et al., 2004;
Pichersky et al., 2006). Moreover, their concentration may depend
on CO2 levels and the metabolic intermediates formed during the
process of photosynthesis (Loreto et al., 1996), though the productivity of plant tissues have a core role in carbon utilizing for
the essential oil anabolism (Sangwan et al., 2001). The ontogenetic
stage of leaves might inuences the glandular trichome density
(more than one type may occur on a single leaf) and the expression of essential oil metabolism, as the developmental stage of
the tissue-leaves is associated to the primary biochemical activities occurring during the essential oil synthesis. For example, the

E. Sarrou et al. / Industrial Crops and Products 94 (2016) 240250

243

Table 1
Essential oil composition and yield of population 1 and 2 of S. fruticosa under the inuence of harvest period.
% Composition4
Nr.

Compound

Monoterpene hydrocarbons
thujene
1

RIL

RRI

April

May

June

July

August

September

October

931

928

0.34 b
0.56 b
3.63 cd
4.82 bc
0.57 d
1.99 c
0.21 c
0.39 d
14.07 a
11.51 a
5.56 a
3.55 bc
0.31 c
0.47 bc
0.35 c
0.64 c
0.98 b
0.93 bc
0.18 de
0.16 c
0.14 d
0.20 e
26.34
25.22

0.30 c
0.44 c
3.86 bcd
4.73 c
1.17 c
4.20 b
0.11 d
0.25 e
9.29 bc
7.06 b
4.37 c
4.78 a
0.35 bc
0.45 c
0.21 e
0.13 e
0.96 b
0.87 c
0.16 ef
0.21 b
0.21 c
0.34 b
20.99
23.46

0.28 c
0.30 d
4.16 ab
5.23 b
1.39 b
4.18 b
0.07 e
0.11 g
7.46 d
5.90 de
3.08 d
3.59 b
0.38 b
0.48 bc
0.33 cd
0.46 d
0.84b
0.74 d
0.13 f
0.12 c
0.23 bc
0.32 bc
18.35
21.43

0.30 c
0.32 d
3.97 bc
5.91 a
1.38 b
5.90 a
0.11 d
0.14 f
7.41 d
6.06 cd
3.03 de
3.38 bc
0.32 bc
0.47 bc
0.52 a
0.59 cd
0.79 b
0.66 e
0.45 a
0.07 d
0.20 c
0.28 d
17.04
23.78

0.29 c
0.61 b
3.47 d
4.60 c
1.39 b
5.22 a
0.20 c
0.67 c
7.46 d
6.02 cde
2.66 f
3.07 c
0.34 bc
0.55 b
0.29 d
0.87 b
0.91 b
1.01 b
0.21 d
0.05 d
0.23 b
0.29 cd
17.45
22.96

0.35 b
0.60 b
4.08 bc
3.91 d
1.61 a
4.05 b
0.36 b
0.82 b
8.72 c
5.78 e
2.97 e
3.08 c
0.42 a
0.47 bc
0.55 a
1.10 a
1.23 a
0.95 b
0.29 c
0.50 a
0.29 a
0.27 d
20.87
21.53

0.48 a
1.06 a
4.53 a
3.55 a
1.59 a
2.39 c
0.48 a
1.07 a
10.23 b
6.22 c
5.44 b
3.29 bc
0.42 a
1.07 a
0.42 b
0.93 b
1.21 a
1.87 a
0.33 b
0.14 c
0.25 b
0.50 a
25.38
22.52

51.75 c
46.16 bcd
0.26 c
0.25 d
0.78 d
1.12 d
1.10 d
0.86 d
1.32 f
4.26 e
0.10 e
0.69 a
0.91 c
0.84 d
0.54 d
1.07 c
3.44 c
2.69 c
nd
nd
nd
nd
0.10 e
0.48 e
60.3
58.42

56.07 ab
47.51 bc
0.52 c
0.24 d
0.95 d
0.56 e
1.43 d
0.91 cd
3.92 c
11.91 c
0.17 d
0.43 b
1.22 b
0.91 cd
0.65 c
0.78 d
4.92 ab
2.81 bc
0.00 e
nd
0.21 a
0.29 b
0.81 d
0.66de
70.87
67.01

58.41 a
50.84 a
1.35 b
0.48c
1.68 c
1.00 d
2.09 c
1.11 bc
4.22 b
12.62 c
0.22 c
0.39 b
1.27 ab
1.09 a
0.67 bc
0.78 d
5.11 ab
3.46 a
0.21 d
nd
0.21 a
0.32 b
2.00 c
0.76 d
77.44
72.85

53.22 bc
45.45 cd
1.41 b
0.41 c
2.35 b
0.90 d
2.79 b
1.06 cd
4.08 b
14.93 a
0.30 b
0.64 a
1.36 a
0.97 bc
0.51 d
0.88 d
5.21 a
2.83 bc
0.00 e
nd
0.21 a
0.38 a
3.29 b
1.26 c
74.73
69.71

55.74 ab
47.80 bc
1.45 b
0.72 b
2.29 b
1.76 c
2.70 b
1.30 b
4.37 a
13.70 b
0.34 a
0.71 a
1.21 b
0.86 d
0.75 b
1.62 b
4.58 b
2.15 e
0.51 c
nd
0.18 b
0.20 c
3.50 b
1.56 b
77.62
72.38

47.31 d
44.70 d
2.25 a
0.51 c
2.84 a
2.31 b
3.20 a
1.60 a
3.77 d
12.02 c
0.33 a
0.67 a
1.27 ab
1.00 b
0.84 a
1.63 b
5.09 ab
2.40 d
1.00 a
nd
0.13 c
0.13 d
5.05 a
2.05 a
73.08
69.02

45.41 d
48.49 ab
1.64 b
1.42 b
2.31 b
3.13 a
2.31 c
1.70 a
2.69 e
8.06 d
0.31 b
0.27 c
1.22 b
1.13 a
0.59 cd
2.18 a
4.83 ab
2.91 b
0.89 b
nd
0.07 d
0.18 cd
3.34 b
2.19 a
65.61
71.66

7.17 a
6.54 a
1.04 a
0.45 a
0.58 a
1.29 a
8.79
8.28

4.95 b
5.14 b
0.86 b
0.42 a
0.28 c
0.38 d
6.09
5.94

1.85 e
2.19 c
0.45 d
0.20 b
0.30 c
0.41 d
2.6
2.8

2.65 d
2.19 c
0.76 b
0.21 b
0.41 b
0.74 bc
3.82
3.14

1.77 e
1.39 d
0.58 cb
0.21 b
0.30 c
0.83 b
2.65
2.43

2.25 de
2.25 c
0.72 bc
0.47 a
0.35 bc
1.10 a
3.32
3.82

3.80 c
1.01 d
1.09 a
0.34 ab
0.35 bc
0.53 cd
5.24
4.03

2.05 a
2.74 a
0.36 a
1.25 a
2.41
3.99
98.84
95.84
1.55 d
1.85 d

0.79 b
1.08 b
0.36 a
0.51 bc
1.15
1.59
99.1
98
4.35 b
3.15 b

0.31 c
0.73 cd
0.12 b
0.43 bc
0.43
1.16
98.82
98.24
4.25 bc
3.58 a

0.37 c
0.73 cd
0.02 c
0.54 bc
0.39
1.27
95.98
97.9
4.60 b
3.30 ab

0.26 c
0.46 d
0.20 b
0.32 c
0.46
0.78
98.18
98.55
4.60 b
3.57 a

0.38 c
0.87 bc
0.20 b
0.68 b
0.58
1.55
97.85
95.92
5.60 a
2.60 c

0.79 b
1.09 cd
0.40 a
0.82 c
1.19
1.9
97.42
96.95
3.75 c
1.60 d

-pinene

939

935

camphene

953

949

sabinene

976

974

-pinene

980

978

myrcene

991

992

-terpinene

1018

1017

p-cymene

1026

1026

-terpinene

1062

1060

10

cis sabinene hydrate

1068

1065

11

-terpinolene

1088

1087

Oxygenated monoterpenes
1.8-cineole
12

1033

1035

13

linalool

1098

1099

14

-thujone

1102

1103

15

-thujone

1114

1115

16

camphor

1143

1142

17

borneol

1165

1163

18

-terpineol

1163

1165

19

terpinen-4-ol

1177

1174

20

-terpineol

1189

1188

21

linalyl acetate

1257

1259

22

bornyl acetate

1285

1280

23

terpinyl acetate

1350

1348

1418

1407

Total

Total
Sesquiterpenes
-caryophyllene
24
25

-humulene

1454

1442

26

caryophyllene oxide

1581

1572

1590

1580

2056

2066

Total
Sesquiterpene alcohol
viridiorol
27
28

manool

Total
Total identied compounds
Essential oil yield5

All data represent the mean values of three independent replicates; Values with the same lowercase letter within columns are statistically different (p 0.05) between the
harvesting periods.
1
order of elution on DB-5 column.
2
RIL: Retention Index Literature.
3
RIL: Relative Retention Index Calculated relative to C7-C22 n-alcanes, on DB-5 column.
4
Percentage of the total peak area.
5
v/w%, on dry plant basis; Components with percentage 0.1% are presented; nd: not detected.

244

E. Sarrou et al. / Industrial Crops and Products 94 (2016) 240250

Table 2
Phenolic content and composition of population 1 and 2 of S. fruticosa methanolic extracts under the inuence of harvest period.
Compounds

Benzoic acid derivatives

p-Hydroxybenzoic acid

2, 6-dihydroxybenzoic acid

3,4-Dihydroxybenzoic acid

3,5-Dimethoxy-4-hydroxybenzaldehyde

Phenolic acids
caffeic acid

ferulic acid

vanillic acid

acetovanillone

coniferyl alcohol

10

Caffeic acid derivatives

rosmarinic acid

11

chlorogenic acid

12

neochlorogenic acid

13

cryptochlorogenic acid

14

Dihydrochalcones
Phloridzin

15

Trilobatin

16

Sieboldin

17

Flavanones
Naringenin

18

Hesperidin

19

Dihydrokaempferol

20

Dihydroquercetin

21

Flavones
Apigenin

22

Apigenin-7-glc

23

Apiin

24

Luteolin

25

Luteolin-7-O-Glc

26

Flavonols
Kaempferol

27

Kaempferol-3-Glc

28

Quercetin

29

Quercetin-3-Glc + Quercetin-3-gal (as que-3-glc)

30

Quercetin-3-Rha

31

Quercetin-3,4-diglucoside

32

Isorhamnetin

33

Isorhamnetin-3-Glc

Concentration mg/g DW
April

May

June

July

August

September

October

0.019 bc
0.015 bc
0.003
0.004
0.051 a
0.006 e
0.003
0.003

0.024 a
0.023 a
0.004
0.009
0.033 a
0.011 de
0.003
0.002

0.021 ab
0.014 bc
0.002
0.005
0.045 a
0.024 b
0.003
0.002

0.019 bc
0.017 b
0.003
0.006
0.033 a
0.020 bc
0.002
0.002

0.016 cd
0.012 c
0.002
0.004
0.030 a
0.016 cd
0.003
0.002

0.015 cd
0.011 c
0.002
0.003
0.051 a
0.023 bc
0.002
0.004

0.014 d
0.017 b
0.001
0.003
0.044 a
0.033 a
0.002
0.004

0.698 a
0.529 a
0.002
0.001
0.005
0.004
0.001
0
0.017
0.02

0.568 b
0.541 a
0.007
0.003
0.012
0.005
0.002
0.001
0.009
0.007

0.593 b
0.537 a
0.006
0.038
0.006
0.002
0.002
0.001
0.004
nd

0.487 d
0.589 a
0.008
0.047
0.007
0.006
0.002
0.001
0.004
nd

0.485 d
0.426 b
0.005
nd
0.006
0.003
0.002
0.001
0.003
nd

0.487 d
0.437 b
0.005
nd
0.004
0.002
0.002
0.001
0.003
nd

0.522 c
0.462 b
0.005
0.005
0.003
0.002
0.001
nd
0.004
nd

41.899 b
60.727 a
1.818 a
0.037 b
0.017 d
0.001 c
0.057 a
0.001

45.055 a
52.416 b
1.663 b
0.015 cd
0.022 c
0.001 c
0.045 b
0.001

29.503 d
38.000 d
0.700 f
0.182 a
0.017 d
0.007 a
0.024 de
0.009

34.182 c
54.101 b
0.881 c
0.009 d
0.028 a
0.001 c
0.035 c
0.002

35.354 c
48.308 c
0.459 g
0.038 b
0.025 b
0.003 b
0.026 d
0.003

26.355 e
30.912 e
0.843 d
0.016 c
0.014 e
0.001 c
0.021 f
0.001

5.567 f
22.781 f
0.776 e
0.33 b
0.007 f
0.001 c
0.013 g
0.002

0.021
0.009 ab
0.004
0.002
0.001
0.001

0.016
0.008 b
0.003
0.002
n.d
n.d

0.014
0.012 b
0.003
0.002
n.d
0.001

0.014
0.008 a
0.003
0.002
n.d
n.d

0.012
0.007 b
0.003
0.001
n.d
n.d

0.016
0.007 b
0.002
0.002
n.d
0.001

0.019
0.006 b
0.3003
0.001
0.001
0.001

0.022 bc
0.011 b
0.269 a
0.349 a
0
0.002
0.001
0.006

0.038 a
0.014 a
0.922 c
0.146 b
0.004
0.001
0.007
0.005

0.024 b
0.006 e
0.464 d
0.077 e
0.007
n.d
0.01
0.001

0.019 d
0.007 de
0.307 e
0.095 d
0.007
n.d
0.009
0.003

0.020 cd
0.009 cd
0.023 e
0.045 f
0.011
n.d
0.016
0.001

0.021 cd
0.009 c
0.087 c
0.044 f
0.011
0.002
0.016
0.004

0.019 d
0.011 b
0.130 b
0.133 c
0.005
0.004
0.007
0.009

0.053 e
0.027 e
0.080 c
0.262 a
0.018 e
0.050 bc
0.121 c
0.174 b
0.068 a
0.249 a

0.142 a
0.085 b
0.084 b
0.101 d
0.039 d
0.015 e
0.214 a
0.168 b
0.052 abc
0.043 d

0.132 ab
0.084 b
0.103a
0.150 b
0.058 b
0.049 c
0.146 b
0.104 e
0.052 abc
0.063 c

0.111 c
0.075 c
0.060 d
0.138 c
0.060 ab
0.054 b
0.152 b
0.139 c
0.045 bc
0.084 b

0.115 bc
0.095 a
0.076 c
0.135 c
0.063 a
0.067 a
0.143 b
0.186 a
0.036 c
0.085 b

0.087 d
0.065 d
0.080 c
0.092 e
0.060 ab
0.046 cd
0.128 c
0.128 d
0.058 ab
0.065 bc

0.038 e
0.060 d
0.060 d
0.084 f
0.045 c
0.042 d
0.104 d
0.131 d
0.054 ab
0.079 bc

0.003 d
0.004 c
0.034 e
0.014 d
0.004 e
0.007 bc
0.025 d
0.031 f
0.002
0.001
0.028 ab
0.027 a
n.d
0.001
n.d
0.010 b

0.025 c
0.006 b
0.178 b
0.069 a
0.022 d
0.004 cd
0.139 c
0.067 b
0.002
0.001
0.023 c
0.015 b
0.002
0.001
0.031 e
0.020 a

0.042 b
0.004 c
0.223 a
0.041 b
0.060 c
0.004 cd
0.251 b
0.052 c
0.002
0.002
0.009 cd
0.005 c
0.006
0.001
0.047 d
nd

0.049 a
0.006 b
0.183 b
0.042 b
0.079 b
0.009 b
0.251 b
0.074 a
0.001
0.001
0.033 a
0.004 c
0.008
0.001
0.065 a
nd

0.054 a
0.004 c
0.179 b
0.017 c
0.078 b
0.003 d
0.283 a
0.040 d
0.001
0.001
0.012 c
0.004 c
0.008
0.001
0.058 b
nd

0.052 a
0.004 c
0.130 c
0.017 c
0.101 a
0.003 d
0.280 a
0.036 e
0.003
0.001
0.011 c
0.006 c
0.012
0.001
0.054 c
nd

0.021 c
0.008 a
0.042 d
0.15 d
0.087 ab
0.022 a
0.149 c
0.032 f
0.002
0.001
0.004 d
0.004 c
0.007
0.002
0.024 f
nd

E. Sarrou et al. / Industrial Crops and Products 94 (2016) 240250

245

Table 2 (Continued)
Compounds

Concentration mg/g DW
April

May

June

July

August

September

October

34

Arbutin

35

Carnosol

n.d
0.001
5.241 c
5.175 c

0.002
0.001
6.753 a
5.982 a

0.002
0.001
7.048 b
5.509 c

0.002
0.001
6.458 b
5.689 c

0.001
0.001
6.355 b
5.567 c

0.001
0.001
6.142 d
4.945 c

n.d
0
5.960 b
5.537 c

36

Coumarins
daphnetin

37

esculin

0.016
0.019
0.005
0.014
50.505
67.794
51.050 c
79.850 a
200.760 c
303.510 b

0.015
0.012
0.005
0.007
56.165
59.808
77.450 a
76.620 a
298.610 a
346.350 a

0.014
0.006
0.003
0.004
39.646
44.999
66.540 b
64.580 b
250.740 b
283.910 b

0.014
0.007
0.004
0.004
43.625
61.244
66.770 b
81.000 a
278.170 ab
153.170 c

0.015
0.009
0.004
0.005
43.982
55.099
64.340 b
78.460 a
278.470 ab
143.790 c

0.012
0.01
0.002
0.005
35.17
36.905
49.550 c
56.510 d
184.950 cd
112.940 cd

0.006
0.01
0.001
0.004
14.043
29.971
41.340 d
57.730 c
150.230 d
84.020 d

Total identied (mg/g DW)

Total phenol content (mg GA/g DW)
Total avonoid content (mg RUT/g DW)

All data represent the mean values of three independent replicates; Values with the same lowercase letter within columns are statistically different (p 0.05) between the
harvesting periods.
n.d: not detected.
Table 3
Correlation coefcient between harvest time and phenolic compounds content of methanolic extracts from two populations of S. fruticosa.

Harvest time

1
2
3
4
5
6
7
a
b

Population 1
signicance
Population 2
signicance

CA1

RA2

CHLA3

LU4

Kmf3glc5

Qu3glc6

CAR7

Total phenols

Total avonoids

0.770a
0.000
0.584a
0.005

0.828a
0.000
0.818a
0.000

0.753a
0.000
0.196
0.394

0.491b
0.024
0.328
0.147

0.120
0.603
0.462b
0.035

0.550a
0.010
0.321
0.378

0.031
0.894
0.203
0.378

0.541b
0.013
0.639a
0.000

0.454b
0.039
0.753a
0.000

CA: caffeic acid.

RA: rosmarinic acid.
CHLA: chlorogenic acid.
LU: luteolin.
Km3glc: kaempferol 3-glucoside.
Qu3glc: quercetin-3-glucoside.
CAR: carnosol.
correlation is signicant at 0.01 level.
correlation is signicant at 0.05 level.

accumulation of borneol and camphor happen more rapidly in

expanding leaves of S. ofcinalis than in mature ones (Croteau et al.,
1981), while new trichomes continue to initiate and develop during
the leaf ontogeny. As trichomes over-mature, subcellular integrity
deteriorates and cellular constituents slowly disappear leaving the
secretory cell empty (Shanker et al., 1999). As previously suggested,
such changes in the composition of the volatiles with the maturation of the organs are directly related to higher rates of cyclization

et al., 1991). In genand dehydration of the compounds (Mnez

eral, plant ontogenetic stage, sage leaf anatomy and physiology
characteristics, in combination to climatic conditions (temperature, humidity and day length) and possible pathogens attack, i.e
fungus, particularly in the months of rainfall, could be the main reason for the volatile metabolite variation (Figueiredo et al., 2008).
In addition, the two populations studied in this work, are originated from regions of Aegean island (population 1) and mainland
(population 2), which are characterized from different environmental conditions (temperature, rainfall, soil characteristics), with
co-existence of biotic and abiotic stress-pressures. Thus the plants
might also adapt to the different environments and present differences in their metabolic proles, indicating their genotype effect.
Several oxygenated compounds of the essential oil, i.e terpineol,
borneol, camphor, and especially the bornyl acetate, linalyl acetate
and terpinyl acetate, even identied in minor amounts in sage oil
contribute highly to the organoleptic characteristics and the essential oils avor, enhancing their commercial value (Sangwan et al.,
2001; Daz-Maroto et al., 2005; Qiao et al., 2008). Although, aand -thujone, are present in S. fruticosa in less amounts than in
S. ofcinalis, previous reports indicate high variation and amounts

Table 4
Correlation coefcient between harvest time and antioxidant activities (FRAP, DPPH,
ABTS) of the methanolic extracts from two populations of S. fruticosa.
FRAP
Harvest time

a
b

Population 1
signicance
Population 2
signicance

0.545
0.011
0.766a
0.000

DPPH

ABTS

0.256
0.262
0.256
0.663

0.421
0.057
0.480b
0.028

correlation is signicant at 0.01 level.

correlation is signicant at 0.05 level.

(>10%) of these constituents, in several populations collected from

the wild, or cultivated (Delamare et al., 2007; Pierozan et al., 2009;
Koliopoulos et al., 2010; Mth et al., 2010). Considering that these
substances have been regarded as severe neurotoxicants, it became
apparent the risk of use and/or consumption of such sage chemotypes in herbal-based products and poses therefore considerable
uncertainties and limitations (EMA, 2009; EMA, 2010). However,
the populations of S. fruticosa investigated in this report, exhibited
rather low amounts of -thujone (0.782.84% and 0.563.13%) and
-thujone (1.13.2% and 0.861.7%) respectively, in all harvesting
stages and may substitute commercial sage (S. ofcinalis), as safer
for use in the pharmaceutical and food industry as well.
3.2. Phenolic analysis
A targeted UPLCMS/MS method was used for the qualitative and quantitative analysis of various phenolic compounds and
their seasonal variation in leaf extracts of the studied accessions

246

E. Sarrou et al. / Industrial Crops and Products 94 (2016) 240250

(Table 2). Thirty seven compounds were identied in the methanolic extracts of S. fruticosa. In previous study nine phenolics have
been reported by Papageorgiou et al. (2008), while Cvetkovikj
et al. (2013) measured up to 53 compounds in different Salvia
species, however distinct identication is only given for twenty
six derivatives, mainly avones but not any avonols. The metabolites of S. fruticosa identied in this study could be classied
in 9 chemical groups: benzoic acid derivatives, coumarins, phenolic acid derivatives, caffeic acid derivatives, dihydrochalcones,
avanones, avones, avonols and diterpenes. In all samples, rosmarinic acid and carnosol were the most abundant compounds,
followed by chlorogenic acid (CHLA), caffeic acid (CA), the avones
apigenin (Ap), Ap 7-O-glucoside (Ap7glc), luteolin (Lu) and Lu
7-O-glucoside (Lu7glc), the avonols kaempferol 3-O-glucoside
(Km3glc), quercetin (Qu) and quercetin 3-O-glucoside (Qu3glc).
Papageorgiou et al. (2008) described one benzoic acid derivative,
namely 3.4 dihydroxybenzoic acid (3.4 di-HBA; syn. protocatechuic acid) in their HPLC analysis of sage aerial parts (leaves).
Several biological activities of 3.4 di-HBA such as antioxidant,
anti-inammatory, neuroprotective, antifungal, antihepatotoxic,
chemoprotective and apoptotic were previously reported (Hur
et al., 2003; An et al., 2006; Yip et al., 2006; Yin et al., 2009).
In this work we identied three new, not yet in sage described
derivatives of the group; 4-hydroxybenzoic acid (4-HBA), 2,6dihydroxybenzoic acid (2.6-di-HBA; syn. -resorcylic acid) and 3.5dimethoxy-4-hydroxybenzaldehyde (syn. syringaldehyde; SA).
Caffeic acid was detected in a similar range as reported previously (Papageorgiou et al., 2008) and shows the tendency to
decrease along the season in both populations. Further phenolic
acid derivatives such as ferulic acid, vanillic acid, acetovanillone
and coniferyl alcohol were also present in both accessions, but
account together to less than 5% of total phenolic acids. In agreement with previous reports rosmarinic acid (RA) was conrmed as
the main phenolic constituent of S. fruticosa (Skoula et al., 2000;
Exarchou et al., 2002; Papageorgiou et al., 2008; Farhat et al., 2013)
and was also found in S. ofcinalis (Pizzale et al., 2002; Dincer
et al., 2012). However, the range of RA concentration observed
in our samples was 230 fold higher than those found previously (Papageorgiou et al., 2008), and an extensive variation of RA
content among the studied populations and the sampling period
was observed. The range of RA content of the accessions studied
in this work is in agreement with Cvetkovikj et al. (2013), who
reported that RA was the major and most variable constituent
among 115 plant samples of various Salvia species. Three related
caffeic acid derivatives, chlorogenic acid, neochlorogenic acid and
cryptochlorogenic acid, were also identied for the rst time in S.
fruticosa leaves, investigated in our study.
Intermediates of the general avonoid pathway were accumulated at the different stages, while the two avanones, naringenin
and hesperidin (4 -methyleriodictyol 7-O-rutinoside) were identied in higher concentration as the later dihydroavonols,
dihydrokaempferol and dihydroquercetin. Derivatives of these two
groups serve as direct precursor for avones and avonols, respectively. Flavones are known to be predominant group of metabolites
in Salvia species and they are represented by Ap and Lu derivatives.
Due to limitation in the actual database of the method used, we
were only able to detect the two aglyca, the 7-O-glucosides and
apiin (Ap 7-O-apiosylglucoside). Cvetkovikj et al. (2013) described
up to twenty avone derivatives occurring in Salvia species.
Known sage avones such as hispidulin, Ap/Lu-rutinosides or
glucuronides were not identied in our study, while Exarhou et al.
(2014) reported the presence of hispidulin (8 mg/g) in ethyl acetate
extracts of S. fruticosa. As for avonols, a broader spectrum was
identied with derivatives of kaempferol, quercetin and isorhamnetin (3 -methylquercetin) as aglyca. Beside the three respective
3-O-glucosides, also the 3-O-rhamnoside (quercitrin) and the 3.4 -

Fig. 1. Structures of newly identied dihydrochalcones (phloridzin, trilobatin) and

coumarins (daphnetin and esculin) in Salvia fruticosa methanolic extracts.

O-diglucoside of Qu (Qu3.4 gluc) was identied. Quercetin, which

was found as the major avonol by Papageorgiou et al. (2008), was
only detected in small amounts in our samples, indicating an efcient glycosylation, while the predominant avonols were Qu3glc
and Km3glc followed by Qu3.4 gluc.
The diterpene carnosol (syn. picrosalvin) was found to be
the second most important metabolite based on overall concentration. This metabolite is a product of auto-oxidation of the
bitter component carnosic acid, which was not detectable in our
extracts, indicating a complete conversion most probably during drying and/or storage process (Zhang et al., 2012).Moreover,
derivatives of two other groups of secondary compounds, not yet
described from S. fruticosa were identied in this study: two dihydrochalcones, namely phloridzin (phloretin 2 -O-glucoside) and
trilobatin (phloretin 4 -O-glucoside), and two coumarins, esculin
(6-O-glucosyl-esculetin) and daphnetin (7,8-dihydroxycoumarin)
(Fig. 1). Dihydrochalcones are well known substances of apples
and were thought to be restricted only in few plant families, but nowadays they are detected more frequently, due to
advances in analytical technique and its sensitivity (Vrhovsek et al.,
2012; Carvalho et al., 2013). Phloridzin showed phytoestrogenic
and putative anti-inammatory activities and demonstrated to
inhibit glucose intestinal absorption (de Bernonville et al., 2010;
Ehrenkranz et al., 2005; Puel et al., 2005). Trilobatin has been suggested as a potential anti-diabetic compound demonstrating strong
inhibitory activity against -glycosidase and moderate inhibitory
activity against -amylase (Dong et al., 2012). Variations in phenolic content were observed under the inuence of harvesting
time and genotype, while both sage populations presented similar
trends on specic compounds accumulation: RA, hesperidin, Ap,
Lu, Km3glc and Qu3glc. Moreover, correlation coefcient analysis
revealed signicant positive correlations between the harvesting
period and the following compounds: CA, RA and CHLA (Table 3).
According to our data, both sage populations exhibited the highest content of RA during spring (AprilMay), followed by summer
harvest (July-August), while a decrease was observed in autumn
(SeptemberOctober). In addition, Ap, Km3glc and Qu3glc level
increased from spring to summer period and decreased from
summer to autumn, while hesperidin varied in opposite to the
rest of discussed compounds. In general, population 1 seems to
contain signicantly higher amounts of benzoic acid derivatives,
avonols, dihydrochalcones and diterpene carnosol, while population 2 exhibited higher concentration of caffeic acid derivatives
and more specically RA. The use of sage extracts in phytotherapy
is highly correlated to the content of RA and the diterpenes carnosol
and carnosic acid, due to their biological activities such as antiox et al., 2003; Qiao et al., 2005), chemoprotective
idant (del Bano
(Ramos et al., 2010), antimicrobial (Moreno et al., 2006), anti-viral
(Swarup et al., 2007), anti-inamatory (Osakabe et al., 2004), anti-

E. Sarrou et al. / Industrial Crops and Products 94 (2016) 240250

247

Fig. 2. The antioxidant activity FRAP (A), ABTS (B) and DPPH (C) of population 1 and 2 of S. fruticosa methanolic extracts, under the inuence of harvest period. Vertical bars
represent mean values of three independent replicates St error; Different letters indicate signicant difference (p 0.05) between the harvesting periods.

Table 5
Correlation coefcient between antioxidant activities (FRAP, DPPH, ABTS) and phenolic content of the methanolic extracts from two populations of S. fruticosa.
DPPH
FRAP

DPPH

ABTS

1
2
3
4
5
6
a
b

Population 1
signicance
Population 2
signicance
Population 1
signicance
Population 2
signicance
Population 1
signicance
Population 2
signicance

0.491
0.024
0.373
0.096

CA: caffeic acid.

RA: rosmarinic acid.
LU: luteolin.
Kmf3glc: kaempferol-3-glucoside.
Qu3glc: quercetin-3-glucoside.
CAR: carnosol.
Correlation is signicant at 0.01 level.
Orrelation is signicant at 0.05 level.

ABTS

Total phenols
b

0.442
0.045
0.480b
0.028
0.579a
0.006
0.508b
0.019

0.952
0.000
0.841a
0.000
0.480b
0.028
0.472b
0.031
0.549a
0.010
0.580a
0.006

Total avonoids
a

0.907
0.000
0.641a
0.002
0.592a
0.005
0.008
0.973
0.551a
0.010
0.506b
0.019

CA1
0.047
0.840
0.729a
0.000
0.179
0.439
0.050
0.839
0.097
0.677
0.340
0.884

RA2

LU3
a

0.680
0.001
0.862a
0.000
0.605a
0.004
0.471b
0.031
0.684a
0.001
0.560b
0.008

Kmf3glc4
a

0.812
0.000
0.375
0.094
0.362
0.107
0.628a
0.002
0.375
0.094
0.705
0.000

0.759
0.000
0.562a
0.008
0.459b
0.036
0.170
0.463
0.453b
0.039
0.392
0.079

Qu3glc5

CAR6

0.184
0.423
0.681a
0.001
0.338
0.134
0.211
0.198
0.198
0.389
0.357
0.112

0.047
0.840
0.537b
0.012
0.179
0.439
0.343
0.128
0.097
0.677
0.425
0.055

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E. Sarrou et al. / Industrial Crops and Products 94 (2016) 240250

allergic (Ito et al., 1998), anti-depression (Takeda et al., 2002) and

anti-hyperglycemic (Azevedo et al., 2011).

3.3. Antioxidant activity

The evaluation of antioxidant and radical scavenging properties of plants, claimed to have medicinal applications, or as food
additives, and can be a useful tool for the valorization of their
overall quality and content of bioactive compounds (Farhat et al.,
2013). It has been previously reported that the chemical complexity of plant extracts, polarity, different functional groups and
chemical behavior may lead to scattered results due to the antioxidant test employed (Kelen and Tepe, 2008). Therefore, in our work
the determination of antioxidant activity of the phenolic extracts
was examined with more than one method. FRAP, DPPH and ABTS
assays appear to be simple, attractive and potentially useful tests,
due to inexpensive reagents, highly reproductive results and fast
procedures. Especially, ABTS and DPPH assays are capable of testing
the antioxidant activity of both hydrophilic and lypophilic compounds (Benzie and Strain, 1996; Snchez-Moreno, 2002).
The results of FRAP antioxidant activity, DPPH and ABTS scavenging activity of the studied methanolic extracts of sage, are
summarized in Fig. 2. All extracts from the two S. fruticosa populations indicated high capacity to scavenge free radicals. In general
both populations demonstrated almost the same trend i.e. slightly
increase from April to May, then roughly decrease from May to
June, recording a maximum on July and/or August, while then
decrease to the lowest level until October. More specically in FRAP
antioxidant and ABTS scavenging activity, high differentiations due
to harvesting time was observed, varying between 31.83-202.93
and 60.94-242.3 M Trolox/g DW and 135.81-326.22 and 199.64312.49 M Trolox/g DW, for population 1 and 2 respectively (Fig. 2A
and B). On the other hand DPPH scavenging activity data indicated
less signicant variations among the harvest time for both populations, varying between 182.99-192.62 and 185.30-192.19 M
Trolox/g DW (Fig. 2C). From our results it could be hypothesized that the kinetics of radical scavenging reactions might differ
between the DPPH and ABTS tests. Our ndings, in combination
with previous mentioned (polarity, different functional groups and
chemical behavior) conrm the difculty to determine the antioxidant activity of a plant tissue or product, by using only a single
method and the difculty to compare the results obtained by different assays testing the antioxidant activity.
Moreover, it seems that there is low correlations between FRAP
and the other two assays used (signicant at 0.05 level), as well
as slight correlation can be observed between DPPH and ABTS
assay (Table 4), which was also previously reported from Miliauskas
et al. (2004). In contrast, high positive correlations was observed
between FRAP and ABTS antioxidant activities and total phenolic/avonoid, RA, Km3glc, Qu3glc and carnosol content (Table 5).
It has been proposed that only avonoids of a certain structure,
specically hydroxyl position in the molecule and especially with
presence of a second hydroxyl group in the ortho- and para-position
increase the efcacy of antioxidants like RA, carnosol and carnosic
acid to determine antioxidant properties, while these properties
depend on their ability to donate hydrogen or electron to a free
radical (Tung et al., 2009; Roby et al., 2013).
Furthermore RA content, is signicantly correlated with all three
assays used, indicating its high contribution to the antioxidant
potential of sage extracts. Our ndings are in agreement with previous reports showing that the capacity of sage extracts to scavenge
free radicals coincides with high total phenol content, but it is not
proportional, while extracts rich in RA had higher antioxidant activity (Exarchou et al., 2002; Miliauskas et al., 2004; Roby et al., 2013).

4. Conclusion
The combined analysis of metabolites (essential oils, phenolics)
and the antioxidant activity determination of the extracts of two
sage populations, harvested over different time, from spring to
autumn, gave new inside into the general metabolite pattern of
the plant. Particularly, several not yet known phenolic compounds
were identied in the extracts, but also into variation, based on the
genetic background and growing season. The use of state to the
art targeted LCMS/MS analysis show strong potential for a fast,
efcient and sensitive analytical identication of phenolic nger
prints of Salvia, including minor constituents. This method can also
be applied for other Lamiaceae plant species, as the database of targeted compounds already comprises more than 140 compounds,
but it can be easily adopted and improved to the metabolite spectrum found in the plant of interest.
Taking into account the use of sage in different products, such as
essential oils, herbal teas, phytomedicines, food supplements etc,
the quality of plant material is important and depends both on its
phytochemical content and composition. Genotype background is
highly affecting the metabolic prole of sage, as population 1 exhibited higher amounts of most of the phenolic compounds identied,
compared to population 2. In addition, considering that: 1) the studied populations of S. fruticosa indicated very low amounts of a- and
b- thujones under all the harvest periods, 2) exhibited signicantly
higher essential oil yield, increased concentrations of oxygenated
terpenes in summer, as well as simultaneously rising biomass production due to rapidly increase of the vegetative growth from
spring to summer and 3) the variation of phenolic compounds
throughout the harvesting period, we may assume that summer is
the most proper period for essential oil production, while spring
collection favors plant material rich in major phenolics like RA.
Therefore, suitable genotype and appropriate harvesting period
may be dened properly for obtaining high quality-standardized
sage product.
Authors contributions
ES performed most of the experimental work together with
interpretation of data, involved in the design of the work and most
of writing and editing; SM contributed in performing metabolitephenolic analysis, supporting interpretation of the data, writing and
editing as well; PC contributed to the conception and design of the
work, involved on writing and editing and supervised the experimental work. All authors read and approved the nal manuscript.
Acknowledgments
Part of this research was funded under the Project Research
& Technology Development Innovation Projects-AgroETAK, MIS
453350, in the framework of the Operational Program Human
Resources Development. It is co-funded by the European Social Fund
through the National Strategic Reference Framework (Research
Funding Program 20072013) and coordinated by the Hellenic
Agricultural Organization DEMETER.
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