Acta Physiol Plant (2011) 33:11031111

DOI 10.1007/s11738-010-0638-z

ORIGINAL PAPER

Drought effects on polyphenol composition and antioxidant

activities in aerial parts of Salvia officinalis L.
Iness Bettaieb Ibtissem Hamrouni-Sellami
Soumaya Bourgou Ferid Limam Brahim Marzouk

Received: 20 July 2010 / Revised: 4 October 2010 / Accepted: 2 November 2010 / Published online: 19 November 2010
Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2010

Abstract Salvia officinalis L. is a medicinal plant Keywords Salvia officinalis L.  Drought  Growth 
containing several compounds with important pharmacological Polyphenols  Antioxidant
activity. In this study, we investigated the effects of water
deficit (moderate and severe water deficits) on the contents
Abbreviations
of total and individual polyphenols of the aerial parts. Also,
C Control
we studied the effect of drought on the antioxidant activity
MWD Moderate water deficit
of methanolic extracts. Our results showed that water
SWD Severe water deficit
deficiency, as estimated by the decrease in water potential,
FC Field capacity
resulted in a reduction of the biomass, plant height and
BHT Butylhydroxytoluene
total chlorophyll contents. In general, drought increased the
HCl Chloridric acid
level of total and individual polyphenols and this increase
IC50 The half maximal inhibitory concentration
was more pronounced under moderate water deficit. These
EC50 The half maximal effective concentration
findings suggest that S. Officinalis is a sensitive species and
ROS Reactive oxygen species
that a severe water deficit could result in a decline in the
DW Dry weight
activity of enzymes involved in the biosynthesis of phe-
FW Fresh weight
nolic compounds. On the other hand, our results showed an
DPPH 2,20 -Diphenyl-1-picrylhydrazyl
enhancement of reducing power and the radical scavenging
GAE Gallic acid equivalent
activity as assessed using the DPPH assay with increasing
K3Fe (CN)6 Potassium ferricyanide
stress severity. Finally, the evaluation of the chelating
capacity of the extracts was found to be altered signifi-
cantly under severe treatment by 39.71%. Based on these
results, it seems that drought tolerance of S. officinalis is
related to the capacity of the plant to modulate its phenolics Introduction
in order to face to oxidative stress caused by water limiting
conditions. Phenolic compounds and flavonoids are among the most
influencial and widely distributed secondary products in the
plant kingdom (Ali and Abbas 2003). Many of them play
important physiological and ecological roles, being involved
Communicated by R. Aroca.
in resistance to different types of stress (Ayaz et al. 2000).
These metabolites have several defence functions and,
I. Bettaieb (&)  I. Hamrouni-Sellami  S. Bourgou  therefore, their biosynthesis in plants is generally induced in
F. Limam  B. Marzouk response to biotic and abiotic stimuli such as UV-B radiation,
Laboratoire des Substances Bioactives Centre
drought, chilling, ozone, heavy metals, and attacks by
de Biotechnologie a la Technopole de Borj-Cedria (CBBC),
BP 901, Hammam-Lif, Tunisia pathogens, wounding, or nutrient deficiency (Dixon and
e-mail: rosainess@yahoo.fr Paiva 1995; Grace 2005).

123
1104
In the past few years, interest in the antioxidant prop-
erties of plant-derived foods and medicinal plants has
increased, since antioxidants contained in plants are
involved in the preservation of human health (Giorgi et al.
2009). In addition to, plant materials containing phenolic
constituents are increasingly of interest for the food
industry as they retard oxidative degradation of lipids and
thereby improve quality and nutritional value of food
(Landry et al. 1995; Rice-Evans et al. 1996). Regarding
these beneficial effects and properties of plant phenolics,
interest is considerably increasing in finding naturally
occurring antioxidants from botanical sources, for use in
the food industry and in preventive medicine to replace
synthetic antioxidants that are being restricted due to
their possible toxicity and carcinogenicity (Namiki 1990;
Velioglu et al. 1998; Hu et al. 2000). Therefore, effective
production of polyphenols in plants has been considered to
be important to supply a lot of polyphenols (Yaginuma
et al. 2002). To increase the production of phenolic com-
pounds in plants, one approach may be submitting desired
plants to biotic and abiotic stresses. In fact, an increase in
phenolic compound biosynthesis and accumulation fre-
quently occurs in plant tissues as a reaction to biotic and
abiotic stresses (Dixon and Paiva 1995). Indeed, polyphe-
nolic compounds participate in the defence against reactive
oxygen species (ROS), which are inevitably produced
when aerobic or photosynthetic metabolism is impaired by
environmental stresses (Sreenivasulu et al. 2000). Thus,
drought stressed plants might represent potential sources of
polyphenols for economical use. Spices used in different
types of food to improve flavours, since ancient times, are
well known for their antioxidant properties (Madsen and
Bertelsen 1995). In various studies, common sage (Salvia
officinalis) belonging to the Lamiaceae family, has been
shown to be among the most potent natural antioxidant of
the common spices (Chipault et al. 1956; Hermann 1981).
It is a perennial woody sub-shrub which grows in the
temperate and warmer zones of the world. It is cultivated
mainly to obtain dried leaves to be used as raw material in
medicine, perfumery, and food industry (Bruneton 1999).
Sage extracts have also been reported to have excellent
properties in inhibiting lipid peroxidation (Lu and Foo
1999). The antioxidative activity of sage has been attrib-
uted mainly to rosmarinic acid (Briekson and Domling
1969; Cuvelier et al. 1996). Sage extracts also contain
flavonoids and other phenolics which may contribute to the
total antioxidant activity (Dapkevicius et al. 1998; Lu and
Foo 2000).
Until now, few studies have been carried out on the
effect of abiotic stresses on primary and secondary
metabolism of S. officinalis. Among these studies, only two
investigations have been carried out on the effect of water
deficit on this species. In the first one, Bettaieb et al. (2009)
123
Acta Physiol Plant (2011) 33:11031111
reported the effect of this constraint on fatty acids and
essential oil composition. In the second study, Munne-
Bosch et al. (2001) evaluated the effect of drought on
diterpenes and tocopherols. After all the above, the purpose
of our investigation was to study the variation of poly-
phenols and antioxidant potential of S. officinalis under
water deficit conditions.
Materials and methods
Plant material and water deficit treatments
Plants used in this work were obtained by cutting propa-
gations. The mother plants were cultivated in a plant
nursery, 35 km northeast of Tunis. Ten-centimetre-length
stems with two nodes and four opposite leaves were cut
from mother plants and transplanted to 10 l pots (4 plants
per pot) filled with agricultural soil containing 0.22, 0.34,
0.05 and 0.08 meq (100 g)-1of dry soil of Na?, K?, Ca2?,
Fe2?, respectively. During 30 days of pretreatment, plants
were irrigated with tap water, and then divided into three
lots subjected to different water levels: 100% control (C),
50% moderate water deficit (MWD) and 25% severe water
deficit (SWD) of field capacity (FC) (Bouyoucos 1983).
This later was determined in the pots by weight. Experi-
ments were carried out in a greenhouse with a 14-h pho-
toperiod (photosynthetic photon flux density, PPFD:
400 mol m-2 s-1) and lasted 2 months from April 2007 to
June 2007. Mean temperature and relative humidity were
30 5C, 55 5% day and 16 2C, 90 5% night,
respectively.
Growth and water potential measurements
At the end of each treatment, the seedling length (the
distance from soil surface to upper end of the longest
leaves) was measured (cm/plant). The fresh (FW) and dry
(DW) biomass of the seedlings was also determined after
oven drying at 100C for the latter. Measurements of aerial
parts water potential were realized for the different treated
plants by means of Scholander pressure chamber (Scholander
et al. 1965).
Estimation of total chlorophyll content
Total chlorophyll was extracted and estimated from fresh
leaves following the standard method of Arnon (1949).
Extraction and determination of total polyphenols
Extracts were obtained by stirring 2.5 g of dry aerial parts
powder with 10 ml of pure methanol for 30 min, were kept
Acta Physiol Plant (2011) 33:11031111
for 24 h at 4C, filtered through a Whatman no. 4 filter
paper, and evaporated under vacuum to dryness and stored
at 4C until analyzed (del Bano et al. 2003). Phenolic
compounds were assayed using the Folin-Ciocalteu
reagent, following Singletons method slightly modified
(Dewanto et al. 2002). Aliquot (0.125 ml) of diluted sam-
ple of extract was added to 0.5 ml of deionized water and
0.125 ml of the Folin-Ciocalteu reagent. The mixture was
shaken and allowed to stand for 6 min, before adding
1.25 ml of 7% Na2CO3 solution. The solution was then
diluted with deionized water to a final volume of 3 ml and
mixed thoroughly. After incubation for 90 min at 23C, the
absorbance versus prepared blank was read at 760 nm. Total
phenolic content of plants (three replicates per treatment) was
expressed as mg gallic acid equivalents (GAE) g-1 DW
through the calibration curve with gallic acid.
Hydrolysis, quantification and identification
of phenolic compounds using HPLC
Dried samples were hydrolysed according to the slightly
modified method of Proestos et al. (2006). Forty millilitres
of methanol containing BHT (1 g l-1) were added to 0.5 g
of dried S. officinalis sample. Then, 10 ml of 6 N HCl were
added. The mixture was stirred carefully and then sonicated
for 15 min and refluxed in a water bath at 90C for 2 h.
The obtained mixture was filtered through a 0.45 lm
membrane filter. Before injection in HPLC, an internal
standard (trans-2-hydroxycinnamic acid) was added to this
mixture in order to quantify phenolic compounds. Phenolic
compounds analysis was carried out using an Agilent Tech-
nologies 1100 series liquid chromatograph (RP-HPLC) cou-
pled with an UV-vis multiwavelength detector. The separation
was carried out on 250 9 4.6 mm, 4 lm Hypersil ODS C18
(Agilent Technology, Palo Alto, CA) reversed phase column
at ambient temperature. The mobile phase consisted of ace-
tonitrile (solvent A) and water with 0.2% sulphuric acid
(solvent B).The flow rate was kept at 0.5 ml min-1. Peaks
were identified by congruent retention times compared with
standards. Analyses were performed in triplicates.
Diphenyl-2-picrylhydrazyl (DPPH) scavenging activity
The effect of methanolic extracts on DPPH degradation
was estimated according to Hanato et al. (1988). 2 ml of
the different extracts was added to 0.5 ml of a
0.2 mmol l-1 DPPH-methanolic solution. The mixture was
shaken vigorously and left standing at room temperature
for 30 min. The absorbance of the resulting solution was
then measured at 517 nm measured after 30 min. The
antiradical activity was expressed as IC50 (mg ml-1), the
antiradical dose required to cause a 50% inhibition. A
lower IC50 value corresponds to a higher antioxidant
1105
activity of plant extract. The ability to scavenge the DPPH
radical was calculated using the following equation:
DPPH scavenging effect % A0  A1 =A0   100
where A0 is the absorbance of the control at 30 min, and A1
is the absorbance of the sample at 30 min.
Reducing power
The method of Oyaizu (1986) was used to assess the reducing
power of S. officinalis aerial parts extracts. Methanol extracts
(1 ml) were mixed with 2.5 ml of a 0.2 M sodium phosphate
buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide
(K3Fe (CN)6), and incubated in a water bath at 50C for
20 min. Then, 2.5 ml of 10% trichloroacetic acid was added to
the mixture that was centrifuged at 650g for 10 min. The
supernatant (2.5 ml) was then mixed with 2.5 ml distilled
water and 0.5 ml of 0.1% ferric chloride solution. The inten-
sity of the bluegreen colour was measured at 700 nm. The
EC50 value (mg ml-1) is the extract concentration at which the
absorbance was 0.5 for the reducing power and was calculated
from the graph of absorbance at 700 nm against extract con-
centration. Ascorbic acid was used as a positive control.
Chelating power
The chelating activity was assessed according to the
method of Zhao et al (2006). The extract (0.1 ml) was
reacted with 2 mM of ferrous chloride (0.05 ml), 5 mM
ferrozine (0.1 ml) and 2.75 ml of distilled water. The
mixture was shaken vigorously and left at room tempera-
ture for 10 min. The absorbance of the solution was then
measured at 562 nm. The scavenging activity was calcu-
lated as follows: PI % = [(Ablank - Asample)/Ablank] 9 100,
where Ablank is the absorbance of the control reaction and
Asample is the absorbance in the presence of plant extract.
Statistical analysis
Data were analyzed according to the One-Way ANOVA
method of the stat. prog. STATISTICA subjected to statistical
analysis using statistical program package STATISTICA.
Mean values (three replicates SD) were statistically com-
pared with Ducan test, at probability level P \ 0.05.
Results and discussion
Effect of drought on plant growth and chlorophyll
content
Plants subjected to severe water deficit presented thinner
stems with fewer dry and smaller leaves than control ones.
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1106
Table 1 Effect of water stress on growth, water potential and chlo-
rophyll content in S. officinalis aerial parts
C MWD SWD
Height (cm) 26 0.03a 20 0.05b 14 0.02c
a b
FW (g) 19.53 1.22 12.63 0.45 4.96 0.04c
a b
DW (g) 4.48 0.06 3.68 0.02 3.30 0.02b
a b
Water Potential -1.60 0.01 -3 0.03 -4.8 0.04c
(MPa)
Chlorophyll content 5.63 0.11a 1.21 0.01b 0.25 0.01c
(mg/g FW)
C control, MWD moderate water deficit, SWD severe water deficit
Values are means of three replications (N = 3 SD). Means with
different letters in the same row symbolize statistically significant
differences between treatments at probability level P \ 0.05,
according to the Duncans multiple range test
These morphological modifications were accompanied by a
reduction in water potential of aerial parts from -1.6 MPa
in control plants to -3 and -4.8 MPa in MWD and SWD
plants, respectively (Table 1). Many investigations have
shown that when plants are subjected to drought, they
exhibit large reductions in relative water content and water
potential (Kyparissis et al. 1995; Scarascia-Mugnozza et al.
1996; Nayyar and Gupta 2006). On the other hand, a
decrease in the studied growth parameters under drought
stress was observed and is positively correlated with the
degree of stress severity. In fact, our results showed that
plant height is reduced of 23.1% for MWD and 46.2% for
SWD as compared to control. As for FW and DW, the
decreases reached 35.3 and 74.6%, respectively, for MWD
and SWD for the first parameter and 17.9 and 26.4%,
respectively, for MWD and SWD for the second parameter.
As reported by Viera et al. (1991), the decrease in growth
parameters of plants under water stress conditions could be
the result of the decrease of chlorophyll content what
results in a decline of the photosynthesis efficiency and
thereby a decrease in the production of biomass. Finally, at
the level of chlorophyll content, our results showed that
total chlorophylls were significantly reduced under water
stress conditions. The decreases reached 78.5% for MWD
and 95.4% for SWD (Table 1). According to Kiani et al.
(2008), water stress, among other changes, has the ability
to reduce the tissue concentrations of chlorophylls and
carotenoids, primarily with the production of ROS in the
thylakoids (Reddy et al. 2004).
Effect of drought on total phenolics content
The total phenolic content of control plants as estimated by
the Folin-Ciocalteu method was of 2.02 mg of GAE/g DW
(Fig. 1). It was significantly increased when plants are
subjected to the water deficit treatment. Thus, under MWD,
total polyphenol content was about 4.07 times higher
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Acta Physiol Plant (2011) 33:11031111
Fig. 1 Effect of water stress on total polyphenol contents of S.
officinalis aerial parts estimated by Folin-Ciocalteu method (FC) and
hydrolysis acid (HA); C control; MWD moderate water deficit; SWD
severe water deficit. *Total phenolic were expressed as mg GAE/g
DW for (FC) method and as mg/g DW for (HA) method. Values are
means of three replications (N = 3 SD). The data marked with
the different capital letter, for the method, and small letter, for the
treatments, in the histograms of each phenolic content share
significant differences at P \ 0.05 (Duncan test)
than the control one. Besides, it increased significantly by
3.21-fold under SWD. On the other hand, the assessment of
phenolic compounds as determined by RP-HPLC was
1.02 mg/g DW in the control samples. An increase in
polyphenol content has been found under the two treat-
ments as determined by this method. Thus, under MWD,
total polyphenol content was about 2.04 times higher than
that observed in control plants. Besides, it increased sig-
nificantly by 31.37% under SWD. According to these
results, the contents of phenolic compounds as assessed by
RP-HPLC are significantly too inferior to those obtained by
the Folin-Ciocalteu method at P \ 0.05. These differences
could be explained by the weak selectivity of the Folin-
Ciocalteu reagent, as it reacts positively with different
antioxidant compounds. Based on the results listed above,
biosynthesis of total polyphenols was induced by both
MWD and SWD treatments. In fact, according to biblio-
graphic data, polyphenol synthesis and accumulation in
plants are generally stimulated in response to biotic/abiotic
stresses (Yaginuma et al. 2002; Ksouri et al. 2007; Giorgi
et al. 2009) such as water deficit (Abreu and Mazzafera
2005). In stressed plants, the accumulation of phenolic
compounds is negatively correlated with the accumulation
of plant biomass. According to Abreu and Mazzafera
(2005), these results could be explained by the fact that
water deficit may be associated with an increase in sec-
ondary metabolites through the reallocation of the assimi-
lated carbon as plant growth is progressively reduced.
Phenolic compounds are known to play important roles in
the resistance of plants to environmental stresses and par-
ticularly in water deficit stress. Under this constraint, the
uptake of carbon dioxide is reduced because of stomatal
closure what results in the exposure of chloroplasts to
Acta Physiol Plant (2011) 33:11031111 1107

excess excitation energy (Smirnof 1993) and an increase in
the formation of ROS (Li and Staden 1988). The latter may
initiate destructive oxidative process such as lipid peroxi-
dation, chlorophyll destruction, protein oxidation and
nucleic acids damage (Scandalios 1993). These ROS are
normally scavenged by a range of antioxidants including
phenolics and antioxidant enzymes (Smirnof 1993). Either
their role as free radical scavengers, phenolics can function
as a filter absorbing radiation and limiting the excitation of
chlorophyll during conditions unfavourable for the photo-
synthetic apparatus (Nogues and Baker 2000). Thus,
according to Hura et al. (2008), water deficit in the leaf
tissue can induce the protective mechanisms involving the
synthesis of phenolic compounds and subsequently, the
neutralization of the radiation by its absorption and trans-
formation into blue fluorescence.
Quantitative and qualitative analysis of phenolic
compounds by RP-HPLC
RP-HPLC analysis showed that aerial parts extracts pre-
sented a chemical profile composed of nineteen identified
phenolics including gallic, cafeic, vanillic, dihydroxyben-
zoic, p-coumaric, chlorogenic, trans-2-hydroxycinnamic,
cinnamic, rosmarinic, ferulic and trans-cinnamic acids as
well as quercetin-3-D-galactoside, quercetin, kaempferol,
catechin, narginin, apigenin, amentoflavone and flavone.
The amounts of the different phenolics identified in
S. officinalis aerial parts are shown in Table 2. Results
showed that phenolic acids constituted the main phenolic
class of the aerial parts extracts (607.61 lg/g DW for C;
1,449.28 lg/g DW for MWD and 837.37 lg/g DW for
SWD). Independently of the applied treatments, rosmarinic
acid was detected as the major phenolic acid in S. offici-
nalis aerial parts with 170.73 lg/g of DW. Besides, dihy-
droxybenzoic acid was the second major phenolic acid at
the level of 143.82 lg/g of DW. Results indicated that,
except cinnamic acid and chlorogenic acid, MWD resulted
in a significant increase of the biosynthesis of the different
phenolic acids and especially the benzoic ones which
resulted in the enhancement of their contents by about
2.89-fold. Therefore, the level of rosmarinic and dihy-
droxybenzoic acids increased, by 4 and 2.4-fold, respec-
tively, as compared to the control. On the other hand,
results demonstrated that also SWD enhanced significantly
the biosynthesis of phenolic acids with the exception of
vanillic, p-coumaric cinnamic, dihydroxybenzoic, trans-
cinnamic, cafeic, ferulic and cinnamic acids. However,

Table 2 Quantitative (lg g-1

Water deficit
DW) changes of aerial parts
polyphenol components in C MWD SWD
S. officinalis as influenced by
water deficit Benzoic acids 296.34 1.29c 857.38 1.33a 537.82 2.45b
1 Gallic acid 21.62 1.32b 35.55 0.04a 37.40 0.03a
2 Cafeic acid 17.80 2.46b 28.98 0.46a 20.75 0.73b
b a
3 Vanillic acid 59.03 1.3 69.80 0.39 37.20 3.5c
c a
4 Rosmarinic acid 170.73 1.63 707.63 1.75 430.34 2.03b
b a
5 p-Coumaric acid 27.16 1.36 47.42 1.53 12.13 3.63c
b a
Cinnamic acids 311.27 2.66 591.90 3.51 299.55 0.34b
6 Chlorogenic acid 17.92 6.73b 22.52 0.84b 42.21 2.4a
c b
7 trans-2-hydroxycinamic acid 36.76 0.02 47.59 0.8 57.87 0.75a
a b
8 Cinnamic acid 24.11 0.34 15.48 2.94 12.70 1.22b
b a
9 Dihydroxybenzoic acid 143.82 0.73 346.35 3.2 112.31 0.73c
b a
10 Ferulic acid 66.01 1.59 119.08 1.03 69.42 1.79b
b a
11 trans-Cinnamic acid 22.65 0.04 40.88 0.73 5.04 0.08c
Flavonoides 349.55 1.76b 563.55 3.80a 319.88 1.25c
a a
12 Quercetin 3-D-galactoside 50.30 2.38 57.16 3.7 30.30 4.74c
C control, MWD moderate water ab a
deficit, SWD severe water deficit 13 Quercetin 50.91 7.34 68.58 1.45 25.44 0.22b
Values are means of three 14 Campherol 126.76 3.44b 274.98 0.62a 135.08 1.73b
replications (N = 3 SD). 15 Catechin 29.33 0.53ab 36.41 1.54a 37.34 0.03a
Means with different letters in 16 Naringin 39.02 3.84a 16.09 1.36b 15.79 0.64b
the same row symbolize b a
17 Apigenin 9.79 0.02 17.78 0.34 6.83 0.03b
statistically significant b a
differences between treatments 18 Amentoflavone 14.01 2.64 33.84 2.98 12.23 1.53b
b a
at probability level P \ 0.05, 19 Flavone 29.43 1.83 58.71 0.53 56.87 1.23a
according to the Duncans P b b
NI 70.91 2.63 85.70 3.12 186.7 1.73a
multiple range test

123
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we noticed that accumulation of phenolic acids under SWD
was less pronounced than that observed under MWD.
According to these results, it seems that S. officinalis is
sensitive to SWD which may affect the enzymes involved
in the biosynthesis of phenolics leading to their partial
inactivation what resulted in a decline of the content of
phenolic compounds under severe water limiting condi-
tions. Analysis of the effect of drought on flavonoids
contents of S. officinalis aerial parts indicated that MWD
improved significantly the accumulation of these metabo-
lites by about 1.6-fold. Excluding, naringin, which content
decreased by 59% and quercetin and catechin that did not
change; moderate stress improved the biosynthesis of all
the flavonoids and led to double the content of campherol,
and amentovlavone as compared to the control. However,
SWD altered slightly the content of flavonoids by 8.48%.
This decrease was expressed mainly by the reduction of the
contents of quercetin 3-D-galactoside, quercetin and narg-
inin by 39.00, 49.11 and 59.53%, respectively, as compared
to the control. These variations were related to the relative
contents of the constituents and not to the presence of new
ones or the absence of particular components.
Changes in the contents of phenolic acids have been
associated with the modification of Phenylalanine ammo-
nia-lyase (PAL) activity, the entry point enzyme into the
phenylpropanoids pathway (Kacperska 1993). The accu-
mulation of phenolic compounds may be related to the
activation of PAL under drought conditions. In general,
PAL catalyses the transformation of L-Phenyalanine into
trans-cinnamic acid, which is the prime intermediary in the
biosynthesis of phenolics (Dixon and Paiva 1995; Giorgi
et al. 2009; Kim et al. 2006). The activity of this enzyme
increases activity in response to stress and is considered by
the most authors to be one of the main lines of cell accli-
mation against stress in plants (Kacperska 1993, Leyva
et al. 1995). According to (Lin et al. 2006), enhancement of
the antioxidant capacity both by increasing contents and
quality of phenolic compounds may play an important role
in the tolerance of plants to abiotic stresses. This suggests
that plants respond as an adaptive mechanism by activating
genes involved in the biosynthesis of antioxidants when-
ever there is an environmental perturbation (Oh et al.
2009). A number of studies have shown that genes
involved in the phenylpropanoid pathway, including PAL,
and those involved in the biosynthesis of tocopherols in a
number of plant species are activated in responses to abi-
otic stresses, including water stress (Leyva et al. 1995;
Collakova and Dellapenna 2003). Furthermore, studies
showing that mutants deficient in secondary metabolites or
plants with blocked PAL activity are sensitive to environ-
mental stresses clearly demonstrate the role of these anti-
oxidants in plant adaptation (Li et al. 1993; Gitz et al.
2004). Based on our results, it seems that the tolerance of
123
Acta Physiol Plant (2011) 33:11031111
S. officinalis aerial parts to drought was associated with
rich polyphenol content of leaves, so the capacity to
accumulate polyphenols contributed to drought tolerance
of this species.
At the level of phenolic acids and under abiotic stresses,
previous data indicated a variability of answers. Thus,
Meot-Duros and Magne (2009) reported an accumulation
of chlorogenic acid in the leaves of Crithmum maritimum
living on sand hills which suffered from both water stress
(due to the nature of sandy substrate) and ionic stress (due
to sea sprays). Accordingly, a wide range of environmental
stresses, including boron and nitrogen deficit, has been
shown to increase chlorogenic acid levels in plants (Del
Moral 1972; Camacho-Cristobal et al. 2004). Besides,
Yaginuma et al. (2002) demonstrate an increase in flavo-
noids content and especially luteolin and luteolin-7-gly-
coside under light and water stresses in the seedlings of
safflower and cucumber. In addition, Abreu and Mazzafera
(2005) reported an increase in the levels of rutin, quercetin
and betulinic acid under drought conditions in the aerial
parts of Hypericum brasiliense.
Either their role as antioxidants and radical scavengers,
phenolic compounds can function as photoprotectors lim-
iting light penetration into the mesophyll cells and in this
way reduce the excitation of chlorophyll during the leaf
water deficit (Nogues et al. 1998; Nogues and Baker 2000).
Other investigations have shown that phenolics in the
chlorophyll-free epidermis can be an obstacle for the light
to reach the leaf by its absorption before transformation
into blue fluorescence (Bilger et al. 1997). According to
Hura et al. (2008), ferulic acid is the main source of blue
fluorescence emissions in resistant maize seedlings sub-
mitted to severe water deficit. Based on these findings, it
seems that the high levels of ferulic acid in moderately
stressed S. officinalis plants (119.08 lg/g-1 DW) as com-
pared to control ones (66.01 lg/g-1 DW) would be an
adaptive response in this species by absorbing UV light and
its transformation into blue fluorescens. This mechanism of
adaptation could protect the plant from the destructive
effects of UV light under drought conditions.
Effect of drought on antioxidant activities
By analysing the antioxidant activity of the different
extracts under the different levels of drought, it was dem-
onstrated that they all had the capacity to scavenge DPPH
free radicals (Table 3). Indeed, DPPH scavenging activity
increased significantly by 17.77 and 61.48% under MWD
and SWD, respectively. On the other hand, the evaluation
of the chelating capacity of the extracts was found to be
altered significantly under SWD by 39.71%. Conversely,
there was no significant effect of moderate stress on the
chelating ability. Finally, the estimation of the reducing
Acta Physiol Plant (2011) 33:11031111
Table 3 Effect of water deficit on antiradical (DPPH) and reducing activity of S. officinalis aerial parts extracts
DPPH scavenging activity IC50 (mg ml-1)
-1
Chelating power IC50 (mg ml )
-1
Reducing power EC50 (mg ml )
C control, MWD moderate water deficit, SWD severe water deficit
Values are means of three replications (N = 3 SD). Means with different letters in the same row symbolize statistically significant differences
between treatments at probability level P \ 0.05, according to the Duncans multiple range test
power showed that both MWD and SWD increased the
reducing ability by 2.05 and 3.14-fold, respectively.
These activities may be directly linked to the content of
phenols, tannins and flavonoids and consequently to their
free radical scavenging activities (Huang et al. 2006).
Drought induces oxidative stress in various plants, in which
ROS, such as superoxide radical (O2-), hydroxy radical
(OH), hydrogen peroxide (H2O2) and alkoxy radical (RO)
are produced (Munne-Bosch and Penuelas 2003) with the
result that antioxidative defence can become overwhelmed
(Foyer et al. 1994). Besides, plant resistance to various
stresses is associated with antioxidant capacity and
increased levels of antioxidants may prevent stress damage
(Bor et al. 2003). The concerted action of low molecular
weight antioxidants like polyphenols (Sgherri et al. 2004)
and flavonoids (Hernandez et al. 2004) can effectively
scavenge harmful radicals and stabilize lipid oxidation. In
this context, a significant positive correlation between
antioxidant activity and phenolic compounds of radish
sprouts was observed (Kim et al. 2006). An increased level
of ROS in the water-depleted plants could lead to an
increased capacity of the scavenging system of ROS, par-
ticularly in reduction power ability. The decrease in che-
lating capacity in reponse to SWD was associated with a
decrease in total polyphenols content but remained superior
to the control one. This is in agreement with the finding of
(Ksouri et al. 2007), who reported that under salinity, there
is a decline in polyphenols content and antioxidant activity
in leaves of the halophyte Cakile maritima. On the other
hand, (Kim et al. 2008) reported that the phenolic com-
pounds and antioxidant activity could be changed by salt
stress, but this is critically dependent on the salt sensitivity
of plants.
Conclusion
In conclusion, our results showed that drought have a
marked influence on the content of pharmacologically
active secondary metabolites in S. officinalis aerial parts.
However, there is a variation depending on the degree of
stress and the analyzed compound. The increase in the
contents of the phenolic compounds agrees with data in the
1109
C MWD SWD
135 4.18a 111.5 2.60b 52.7 0.88c
b b
3.45 0.08 3.70 0.04 4.82 0.03a
a b
1.07 0.02 0.52 0.01 0.34 0.01c
literature indicating that this is probably a response to the
generation of ROS. These results also show that drought
can be successfully used to enhance the health-promoting
phytochemicals in sage and it is possible to increase the
content of pharmacologically desirable compounds by
manipulating agricultural techniques.
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