Acta Physiol Plant (2015) 37:132

DOI 10.1007/s11738-015-1879-7

ORIGINAL PAPER

Physio-chemical and antioxidant proling of Salvia sclarea L.

at different climates in north-western Himalayas
Tarandeep Kaur1,3 Hilal A. Bhat1 Rohini Bhat1,3 Arun Kumar1
Kushal Bindu2 Sushma Koul1 Dhiraj Vyas1,3

Received: 1 August 2014 / Revised: 11 May 2015 / Accepted: 3 June 2015 / Published online: 21 June 2015
Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2015

Abstract Salvia sclarea Linn. commonly known as clary

sage, is an important medicinal herb with high market
demand. To assess properties suitable for commercial
exploitation, physiological and biochemical studies were
conducted at different climatic zones in the Western
Himalayas. These include Jammu (subtropical; 305 m),
Srinagar (temperate; 1730 m) and Leh (cold arid; 3505 m)
environment. Antioxidant capacity based on radical scavenging and DNA protecting activity of the plants growing
at three locations was found to be highest in Srinagar. The
cellular damage in terms of lipid peroxidation was found
signicantly (p B 0.05) higher in Jammu as compared to
Srinagar and Leh. SOD and GR showed signicant
(p B 0.05) differences between all three climatic zones.
High expression of GR at higher altitudes is also corroborated by higher reduced state of glutathione. Signicant
(p B 0.05) increase in oral characteristics like inorescence and spike length was observed at Leh. Chemical
investigation of essential oil revealed the increased percentage of linalool and sclareol, two commercially
important constituents, in Leh. 52.9 and 39.4 % increase
was observed in the metabolic content of sclareol in Leh as
Communicated by L. Bavaresco.
& Dhiraj Vyas
dhirajvyas@rediffmail.com
1

Biodiversity and Applied Botany Division, Indian Institute of

Integrative Medicine (CSIR), Canal Road, Jammu 180001,
India

Instrumentation Division, Indian Institute of Integrative

Medicine (CSIR), Canal Road, Jammu 180001, India

Academy of Scientic and Innovative Research, Indian

Institute of Integrative Medicine (CSIR), Canal Road,
Jammu 180001, India

compared to their values in Jammu and Srinagar, respectively. Higher oral biomass and qualitative increase in
essential oil suggest that cold arid Himalayan region can be
exploited for commercial cultivation of clary sage.
Keywords Antioxidant enzymes  Essential oil  Light
response curve  Radical scavenging activity  Redox
metabolites  Sclareol

Introduction
Salvia sclarea Linn. (family Lamiaceae) is an important
plant native to the Northern Mediterranean region, and is
largely cultivated in Europe (France, Hungary, Bulgaria,
Turkey) and North America. It is also known as clary
sage and grown for its essential oil that has been traditionally used in food, avor, pharmaceutical, cosmetic
industry (Kumar et al. 2013; Gross et al. 2013; Pesic and
Bankovic 2003) and widely used in aromatherapy (Setzer
2009). Its oral extracts are widely used as avor additive
for soft and alcoholic beverages, extensively used in frozen
dairy desserts, candy, baked goods, gelatins, puddings,
condiments and salads. It is also consumed as tea in Turkey, where it is known as misk sage tea (Yalcin et al.
2011). Clary sage seeds have been found rich in fatty acids
and contained high levels of antioxidant and antiradical
activities, making them ideal for use as nutraceuticals
(Tulukcu et al. 2012). Recent reports suggest that clary
sage is found to have neuro-protective (Asadi et al. 2010),
anti-microbial (Kuzma et al. 2007), anti-depressant (Seol
et al. 2010) and anti-cancer (Noori et al. 2010) activities.
Most of the health benetting properties in any plant/plant part is attributed to the antioxidative potential of the
constituents (Halliwell 2011). Hence, there is increasing

123

132 Page 2 of 10

interest in naturally occurring antioxidants for use in food

and pharmaceutical industry to replace the synthetic
antioxidants (Kaur et al. 2013a). Earlier, several reports on
the evaluation of antioxidant activities of Salvia sclarea
have suggested it to be an excellent source of antioxidants
(Tepe et al. 2006; Miliauskas et al. 2004). It was even
argued that its antioxidant potential is comparable to atocopherol (Couladis et al. 2003).
The yield and composition of clary sage essential oil
have been determined in inorescence at the full owering
stage and its main constituents include linalyl acetate,
linalool, and germacrene D (Dzamic et al. 2008; Carrubba
et al. 2002). Clary sage, however, is cultivated for the
extraction of sclareol, a diterpene diol that mainly accumulates in the glandular secretory trichomes of ower
calyces (Laville et al. 2012). Sclareol is used as a starting
product in industrial synthesis of Ambrox, a basic ingredient of most modern amber-based fragrances. As far as
yield and quality of essential oil constituents in Salvia
sclarea is concerned, varying degree of differences have
been reported under normal and stressed conditions (Dzamic et al. 2008; Ben Taarit et al. 2011; Verma 2010;
Sharma and Kumar 2012; Paknejadi et al. 2012). This has
largely been attributed to the cultivation conditions as with
all aromatic plants (Peana et al. 1999), and led to variable
production of sclareol. Hence, a need is always felt for
sites/conditions producing higher essential oil content.
Since genes and enzymes responsible for sclareol
biosynthesis have not been described fully (Caniard et al.
2012), standardization/optimization of cultivation conditions/sites remain the most viable option for increasing its
yield. We have recently shown this strategy to be effective
for secondary metabolite production in Withania somnifera
(Kumar et al. 2012). The cropping technique recommended
for Salvia sclarea to obtain high biomass, requires rather
high amounts of water and nutrients (Bruni 1999), but its
natural arido-resistant characteristics have allowed
exploitation in other environments such as the semi-arid
Mediterranean (Carrubba et al. 2002). As such genus Salvia
consists of more than 900 species that are present widely in
subtropical and temperate regions (Walker and Sytsma
2007), thus, making it quite adaptive to a larger area.
Keeping in view the use of clary sage in food and avor
industry, any improvement in quantity and quality of the
essential oil would be desirable. Environmental conditions
are known to play a key role in dening distribution of
plants. The current climate change scenario also governs
changes in environmental factors including temperature,
light intensity and partial pressure of gases that will dene
new physiological and metabolic status in plants. However,
knowledge of how species adapt due to environmental
changes is still relatively limited. The present investigation
was therefore envisaged to look for newer cultivation sites,

123

Acta Physiol Plant (2015) 37:132

and to understand antioxidant and biochemical response to

the environmental conditions at these sites. Also, most of
the studies on this aspect have been conducted in
Mediterranean region and to the best of our knowledge is
the rst report on changes in antioxidant potential and
essential oil at different climates of the Western Himalayas. Results thus obtained would help to develop these as
cultivation sites in future. Baseline data, thus generated
would help in understanding biochemical adaptation and
promote Salvia sclarea at newer locations for food and
aroma industry.

Materials and methods

Planting material
Salvia sclarea L. seeds were procured from the seed bank
of Indian Institute of Integrative Medicine (IIIM), Jammu,
where these were stored under standard conditions. Same
set of seeds was sown at three different chosen locations
viz. Jammu (32430 N, 74540 E, 305 m asl), Srinagar
(34500 N, 74470 E, 1730 m asl) and Leh (34100 N,
77400 E, 3505 m asl) representing sub-tropical, temperate
and cold arid climate, respectively. Sowing of seeds was
done in the main growing season for optimum plant
growth, i.e., January (Jammu), March (Srinagar) and May
(Leh).
Morpho-physiological characteristics
Plant morphological characteristics like height, leaf
dynamics, as well as oral characters like number of spikes
per plant and inorescence length, were determined in
randomly selected plants (n C 25) using standard procedures. Readings were taken at owering stage in all the
three locations during the month of May (Jammu), July
(Srinagar) and September (Leh). Physiological robustness
of the plants at different locations was determined by light
response curves. Red/blue LED light source was used for
measurement of leaf photosynthesis of different photosynthetic photon ux density (PPFD) levels ranging from
high (3000 lmol m-2 s-1) to low (0 lmol m-2 s-1) at
250 lmol m-2 s-1 intervals using 6400 XT photosynthesis
system (LI-COR Inc., Lincoln NE, USA) at an ambient
CO2 concentration of 380 lmol mol-1.
Healthy leaves from a similar leaf position were illuminated at highest PPFD till they attained steady state
levels, and irradiance was then changed in a stepwise
reduction. The light response curve was tted using the
rectangular hyperbola MichaelisMenten based model to
describe the relationship between photosynthesis and light
(Henley 1993). Initial slope of the light response curve or

Acta Physiol Plant (2015) 37:132

apparent quantum yield (/i) and light-saturated net rate of

photosynthesis (Pmax) were estimated from light response
curves using the solver function of Microsoft Excel in
routines provided by Lobo et al. (2014).
Antioxidant analysis
One gram of leaves from all three locations was crushed
using liquid nitrogen. Extracts were prepared using 10 mL
of 80 % methanol at 40 C by continuous stirring for 8 h.
This was repeated twice; extracts were pooled, ltered, and
dried using rotary vacuum concentrator. The dried extracts
were nally dissolved in methanol and diluted for various
working concentrations for antioxidant assays. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and
reducing power assay was determined as described earlier
(Guleria et al. 2011). Super oxide anion (O2-) scavenging
activity was performed based on NBT reduction assay
essentially as described earlier (Vyas and Kumar 2005).
For hydroxyl radical induced DNA breakage in plasmid
pUC 18, 5 lL of the above mentioned extract was mixed
with freshly prepared Fentons reagent (50 mM phosphate
buffer, 1.6 mM FeSO4, 3 mM EDTA and 3 mM H2O2) and
1 lL of pUC 18 plasmid (250 ng). The reaction mixture
was incubated for 30 min at 37 C and plasmid DNA
products were visualized (Kaur et al. 2013a).
Redox metabolites
Reduced glutathione (GSH), oxidized glutathione (GSSH)
and pyridine molecules [NAD?(H) and NADP?(H)] were
measured based on preferential acidalkali destruction of
reduced-oxidized forms using coupled recycling assay as
described earlier (Kaur et al. 2013b). Briey, 500 lL, acid
crushed supernatant was neutralized (pH 56) with 0.2 N
NaOH. 20 lL of neutralized sample was incubated in
assay buffer consisting of 0.2 M NaH2PO4 (pH 7.5),
0.5 mM EDTA, 0.5 mM NADPH and 0.6 mM DTNB
(5,50 -dithiobis-2-nitrobenzoic acid). The reaction was initiated by the addition of 0.2 units of glutathione reductase
and increase in A412 was monitored for 5 min to obtain
GSH. Pre-treatment of extract aliquots with 2-vinylpyridine was used to get the oxidized form. Experimental
rates were derived from standard curve made for
0500 pmol of GSH using an initial change of rate. For
NAD, the assay buffer consisted of 0.1 M Hepes (pH 7.5),
2 mM EDTA, 0.12 mM DCPIP (2,6-dichlorophenol
indophenol), 2 mM PMS (phenazine methosulfate) and 25
units alcohol dehydrogenase (ADH). The reaction was
started using appropriate volume of absolute ethanol. For
NADP, the assay buffer had 1 mM glucose-6-phosphate
and 2 units of glucose 6-phosphate dehydrogenase instead
of ethanol and ADH.

Page 3 of 10

132

Antioxidant enzymes activity

Activity of antioxidant enzymes, namely superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase
(CAT) and glutathione reductase (GR) was analyzed using
established protocols as published earlier (Abrol et al.
2012). The damage incurred to cells by radical mediated
lipid peroxidation was also assessed in a plate reader using
malondialdehyde content following exposure to thiobarbituric acid (Heath and Packer 1968).
Analysis of marker compounds
For identication and quantication of marker compounds
sclareol and linalool, fresh inorescences (500 g) above
owering nodes were harvested. The oil was extracted
from inorescences by hydro-distillation in a Clevengertype apparatus. Oils were dried over anhydrous sodium
sulfate and stored in sealed dark glass vials at low temperature (0 to -4 C) prior to analysis.
GCMS analysis of essential oils was performed on a
Varian mass spectrometer-4000 series system tted with a
CP-SIL 8 CB column (30 9 0.32 mm ID, lm thickness
1 lm) (Verma et al. 2010). Temperature programming of
column oven was performed at 60 to 240 C with
3 C min-1 rising rate. Helium was used as the carrier gas
at a ow rate of 1 ml min-1. Mass spectra were recorded
over 50400 amu range at one scan per second with E.I. at
1570 eV. Identication of peaks was carried out by
comparison with authentic reference compounds and
comparison of Kovat retention indices from C9 to C21
alkanes with literature values, and comparison of the mass
spectra with those reported in the NIST and WILEY online
libraries and those of published in literature.
Statistical analysis
All the physiological and biochemical parameters were
conducted in triplicate on three or more biological replicates (n C 3). One way analysis of variance followed by a
Tukey HSD post hoc test was conducted to identify the
signicant differences (p B 0.05) between various locations. ANOVA was performed using IBM SPSS statistics
program version 20 (SPSS Inc., USA).

Results and discussion

Due to difference in environmental conditions, the growing
vegetative season varies from January to March, March to
June and May to July for Jammu, Srinagar, and Leh,
respectively. Antioxidant and physiochemical measurements were taken 60 days after plantation. After owering,

123

132 Page 4 of 10

Acta Physiol Plant (2015) 37:132

harvesting of inorescence at all three locations were done

for estimation of oil content during the rst week of May
(Jammu), July (Srinagar) and September (Leh). Weather
conditions pertaining to the growth period at all three
locations are shown in Table 1.
Morpho-physiological traits at different locations
The mean performance of vegetative and oral characteristics is given in Table 2. Our results showed that the mean
vegetative biomass of the plant was highest at Srinagar.
This was also evident from plant height and number of
leaves per plant at this location. Leaf area was found signicantly lower (p B 0.05) at Leh. Leaf area was found to
be reduced by about 48.6 % in Salvia plants grown at Leh
(3505 m) with regards to those grown at Jammu (305 m).
As far as leaf size is concerned, upland plants have been
known to have smaller leaves, which have been attributed
to lower air temperature at higher elevations (Kao and
Chang 2001). Although light is needed for photosynthesis,
Table 1 Climatic conditions at
three different experimental
locations

the absorption of high light can lead to increased production of highly reactive intermediates and byproducts that
can potentially cause photo-oxidative damage and inhibit
photosynthesis (Li et al. 2009). The smaller leaf area might
also act as an adaptive strategy to avoid absorption of light
photons. However, the oral portion in terms of the number
of spikes, spike length and inorescence length was found
signicantly higher (p B 0.05) at Leh than Srinagar and
Jammu. The length of inorescence was about 35 % higher
in Leh than in Srinagar suggesting oral supremacy in cold
arid climate. This is of particular importance as inorescence is used for extraction of essential oil in this crop.
Unlike Salvia, reduction in the number of owers was
found in Hypericum during elevational gradient (Roblek
et al. 2008).
Photosynthetic parameters derived from the light
response curve show several climate dependent trends (Fig. 1). At all light levels, photosynthesis of plants
from Srinagar was greater than that of plants growing in the
other two sites. This effect was noteworthy at saturating

Jammu (sub-tropical)
Altitude (meter above sea level)
Longitude

Srinagar (temperate)

Leh (cold arid)

305

1730

3505

32430 N

34500 N

34100 N

7454 E

7447 E

77400 E

Max.

25.6 2.3

21.5 3.2

20.6 1.7

Min.

9.6 1.6

8.7 1.3

6.6 2.2

Max.

38.2 1.9

31.0 2.4

23.0 2.2

Min.

21.1 2.9

18.0 2.5

7.5 1.6

Latitude

Mean temperature (C)

Vegetative

Flowering

Mean relative humidity (%)

Vegetative

66.6 5.6

64.0 5.8

53.8 6.3

Flowering

29.5 4.9

64.5 6.3

59.7 4.6

56.7
28

70.5
59.5

9.0
12.0

Mean precipitation (mm)

Vegetative
Flowering

Table 2 Morphometric and

oral characteristics of Salvia
sclarea cultivated at different
locations

Characters

Jammu

Srinagar

Plant height (cm)

44.8 0.8

105.18 7.61

Average number of leaves/plant

Average leaf area (cm2)

15.18 0.5c
268.6 22.8a

44.20 5.57a
244.0 19.5a

28.49 2.95b
108.6 5.5b

No. of spikes/plant

4.66 1.20c

Spike length (cm)

Leh
a

93.11 2.5b

15.33 4.42b

31.03 1.87a

12.03 0.72

34.63 1.36

50.34 4.98a

Inorescence length (cm)

13.34 0.91

50.0 3.25

76.16 1.90a

Essential oil (%) FW inorescence

0.130.23

0.210.28

0.290.35

Each value is a mean of 25 individual replications. Different alphabets (a, b, c) represents statistically
signicant values (p B 0.05) as determined by Tukeys pairwise comparison test

123

Acta Physiol Plant (2015) 37:132

Page 5 of 10

132

Photosynthesis ( mol CO2 m-2 s-1)

40
Pmax = 32.3
i = 0.0913

30
Pmax = 20.7
i = 0.0904

20
Pmax = 18.4
i = 0.0957

10

0
-500

500

1000

1500

2000

PPFD ( mol Photon

2500

3000

3500

m-2s-1)

-10

Antioxidant and redox characteristics

The better photosynthetic rate of plants at Srinagar ideally
makes them less vulnerable to the escape of electrons and,
thereby, lesser potential oxidative damage. In fact, plants
grown at Srinagar showed lesser lipid peroxidation as
compared to the other two locations (Fig. 2A). GR had
signicantly (p B 0.05) higher activities in Srinagar than
Jammu and Leh (Fig. 3D). Its activity decreased to 68.8
and 39.7 % in Jammu and Leh, respectively. GR maintains
the balance towards GSH, and can well be used as a marker
to assess biochemical response of the plant towards environmental conditions (Foyer and Noctor 2005). SOD, the

0.075

Lipid Peroxidaon (A 532)

light levels. Pmax of plants grown at temperate region was

75 % more than sub-tropical plants of Jammu and 56 %
more than those of cold arid plants of Leh. Initial slopes of
the light response curves representing quantum photosynthetic efciency from Jammu, Srinagar and Leh were
0.0957, 0.0913 and 0.0904, respectively. Photosynthetic
efciency follows the altitude dependent trend along with
increasing light intensity. Higher entrapment of photons
and lower photosynthetic rates make an ideal case of
leakage of electrons to alternative sources such as O2.
There are contrasting reports on the net assimilation rates
of CO2 assimilation in plants from different altitudes (Shi
et al. 2006); however, in case of Salvia in Srinagar, in this
study, better photosynthetic capacity results in higher
biomass and vegetative growth (Koul et al. 2013).

of leaf photosynthesis at different PPFD levels ranging from high

(3000 lmol m-2 s-1) to low (0 lmol m-2 s-1) at 250 lmol m-2 s-1
intervals using 6400 XT photosynthesis system at an ambient CO2
concentration of 380 lmol mol-1. Values are mean of at least three
independent light curves at each location (n C 3)

A
a

0.05
b
b

0.025

0
0.5

Reducing Power (A 700)

Fig. 1 Photosynthetic light response curves of Salvia sclarea grown

at three cultivation sites of Jammu (closed triangle), Srinagar (closed
circle) and Leh (closed square). Pmax light-saturated net rate of
photosynthesis, /i initial slope of the light response curve or apparent
quantum yield. Redblue LED light source was used for measurement

B
a
b

0.25

Jammu

Srinagar

Leh

Fig. 2 Lipid per oxidation mediated cellular damage (A), and

reducing power (B) in leaf extract of Salvia sclarea plants grown at
three cultivation sites of Jammu, Srinagar and Leh. All the values are
mean of at least three independent readings (n C 3). Different
alphabets (a, b, c) represents statistically signicant values (p B 0.05)
as determined by Tukeys pairwise comparison test

rst enzyme that catalyses the dismutation of superoxide

anion, decreases progressively along the altitude (Fig. 3A).
Higher activity of SOD at Jammu during this study does

123

A
a

SOD (Units mg-1 FW)

0.4

0.3

0.2
c
0.1

APX (mol of ASC oxidised min-1 mg-1 FW)

10

7.5
a

b
b

2.5

Jammu

Srinagar

Leh

GR (mol of DTNB reduced mg-1 FW)

0.5

Acta Physiol Plant (2015) 37:132

CAT (mol H2O2 decomposed min-1 mg-1 FW)

132 Page 6 of 10
0.5

0.4

0.3

a
a
a

0.2

0.1

30

a
20
b
10

Jammu

Srinagar

Leh

Fig. 3 Activities of key antioxidant enzymes namely SOD (A), CAT

(B), APX (C) and GR (D) in leaf extracts of Salvia sclarea grown at
three cultivation sites of Jammu, Srinagar and Leh. Values are
mean SD (n C 3). 1 unit of superoxide dismutase is described as

the amount of enzyme required to get 50 % inhibition in the reduction

of nitro blue tetrazolium. Different alphabets (a, b, c) represents
statistically signicant values (p B 0.05) as determined by Tukeys
pairwise comparison test

not corroborate with the fact that lipid peroxidation was

also found highest here. It therefore suggests a buildup of
the product of dismutation, H2O2, which is not effectively
removed from the cytoplasm. Catalase, which is mainly
responsible for scavenging H2O2 from the cytoplasm
(Ishikawa and Shigeoka 2008) do not show signicant
difference in their activities at all the three cultivation sites
(Fig. 3B).
The maximum value of antioxidant capacity was found
in plants grown at Srinagar. Percentage radical scavenging
activity based on DPPH assay was found to be 71.9, 89.5
and 80.4 % for Jammu, Srinagar and Leh, respectively
(Fig. 4A). Thus, it was found to be 24.4 % higher at Srinagar than at Jammu. O2- scavenging activity was performed to check the ability of plant extracts to quench free
radicals. Leaf extract from Srinagar have shown remarkable superoxide radical scavenging. Percentage inhibition
for O2- scavenging activity was found to be as: 91.0 %
(Srinagar) [55.4 % (Leh) [16.3 % (Jammu) (Fig. 4B).
Apart from singlet oxygen, superoxide radicals are the rst
products of oxidative chemistry that upon reaction, can

further give rise to deleterious hydroxyl radical (Halliwell

2012). The superoxide radical scavenging activity in Srinagar is in fact higher than that shown by the leaves of
Lepidium latifolium at Leh (Kaur et al. 2013a), which
makes it an effective antioxidant. Signicant differences
obtained at three locations suggest that antioxidant status
mediated by phenols and avonoids among other factors
might play an important role in plantenvironment interactions (Appel 1993). Effects of phenols and avonoids are
generally associated with their redox properties, which
allow them to act as reducing agents, hydrogen donors, and
singlet oxygen quenchers (Soobrattee et al. 2005).
A signicant amount of DNA protective activity was
observed among the leaf extracts of Salvia sclarea. As
observed in Fig. 5, supercoiled and open circular forms are
clearly visible where extracts have been added, in comparison to the control samples where no extracts have been
added. When DNA protective activity was compared
among the extracts from different sites, the total amount of
DNA (both the forms) was found higher in Srinagar and
Leh. This is in accordance with the DPPH radical

123

Acta Physiol Plant (2015) 37:132

A
a
a

75

B
50

25

Superoxide Radical Scavenging Acvity

(% Inhibion)

Plasmid Control

100

B
a

b
50

25

Jammu

Srinagar

Leh

Fig. 4 Antioxidant activity of methanolic plant extracts of Salvia

sclarea grown at three cultivation sites of Jammu, Srinagar and Leh.
A, % Inhibition of DPPH free radicals scavenging activity; B,
superoxide scavenging activity. All the values are mean of at least
three independent readings (n C 3). Different alphabets (a, b, c)
represents statistically signicant values (p B 0.05) as determined by
Tukeys pairwise comparison test

scavenging activity as observed earlier. These results

suggest that the order of antioxidant capacity of the
plants growing in these three locations would be
Srinagar [ Leh [ Jammu.
Total glutathione metabolic content in the present study
decreased by 7.9 and 10.2 %, respectively, for Srinagar and
Leh vis-a`-vis Jammu. Although the total glutathione was
higher in Jammu, the percentage of reduced form that tilts
the balance toward the reducing environment was higher in
Srinagar (86.5 %) and Leh (83.7 %) as compared to
Jammu (70.7 %). GR helps in maintaining the reduced
form (GSH) of glutathione at higher altitudes, thereby,
maintaining the intracellular redox homeostasis and growth
(Kaur et al. 2013b). This is also evident from the morphological parameters (Table 2). The depletion in reduced
form has often been associated with increased sensitivity
towards oxidative stress (Noctor et al. 1998; Foyer and
Noctor 2005), as seen in the lipid peroxidation values
(Fig. 2A). The reduced form of NAD was also found signicantly (p B 0.05) higher in Srinagar (62.9 %) and Leh

Leh

150000

100000

50000

75

Jammu Srinagar

132

Linear form
Nicked form
Circular form

Integrated Density Value

DPPH Radical Scavenging Acvity

(% Inhibion)

100

Page 7 of 10

Plasmid

Control

Jammu

Srinagar

Leh

Fig. 5 DNA protective activity of methanolic plant extracts of Salvia

sclarea grown at three cultivation sites Jammu, Srinagar and Leh. A,
1.2 % agarose gel stained with ethidium bromide visualized under
UV whereas; B, integrated density values (IDV) of bands using
densitometry analysis

(92.2 %) suggesting the ability of Salvia sclarea to maintain the reducing environment in potentially oxidizing
conditions (Fig. 6B). At higher altitudes, the total pool of
NADP, however, declines due to increasing light intensities
that causes progressive over-reduction at the reducing side
of PS I in chloroplast (Allan et al. 2009). Figure 6C
shows that NADP pool was found signicantly (p B 0.05)
higher in Jammu (14.24 pmol mg-1) than Srinagar
(6.55 pmol mg-1) and Leh (4.90 pmol mg-1).
An increase in metabolic content of NAD pool was
observed in higher altitudes with respect to lower altitude
(Fig. 6B). Recent studies on role of NAD in mediating
signaling and post-translational modications of target
proteins suggest that higher altitudes warrant the need for
higher metabolic reconguration to adaptation of plants
(Ying et al. 2003; Allan et al. 2009). Metabolic content of
these pyridine nucleotides suggests that Salvia experiences
stressful environment at higher altitudes; however, due to
its robust redox and antioxidant mechanism, it maintains
better growth at these locations.
Cold arid region shows better quantity and quality
of essential oil in S. sclarea
Total yield (%) of essential oil obtained from hydro-distillation of ower inorescence at Jammu, Srinagar and
Leh were found to be in the range 0.130.23, 0.210.28 and
0.290.35 %, respectively, on fresh weight basis of the
inorescence. This showed direct proportionality with
altitudinal gradient showing the maximum oil percent

123

Glutathione (Pico moles mg-1 FW)

132 Page 8 of 10

500

400

300

a,b
a

200

b
a

100

b
0
30

NAD (Pico moles mg-1 FW)

Acta Physiol Plant (2015) 37:132

B
a

a,b
20

10

NADP (Pico moles mg-1 FW)

20

C
a

15

a
10

b
b

a
0

Jammu

b
c

b
Srinagar

b
Leh

Fig. 6 Metabolic content of different redox buffer in various states of

oxidation and reduction. Panel represents GSH (A), NAD (B) and
NADP (C) content in leaves of Salvia sclarea grown at three different
cultivation sites of Jammu, Srinagar and Leh. Black bar represents
total metabolic content of the metabolites; white bar represents the
oxidized forms and grey bars represent reduced forms of the
metabolites. All the values are mean of at least three independent
readings (n C 3). Different alphabets (a, b, c) represents statistically
signicant values (p B 0.05) as determined by Tukeys pairwise
comparison test

(0.35 %) at the highest altitude (Table 2). Similarly, in

another study on Salvia sclarea, the percentage essential oil
component was found highest in the plants grown at the
higher altitude, and the lower temperatures during the
cropping season was found to be the reason for this
(Yaseen et al. 2014). Earlier, the location had no signicant
effect on growth characteristics of the two thyme species,
but the quantity and quality of their essential oils were
different in different regions (Pirbalouti et al. 2013).

123

Phenological changes have shown to alter the oil percentage in Salvia (Pesic and Bankovic 2003). Due to environmental conditions, the overall growth period varies at all
the three locations with Leh showing smallest growth
period followed by Srinagar. The maturity of the stages
would therefore be in order of Leh [ Srinagar [ Jammu.
Earlier, the initial seed ripening stage has shown 2.73 times
more oil than full owering stage (Carrubba et al. 2002).
The oil percentage was also corroborated with the higher
oral biomass along the altitude (Table 2), thereby,
increasing the quantitative yield of oil per unit area.
The percentage of two marker compounds sclareol and
linalool were analyzed in essential oil of three locations for
qualitative determination of its components (Fig. 7A). This
ecotype of Salvia sclarea grown in the Western Himalayan
region was found to contain higher amounts of monoterpenoid alcohol, linalool, than obtained in the Mediterranean region (Souleles and Argyriadou 1997; Carrubba
et al. 2002). Percentage of Linalool was found to be 30.50,
41.83 and 46.56 % in Jammu, Srinagar and Leh, respectively (Fig. 7B). Another marker compound, sclareol was
39.2 and 52.8 % higher in Leh (2.83 %) than at Srinagar
(2.03 %) and Jammu (1.85 %), respectively (Fig. 7C). It
was found that both of these compounds were highest at
Leh. Earlier literature suggests contrasting reports on the
essential oil components in Salvia sps. It was found to be
affected by cultivation sites and season (Perry et al. 1999;
Souleles and Argyriadou 1997; Pesic and Bankovic 2003;
Dzamic et al. 2008), whereas no such deviation was
observed in plants grown in Southern Uzbekistan (Dzumayev et al. 1995). In our study, however, we found that
there is a signicant increase in the percentage of linalool
and commercially important sclareol in Leh. It has been
earlier reported that Salvia sclarea is a xerophytic biennial
plant and fares well in semi-arid conditions (Carrubba et al.
2002), which supports our data.
In conclusion, this study revealed that environmental
conditions at three distinct cultivation sites greatly vary the
antioxidant and physio-chemical response in S. sclarea L.
Photosynthetic efciency as suggested by initial slope and
saturation points of light curves suggest that light intensities are optimally utilized in photochemistry in Srinagar.
This leads to lesser damage caused by lipid peroxidation
and, thereby, maintaining higher vegetative biomass.
Although, Srinagar (temperate) was found for the most
suitable site based on the photosynthetic light response and
vegetative growth, the goal of the study was to nd locations that produce higher quantity and better quality of
essential oil. High oral density and the content of two
commercially important marker compounds, linalool and
sclareol, were found signicantly higher at Leh (cold arid
region). In view of climate change scenario, these results
have wider implications and suggest that cold arid

Acta Physiol Plant (2015) 37:132

Page 9 of 10

132

50

a,b

25

Sclareol (% of total essenal oil)

Linalool (% of total essenal oil)

75

4
3
2

a
b

1
0

Jammu

Srinagar

Leh

Jammu

Srinagar

Leh

Fig. 7 Analysis of marker chemical compounds in Salvia sclarea

grown at three different cultivation sites of Jammu, Srinagar and Leh
using GCMS. Representative sample of chromatogram showing
various compound peaks (A); percentage linalool content in essential
oil analysis (B); percentage sclareol content in essential oil analysis of

Salvia sclarea (C). All the values are mean of at least three
independent readings (n C 3). Different alphabets (a, b, c) represents
statistically signicant values (p B 0.05) as determined by Tukeys
pairwise comparison test

Himalayan region can be exploited for commercial cultivation of clarysage.

investigation of neuroprotection by six Salvia species from Iran:

a comparative study. Food Chem Toxicol 48(5):13411349
Ben Taarit M, Kamel M, Karim H, Brahim M (2011) Physiological
changes and essential oil composition of clary sage (Salvia
sclarea L.) rosette leaves as affected by salinity. Acta Physiol
Plant 33:153162
Bruni A (1999) Farmacognosia Generale e Applicata. Piccin (ed),
Padova, pp 377378
Caniard AM, Zerbe P, Legrand S, Cohade A, Valot N, Magnard JL,
Bohlmann J, Legendre L (2012) Discovery and functional
characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume
manufacture. BMC Plant Biol 12:119
Carrubba A, Torre R, Piccaglia R, Marotti M (2002) Characterization
of an Italian biotype of clary sage (Salvia sclarea L.) grown in a
semi-arid Mediterranean environment. Flavour Frag J 17:191194
Couladis M, Tzakou O, Verykokidou E, Harvala C (2003) Screening
of some Greek aromatic plants for antioxidant activity. Phytother
Res 17(2):194195
Dzamic AN, Sokovic M, Ristic M, Grujic-Jovanovic S, Vukojevic J,
Marin PD (2008) Chemical composition and antifungal activity
of Salvia sclarea (Lamiaceae) essential oil. Arch Biol Sci
60:233237
Dzumayev KK, Tsibulskaya IA, Zenkevich IG, Tkachenko KG,
Satzyperova IF (1995) Essential oils of Salvia sclarea (L.)
produced from plants grown in Southern Uzbekistan. J Essen Oil
Res 7:597604
Foyer CH, Noctor G (2005) Oxidant and antioxidant signalling in
plants: a re-evaluation of the concept of oxidative stress in a
physiological context. Plant Cell Environ 28:10561077
Gross M, Nesher E, Tikhonov T, Raz O, Pinhasov A (2013) Chronic
food administration of Salvia sclarea oil reduces animals
anxious and dominant behavior. J Med Food 16(3):216222

Author contribution statement D.V. and S.K. designed

the research; T.K., H.A.B., R.B., K.B. and A.K. performed
the research; D.V., T.K. and A.K. analyzed the data, and
D.V. and T.K. wrote the paper.
Acknowledgments Authors thank the Director, IIIM, Jammu for
providing necessary facilities to carry out the work. Authors are
grateful to the Council of Scientic and Industrial Research (CSIR),
Government of India, for nancial support under CSIR- networking
project (BSC-0109) on Plant Diversity: Studying adaptation biology
and understanding/exploiting medicinally important plants for useful
bioactives (SIMPLE). TK, HAB and RB acknowledge the nancial
assistance provided by CSIR in form of JRF/SRF fellowship.

References
Abrol E, Vyas D, Koul S (2012) Metabolic shift from secondary
metabolite production to induction of anti-oxidative enzymes
during NaCl stress in Swertia chirata Buch.-Ham. Acta Physiol
Plant 34:541546
Allan WL, Clark SM, Hoover GJ, Shelp BJ (2009) Role of plant
glyoxylate reductases during stress: a hypothesis. Biochem J
423:1522
Appel HM (1993) Phenolics in ecological interactions: the importance of oxidation. J Chem Ecol 19(7):15211552
Asadi S, Ahmadiani A, Esmaeili MA, Sonboli A, Ansari N,
Khodagholi F (2010) In vitro antioxidant activities and an

123

132 Page 10 of 10
Guleria S, Tiku AK, Singh G, Vyas D, Bhardwaj A (2011)
Antioxidant activity and protective effect against plasmid DNA
strand scission of leaf, bark, and heartwood extracts from Acacia
catechu. J Food Sci 76:959964
Halliwell B (2011) Free radicals and antioxidantsquo vadis? Trends
Pharmacol Sci 32(3):125130
Halliwell B (2012) Free radicals and antioxidants: updating a personal
view. Nutr Rev 70(5):257265
Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts
I. Kinetics and stoichiometry of fatty acid peroxidation. Arch
Biochem Biophys 12:189198
Henley WJ (1993) Measurement and interpretation of photosynthetic
light-response curves in algae in the context of photoinhibition
and diel changes. J Phycol 29:729739
Ishikawa T, Shigeoka S (2008) Recent advances in ascorbate
biosynthesis and the physiological signicance of ascorbate
peroxidase in photosynthesizing organisms. Biosci Biotechnol
Biochem 72:11431154
Kao WY, Chang KW (2001) Altitudinal trends in photosynthetic rate
and leaf characteristics of Miscanthus populations from central
Taiwan. Aus J Bot 49:509514
Kaur T, Hussain K, Koul S, Vishwakarma R, Vyas D (2013a)
Evaluation of nutritional and antioxidant status of Lepidium
latifolium Linn.: a novel phytofood from Ladakh. PLoS One
8(8):19
Kaur T, Bhat HA, Raina A, Koul S, Vyas D (2013b) Glutathione
regulates enzymatic antioxidant defence with differential thiol
content in perennial pepperweed and helps adapting to extreme
environment. Acta Physiol Plant 35(8):25012511
Koul S, Kaur T, Bhat R, Bindu K, Kumar A, Kitchlu S, Vyas D
(2013) Morpho-chemical characteristics of Salvia sclarea L. at
two different locations in Jammu and Kashmir. Res Rev Bio
Biosci 1:17
Kumar A, Abrol E, Koul S, Vyas D (2012) Seasonal low temperature
plays an important role in increasing metabolic content of
secondary metabolites in Withania somnifera (L.) dunal and
affects the time of harvesting. Acta Physiol Plant 34:20272031
Kumar R, Sharma S, Pathania V (2013) Effect of shading and plant
density on growth, yield and oil composition of clary sage
(Salvia sclarea L.) in north western Himalayas. J Essen Oil Res
25(1):2332
Kuzma , Roz_ alski M, Walencka E, Roz_ alska B, Wysokinska H
(2007) Antimicrobial activity of diterpenoids from hairy roots of
Salvia sclarea L.: salvipisone as a potential anti-biolm agent
active against antibiotic resistant Staphylococci. Phytomedicine
14(1):3135
Laville R, Castel C, Filippi JJ, Delbecque C, Audran A, Garry PP,
Legendre L, Fernandez X (2012) Amphilectane diterpenes from
Salvia sclarea: biosynthetic considerations. J Nat Prod 75:121126
Li Z, Wakao S, Fischer BB, Niyogi KK (2009) Sensing and
responding to excess light. Annu Rev Plant Biol 60:239260
C, Pereira WE,
Lobo FDA, de Barros MP, Dalmagro HJ, Dalmolin A
de Souza EC, Vourlitis GL, Ortz CR (2014) Erratum to: tting
net photosynthetic light-response curves with Microsoft Excel
a critical look at the models. Photosynthetica 52:479480
Miliauskas G, Venskutonis PR, Van Beek TA (2004) Screening of
radical scavenging activity of some medicinal and aromatic plant
extracts. Food Chem 85(2):231237
Noctor G, Arisi ACM, Jouanin L, Kunert KJ, Rennenberg H, Foyer
CH (1998) Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants. J Exp Bot
49:623647
Noori S, Hassan ZM, Mohammadi M, Habibi Z, Sohrabi N,
Bayanolhagh S (2010) Sclareol modulates the Treg intra-tumoral
inltrated cell and inhibits tumor growth in vivo. Cell Immunol
263(2):148153

123

Acta Physiol Plant (2015) 37:132

Paknejadi M, Fatemeh F, Yousefzadi M (2012) Antimicrobial
activities of the essential oils of ve Salvia species from Tehran
province, Iran. J Paramed Sci 3:1218
Peana AT, Moretti MD, Juliano C (1999) Chemical composition and
antimicrobial action of the essential oils of Salvia desoleana and
S. sclarea. Planta Med 65:752754
Perry NB, Anderson RE, Brennan NJ, Malcolm H, Douglas A, Heaney
J, McGimpsey JA, Bruce M (1999) Essential oils from dalmatian
sage (Salvia ofcinalis L.): variations among individuals, plant
parts, seasons, and sites. J Agr Food Chem 47:20482054
Pesic PZ, Bankovic VM (2003) Investigation on the essential oil of
cultivated Salvia sclarea (L.). Flav Frag J 18:228230
Pirbalouti AG, Hashemib M, Ghahfarokhic FT (2013) Essential oil and
chemical compositions of wild and cultivated Thymus daenensis
Celak and Thymus vulgaris L. Ind Crops Prod 48:4348
Roblek M, Germ M, Sedej TT, Gaberscik A (2008) Morphological
and biochemical variations in St. Johns wort Hypericum
perforatum L. growing over altitudinal and UV-B radiation
gradients. Period Biol 110(3):257262
Seol GH, Shim HS, Kim PJ, Moon HK, Lee KH, Shim I, Suh SH, Min
SS (2010) Antidepressant-like effect of Salvia sclarea is
explained by modulation of dopamine activities in rats.
J Ethnopharmacol 130(1):187190
Setzer WN (2009) Essential oils and anxiolytic aromatherapy. Nat
Prod Commun 4(9):13051316
Sharma S, Kumar R (2012) Effect of nitrogen on growth, biomass and
oil composition of clary sage (Salvia sclarea L.) under mid hills
of north western Himalayas. Indian J Nat Prod Res 3:7983
Shi Z, Liu S, Liu X, Centritto M (2006) Altitudinal variation in
photosynthetic capacity, diffusional conductance and d13C of
buttery bush (Buddleja davidii) plants growing at high elevations. Physiol Plant 128:722731
Soobrattee MA, Neergheen VS, Luximon-ramma A, Aruoma OI,
Bahorun T (2005) Phenolics as potential antioxidant therapeutic
agents: mechanism and actions. Mutat Res 579:200213
Souleles C, Argyriadou N (1997) Constituents of the essential oil of
Salvia sclarea growing wild in Greece. Pharm Biol 35:218222
Tepe B, Sokmen M, Akpulat HA, Sokmen A (2006) Screening of the
antioxidant potentials of six Salvia species from Turkey. Food
Chem 95(2):200204
Tulukcu E, Yalcin H, Ozturk I, Sagdic O (2012) Changes in the fatty
acid compositions and bioactivities of clary sage seeds depending on harvest year. Ind Crop Prod 39:6973
Verma RS (2010) Chemical investigation of decanted and hydrophilic
fractions of Salvia sclarea essential oil. Asian J Trad Med
5:102108
Verma MK, Anand R, Chisti AM, Kitchlu S, Chandra S, Shawl AS,
Khajuria RK (2010) Essential oil composition of Artemisia
dracunculus L. (Tarragon) growing in KashmirIndia. J Essn Oil
Bear Plant 13:331335
Vyas D, Kumar S (2005) Purication and partial characterization of a
low temperature responsive Mn-SOD from tea [Camellia
sinensis(L.) O. Kuntze.]. Biochem Biophys Res 329:831838
Walker JB, Sytsma KJ (2007) Staminal Evolution in the Genus Salvia
(Lamiaceae): molecular phylogenetic evidence for multiple
origins of the stamina lever. Ann Bot 100:375391
Yalcin H, Ozturk I, Hayta M, Sagdic O, Gumus T (2011) Effect of
gamma-irradiation on some chemical characteristics and volatile
content of linseed. J Med Food 14(10):12231228
Yaseen M, Singh M, Ram D, Singh K (2014) Production potential,
nitrogen use efciency and economics of clarysage (Salvia
sclarea L.) varieties as inuenced by nitrogen levels under
different locations. Ind Crops Prod 54:8691
Ying W, Garnier P, Swanson RA (2003) NAD? repletion prevents
PARP-1-induced glycolytic blockade and cell death in cultured
mouse astrocytes. Biochem Biophys Res 308:809813