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Antioxidant and anticholinesterase evaluation of selected
Turkish Salvia species
Ilkay Orhan
a,*
, Murat Kartal
b
, Qamar Naz
c
, Asma Ejaz
c
,Gu
¨lderen Yilmaz
d
,
Yu
¨ksel Kan
e
, Belma Konuklugil
b
, Bilge S
ßener
a
, M. Iqbal Choudhary
c
a
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey
b
Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06110 Ankara, Turkey
c
H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, 75270 Karachi, Pakistan
d
Department of Pharmaceutical Botany, Faculty of Pharmacy, Ankara University, 06110 Ankara, Turkey
e
Department of Field Crops, Faculty of Agriculture, Selc¸uk University, 42070 Konya, Turkey
Received 24 November 2005; received in revised form 18 July 2006; accepted 13 October 2006
Abstract
Since Salvia species (Lamiaceae) have been recorded to be used against memory loss in European folk medicine, we herein examined
in vitro anticholinesterase and antioxidant activities of 56 extracts prepared with petroleum ether, chloroform, ethyl acetate and meth-
anol obtained from 14 Salvia species (Salvia albimaculata Hedge and Hub, Salvia aucheri Bentham var. canescens Boiss and Heldr, Salvia
candidissima Vahl. ssp. occidentalis,Salvia ceratophylla L., Salvia cryptantha Montbret and Bentham, Salvia cyanescens Boiss and Bal.,
Salvia frigida Boiss, Salvia forskahlei L., Salvia halophilaHedge, Salvia migrostegia Boiss and Bal., Salvia multicaulis Vahl., Salvia sclarea
L., Salvia syriaca L., Salvia verticillata L. ssp. amasiaca) growing in Turkey. The antioxidant activities were assessed by both chemical
and enzymatic methods against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and xanthine/xanthine oxidase (XO) system
generated superoxide anion radical inhibition. Anticholinesterase effect of the extracts was tested against both acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) at concentrations of 0.2 and 1 mg/ml using a microplate-reader assay based on the Ellman
method. Most of the extracts did not show any activity against AChE at 0.2 mg/ml, while the chloroform extracts had noticeable inhi-
bition against BChE between 47.7% and 74.7%. The most active extracts at 1 mg/ml for AChE inhibition were observed to be petroleum
ether extract of Salvia albimaculata (89.4%) and chloroform extract of Salvia cyanescens (80.2%), whereas ethyl acetate extracts of Salvia
frigida and Salvia migrostegia, chloroform extracts of Salvia candidissima ssp. occidentalis and Salvia ceratophylla, as well as petroleum
ether extract of Salvia cyanescens were found to inhibit potently BChE (92.2%, 89.6%, 91.1%, 91.3%, and 91.8%, respectively). Partic-
ularly, the ethyl acetate and methanol extracts were observed to be highly active against both DPPH and XO. Our data indicates that
nonpolar extracts of Salvia species for anticholinesterase activity and the polar extracts for antioxidant activity are worth further phy-
tochemical evaluation for identifying their active components.
2006 Elsevier Ltd. All rights reserved.
Keywords: Salvia; Lamiaceae; Acetylcholinesterase; Butyrylcholinesterase; Alzheimer’s disease; Antioxidant; DPPH; Free radical; Xanthine oxidase
1. Introduction
Alzheimer’s disease (AD) is a degenerative neurological
disorder characterized by senile plaques containing amy-
loid bprotein and loss of cholinergic neuromediators in
the brain (Lawrence & Shakian, 1998; Whitehouse et al.,
1982). The most remarkable biochemical change in AD
patients is a reduction of acetylcholine (ACh) levels in
the hippocampus and cortex of the brain (Jaen, Gregor,
Lee, Davis, & Emmerling, 1996). Therefore, inhibition of
acetylcholinesterase (AChE), the enzyme responsible for
hydrolysis of ACh at the cholinergic synapse, is currently
0308-8146/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2006.10.030
*
Corresponding author. Tel.: +90 312 2023186; fax: +90 312 2235018.
E-mail address: iorhan@gazi.edu.tr (I. Orhan).
www.elsevier.com/locate/foodchem
Food Chemistry 103 (2007) 1247–1254
Food
Chemistry
the most established approach to treating AD (Schneider,
1996; Tariot et al. galanthamine USA-10 study group,
2000). While AChE is found in all excitable tissue, whether
nerve or muscle, in most erythrocytes and in placental tis-
sue, BChE is present more commonly in the body including
within the central and peripheral nervous system, liver and
plasma (Massoulie, Pezzementi, Bon, Krejci, & Vallette,
1993). On the other hand, oxidative stress, caused by reac-
tive oxygen species (ROS), is known to cause the oxidation
of biomolecules leading to cellular damage. It is also spec-
ulated to be pathologically important in various neurode-
generative processes including cognitive deficits that
occur during normal cerebral aging, Alzheimer’s (AD),
and Parkinson’s diseases (Bastianetto & Quirion, 2002;
Behl & Moosman, 2002; Butterfield et al., 1999; Gray
et al., 2003; Jenner, 1996; Smith et al., 1996). Nowadays,
the most accepted theory about the disturbing effect of free
radicals in the process of aging was reported by Harman
(1956). Later on, it was also reported that oxidative stress
is associated with the pathogenesis of AD and cellular
characteristics of this disease are either causes or effects
of oxidative stress (Smith, Harris, Sayre, & Perry, 1997;
Smith et al., 1996; Vina, Lloret, Orti, & Alonso, 2004).
These evidences clearly show that oxidative stress, an early
event in AD, may play a key pathogenic role in the disease
(Zhu et al., 2004). Interestingly, intake of polyphenols
through diets rich in fruits, vegetables and beverages such
as red wine was stated to reduce incidence of certain age-
related neurological disorders including macular degenera-
tion and dementia (Bastianetto & Quirion, 2002;
Commenges et al., 2000). Therefore, these data suggest that
high dietary or supplemental consumption of antioxidants
in people may reduce the risk of AD.
On the other hand, Salvia species (sage) were reported to
be used for memory-enhancing purposes in European folk
medicine (Perry, Bollen, Perry, & Ballard, 2003). For this
purpose, we aimed to examine in vitro acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) inhibitory activ-
ities of 14 Salvia species (Salvia albimaculata Hedge and
Hub, Salvia aucheri Bentham var. canescens Boiss and
Heldr, Salvia candidissima Vahl. ssp. occidentalis,Salvia
ceratophylla L., Salvia cryptantha Montbret and Bentham,
Salvia cyanescens Boiss and Bal., Salvia frigida Boiss,
Salvia forskahlei L., Salvia halophilaHedge, Salvia migros-
tegia Boiss and Bal., Salvia multicaulis Vahl., Salvia sclarea
L., Salvia syriaca L., Salvia verticillata L. ssp. amasiaca)
growing in Turkey by the Ellman method. Besides, this
study was also designed to evaluate the antioxidant capac-
ity of the aforementioned Salvia species by the 1,1-diphe-
nyl-2-picrylhydrazyl (DPPH) radical-scavenging method
and xanthine/xanthine oxidase (XO) system generated
superoxide anion radical inhibition assay.
2. Materials and methods
2.1. Plant materials
Fourteen Salvia species (S. albimaculata Hedge and
Hub, S. aucheri Bentham var. canescens Boiss and Heldr,
S. candidissima Vahl. ssp. occidentalis,S. ceratophylla L.,
S. cryptantha Montbret and Bentham, S. cyanescens Boiss
and Bal., S. frigida Boiss,S. forskahlei L., S. halophila
Hedge, S. migrostegia Boiss and Bal., S. multicaulis Vahl.,
S. sclarea L., S. syriaca L., S. verticillata L. ssp. amasi-
aca) were collected throughout Turkey. Collection sites,
dates, and herbarium numbers (AEF) are listed in Table
1. The plants were identified by Prof. Dr. H. Duman
from the Department of Biology, Faculty of Art and Sci-
ence, Gazi University, Ankara, Turkey and Dr. G. Yil-
maz from the Department of Pharmaceutical Botany,
Faculty of Pharmacy, Ankara University, Ankara, Tur-
key. The voucher specimens were preserved at the Her-
barium of the Faculty of Pharmacy, Ankara University,
Ankara, Turkey. Only, S. forskahlei was identified by
Dr. S. Terzioglu of Department of Forest Botany, Fac-
ulty of Forestry, Karadeniz Technical University, Trab-
zon, Turkey. The voucher specimen of S. forskahlei is
preserved at the Herbarium of the Department of Phar-
macognosy, Faculty of Pharmacy, Gazi University,
Ankara, Turkey (Table 1).
Table 1
Collection sites, dates and herbarium numbers of Turkish Salvia species
Salvia species examined Collection site Collection date Herbarium number
S. albimaculata Hedge and Hub Balkusan-Ermenek, Konya June, 2005 AEF 23520
S. aucheri Bentham var. canescens Boiss and Heldr Ermenek-Gu
¨lnar, Konya June, 2005 AEF 23525
S. candidissima Vahl. ssp. occidentalis Ermenek, Konya June, 2005 AEF 23522
S. ceratophylla L. Ergani-Maden, Diyarbakir June, 2005 AEF 23559
S. cryptantha Montbret and Bentham Beynam, Ankara June, 2005 AEF 23614
S. cyanescens Boiss and Bal. Beynam, Ankara June, 2005 AEF 23620
S. frigida Boiss Ermenek, Konya June, 2005 AEF 23528
S. forskahlei L. Su
¨mela Monestry, Trabzon August, 2001 GUE 1987
S. halophila Hedge Karakulluk, Konya June, 2005 AEF 23649
S. migrostegia Boiss and Bal. Ermenek-Tekecati, Konya June, 2005 AEF 23523
S. multicaulis Vahl. Ergani-Maden, Diyarbakir June, 2005 AEF23561
S. sclarea L. Bozkir, Konya June, 2005 AEF 23521
S. syriaca L. Hadim-Bozkir, Konya June, 2005 AEF 23530
S. verticillata L. ssp. amasiaca Cubuk-Karago
¨l National Park, Ankara July, 2005 AEF 23552
1248 I. Orhan et al. / Food Chemistry 103 (2007) 1247–1254
2.2. Preparation of crude extracts
Each plant material was dried in shade at room temper-
ature and then ground to a fine powder in a mechanic grin-
der and approximately 6.0 g weighed accurately on a digital
balance (Mettler Toledo AG245). Then, each plant was
successively extracted with petroleum ether (PE), chloro-
form (CHCl
3
), ethyl acetate (EtOAc), and then methanol
(MeOH). After filtration of each solvent, the organic
phases were independently concentrated under vacuum
by evaporating to dryness. Yields of the crude extracts
obtained a given in Table 2.
2.3. Determination of AChE and BChE inhibitory activities
AChE and BChE inhibitory activities were measured by
slightly modifying the spectrophotometric method devel-
oped by Ellman, Courtney, Andres, and Featherstone
(1961). Electric eel AChE (Type-VI-S, EC 3.1.1.7, Sigma)
and horse serum BChE (EC 3.1.1.8, Sigma) were used,
while acetylthiocholine iodide and butyrylthiocholine chlo-
ride (Sigma, St. Louis, MO, USA) were employed as sub-
strates of the reaction. 5,50-Dithio-bis(2-nitrobenzoic)acid
(DTNB, Sigma, St. Louis, MO, USA) was used for the
measurement of the cholinesterase activity. All the other
reagents and conditions were same as described in previous
publications (Atta-ur-Rahman, Parveen, Khalid, Farooq,
& Choudhary, 2000; Orhan, S
ßener, Choudhary, & Khalid,
2004). Briefly, in this method, 140 ll of 0.1 mM sodium
phosphate buffer (pH 8.0), 20 ll of DTNB, 20 ll of test
solution and 20 ll of AChE/BChE solution were added
by multichannel automatic pipette (Gilson pipetman,
France) in a 96-well microplate and incubated for 15 min
at 25 C. The reaction was then initiated with the addition
of 10 ll of acetylthiocholine iodide/butyrylthiocholine
chloride. The hydrolysis of acetylthiocholine iodide/buty-
rylthiocholine chloride was monitored by the formation
of the yellow 5-thio-2-nitrobenzoate anion as a result of
the reaction of DTNB with thiocholines, catalyzed by
enzymes at a wavelength of 412 nm utilizing a 96-well
microplate-reader (Spectramax Plus-384, Molecular
Devices, CA, USA). The measurements and calculations
were evaluated by using Softmax PRO 4.3.2.LS software.
Percentage of inhibition of AChE/BChE was determined
by comparison of rates of reaction of samples relative to
blank sample (ethanol in phosphate buffer pH=8) using
the formula (ES)/E ·100, where Eis the activity of
enzyme without test sample and Sis the activity of enzyme
with test sample. The experiments were done in triplicate.
Galanthamine, the anticholinesterase alkaloid-type of drug
isolated from the bulbs of snowdrop (Galanthus sp.), was
purchased from Sigma (St. Louis, MO, USA) and was used
as reference.
2.4. Antioxidant activity
2.4.1. DPPH free radical-scavenging assay
The antiradical activity of the plant extracts and the refe-
rence were assessed on the basis of the radical-scavenging
effect of the stable 1,1-diphenyl-2-picrylhydrazyl radical
(DPPH) free radical (Lee et al., 1998). The concentration
of DPPH was kept as 300 lM. The extracts and reference
were dissolved in dimethylsulfoxide (DMSO), an effective
solvent that is miscible in all proportions with water, while
the DPPH solution was prepared in ethanol. Ten microli-
tres of each extract and reference was allowed to react with
200 ll of stable free radical DPPH at 37 C for 30 min in a
96-well microtiter plate. After incubation, decrease in
absorption for each solution was measured at 515 nm using
an ELISA microplate-reader (Spectra MAX-340 Molecular
Devices, USA). The corresponding blank readings were
also taken and the remaining DPPH was calculated. Per-
cent radical-scavenging activity by samples was determined
in comparison with a DMSO treated control group. Butyl-
ated hydroxyanisol (BHA), a widely used antioxidant for
long preservation of food products, was used as reference.
Inhibition of free radical DPPH in percent (I%) was calcu-
lated in following way:
I%=(1A
sample
/A
blank
)·100, where A
blank
is the
absorbance of the control reaction (containing all reagents
except the test sample), and A
sample
is the absorbance of the
extracts/reference.
2.4.2. Xanthine oxidase (XO) inhibition assay
The xanthine–xanthine oxidase (X–XO) reaction is a
suitable system to generate superoxide anion (O:
2Þ. Com-
pounds/extracts which interact with XO can affect the
kinetics of reaction of oxidation of xanthine to uric acid
(Fridovich, 1970). The xanthine oxidase (XO) inhibition
assay was assessed in phosphate buffer (0.1 M, pH = 7.5).
Twenty microliters XO (0.003 unit/well) and various con-
centrations of test samples in 10 ll of DMSO were mixed
in a 96-well microplate and pre-incubated for 10 min at
room temperature. The reaction was initiated by adding
20 ll of 0.1 mM xanthine and uric acid formation was
measured spectrophotometrically at 295 nm by using
Table 2
Percentage yields (w/w) of PE, CHCl
3
, EtOAc, and MeOH extracts of
Turkish Salvia species
Salvia species examined PE CHCl
3
EtOAc MeOH
S. albimaculata 1.13 2.75 1.13 4.38
S. aucheri var. canescens 1.63 2.77 1.14 2.77
S. candidissima ssp. occidentalis 2.59 1.13 1.46 10.84
S. cetrophylla 7.63 1.30 0.49 2.92
S. cryptantha 3.02 2.86 0.79 9.05
S. cyanescens 2.04 2.04 0.63 7.23
S. frigida 7.65 1.83 0.67 2.99
S. forskahlei 0.63 1.10 0.31 5.98
S. halophila 0.47 0.31 0.31 20.41
S. migrostegia 4.45 1.15 0.66 6.42
S. multicaulis 2.07 6.21 0.64 6.05
S. sclarea 3.15 3.15 1.57 6.61
S. syriaca 1.15 2.30 0.66 5.75
S. verticillata ssp. amasiaca 0.48 1.75 0.63 11.75
I. Orhan et al. / Food Chemistry 103 (2007) 1247–1254 1249
Molecular Devices, Spectramax 384 (Lee et al., 1998). Allo-
purinol, a xanthine oxidase inhibitor also known as 1,5-
dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, was used as
the reference compound.
2.4.3. Statistical analysis of data
Data obtained from in vitro experiments were expressed
as mean standard error (±SEM). Statistical differences
between the treatments and the control were evaluated by
ANOVA test. P< 0.05 was considered to be significant
[
*
P< 0.05;
**
P< 0.01;
***
P< 0.001].
3. Results
3.1. Acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) inhibitory activity
At 0.2 mg/ml; PE, EtOAc and MeOH extracts did not
exhibit any activity against AChE, while the most active
extract was found to be the chloroform extract of S. cryp-
tantha (74.7%), followed by S. aucheri var. canescens
(47.72%) (Table 3). Against BChE at the same concentra-
tion, none of the methanolic extracts were active, while
PE extracts of S. cyanescens and S. cryptantha were
observed to be the most effective (67.1% and 82.9%, respec-
tively). The EtOAc extracts had inhibition under 50%,
except S. cyanescens (63.3%). However, among the CHCl
3
extracts, S. sclarea and S. syriaca did not show any inhibi-
tion at all, while the rest had inhibition over 50%, excepting
S. halophila and S. migrostegia (Table 3). Only two PE
extracts belonging to S. albimaculata and S. cryptantha dis-
played remarkable inhibition over 50% (89.4% and 71.8%,
respectively) at 1 mg/ml against AChE (Table 4). The PE
extract of S. albimaculata (89.4%) and the CHCl
3
extract
of S. cyanescens (80.2%) were revealed to be the most active
extracts for AChE inhibition, whereas the EtOAc extracts
of S. frigida and S. migrostegia, the CHCl
3
extracts of
S. candidissima ssp. occidentalis and S. ceratophylla, as well
as PE extract of S. cryptantha appeared to inhibit potently
BChE (92.2%, 89.6%, 91.1%, 91.3%, and 92.0%, respec-
tively). However, the MeOH extracts were found to be
completely inactive against AChE at 1 mg/ml. The PE
extracts were effective against BChE, excluding S. cerato-
phylla and S. migrostegia. With the exception of S. multi-
caulis and S. cryptantha, the rest of the CHCl
3
extracts
had significant BChE inhibitory activity. The EtOAc
extracts, excluding S. ceratophylla and S. halophila, were
also observed to be highly effective, while all of the MeOH
extracts, apart from S. frigida, were shown to be totally
ineffective against BChE.
3.2. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical-
scavenging and xanthine oxidase (XO) inhibitory activity
The PE, CHCl
3
, EtOAc, and MeOH extracts of the
above mentioned Salvia species were also screened for their
antioxidant capacity by both chemical and enzymatic
methods. Butylated hydroxyanisole (BHA) for DPPH
Table 3
Percentage of inhibitions ±SEM
a
of Turkish Salvia species against AChE and BChE at 0.2 mg/ml
Salvia species used Inhibition % against AChE Inhibition % against BChE
PE CHCl
3
EtOAc MeOH PE CHCl
3
EtOAc MeOH
S. albimaculata 17.2 ± 1.11 8.4 ± 0.86 NI NI 31.4 ± 1.10
*
83.8 ± 1.61
*
30.8 ± 0.62 25.3 ± 0.67
S. aucheri var.
canescens
NI
b
47.7 ± 3.15
*
NI NI 13.4 ± 0.46 87.3 ± 2.48 10.7 ± 1.06
*
20.9 ± 1.82
*
S. candidissima ssp.
occidentalis
NI 26.5 ± 0.71 NI NI 43.4 ± 2.73
**
74.8 ± 2.09 37.9 ± 4.23
**
NI
S. ceratophylla NI 7.4 ± 0.42 NI NI NI 57.4 ± 2.58 30.7 ± 1.13
*
27.0 ± 1.76
*
S. cyanescens NI 41.3 ± 2.02 NI NI 67.1 ± 0.52
***
81.3 ± 1.83
*
63.3 ± 4.05
**
NI
S. cryptantha 20.9 ± 1.12 74.7 ± 0.36 NI NI 82.9 ± 1.06
*
66.3 ± 1.33
*
31.9 ± 1.55
*
NI
S. frigida NI NI NI 46.1 ± 3.54
*
11.7 ± 0.38 67.8 ± 5.23
**
48.8 ± 4.49
**
NI
S. forskahlei NI NI NI NI 36.9 ± 1.02 46.0 ± 8.97
***
8.1 ± 1.66
*
17.6 ± 1.46
*
S. halophila NI NI NI NI 13.9 ± 0.71 39.8 ± 2.39
*
15.5 ± 0.66 NI
S .migrostegia NI NI NI NI NI 26.0 ± 2.32
*
38.2 ± 1.78
*
NI
S. multicaulis NI NI NI NI 23.9 ± 2.06
*
78.2 ± 5.11
**
42.4 ± 1.89
*
NI
S. sclarea NI 16.9 ± 8.52
**
NI NI 33.1 ± 0.73 59.9 ± 2.62
**
45.8 ± 1.53
*
34.8 ± 2.87
**
S. syriaca NI NI NI NI 36.4 ± 0.94 NI 32.9 ± 0.94 NI
S. verticillata ssp.
amasiaca
10.5 ± 0.61 NI NI NI 45.2 ± 5.13
**
NI NI NI
Galanthamine 100.05 ± 0.64 98.60 ± 0.59
P> 0.05.
a
Values were expressed as mean ±SEM (n= 3).
b
NI = No inhibition.
*
P< 0.05.
**
P< 0.01.
***
P< 0.001.
1250 I. Orhan et al. / Food Chemistry 103 (2007) 1247–1254
scavenger activity and allopurinol for XO inhibition were
used as reference drugs. While all of the extracts were
tested against DPPH, only the EtOAc and MeOH extracts
were screened against XO. As listed in Table 5, the results
showed that the EtOAc extracts had high antioxidant
activity against XO, ranging between 66.1% and 162.4%,
whereas the MeOH extracts belonging to S. albimaculata,
S. aucheri var. canescens,S. candidissima ssp. occidentalis,
S. ceratophylla,S. cyanescens,S. cryptantha,S. frigida,
S. migrostegia,S. multicaulis,S. sclarea, and S. syriaca pos-
sessed remarkable inhibition over 50% against XO. The PE
extracts of selected Salvia species did not seem to have
DPPH scavenger activity, excepting S. cryptantha
(74.6%). Only half of the CHCl
3
extracts displayed scav-
enging effect on DPPH over 50%. The most noticeable
CHCl
3
extracts in the DPPH assay belonged to S. syriaca
(92.2%), S. frigida (82.7%), and S. cryptantha (71.1%),
which had inhibition rates close to that of BHA. The
EtOAc extracts were highly active against DPPH, exclud-
ing S. syriaca, while all of the MeOH extracts exhibited
quite noticeable DPH radical-scavenging activity, which
were identical to that of BHA.
4. Discussion
Up to date, a number of studies on AChE inhibitory
activity of several Salvia species have been reported.
Among these, the essential oil and ethanolic extract of
S. officinalis along with the essential oil of S. lavandulaefo-
lia have been shown to possess anticholinesterase activity
(Perry, Court, Bidet, Court, & Perry, 1996). Moreover,
the essential oil as well as its major components, a-pinene,
1,8-cineole, and camphor were determined to have uncom-
petitive and reversible acetylcholinesterase inhibitory activ-
ity (Perry, Houghton, Theobold, Jenner, & Perry, 2000).
The activity of the essential oil was concluded mainly to
be due to its monoterpenoids. In another study, the acetone
extract of the dried root of S. miltiorrhiza was subjected to
activity-guided isolation for its acetylcholinesterase inhibi-
tory activity by the Ellman method and afforded four diter-
penes; dihydrotanshinone, cryptotanshinone, tanshinone I,
and tanshinone IIA (Ren, Houghton, Hider, & Howes,
2004). On the other hand, the essential oils of S. fruticosa,
S. lavandulaefolia,S. officinalis and S. officinalis var. purpu-
rea were screened for their anti-BChE activity by the Ell-
man method and a time-dependent increase was shown in
the inhibition of BChE by the oils of S. fruticosa and
S. officinalis var. purpurea. In this study, it was concluded
that synergy was the most possible interaction for anti-
BChE activity of the oils and their constituents (Savelev,
Okello, & Perry, 2004).
All these data indicate that the terpenoids, monoter-
penes in particular, may have anticholinesterase activity
which prompted us to investigate the above-mentioned
Salvia species for their terpenoid-derivative compounds.
For this purpose; we analyzed the extracts by thin layer
Table 4
Percentage of inhibitions ±SEM
a
of Turkish Salvia species against AChE and BChE at 1 mg/ml
Salvia
species used
Inhibition %
against AChE
Inhibition %
against BChE
PE CHCl
3
EtOAc MeOH PE CHCl
3
EtOAc MeOH
S. albimaculata 89.4 ± 2.07
*
NI
b
51.7 ± 3.22
***
38.9 ± 3.22
***
73.9 ± 0.76
***
87.9 ± 0.22 69.8 ± 1.99
***
27.4 ± 1.32
***
S. aucheri var.
canescens
27.3 ± 0.98
***
64.5 ± 1.03
***
53.4 ± 1.59
***
39.9 ± 1.17
***
59.9 ± 378
***
77.6 ± 3.78
***
69.6 ± 2.15
***
12.6 ± 1.05
***
S. candidissima
ssp.
occidentalis
39.4 ± 4.31 48.6 ± 5.13
***
46.1 ± 1.28
***
NI 55.6 ± 0.28 91.1 ± 1.98 77.8 ± 0.93
***
NI
S. ceratophylla NI 30.8 ± 5.25
***
19.3 ± 1.57
***
27.8 ± 2.82
***
38.8 ± 4.94
***
91.3 ± 1.63 29.2 ± 0.77
***
34.9 ± 6.50
***
S. cyanescens 37.7 ± 5.35 80.2 ± 4.35
***
51.2 ± 3.78
***
9.0 ± 0.88
***
67.4 ± 3.59
***
91.8 ± 0.54 56.9 ± 1.03
***
13.1 ± 0.70
***
S. cryptanhta 71.8 ± 2.62
***
24.9 ± 1.65 73.3 ± 2.55
***
47.2 ± 5.18
***
92.0 ± 0.41 NI 53.6 ± 0.67
***
36.3 ± 2.79
***
S. frigida 6.2 ± 0.24
***
53.7 ± 2.25
***
59.5 ± 0.45
***
32.6 ± 0.01
***
54.9 ± 1.95
***
77.8 ± 0.21
***
92.2 ± 0.29 59.9 ± 2.30
***
S. forskahlei 25.2 ± 4.46
***
41.3 ± 2.91 47.0 ± 2.31
***
35.8 ± 2.46
***
69.3 ± 1.65
***
60.2 ± 4.42
***
62.9 ± 0.67
***
46.7 ± 3.69
***
S. halophila 18.9 ± 1.21
***
NI 36.1 ± 1.21
***
NI 50.9 ± 4.20
***
53.9 ± 2.16
***
37.2 ± 3.88
***
NI
S. migrostegia NI 36.4 ± 5.45
***
37.1 ± 3.15
***
23.6 ± 0.61
***
22.1 ± 2.70
***
62.5 ± 1.31
***
89.6 ± 0.67
*
32.6 ± 3.40
***
S. multicaulis 21.4 ± 3.91
***
NI NI 47.7 ± 3.58
***
68.8 ± 3.80
***
NI 64.3 ± 1.02
***
36.2 ± 0.93
***
S. sclarea 25.8 ± 4.51
***
55.3 ± 0.98
***
33.5 ± 4.94
***
25.3 ± 1.86
***
52.6 ± 2.92
***
59.9 ± 0.50
***
75.7 ± 1.83
***
15.1 ± 1.76
***
S. syriaca 33.4 ± 2.98 66.9 ± 2.49
***
49.8 ± 2.41
***
12.1 ± 1.22
***
63.5 ± 2.12
***
87.3 ± 1.99 70.9 ± 2.69
***
12.3 ± 1.10
***
S. verticillata
ssp. amasiaca
45.6 ± 4.17 NI NI 39.1 ± 3.10
***
85.0 ± 53.10
*
55.7 ± 0.55
***
53.3 ± 5.50
***
72.0 ± 2.99
***
Galanthamine 99.87 ± 0.31 80.31 ± 1.14
P> 0.05.
a
Values were expressed as mean ± SEM (n= 3).
b
NI = No inhibition.
*
P< 0.05.
**
P< 0.01.
***
P< 0.001.
I. Orhan et al. / Food Chemistry 103 (2007) 1247–1254 1251
chromatography (TLC) on silica gel, whose spots were
revealed by vanillin–sulphuric acid reagent and the vio-
let colored-spots occurred after application of this
reagent that pointed toward the presence of terpenoids
in the CHCl
3
and EtOAc extracts of the Salvia species.
At 1 mg/ml concentration (Table 4), it was observed
that, as the polarity increased, anticholinesterase effects
of the PE extracts belonging to S. albimaculata and
S. crypthantha gradually decreased. This may be most
likely due to anticholinesterase activity of nonpolar
compounds found in high amounts within these
extracts, which is in accordance with our supposition
that the MeOH extracts, containing the polar com-
pounds, exerted the least inhibitory activity at both con-
centrations, with the exception of S. verticillata ssp.
amasiaca.
On the other hand, the role of oxidative stress in the
pathogenesis of diseases such as macular degeneration,
certain types of cancer, and Alzheimer’s disease (AD)
has received substantial attention. For that reason, we
also aimed to look into antioxidant capacities of selected
Salvia species (sage) as well as their anticholinesterase
activity. Our literature survey highlighted that there have
been a number of studies on the antioxidant potential of
various Salvia species. In earlier studies, sage was shown
to contain phenolic compounds for the most part and
the antioxidant activity of the plant was mainly attributed
to carnosic and rosmarinic acids (Cuvelier, Richard, &
Berset, 1996). In Lu and Foo’s study (2001), the sage poly-
phenols, including flavone glycosides and some rosmarinic
acid derivatives, from S. officinalis were found to display
potent antioxidant activity against DPPH and superoxide
anion radicals.
Polyphenolic compounds from various Salvia species
have been reported to have potent antioxidative effect
(Lu & Foo, 2001; Madsen & Bertelsen, 1995; Whu, Lee,
Ho, & Chang, 1982). For instance; salvianolic acid, a ros-
marinic acid dimer isolated from S. officinalis, had a very
strong free radical-scavenging activity for DPPH and
superoxide anion radicals (Lu & Foo, 2001). The same
researchers investigated the antioxidant capacity of ros-
marinic acid, salvianolic acids K and I, sagecoumarin
and sagerinic acid isolated from the same plant as well as
a number of flavon glycosides such as luteolin 7-glucoside,
7-glucuronide, 30-glucuronide, 6-hydroxyluetolin 7-gluco-
side, and apigenin 6,8-di-C-glucoside (Lu & Foo, 2001).
Consequently, the flavonoid glycosides were found to pos-
sess weaker DPPH-scavenging activity. Interestingly, b-
sitosterol isolated from S. plebeia was also found to be a
strong antioxidant by the oxidative stability instrument
(OSI) (Weng & Wang, 2000). Therefore, it made us think
that the high antioxidative property of PE extract of S.
cryptantha against DPPH in our study could be related
to its sterol content. Moreover, beyond that, we have just
started to analyze our 14 Salvia species by reversed-phase
HPLC for their caffeic, rosmarinic, chlorogenic, and gallic
acids by a new method to be validated, which, later on,
may allow us to be able to make a correlation between their
antioxidant capacity and quantity of these phenolic acids,
known as the strong antioxidant components.
Table 5
Percentage of inhibitions ±SEM
a
of Turkish Salvia species against DPPH and xanthine oxidase (XO)
Salvia species screened Inhibition % against XO Inhibition % against DPPH
EtOAc MeOH PE CHCl
3
EtOAc MeOH
S. albimaculata 81.9 ± 1.12 95.8 ± 1.08 7.7 ± 0.98
***
15.1 ± 1.34
***
83.1 ± 2.53
*
89.4 ± 0.92
S. aucheri var. canescens 81.4 ± 1.38 94.4 ± 0.92 34.3 ± 4.94
***
8.4 ± 1.17
***
90.9 ± 0.54 92.0 ± 0.63
S. candidissima ssp. occidentalis 90.5 ± 1.97 69.4 ± 1.22 21.2 ± 1.89
***
59.9 ± 4.29
***
92.4 ± 0.67 87.7 ± 1.07
S. ceratophylla 90.1 ± 2.49 81.7 ± 2.16 15.9 ± 2.03
***
53.3 ± 1.78
***
88.9 ± 0.84 84.8 ± 1.11
S. cyanescens 77.8 ± 0.97 72.6 ± 5.73 30.6 ± 3.06
***
55.3 ± 3.30
***
51.8 ± 1.51
***
87.2 ± 2.58
S. cryptanhta 66.3 ± 1.18 74.4 ± 7.12 74.6 ± 2.13
***
71.1 ± 0.91
***
82.5 ± 0.75
*
91.5 ± 1.07
S. frigida 93.2 ± 3.47 80.8 ± 0.69 18.9 ± 3.93
***
82.7 ± 1.71 80.1 ± 2.80
**
91.5 ± 0.09
S. forskahlei 82.6 ± 2.68 46.8 ± 1.19
***
4.8 ± 0.51 28.3 ± 0.83
***
82.2 ± 0.38
*
93.5 ± 1.18
S. halophila 75.4 ± 1.74 18.4 ± 2.61
***
NI 34.6 ± 0.42
***
90.49 ± 0.97 83.9 ± 1.32
S. migrostegia 91.5 ± 0.91 74.3 ± 1.81 17.8 ± 1.36
***
22.2 ± 0.73
***
86.4 ± 0.79 89.7 ± 1.27
S. multicaulis 100.4 ± 8.96 73.3 ± 3.45 4.9 ± 0.96
***
26.8 ± 2.31
***
85.3 ± 3.32 92.6 ± 0.38
S. sclarea 90.5 ± 1.58 87.2 ± 1.78 22.6 ± 0.73
***
54.2 ± 3.93
***
72.8 ± 2.66
***
87.8 ± 1.13
S. syriaca 83.2 ± 0.99 70.8 ± 2.32 13.0 ± 0.43
***
92.2 ± 0.28 36.7 ± 4.91
***
90.7 ± 1.00
S. verticillata ssp. amasiaca 66.1 ± 0.98 45.4 ± 2.27
***
18.0 ± 0.42
***
30.5 ± 1.37
***
72.8 ± 0.42
***
93.6 ± 0.67
Reference drugs
BHA (for DPPH) – – 92.75 ± 0.21
Allopurinol (for XO) 80.52 ± 0.85 – – – –
P> 0.05.
a
Values were expressed as mean ±SEM (n= 3).
b
NI = No inhibition.
*
P< 0.05.
**
P< 0.01.
***
P< 0.001.
1252 I. Orhan et al. / Food Chemistry 103 (2007) 1247–1254
5. Conclusion
According to data we obtained, out of 56 extracts from 14
Salvia species, only 2 extracts against AChE and 13 extracts
against BChE had noticeable activity at 0.2 mg/ml, whereas
13 extracts against AChE and 37 extracts against BChE
showed remarkable activity at concentration of 1 mg/ml.
From these results, we can point out that the nonpolar Salvia
extracts, including PE and CHCl
3
, seemed to be much more
effective then EtOAc and MeOH extracts at both concentra-
tions. Besides, the Salvia extracts studied were more active
against BChE than AChE (Tables 3 and 4). To the best of
our knowledge, this is the first evaluation of the AChE and
BChE inhibitory activities of Turkish Salvia species.
The difference observed between the AChE and BChE
inhibitory activities of Turkish Salvia species seems to be
due to their different phytochemical contents. Since it is
evident that preventive and symptomatic treatment of
AD needs a multitarget drug strategy, it is quite rational
to examine the phytochemistry of the Salvia species hav-
ing both anticholinesterase and antioxidant activities.
Consequently, ingredients of the active extracts, which
are primarily expected to be terpenoid-type compounds,
should be identified and activity of the single compounds
should also be compared with that of the crude extract to
reveal possible synergistic interaction. Thus, the further
work on identification of the active component(s) of the
Salvia species effective in both assays is in progress in
our laboratory.
Acknowledgements
The authors Prof.Dr. Hayri Duman of Department of
Biology, Faculty of Art and Science, Gazi University, An-
kara (Turkey) and Assoc. Prof. Salih Terzioglu of Depart-
ment of Forest Botany, Faculty of Forestry, Karadeniz
Technical University, Trabzon (Turkey) for identification
of the plants. This study was financially supported by the
Islamic Development Bank-Young Researchers Support
Project (screening new antioxidant compounds from
medicinal plants growing in Turkey).
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