Chiang Mai J. Sci. 2013; 40(1)
49
Chiang Mai J. Sci. 2013; 40(1) : 49-59
http://it.science.cmu.ac.th/ejournal/
Contributed Paper
Antimicrobial and Antioxidant Activities of Methanol,
Dichloromethane and Ethyl Acetate Extracts of
Scutellaria lindbergii Rech.f.
Bibi Sedigheh Fazly Bazzaz*[a], Atefeh Arab [b], Seyed Ahmad Emami [c], Javad Asili [c],
Mohamad Hasanzadeh-Khayyat [d] and Amirhossein Sahebkar [a]
[a] Biotechnology Research Center and School of Pharmacy, Mashhad University of Medical Sciences,
PO Box: 91775-1365, Mashhad, Iran.
[b] School of Pharmacy, Mashhad University of Medical Sciences, PO Box: 91775-1365, Mashhad, Iran.
[c] Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences,
PO Box: 91775-1365, Mashhad, Iran.
[d] Pharmaceutical Research Center and School of Pharmacy, Mashhad University of Medical Sciences,
PO Box: 91775-1365, Mashhad, Iran.
*Author for correspondence; e-mail: [email protected]
Received: 11 January 2012
Accepted: 17 April 2012
ABSTRACT
Plants belonging to genus Scutellaria (Lamiaceae) possess different
pharmacological properties. S.lindbergii Rech.f is a species from this genus that is found
in Iran and Afghanistan. The present study aimed to determine total phenolic and
flavonoid contents together with antimicrobial and antioxidant activities of methanol,
ethyl acetate and dichloromethane extracts of the aerial parts of S.lindbergii. Total
flavonoid contents were detected using 2% aluminum chloride and expressed as quercetin
equivalents (QE). Total phenolics were determined using Folin-Ciocalteu method and
expressed as tannic acid equivalent (TAE). The antimicrobial activities of the three
mentioned extracts were determined against standard strains of Staphylococcus aureus,
Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans by broth
macro-dilution method. Antioxidant activities of the extracts were determined using
two model systems, the thiobarbituric acid reactive species (TBARS) and 2,2′-diphenyl1-picrylhydrazyl (DPPH) radical scavenging assays. Vitamin E was used as standard in
TBARS assay while vitamin C and BHT served as standards in DPPH assay. Total
flavonoid and phenolic contents ranged between 33.72-82.32 mg QE/g of dry material
and 47.12-80.64 TAE/g, respectively. The results showed that MIC and MCC of the
methanol extract for the most sensitive strain, S. aureus, were 6.25 mg/mL. In DPPH
assay, the methanol extract was more effective than the two other extracts. The IC50s for
radical scavenging activity of methanol, ethyl acetate and dichloromethane extracts were
3.03, 4.69 and 16.73 mg/mL respectively. In TBARS method, ethyl acetate extract was
more effective than the two other extracts. The highest antioxidant activity of ethyl
acetate extract was 36.0% at 4 mg/mL, whereas those of methanol extract was 37.0% at
16 mg/mL and dichloromethane extract was 33.0% at 8 mg/mL. In antimicribial assays,
ethyl acetate extract was found as the most effective extarct and S. auerus as the most
sensitive strain. In conclusion, S. lindbergii extracts were found to possess moderate
antioxidant and antibacterial capacity.
Keywords: antioxidant activity, antimicrobial activity, DPPH assay, Scutellaria lindbergii,
TBARS assay
50
1. INTRODUCTION
Some medicinal plants contain large
amounts of antimicrobial and antioxidant
compounds such as phenols and flavonoids.
Phenolic compounds are a group of plant
metabolites that have numerous beneficial
activities such as anti-inflammatory, antibacterial, anti-mutagenic, anti-viral and
antioxidant properties [1].
Flavonoids are also common
ingredients of plants, and many of them
(e.g. quercetin, luteolin and catechins), are
better antioxidants than the widely used
nutrient antioxidants (vitamin C and
vitamin E) [2]. In addition, some flavonoids
such as apigenin and luteolin are active
against methicillin-resistant Staphylococcus
aureus (MRSA) [3].
There have been some reports on the
adverse effects of synthetic antioxidants
such as butylated hydroxytoluene, butylated
hydroxyanisole, propyl gallate and tertiary
butyl hydroquinone [1, 4]. Therefore, finding
natural antioxidants to be used in foods and
pharmaceutical products is an interesting
and on demand field of investigation. Several
lines of evidence have supported the
effectiveness of natural products in lessening
inflammation and oxidative stress. Hence,
these products could be successfully
exploited for the treatment of a variety of
disorders [5-9].
Genus Scutellaria (Lamiaceae) contains
around 300 species. This genus consists of
20 species and 2 hybrids in Iran, with
10 species and 2 hybrids endemic to the
country [10]. The plants of this genus have
numeorus medicinal properties. To name a
few are antioxidant [2,11], antimicrobial
[3,12], cytotoxic, anti-inflammatory [13]
and anti-tumor effects [14,15]. It is a species
from this genus that is commonly found
in Iran and its neighbor Afghanistan. S.
lindbergii is a newly discovered plant which
Chiang Mai J. Sci. 2013; 40(1)
has been reported for the first time in the
world in 1982 [16], and in Iran in 2001 [17].
This plant has been shown to possess
cytotoxic and apoptotic activities against
different cancer cell lines [18]. Although
there have been some studies on the
pharmacologic effects of Scutellaria
growing in different parts of the world
[2,3,12,14], there has been no previous
report concerning antimicrobial and
antioxidant effects of S. lindbergii.
Therefore, the present study aimed to assess
the antioxidant and antimicrobial activities
of different (methanol, dichloromethane
and ethyl acetate) extracts of S. lindbergii.
2. MATERIALS AND METHODS
2.1 Plant Material
Aerial parts of mature S. lindbergii
were collected in July 2008 from the Kang
mountain at the height of 1800 m, near
Mashhad, Razavi Khorasan province,
north-east of Iran. The plant was identified
by Mr. M. R. Joharchi at the Herbarium
of Ferdowsi University of Mashhad
(Mashhad, Iran). A voucher specimen of
the plant (herbarium no. 8642) was
deposited at the Herbarium of Ferdowsi
University of Mashhad.
2.2 Preparation of Plant Extracts
In order to prepare various extracts
from S. lindbergii, the aerial parts of plant
were dried under shade at room
temperature and were powdered by an
electric blender (Toosshekan Khorasan,
Iran). About 150 g of the material was
macerated separately in methanol,
dichloromethane and ethyl acetate solvents
at room temperature for 48 hrs. Extraction
with the respective solvent was performed
3 times, each time with ~1000 mL of
solvent. All extracts were concentrated
Chiang Mai J. Sci. 2013; 40(1)
by rotary vacuum evaporator (Buchi,
Switzerland). The condensed extracts were
weighted and kept at -20°C until used.
2.3 Determination of Total Flavonoid
Content
The total flavonoid content of
S. lindbergii in each extract was determined
spectrophotometrically according to the
Lamaison and Carnat method [19]. This
method is based on the formation of a
complex of flavonoid-aluminium, with
maximum absorption at 430 nm. In this
method, 1 mL of sample (containing 125 μg
of the extract) was mixed with 1 mL of 2%
aluminium chloride methanol solution.
After incubation at room temperature for
15 min, the absorbance of the reaction
mixture was measured at 430 nm with a
UV-Vis spectrophotometer (Cecil, England).
The same procedure was repeated for all the
quercetin standard solutions (0, 1.25, 2.5, 5.0,
10.0 and 20.0 μ g/mL) [1]. The total
flavonoid content of each extract was
determined in accordance with the equation
(1) that was obtained by calibration curve
as quercetin equivalent (QE, mg/g dry
material).
Equation (1):
y = 0.0316x + 0.0118 (r2 = 0.9978)
Where x is the absorbance at 430 nm and
r2 is regression coefficient.
2.4 Determination of Total Phenolic
Compounds
Total phenolic contents were
determined using the Folin-Ciocalteu
method as described elsewhere [20, 21].
Briefly, each sample (100 μL) and standard
(tannic acid) were diluted with distilled
water to the final volume of 0.5 mL.
Afterwards, Folin-Ciocalteu reagent (0.25
mL) and sodium carbonate solution (7.5%;
51
1.25 mL) were added to each tube,
respectively. Following vortexing, the
absorbances of all samples and standards
were measured at 725 nm using a UV-Vis
spectrophotometer (Cecil, UK) after 40
min. Total phenolic content was expressed
as tannic acid equivalent (TAE) calculated
from the calibration curve.
2.5 Antioxidant Activity
2.5.1 TBARS Assay
TBARS assay was used to measure the
potential antioxidant capacity of extracts in
lipid media [4, 22]. TBARS method is based
on the reaction of thiobarbituric acid (TBA)
with malondialdehyde (MDA) and other
lipid peroxidation products which produce
the pink pigment that has absorbance at
532 nm [4].
Briefly, 0.5 mL of 10% (w/v) egg yolk
homogenate (lipid rich media) and 0.1 mL
of sample solution to be tested in methanol
(concentrations of stock solutions were
0.5, 1, 2, 4, 8, 16 and 32 mg/mL), were
prepared immediately before use, added to
a test tube and made up to 1.0 mL with
distilled water. Then, 50 μL of 2,2′-azobis
(2-amidinopropane) dihydrochloride
solution (0.07 M) in water was added to
induce lipid peroxidation. Afterwards, 1.5
mL of 20% acetic acid (pH 3.5) and 1.5 mL
of 0.8% (w/v) thiobarbituric acid in 1.1%
(w/v) sodium dodecyl sulfate solution were
added and the mixture was vortexed. The
resulting mixture was heated at 95°C for
60 min. After cooling, 5.0 mL of butan-1ol was added to each tube, then extensively
vortexed and centrifuged (Hettich,
Germany) at 2,500 g for 10 min. The
organic upper layer was separated and the
absorbance of this layer was measured at
532 nm. Butan-1-ol was used as blank. All
values were expressed as the percentage of
antioxidant index (AI %) using equation (2):
52
Chiang Mai J. Sci. 2013; 40(1)
(2): AI% = (1 – AT/AC) × 100
Where AC is the absorbance of the
control (containing all reagents except the
test sample) and AT is the absorbance of the
test sample.
Vitamin E was used as positive control
at concentrations similar to the extracts i.d.
0.5, 1, 2, 4, 8, 16, 32 and 64 mg/mL. All
tests were carried out in quadruplicate.
2.5.2 2,2-diphenyl-1-picrylhydrazyl
(DPPH) Assay
The method is based on the reduction
of DPPH methanol solution in the
presence of a hydrogen donating
antioxidant in the media [23].
Each plant extract, 100 μL, at various
concentrations (0.5, 1, 2, 4, 8, 16 and 32 mg/
mL) in methanol was separately added
to 3.9 mL of freshly prepared DPPH
methanol solution (0.1 mM). The mixtures
were shaken vigorously and then incubated
for 30 min at room temperature in a dark
place until their absorbances were measured
at 517 nm [23]. Methanol was used as blank.
The antioxidant index (AI%) was calculated
based on the inhibition of free radical
DPPH generation as follows [equation (3)]:
(3): AI% = [A0 – A) / A 0] × 100
Where A0 is the absorbance of the control
(containing all reagents except the test
compound) and A is the absorbance of the
test compound. Vitamin C and butylated
hydroxytoluene (BHT) were used as positive
controls (at concentrations similar to the
extracts i.d. 0.5, 1, 2, 4, 8, 16, 32 and 64 mg/
dL).
2.6 Statistical Analysis
The results were presented as the means
± SD. Instat 3 for Windows XP was used to
analyze data. Mean values were compared
using one-way analysis of variance (ANOVA)
with Tukey-Kramer test (for post-hoc
multiple comparisons). A p-value < 0.05 was
considered as statistically significant. IC50
values were calculated using the CalcuSyn
program for Windows based on the
method described by Chou and Talalay
[24]. In this program, the median-effect
equation was used to produce dose-effect
curves. (IC50 value is the concentration at
which DPPH radicals are scavenged by
50%).
2.7 Antimicrobial Activity
2.7.1 Test Microorganisms
The extracts were screened against
microorganisms, all purchased from
American Type Culture Collection
(ATCC). Test microorganisms included 2
Gram-positive bacteria, Staphylococcus
aureus (ATCC 29737) and Bacillus cereus
(ATCC 10876), 2 Gram-negative bacteria,
Pseudomonas aeruginosa (ATCC 9027) and
Escherichia coli (ATCC 8739) and the
fungus, Candida albicans (ATCC 10231).
2.7.2 Determination of Minimum Inhibitory Concentration (MIC)
A modified broth macro-dilution
method in 24-well plate was used to
determine the antimicrobial activity of
extracts [25, 26]. To prepare the initial
concentration of each extract (200 mg/mL),
0.8 g of each dried extract was dissolved in
4 mL solvent [1 mL of dimethylsulfoxide
(DMSO; Merck) + 3 mL of sterile distilled
water and a drop of tween 80 (as cosolvent)]
with the aid of a bath sonicator (Kerry,
England) and mild heating (35-40°C). One
mL of diluted extract together with 1 mL
of sterile Mueller-Hinton broth (2X MHB;
HiMedia, India) were infused into wells of
macro- plate and then serially diluted (50%
Chiang Mai J. Sci. 2013; 40(1)
with MHB). The resulting mixture was
eventually homogenized. Microbial
suspensions (1×108 cells/mL) were prepared
and compared to that of 0.5 McFarland
standard tube. These suspensions were
further diluted to obtain a concentration
of 106 colony-forming units (CFU)/mL for
bacteria and fungus. To each well of macroplate, 0.1 mL of diluted inoculums was
added. For each strain, the sufficiency of
growth conditions, the effects of positive
controls (ketoconazol; 5 μg /mL for fungus
and gentamicin; 5 μg/mL for bacteria) and
sterility of the medium were tested in two
wells. Plates were incubated for 24 hrs at
37°C for the bacteria and 48 hrs at 25°C
for C. albicans. In order to determine the
growth or lack of growth of the
microorganisms, 0.5 mL of MTT (2,3,5triphenyl tetrazolium chloride; Sigma) (5
mg/mL in sterile distilled water) was added
to each well and plates were incubated at
37°C for bacteria and 25°C for fungus for
3 more hrs [27]. The results of were
expressed as the lowest concentration of
plant extract able to inhibit any red dye
production. This concentration represents
the MIC of each extract [27]. The
experiment was performed in triplicate.
53
2.7.3 Determination of Minimum Cidal
Concentration (MCC)
Cidal concentration of each extract
was determined as described by Rios et al
[28]. Sample extract, 100 μL, from each
well of macro-plate without any red dye
production was sub-cultured onto the
Mueller Hinton agar plates (HiMedia,
India) and incubated at 37°C for 24 hrs (for
bacteria) or at 25°C for 48 hrs (for fungus).
The lowest concentration of extract with
no growth of microorganism was
considered as MCC [26].
3. RESULTS AND DISCUSSION
The yield values (w/w%) for the
methanol, dichloromethane and ethyl
acetate extracts of S. lindbergii were 10%,
1.67% and 1.67%, respectively. Total
flavonoid and phenolic contents of each
extract were determined spectrophotometrically. It was found that the amount
of flavonoids in the aerial parts of S.
lindbergii varies among different extracts.
Dichloromethane extract contained higher
flavonoid content, compared to ethyl
acetate and methanol extracts. As for the
total phenolic content, methanol extract
contained the highest amount, followed by
ethyl acetate and dichloromethane extracts
(Table 1).
Table 1. Total phenolic and flavonid contents, and radical scavenging activity of tested
extracts.
Methanol
Dichloromethane Ethylacetate Vit C BHT
Total phenolic content
80.64
47.12
74.71
(TAE/g dried extract)
Total flavonoid content
33.72
82.32
69.67
(QE/g dried extract)
IC50 for DPPH radical
3.03
16.73
4.69
0.15
0.32
scavenging activity
(mg/mL)
TAE: tannic acid equivalent; QE: quercetin equivalent.
54
Chiang Mai J. Sci. 2013; 40(1)
Since different antioxidant components
are present in plant tissues, it is hard to
quantify each antioxidant component
separately. Therefore, in many studies,
several intermediate extractions are used to
ensure a maximum extraction of the
available antioxidants [29]. In the present
study, solvents with different polarities
(including methanol, ethyl acetate and
dichloromethane) were used for extraction.
Methanol is capable of extracting a wide
range of polar and rather non-polar
compounds such as alkaloids, sterols,
flavonoids and carbohydrates [30].
Flavonoids with hydroxyl group are
soluble in this solvent while lipophilic
flavonoids
are
extracted
by
dichloromethane. Monoglycosylated
compounds are extracted by ethyl acetate
[31].
In order to evaluate the antioxidant
activity of plant extracts, it is important to
use different methods to obtain a reliable
assessment on their antioxidant activities
[29]. In the present study, antioxidant
activities of methanol, ethyl acetate,
dichloromethane extracts of S. lindbergii
were determined by two different methods
namely DPPH and TBARS.
Results of TBARS assay indicated that
the highest antioxidant activity of
methanol, dichloromethane and ethyl
acetate extracts of S. lindbergii were at 16,
8 and 4 mg/mL, respectively (Table 2).
According to the results, AI% of the three
extracts, at all the tested concentrations,
was significantly lower than those of
vitamin E as positive control (p < 0.001).
In this method, the highest AI% was
observed from the ethyl acetate extract
amounting to 35.80% at 4 mg/mL
concentration.
According to the findings of DPPH
test (Table 3), vitamin C and BHT showed
the highest antioxidant activity while the
activity of the S. lindbergii extracts was
Table 2. Antioxidant activity of the methanol, dichloromethane and ethyl acetate extracts
from S. lindbergii in TBARS assaya.
Concentration
( mg/mL)
Samples
Vitamin E
AI%b
0.5
45.4±
0.4
12.7±
Methanol extract
0.1*
Dichloromethane extract 15.3±
0.8*
18.2±
Ethyl acetate extract
0.6*
1
2
4
8
16
32
58.9±
0.5
14.0±
0.7*
20.5±
0.7*
20.8±
0.9*
63.6±
0.4
16.3±
0.8*
20.9±
0.6*
32.6±
0.9*
77.4±
0.7
17.2±
0.5*
24.4±
0.8*
35.8±
1.0*
80.4±
0.3
25.6±
0.7*
33.2±
0.9*
24.8±
0.2*
85.1±
0.05
37.4±
0.4*
21.4±
0.9*
20.3±
0.5*
90.4±
0.02
22.1±
0.6*
20.3±
0.9*
16.4±
0.9*
Results were reported as mean ± SD of four different experiment
Antioxidant effectiveness was expressed as antioxidant index
Comparison with vitamin E: *p < 0.001.
a
b
Chiang Mai J. Sci. 2013; 40(1)
55
Table 3. Antioxidant activity of the methanol, dichloromethane and ethyl acetate extracts
from S.lindbergii in DPPH assay.a
Concentration
( mg/mL)
Samples
Vitamin C
AI%b
0.5
96.4±
0.1
BHT
61.7±
0.1
4.7±
Methanol extract
0.2*#
Dichloromethane extract 9.1±
0.7*#
12.0±
Ethyl acetate extract
0.5*#
1
2
4
8
96.5±
0.1
84.5±
0.4
9.2±
0.4*#
10.0±
0.5*#
13.6±
0.7*#
96.6±
0.04
92.9±
0.2
22.1±
0.8*#
21.6±
0.9*#
27.1±
0.9*#
96.6±
0.1
94.6±
0.1
49.3±
0.8*#
31.1±
0.6*#
42.8±
1.1*#
96.7±
0.04
94.7±
0.1
92.9±
*#60.2
34.0±
0.9*#
66.7±
1.0*#
16
32
96.7± 96.8±
0.04
0.05
95.5± 95.7±
0.1
0.3
92.4± 89.0±
0.5*# 0.8*#
48.0± 40.0±
0.8*# 1.0***#
61.0± 29.2±
0.7*# 0.9*#
Results were reported as mean ± SD of four different experiments.
Antioxidant effectiveness was expressed as antioxidant index
Comparison with vitamin C: *p < 0.001; Comparison with BHT: #p < 0.001.
a
b
significantly lower at all tested concentrations (p < 0.001). Among the three
tested extracts, methanol extract showed the
strongest activity (AI% = 92.87 at 8 mg/
mL) compared to other extracts. The
highest AI% for ethyl acetate and
dichloromethane extracts were 66.72% at
8 mg/mL and 48.04% at 16 mg/mL,
respectively. The AI% of the methanol
extract was significantly higher than those
of other extracts at most tested concentrations (p < 0.001). Aside from AI%, IC50
values (50% inhibitory concentration) were
calculated for each extract. Methanol
extract reduced the stable radical DPPH
to 1,1-diphenyl-2-picrylhydrazine with an
IC50 value of 3.03 mg/mL while IC50 values
for ethyl acetate and dichloromethane
extracts were higher. The IC50 of none of
the extracts was comparable to those of
standard reference compounds vitamin C
and BHT (Table 1). In both assays, the
antioxidant activity was decreased at higher
concentrations. There is ample evidence
indicating dual pro-oxidant/antioxidant
activity of polyphenols, flavonoids and
phenolic acids which could be regarded as
a double-edge sword. Among several
factors that have been put forward for
explaining the pro-oxidant effects of these
phytochemicals, is the high concentration.
There are several reports on the prooxidant effects of phenolics and flavonoids
at higher doses/concentrations [32].
According to the present results,
antioxidant activity of extracts in DPPH
test is dependent on the polarity of extracts.
The more polar extract (methanol), had the
highest antioxidant activity. The difference
in the antioxidant activity of extracts may be
described by the difference in the total
phenolic and flavonoid content [23,29].
To the authors knowledge, this is the
first report on the antimicrobial activity of
S. lindbergii. In a study by Sato et al., apigenin
and luteolin were isolated from S. barbata
56
and their anti-bacterial activity was studied.
The results showed that these flavonoids
were toxic against MRSA and methicilinsensitive S. aureus [3]. In another investigation,
antimicrobial activity of S. baicalensis was
studied against S. aureus, Salmonella typhimurium
and Vibrio parahaemolyticus. The growth
of these microorganisms was inhibited
significantly by S. baicalensis extract [33]. In
the present study, MICs and MCCs of
S. lindbergii extracts were evaluated. The
growth of microbial strains was inhibited by
Chiang Mai J. Sci. 2013; 40(1)
the extracts with MICs ranging from 6.25
to 100 mg/mL, depending on the susceptibility of the test microorganism (Tables 4
and 5). As for gentamicin, observed MICs
were 0.03, 0.3, 5 and 1.25 μg/ml against S.
aureus, B. cereus, E. coli and P. aeruginosa,
respectively. Inhibition of C. albicans
growth was also confirmed by the single
tested concentration of ketoconazole (5 μg/
mL). Experimental studies carried out in
species of Scutellaria have identified
phenols and flavonoids as the antimicrobial
Table 4. Minimum inhibitory concentrations (MIC, mg/mL) of the methanol,
dichloromethane and ethyl acetate extracts of S. lindbergii.
Micro-organism
S.aureus
B.cereus
E.coli
P.aeruginosa
C.albicans
Extract
6.25
25
25
50
100
Methanol
12.5
25
50
50
100
Dichloromethane
6.25
12.5
50
25
100
Ethyl acetate
Table 5. Minimum cidal concentrations (MCC) mg/mL of the methanol, dichloromethane
and ethyl acetate extracts of S. lindbergii.
Micro-organism
S.aureus
B.cereus
E.coli
P.aeruginosa
C.albicans
Extract
Methanol
6.25
50
50
100
>100
Dichloromethane
12.5
50
50
100
>100
Ethyl acetate
12.5
25
50
50
>100
constituents of S. barbata [3]. Moreover,
studies on S. luteocoerulea have indicated
low flavonoids content but moderate
amounts of saponins and tannins, and low
anti-bacterial activity against S. aureus [34].
Therefore, it might be speculated that
antimicrobial effect of S. lindbergii is related
to its flavonoids.
Surprisingly, there has been almost no
previous report on the phytochemical
analysis of S. lindbergii extract. The reason is
that S. lindbergii is a newly discovered species
and reported for the first time in the world
in 1982 [16], and in Iran in 2001 [17].
However, it is likely that S. lindbergii
contains baicalin, bicalein and wogonin,
the famous flavonoids that are found in other
Scutellaria species such as S. baicalensis.
These flavonoids have been extensively
studied for their activity and are responsible at least in part - for many of the biological
properties of Scutellaria species. Some of
these properties include antioxidant,
anti-inflammatory, antimicrobial, antithrombotic, anti-cancer, cardioprotective
and neuroprotective actions [15].
There is a report that indicates most of
the clinically used antibiotics are active at
a concentration of 10 μg/mL and if a pure
substance is not active at 100 μg/mL, it
Chiang Mai J. Sci. 2013; 40(1)
probably will not be clinically useful as
antibiotic. [26]. Among tested extracts, ethyl
acetate showed the best bacteriostatic as
well as bacteriocidal effects. This is consistent
with what has been previously reported for
S. baicalensis. The observed antimicrobial
activities could be speculated to be exerted
by baicalin, baicalein and wogonin as these
phytochemicals have been reported to
inhibit the growth of several bacterial, fungal
and viral strains. Moreover, baicalin and
baicalein have synergism (through different
mechanisms) with several antibiotics such
as tetracycline, ampicillin, amoxycillin,
benzylpenicillin, cefotaxime and methicillin,
thus potentiating their efficacy against
different types of bacteria, notably MRSA [35].
Since the aforementioned flavonoids are not
very polar, they tend to accumulate in the
ethyl acetate rather than the more polar
methanol extract. This is consistent with the
higher antioxidant and antimicrobial
properties of the ethyl acetate extract.
57
vit C and BHT), they showed degrees of
biological activity which deserves attention in
several ways. First, individual phytochemicals
may be responsible for the observed activities.
If so, it may be interesting to find such
phytochemicals and test their activity as
pure compounds rather than in the extract
that is a mixtures containing low amount of
compounds and also source of potential
antagonism. Besides, performing a sequential
extraction and successive partitioning of
the obtained extracts would give a better
understanding on the true biological activity
and nature of active compounds within the
plant. Second, while the tested extracts are
not much potent to be used as drugs, their
consumption as supplements or adjunct
drugs (provided that their safety is confirmed)
could confer health benefits. For these
reasons, it is recommended that further
phytochemical analysis of the extracts along
with additional tests could be helpful for a
better judgement on the plant’s antioxidant
and antimicrobial activity.
4. CONCLUSIONS
In summary, the present findings
indicated moderate antioxidant activity of
S. linbergii extracts which was higher for
methanol and ethyl acetate extracts in the
DPPH and TBARS assays, respectively.
S. lindbergii extracts also showed moderate
antimicrobial effects against S. aureus, B. cereus,
E. coli, P. aeruginosa and C. albicans. To our
knowledge, there has been no previous study
on the antioxidant and antimicrobial
properties of this plant. While the current
investigation is subjected to limitations
such as lack of enough phytochemical
identification of plant bioactives and lack of
bioassay guided fractionation, investigation
on this plant, though preliminary, is very
novel. While S. lindbergii extracts were not
found to be as potent antioxidant and
antimicrobials as positive controls used (e.g.
ACKNOWLEDGEMENTS
This research was supported by a grant
from the Research Council of the Mashhad
University of Medical Sciences, Mashhad, Iran.
REFERENCES
[1] Djeridane A., Yousfi M., Nadjemi B.,
Boutassouna D., Stocker P. and Vidal
N., Antioxidant activity of some
algerian medicinal plants extract
containing phenolic compounds, Food
Chem., 2006; 97: 654-660.
[2] Gao Z., Huang K., Yang X. and Xu
H., Free radical scavenging and
antioxidant activities of flavonoids
extracted from the radix of Scutellaria
baicalensis Georgi, Biochim. Biophys.
Acta, 1999; 1472: 643-650.
58
[3] Sato Y., Suzaki S., Nishikawa T.,
Kihara M., Shibata H. and Higuti T.,
Phytochemical flavones isolated
from Scutellaria barbata and
antibacterial
activity
against
methicillin-resistant Staphylococcus
aureus, J. Ethnopharmacol., 2000; 72:
483-488.
[4] Kulisic T., Radonic A., Katalinic V.
and Milos M., Use of different
methods for testing antioxidative
activity of oregano essential oil, Food
Chem., 2004; 85: 633-640.
[5] Burits M., Asres K. and Bucar F., The
antioxidant activity of the essential oils
of Artemisia afra, Artemisia abyssinica
and Juniperus procera, Phytother. Res.,
2001; 15: 103-108.
[6] Lakshmanashetty R.H., Nagaraj V.B.,
Hiremath M.G. and Kumar V., In
vitro antioxidant activity of Vitex
negundo L. leaf extracts, Chiang Mai
J. Sci., 2010; 37: 489-497.
[7] Fazly Bazzaz B.S., Hassanzadeh M.K.,
Emami S. A., Asili J., Sahebkar A. and
Javadi Neishbory E., Antioxidant and
antimicrobial activity of methanol,
dichloromethane, and ethyl acetate
extracts of Scutellaria litwinowii,
ScienceAsia, 2011; 37: 327-334.
[8] Budrat P. and Shotipruk A.,
Extraction of phenolic compounds
from fruits of bitter melon
(Momordica charantia ) with subcritical
water extraction and antioxidant
activities of these extracts, Chiang Mai
J. Sci., 2008; 35: 123-130.
[9] Sahebkar A. and Iranshahi M.,
Biological activities of essential oils
from the genus Ferula (Apiaceae),
Asian Biomed., 2010; 4: 835-847.
[10] Ghahreman A. and Attar F.,
Biodiversity of Plant Species in Iran,
Tehran University Publication,
Tehran, 1999; 1: 384.
Chiang Mai J. Sci. 2013; 40(1)
[11] Schinella G.R., Tournier H.A., Prieto
J.M., Mordujovich de Buschiazzo P.
and R os J.L., Antioxidant activity of
anti-inflammatory plant extracts, Life
Sci., 2002; 70: 1023-1033.
[12] Yu J., Lei J., Yu H., Cai X. and Zou
G., Chemical composition and
antimicrobial activity of the
essential oil of Scutellaria barbata,
Phytochem., 2004; 65: 881-884.
[13] Kim H.M., Moon E.J., Li E., Kim
K.M., Nam S.Y. and Chung C.K. The
nitric oxide-producing activities of
Scutellaria baicalensis, Toxicol., 1999;
135: 109-115.
[14] Himeji M., Ohtsuki T., Fukazawa H.,
Tanaka M., Yazaki S. and Ui S., et al.,
Difference of growth-inhibitory effect
of Scutellaria baicalensis-producing
flavonoid wogonin among human
cancer cells and normal diploid cell,
Cancer Lett., 2006; 245: 269-274.
[15] Li-Weber M., New therapeutic aspects
of flavones: the anticancer properties
of Scutellaria and its main active
constituents Wogonin, Baicalein and
Baicalin, Cancer Treat. Rev., 2009; 35:
57-68.
[16] Rechinger K.H., Scutellaria in
Rechinger K.H., (ed.) Flora Iranica
1982; 150: 44-84. Akademische Drucku. Verlagsanstalt. Graz.
[17] Attar F. and Joharchi M.R., New
plant records from Iran, Iran J. Botany
2002; 9: 223-228.
[18] Tayarani-Najaran Z., Mousavi S.H.,
Asili J. and Emami S.A., Growthinhibitory effect of Scutellaria
lindbergii in human cancer cell lines,
Food Chem. Toxicol., 2010; 48: 599604.
[19] Lamaison J.L.C., Carnet A. Teneurs
en principaux flavonoids des fleurs
de Crataegeus monogyna Jacq et
de Crataegeus laevigata (Poiret D.C.)
Chiang Mai J. Sci. 2013; 40(1)
en fonction de la vegetation, Pharm.
Acta Helv., 1990; 65: 315-320.
[20] Lister E. and Wilson P., Measurement
of total phenolics and ABTS assay for
antioxidant activity (personal
communication), 2001, Crop Research
Institute, Lincoln, New Zealand.
[21] Bazzaz B.S.F., Khayat M.H., Emami
S.A., Asili J., Sahebkar A. and
Neishabory E.J., Antioxidant and
antimicrobial activity of methanol,
dichloromethane, and ethyl acetate
extracts of Scutellaria litwinowii,
ScienceAsia, 2011; 37: 327-334.
[22] Duffy C.F. and Power R.F.,
Antioxidant and antimicrobial
properties of some Chinese plant
extracts, Int. J. Antimicrob. Agents,
2001; 17: 527-529.
[23] Saha K., Lajis N.H., Israf D.A.,
Hamzah A.S., Khozirah S. and
Khamis S., et al., Evaluation of
antioxidant and nitric oxide inhibitory
activities of selected Malaysian
medicinal plant, J. Ethnopharmacol.,
2004; 92: 263-267.
[24] Chou T.C. and Talalay P.,
Quantitative analysis of dose-effect
relationships: the combined effects of
multiple drugs or enzyme inhibitors,
Adv. Enzyme Regul., 1984; 22: 27-55.
[25] Rios J.L., Recio M.C. and Villar A.,
Screening methods for natural
products with antimicrobial activity,
J. Ethnopharmacol., 1988; 23: 127-149.
[26] Eloff J.N., A sensitive and quick
microplate method to determine the
minimal inhibitory concentration of
plant extract for bacteria, Planta Med.,
1998; 64: 711-713.
[27] Singh R., Singh S., Kumar S. and
Arora S., Evaluation of antioxidant
potential of ethyl acetate extract/
59
fractions of Acacia auriculiformis A.
Cunn., Food Chem. Toxicol., 2007; 45:
1216-1223.
[28] Rios J.L., Recio M.C. and Villar A.,
Screening methods for natural
antimicrobial
products
with
antimicrobial activity: a review of the
literature, J. Ethnopharmacol., 1988;
23: 127-149.
[29] Leite P., Raphael J. and Vieira C.,
Antimicrobial activity of Indigofera
suffruticosa. Evid. Based Complement,
Alternat. Med., 2006; 3: 261-265.
[30] Markham R.K., Techniques of
Flavonoid Identification. London:
Academic Press, 1982; 15: 45.
[31] Blois M.S., Antioxidant determinations by the use of a stable free radical,
Nature, 1958; 181: 1199-1200.
[32] Yordi E.G., P rez E.M., Matos M.J.
and Villares E.U., Antioxidant and
Pro-Oxidant Effects of Polyphenolic
Compounds and Structure-Activity
Relationship Evidence, In: Nutrition,
Well-Being and Health. Bouayed J.
(ed.), 2012; InTech, Croatia, 23-48.
[33] Woo I.T., Park K.N. and Lee S.H.,
Antimicrobial activities of Scutellaria
baicalensis Georgi against various
pathogens and spoilage bacteria
isolated from Tofu, J. Korean Soc. Food
Sci. Nutr., 2007; 36: 470-475.
[34] Fazly Bazzaz B.S. and Haririzadeh G.,
Screening of Iranian plants for
antimicrobial activity, Pharm. Biol.,
2003; 41: 573-583.
[35] B ach-Olszewska Z. and LamerZarawska E., Come back to roottherapeutic activities of Scutellaria
baicalensis root in aspect of innate
immunity regulation-part I., Adv.
Clin. Exp. Med. 2008; 17: 337-45.