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Medicinal and Aromatic Plant Science and Biotechnology ©2013 Global Science Books
In Vitro Biological Activities of the
Components from Silene wallichiana
Nilufar Z. Mamadalieva1* • Dilfuza Egamberdieva2 • Antonio Tiezzi3
1 Laboratory of the Chemistry of Glycosides, Institute of the Chemistry of Plant Substances AS RUz, Mirzo Ulugbek Str. 77, Tashkent 100170, Uzbekistan
2 Department of Biotechnology and Microbiology, National University of Uzbekistan, Tashkent 100174, Uzbekistan
3 Department of Agriculture, Forests, Nature and Energy, Laboratory of Plant Cytology and Biotechnology, Tuscia University, Viterbo 01100, Italy
Corresponding author: * nmamadalieva@yahoo.com
ABSTRACT
Methanol, butanol, chloroform and water extracts and individually six phytoecdysteroids (viticosterone E, 20-hydroxyecdysone-22benzoate, 2-deoxy-20-hydroxyecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and integristerone A) isolated from a Silene wallichiana
Klotsch. plant were evaluated for their antibacterial and antiproliferative properties. The methanolic extract inhibited the growth of
Acinetobacter sp., Enterococcus faecalis, Klebsiella oxytoca, Pantoea agglomerans, Proteus rettgeri, Pseudomonas aeruginosa and
Staphylococcus aureus at an MIC of 2.5 mg/ml, while Escherichia coli and Klebsiella pneumoniae were inhibited at an MIC of 1.25
mg/ml. The proliferation of cancer cells was potently inhibited by the chloroform extract (IC50 28.53 ± 1.98 μg/ml in HeLa, 26.34 ± 2.34
μg/ml in HepG-2 cells). Aqueous and butanol extracts exhibited good antioxidant activity with IC50 values of 24.83 ± 2.82 and 46.32 ±
3.73 μg/ml.
_____________________________________________________________________________________________________________
Keywords: antibacterial, antioxidant, cytotoxic activity, phytoecdysteroids, plant extract
INTRODUCTION
The Silene genus (Caryophyllaceae) comprises more than
700 different species and its taxonomy appears to be very
complex (Grauter 1995). In Central Asia, 84 species plants
of the Silene genus grow (Bondarenko 1971). Chemical
investigations of this genus have led to the isolation of
ecdysteroids (Zibareva 1999), triterpene saponins (Glensk
et al. 1999; Lacaille-Dubois et al. 1999), flavonoids (Zemtsova and Dzhumyrko 1976), polysaccharides (Ovodova et
al. 2000), amino acids (Terrab et al. 2007), terpenes, nitrogen-containing compounds (Dotterl et al. 2007), organic
acids and microelements (Eshmirzayeva et al. 2005; Arnetoli et al. 2008). Silene spp. Plants are characterized by both
a large number of ecdysteroid-containing species and by the
highest diversity of ecdysteroid derivatives, such as acetates,
benzoates, glucosides, galactosides, xylosides, sulfates, etc.
(Lafont et al. 2002). Given the complexity of ecdysteroid
cocktails existing in many Silene species, it has been proposed that ecdysteroids have a chemotaxonomic value in
this genus (Zibareva et al. 2009). The qualitative and quantitative composition of ecdysteroid cocktails depends considerably on the plant species, but possibly also on soil climatic conditions and on the developmental stage of the
plant. Detailed investigations on biological and pharmacological activities of ecdysteroids isolated from the genus
Silene have been carried out and anabolic (Syrov 1984),
adaptogenic, tonic (Syrov and Kurmukov 1977), cardiotonic (Kurmukov and Yermishina 1991), antioxidant (Kuzmenko et al. 1997), antifeedant and insect growth-inhibition
(Kubo and Klocke 1983) properties of phytoecdysteroids
have been reported.
However, the in vitro biological actions of Silene plants
are not yet fully studied and in order to enlarge our knowledge on the biological and pharmacological properties of
this family of chemical compounds, we decided to carry out
investigations in order to evaluate possible in vitro biological activities of phytoecdysteroids and extracts of Silene
Received: 21 January, 2012. Accepted: 30 October, 2012.
wallichiana Klotzsch. Previous studies (Saatov et al. 1987;
Mamadalieva et al. 2000) reported that S. wallichiana is an
ecdysteroid-rich resource and the following phytoecdysteroids were isolated from this plant: viticosterone E, 20hydroxyecdysone-22-benzoate, 2-deoxyecdysone-22-benzoate, viticosterone E-22-benzoate, 2-deoxy-20-hydroxyecdysone, 2-deoxyecdysone, 20-hydroxyecdysone, 3-benzoate-2-deoxy-20-hydroxyecdysone, 22-benzoate-2-deoxy20-hydroxyecdysone, integristerone A and 25-acetate-2deoxy-20-hydroxyecdysone.
In the present study we report on the antimicrobial,
antioxidant and cytotoxic activities of the aerial parts of
several extracts (methanol, chloroform, butanol and water)
from S. wallichiana in comparison with six major isolated
phytoecdysteroids, namely viticosterone E (1), 20-hydroxyecdysone-22-benzoate (2), 2-deoxy-20-hydroxyecdysone
(3), 2-deoxyecdysone (4), 20-hydroxyecdysone (5), and
integristerone A (6) (Fig. 1).
MATERIALS AND METHODS
Chemicals and reagents
Cell culture media, supplements, dimethyl sulfoxide (DMSO),
quercetin, MTT, and doxorubicin ( 98%) were purchased from
Gibco, Invitrogen (Italy) and Sigma (Milan, Italy).
HPLC analysis
Authentical phytoecdysteroids 1-6 were obtained from the Institute of the Chemistry of Plant Substances, Tashkent, Uzbekistan.
The purity of the compounds 1-6 were > 95%, as determined by
HPLC using a high performance liquid chromatograph LC10ATvp connected to a UV-VIS detector SPD-10Avp (Shimadzu
Co, Kyoto, Japan). Samples were diluted to 1 mg/ml, filtered
through a 0.22 μm filter and 20 μl was injected into a column
(Nucleosil 100-5 C18, 250 mm × 4 mm in size (Macherey-Nagel
GmbH & Co, KG)). Elution was carried out by mobile phase A
Original Research Paper
Medicinal and Aromatic Plant Science and Biotechnology 7 (1), 1-6 ©2013 Global Science Books
Al2O3 column (0.5 kg) with elution by system A and 125 mg of
compound 1 was isolated. The yield of this phytoecdysteroid was
0.005%. Yield was calculated based on the air-dried weight of
plant material, C29H46O8, mp 194-196°C, using acetone (Saatov et
al. 1987).
Further elution of the column with system A gave 2 (1 g,
0.04%), C34H48O8, mp 203-205°C (MeOH-H2O). Elution of the
column with system B gave compounds 3 (2 g, 0.08%, C27H44O6,
mp 254-256°C (EtOH-H2O)) and 4 (4 g, 0.16%, C27H44O5, mp
234-235°C (EtOH-H2O)) (Mamadalieva et al. 2000). Elution of
the column with system C gave 5 (2 g, 0.08%), C27H44O7, mp 241242°C (methanol-acetone). Subsequent elution of the column with
the same system gave 6 (425 mg, 0.017%), C27H44O8, mp 246248°C (ethyl acetate-methanol) (Mamadalieva et al. 2000). The
isolated phytoecdysteroids were used for biological assays.
R4
R3
21
22
24 26
18
20
23
R1
11
19
13
OH
25
R5
27
17
R2
OH
5
7
HO
H
Phytoecdysteroid
1
2
3
4
5
6
15
9
1 10
3
R1
H
H
H
H
H
OH
O
R2
OH
OH
H
H
OH
OH
R3
OH
OH
OH
H
OH
OH
R4
OH
COOC6H5
OH
OH
OH
OH
R5
COOCH3
OH
OH
OH
OH
OH
Antibacterial activity
Microorganisms. The methanol and butanol extracts were individually tested against the following clinical microorganisms: Klebsiella oxytoca 6653, K. pneumoniae 40602, K. aerogenes
NCTC8172, Citrobacter freundii 82073, Staphylococcus aureus
MRSA16, Enterococcus faecalis NCTC775, Proteus rettgeri
NCIMB9570, Pseudomonas aeruginosa NCTC6749, Escherichia
coli NCTC9001, Enterobacter hormaechei T2, Acinetobacter sp.
T132, Pantoea agglomerans T26, and Bacillus cereus T80. Reference strains and clinical isolates were obtained from the Department of Microbiology, Manchester Metropolitan University, UK,
the National Culture Type Collection (NCTC), UK, and from the
culture collection of National University of Uzbekistan.
Strains were maintained at 37°C on Columbia agar slants supplemented with 5% horse blood (v/v) (Oxoid, Basingstoke, UK).
The antibacterial activity of the extracts and individual phytoecdysteroids was carried out by the disc diffusion test (Kim et al. 1995).
Microorganisms were grown overnight at 30°C in Mueller-Hinton
Broth (Oxoid), supplemented with 5% horse blood and 100 l of
suspension containing 106 CFU/ml of bacteria spread on the surface of agar plates. Plant extracts (5 mg/ml) and compounds (1
mg/ml) were first dissolved in dimethylsulfoxide (DMSO). Sterile
filter discs (6 mm in diameter) were impregnated with 15 μl of
each extract (0.6-5 mg/ml) and of each individual phytoecdysteroid concentrations (0.06-1 mg/ml) and placed on the surface of
inoculated plates. The plates were incubated at 37°C for 24 h. The
assessment of antibacterial activity was based on the measurement
of inhibition zones formed around the discs. Five discs per plate
were used and each test was run in triplicate. The diameter of the
zone was measured and recorded. Ampicillin (10 μg/ml) and tetracyclin (30 μg/ml) were used as positive controls.
Fig. 1 The chemical structure of the phytoecdysteroids studied in vitro
tests.
(water) and solvent B (acetonitrile) and the gradient profile was as
follows: from 0% B to 5% B in 8 min, from 5% B to 85% B from
8-30 min, from 95% B to 100% B% from 30-35 min and at 100%
B% until 40 min. Flow rate was 1 ml/min and detection was at 247
nm and 200 nm (Abdukadyrov et al. 2005).
Plant material
S. wallichiana was obtained from the Syrkhandaryo region of
Uzbekistan in June 2008 and identified by Dr A.M. Nigmatullaev
and deposited in the Laboratory of Herbal Plants, Institute of the
Chemistry of Plant Substance, Tashkent (voucher specimen No.
2006231).
Extract preparation for bioassays
S. wallichina (syn. S. vulgaris, Oberna wallichiana Klotzsch) is a
hairless, perennial forb that grows up to 91 cm tall from a woody
rootstock. Stems are branched from the base, smooth, and swollen
at the nodes. Leaves are sessile, smooth, ovate or lanceolate, glaucous, pale green, 31 to 82 mm long, and 12 to 31 mm wide.
Flowers are 12 mm in diameter and are borne in terminal clusters
of 5 to 30. They are composed of 5 united and deeply notched
petals, 10 stamens, and 3 styles. Calyxes are initially slender but
develop into greatly inflated, often purplish, papery, sac-like structures that surround the bulbous fruits. Fruits open at the toothed
tops of the calyxes. Seeds are numerous, small, and grayish
(Douglas and MacKinnon 1998; Royer and Dickinson 1999; Whitson et al. 2000; Klein 2011).
The aerial parts of S. wallichiana were dried at room temperature and reduced to a coarse powder. After grinding, 10 g of plant
material was extracted separately with 50 ml of different solvents
(methanol, chloroform and water) for 24 h in the dark and at room
temperature. Solvents were evaporated under reduced pressure to
provide 0.71 g of the methanolic (7.0% of air-dried weight of the
plant), 0.41 g of the chloroform (4.1%) and 0.29 g of the aqueous
(2.9%) extracts. The butanol extract was prepared as reported by
Saatov et al. (1987). The obtained residues were than used for in
vitro screening of antibacterial, antioxidant or antiproliferative
tests.
Cytotoxic activity
1. Cell cultures
HeLa (cervical cancer) and HepG-2 (hepatic cancer) cell lines
were supplied by the Laboratory of Plant Cytology and Biotechnology, Tuscia University, Italy. Cancer cells were maintained in
DMEM complete media (L-glutamine supplemented with 10%
heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100
μg/ml streptomycin (Invitrogen, Italy) in addition to 10 mM of
non-essential amino acids (Invitrogen, Italy). Cells were grown at
37°C in a humidified atmosphere of 5% CO2. All experiments
were performed with cells in the logarithmic growth phase.
2. MTT assay
Isolation of phytoecdysteroids
Sensitivity to drugs was determined in triplicate using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell
viability assay (Mosmann 1983). The extracts and phytoecdysteroids were dissolved in DMSO and diluted in the medium at
final concentrations ranging from 0.977 to 500 μg/ml for extracts
and from 0.977 to 500 μM for phytoecdysteroids, in 96-well plates
(Greiner Labortechnik). Wells containing the solvent and wells
without the solvent were included in the experiment. Cells (2 ×
104 cells/well of exponentially growing cells of each individual
HeLa and HepG-2 cell lines) were seeded in a 96-well plate (Grei-
Air-dried material (2.5 kg) of S. wallichiana was exhaustively
extracted with ethanol (10 L). The extract was concentrated and
diluted with water. The resulting precipitate was removed. The
ethanol was evaporated. The aqueous solution was treated first
with CHCl3 and then with ethyl acetate. The solvents were evaporated under vacuum. A solvent system of CHCl3 - CH3OH [15:1
(A), 9:1 (B), 4:1 (C)] was used for TLC and column chromatography.
The ethyl acetate extract (15 g) was chromatographed on an
2
Biological activities of Silene wallichiana. Mamadalieva et al.
0.25
0.5
0.5
0.25
-
0.5
0.5
-
Tetracyclin
0.25
0.25
0.25
0.5
0.5
Ampicillin
Viticosterone E (1)
2.5
1.25
-
Integristerone A (6)
Water extract
2.5
2.5
1.25
-
20-Hydroxyecdysone (5)
Chloroform extract
2.5
2.5
2.5
2.5
1.25
2.5
-
2-Deoxyecdysone (4)
Butanol extract
2.5
1.25
2.5
2.5
1.25
2.5
2.5
2.5
2.5
2-Deoxy20-ydroxyecdysone (3)
Methanol extract
Acinetobacter sp. T132
B. cereus T80
C. freundii 82073
E. coli NCTC9001
E. faecalis NCTC775
E. hormaechei T2
K. oxytoca 6653
K. pneumoniae 40602
K. aerogenes NCTC8172
P. agglomerans T26
P. rettgeri NCIMB9570
P. aeruginosa NCTC6749
S. aureus MRSA16
20-Hydroxyecdysone22-benzoate (2)
Table 1 Minimum inhibitory concentrations (MIC) of the extracts and phytoecdysteroids of S. wallichiana against different pathogens using the disc
diffusion test.
MIC (mg/ml)
Bacterial strain
0.5
0.5
-
0.5
0.5
0.25
-
0.5
-
2.5
0.25
2.5
1.25
0.5
1.25
2.5
2.5
0.25
0.25
0.5
1.25
1.25
0.5
1.25
1.25
1.25
1.25
2.5
1.25
1.25
reported in Table 1. The Acinetobacter sp, Enterococcus
faecalis, Klebsiella oxytoca, Pantoea agglomerans, Proteus
rettgeri, Pseudomonas aeruginosa and Staphylococcus
aureus strains were inhibited by the methanol extract of S.
wallichiana at MIC = 2.5 mg/ml, while Escherichia coli
and Klebsiella pneumoniae was inhibited at MIC = 1.25
mg/ml. The butanol extract of S. wallichiana showed activity against the pathogenic bacterium Acinetobacter sp., E.
coli, K. pneumoniae, P. agglomerans, P. aeruginosa (MIC =
2.5 mg/ml), and P. rettgeri (MIC =1.25 mg/ml), although
with weaker action respect to the methanol extract. The
chloroform extract had minimum activity against all bacterial strains and only inhibited Citrobacter freundii, E. coli
(MIC = 2.5 mg/ml) and P. aeruginosa (MIC = 1.25 mg/ml).
The S. wallichiana aqueous extract also showed low antimicrobial activity against two strains only, E. coli and P.
aeruginosa (MIC = 2.5 and 1.25 mg/ml, respectively).
Pure phytoecdysteroids exhibited very low activity
against the bacteria. Among the tested compounds, phytoecdysteroid 1 showed in vitro activity against E. coli, K.
pneumoniae, K. aerogenes, P. rettgeri (MIC = 0.25 mg/ml),
and S. aureus and P. rettgeri (MIC = 0.5 mg/ml). Compound 2 showed antibacterial activity against B. cereus, K.
aerogenes (MIC = 0.25 mg/ml) and E. coli and K. pneumoniae (MIC = 0.5 mg/ml). Phytoecdysteroids 3 and 4
showed low antimicrobial activity against E. coli and P.
rettgeri (MIC = 0.5 mg/ml). Phytoecdysteroid 6 only inhibited the growth of K. aerogenes NCTC8172. In contrast,
phytoecdysteroid 5 promoted the growth of P. rettgeri (MIC
= 0.25 mg/ml), E. coli and K. pneumoniae (MIC = 0.5
mg/ml) (Table 1). From these results we observed that
phytoecdysteroids show weak antibacterial activity compared medicinal plant extracts tested. In addition, this finding was consistent with the previous results of Ahmad et al.
(1996) and Shirshova et al. (2006), who claimed that most
likely such compounds are not the major molecules responsible for the antibacterial activity of the plant extracts.
Ahmad et al. (1996) and Shirshova et al. (2006) reported
that some natural phytoecdysteroids, including 20-hydroxyecdysone, inokosterone, and ecdysone, did not exhibit antimicrobial activity with respect to most standard test microbe cultures. However, introduction of the acetyl group
into the 20-hydroxyecdysone molecule significantly increased the antibacterial activity with respect to microbes
inducing inflammatory and purulent processes (Shirshova et
al. 2006). In our case, besides ecdysteroids, extracts exhibiting antibacterial activity were related to the chemical
ner Labortechnik), cultivated for 24 h then incubated with various
concentrations of tested samples at 37°C for 24 h and then with
0.5 mg/ml MTT for 4 h. The formazan crystals that formed were
dissolved in 100 μl DMSO. The absorbance was detected at 595
nm with a TECAN Sunrise Reader. The cell viability rate (%) of
three independent experiments was calculated by the following
formula (Zeytinoglu et al. 2008):
Cell viability rate (%) = ((OD of treated cells OD of media
(blank) / (OD of control cells OD of media (blank)) × 100 %
DPPH radical-scavenging (antioxidant) activity
The antioxidant and radical scavenging activities of the isolated
compounds and extracts were evaluated according to Brand-Williams et al. (1995) using diphenyl picryl hydrazyl (DPPH). Equal
volumes of sample solutions containing 0.02-10 mg/ml of sample
and 0.2 mM methanolic solution of DPPH were pipetted into 96well plates. The absorbance was measured against a blank at 517
nm using a TECAN Sunrise Reader after incubation in the dark for
30 min at room temperature and compared with DPPH control
after background subtraction. Quercetin was used as a positive
control. The percent inhibition was calculated from three different
experiments using the following equation:
RSA (%) = [(Abs517 control - Abs517 sample)/ Abs517 control] × 100
where RSA = radical scavenging activity; Abs517 = absorption at
517 nm; control = non-reduced DPPH.
Statistical analysis
All experiments were carried out three times unless mentioned in
the procedure. Continuous variables were presented as mean ± SD.
IC50 values were calculated using Student’s t-test followed by
Dunn’s post-hoc multiple comparison test when the significance
value was < 0.05 using the same significance level.
RESULTS AND DISCUSSION
Antibacterial activity
Powder extracts (methanol, chloroform, butanol and water)
and individual phytoecdysteroids 1-6 of S. wallichiana were
tested in order to evaluate their antibacterial activity. The
plant extracts were tested at various concentrations ranging
from 0.6 – 5 mg/ml and the evaluated MIC values are
3
Medicinal and Aromatic Plant Science and Biotechnology 7 (1), 1-6 ©2013 Global Science Books
Table 2 Antiproliferative activities of ecdysteroids and extracts isolated from S. wallichiana on HeLa and HepG-2 cell lines. The data are represented as
IC50 values (mean ± SD).
IC50 of extracts (ȝg/ml) and compounds (ȝM)
Sample
HeLa
HepG-2
Extracts
Methanol
70.81 ± 1.73 de
74.56 ± 2.92 de
Butanol
86.08 ± 2.30 d
80.21 ± 7.12 d
Chloroform
28.53 ± 1.98 e
26.34 ± 2.34 e
Water
103.72 ± 1.84 c
98.47 ± 3.92 cd
Phytoecdysteroids
Viticosterone E (1)
98.27 ± 6.25 cd
88.96 ± 5.77 d
20-Hydroxyecdysone-22-benzoate (2)
127.97 ± 1.34 b
106.76 ± 7.81 c
2-Deoxy-20-hydroxyecdysone (3)
174.88 ± 9.10 a
195.61 ± 7.26 a
2-Deoxyecdysone (4)
171.25 ± 8.13 a
184.48 ± 9.49 ab
20-Hydroxyecdysone (5)
175.02 ± 6.34 a
130.26 ± 2.87 bc
Integristerone A (6)
158.75 ± 4.52 ab
142.67 ± 5.87 b
Control
Doxorubicin (μg/ml)
1.07 ± 0.11
0.39 ± 0.04
Doxorubicin (μM/ml)
1.84 ± 0.19
0.67 ± 0.07
nature of the solvents which play a key role in the extraction of different chemical compounds from the powder of S.
wallichiana.
Compared with antibiotics (positive control), the disk
diffusion method showed that all plant extracts had antimicrobial properties. The aqueous extract showed weak
antimicrobial activity against the tested microorganisms.
The antimicrobial activity of the chloroform extract of S.
wallichiana against bacterial strains confirmed the results
reported by us and by other authors (Kucukboyaci et al.
2010; Mamadalieva et al. 2010). Both chloroform and
aqueous extracts were more active only for P. aeruginosa
than methanol and butanol extracts. The methanol and
butanol extracts of S. wallichiana contains flavonoids and
triterpene glycosides (unpublished data), and flavonoids
might be responsible for part of the antimicrobial activity,
as well as several other classes of alcohol-soluble plant
molecules (Cowan 1999). The methanol extract some of
Silene species contains a wider range of components, especially lipids, essential oils, flavonoids, steroids, carbohydrates, microelements, proteins and amino acids (LacailleDubois et al. 1999; Jürgens 2004; Eshmirzayeva et al.
2005; Dotterl et al. 2007; Terrab et al. 2007). Its higher
activity than that of the butanol extract can be explained by
the fact that some polar compounds present in the methanol
extract (which are removed during the preparation of the
butanol extract) display a maintain effect towards the antimicrobial components. As a consequence, this raises a question about the evaluation of the activity of crude methanolic
plant extracts, as used classically during screening tests,
which might underscore their actual antimicrobial potency.
Compared to doxorubicin, the extracts and phytoecdysteroids showed moderate antiproliferative activity.
In the experiments phytoecdysteroids displayed mild
antiproliferative activity against both cancer cell lines while
the chloroform extract was more active. Some phytoecdysteroids isolated from Ajuga species showed antitumour
activities in a mouse-skin model in vivo in a two-stage carcinogenesis trial, using 7,12-dimethylbenz[a]anthracene as
initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA)
as the promoter (Takasaki et al. 1999). Also, El-Mofti (1987,
1994) reported that ecdysone was able to induce neoplastic
lesions in toads and mice; other researchers reported that
ecdysteroid-containing extract of Silene viridiÀora exerted
antitumour activity in vivo in mice (Zibareva 2003). However, Lagova and Valueva (1981) reported that 20-hydroxyecdysone was mainly ineffective in preventing tumour
growth in mice, but it stimulated the growth of mammary
gland carcinomas. Most likely, since ecdysteroids structurally resemble sex hormones, they may bind to steroid
hormone receptors in mammals and stimulate the growth of
hormone-dependent tumours.
Flavonoids, triterpene saponins, ecdysteroids, and polysaccharides are common constituents in the genus Silene. In
particular, saponins may be responsible for the antiproliferative effects of the extracts. The saponins jenisseensosides C
and D from Silene fortunei stimulated the proliferation of
Jurkat tumor cells at low concentrations (10-3 – 10-1 μM); at
high concentrations (10 μM) they were cytotoxic and apparently induced apoptosis (Gaidi et al. 2002). Our results
suggest that the chemical contents of chloroform extract
and the mechanism of these substances as they affect tumor
cells needs to be further elucidated.
Antiproliferative activity
Antioxidant activity
The antiproliferative activity of the extracts, six individual
isolated phytoecdysteroids and doxorubicin (as the positive
control) were tested against HeLa and HepG-2 cell lines.
The IC50 values are shown in Table 2. Experimental results
revealed that the chloroform extract of S. wallichiana possesses significant antiproliferative activity which potently
inhibited cell growth in all cells tested (IC50 28.53 ± 1.98
μg/ml in HeLa, 26.34 ± 2.34 μg/ml in HepG-2 cells). The
proliferation of malignant cells was more strongly inhibited
by methanol extract (IC50 70.81 ± 1.73 μg/ml in HeLa,
74.56 ± 2.92 μg/ml in HepG-2 cells) than by butanol (86.08
± 2.30 μg/ml in HeLa, 80.21 ± 7.12 μg/ml in HepG-2 cells).
In this case water extract showed weak antiproliferative
activities against HeLa and HepG-2 cell lines, with IC50
values of 103.72 ± 1.84 and 98.47 ± 3.92 μg/ml.
Viticosterone E (1) was most active in HeLa and HepG2 cells (IC50 = 98.27 ± 6.25 and 88.96 ± 5.77 μM, respectively), while other phytoecdysteroids 2-6 were weakly
active against human cancer cells (IC50 > 106.76 ± 7.81 μM).
The antioxidant and radical scavenging activities of the isolated compounds, extracts and quercetin (as a positive control) are summarized in Table 3. The activity of the positive
control was 3.37 μg/ml. Maximum scavenging activity of
DPPH was observed with the aqueous extract at IC50 =
24.83 μg/ml, followed by the activity of the butanol, methanol, and chloroform extracts with IC50 = 46.32, 131.55, and
153.31 μg/ml, respectively. All ecdysteroids had weak
DPPH radical scavenging activity with IC50 values greater
than 100 μM. The effectiveness of phytoecdysteroids as
DPPH radical scavengers ranged in the following ascending
order: 2-deoxyecdysone (137.68 μg/mL) > 20-hydroxyecdysone (144.75 μg/mL) > 2-deoxy-20-hydroxyecdysone
(157.29 μg/mL) > 20-hydroxyecdysone-22-benzoate
(168.33 μg/mL) > integristerone A (178.98 μg/mL) >
viticosterone E (181.23 μg/mL).
Our results coincide with what was found in another
study (Miliauskas et al. 2005). In their study identified
4
Biological activities of Silene wallichiana. Mamadalieva et al.
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Table 3 Antioxidant activity of pure isolated phytoecdysteroids and
extracts of S. wallichiana using the DPPH* radical scavenging assay. The
data are represented as IC50 values (mean ± SD).
Sample
IC50 (ȝg/ml)
Extracts
Methanol extract
131.55 ± 9.43 d
Butanol extract
46.32 ± 3.73 e
Chloroform extract
153.31 ± 12.65 bc
Water extract
24.83 ± 2.82 e
Phytoecdysteroids
Viticosterone E (1)
181.23 ± 15.34 a
20-Hydroxyecdysone-22-benzoate (2)
168.33 ± 11.02 ab
2-Deoxy-20-hydroxyecdysone (3)
157.29 ± 16.72 b
2-Deoxyecdysone (4)
137.68 ± 3.56 cd
20-Hydroxyecdysone (5)
144.75 ± 11.53 c
Integristerone A (6)
178.98 ± 11.51 a
Control
Quercetin (positive control)
3.37 ± 0.77
radical scavenging compounds in extracts of Rhaponticum
carthamoides which tested against DPPH radical showed
weak radical scavenging activity. Our TLC investigations
showed that polar extracts of S. wallichiana such as methanol, water and butanol contained a high amount of phytoecdysteroids (unpublished data). And this seemed that presence of phytoecdysteroids in these extracts might reduce
their antioxidant activity. It is known that the presence of
the ortho arrangement of two hydroxyl groups on the
aromatic ring and 2,3-double bond in conjugation with 4oxo function is essential for the antiradical activity of flavonoids. More effective is the ortho-arrangement of hydroxyl groups on the aromatic ring B (quercetin) (Harborne
and Williams 2000). Ecdysteroids are polyhydroxylated
steroids that contain a 7,8-double bond and a 6-oxo function.
This explanation seems to be a more forceful argument
since the structure of ecdysteroid molecules is unlikely to
exert an antioxidant effect compared to common antioxidative flavonoids.
In conclusion, we suggest that further studies should be
performed on the isolation and identification of the active
nonpolar compounds of the chloroform extract of S. wallichiana. Also, the aqueous extract should be further studied
in detail to isolate individual chemical constituents responsible for the antioxidant activity. These results may provide
a starting point for investigations to exploit new natural
antimicrobial, cytotoxic and antioxidant substances.
ACKNOWLEDGEMENTS
The authors thank Dr. Jaime A. Teixeira da Silva for significant
improvements to grammar.
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