Flavonoid constituents and biological screening of Astragalus bombycinus Boiss

Article abstract of DOI:10.1080/14786419.2012.701213

Two new flavonoid compounds were isolated from Astragalus bombycinus Boiss. and identified as quercetin-3,7-di-O-β-glucopyranoside 40-O-β-rhamnopyranoside and 5,20,40-trihydroxy-flavone-8-C-β- arabinopyranoside-7-O-β-glucopyranoside. In addition, apigenin

Full text of DOI:10.1080/14786419.2012.701213

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Flavonoid constituents and biological
screening of Astragalus bombycinus
Boiss.
a
a
a
Lamyaa F. Ibrahim , Mona M. Marzouk , Sameh R. Hussein
,
a
b
a
Salwa A. Kawashty , Khaled Mahmoud & Nabiel A.M. Saleh
a
Department of Phytochemistry and Plant Systematics, National
Research Centre, Dokki – 12644, Cairo, Egypt
b
Department of Pharmacognasy, National Research Centre, Dokki
–
12311, Cairo, Egypt
Version of record first published: 06 Jul 2012.
To cite this article: Lamyaa F. Ibrahim , Mona M. Marzouk , Sameh R. Hussein , Salwa A. Kawashty ,
Khaled Mahmoud & Nabiel A.M. Saleh (2013): Flavonoid constituents and biological screening of
Astragalus bombycinus Boiss., Natural Product Research: Formerly Natural Product Letters, 27:4-5,
386-393
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Natural Product Research, 2013
Natural Product Research
Vol. 27, Nos. 4–5, 386–393, http://dx.doi.org/10.1080/14786419.2012.701213
2
012, 1–8, iFirst
Flavonoid constituents and biological screening of Astragalus
y
bombycinus Boiss.
a
Lamyaa F. Ibrahim , Mona M. Marzouk , Sameh R. Hussein , Salwa A. Kawashty ,
a
a
a
b
a
Khaled Mahmoud and Nabiel A.M. Saleh *
a
Department of Phytochemistry and Plant Systematics, National Research Centre, Dokki – 12644,
Cairo, Egypt; Department of Pharmacognasy, National Research Centre, Dokki – 12311, Cairo,
Egypt
b
(
Received 6 November 2011; final version received 24 May 2012)
Two new flavonoid compounds were isolated from Astragalus bombycinus Boiss.
0
and identified as quercetin-3,7-di-O-�-glucopyranoside 4 -O-ꢀ-rhamnopyranoside
0
0
and 5,2 ,4 -trihydroxy-flavone-8-C-ꢀ-arabinopyranoside-7-O-�-glucopyranoside.
In addition, apigenin, apigenin-7-O-�-glucopyranoside, apigenin 7-O-gentobio-
side, luteolin, luteolin-7-O-�-glucopyranoside, quercetin-3,7-di-O-�-glucopyrano-
side, quercetin-3-O-�-glucopyranoside-7-O-ꢀ-rhamnopyranoside and daidzein
were also isolated and identified. The structure elucidation of the isolated
compounds was performed by chromatographic, chemical and spectroscopic
methods. Antioxidant and cytotoxic activities were also determined for the four
consecutive extracts of the plant.
Keywords: Astragalus bombycinus Boiss.; Fabaceae (Leguminosae); new
flavonoids; antioxidant; cytotoxic activities
1
. Introduction
Astragalus L. is the largest genus belonging to the family Fabaceae (Leguminosae). It is
comprised of 2000 species distributed mainly in the northern temperate regions and
tropical African mountains, with 32 species indigenous to Egypt (Boulos, 1999).
Astragalus is a precious herbal plant in Chinese traditional medicine which has the
properties of intensifying phagocytosis of reticulo-endothelial systems, stimulate pituitary–
adrenal cortical activity and restore depleted red blood cell formation in bone marrow
(
Cassileth et al., 2009). Earlier investigations performed on Astragalus species resulted in
the isolation of three major classes of compounds; polysaccharides, saponins and
flavonoids (El-Sebakhy et al., 1994; He & Findlay, 1991; Isaev, Gorovits, & Abubakirov,
1
988; Tang & Eisenbrand, 1992). On the basis of these reports and as a part of our ongoing
research of new bioactive compounds from Egyptian species, we have investigated the
whole plant of Astragalus bombycinus Boiss., a selected species previously not subjected to
chemical or biological studies. This study reports the isolation and structure elucidation of
two new flavonoid compounds 1 and 2, along with eight known compounds for
the first time. The free radical scavenging activity of different extracts was measured
*
Corresponding author. Email: nabsaleh@gmail.com
This article is a contribution for the special issue on the 70th birthday of Prof. Dr Atta-ur-Rahman.
y
*
Corresponding author. Email: nabsaleh@gmail.com
I† SSN 1478–6419 print/ISSN 1478–6427 online
This article is a contribution for the special issue on the 70th birthday of Prof. Dr Atta-ur-Rahman.
*
Corresponding author. Email: xxxxxxxxxxxxxx
�
http://dx.doi.org/10.1080/14786419.2012.701213
2012 Taylor & Francis
©
2013 Taylor & Francis
h© tt 2p 0: /1/ 3w Tw a wy l. to ar n&d f Fo rna lni nc ie s.com

2
L.F. Ibrahim et al.
Natural Product Research
387
.
by 1,1-diphenyl-2-picryl-hydrazil (DPPH ) as
a
model of antioxidant activity.
Furthermore, the cytotoxic effect against four human cell lines; lung carcinoma A549,
breast carcinoma MCF7, colon carcinoma HCT116 and liver carcinoma HEPG2 cancer
cell lines was also evaluated.
2
2
. Results and discussion
.1. Identification of isolated compounds
0
Two new flavonoid compounds; quercetin-3,7-di-O-�-glucopyranoside-4 -O-ꢀ-rhamno-
0
0
pyranoside (1) and 5,2 ,4 -trihydroxy-flavone-8-C-ꢀ-arabinopyranoside-7-O-�-glucopyra-
noside (2) were isolated from A. bombycinus Boiss. extract along with eight known
flavonoid compounds (Figure 1). These were identified as apigenin (3), apigenin-7-O-�-
glucopyranoside (4), apigenin 7-O-gentobioside (5), luteolin (6), luteolin-7-O-�-glucopyr-
anoside (7), quercetin-3,7-di-O-�-glucopyranoside (8), quercetin 3-O-�-glucopyranoside-7-
O-ꢀ-rhamnopyranoside (9) and daidzein (10). Their structure elucidation was carried out
through R -values, colour reactions, chemical investigations (enzymatic hydrolysis,
f
complete and mild acid hydrolysis) and physical investigations (UV, NMR and MS)
(
Agrawal & Bansal, 1989; Mabry, Markham, & Chari, 1982; Mabry, Markham, &
Thomas, 1970; Markham, 1982; Markham & Geiger, 1994). Spectral data of the known
flavonoids were in good accordance with those previously published (Agrawal & Bansal,
1
989; Markham & Geiger, 1994).
Compound 1 was obtained as an amorphous yellow powder. The negative-ion ESIMS
�
showed a molecular ion peak [M � H] at m/z 771.26, corresponding to C H O . The
3
3
40 21
UV spectral data after using various shift reagents indicated a skeleton of a flavonol with a
0
free hydroxyl group at C-5 position, while those at C-3, C-7 and C-4 are substituted.
Investigation of the hydrolysate at different time intervals during partial acid hydrolysis
ꢀ
(
0.1 N HCl, 1 h, 100 C) revealed the presence of two intermediates which were identified
by TLC and comparison with authentic samples of quercetin-3,7-di-O-�-glucopyranoside
and quercetin-7-O-�-glucopyranoside before yielding the corresponding quercetin agly-
cone and glucose and rhamnose as sugar moieties. Precise PC investigation of the
hydrolysate resulting from �-glucosidase enzymatic hydrolysis revealed the presence of
0
quercetin 4 -rhamnopyranoside (Co-chromatography, UV and NMR) which indicated
1
that glucosylation sites were at positions C-3 and C-7. The H-NMR spectrum of
compound 1 showed the presence of a quercetin skeleton; in which ring A had two meta-
coupled protons for H-6 and H-8 indicated by their downfield shift at 6.40 and 6.72,
respectively, due to the occupation at C-7. The aromatic protons of the B-ring appeared as
0
a doublet of doublet (ꢁ 7.66, J ¼ 2.0, 8.5 Hz) assigned to H-6 and a doublet (ꢁ 7.64,
0
0
J ¼ 2.0 Hz) assigned to H-2 along with another doublet (ꢁ 7.12, J ¼ 8.5 Hz) for H-5 with a
0
downfield shift which indicated that the hydroxyl group at C-4 is substituted (Markham &
Geiger, 1994). Three anomeric proton signals resonated at ꢁ 5.43 and 5.04 (J ¼ 7.8 Hz)
corresponding to two �-configuration glucose units at C-3 and C-7, respectively, and ꢁ 5.40
0
(
J ¼ 1.5 Hz) for an ꢀ-configuration rhamnose unit at C-4 position. A doublet signal at
ꢁ
0.81(J ¼ 6.0 Hz), corresponded to the methyl group of the rhamnose moiety (Markham &
1
3
Geiger, 1994). The C-NMR spectrum confirmed the presence of 15 carbon signals
assigned for the quercetin aglycone and 18 carbon signals for the sugar units with respect
0
to the chemical shift of C-3 at 133.5, C-7 at 162.8 and C-4 at 146.4 confirming the
0
glycosylation sites. The position of the rhamnose unit was assigned to C-4 on the basis of
0
HMBC correlation between the anomeric proton of rhamnose at ꢁ 5.40 and C-4 at 146.4.
It was also proved that the two �-glucopyranoside moieties are attached to C-3 and C-7
0
0
000
showing correlations between the anomeric protons of H-1 at ꢁ 5.43 and H-1 at ꢁ 5.04

3
88
L.F. Ibrahim et al.
Natural Product Research
3
Figure 1. Compounds 1–10 isolated from A. bombycinus.
with C-3 at 133.5 ppm and C-7 at 162.8 ppm, respectively. On the basis of the previously
0
discussed data, compound 1 was identified as quercetin-3,7-di-O-�-glucopyranoside-4 -O-
ꢀ
-rhamnopyranoside.
Compound 2 was isolated as a yellow powder. The negative-ion ESIMS spectrum
�
showed a negative-ion peak [M � H] at 579.24 m/z corresponding to a molecular formula
C H O . UV spectral data with diagnostic shift reagents indicated a flavone nucleus
2
6
28 15
0
with a substituted hydroxyl group at C-7 while those at C-5 and C-4 are free in addition to
the absence of any ortho-dihydroxyl groups (Mabry et al., 1970). Complete acid hydrolysis
ꢀ
(
2 N HCl, 1 h, 100 C) gave glucose along with an unknown component which was isolated

4
L.F. Ibrahim et al.
Natural Product Research
389
and subjected to ESIMS showing an ion peak of 417 m/z suggesting a structure of a mono-
C-pentoside of tetra-hydroxy-flavone. For identification of the C-linked pentose sugar,
ferric chloride degradation was applied and the degradation product revealed the presence
1
of arabinose moiety after comparison with an authentic sample. The H-NMR spectrum
of 2 showed signals for ring A and C protons resonated at � 6.31 (d, J ¼ 2.0 Hz) and � 6.69
(
1H, s) assigned to H-6 and H-3, respectively. Thus confirming the flavone nucleus as well
as the occupation of positions 7 and 8 of compound 2 (Markham, 1982). In ring B an ABX
system was present; an ortho-coupled proton doublet at � 7.32 (J ¼ 8.5 Hz), a meta-coupled
doublet at � 6.41 (J ¼ 2.0 Hz) and an ortho–meta-coupled doublet of doublet at � 6.35
0
0
0
(
J ¼ 2.0, 8.5 Hz) assigned to H-6 , H-3 and H-5 , respectively (Souleles, 1990). The
spectrum further revealed the presence of two anomeric signal protons of sugar units
resonated at � 5.01 (J ¼ 7.9 Hz) and � 4.57 (J ¼ 8.6 Hz) indicating the 7-O-ꢀ-glucopyranose
1
3
and 8-C-ꢁ-arabinopyranose units, respectively. The C-NMR spectrum confirmed the
C-linked substituent at C-8 from the downfield shift at � 104.9. The upfield signal at � 102.8
0
0
for C-3 supported the B-ring structure of 2, as the C-3 is oxygenated at both ortho
positions (Mabry et al., 1982). The signals for the two sugar units resonated at their
expected range for 7-O-glucopyranoside at � 100.2 and 8-C-arabinopyranoside at � 74.7.
The results were found to be in agreement with the proposed structure as
0
0
5
,2 ,4 -trihydroxy-flavone-8-C-ꢁ-arabinopyranoside-7-O-ꢀ-glucopyranoside.
0
2
.1.1. Quercetin-3,7-di-O-ꢀ-D-glucopyranoside-4 -O-ꢁ-L-rhamnopyranoside (1)
Yellow amorphous powder R -values: 0.74 (H O), 0.80 (HOAc), 0.22 (BAW). UV/Vis ꢂ
max
f
2
(
(
2
6
MeOH) nm: (MeOH) 254, 266, 352; (þNaOMe) 275, 418; (þAlCl ) 273, 299, 334, 406;
3
þAlCl /HCl) 271, 300, 348, 403; (þNaOAc) 257, 270sh, 356, 413sh; (NaOAc/H BO )
3
3
3
1
58, 375. H-NMR (500 MHz, DMSO-d , � (ppm), J (Hz)): 7.66 (1H, dd, J ¼ 2.0, 8.5, H-
6
0
0
0
); 7.64 (1H, d, J ¼ 2.0, H-2 ); 7.12 (1H, d, J ¼ 8.5, H-5 ); 6.72 (1H, d, J ¼ 2.0, H-8); 6.4
00
0000
(
1H, d, J ¼ 2.0, H-6); 5.43 (1H, d, J ¼ 7.2, H-1 ); 5.40 (1H, d, J ¼ 2.0, H-1 ); 5.04 (1H, d,
J ¼ 7.5, H-1 ); 3–4 (m, sugar protons overlapped with –OH proton signals); 0.81 (1H,
0
00
0
000 13
d, J ¼ 6.0, H-6 ). C-NMR (125 MHz, DMSO-d , ppm): 178.1 (C-4), 162.8 (C-7), 161.4
6
0
0
0
(
(
(
(
(
C-5), 157.6 (C-9), 156.6 (C-2), 146.4 (C-4 ), 145.4 (C-3 ), 133.5 (C-3), 122.6 (C-6 ), 121.2
0
0
0
00
000
C-1 ), 115.8 (C-5 ), 115.7 (C-2 ), 106.2 (C-10), 100.7 (C-1 ), 99.9 (C-1 ), 98.9 (C-6), 98.5
000
0000
00
000
00
0000
C-1 ), 95.5 (C-8), 77.5 (C-5 ), 77.0 (C-5 ), 76.3 (C-3 ), 76.2 (C-3 ), 74.2 (C-2 ), 74.1
000
00
0000
0000
0000
0000
00
000
C-2 ), 73.1 (C-2 ), 71.8 (C-4 ), 70.0 (C-3 ), 69.9 (C-5 ), 69.8 (C-4 ), 69.5 (C-4 ), 60.9
�
0000
00
C-6 ), 60.8 (C-6 ), 18.1 (C-6 ). Negative ESIMS: m/z 771.26 [M � H] .
0
0
2
.1.2. 5,2 ,4 -trihydroxy-flavones-8-C-ꢁ-arabinopyranoside-7-O-ꢀ-glucopyranoside (2)
(MeOH)
Yellow powder R -values: 0.46 (H O), 0.68 (HOAc), 0.32 (BAW). UV/Vis ꢂ
max
f
2
nm: (MeOH) 267, 334; (þNaOMe) 270, 396; (þAlCl ) 277, 299sh, 345, 393; (þAlCl /HCl)
3
3
1
2
77, 344, 392; (þNaOAc) 264, 398; (NaOAc/H BO ) 286, 343. H-NMR (500 MHz,
3
3
0
0
DMSO-d , � (ppm), J (Hz)): 7.32 (1H, d, J ¼ 8.5, H-6 ); 6.41 (1H, d, J ¼ 2.0, H-3 ); 6.35
6
0
(
1H, dd, J ¼ 2.0, 8.5, H-5 ); 6.69 (1H, s, H-3); 6.31 (1H, d, J ¼ 2.0, H-6); 4.57 (1H, d,
00
000
J ¼ 8.6, H-1 ); 5.01 (1H, d, J ¼ 7.9, H-1 ); 3–4 (m, sugar protons overlapped with –OH
1
3
proton signals). C-NMR (125 MHz, DMSO-d , ppm): 181.4 (C-4), 163.8 (C-2), 162.6
6
0
0
0
0
(
(
(
(
C-7), 161.6 (C-4 ), 160.8 (C-5), 155.2 (C-2 ), 152.2 (C-9), 130.3 (C-6 ), 116.9 (C-1 ), 107.6
0
0
000
000
C-5 ), 105.7 (C-10), 104.9 (C-8), 103.1 (C-3), 102.8 (C-3 ), 100.2 (C-1 ), 99.5 (C-6), 76.9
00
0
00
000
00
00
00
000
C-5 ), 76.5 (C-3 ), 74.7 (C-1 ), 74.3 (C-2 ), 72.9 (C-2 ), 71.3 (C-3 ), 70.2 (C-4 ), 69.2
�
00
000
C-4 ), 68.1 (C-5 ), 60.7 (C-6 ). Negative ESIMS; m/z 579.24 [M � H] .

3
90
L.F. Ibrahim et al.
Natural Product Research
5
2
.2. In vitro cytotoxic activity
Ethyl acetate, butanol, methanol and aqueous extracts of A. bombycinus were tested for
cytotoxic activities on four human carcinoma cell lines; lung (A549), breast (MCF7), colon
(
showed no evident cytotoxicity against HCT116 and HEPG2 cell lines, but selective
HCT116) and liver (HEPG2) using the MTT assay. The results indicated that all extracts
�
1
cytotoxicity, particularly against breast cancer cells at 100 mg mL
6
(butanol extract
8.3%, ethyl acetate extract 56.8%, water extract 45.3% and methanol extract 23.1%).
The same extracts exhibited weak effect against lung carcinoma cell line with cell viability
ranged from 6.2% to 24.5% (Table 1).
2
.3. DPPH radical scavenging activity
Ethyl acetate, butanol, methanol and aqueous extracts of A. bombycinus exhibited weak
�1
activities on scavenging DPPH free radicals with inhibition at 100 mg mL of 11.5%,
8
.5%, 12.2% and 8.2%, respectively.
3
3
1
. Experimental
.1. General experimental procedure
1
D- and 2D-NMR measurements ( H, C and HMQC) were recorded using a Jeol
13
1
13
EX-500 spectrometer: 500 MHz ( H-NMR), 125 MHz ( C-NMR). The chemical shifts are
expressed in (ppm) and the coupling constants are in Hz. The mass spectra (ESIMS) were
measured on LCQ Advantage Thermo Finnigan spectrometer: m/z (rel. %) and the UV
spectra were recorded on UV spectrophotometer (Shimadzu UV-240). Column chroma-
tography was carried out on a Polyamide 6S (Riedel-De-Haen AG, Seelze Haen AG,
Seelze Hanver, Germany) and Sephadex LH-20 (Pharmacia). The paper chromatography
Table 1. In vitro cytotoxic activity of A. bombycinus extracts using MMT assay.
%
Inhibition of cell viability
�1
Extract concentration (mg mL
)
Extracts
Cancer cell lines
12.5
25
50
100
Ethyl acetate
A-549
0.8
3.5
0
7.3
11
0
0
11.2
24.6
0
24.5
56.8
0
MCF-7
HCT116
HepG2
0
0
0
Butanol
Methanol
Water
A-549
10.1
15.6
0
15.1
23.9
0
22.7
43.9
0
27.8
68.3
0
MCF-7
HCT116
HepG2
0
0
0
0
A-549
0
1.6
0
0.6
4.5
0
2.9
12.3
0
6.2
23.1
0
MCF-7
HCT116
HepG2
0
0
0
0
A-549
3.1
8.5
0
5.2
14.2
0
9.5
24
0
3.5
17
45.3
0
MCF-7
HCT116
HepG2
0
0
17

6
L.F. Ibrahim et al.
Natural Product Research
391
(Whatman no. 1 and 3 MM) was performed using solvent systems: (1) H O, (2) 15%
HOAc (H O–HOAc 85 : 15), (3) BAW (nBuOH–HOAc–H O 4 : 1 : 5, upper layer) and
2
2
2
(
4) (C H –nBuOH–H O–pyridine 1 : 5 : 3 : 3, upper layer). Solvents 3 and 4 were used for
6 6 2
sugar analysis. The compounds were detected by their UV absorption at a wavelength of
54 nm. Laminar flow cabinet biosafety class II level (Baker, SG403INT, Sanford, ME,
2
USA). Water jacketed Carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR,
USA). Microplate multi-well reader (Bio-Rad Laboratories Inc., model 3350, Hercules,
CA, USA).
3
.2. Plant material
A fresh sample of the whole plant of A. bombycinus Boiss. was collected from Suez–Cairo
desert road, 31 km on the 4 March 2010. Authentication was performed by Dr Mona M.
Marzouk and Dr Sameh R. Hussein. A voucher specimen (s.n. 519) was deposited in the
herbarium of the National Research Centre (CAIRC).
3
.3. Cell culture and sample treatment
The cell line under investigation was human breast adenocarcinoma (MCF7), human
hepatocellular carcinoma cell line (HEPG 2), human lung carcinoma (A549) and human
colon cell line (HCT116). They were purchased from American Tissue Culture Collection.
HEPG2, MCF7 and HCT116 cells lines were cultured in RPMI 1640 medium while A549
cell line was cultured in DMEM media. Media are supplemented with 1% antibiotic–
�
1
�1
antimycotic mixture (10,000 U mL potassium penicillin, 10,000 mg mL streptomycin
�
1
sulphate and 25 mg mL amphotericin B), 1% L-glutamine and 10% fetal bovine serum.
4
According to the cells growth profile, cells were seeded with a density of 1 ꢀ 10 cell per
well. This number was sufficient to give a reliable reading with the MTT assay, which
corresponded well with the cell number and was the one that gave exponential growth
throughout the incubation period with the tested sample.
3
.4. Extraction and isolation
The ground air-dried whole plant of A. bombycinus (950 g) was defatted with light
ꢁ
petroleum ether (40–60 C) and extracted four times at room temperature with 70%
methanol/water. The aqueous methanol extract was evaporated under reduced pressure
and temperature to obtain a residue of 187 g (19.86%). The residue was dissolved in
distilled water (500 mL). The aqueous solution was successively extracted with EtOAc,
n-butanol and methanol (3 ꢀ 500 mL) yielding 48.4 (5.094%), 36.8 (5.978%) and 22.6 g
(
(
2.378%), respectively. The remainder was concentrated till dryness affording 43.3 g
4.557%). The EtOAc extract was fractionated using polyamide 6 S column chromatog-
2
raphy (50 ꢀ 2.5 cm ), eluting with MeOH/H O mixtures of decreasing polarities to yield
2
fractions containing the aglycones and mono-glycosides; compound 3 (27 mg, 0.0028%),
4
(56 mg, 0.0059%), 6 (22 mg, 0.0023%) and 7 (38 mg, 0.0040%). The butanol extract was
2
applied to a silica gel column (45 ꢀ 2.0 cm ) using CH Cl : MeOH (2 : 3) followed by
2
2
elution technique on preparative paper chromatography (PPC) using H O, 15% HOAc
2
and BAW, as eluents to afforded compound 1 (15 mg, 0.0016%), 8 (19 mg, 0.0020%),
9
(28 mg, 0.0029%) and 10 (23 mg, 0.0024%). The methanol and aqueous extracts
afforded compound 2 (12 mg, 0.0013%) and 5 (36 mg, 0.0038%). Final purification
for all compounds was achieved by a combination of PPC and repeated Sephadex
LH-20 column.

3
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L.F. Ibrahim et al.
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3
.5. In vitro cytotoxic activity
The antiproliferative activity of plant extracts was measured using MTT
3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Promega, USA).
(
The assay detects the reduction of MTT by mitochondrial dehydrogenase to blue
formazan product, which reflects the normal function of mitochondria and cell viability.
4
Exponentially growing cells were washed and seeded at 1 � 10 cells per well (in 200 mL of
growth medium) in 96 well microplates. After 24 h incubation, a partial monolayer was
formed then the media was removed and 100 mL of the medium containing different
concentrations of A. bombycinus extracts (initially dissolved in DMSO) were added and
re-incubated for 48 h. Then, 100 mL of the medium were aspirated and 40 mL of the MTT
solution were added in each well. After 4 h contact with the MTT solution, blue crystals
were formed. 150 mL of the SDS stop solution were added and further incubated for 1 h.
Reduced MTT was assayed at 595 nm using a microplate reader (Thabrew, Hughes, &
McFarlane, 1997). Control groups received the same amount of DMSO (0.1%). Untreated
cells were used as a negative control while cells treated with doxorubicin were used as a
ꢀ1
positive control at a concentration of 100 mg mL . Stock solutions of the plant extract
were dissolved in DMSO then diluted with the medium and sterilised using 0.2 mm
membrane filters. The final dilution of extracts used for treating the cells contained no
more than 0.1% DMSO. The percentage of change in viability was calculated according to
the formula [1 ꢀ (reading of extract/reading of negative control)] � 100.
3
.6. DPPH radical scavenging activity
The free radical scavenging activity of the different extracts (aqueous, ethyl acetate,
.
methanol and butanol) was measured by DPPH using the method of Shimada et al.
.
1992) and Fan et al. (2009). A solution of DPPH in methanol (0.1 mM) was prepared.
(
One millilitre of this solution was added to 3 mL of extract solution at different
concentrations (25–100 mg mL). The mixture was shaken vigorously and allowed to stand
at room temperature for 30 min. The absorbance was then measured at 517 nm. DPPH
.
.
scavenging effect (%) ¼ [A ꢀ (A ꢀ A )/A ] � 100, where A is the absorbance DPPH
0
1
0
0
.
without sample, A the absorbance sample with DPPH and A the absorbance of the
1
.
sample without DPPH (Oktay, Gu
¨ ¨
lc¸ in, & Kufrevio, 2003).
4
. Conclusion
In this study, two new flavonoid glycosides 1 and 2, along with eight known flavonoids,
were characterised for the first time from A. bombycinus. The results of the preliminary
biological assay revealed that all tested extracts exhibited weak antioxidant activity. While
the butanol extract is the only extract which exhibited the most effective cytotoxic activity
against breast carcinoma cell line (MCF7).
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