Two new flavonol glycosides and biological activities of Diplotaxis harra (Forssk.) Boiss.

Article abstract of DOI:10.1080/14786419.2013.825914

Two new flavonol glycosides, isorhamnetin 3-O-β-glucopyranoside- 4′-O-β-xylopyranoside (1) and kaempferol 3-O-β-glucopyranoside -4′-O-β-xylopyranoside (2), were isolated from the defatted aqueous methanol extract of the whole plant Diplotaxis harra along with 12 known flavonols (3-14). They were characterised by chemical and spectral methods. The 70% aqueous methanol, chloroform and defatted aqueous methanol plant extracts exhibited significant antioxidant effects (nitroblue tetrazolium reduction method). Their cytotoxic activity was carried out against 11 tumour cell lines (sulphorhodamine B assay). The three extracts expressed the greatest antiproliferative activity against colon 38, P388 and MKN-28 with GI50 (0.45, 0.4, 0.07 g/mL) and against P388 [3-(4,5-dimethylthiazol-2yl)-2,5- diphenyltetrazolium bromide assay] with IC50 (0.26, 0.24, 0.25 g/mL), respectively. The chloroform extract showed the highest activity as eukaryotic DNA topoisomerase II inhibitors of P388 with IC50 0.24 g/mL. Antiviral screening of the extracts and the pure compounds against foot-and-mouth disease virus types A and O revealed a prominent inhibition of its cytopathic effect. 2013

Full text of DOI:10.1080/14786419.2013.825914

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Natural Product Research: Formerly
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Two new flavonol glycosides and
biological activities of Diplotaxis harra
(
Forssk.) Boiss.
a
b
a
Mona E.S. Kassem , Manal S. Afifi , Mona M. Marzouk & Manal A.
c
Mostafa
a
Department of Phytochemistry and Plant Systematics, National
Research Centre, Cairo, Egypt
b
Department of Pharmacognosy, Faculty of Pharmacy, Misr
International University, Cairo, Egypt
c
Department of Foot and Mouth Disease Vaccine Research,
Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo,
Egypt
Published online: 20 Aug 2013.
To cite this article: Mona E.S. Kassem, Manal S. Afifi, Mona M. Marzouk & Manal A. Mostafa
(
2013) Two new flavonol glycosides and biological activities of Diplotaxis harra (Forssk.)
Boiss., Natural Product Research: Formerly Natural Product Letters, 27:24, 2272-2280, DOI:
0.1080/14786419.2013.825914
1
To link to this article: http://dx.doi.org/10.1080/14786419.2013.825914
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Natural Product Research, 2013
Vol. 27, No. 24, 2272–2280, http://dx.doi.org/10.1080/14786419.2013.825914
Two new flavonol glycosides and biological activities of Diplotaxis harra
(
Forssk.) Boiss.
a
Mona E.S. Kassem , Manal S. Afifi , Mona M. Marzouk * and Manal A. Mostafa
b
a
c
a
Department of Phytochemistry and Plant Systematics, National Research Centre, Cairo, Egypt;
Department of Pharmacognosy, Faculty of Pharmacy, Misr International University, Cairo, Egypt;
b
c
Department of Foot and Mouth Disease Vaccine Research, Veterinary Serum and Vaccine Research
Institute, Abbasia, Cairo, Egypt
(
Received 26 March 2013; final version received 4 June 2013)
0
anoside (1) and kaempferol 3-O-b-glucopyranoside -4 -O-b-xylopyranoside (2), were
Two new flavonol glycosides, isorhamnetin 3-O-b-glucopyranoside-4 -O-b-xylopyr-
0
isolated from the defatted aqueous methanol extract of the whole plant Diplotaxis harra
along with 12 known flavonols (3–14). They were characterised by chemical and
spectral methods. The 70% aqueous methanol, chloroform and defatted aqueous
methanol plant extracts exhibited significant antioxidant effects (nitroblue tetrazolium
reduction method). Their cytotoxic activity was carried out against 11 tumour cell lines
(
sulphorhodamine B assay). The three extracts expressed the greatest antiproliferative
activity against colon 38, P388 and MKN-28 with GI50 (0.45, 0.4, 0.07 mg/mL) and
against P388 [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay]
with IC50 (0.26, 0.24, 0.25 mg/mL), respectively. The chloroform extract showed the
highest activity as eukaryotic DNA topoisomerase II inhibitors of P388 with IC50
0.24 mg/mL. Antiviral screening of the extracts and the pure compounds against foot-
and-mouth disease virus types A and O revealed a prominent inhibition of its
cytopathic effect.
Keywords: Diplotaxis harra; flavonols; antioxidant; cytotoxic; antiviral activity
1
. Introduction
Diplotaxis harra (Forssk.) Boiss. is an annual desert plant belonging to the Brassicaceae family
and widely distributed in Egypt (Boulos 1999). It is locally known as Harra, and is used in folk
medicine as antibacterial, antifungal, anti-inflammatory and anticancer (Hartwell 1982). The
non-methylated fatty acids and steroids from D. harra showed strong activity against bacteria
than fungi (Hashem & Saleh 1999). Leaf flavonoids of 13 Diplotaxis species were reported to be
glycosides of kaempferol, quercetin and isorhamnetin (Sanchez-Yelamo & Martinez-Laborde
1
of kaempferol, quercetin and isorhamnetin, isorhamnetin 3-O-a-rhamnoside, isorhamnetin 3-O-
991). Quercetin, isorhamnetin 7-O-b-glucoside, apigenin 7-O-a-rhamnoside, 3-O-b-glucoside
0
rutinoside and isorhamnetin 3-O-b-glucoside 4 -O-a-rhamnoside were reported on aerial parts
of D. harra (Atta et al. 2011; Mohammed et al. 2011). Despite the widespread use of D. harra as
medicinal herb, we aimed to identify the active compounds from the whole plant and evaluated
its antioxidant and cytotoxic activities as well as the antiviral activity against foot-and-mouth
disease viruses (FMDV).
*
Corresponding author. Email: monakalil66@hotmail.com
q 2013 Taylor & Francis

Natural Product Research
2273
2
2
. Results and discussion
.1. Identification of isolated compounds
The phytochemical investigation of the defatted aqueous methanol extract (DAME) of the
whole plant of D. harra afforded 14 flavonols (Figure 1). Compound 1 was isolated as a yellow
amorphous powder. The positive-ion ESI-MS showed a molecular ion peak at m/z 611.38
þ
[
indicated a flavonol skeleton with free hydroxyl groups at C-5 and C-7, whereas those at C-3
M þ H] , corresponding to C H O . The UV spectral data using various shift reagents
2
7 30 16
0
and C-4 are substituted (Mabry et al. 1970). Complete acid hydrolysis (2 N HCl, 1 h, 1008C)
yielded glucose and xylose as the sugar moieties [co-paper chromatography (Co-PC)] and
1
isorhamnetin (Co-PC, EI-MS and UV). The H NMR spectrum of 1 showed the aromatic
protons of ring A with two meta coupled protons for H-6 and H-8 at 6.23 and 6.47,
respectively. The aromatic protons of the B-ring appeared as a doublet (d7.97, J ¼ 2.0 Hz)
0
0
assigned to H-2 and doublet of doublet at (d7.54, J ¼ 2.0, 8.5 Hz) assigned to H-6 along with
0
another doublet (d7.24, J ¼ 8.5 Hz) for H-5 with a downfield shift indicating the substitution
0
of the hydroxyl group at C-4 (Park & Lee 1996). The spectrum also showed a singlet at 3.84,
attributed to OCH of isorhamnetin skeleton. In addition to two anomeric doublets at d5.58
3
(
J ¼ 7.5 Hz) and 5.37 (J ¼ 4.6 Hz) corresponding to b-glucopyranose and b-xylopyranose
0
13
units at C-3 and C-4 , respectively (Markham & Geiger 1994). The C NMR spectrum
confirmed the presence of 16 carbon signals assigned for the isorhamnetin and 11 signals for
0
the sugar units. The chemical shift of C-3 at 133.3 and C-4 at 148.1 suggested their
glycosylation (Agrawal 1989) which is confirmed by HMBC data, this showed the correlation
0
0
000
0
from H-1 (d5.58) to C-3 (133.3) and from H-1 (d5.37) to C-4 (148.1). On the basis of the
0
above data, 1 was identified as isorhamnetin 3-O-b-glucopyranoside-4 -O-b-xylopyranoside.
Compound 2 was obtained as a yellow powder with a molecular formula C H O as
2
6 28 15
2
determined by the negative-ion ESI-MS at m/z 579.45 [M–H] . The UV spectral data using
diagnostic shift reagents indicated a flavanol skeleton with free hydroxyl groups at C-5 and
0
C-7, whereas those at C-3 and C-4 are substituted (Mabry et al. 1970). Complete acid
1
hydrolysis produced glucose, xylose and kaempferol (Co-PC). Its H NMR spectrum showed
0
0
0
0
two doublet protons at d 8.13 and d 7.19 (each J ¼ 9.0 Hz), assigned to H-2 ,6 and H-3 ,5 ,
0
respectively; this downfield chemical shift confirmed that C-4 was substituted (Yoshitama
et al. 1993). Two doublets at d 6.47 and d 6.23 (each J ¼ 2.0 Hz) were assigned to H-8 and
H-6, respectively. The spectrum also revealed two protons at d 5.47 (J ¼ 7.2 Hz) and d 5.32
00
000
(
respectively (Markham & Geiger 1994). In addition, the C NMR spectrum confirmed the
J ¼ 4.6 Hz), attributed to H-1 and H-1 of the b-glucopyranose and b-xylopyranose units,
1
3
0
substitution of C-3 (134.1) and C-4 (160.8). Therefore, 2 seems to be the kaempferol analogue
0
of 1; kaempferol 3-O-b-glucopyranoside-4 -O-b-xylopyranoside. Compounds (3–14) were
elucidated by comparing their spectral data with those published previously (Agrawal 1989;
0
00
Markham & Geiger 1994; Shang et al. 2006). They were identified as isorhamnetin 3-O-[2 -O-
0
acetyl-b-glucopyranosyl (1 ! 6)-b-glucopyranoside] (3), isorhamnetin 3,4 -di-O-b-glucoside
(
4), isorhamnetin 3,7-di-O-b-glucoside (5), isorhamnetin 3-O-b-glucoside (6), isorhamnetin
-O-a-rhamnoside (7), isorhamnetin 7-O-b-glucoside (8), quercetin 3-O-b-glucoside (9),
3
quercetin 7-O-b-glucoside (10), quercetin 3-O-a-rhamnoside (11), kaempferol 3-O-b-
glucoside (12), isorhamnetin (13) and quercetin (14).
2
.2. Antioxidant assay
The 70% aqueous methanol extract (AME), chloroform extract (CE) and DAME showed high-
antioxidant activities (at a dose of 50 mg/mL) with percentage scavenging activity; 62.66, 31.15
and 51.66, respectively, relative to ascorbic acid (22.2%).

2
274
M.E.S. Kassem et al.
Figure 1. Chemical structure of compounds 1–14.

Natural Product Research
2275
2
.3. Cytotoxic assay
2
.3.1. Growth-inhibitory effect
The results showed that AME, CE and DAME exhibited the most potent activities on MKN-28,
colon 38 and P388, respectively, with GI50 (0.07, 0.45 and 0.4 mg/mL), TGI (0.65, 2.66 and
3.90 mg/mL) and LC50 (5.89, 6.18 and 29.56 mg/mL), respectively (Table 1). They were also
tested against P388 using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT)
assay with IC50 (mg/mL); 0.26, 0.24 and 0.25, respectively (Table 2).
2
.3.2. Inhibition of topoisomerase II activity
All extracts showed inhibition of topoisomerase II activity with lower concentration than that of
etoposide. Among them, CE showed the highest activity where CE and etoposide show IC50
values of 0.24 and 0.30 mg/mL, respectively, which means that CE exhibited 1.182 times greater
inhibitory effect than etoposide (Table 2).
2
.4. Antiviral assay
The extracts and the pure flavonoids were found to be safe for baby hamster kidney cell culture
BHK-21). All of them showed antiviral effect against FMDV (types A and O) as indicated by
(
the inhibition of the virus cytopathic effect (Table 3). The antiviral effect was varied from one
sample to another depending on the active dilution. The samples which showed antiviral effect
when they added to the infected cell culture before, after and simultaneously with the virus
infection (1, 2, 8, 11 and DAME) were of choice to follow up such effect as virus inactivators.
These samples began to show their action as inactivators by the first hour post starting of the
process for either types of FMDV, although the required periods for complete inactivation were
varied according to the used concentration. The study revealed that the highest concentration of
the tested materials the faster inactivation of FMDV (types A and O). Such materials did not
affect passively the antigenicity of FMDV as confirmed by the complement fixation test, which
revealed the same results obtained before and after virus inactivation (titre 1/8) with different
concentrations used.
Table 1. Cytotoxicity of AME, CE and DAME (SRB assay).
AME
TGI
CE
DAME
TGI
Cell line
GI50
LC50
GI50
TGI
LC50
GI50
LC50
a
a
a
Colon 38
WIDR
0.41
0.43
0.43
9.00
5.89
0.55
60.9
5.11
24.44
225.25
5.89
6.90
8.66
9.87
0.45
1.57
0.76
3.71
1.52
1.63
1.42
1.56
0.51
1.88
1.42
2.66
9.28
7.89
23.66
7.44
5.66
3.14
5.66
4.88
5.78
12.45
6.18
56.78
24.11
114.06
58.66
24.30
14.08
16.09
25.00
38.16
67.45
6.44
0.54
1.17
1.51
2.60
0.68
0.98
4.67
50.78
4.89
16.19
14.36
19.56
5.88
5.66
56.78
3.90
11.66
43.67
124.9
38.90
155.89
100.78
112.98
53.89
40.89
208.88
29.56
110.89
307.78
MKN-1
MKN-7
MKN-28
MKN-74
HGC-27
GT3TKB
P388
8.78
56.90
0.65
a
a
a
0.07
0.07
0.09
0.09
1.88
5.66
0.065
0.71
0.87
0.89
12.77
6.55
0.89
a
a
a
34.99
18.12
7.88
0.40
1.71
5.78
Lx-1
A549
Note: Concentration (mg/mL) required to inhibit cell growth by 50% (GI50), concentration (mg/mL) required to produce
total growth inhibition (TGI), concentration (mg/mL) required to cause net cell killing (LC50).
The most active extract with the least GI50 concentration.
a

2
276
M.E.S. Kassem et al.
Table 2. IC50 of AME, CE, DAME and etoposide (MTT assay) inhibited the topoisomerase II on DNA-
relaxing activity of P388 topoisomerase II.
Tested extracts
IC50 (mg/mL)
Topoisomerase II inhibition relative potency
AME
CE
DAME
Etoposide
0.26
0.24
0.25
0.30
1.11
1.182
1.125
1
Notes: Supercoiled pBR-322 DNA was incubated with topoisomerase II purified from P388 cells in the presence or
absence of an inhibitor. Concentration (mg/mL) that causes 50% inhibition per unit enzyme (IC50).
Table 3. The safe samples dilution on BHK-21 cells, the preliminary antiviral activity against FMDV
strains (O and A) and their antiviral effect according to the time of their addition to the infected cell culture.
The antiviral effect according to the time of their
addition to the infected cell culture
Safe sample
dilution
(non-toxic)
Preliminary
antiviral activity
against FMDV
Simultaneously
with VI
a
Sample
Before VI
After VI
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
4
4
1
2
2
2
3
2
2
3
5
1
2
3
4
5
6
8
10
10
10
10
10
10
10
10
10
10
10
10
10
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
2
2
2
2
þ
þ
2
2
þ
2
þ
þ
þ
2
2
2
2
þ
þ
2
2
2
2
þ
þ
þ
þ
2
2
þ
þ
þ
2
2
þ
þ
þ
11
13
14
AME
CE
DAME
a
Antiviral effect ¼ no cytopathic effect (CPE), positive antiviral effect determined by inhibition of the cytopathic effect
of either type of FMDV. VI, virus infection.
3
3
. Experimental
.1. General
NMR experiments were recorded on Jeol EX-500 spectrometer. EI-MS was measured on
Finnigan-Mat SSQ 7000 spectrometer and ESI-MS was measured on LCQ Advantage Thermo
Finnigan spectrometer. UV spectra were recorded on Shimadzu model-2401 CP spectropho-
tometer. Column chromatography (CC) was carried out on Polyamide 6S (Riedel-De-Haen AG,
Seelze Haen AG, Seelze Hanver, Germany) and Sephadex LH-20 (Pharmazia, Uppsala,
Sweden). Paper chromatography (PC, descending) Whatman No. 1 and 3 mm papers, using
solvent systems (1) H O, (2) 15% HOAc (H O:HOAc, 85:15), (3) BAW (n-BuOH:HOAc:H O,
2
2
2
4
:1:5, upper layer), (4) BBPW (C H :n-BuOH:pyridine:H O, 1:5:3:3, upper layer).
6 6 2
3
.2. Plant material
D. harra was freshly collected and identified by Dr Mona M. Marzouk in 20 March 2007 from
Cairo–Suez desert road at 35 km. A voucher specimen (no. 808) has been deposited in the
herbarium of National Research Centre, Cairo, Egypt.

Natural Product Research
2277
3
.3. Extraction and isolation
The air-dried powdered whole plant (1.2 kg) was extracted with 70% MeOH (3 £ 4 L). The
solvent was evaporated under reduced pressure at 508C. The dried aqueous methanol
extract; AME (228 g) was defatted with CHCl (3 £ 1 L) afforded CE (32 g). The DAME
3
(
followed by H –MeOH mixtures of decreasing polarity. A total of 42 fractions were
180 g) was then subjected to polyamide CC (5.5 cm £ 125 cm) eluted with H O and
2
2
collected, each 400 mL; these were controlled by PC to give four main fractions (A–D).
Fraction A was chromatographed on Sephadex column using H O:MeOH (1:1) for elution
2
yielding three sub-fractions, each was subjected to preparative paper chromatography (PPC)
using BAW and followed by Sephadex column using methanol to yield compounds 1
(
gradient elution with H –MeOH, gave two major sub-fractions: B1 and B2. Repeated CC
23 mg) 2 (18 mg) and 3 (38 mg). CC of fraction B on polyamide column (2.5 cm £ 45 cm),
2
on Sephadex of B1 using MeOH:C H :H O (60:38:2), followed by PPC using BAW,
6
6
2
afforded compounds 4 (12 mg) and 5 (25 mg). Compounds 6 (31 mg) and 7 (10 mg) were
obtained from B2 by repeated CC on Sephadex LH-20 using saturated BuOH. Fraction C
was applied on PC using BAW which yielded compounds 8 (26 mg), 9 (10 mg) and 11
(18 mg). Fraction D yielded compounds 10 (22 mg), 12 (8 mg), 13 (16 mg) and 14 (36 mg)
by separation on PC using 15% HOAc and followed by BAW two times.
0
3
.3.1. Isorhamnetin 3-O-b-glucopyranoside-4 -O-b-xylopyranoside (1)
þ
A bright yellow powder, R 0.34 (BAW). Positive ESI-MS; m/z 611.38 [M þ H] . UV
f
spectral data, l
(nm): (MeOH) 255 sh, 262, 356; (þNaOMe) 270, 330 sh, 419 (dec.);
3
max
(
þAlCl ) 263, 300 sh, 359 sh, 402; (þAlCl /HCl) 264, 301 sh, 360 sh, 401; (þNaOAc) 272,
3
1
3
7
6
20, 406; (NaOAc/H BO ) 254, 266 sh, 357. H NMR (500 MHz, DMSO-d , d, ppm, J/Hz:
3 3 6
0
0
0
.97 (1H, d, J ¼ 2.0, H-2 ); 7.54 (1H, dd, J ¼ 2.0, 8.5, H-6 ); 7.24 (1H, d, J ¼ 8.5, H-5 );
0
0
.47 (1H, d, J ¼ 2.0, H-8); 6.23 (1H, d, J ¼ 2.0, H-6); 5.58 (1H, d, J ¼ 7.5, H-1 ); 5.37 (1H,
0
00
000
d, J ¼ 4.6, H-1 ), 3.84 (3H, s, OCH ); 3.69 (1H, dd, J ¼ 5.3, 11.5, H-5 ); 3.61 (1H, dd,
3
00
00
00
00
J ¼ 5.2, 11.6, H-6 ); 3.41–3.48 (2H, m, H-3 , H-4 , overlapped); 3.38 (1H, m, H-5 ); 3.29
00 000 000
000 13
(
1H, m, H-2 ); 3.19–3.25 (2H, m, H-3 , H-4 , overlapped); 3.08 (1H, m, H-2 ). C NMR
1
1
1
25 MHz, DMSO-d , ppm, 177.4 (C-4), 164.2 (C-7), 161.1 (C-5), 156.3 (C-2), 155.6 (C-9),
6
0
0
0
0
0
0
48.1 (C-3 ), 148.1 (C-4 ), 133.3 (C-3), 123.5 (C-6 ), 121.2 (C-1 ), 114.4 (C-5 ), 113.4 (C-2 ),
000
00
00
00
00
04.1 (C-10), 100.7 (C-1 ), 99.4 (C-1 ), 98.7 (C-6), 93.9 (C-8), 76.9 (C-5 ), 76.8 (C-3 ), 76.3
000
0
00
00
000
00
000
(
C-3 ), 74.2 (C-2 ), 73.1 (C-2 ), 69.7 (C-4 ), 69.4 (C-4 ), 66.4 (C-5 ), 60.5 (C-6 ), 55.6
OCH3).
(
0
3
.3.2. Kaempferol 3-O-b-glucopyranoside -4 -O-b-xylopyranoside (2)
2
A pale yellow powder, R 0.32 (BAW). Negative ESI-MS; m/z 579.45 [M–H] . UV spectral
f
data, lmax(nm): (MeOH) 262, 352; (þNaOMe) 270, 328sh, 409 (dec.); (þAlCl ) 263, 300, 354,
3
1
4
00; (þAlCl /HCl) 264, 301, 356, 399; (þNaOAc) 272, 388; (NaOAc/H BO ) 266, 353. H
3
3
3
0
0
NMR (500 MHz, DMSO-d , d, ppm, J/Hz: 8.13 (2H, d, J ¼ 9.0, H-2 , 6 ); 7.19 (2H, d, J ¼ 9.0,
6
0
0
00
H-3 , 5 ); 6.47 (1H, d, J ¼ 2.0, H-8); 6.23 (1H, d, J ¼ 2.0, H-6); 5.47 (1H, d, J ¼ 7.2, H-1 ); 5.32
000
000
00
(
1H, d, J ¼ 4.6, H-1 ); 3.68 (1H, dd, J ¼ 5.4, 11.4, H-5 ); 3.62 (1H, dd, J ¼ 5.4, 11.5, H-6 );
00 00 00 000
3
H-3 ); 3.04 (1H, m, H-2 ), 2.98 (1H, m, H-2 ). C NMR 125 MHz, DMSO-d , ppm, 178.1
.54 (1H, m, H-4 ); 3.36-3.44 (2H, m, H-3 , H-5 , overlapped); 3.31 (1H, m, H-4 ); 3.16 (1H, m,
0
00
00
000 13
6
0
0
(C-4), 162.5 (C-7), 161.4 (C-5), 160.8 (C-4 ), 158.6 (C-2), 156.5 (C-9), 134.1 (C-3), 131.2 (C-2 ,
0
C-6 ), 121.3 (C-1 ), 116.2 (C-3 , C-5 ), 106.2 (C-10), 100.9 (C-1 ), 99.9 (C-1 ), 98.9 (C-6), 93.9
0
0
0
00
000
00 00 000 00 000 000 00
000 00
5.8 (C-5 ), 60.6 (C-6 ).
(
C-8), 77.6 (C-5 ), 76.6 (C-3 ), 76.5 (C-3 ), 74.5 (C-2 ), 73.8 (C-2 ), 70.1 (C-4 ), 69.9 (C-4 ),
6

2
278
M.E.S. Kassem et al.
3
.4. Antioxidant assay
Superoxide radicals were generated by xanthine/xanthine oxidase and measured by the nitroblue
tetrazolium reduction method (Chung et al. 2004).
3
.5. Cytotoxicity activity
3
.5.1. Cell lines used for anti-tumour activity test
Murine colon, colon 38; murine leukaemia, P388 and Lung human, LX-1 cancer cell lines were
obtained from the Cancer Chemotherapy Centre (Tokyo, Japan). Human gastric cancer cell
lines, MKN-1, MKN-7, MKN-28 and MKN-74, were obtained from Immuno Biology
Laboratory (Gunma, Japan) and HGC-27 and GT3TKB were from Riken Cell Bank (Ibaraki,
Japan). Lung human, A549 and colon, WIDR cancer cell lines were obtained from Dainippon
Pharmaceutical, Co. Ltd (Osaka, Japan).
3
.5.2. Growth-inhibitory effect
The cytotoxicity of AME, CE and DAME against 11 cancer cell lines was evaluated using
sulphorhodamine B (SRB) assay (Skehan et al. 1990). Three dose response parameters for the
extracts, GI , TGI and LC , were calculated (Table 1) (Nakamura et al. 2002). Their
5
0
50
cytotoxicity on P388 cell line was also determined by MTT assay for assessment of cell viability
Table 2) (Hsiang et al. 1989).
(
3
.5.3. Inhibition of DNA topoisomerase II activity
The inhibitory effects on DNA topoisomerase II activity were evaluated by relaxation assay of
supercoiled DNA (Saijo et al. 1990) in comparison with etoposide as reference drug (Table 2).
3
.6. Antiviral activity
.6.1. Viral material
3
Serotypes O (1/3/93) and A (A/Egypt/2006) of FMDV and BHK-21 clones, using Eagle’s
medium with 10% new born bovine serum, were obtained from Veterinary Serum and Vaccine
Research Institute, Abbasia, Cairo, Egypt.
3
.6.2. Detection of material cytotoxicity
Three-day BHK-21 cell cultures in micro-titre plates were used to detect the toxicity of the tested
samples. Ten-fold dilutions were prepared from each sample in phosphate buffer saline (PBS).
Then 25 mL of each concentration was inoculated in each of the 5 wells of 96-well tissue culture
plates.
3
.6.3. Investigation of in vitro antiviral effect
8
BHK-21 cell culture plates were infected with FMDV strains using 25 mL/well of 10 TCID50 of
the used virus for 1 h. Then the plates were washed twice with PBS. Each one of the prepared
concentrations was added to each of five tissue culture wells with 150 mL of maintenance
medium. The cytopathic effect on BHK-21 cells of each sample was observed under light
microscope (Lefevre & Diallo 1990).

Natural Product Research
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3
.6.4. Complement fixation test
Complement fixation test (CFT) was carried out according to Health protection Agency (2009)
using the complement obtained from normal healthy Guinea pigs and the sheep RBCs from
normal healthy sheep for preparation of the haemolytic system. It was used as an indicator in
CFT consisting of four minimum haemolytic doses of Hemolysin and equal volume of 2% sheep
RBCs in PBS diluents and synthesised at 378C for 15 min.
4
. Conclusion
In the present study, to the best of our knowledge, compounds 1 and 2 had not been isolated from
plants before. In addition six flavonols; 3–5, 10, 11 and 13 were isolated for the first time from
D. harra. Biological investigation of extracts demonstrated promising antioxidant and anti-
cancer properties. They are targets for DNA topoisomerase, and their cytotoxicity is dependent
on tumour cell type. These activities could be presumably ascribed in part to the flavonoids as
well as other constituents of the plant. The pure compounds (1, 2, 8, 11) and DAME are of choice
as virus inactivators in the antiviral assay on FMDV, and their activity is concentration
dependent. Therefore, this study provides the biochemical rationale for further chemical and
biological analysis.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S10.
Acknowledgements
The phytochemical work was financially supported by the Phytochemistry and Plant Systematics
Department, National Research Centre, Egypt, project ‘S90203’. The authors thank the Univet Ireland Ltd
(Head Quarter & Factory Tullyvin, Coothill, County Cavan, Ireland) and Dr Mohamed M. Abdalah for the
evaluation of the growth-inhibitory effects and the topoisomerase II activity.
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