Cytotoxic ellagitannins from Reaumuria vermiculata

Article abstract of DOI:10.1016/j.fitote.2012.06.007

Three ellagitannins and one disulfated flavonol were isolated from the aerial parts of Reaumuria vermiculata L. Besides that, 16 known compounds were characterized as well. The structures of all compounds were elucidated on the basis of spectroscopic data including 1D and 2D NMR and ESI HR-FTMS. The in vivo antioxidant activity using the oxygen radical absorbance capacity (ORAC) method, of the extract, its column fractions and two of the isolated ellagitannins was accomplished. In addition, a possible cytotoxicity of the extract and two of the new ellagitannins on HaCaT human keratinocytes and the activity of both compounds against the prostate cancer cell line (PC-3) were also assessed, whereby a potent cytotoxicity with IC₅₀ less than 1 μg/ml was determined for both compounds. Besides, the extract exhibited a potential cytotoxic effect against four different solid tumor cell lines, namely liver (Huh-7), colorectal (HCT-116), breast (MCF-7) and prostate (PC-3). The IC₅₀s were found to be substantially low (ranged from 1.3 ± 0.15 to 2.4 ± 0.22 μg/ml) with relatively low resistance possibility reaching to 0% in the case of Huh-7 cell.

Full text of DOI:10.1016/j.fitote.2012.06.007

Fitoterapia 83 (2012) 1256–1266
Contents lists available at SciVerse ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Cytotoxic ellagitannins from Reaumuria vermiculata
a,
b
a
c
Mahmoud A. Nawwar ⁎, Nahla A. Ayoub , Mohamed A. El-Rai , Fatma Bassyouny ,
c
d
e
e
Eman S. Mostafa , Ahmed M. Al-Abd , Manuela Harms , Kristian Wende ,
Ulrike Lindequist , Michael W. Linscheid
Department of Phytochemistry and Plant Systematics, National Research Center, Dokki, Cairo, Egypt
Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt
Pharmaceutical Research Department, Center of Excellence for Advanced Sciences, National Research Center, Cairo, Egypt
Department of Pharmacology, National Research Center, Dokki, Cairo, Egypt
e
f
a
b
c
d
e
f
Institute for Pharmacy, Pharmaceutical Biology, Ernst-Moritz-Arndt-University, Greifswald, D-17487, Germany
Laboratory of Applied Analytical and Environmental Chemistry, Humboldt-Universitaet zu Berlin, Department of Chemistry, 12489 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 May 2012
Received in revised form 13 June 2012
Accepted 15 June 2012
Available online 26 June 2012
Three ellagitannins and one disulfated flavonol were isolated from the aerial parts of Reaumuria
vermiculata L. Besides that, 16 known compounds were characterized as well. The structures of all
compounds were elucidated on the basis of spectroscopic data including 1D and 2D NMR and ESI
HR-FTMS. The in vivo antioxidant activity using the oxygen radical absorbance capacity (ORAC)
method, of the extract, its column fractions and two of the isolated ellagitannins was accomplished.
In addition, a possible cytotoxicity of the extract and two of the new ellagitannins on HaCaT human
keratinocytes and the activity of both compounds against the prostate cancer cell line (PC-3) were
also assessed, whereby a potent cytotoxicity with IC50 less than 1μg/ml was determined for both
compounds. Besides, the extract exhibited a potential cytotoxic effect against four different solid
tumor cell lines, namely liver (Huh-7), colorectal (HCT-116), breast (MCF-7) and prostate (PC-3).
The IC50s were found to be substantially low (ranged from 1.3± 0.15 to 2.4± 0.22μg/ml) with
relatively low resistance possibility reaching to 0% in the case of Huh-7 cell.
Keywords:
Reaumuria vermiculata
Tamarixetin 3,7‐di-sulfate
3
‐Methoxyellagic acid 4,4′-di-sulfate
Vermiculatin
Antioxidant
Cytotoxicity assessment
©
2012 Elsevier B.V. All rights reserved.
1
. Introduction
Reaumuria species existing in Egypt. Many of these plants
grow on saline soils, tolerating up to 15,000ppm soluble salt
and can also tolerate alkaline conditions. In view of this fact,
the capability of these plants on synthesizing and accumulating
sulfate conjugates of flavonols phenyl propanoids and other
phenolics [6c), d), g), m)], is thus not all that surprising. These
compounds are produced via the sulfotransferase (SOT)
enzymes [8] which catalyze the sulfonation of a wide range of
compounds, including flavonoids and phenols, and produce
the more water-soluble sulfate esters and sulfate conju-
gates. It is hypothesized that sulfonation, via SOTs, enzymes,
affects the biological activity of certain compounds, thereby
modulating physiological processes such as growth, develop-
ment, and adaptation to salinity stress [9–11]. Besides, the
Tamaricaceae plants are capable of synthesizing and accumu-
lating a wide variety of phenolics, including lignans [6e)],
phenolic glycerides [6i)], gallotannins [6k)], monomeric and
oligomeric ellagitannins [5,6l)] and flavonol glucuronoids
Laboratory investigations show that the biological activities
of some plant phenolics, specially ellagitannins and flavo-
noids are most probably due to their strong antioxidant
properties [1,2]. Several plant families are known to be rich
in ellagitannins and flavonoids. Among these families, the
Tamaricaceae includes species which provide extracts rich
in ellagitannins and flavonoids. Egyptian Tamaricaceous
plants belong to two definite genera, namely, Tamarix and
Reaumuria [3]. While almost all parts of the existing five
Tamarix species and of the species Reaumuria hirtella have
been comprehensively studied for their phenolics [4–6],
only preliminary phytochemical investigations [7] have
been carried out on Reaumuria vermiculata, one of the four
⁎
Corresponding author. Tel.: +20 2 2752711; fax: +20 2 3370931.
E-mail address: mahmoudnawwarhesham@yahoo.com (M.A. Nawwar).
0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2012.06.007

M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
1257
[
6g)]. Tamaricaceous plants have been used as folk medicines
mainly as anti-inflammatory, antiseptic and anti-pyretic agents
12]. Among these plants, R. vermiculata L. (=Reaumuria
and slit width: 0.5nm. Paper chromatographic analysis (PC)
was carried out on Whatman No. 1 paper, using solvent
[
2
systems: (1) H O; (2) 6% HOAc; (3) BAW (n-BuOH–HOAc–
mucronata Jaub. & Spach), known in English as saltwort-leaved
Reaumuria is an elegant little shrub which grows wild in the
Mediterranean coastal strip of Egypt. Its leaves are numerous,
scattered, sessile from a quarter to three quarters of an inch
long, 4 spreading linear-a whapped, acute, fleshy, smooth,
glaucous, convex beneath, flat above, and dotted on both sides
with minute depression. The white flowers are terminal and
solitary. Calyx leaves are ovate with a narrow entire membra-
nous edge. The petals terminate in three slight lops. Its capsule is
brown, very smooth and somewhat shining, and its valves are
reflexed after the seeds are discharged [3]. In Egypt, the plant is
used as a cure from itch and bruises by being applied externally
or taken internally in the form of a decoction [12]. The plant has
been reported previously to contain kaempferol 3,7-disulfate,
on the basis of chromatographic, electrophorotic, and UV
spectral analysis and results of hydrolysis [7]. The present
study has been undertaken to investigate in detail the
constitutive phenolics of R. vermiculata aqueous methanol
aerial part extract in association with its antioxidant capability,
cytotoxicity and anticancer activity. During the course of this
work, we were able to isolate and determine the structure of
twenty phenolics including four metabolites which have not
been reported previously to occur as natural products, namely,
tamarixetin 3,7-disulfate (2), 3-methoxyellagic acid 4,4′-
disulfate (4), 2-O-dehydrodigallic acid monocarboxyloyl-3-O-
galloyl-(α/β)-glucose (9) and the ellagitannin dimer, verma-
culitin (13). On the other hand, the activity of the extract, its
column fractions and the two isolated ellagitannins 9 and 13 for
the radical scavenging capacity using the DPPH method [13]
and the ORAC assay [14] has been evaluated. In addition, the
possible cytotoxicity of the extract, and the two ellagitannins 9
and 13 against HaCaT human keratinocytes, the potential
cytotoxic effect of the extract against four different solid
tumor cell lines and the cytotoxic effect of metabolites 9 and
H O, 4:1:5, upper layer).
2
2.2. Plant materials
Collection of the fresh aerial parts of R. vermiculata was
made at the Mediterranean costal region, 5km west of
Mersa-Matrooh, Egypt, on April, 2010. Authentication was
performed by Dr. S. Kawashty, Prof. of Botany at the National
Research Center (NRC) of Cairo. Voucher specimen was
deposited at the herbarium of the NRC.
2.3. Preparation of extract
The fresh aerial parts of R. vermiculata, dried in the shade in
an air draft at room temp. (2kg) were comminuted to powder
and homogenized with a MeOH/H O (3:1) mixture, filtered,
2
and dried in vacuum to yield a dark brown amorphous powder
(290g).
2.4. Isolation and identification of phenolics
A portion (97.5g) of the aqueous MeOH extract thus
obtained, was applied to a Sephadex LH-20 (850g) column
(100×7.5cm) and eluted with H O followed by H O/MeOH
2 2
mixtures of decreasing polarities, whereby, twelve fractions
(I–XII) were individually desorbed, dried in vacuum and
subjected to two dimensional paper chromatography (2DPC).
Compounds 1 (87mg) and 2 (54mg) were isolated pure from
1.36g of fraction II (eluted from the major column with 10% aq.
MeOH), by column fractionation (CF) over 15g of Sephadex
2
LH-20 using a MeOH/H O mixture for elution. Compounds 3
(64mg) and 4 (92mg) were separated pure from 698mg of
fraction III (eluted with 20%), by fractionation on a Sephadex
LH-20 (13g) column using 40% aqueous MeOH for elution.
Compounds, 5 (102mg), 6 (92mg) and 7 (54mg) were
individually separated pure from 601mg of fraction IV (eluted
with 30%) by prep. PC, using n-BuOH water saturated as
solvent. Compounds 8 (44mg), 9 (103mg) and 10 (88.7mg)
were isolated pure from 767mg of fraction V (eluted with 40%)
by applying a polyamide column (35g) fractionation and
gradient elution with water/MeOH mixture of decreasing
polarities, whereby, three successive subfractions were indi-
vidually eluted, subfraction i, by 20%; ii, by 30% and iii, by 50%
aq. MeOH, respectively. Purification by prep. PC using BAW
(n-BuOH–HOAc–H2O, 4:1:5, upper layer) as solvent afforded
pure samples of compounds 8 (48mg) from i, 9 (33mg) from ii
and 10 (17mg) from iii. 2DPC and CoPC of fraction VI proved
the presence of compound 9 as a main constituent, among
other minors. This fraction was therefore not further processed.
Compound 11 (91mg) was obtained from 372mg of fraction
VII (eluted with 60%) through repeated (thrice) crystallization
from aq. MeOH (50%). Sephadex LH-20 column of 286mg of
1
2
2
3 against prostate cancer cell line were also assessed [15].
. Experimental
.1. General experimental procedures
1
H NMR spectra were measured using a Jeol ECA 500MHz
1
NMR spectrometer, at 500MHz. H chemical shifts (δ) were
measured in ppm, relative TMS and 13C NMR chemical shifts
6
to DMSO-d and were converted to TMS scale by adding 39.5.
High resolution ESI mass spectra were measured using a
Finnigan LTQ FT Ultra mass spectrometer (Thermo Fisher
Scientific, Bremen, Germany) equipped with a Nanomate ESI
interface (Advion). An electrospray voltage of 1.7kV (+/−)
and a transfer capillary temperature of 200°C were applied.
Collision induced dissociation (CID) was performed in the ion
trap using a normalized collision energy of 35%, activation
time of 30ms, 0.25 activation Q and a precursor ion isolation
width of 2amu. High resolution product ions were detected
in the Fourier transform ion cyclotron resonance (FTICR) cell
of the mass spectrometer. UV recording was made on a
Shimadzu UV–Visible-1601 spectrophotometer. Flame atomic
absorption analysis was performed on a Varian Spectra-AA220
instrument, lamp current: 5mA, fuel: acetylene, oxidant: air,
2
fraction VIII (eluted with 70%), using n-BuOH saturated with H O
for elution yielded a pure sample of compound 12 (122mg).
Compound 13 was obtained from 717mg of fraction IX (eluted
with 80%) through an MCI gel (CHP-20P) column fractionation,
using gradient elution with H
polarities. The obtained subfractions were examined by 2DPC
2
O/MeOH mixtures of decreasing

1258
M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
(
two dimensional paper chromatography). The major sub-
122.56 (C‐1′), 114.80 (C‐2′), 146.00 (C‐3′), 149.10 (C‐4′),
111.50 (C‐5′), 126.40 (C‐6′), 56.1 (C-OMe).
fraction vii, eluted with 90% was dried under vacuum at 45°C,
dissolved in acetone, and filtered and the filtrate was treated
with diethyl ether in excess to yield an off-white precipitate.
Repeated precipitation (thrice) afforded a pure sample (189mg)
of 13. The material, 722mg of fraction X (eluted with 90%) was
fractionated over a polyamide-s6, using a solvent mixture of
2.4.2. 3-Methoxy ellagic acid 4,4′-di-sulfate (4)
R -values: 0.72 (H O), 0.60 (HOAc), 0.33 (BAW). Electro-
f 2
phoretic mobility: 5.0cm UV λmax nm in MeOH: 248, 335, 349.
Complete acid hydrolysis (14mg in 5ml, 0.1N aq. HCl, at 100°C
MeOH:Benzen:H
collected crude materials were separately purified by crystalli-
zation from EtOH, thus yielding pure samples of compounds, 14
2
O (60:38:2) for elution, and the individually
for 15min) yielded Na
analysis) and ellagic acid 3-methyl ether 4a, filtered on from the
cooled hydrolysate: R -values: 0.00 (H O), 0.07 (HOAc), 0.71
2 4 2
SO (BaCl test and atomic absorption
f
2
1
(33mg), 15 (19mg) and 16 (46mg). Compounds 17 (26mg),
(BAW); UV λmax nm in MeOH: 250, 346, 363. H NMR of 4a: δ
ppm: 7.47 (1H, s, H‐5), 7.42 (1H, s, H‐5′), 4.00 (3H, s, OMe‐3).
and 18 (19mg) were individually isolated from 167mg of
fraction XI (eluted with MeOH) through repeated fractional
crystallization (thrice). The material, 123mg of fraction XII (also,
eluted with MeOH) was subjected to repeated prep. PC, using
13
C NMR of 4a: δ ppm: 112.36 (C‐1), 141.98 (C‐2), 140.64 (C‐3),
148.63 (C‐4), 111.84 (C‐5), 112.66 (C‐6), 159.40 (C‐7), 112.98
(C‐1′), 136.63 (C‐2′), 140.21 (C‐3′), 152.63 (C‐4′), 110.83 (C‐5′),
107.85 (C‐6′), 159.30 (C‐7′), 61.45 (OMe‐3). ESI-FTMS (negative
ions) of the parent compound 4: HR ICR MS: m/z=m/2=
30% of aq. acetic acid as solvent, whereby pure samples of
compounds 19 (25mg) and 20 (34mg) were isolated pure.
2
−
2
36.9605=[M−2Na] , corresponding to the molecular for-
mula C15 (calc. 236.9601). Controlled acid hydrolysis of
4 (22mg, aq. 10% AcOH, 100°C, 10min) yielded 4b: R -values:
0.42 (H O), 0.36 (HOAc), 0.39 (BAW). Electrophoretic mobility:
6 14 2
H O S
2
.4.1. Tamarixetin 3,7-disulfate (2)
A light yellow amorphous powder, R
.73 (HOAc), 0.20 (BAW). Electrophoretic mobility: 5.6cm, on
O–
f
f
-values: 0.85 (H
2
O),
2
1
0
2.0cm, UV λmax nm in MeOH: 250, 345, 369; H NMR: δ ppm:
8.04 (1H, s, H‐5), 7.47 (1H, s, H‐5′), 4.1 (3H, s, OMe‐3); H NMR
1
Whatman No. 3 MM paper, buffer solution of pH 2, H
2
HCOOH–AcOH (89:8.5:2.5), 1 and 1/2h, 50V/cm. UV λmax nm in
MeOH: 252, 267, 290, 349; NaOMe: 250, 265 (shoulder), 342;
and HMBC of 4: δ ppm: 8.15 (1H, s, H‐5, HMBC: cross peaks with
C-1, C-3, C-4 and C-6); 8.05 (1H, s, H‐5′, HMBC: cross peaks with
C-1′, C-3′, C-4′ and C-6′); 4.10 (3H, s, OMe‐3, HMBC: cross peak
with C-3). 13C NMR of 4: δ ppm: 117.3 (C‐1), 141.9 (C‐2), 144.4
(C‐3), 146.1 (C‐4), 118.6 (C‐5), 109.4 (C‐6), 161.0 (C‐7), 115.8
(C‐1′), 139.0 (C‐2′), 157.6 (C‐3′), 146 (C‐4′), 118.2 (C‐5′), 113.0
(C‐6′), 161.8 (C‐7′), 61.95 (C‐OMe-3).
NaOAc: 250, 268, 350; NaOAc–H
00, 350 (shoulder), 396; AlCl +HCl (30min): 253, 267, 368.
Complete acid hydrolysis of 2 (14mg in 5ml, 0.1N aq. HCl, at
00°C for 15min) yielded Na SO (BaCl test and atomic
absorption analysis) and quercetin 4′-methyl ether, tamarixetin
3 3 3
BO : 255, 268, 350; AlCl : 272,
3
3
1
2
4
2
(
5mg), filtered from the cooled hydrolysate: R
O), 0.17 (HOAc), 0.83 (BAW); UV λmax nm in MeOH: 238,
55, 268, 369; NaOAC: 253 (inflection), 273, 312, 360
shoulder); NaOAc–H BO : 255, 265 (inflection), 368; AlCl
68, 301 (inflection), 363, 430; AlCl +HCl: 268, 301 (inflec-
f
-values: 0.08
(H
2
2.4.3. 2-O-dehydrodigallic acid monocarboxyloyl-3-O-galloyl-
(α/β)-glucose (9)
2
(
2
3
3
3
:
R
f
-values: 0.39 (H
2
O), 0.32 (HOAc), 0.14 (BAW); UV λmax nm
−
3
in MeOH: 272; negative ESIMS, [M−H] =633, corresponding
to m/r 634 and molecular formula of C27H O18. Complete acid
22
1
tion), 362, 426; NaOMe: 268, 422. H NMR: δ ppm: 6.22 (1H, d,
J=2Hz, H-6); 6.45 (1H, d, J=2Hz, H-8); 7.08 (1H, d, J=8Hz,
H-5′); 7.65 (m, H-2′ and H-6′); 3.81 (s, Me-4′). Controlled acid
hydrolysis (18mg, 10% aq. AcOH, 15min, 100°C) yielded
hydrolysis (52mg in 10ml aq. 1.5N HCl, 100°C, 5h) of 9 yielded
glucose (CoPC), and gallic and dehydrodigallic acids; gallic acid:
R -values: 0.53 (H O), 0.53 (HOAc), 0.78 (BAW); UV λmax nm in
f 2
1
13
intermediates 2a (6mg) and 2b (4mg). 2a: R
f
-values: 0.46
O), 0.40 (HOAc), 0.33 (BAW); electrophoretic mobility:
.5cm. UV λmax nm in MeOH 250, 265, 363 (shoulder); NaOAC:
52, 264, 364; NaOAc+H BO : 254, 267, 358; AlCl : 263, 300,
45, 420; AlCl +HCl: 265, 365, 405; NaOMe: 253, 356, 410. H
MeOH: 272; H NMR: δ ppm: 6.96 (1H, s, H‐2 and H‐6);
NMR: δ ppm: 120.6 (C‐1), 108.8 (C‐2 and C‐6), 145.5 (C‐3 and
C‐5), 138.1 (C‐4); dehydrodigallic acid: R -values: 0.55 (H O),
C
(H
2
2
2
3
f
2
1
3
3
3
0.60 (HOAc), 0.78 (BAW); UV λmax nm in MeOH: 272; H NMR:
δ ppm: 7.03 (1H, d, J=2.5Hz, H‐2), 6.5 (1H, d, J=2.5Hz, H‐6),
6.9 (1H, s, H‐6′); 13C NMR: δ ppm: 120.6 (C‐1), 111.3 (C‐2), 148
(C‐3), 139.6 (C‐4), 146.1 (C‐5), 107.1 (C‐6), 168.2 (C‐7), 115.7
(C‐1′), 136.6 (C‐2′), 140.0 (C‐3′), 139.7 (C‐4′), 143.0 (C‐5′),
109.0 (C‐6′), 167.1 (C-7′). Controlled acid hydrolysis (25mg of
1
3
NMR: δ ppm: 6.55 (1H, d, J=2Hz, H-6); 6.94 (1H, d, J=2Hz,
H-8); 6.88 (1H, d, J=8Hz, H-5′); 7.92 (1H, d, J=2Hz, H-2′); 7.65
(
0
2
1H, dd, J=8Hz, J=2Hz, H-6′), 3.85 (s, Me-4′). 2b: R
.54 (H
2
f
-values:
O), 0.45 (HOAc), 0.56 (BAW); electrophoretic mobility:
.7cm. UV λmax nm in MeOH: 252 (inflection), 267, 343;
BO : 254, 267,
+HCl: 254, 268, 390;
9, aq. 1N HCl, 100°C, 3h) gave intermediate 9a: R
(H O), 0.72 (AcOH), 0.33 (BAW); negative ESIMS: m/z=331,
f
-values: 0.55
NaOAC: 255 (inflection), 272, 388; NaOAc+H
45; AlCl : 268, 274, 300, 412; AlCl
3
3
2
[M−H] , Mr=332; λmax in MeOH at 273nm. 1H NMR of 9:
α-glucose moiety: δ ppm: 5.63 (t, J=9Hz, H‐3‐α), 5.25 (d, J=
3.5Hz, H‐l‐α), 4.82 (dd, J=9Hz and 3.5Hz, H‐2‐α), 3.6–3.95 (m,
H‐4, H‐5 and H‐6 in this moiety); β-glucose moiety: δ ppm: 5.2
(t, J=8Hz, H‐3‐β), 4.96 (t, J=8Hz, H‐2‐β), 4.38 (d, J=8Hz,
H‐1‐β) 3.95 (m, H‐5‐β), 3.6–95 (m, H‐4 and 2H‐6); galloyl
moieties: 7.04, 6.99 (each s, H‐2 and H‐6 in both moieties);
dehydrodigalloyl moieties: δ ppm: 7.14, 7.13 (each d, J=2.5Hz,
H‐2 in both moieties), 6.66, 6.64 (each d, J=2.5Hz, H‐6 in both
moieties), 6.96, 6.94 (each s, H‐6′ in both moieties); COSY
results (see Results and discussion part).
−
3
3
3
1
NaOMe: 269, 320, 389. H NMR: δ ppm: 6.20 (1H, d, J=2Hz,
H‐6); 6.40 (1H, d, J=2Hz, H-8); 7.10 (1H, d, J=8Hz, H-5′); 7.62
(
2
m, H-2′ and H-6′), 3.83 (s, Me-4′). ESI-FTMS (negative ions) of
2−
: m/z=m/2=236.97871=[M−2Na] , calc: 236.97869 cor-
1
responding to a molecular formula of C16
δ ppm: 6.59 (1H, d, J=2Hz, H-6); 6.98 (1H, d, J=2Hz, H-8);
.90 (1H, d, J=8Hz, H-5′); 7.94 (1H, d, J=2Hz, H-2′); 7.66 (1H,
10 13 2
H O S . H NMR of 2:
6
13
dd, J=8Hz, J=2Hz, H-6′), 3.84 (s, Me-4′). C NMR of 2: δ ppm:
58.72 (C‐2), 132.34 (C‐3), 179.69 (C‐4), 161.00 (C‐5), 103.05
C‐6), 158.72 (C‐7), 98.59 (C‐8), 155.81 (C‐9), 106.98 (C‐10),
1
(

M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
1259
1
3
C NMR of 9: δ ppm: α-glucose moiety: 89.79 (C‐l), 72.07
C‐2), 71.69 (C‐3), 68.53 (C‐4), 73.07 (C‐5), 60.88 (C-6);
β-glucose moiety: 94.58 (C-l), 73.25 (C-2), 77.08 (C-3), 68.35
C-4), 76.33 (C-5), 60.88 (C‐6); galloyl moieties: 123.40, 122.80
C-l), 109.55, 109.45 (C-2 and C-6), 145.08, 145.03 (C-3 and C-5),
38.61, 138.39 (C-4), 166.86, 167.45, (C_O); dehydrodigalloyl
moieties: 120.13, 119.88 (C-1), 111.42, 1141 (C-2), 146.99,
46.76 (C-3), 139.96 (C-4), 145.24, 145.03 (C-5), 106.97, 106.88
C-6), 164.42, 163.82 (C_O, 7), 113.41, 113.12 (C-1′), 136.48,
2.5.2. Determination of oxygen radical absorbance capacity by
ORAC assay
(
ROS are generated by the thermal decomposition of
[2,2″-azobis(2-amidinopropane) dihydrochloride (AAPH)] and
over time quench the signal of the fluorescent probe fluorescein.
The subsequent addition of antioxidants reduces the quenching
by preventing the oxidation of the fluorochrome. Briefly,
6- hydroxy-2,5,7,8-tetra-methylchroman-2‐carboxylic acid
(Trolox) which was used as the positive control and test
compounds 13 and 9 were dissolved/diluted in a phosphate
buffered saline (10mM, pH 7.4). In each well of a 96 well plate
(
(
1
1
(
1
1
37.13 (C-2′), 1140.07, 139.82, (C-3′), 140.07 (C-4′), 142.48,
42.49 (C-5′), 109.00, 109.01 (C-6′), 166.88, 167.37 (C_O, 7′).
150μl of 10nM fluorescein, 25μl of Trolox (0.2–3.13μM) or 25μl
of the test compound was pipetted in quadruplicate. The plate
was allowed to equilibrate at 37°C for 30min. After incubation,
fluorescence measurements (Ex. 485nm, Em. 520nm) were
taken every 90s to determine the background signal. After
3cycles, 25μl (240mM) of AAPH was injected in each well.
Measurements were continued for 90min. The half life time of
fluorescein was determined using MS Excel software.
2
.4.4. Vermiculatin (13)
An off-white amorphous powder, R
f
-values: 0.42 (H
+34 (MeOH, c=1). UV λmax MeOH
nm: 224, 278. Complete acid hydrolysis of 13 (62mg, 2N aq. HCl,
00°C, 5h) yielded glucose (CoPC), and gallic and dehydrodi-
2
O), 0.38
25
(HOAc), 0.16 (BAW). [α]
D
1
1
13
gallic acids (CoPC, H and C NMR). Controlled aqueous
hydrolysis of vermiculatin (65mg, 20ml H O, 100°C, 12h)
yielded 13a, (2DPC), separated pure (17mg) by MCI-gel
CHP-20P) column fractionation of the dried aq. hydrolysate
and elution with H O/MeOH mixtures of decreasing polarities.
3a was identified to be 2-O-dehydrodigalloyl-3-O-galloyl-(α/
2
2.5.3. Viability/cytotoxicity, MTT assay
(
The spontaneously transformed non-tumorigenic human
keratinocyte cell line HaCaT (kindly provided by Prof. Fusenig
of the German Cancer Research Centre, Heidelberg, Germany)
was used for determination of the viability and metabolic activity
of non-tumorigenic cells under the influence of the test samples.
Cell culture plastics and medium supplements were obtained
from Biochrom AG (Berlin, D), except if otherwise stated. Cells
2
1
1
13
β)-glucose (CoPC against compound 9, H and C NMR).
FTESI-MS (positive ions) of the parent compound, 13: m/z=
+
1139 [M+Na] , HR-ICRMS: m/z=1139.1409 corresponding to
1
the molecular formula C47
36
H O31Na; calculated: 1139, 1395. H
and 13C NMR data of 13: Table 1.
2
were cultured in a growth medium at 37°C with 5% CO in a
humidified atmosphere. Growth medium (RPMI 1640) was
supplemented with 8% heat inactivated fetal calf serum (FCS,
Sigma, Taufkirchen, D) and antibiotics (penicillin 100unitsml,
streptomycin 100μg/ml). The medium was changed every
3days. Cells were subcultured routinely using EDTA (0.05% in
phosphate buffered saline, PBS) and trypsin/EDTA (0.05%/0.02%
in PBS). For the experiments, the growth medium was replaced
by RPMI 1640 containing 0.01% bovine serum albumine (BSA,
Sigma Taufkirchen, D, BSA medium) and penicillin/streptomycin
(BSA medium). HaCat cells between passages 50 and 70 were
plated in 96 well plates in growth medium in a density of 2×104
cells per well. After 24h the medium was replaced by the BSA
medium and the cells were incubated with different con-
centrations of extract and compounds 13 and 9 for 72h. After
incubation the cells were observed under the microscope for cell
integrity and were treated with MTT solution (in BSA medium,
final concentration of 0.5μg/ml). Formazan crystals were
dissolved with DMSO and the optical density was measured
with a multi well plate reader (BMG Omega, BMG Labtech,
Offenburg, D) at 550nm. Cell viability was expressed as the
percentage of vehicle control. Data were analyzed by Mann–
Whitney test using SPSS13. A pb0.05 value was considered
significant. Results were given as mean+SD for 4 independent
experiments with 6 replicates for each measurement. As a
positive control, 100μg/ml of Aloe vera extract [unformulated,
freeze-dried A. vera inner gel powder (AV, supplied by Aloe Vera
of America Inc., USA or Rainbow Naturprodukte GmbH, D)] was
used.
2
2
.5. Biological methods
.5.1. Determination of radical scavenging activity by DPPH
assay
Radical scavenging activity of plant extract against the
stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl,
Sigma-Aldrich Chemie, Steinheim, Germany) was determined
spectrophotometrically by the DPPH assay. When DPPH reacts
with an antioxidant compound, which can donate hydrogen, it
is reduced. The changes in color (from deep‐violet to light‐
yellow) were measured at 517nm. The determination was done
by a slightly modified method of Brand-Williams et al. [13],
where the extract solution was prepared by dissolving 0.025g of
the dry extract in 10ml of methanol. The solution of DPPH in
methanol (6×10 M) was freshly prepared, before UV mea-
surements. 3ml of this solution was mixed with 9 different
concentrations of the samples. The resulting solutions were kept
in the dark for 30min at room temperature and then the
decrease in absorbance was measured.
Absorbance of the blank sample containing the same
amount of methanol and DPPH solution was prepared and
measured. The experiment was carried out in triplicate.
Radical scavenging activity was calculated by the following
formula: % inhibition=[(AB−AA)/AB]×100, where: AB is
the absorbance of the blank sample and AA is the absorbance
of the tested samples. IC50 values, the concentration of the
substrate that causes 50% loss of the DPPH activity (color),
were calculated for the standard and the extract from a
graph plotted for the % inhibition against the concentration
in μg/ml.
−5
2.5.4. Culture of solid tumor cells
Human hepatocellular carcinoma cell line, Huh-7, colorectal
adenocarcinoma cell line (HCT-116), breast adenocarcinoma cell

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M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
Table 1
NMR spectral data of vermiculatin (13) in DMSO-d
6
(500MHz for 1H and 125.7MHz for 13C).
δ
H
(J, Hz)
δ
C
(ppm) a,b
Long range 1H–13C correlationsc
Glucose I
1
2
3
4
5
6
4.80 (d, J=8.5Hz)
92.8 d
71.8 d
74.0 d
69.8 d
77.3 d
60.77 t
H-1 to C-2, C-3, C-5
4.75 (dd, J=8.5 and 9.5Hz)
3.72 (t, J=9.5Hz)
3.35 (t, J=9.5Hz)
3.6 (m)
H-4 to C-1, C-3
3.68 (dd, J=4 and 12Hz)
3.82 (dd, J=2 and 12Hz)
Glucose II
1
2
3
4
5
6
″″
″″
″″
″″
″″
″″
4.96 (d, J=8.5Hz)
5.01 (dd, J=8.5 and 9.5Hz)
5.29 (t, J=9.5Hz)
3.68 (t, J=9.5Hz). 69.4 d
3.46 (m)
3.78 (dd, J=4 and 12Hz)
92. 6 d
69.2 d
75.7 d
H-1″″ to C-2″″, C-3″″, C-5″″
H-5″″ to C-1″″, C-3″″
77.4 d
60.77 t
3.70 (dd, J=2 and 12Hz)
Dehydrodigallic-dicarboxyloyl moieties
1
2
″ and 1″′
″ and 2″′
118.63, 117.65 all s
7.02 and 7.05 (both d and d, J=2.5Hz, H-2″, 111.40, 111.45 both d
H-2″ to C-4″, C-6″, C-7″; H-2″′ to C-4″′, C-6″′, C-7′′′
H-2″′)
3
4
5
6
″ and 3″′
″ and 4″′
″ and 5″′
″ and 6″′
147.55, 147.61 all s
139.04 s
145.17, 145.18 all s
104.96, 104.99 all s
Both d
′
6.02 and 5.99 (both d and d
J=2.5Hz, H-2″, H-2″′)
H-6″ to C-2″, C-4″, C-7″; H-6″′ to C-″′2 and C-4″′
C-7″
8
9
1
1
1
1
″ and 8″′
″ and 9″′
0″ and 10″′
1″ and 11″
2″ and 12″′
3″ and 13″
113.10, 112.98 all s
135.24, 135.09 all s
138.99, 139.04 all s
139.05 s
142.32 s
109.33 d
7.22 and 7.16 (both s)
H-13″ to C-9″, C-11″, C-14″; H-13′″ to C-9″′, C-11″′,
C-14″′
C_O in both moieties
″ and 7″′ and 14″ and
4″′
7
164.20, 163.91, 163.80, 163.73
all s
1
Galloyl moiety
1
2
3
4
7
″″″
119.62 s
109.46 d
145.10 s
138.68 s
166.37
″″ and 6″″
″″ and 5″″
C-4″″, C-6″″′, C-7″″
″″
″″
a
b
c
Assignments by HMBC and HMQC.
Multiplicities by DEPT.
H–C.
line, MCF-7, and prostate adenocarcinoma cell line, PC-3, were
obtained from Vaccera (Giza, Egypt). Cells were maintained in
RPMI-1640 supplemented with 100μg/ml of streptomycin,
overnight, Tris–HCl was used to dissolve the SRB-stained cells
and the color intensity was measured at 540nm.
100units/ml of penicillin and 10% heat-inactivated fetal bovine
2.5.5.1. Data analysis. The dose response curve of the com-
serum in a humidified, 5% (v/v) CO atmosphere at 37°C.
2
pounds was analyzed using the Emax model (Eq. (1)).
2
.5.5. Cytotoxicity assays against tumor cells
The cytotoxicity of the Reaumuria extract and two of the new
ꢀ
ꢁ
m
½
Dꢁ
%
cell viablility ¼ ð100−RÞ ꢀ 1−K
þ R
ð1Þ
m
d
m
þ ½Dꢁ
compounds, 9 and 13 was tested against tumor cells by SRB
assay as previously described [15]. Exponentially growing cells
were collected using 0.25% trypsin–EDTA and plated in 96-well
plates at 1000–2000 cells/well. Cells were exposed to the crude
extract or purified compound for 72h and subsequently fixed
with TCA (10%) for 1h at 4°C. After several washing, cells were
exposed to 0.4% SRB solution for 10min in a dark place and
subsequently washed with 1% glacial acetic acid. After drying
where [R] is the residual unaffected fraction (the resistance
fraction), [D] is the drug concentration used, [K ] is the drug
concentration that produces a 50% reduction of the maximum
inhibition rate and [m] is a Hill-type coefficient. IC50 was defined
as the drug concentration required to reduce fluorescence to
d

M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
1261
5
0% of that of the control (i.e., K
d
=IC50 when R=0 and Emax
=
confirming the structure of compound (2) as tamarixetin
3,7-di-sodium sulfate. The achieved structure of 2 was
finally confirmed by 13C NMR spectroscopy. From the received
spectra, the presence of sulfate substituents attached to
positions 3 and 7 was concluded from the upfield shifts of
these carbon signals and from the accompanying downfield
shift of the corresponding ortho and para-carbon signals as
well, all in comparison with the corresponding chemical shift in
the spectrum of the aglycone (see Experimental). Similar
shifts are well-known from the work of Markham et al.
[19]. The structure of 2 is therefore finally confirmed as
tamarixetin 3,7-disodium sulfate, a flavonoid which repre-
sents to the best of our knowledge a new natural product.
It should be noted however, that this compound was
actually obtained previously by semi-synthetic method
[20]. The synthetic product showed 13C NMR chemical
100−R).
2
.5.5.2. Statistical analysis. Data are presented as mean± SD.
. Results and discussion
3
Following column chromatographic fractionation of the
aqueous methanol extract obtained by homogenizing the
aerial parts of R. vermiculata in aqueous methanol (75%), 20
compounds (1–20) were isolated. Conventional and spectral
analyses mainly by NMR spectroscopy and by mass spec-
trometry indicated that four of these compounds are new
natural products (2, 4, 9 and 13).
Compound 2, was isolated as an off-white amorphous
powder which exhibited a chromatographic (dark purple
spot on PC turning dull yellow when fumed with ammonia,
dull yellow when sprayed with Natur-Stuff) and anionic
character on electrophoretic analysis similar to those of
anionic flavonols [6m), n),16]. UV absorption maxima in
MeOH and after addition of diagnostic shift reagents [17,18]
shifts in DMSO-d
product.
6
similar to those recorded for the natural
3 3
showed no shift with NaOAc or with NaOAc/H BO , a small
shift with NaOMe and a 28nm shift with HCl. These data
were consistent with 3,7,4′-trisubstituted quercetin struc-
ture. Complete hydrolysis of 2 (0.1N aq. HCl at 100°C for
1
5min) yielded quercetin 4′-methyl ether, tamarixetin
1
13
(
CoPC, UV, H and C NMR). The aqueous acidic hydrolysate
gave a white ppt. with aq. BaCl to prove the presence of the
SO group. Atomic absorption analysis confirmed that the SO
2
4
4
radical(s) exists in the molecule of 2 as sodium sulfate (S). On
controlled acid hydrolysis (10% aq. AcOH, 15min, 100°C) 2
yielded two intermediates, 2a (major, dull yellow spot on PC
under UV light) and 2b (minor, dark purple spot on PC under
UV light). Intermediates 2a and 2b were individually
separated by preparative paper chromatography (prep. PC).
Their chromatographic and electrophoretic properties, UV
Compound 2: Tamarixetin 3,7-di-sodium sulfate.
1
absorption and H NMR data proved a 7,4′-disubstituted
quercetin structure for 2a and a quercetin 3,4′-disubstituted
structure for 2b, a result which, when incorporated with
the above given analytical data proved that 2 is tamarixetin
Compound 4, an amorphous faint yellow powder which
showed chromatographic properties (rosy buff spot on PC
under UV light turning yellow on fuming with ammonia) and
UV spectral maxima, in MeOH (λmax in MeOH: 248, 335, 349)
closely similar to those of ellagic acid derivatives [6h), m)]. 4
exhibited an-ionic characters and yielded on complete acid
hydrolysis (aq. 0.1N HCl, 100°C, 15min) compound 4a,
which precipitated pure from the cold hydrolysate and was
identified to be ellagic acid 3-monomethyl ether through
CoPC, UV, 1H and C NMR analysis [20]. The hydrolysate
proved by chromatographic analysis, to be free of any sugar,
3
,7-disodium sulfate. Further support for this view was
1
obtained through H NMR spectral analysis. The spectrum
revealed in the aromatic region a pattern of signals though
similar to that of the aglycone, tamarixetin (see Experimental),
yet a distinction could be made through the recognition of the
downfield shift of the proton signals of H‐6 and H‐8 (δ ppm
6
.60 and 6.99, respectively), in comparison with the signals at
13
δ ppm 6.22 and 6.45 of the corresponding protons in the
spectrum of the free aglycone due to substitution at C‐7 of the
aglycone. Substitution at the C‐3 of the aglycone was quite
obvious from the dark purple coloration of the spot of 2 on PC.
Further confirmation of the structure was received through the
mass spectra of 2. The spectra (negative ions) exhibited a
doubly charged Mr ion at m/z=m/2=237.33=[M−2Na]−
at low resolution in the ion trap corresponding to Mr=
2
but gave a white precipitate when treated with aq. BaCl and
proved to contain sodium ions by flame atomic absorption,
thus confirming the presence of sodium sulfate(s) in the
molecule of 4. On using ESI MS and detection of negative ions,
compound 4 exhibited a doubly charged molecular ion at m/z=
2
2−
237.3 [M−2Na] , corresponding to an Mr of 520Da. The
[
(237.33×2)+2Na]=520.66. MSMS analysis of the 237.33 ion
showed a doubly charged ion at m/z=m/2=197.1 indicating a
loss of neutral SO from the doubly charged ion. High
resolution data obtained in the FTICR cell confirmed the
formula C16 from the negative doubly charged ion
at m/z=236.9787=[M−2Na]
MSMS analysis of that ion showed loss of a neutral SO unit
3
indicating a sulfate moiety; the structural formulas were
established on the basis of high resolution FTICR MS which
confirmed a molecular structure of a di-sulfated ellagic acid
monomethylether. 4 gave on controlled acid hydrolysis (10% aq
AcOH, 100°C, 10min), an intermediate 4b, which was separated
3
10 13 2
H O S
−
2
3
and the loss of SO thus

1262
M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
−
pure by prep. PC, using H
properties, UV absorption maxima and electrophoretic mobility
of 4b (see Experimental) suggested it to be an ellagic acid
2
O as solvent. Chromatographic
corresponding to a molecular ion [M−H] at m/z 633. Complete
acid hydrolysis (aq. 1.5N HCl, 100°C, 5h) yielded glucose (CoPC),
dehydrodigallic acid and gallic acid. MCI-gel (CHP-20P) column
fractionation of the dried EtOAc extract of the hydrolysate and
3-methyl ether substituted at either the 4-OH or the 4′-OH by
1
sodium sulfate. This view was supported by H NMR analysis of
b. The received spectrum showed a pattern of resonances (δ
elution with H
2
O/MeOH mixtures of decreasing polarities
yielded individual samples of gallic and dehydrodigallic acids.
The identity of both acids was achieved through CoPC, UV, H
4
1
ppm: 8.04 (1H, s, H‐5), 7.47 (1H, s, H‐5′), 4.1 (3H, s, OMe‐3))
which proved that the substituent at either OH‐4 or OH‐4′ has
been released so that the resonance of the vicinal proton, H‐5 or
H‐5′, was shifted upfield to a δ ppm of 7.47. Comparison of the 1D
NMR data proved that 4 contained two sulfate substituents, one
at OH‐4 and the second at OH‐4′. This followed from the
locations of the resonances of H‐5 and H‐5′ at δ ppm 8.15 and
and 13C NMR data [6e)]. On controlled acid hydrolysis (1N aq.
HCl, 100°C, 3h) 9 gave, besides glucose, and gallic and
dehyrodigallic acids, an intermediate 9a with chromatographic
properties similar to those reported for galloyl esters. 9a was
separated from an EtOAc extract of the hydrolysate by prep. PC
and was shown to have an Mr of 332 (negative ESIMS, [M−
−
8.06, which are recognizably lowfield in comparison with those
H] : m/z=331) and a λmax in MeOH at 273nm. These data
show 9a to be a monogalloyl glucose. The H NMR spectrum of
1
of the corresponding resonances in the spectrum of the free
ellagic acid 3-methyl ether [21]. The 13C NMR data of 4 confirmed
its achieved structure. Substitution with sulfates at C‐4 and C-4′
is recognized from the upfield resonances of these carbons (δ
9 revealed two distinct patterns of proton resonances belong-
ing to substituted α and β-glucose anomers. Each pattern was
found to contain well separated resonances of the seven-spin
system belonging to a distinct glucose anomer, thus proving
absence of substitution at the glucose anomeric hydroxyl
group. The presence, in this spectrum, of the resonance of the
diagnostic glucose proton, H‐2‐α, downfield at 4.82 (dd, J=
9Hz and 3.5Hz, H‐2‐α) proved acylation at this hydroxyl
group. Measurement of the COSY spectrum for 9 confirmed this
finding and proved additional acylation at the 3-hydroxyl of
the glucose moiety, whose proton was found to be resonating
downfield at δ 5.36 (t, J=9Hz, H‐3‐α), 5.2 (t, J=8Hz, H‐3‐β),
while the resonance of the H-2‐β proton was found to resonate
at δ 4.96 (t, J=8Hz, H‐2‐β). The location of other glucose
proton resonances, comparatively upfield proved the
absence of acylation at all the other glucose hydroxyl
groups except numbers 2 and 3. These and the above given
data (specifically results of controlled acid hydrolysis)
146.12 and 146.01ppm), in comparison with those of the
corresponding resonances in the spectrum of the parent
compound 4a. The accompanying downfield shift of the
resonances of the β-carbons C‐3, C‐5, C‐3′, and C‐5′ are in
consistency with this view (see Experimental). Unambiguous
assignments of the remaining carbon resonances were based on
the results of the HMQC and HMBC spectral analyses. In the
3
HMBC spectrum, J correlations of proton H‐5 at 8.15 to C‐1
(117.3), C‐3 (144.4), and C‐7 (161.0) and of H‐5′ at δ 8.06 to
carbons C‐1′ (115.8), C‐3′ (157.6), and C‐7′ (161.8) were
recognized. Correlations of the methoxyl protons (δ 4.1) to the
aromatic C‐3 (δ 144.42) and of the same downfield aromatic
proton (δ 8.15) to the same C-3 carbon confirmed that the site of
attachment of one of the sulfate substituent is at the C-4 position
3
of the 3-methoxyellagic acid moiety. The recorded J correlations
of H‐5′ (δ 8.06) allowed positioning of the second sulfate
substituent at C‐4′. The recognizable J correlation of this proton
to the same C‐4′ carbon was in accordance with this conclusion.
The complete structure of compound 4 was therefore, deter-
mined to be ellagic acid 3-methyl ether 4.4′-di-sodium sulfate.
would suggest
monocarboxyloyl-(α/β)-glucose structure for 9. A reversed
substitution, would produce on controlled hydrolysis
3-O-dehydrodigallic acid monocarboxyloyl glucose as it is well
known that an ester linkage at the glucose 2-OH is more labile
and more easily hydrolyzed than the ester linkage at position no.
a 3-O-galloyl-2-O-dehydrodigallic acid
2
a
3
[4]. This was confirmed by the measurement of a long range
shift correlation, HMBC spectrum of 9, which revealed two cross
peaks correlating the galloyl carbonyl carbon resonances in each
of the α- and β-anomer to the resonances of the glucose H-3
proton resonances. Cross peaks correlating to the dehydrodigallic
acid monocarboxyloyl carbonyl carbon signals in both anomers
to the resonances of the H-2 glucose protons were also
recognized. The minor 2J correlations between H-2 and the
3
etherified C-3 carbon and the detectable
J cross peaks
correlating H-6′ to the free carboxylic carbonyl carbon in the
dehydrodigalloyl moiety lent further support to the structure of
9
as 2-O-dehydrodigallic acid monocarboxyloyl-3-O-galloyl-(α/
13
β)-glucose. The C NMR data of 9 further confirmed its
achieved structure. The α- and β-anomers were recognized,
in the recorded spectrum from the resonances at δ 89.79
and 94.58ppm, respectively. The acylation by dehydrodi-
gallic acid mono-carboxyloyl and galloyl of the glucose OH
groups at positions 2 and 3 follows from the upfield shift of
the resonances of glucose C-1 and C-4 compared to the
corresponding resonances in free D-glucopyranose [6f)].
These β-effects are in agreement with those reported for
similarly substituted glucopyranose [6f)]. The upfield shifts of
Compound 4: ellagic acid 3-monomethyl ether 4,4′-di-sodium sulfate.
Compound 9, a white amorphous powder, was found to
possess chromatographic properties, color reactions (dark blue
spot on PC under short UV, intense blue with FeCl
KIO [22]) and UV spectra data (λmax in MeOH: 273nm)
consistent with gallotannins. It exhibited an Mr of 634,
3
, pink with
3

M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
1263
1
13
the resonances of C‐2 and C‐3 are caused by both α- and
β-effects which brought these resonances to δ ppm 72.07 (C‐
through CoPC, H and C NMR. The positive ion FTESIMS spectra
+
of 13 exhibited a molecular ion at m/z=1139 [M+Na] ,
2
‐α), 71.69 (C‐3‐α), 73.25 (C‐2‐β), and 77.08 (C‐3‐β). The
corresponding to an Mr of 1116Da. The accurate MSMS
analysis of that ion revealed a fragmentation pattern consistent
with the proposed structure. The ions found are: m/z: 969.1151
presence of only one dehydrodigallic acid monocarboxyloyl
moiety and one galloyl moiety in 9 follows from the four
carbonyl carbon resonances of the dehydrodigallic acid mono-
carboxyloyl moiety (two for each anomer) and the two galloyl
carbonyl carbon (one for each anomer) resonances at δ ppm
+
+
+
[M+Na ‐gallic acid-H] , m/z 825.0729 [M+Na-314] which
is due to breaking of the bonds of oxygen at C‐1″″ and C‐2″″, m/
+
z 843.0712 [M+Na-314-O] (breaking of the bonds at C‐1″″
+
164.42, 163.82, 166.88, and 167.37 (dehydrodigallic acid mono-
and C‐14″′), m/z 657.0679 [M−Na ‐galloyl-dehydrodigalloyl
+
carboxyloyl C_O) and at 166.86, and 167.45 (galloyl C_O).
Esterification of only one of the carboxyl groups of the
dehydrodigallic acid monocarboxyloyl moiety followed from
the recognizable upfield shift of the esterified carbonyl (δ 164.42,
glucose] (breaking of the bonds at C‐9″and C‐14″′), m/z
505.0580 and m/z 487.0475 are due to breaking of the bonds
at C-9″ and C‐5″′ or C‐9″′, respectively, thus suggesting
a
molecular structure consisting of two glucose, two
1
63.82 in the α- and β-anomers). Assignment of the remaining
dehydrodigallic acid dicarboxyloyl and one galloyl moieties
incorporated together in the dimeric cyclic structure given for
vermiculatin. The strong affinity to sodium (all fragments bear
the sodium ion) adds evidence to the cyclic structure which
looks like a cage compound. Structure elucidation of 13 was
carbon resonances were aided by comparison with the previ-
ously reported data of analogous compounds [6k)]. Furthermore,
the measured δ values of the glucose carbon resonances
confirmed that the sugar core exists in the pyranose form.
Consequently, 9 is a 2-O-dehydrodigallic acid monocarboxyloyl-
achieved by 1H and C NMR spectroscopy, including 1H– H
COSY, DEPT, HMQC and HMBC spectroscopy, which allowed
the full assignment of all carbon and proton resonances.
13
1
3-O-galloyl-(α/β)glucopyranose, which has not been reported
previously in nature.
1
13
The H, C NMR and HMBC spectra (Table 1) unambiguously
identified 13 as an ellagitannin with two dehydrodigallic
dicarboxyloyl moieties whose four carbonyl resonances
(
1
HMBC) were located at δ ppm 164.20, 163.91, 163.80, and
63.73. This upfield location (in comparison with the corre-
sponding resonances in the spectrum of free dehydrodigallic
acid, is obviously due to esterification with the alcoholic
glucose groups [21]. The presence of the galloyl moiety in the
molecule of 13 was concluded from its characteristic reso-
nances in the 1H and C NMR spectra (Table 1). The number
13
13
and characteristic shifts of the C sugar signals indicated
the presence of two glucose systems in the pyranose form.
The assignment of their 1H and C signals followed directly
13
13
from the COSY and HMQC spectra. In all cases the C shifts of
their C‐6 and C‐4 signals indicated that these positions were
un-substituted. This was also the case with one of the C‐3 signals.
All sugars had β-glycosidic linkages from the magnitude of the
vicinal proton couplings of the anomeric protons in the 1
H
spectrum. That one of the glucose moiety has esterified OH-1 and
OH-2 groups, while the second glucose moiety has its OH-1,
OH-2 and OH-3 groups esterified was readily identified from the
low field shift of the geminal proton resonances of these OH
groups [glucose‐I: 4.80 (d, J=8.5Hz, H-1), 4.75 (dd, J=8.5
and 9.5Hz, H-2); glucose‐II: 4.96 (d, J=8.5Hz, H-1″″), 5.01 (dd,
J=8.5 and 9.5Hz, H-2″″), 5.29 (t, J=9.5Hz, H-3″″)] and
unambiguously confirmed by a long-range correlation between
this proton and the carbonyl carbons C‐7″′, C‐14″, C-7″, C‐14″′
and C‐7″″′, respectively, in the HMBC spectrum. This latter
spectrum also allowed the interconnectivity of the two glucoses,
the two dehydrodigallicdicaboxyloyl and the galloyl units to be
unambiguously determined. Proton H‐1 (δ 4.80) showed a
correlation with CO‐7″′ (δ 163.80), H‐2 (δ 4.75) with CO‐14″ (δ
163.91), H‐1″″ (δ 4.96) with CO‐7″ (δ 163.73), H‐2″″ (δ 5.01)
with CO-14″′ (164.20) and H-3″″ with CO-7″″′(166.37).
Similarly for the assignments of the aromatic carbon moieties
a long-range correlation between H‐2″, H‐2″′ (δ 7.02 and 7.05),
H‐6″, H-6″′ (δ 6.02 and 5.99) and H‐13″, H‐13″′ (δ 7.22 and
7.16) in the two dehydrodigallic acid carboxyloyl moieties and
H‐1″″′ and H‐5″″′ in the galloyl moieties to the carbons existing
in the corresponding rings (see Table 1) was observed. Hence,
Compound 9: 2-O‐dehydrodigallic acid monocarboxyloyl-3-O-galloyl-(α/β)
glucopyranose.
Compound 13 was isolated as an amorphous off-white
powder which possesses galloyl ester-like characters (in-
tense blue color with FeCl , rosy red color with KIO ) [22] and
3 3
UV spectral maximum in MeOH at 224, and 278nm.
Complete acid hydrolysis of 13 (2N aq. HCl, 100°C, 5h)
yielded glucose (CoPC), and gallic and dehydrodigallic acids.
Both acids were individually isolated pure through MCI gel
column fractionation of an EtOAc extract of the hydrolysate,
using n-BuOH water saturated for elution and were identified
1
13
by CoPC, H and C NMR (see Experimental). Controlled
aqueous hydrolysis of 13 (H O, 100°C, 12h) yielded among
2
other minors, a major intermediate, 13a, (2DPC), separated
pure by MCI-gel (CHP-20P) column fractionation of the
dried aq. hydrolysate and elution with H O/MeOH mixt-
2
ures of decreasing polarities. 13a was identified to be 2-O-
dehydrodigallic monocroxyloyl-3-O-galloyl-(α/β)glucopyranose

1264
M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
Compound 13: vermiculatin.
the structure of 13 was determined as a dimeric cyclic
ellagitannin, which we named vermiculatin.
undissolved substance particles (Fig. 2). Cell viability due to
tested concentrations of all test samples was well above the IC50
limit, so the latter could not be estimated. The positive control
led to an increased cell viability of about 150%. The results of the
biological investigation of the extract and two of the isolated
novel compounds confirm the antioxidant potential of plant
phenolics. The cytotoxicity is neglectable, and small concen-
trations of the extract have yet a stimulating effect on the
investigated skin cells. The results substantiate the ethnome-
dicinal use of R. vermiculata.
In addition, the known compounds, 2,6-digalloyl glucose
(
1), kaempferol 3,7-disodium sulfate (3), quercetin 3,7-dis-
odium sulfate (5), gallic acid (6), 3‐mono methoxy ellagic
-sodium sulfate (7), nilocitin (8), tamarixellagic acid (10),
4
dehydrodigallic acid (11), decarboxy dehydrodigallic acid (12),
quercetin (14), kaempferol (15), tamarixetin (16), ellagic acid
(
3
(
17), ellagic acid 3-monmethyl ether (18), ellagic acid
,3′-dimethyl ether (19), and 6-hydroxy7-methoxycoumarine
20) were also isolated and identified by applying the
conventional and spectral methods of analysis.
3.3. Cytotoxicity against tumor cell lines
3
3
.1. Antioxidant activity
SRB-U assay was used to assess the cytotoxicity of the
Reaumuria extract against four different solid tumor cell lines.
The cytotoxicity parameters, IC50 and R-fraction were calculat-
ed using the Emax model as described in the Biological methods
section. The Reaumuria extract showed comparable potencies
against all solid tumor cell lines used with IC50 ranging from
.1.1. DPPH assay
The radical scavenging activity of the crude extract was
first determined in the DPPH assay. The IC50 of the extract is
.8μg/ml which is in a similar range as that for the positive
control ascorbic acid with 1.8μg/ml.
5
1
.3± 0.15 to 2.4± 0.22μg/ml (Table 2). The cytotoxicity pattern
(
dose–response profile) of Reaumuria extract was sharp in
3
.1.2. ORAC assay
Because of this high activity the antioxidative capacity
HCT-116, MCF-7 and PC-3 cell lines (Fig. 3-A) with a relatively
was furthermore investigated in the ORAC assay. It demon-
strated a moderate anti-oxidant capacity of the crude extract
and higher activities of the isolated compounds 9 and 13
(
Fig. 1).
3
.2. Cytotoxicity: non tumorigenic cells
The R. vermiculata crude extract diminished the viability of
the non tumorigenic HaCaT cells only in concentrations higher
than 3.1μg/ml and only in a moderate degree. In opposite, a
slight increase in HaCaT cell viability was observed at the
lowest concentration tested. There is also a moderate dose
dependent cytotoxic effect in the higher concentrations. The
higher bar seen at a concentration of 25, 50 and 100μg/ml for
compound 13 and at a concentration of 100μg/ml for compound
Fig. 1. Antioxidant activity of R. vermiculata samples measured in the ORAC
assay: half life of fluorescein after protection with 12.5μg/ml test sample.
The positive control is Trolox (3.13μM).
9
is probably not due to an increased cell viability but rather to

M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
1265
1
1
20
00
Reaumuria extract
compound 9
*
compound 13
*
*
*
*
*
*
*
*
*
*
*
*
8
6
4
2
0
0
0
0
0
*
*
*
*
*
*
*
vehicle
0.8
1.6
3.1
6.3
12.5
25
50
100
concentration [µg/ml]
Fig. 2. Viability of HaCaT keratinocytes after treatment with Reaumuria extract, 2-Odehydrodigallicacid monocarboxyloyl-3-O-galloyl-(α/β)glucopyranose (9) or
vermiculatin (13) measured in the MTT assay. Experiments were carried out in 4 independent experiments with 6 replicates.
higher Hill-type co-efficient ranging from 4.22 to 8.47. On the
other hand, the dose response pattern was gradual with
relatively lower Hill-type co-efficient (2.38) in Huh-7 liver
cancer cell line. Reaumuria extract possesses the highest IC50 in
Huh-7. However, the resistant fraction of Huh-7 was the lowest
cancer cell line. The cytotoxicity pattern (dose–response profile)
of both compounds in PC-3 cell was much gradual than that of
the total extract (Fig. 3-B) with hill-type co-efficients of 0.5 and
0.8, respectively. Compounds 9 and 13 possess IC50s of 1.5± 0.33
(
0%) among all tested cell lines. Other cell lines showed
substantial R- fraction ranging from 0.88± 0.2 to 2.4± 0.6
Table 2). The types of cancers examined herein were selected
A) 120
(
1
00
based on clear epidemiological evidence and high health related
problem. Liver cancer constitutes a national health problem in
Egypt and Middle East. Colorectal cancer is the third most
commonly diagnosed cancer in males and the second in females.
Breast cancer is the most frequently diagnosed cancer and the
leading cause of cancer death in females worldwide. Prostate
cancer is the second most frequently diagnosed cancer and the
sixth leading cause of cancer death in males [23]. The observed
IC50s of the Reaumuria extract represents a promise for universal
potencies against different types of cancers with high deterio-
rating health damage.
8
0
Huh-7
HCT-116
MCF-7
PC-3
60
40
2
0
0
0.01
0.1
1
10
100
Concentration (µg/ml)
According to the cytotoxic profile of the crude extract,
promisingly low IC50 and R-fraction in addition to a reasonable
value of hill type co-efficient were noticed in the case of prostate
cancer cell line (PC-3) which warranted further investigation.
The very high value of hill-type co-efficient suggests non
specificity in cytotoxic molecular target and vice versa [24,25].
Compounds 9 and 13 were therefore, tested against the prostate
B) 120
00
1
Cpd # 4
8
6
4
0
0
0
Cpd # 13
Table 2
20
0
Cytotoxicity of Reaumuria extract against different solid tumor cell lines.
Cell line
Tumor
origin
Cytotoxicity parameter
0
.01
0.1
1
10
100
IC50
R-fraction
(%)
Hill-type
co-efficient
Concentration (µg/ml)
(μg/ml)
Fig. 3. Dose–response curve of Reaumuria extract and its derived purified
compounds against solid tumor cell lines. Huh-7 (○), HCT-116 (●), MCF-7
Huh-7
HCT-116
MCF-7
PC-3
Liver
Colorectal
Breast
2.4± 0.22
1.8± 0.94
1.3± 0.15
1.5± 0.17
0.0± 0.0
2.2± 0.58
0.88± 0.2
2.4± 0.6
5.59
2.38
8.47
4.22
(
▼), and PC-3 (Δ) cells were exposed to the crude extract for 72h (A). PC-3
cell line was exposed to 9 (○) and 13 (●). Cell viability was determined
Prostate
using SRB-U assay and data are expressed as mean± S.D. (n=3).

1266
M.A. Nawwar et al. / Fitoterapia 83 (2012) 1256–1266
h
i
Nawwar M, Souleman A. 3,4,8,9,10-Pentahydroxy‐dibenzo[b,d]pyra-
Table 3
n-6-one from Tamarix nilotica. Phytochemistry 1972;23:2966–7.
Barakat H, Nawwar M, Buddrus J, Linscheid M. Niloticol. A phenolic
glyceride and two phenolic aldehydes from the roots of Tamarix
nilotica. Phytochemistry 1972;26:1837–8.
Souleman A, Barakat H, El-Mousallami A, Marzouk M, Nawwar M.
Phenolic constituents of the bark of Tamarix aphylla. Phytochemistry
Cytotoxicity of 9 and 13 of the Reaumuria extract against prostate cancer cell
lines (PC-3).
Compound
Cytotoxicity parameter
j
IC50 (μM)
R-fraction (%)
Hill-type co-efficient
1972;30:3763–6.
9
1.5± 0.33
0.0± 0.0
0.49
0.8
k
Nawwar M, Hussein S, Buddrus J, Linscheid M. Tamarixellagic acid,
an ellagitannin from the galls of Tamarix aphylla. Phytochemistry
13
0.45± 0.09
4.1± 0.94
1972;35:1349–54.
l
Nawwar M, Hussein S. Gall polyphenolics of Tamarix aphylla.
Phytochemistry 1972;36:1035–7.
and 0.54± 0.09μM, respectively and resistant fractions of 0.0%
and 4.1%, respectively (Table 3).
m
Souleman A, Barakat H, Hussein S, El-Mousallami A, Nawwar M.
Unique phenolic sulphate conjugates from the flowers of Tamarix
amplixuicaulis. Nat Prod Sci 1972;4:245–52.
n
El Mousallamy A, Hussein S, Nawwar M. Polyphenolic metabolites of
the flowers of Tamarix tetragyna. Nat Prod Sci 1972;6:193–8.
Acknowledgment
[
7] Nawwar M, El-Sherbieny A, Meshaal S. Flavonoids of Reumuria mucronata
and Thymelea hirsutum. Phytochemistry 1977;16:1319–20.
We are indebted to AvH (Alexander von Humboldt)
foundation for the donation of a Schimadzu UV–Visible-1601
spectrophotometer and an 8001-Kruess polarimeter to
Mahmoud Nawwar. We thank the DAAD and the STDF, Cairo,
Egypt for financing the stay of Reham El-Sharawy and Mahmoud
Nawwar in Germany through the GESP project, ID: 1399.
[
8] Klein M, Papenbrock J. The multi-protein family of Arabidopsis
sulphotransferases and their relatives in other plant species. J Exp Bot
2004;55:1809–20.
[9] Varin L, Marsolais F, Richard M, Rouleau M. Biochemistry and molecular
biology of plant sulphotransferases. FASEB J 1997;11:517–25.
10] Yang P, Matsubayashi Y, Nakamura K, Sakagami Y. Oryza sativa PSK
gene encodes a precursor of phytosulphokine-a, a sulphated peptide
growth factor found in plants. Proc Natl Acad Sci U S A 1999;96:
[
[
13560–5.
References
11] Marsolais F, Gidda K, Boyd J, Varin L. Plant soluble sulphotransferases:
structural and functional similarity with mammalian enzymes. Recent
Adv Phytochemistry 2000;34:433–56.
[1] Quideau S, editor. Chemistry and biology of ellagitannins—an under-
estimated class of bioactive plant polyphenols. World Scientific Publishing
Co. Pte. Ltd; 2008. p. 203–55.
[12] Boulos L. Medicinal plants of North Africa. Michigan: Reference
Publications; 1983. p. 101.
[
[
[
2] Croftk D. The chemistry and biological effects of flavonoids and
phenolic acids. Ann New York Acad Sci 2006.
3] Bolous L. Flora of Egypt, 2. Cairo, Egypt: Al Hadara Publishing; 1999.
p. 124.
4] Ahmed FA, Yoshida T, Okuda T. Tannins of Tamaricaceous plants, V,
new dimeric, trimeric and tetrameric ellagitannins from Reaumuria
hirtella. Chem Pharm Bull 1994;42(2):246–53.
[13] Brand-Williams W, Cuvelier M. Use of a free radical method to evaluate
antioxidant activity. Food Sci Technol 1995;28:25–30.
[14] Ou B, Hampsch-Woodill M, Prior R. Development and validation of an
improved oxygen radical absorbance capacity assay using fluorescein
as the fluorescent probe. J Agric Food Chem 2001;49:4619–926.
[15] Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al.
New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl
Cancer Inst 1990;82:1107–12.
[16] Barron D, Varin L, Ibrahim R, Harborne J, Williams C. Sulphated
flavonoids an update. Phytochemistry 1988;27:2375–95.
[17] Harborne J, Williams C. The flavonoids. In: Harborne J, Mabry J, Mabry
H, editors. London: Chapman & Hall; 1975. p. 37–441.
[5] Orabi NAA, Taniguchi S, Yoshimura M, Yoshida T, Kishina K, Sakagami
H, et al. Hydrolyzable tannins of Tamaricaceous plants. III. Hellinoyl and
macrocyclic-type ellagitannins from Tamarix nilotica.
010;73(5):870–9.
J Nat Prod
2
[6] a Ishak M, El-Sissi H, Nawwar M, El-Sherbieny A. Tannins and
polyphenolics of the galls of Tamarix aphylla. Planta Med 1972;21:
[18] Mabry J, Markham R, Thomas B. The systematic identification of
flavonoids. Heidelberg: Springer; 1970.
246–9.
b
c
El-Sissi H, Nawwar M, Saleh N. Plant constituents of Tamarix nilotica
leaves. Experientia 1972;29:1064.
[19] Markham K, Ternai B, Stanley R, Geiger H, Mabry T. Carbon-13 NMR
studies of flavonoids-III. Tetrahedron 1978;34:1389–97.
[20] Barron D, Ibrahim R. Tetrahedron 1987:5197–202.
[21] Hussein S, Hashim A, El-Sharawy R, Seliem M, Linscheid M, Lindequist
U, et al. Ericifolin: an eugenol 5-O-galloylglucoside and other phenolics
from Melaleuca ericifolia. Phytochemistry 2007;68:1464–70.
[22] Haddock A, Gupta RK, Al-Shafi SM, Haslam E. The metabolites of gallic
acid and hexahydroxydiphenic acid in plants. Part 1. Introduction,
naturally occurring galloyl esters. J Chim Soc Perkin Trans 1 1982:
2515–24.
[23] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Cancer C A J Clin
2011:69–90.
[24] Levasseur L, Slocum H, Rustum Y, Greco W. Cancer Res 1998:5749–61.
[25] Au J, Li D, Gan Y, Gao X, Johnson A, Johnston J, et al. Cancer Res 1998:
2141–8.
Saleh N, El-Sissi H, Nawwar M. A rhamnetin glucuronide trisulphate
from the leaves of Tamarix aphylla. Phytochemistry 1972;14:312–3.
El-Ansary M, Nawwar M, El Sherbieny A, El-Sissi H. A sulphated
kaempferol 7,4′-dimethyl ether and a quercetin isoferulylglucuro-
nide from the flowers of Tamarix aphylla. Phytochemistry 1972;15:
d
231–2.
e
f
Nawwar M, Buddrus J, Bauer H. Dimeric phenolic constituents from
the roots of Tamarix nilotica. Phytochemistry 1972;21:1755–8.
Nawwar M, Souleman A, Buddrus J, Bauer H, Linscheid M. Polyphe-
nolic constituents of the flowers of Tamarix nilotica: the structure of
nilocitin, a new digalloylglucoside. Tetrahedron Lett 1972;25:49–52.
Nawwar M, Souleman A, Buddrus J, Linscheid M. Flavonoids of the
flowers of Tamarix nilotica. Phytochemistry 1972;23:2347–9.
g