Anthraquinone glycosides from Cassia roxburghii and evaluation of its free radical scavenging activity

Article abstract of DOI:10.1016/j.carres.2012.07.020

The methanolic extract of the leaves of Cassia roxburghii DC., was investigated for its anthraquinone glycosides and antioxidant activity. Two new anthraquinone glycosides named emodin 1-O-β-d-glucopyranosyl-(1→2)- glucopyranoside (1) and aloemodin 8-O-β-

Full text of DOI:10.1016/j.carres.2012.07.020

Carbohydrate Research 360 (2012) 47–51
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Anthraquinone glycosides from Cassia roxburghii and evaluation of its free
radical scavenging activity
b
b
c
c
Sayed A. El-Toumy a, , Sahar S. El Souda , Tahia K. Mohamed , Iñaki Brouard , Jame Bermejo
⇑
a Chemistry of Tannins Department, National Research Center, El-Bohouth St., Dokki, 12622 Cairo, Egypt
b Chemistry of Natural Compounds Department, National Research Center, El-Bohouth St., Dokki, 12622 Cairo, Egypt
c Instituto de Productos Naturales y Agrobiología, Av. Astrofisico F. Sanchez 3, 38206 La Laguna, Tenerife, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2012
Received in revised form 28 July 2012
Accepted 30 July 2012
Available online 8 August 2012
The methanolic extract of the leaves of Cassia roxburghii DC., was investigated for its anthraquinone gly-
cosides and antioxidant activity. Two new anthraquinone glycosides named emodin 1-O-b- -glucopyran-
osyl-(1?2)-glucopyranoside (1) and aloemodin 8-O-b- -glucopyranosyl-(1?6)-glucopyranoside (2)
along with aloemodin 8-O-b- -glucopyranoside (3), emodin (4), aloemodin (5) and one flavonoid, quer-
cetin-3-O- -rhamnopyranoside, were isolated from the leaves of C. roxburghii. Structures of the isolated
D
D
D
a
-L
compounds were established by UV, HRESI-MS, and 1D/2D 1H/13C NMR spectroscopy. The total extract
and some isolated compounds were determined against DPPH (2,2-diphenyl-1-(2,4,6-trinitrophe-
nyl)hydrazinyl radical, for their free radical scavenging activity, the total alcoholic extract showed strong
antioxidant activity while the two new compounds showed weak antioxidant activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cassia roxburghii
Anthraquinones
Free radicals scavenging activity
The use of natural products with therapeutic properties is an
ancient as human civilization and for a long time. Recently, herbal
medicines have increasingly been used to treat many human dis-
eases.1 Natural antioxidants, especially phenolics and flavonoids
are safe; they protect the human body from free radicals and retard
the progress of many chronic diseases as well as lipid oxidative
rancidity in foods.2 Numerous studies were carried out on plants
with antioxidant properties.3,4 However, there is still great interest
in finding new antioxidants from natural sources.
The Cassia genus (Family: Fabaceae) represents one of the larg-
est and most diverse group of flowering plants, including herbs to
trees. Plants of the genus Cassia are widely distributed in most trop-
ical and subtropical countries. Cassia species have biological and
medical activities such as hepatoprotective, antibacterial, antioxi-
dant, antitumor, antidiabetic, and antiparasitic.5–11 Species of the
genus Cassia have been also used as laxative, purgative, antipyretic,
antiviral, antibacterial, antifungal, as well as anti-inflammatory
agents.12–14 Among those Cassia roxburghii is one of the medicinal
plants used in ethnomedicine for the treatment of various liver ail-
ments.15 Phytochemical analysis of certain Cassia species led to the
isolation of flavonoids, anthraquinones, proanthocyanidins, and
condensed tannins.16–18 Previous phytochemical investigation of
C. roxburghii seeds have been isolated of chrysophanol, physcion,
Plants have played a major role in the introduction of new ther-
apeutic agents. To the best of our knowledge, there is no scientific
literature on the constituents and antioxidant activity of C. rox-
burghii leaves. The present study deals with the isolation and
identification of anthraquinones glycosides from the leaves of
C. roxburghii and evaluation of its free radical scavenging activity.
The aqueous alcoholic extract MeOH–H2O (7:3) of C. roxburghii
leaves was subjected to extensive repeated column chromatogra-
phy on Polyamide 6 and Sephadex LH-20 resulted in isolation of
two new anthraquinone glycosides 1 and 2 (Fig. 1), together with
aloemodin 8-O-b-
D-glucopyranoside, emodin, aloemodin, and one
flavonoid, quercetin-3-O-
a-L-rhamnopyranoside.
Compound 1 was obtained as orange amorphous powder. It was
positive to the Bornträger reaction, revealing that it was a hydroxyl
anthraquinone compound.20 The UV spectrum gave the absorption
maxima at 221, 271, and 415 nm. Its molecular formula was estab-
lished as C27H30O15 from its HRESI-MS including a pseudomolecu-
lar ion at m/z 617.1487 [M+Na]+. The IR spectrum of the compound
1
showed characteristic absorption bands from OH (broad,
3442 cmꢀ1) and ,b-unsaturated ketone (1675 and 1625 cmꢀ1).
a
On acid hydrolysis compound 1 afforded emodin and glucose,
which identified by chromatography with authentic samples emo-
din and D
-glucose. The 1H NMR spectrum showed two sets of two
emodin, 1,3-dihydroxy-2-methyl anthraquinone 8-O-
a
-
L
-arabino-
meta coupled aromatic hydrogens at d 7.16 (d, J = 1.8 Hz, H-2) and
d 7.47 (d, J = 1.8 Hz, H-4); 6.91 (d, J = 2.1 Hz, H-5) and d 7.28 (d,
J = 2.1 Hz, H-7) suggesting two sets a tetrasubstituted aromatic
ring in addition to singlet for methyl group at d 2.4. Also, 1H
NMR spectrum revealed a singlet at d 8.54, assigned to a hydroxyl
group H-bonded to a carbonyl. The spectrum also showed two
pyranoside, physcion 8-O-
a-
L-xylopyranoside, and emodin O-
a-L-
arabinopyranoside.19
⇑
Corresponding author.
E-mail address: sayedeltomy@yahoo.com (S.A. El-Toumy).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.07.020

48
S. A. El-Toumy et al. / Carbohydrate Research 360 (2012) 47–51
HO
OH
OH
OH
O
HO
O
1``
HO
O
OH
1`
OH
O
O
13
14
12
2
7
11
HO
CH3
5
4
O
1
O
10
9
5
4
CH2OH
11
12
14
2
7
13
O
O
OH
1`
OH
OH
O
O
HO
1``
O
HO
OH
HO
OH
2
Figure 1. The structures of compounds 1 and 2.
distinct signals for anomeric protons, two hexose anomeric proton
resonances at d 5.30 (1 H, d, J = 7.28 Hz) and a proton resonance at
d 4.46 (1H, d, J = 7.28 Hz), indicating the presence of two sugars
tern of 1H–13C correlation was observed between d 5.30 (H-10) with
d 160.81 (C-1) and d 4.46 (H-100) in addition to d 83.07 (C-200) indi-
cating a glucosyl moiety is attached to C-1 and the link is glucose
(1?2). Based on the above data, compound 1 is deduced to be
with b-configuration. The b-
specific optical rotation [
D
-configuration was deduced from
a
]
D
+71.3 and confirmed by the acid
emodin 1-O-b-D-glucopyranosyl-(1?2)-glucopyranoside. To the
hydrolysis with the comparison of
D
-glucose. The DEPT spectrum
best of the authors’ knowledge, this is the first report of isolation
of this compound from any natural source.
of compound 1 revealed the presence one methyl at d 21.74, four
methane aromatic carbons. The downfield signals at (d 186.79
and d 182.55) were assigned to the two carbonyl carbons. Assign-
ments of the aglycone carbons were aided by comparison with the
reported values for emodin 1-O-b-glucoside.21 The application of
HMQC and HMBC experiments (Fig. 2) led to full assignments of
the 1H and 13C NMR chemical shifts of compound 1. From HMBC
spectrum, the signals at d 7.47 and 6.91 simultaneously had corre-
lation with the carbon at d 182.55 indicating that they were located
at H-4 and H-5 respectively. In the HMBC spectrum (Fig. 2) the pat-
Compound 2 was obtained as yellowish-orange powder, which
gave a positive Bornträger’s test for anthraquinone derivatives.20
The UV spectrum gave the absorption maxima at 219, 269, and
408 nm. Its molecular formula was established as C27H30O15 by
positive HR-ESI-MS at m/z 617.1495 [M+Na]+. The IR spectrum of
the compound 2 showed characteristic absorption bands from
OH (broad, 3410 cmꢀ1
) and a,b-unsaturated ketone (1645,
1638 cmꢀ1). On acid hydrolysis compound 2 afforded aloemodin
and glucose, which identified by chromatography with authentic

S. A. El-Toumy et al. / Carbohydrate Research 360 (2012) 47–51
49
HO
OH
OH
OH
O
HO
O
HO
O
OH
OH
O
O
HO
CH3
O
1
O
CH2OH
O
O
OH
OH
O
OH
O
HO
O
HO
OH
HO
OH
2
Figure 2. HMBC correlations H–C for 1, 2.
samples aloemodin and
D
-glucose. The 1H NMR spectrum showed
and d 182.74), five quaternary sp3 carbons, five methine aromatic
carbons, and one sp3 methylene group. The application of HMQC
and HMBC experiments (Fig. 2) led to full assignments of the 1H
and 13C NMR chemical shifts of compound 2. Both hydrogens at d
7.60 (H-4) and d 7.87 (H-5) showed long range correlations with
the carbon at d 182.74 establishing this ketone at C-10. The cross
peak in the HMQC between the hydrogens at d 4.60 (s, 2H) and
the carbon at d 64.4 suggested a hydroxymethyl group at C-3. The
HMBC and HMQC data confirmed the location of the hydroxy-
methyl group at C-3 through the observed correlations among H-
2 and C-1, C-3, and C-4. In the HMBC spectrum (Fig. 2) the pattern
two meta coupled aromatic hydrogens at d 7.24 (d, J = 1.2 Hz, H-
2) and d 7.60 (d, J = 1.2 Hz, H-4), suggesting a 1,3,5,6-tetrasubsti-
tuted aromatic ring. The presence of a 1,2,3-trisubstituted aromatic
ring was evident from the signals at d 7.83 (dd, J = 8.2, 1.2 Hz, H-7),
d 7.86 (t, J = 8.2, H-6) and d 7.87 (dd, J = 8.2, 1.2 Hz, H-5). The 1H
NMR spectra of compound 2 indicated the presence of a methylene
bearing a hydroxyl group at d 4.60 (2 H, s). The 1H NMR spectrum
also showed two distinct signals for anomeric protons, two hexose
anomeric proton resonance at d 5.10 (1 H, d, J = 7.28 Hz) and a pro-
ton resonance at d 4.22 (1 H, d, J = 7.28 Hz), indicating the presence
1
of two sugars with b-configuration. The b-
duced from specific optical rotation [ +48.4 and confirmed by
D
-configuration was de-
of H–13C correlation was observed between d 5.10 (H-10) with d
a
]
D
159.59 (C-8) and d 4.22 (H-100) with d 69.1 (C-600) indicating a gluco-
syl moiety is attached to C-8 and the link is glucose (1?6). Based on
the acid hydrolysis with the comparison of D-glucose.
The 13C NMR spectrum of compound 2 (Table 2) showed 15 car-
bon signals of the anthraquinone, two carbonyl carbons (d 187.87
the above data, compound 2 is deduced to be aloemodin 8-O-b-
D-
glucopyranosyl-(1?6)-glucopyranoside. To the best of the authors0

50
S. A. El-Toumy et al. / Carbohydrate Research 360 (2012) 47–51
Table 1
knowledge, this is the first report of isolation of this compound
from any natural source.
1H NMR (400 MHz) assignments [d (ppm), J in Hz] of compounds 1and 2
The other three known compounds (3–5) were identified as alo-
emodin 8-O-b-glucoside 3, emodin 4, aloemodin 5 and one flavo-
noid, quercetin-3-O-a-L-rhamnopyranoside by comparison of
Position
1
2
2
4
5
6
7.16 d (1.8)
7.47 d (1.8)
6.91 d (2.1)
7.24 d (1.2)
7.60 d (1.2)
7.87 dd, (8.2, 1.2)
7.86 t (8.2)
7.83 dd, (8.2, 1.2)
their 1D and 2D NMR spectral data with the reported data in the
literature.21–23
7
7.28 d (2.1)
2.4 s
The total alcoholic extract and pure compounds were tested for
their free radical scavenging activity on DPPH (2,2-di(phenyl)-
(2,4,6-trinitrophenyl)iminoazanium). The alcoholic extract and
3-CH3
3-CH2OH
10
100
4.60 s
5.30 d (7.2)
4.46 d (7.2)
3.72–3.26 m
5.11 d (7.28)
4.23 d (7.28)
3.70–3.19 m
quercetin-3-O-
dant scavenging activity toward DPPH, with IC50 values of
37.96 0.42 and 29.49 0.98 g/mL, respectively, while the two
a-L-rhamnopyranoside exhibited potent antioxi-
The rest sugar protons
l
new compounds exhibited very weak antioxidant scavenging
activity against DPPH, with IC50 values of 138.01 2.82 and
132.01 2.82 lg/mL and the remaining compounds showed mod-
Table 2
13C NMR data of compounds 1 and 2 in DMSO-d6 at 100 MHz
erate activity (Table 3). Ascorbic acid was used as a positive
Atom
1
2
control.
1
2
3
4
5
6
7
8
160.81
124.61
147.18
119.56
108.35
162.21
107.39
164.65
186.79
182.55
136.86
113.08
114.76
132.36
21.75
160.06
12159
1. Experimental
152.53
116.11
121.01
136.66
123.56
158.59
187.87
182.74
132.85
116.11
121.18
135.20
62.47 (3-CH2OH)
1.1. General methods
1H NMR and 13C NMR spectra were obtained on Bruker AMX-
400, Avance 400, and Avance 300 spectrometers with standard
pulse sequences operating at 400, 300 MHz in 1H NMR and 100,
75 MHz in 13C NMR. Chemical shifts are given in d values (ppm)
using tetramethylsilane as the internal standard. IR spectra were
recorded with Nexus 670 FT-IR FT-Ramen spectrometer as potas-
sium bromide disks. UV spectra were recorded with Schimadzu
UV-1601. HRESI-MS was taken on a Micromass Autospec (70 eV)
spectrometer. Optical rotation values were measured by the use
of an ATAGO POLAX-D polarimeter. Column chromatography (CC)
was carried out on Polyamid 6S and Sephadex LH20.
9
10
11
12
13
14
3-CH3
Glucosyl
10
98.58
83.07
75.62
69.38
79.53
60.69
101.07
74.10
76.96
70.60
77.14
69.10
20
30
40
50
60
1.2. Plant materials
Glucosyl
100
200
300
400
500
600
104.70
75.48
76.85
69.24
77.50
60.03
103.96
73.79
76.74
70.30
77.32
61.52
Leaves of C. roxburghii were collected in April 2007 from the Or-
man Garden. Identification of the plants was confirmed by the
Department of Flora Agricultural Museum, Ministry of Agriculture
and Herbarium of the Department of Botany, Faculty of Science,
Cairo University. Voucher Specimens were kept in herbarium, Na-
tional Research Centre, El-Tahrir Str., Dokki, Cairo, Egypt.
vonoid a quercetin-3-O-a-L-rhamnopyranoside. Using the same
procedures fractions 4 and 5 gave chromatographically pure sam-
1.3. Extraction and isolation
ples emodin 4 and aloemodin 5.
The leaves of C. roxburghii (1.25 g) were crushed and extracted
with the aqueous alcoholic (MeOH–H2O, 7:3) by soaking at room
temperature and the methanol extract was evaporated under re-
duced (diminished) pressure then defatted using successive
extraction by pet ether then chloroform; the residue was extracted
with n-BuOH, affording a dry extract (40 g), which was purified by
chromatography on Polyamide 6 CC. The column was eluted with
H2O and with H2O–EtOH step gradient and 4 fractions (1 L, each)
were collected. Separation of fraction 2 on Sephadex LH-20 CC
using n-BuOH saturated with H2O gave three subfractions I, II,
and III, further purification of II on Sephadex CC using MeOH–
H2O (3:7) afforded the new compound 1 (18 mg), while subfrac-
tions I and III have been separated on paper chromatography then
purified on Sephadex CC using MeOH–H2O (1:1) as eluent, gave
aloemodin 8-O-b-glucoside 3. Consecutive CC on Sephadex (n-
BuOH satd. with H2O for elution) of fraction 3 gave two subfrac-
tions, then one major of them has been applied on TLC Silica Gel
plates using EtOAc–methanol–H2O (100:13.5:10) to afford the
new compound 2 (20 mg). Separation of fraction 1 on preparative
paper chromatography using n-BuOH–HOAc–H2O (4:1:5) gave fla-
1.4. Acid hydrolysis of compounds 1 and 2
A solution of 5 mg of 1 in 10 mL MeOH–H2O (1:1) mixed with
5 mL 2 N HCl was heated at reflux at 60 °C for 6 h. After evapora-
tion of the organic solvent under vacuum and threefold extraction
of the aqueous phase with ethyl acetate, the aglycone was identi-
fied as emodin by NMR and by comparison with an authentic sam-
ple. The neutralized aqueous portion resulted in TLC detection of
glucose by comparison with standard sugar (TLC: n-BuOH–
EtOAc-i-PrOH–HOAc–H2O, 7:20:12:7:6 aniline phthalate spray re-
agent). Using the same procedure compound 2 gave aloemodin and
glucose, which identified by comparison with authentic sample.
1.5. Identification
1.5.1. Emodin 1-O-b-D-glucopyranosyl-(1?2)-glucopyranoside
(1)
Orange amorphous powder; Rf = 0.66 and 0.70 (cellulose paper)
in n-BuOH–HOAc–H2O (upper layer, 4:1:5) and 15% HOAc, respec-

S. A. El-Toumy et al. / Carbohydrate Research 360 (2012) 47–51
51
Table 3
DPPH radical scavenging activity of the total extract and compounds
a
Compound
DPPH free radical scavenging activity IC50 (lg/mL)
Alcoholic extract
Emodin 1-O-b-D-glucoside (2?1)glucopyranoside
Aloemodin 8-O-b-
Aloemodin 8-O-b-
Emodin
Aloemodin
Quercetin-3-O-a-L-rhamnopyranoside
Ascorbic acid
37.96 0.42
138.01 2.82
132.01 2.82
96.32 0.32
71.65 0.42
86.42 0.92
29.49 0.98
7.90 0.21
D
-glucoside (6?1)glucopyranoside
D-glucopyranodide
a
Values of IC50 were calculated as mean of triplicate determination standard deviation, concentrations in
required scavenging the DPPH radical (100 g/mL) by 50%.
lg/mL
l
tively; [a] +71.3 (c, 0.1, MeOH); UV kmax nm: 221, 271, 282, 415,
D
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