Anthraquinones and stilbenes from the roots and rhizomes of Rhubarb

Article abstract of DOI:10.1080/10286020.2011.613828

Two new anthraquinone glucosides [aloe-emodin 8-O-β-D-(6′- galloyl)glucopyranoside (1) and rhein 8-O-β-D-(6′-galloyl) glucopyranoside (2)], together with 16 known compounds (3-18), were isolated from the roots and rhizomes of Rheum hotaoense C.Y. Cheng et C.T. Kao. Their structures were elucidated on the basis of extensive investigation of 1D and 2D NMR, HR-ESI-MS, and chemical evidence. In addition, the free-radical-scavenging activity was tested using the 1,1-diphenyl-2-picrylhydrazyl assay.

Full text of DOI:10.1080/10286020.2011.613828

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Anthraquinones and stilbenes from the
roots and rhizomes of Rhubarb
Wen-Hao Chen a , Juan Chen a & Yan-Ping Shi a
a Key Laboratory of Chemistry of Northwestern Plant Resources
and Key Laboratory for Natural Medicine of Gansu Province,
Lanzhou Institute of Chemical Physics, Chinese Academy of
Sciences , Lanzhou, 730000, China
Published online: 10 Oct 2011.
To cite this article: Wen-Hao Chen , Juan Chen & Yan-Ping Shi (2011) Anthraquinones and stilbenes
from the roots and rhizomes of Rhubarb, Journal of Asian Natural Products Research, 13:11,
1036-1041, DOI: 10.1080/10286020.2011.613828
To link to this article: http://dx.doi.org/10.1080/10286020.2011.613828
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Journal of Asian Natural Products Research
Vol. 13, No. 11, October 2011, 1036–1041
Anthraquinones and stilbenes from the roots and rhizomes of Rhubarb
Wen-Hao Chen, Juan Chen and Yan-Ping Shi*
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural
Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences,
Lanzhou 730000, China
(Received 3 June 2011; final version received 9 August 2011)
Two new anthraquinone glucosides [aloe-emodin 8-O-b-D-(60-galloyl)glucopyrano-
side (1) and rhein 8-O-b-D-(60-galloyl)glucopyranoside (2)], together with 16 known
compounds (3–18), were isolated from the roots and rhizomes of Rheum hotaoense
C.Y. Cheng et C.T. Kao. Their structures were elucidated on the basis of extensive
investigation of 1D and 2D NMR, HR-ESI-MS, and chemical evidence. In addition, the
free-radical-scavenging activity was tested using the 1,1-diphenyl-2-picrylhydrazyl
assay.
Keywords: Rheum hotaoense; rhubarb; anthraquinones; stilbenes; DPPH assay
1. Introduction
exhibit potent antioxidant [3] and anti-
allergic activities [4].
Rhubarb is an important herbal medicine
for the treatment of blood stagnation
syndrome and is used as a purgative agent
in Chinese, Japanese, and Korean tra-
ditional medicines. In the Chinese Pharma-
copoeia, rhubarb consists of the roots and
rhizomes of Rheum officinale Baill., Rheum
palmatum L., and Rheum tanguticum
Maxim. ex Balf., all of which belong to
Sect. Palmata [1]. Aside from Sect.
Palmata, species from Sect. Rheum are
also used for rhubarb drugs in local areas of
China. These unofficial species include
Rheum hotaoense C.Y. Cheng et C.T. Kao,
Rheum franzenbachii Munt., and Rheum
emodi Wall. They exhibit much weaker
purgative activities than official species
due to the absence of sennosides and due to
the relatively low content of anthraqui-
nones [2]. Stilbenes, which are the major
constituents of unofficial rhubarbs, were
also different among the species, and
The chemical profiles of six rhubarb
species (including official and unofficial)
were analyzed by Ye et al. [5] using the
HPLC/DAD/ESI-MSn method, and it was
found that their phenolic patterns showed
significant difference, and the precise
structures have to be determined further.
In this study, the roots and rhizomes of R.
hotaoense C.Y. Cheng et C.T. Kao were
examined chemically leading to the iso-
lation and structural elucidation of two new
anthraquinone glucosides (1 and 2)
together with 16 known compounds (3–
18; Figure 1). Furthermore, the free-
radical-scavenging activity on 1-diphenyl-
2-picrylhydrazyl (DPPH) of the crude
fractions and compounds 1–3 was also
described.
2. Results and discussion
Compound 1 was obtained as a yellow
amorphous powder, and its molecular
*Corresponding author. Email: shiyp@licp.cas.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.613828
http://www.tandfonline.com

Journal of Asian Natural Products Research
1037
O
5
4
10a
8a
OH
O
O
OR
4a
9a
OH
OGlc
O
R
3
2
6
7
10
9
HO
3''
O
C
6'
1''
8
1
O
HO
CH3
O
OH
R
O
O
1'
OH
O
HO
OH
1 R = CH2OH
2 R = COOH
3 R = CH3
4 R = CH2OH
5 R = CH3
6 R = H
OH
OH
7 R = Glc
OH
O
O
OH
O
O
OH
HO
HO
R
R
R
CH3
HO
8 R = CH2OH
9 R = COOH
10 R = OCH3
11 R = OH
12 R = H
13 R = COOH
RO
HO
RO
HO
OH
OGlc
OH
OCH3
OCH3
HO
HO
17 R = H
18 R = Glc
15 R = H
16 R = Glc
14
Figure 1. Chemical structures of compounds 1–18.
formula of C28H24O14 was determined
from the negative-ion HR-ESI-MS (m/z
583.1084 [M 2 H]2). The IR spectrum
showed absorption bands ascribable to
hydroxyl, free and chelated carbonyl, and
aromatic functions at 3465, 1692, 1628,
1469, and 1081 cm21. The UV spectrum of
1 exhibited absorption maxima at 218,
256, 280, and 410 nm, indicating the
presence of an anthraquinone system [3].
The 1H NMR spectrum showed signals
due to oxygenated methylene protons [dH
4.60 (2H, s, 3-CH2OH)], an anomeric
proton [dH 5.25 (1H, d, J ¼ 7.6 Hz, Glc-10-
H)], a galloyl group [dH 6.98 (2H, s,
galloyl-200, 600-H)], two aromatic protons
[dH 7.25, 7.62 (each 1H, both s, 2-H, 4-H)],
a 1,2,3-trisubstituted benzene ring [dH 7.69
(1H, d, J ¼ 7.6 Hz, 7-H); 7.70 (1H, dd,
J ¼ 7.2, 7.6 Hz, 6-H); 7.82 (1H, d,
J ¼ 7.2 Hz, 5-H)], and a chelated hydroxyl
proton [dH 12.86 (1H, br s, 1-OH)]. These
data together with the 13C NMR spectral
data (Table 1) for 1 were similar to those
of chrysophanol 8-o-b-D-(60-galloyl)glu-
copyranoside (3), previously isolated from
a Korean rhubarb Rheum undulatum L. [3].
The major differences between the two
compounds were that the methyl group in
3 was replaced by the oxygenated
methylene resonances (dH 4.60, dC 62.1)
in 1. This was further confirmed by the
HMBC correlations from the oxygenated
methylene at dH 4.60 to C-2 (dC 120.8),
C-3 (dC 152.3), and C-4 (dC 116.1). The
b-glucopyranosyl unit could be located at
C-8 on the basis of the HMBC correlation
observed between the anomeric proton
signal at dH 5.25 and the carbon resonance
at dC 158.0 (C-8). The negative ESI-MS
showed significant fragment ion at m/z 431
[M 2 C7H5O4]2, ascribable to the loss of
the galloyl unit. Furthermore, the linkage
of the galloyl to the glucopyranosyl unit
was established at C-60 by the downfield
shift of C-60 (dC 63.4) and H-60 (dH 4.28,
4.50), as well as the HMBC correlation
between H-60 and C-700 (dC 165.7)

1038
W.-H. Chen et al.
Table 1. 1H and 13C NMR spectral data of 1 and 2 (d values, in DMSO-d6)a.
1
2
No.
dH (mult, J in Hz)
dC
dH (mult, J in Hz)
dC
1
2
3
4
5
6
7
8
9
10
4a
8a
9a
10a
161.7 s
120.8 db
152.3 s
116.1 d
120.7 db
135.8 d
122.3 d
158.0 s
187.6 s
182.1 s
132.3 s
120.7 s
115.5 s
134.9 s
161.3 s
124.0 d
140.5 s
118.5 d
120.7 d
136.0 d
122.3 d
158.1 s
187.6 s
181.8 s
132.6 s
119.4 s
118.9 s
134.8 s
7.25 (s)
7.74 (s)
7.62 (s)
8.12 (s)
7.82 (d, 7.2)
7.70 (dd, 7.2, 7.6)
7.69 (d, 7.6)
7.84 (d, 7.2)
7.74 (dd, 7.2, 8.4)
7.68 (d, 8.4)
1-OH
3-CH2OH
12.86 (br s)
4.60 (s)
12.73 (br s)
62.1 t
3-COOH
10
166.5 s
100.3 d
73.3 d
76.5 d
69.8 d
74.1 d
63.5 t
119.4 s
108.8 d
145.7 s
138.7 s
165.8 s
5.25 (d, 7.6)
3.55 (m)
3.42 (m)
3.40 (m)
3.85 (m)
4.50 (m), 4.28 (m)
100.3 d
73.3 d
76.5 d
69.8 d
74.1 d
63.4 t
119.4 s
108.8 d
145.6 s
138.6 s
165.7 s
5.26 (d, 7.6)
3.54 (m)
3.42 (m)
3.40 (m)
3.85 (m)
4.49 (m), 4.24 (m)
20
30
40
500
600
100
2 , 600
300, 500
400
6.98 (s)
6.97 (s)
700
a Assignments were based on DEPT135, HSQC, and HMBC experiments.
b Assignments (within the same column) may be interchangeable.
(Figure 2). Alkaline hydrolysis of 1 with
1.0% NaOMe/MeOH furnished 4, which
was identified by thin layer chromoto-
graphy (TLC). Further detailed analysis
of the 1H, HSQC, and HMBC NMR
spectroscopic data allowed unambiguous
assignments for all of the 1H and 13C NMR
signals of 1. Thus, the structure of 1 was
elucidated as aloe-emodin 8-o-b-D-(60-
galloyl)glucopyranoside.
Compound 2 was also obtained as
a yellow amorphous powder, and its
O
O
COOH
CH2OH
HO
HO
HO
HO
O
O
C
O
HO
C O
O
OH
O
OH
O
O
O
O
OH
OH
OH
OH
HO
OH
OH
2
1
Figure 2. Key HMBC (H ! C) correlations of 1 and 2.

Journal of Asian Natural Products Research
1039
Table 2. DPPH radical scavenging activities of fractions and compounds 1–3.
Sample
SC50 (mg/ml)
Sample
SC50 (mM)
EtOAc-soluble fraction
n-BuOH-soluble fraction
30% EtOH-eluted fraction
70% EtOH-eluted fraction
8.8
15.5
6.9
1
2
3
13.6
12.7
11.1
9.3
14.7
Quercetin
molecular formula of C28H22O15 was
determined from the negative-ion HR-
ESI-MS (m/z 597.0874 [M 2 H]2). The
1H and 13C NMR (Table 1) spectra closely
resembled those of 1, except for the
presence of the signal for an additional
carboxyl group (dC 166.5, s), and the
absence of the oxygenated methylene
resonances at C-3 as found in 1, suggesting
that the carboxyl group is linked to C-3 in 2.
This was further confirmed by the HMBC
correlations from H-2 and H-4 to the
carbonyl group (dC 166.5) (Figure 2). Thus,
the structure of 2 was elucidated as rhein 8-
o-b-D-(60-galloyl)glucopyranoside.
The known compounds (3–18) were
determined to be chrysophanol 8-o-b-D-
(60-galloyl)glucopyranoside (3) [3], aloe-
emodin 8-o-b-D-glucopyranoside (4) [6],
chrysophanol 8-o-b-D-glucopyranoside
(5) [7,8], chrysophanol (6) [6,7], chryso-
phanol 1-o-b-D-glucopyranoside (7) [8],
aloe-emodin (8) [6], rhein (9) [6], physcion
(10) [7], emodin (11) [6,7], pyrogallic acid
(12), gallic acid (13) [9], piceatannol 30-o-
b-D-glucopyranoside (14) [7], desoxyrha-
pontigenin (15) [7], desoxyrhaponticin
(16) [7], rhapontigenin (17) [7], and
rhaponticin (18) [7], respectively. Com-
pounds 5 and 7 were isolated as an
approximately 1:1 mixture of isomers,
which was different from the ratio of 5:3
(or 3:5) reported in the literature [10].
The DPPH radical scavenging activity
of the EtOAc- and n-BuOH-soluble, 30%
and 70% EtOH-eluted fractions and com-
pounds 1–3 were examined. The 30%
EtOH-eluted fraction showed the most
potent scavenging activity (SC50
6.9 mg/ml) compared with all the other
fractions. As shown in Table 2, compounds
1 and 2 were slightly less potent than the
standard quercetin. In agreement with
previous study, compound 3 showed potent
radical scavenging activity. These results
confirmed that the galloyl moiety enhanced
the activity [3].
3. Experimental
3.1 General experimental procedures
UV and IR spectra were recorded on a
Shimadzu UV-240 spectrophotometer and a
Nicolet NEXUS-670 FT-IR spectropho-
tometer, respectively. Optical rotations
were recorded on a Perkin-Elmer 341
polarimeter with a 1 dm cell. NMR spectra
were recorded on a Bruker AVANCE III-
400 spectrometer with TMS as an internal
reference (d). ESI-MS and HR-ESI-MS
were measured on a Watersw ACQUITYTM
TQ Detector and a Bruker APEX-II mass
spectrometer, respectively. D101 macro-
porous adsorptive resins (granularity: 0.30–
1.25mm) were supplied by the Cangzhou
Baoen Chemical Industry Co., Ltd (Cangz-
hou, China). DPPH was obtained from
Sigma-Aldrich Chemical Co. (St Louis,
MO, USA). Silica gel (200–300 mesh) used
for column chromatography (CC) and silica
gel GF254 (10–40mm) for TLC were both
supplied by the Qingdao Marine Chemical
Factory (Qingdao, China). TLC was
detected at 254 nm and spots were visual-
ized by spraying with 5% H2SO4 in
C2H5OH (v/v) followed by heating.
3.2 Plant material
The roots and rhizomes of R. hotaoense
C.Y. Cheng et C.T. Kao were purchased in

1040
W.-H. Chen et al.
July 2009 from Huanghe Medicinal
Material Market in Gansu, China, and
were identified by adjunct Professor Huan-
Yang Qi of Key Laboratory of Chemistry
of Northwestern Plant Resources and Key
Laboratory for Natural Medicine of Gansu
Province, Lanzhou Institute of Chemical
Physics, where a voucher specimen
(ZY2010DH01) is deposited.
CHCl3ZMeOH as solvent, to afford five
fractions [Fr1 (10:1, 18 g), Fr2 (5:1, 45 g),
Fr3 (3:1, 36 g), Fr4 (2:1, 26 g), and Fr5
(1:1, 128 g)] and 18 (12.5 g). Fraction Fr2
was chromatographed on silica gel with a
CHCl3ZMeOH gradient system to give
three subfractions. The subfractions 2
(8.6 g) and 3 (15.5 g) were further separ-
ated by repeated chromatography over
silica gel (CHCl3ZMeOH, 4:1) which
afforded 14 (27 mg) and 4 (36 mg),
respectively. Fraction Fr3 was subjected
to silica gel CC with CHCl3ZMeOH (3:1)
as eluant followed by recrystallization to
yield 1 (25 mg). Fraction Fr4 was subjected
to silica gel CC with CHCl3ZMeOH (2:1)
as eluant followed by recrystallization to
yield 2 (31 mg). Likewise, a similar
isolation procedure adopted for the 70%
EtOH fraction (180 g) afforded six com-
pounds in the following order: 8 (10 mg),
17 (14 mg), 5 and 7 as a mixture (1:1,
2.7 g), 16 (1.2 g), and 3 (31 mg).
3.3 Extraction and isolation
The air-dried powders of roots and
rhizomes of R. hotaoense C.Y. Cheng et
C.T. Kao (4.5 kg) were extracted with 95%
EtOH (3 £ 2 h) at 608C. The crude extract
was concentrated in vacuo to afford a dark
brown residue, which was suspended in
H2O and fractionated by successive parti-
tioning with EtOAc and n-BuOH. The
n-BuOH-soluble fraction was dissolved in
H2O and subjected to CC over D101 and
eluted with H2O and 30% and 70% EtOH,
successively. The EtOAc-soluble fraction
(220 g) was subjected to silica gel CC,
using a step gradient-elution technique,
employing mixtures of petroleum ether–
acetone as solvent, to afford six fractions
[Fre 1 (20:1, 10 g), Fre 2 (10:1, 15 g), Fre 3
(5:1, 36 g), Fre 4 (3:1, 45 g), Fre 5 (2:1,
32 g), and Fre 6 (1:1, 20 g)] according to
TLC analysis. Fraction Fre 2 was chro-
matographed on silica gel with petroleum
ether–EtOAc gradient system to give three
subfractions (Fre 2.1–Fre 2.3). Further
purification of subfraction Fre 2.1 (2.4 g)
through repeated chromatography with
petroleum ether–acetone (8:1) as eluant
over silica gel yielded 6 (100 mg). Fre 2.2
(5.2 g) was subjected to silica gel CC with
petroleum ether–EtOAc (5:1) as eluant
followed by recrystallization to yield 11
(25 mg), 8 (100 mg), and 10 (10 mg),
respectively. Fre 3, Fre 4, and Fre 5 were
separated using the same procedure as Fre
2 to afford 15 (13 mg), 12 (22 mg), 17 (1 g),
9 (20 mg), and 13 (15 mg), respectively.
The 30% EtOH fraction (300 g)
was subjected to silica gel CC with
3.3.1 Aloe-emodin 8-o-b-D-(60-
galloyl)glucopyranoside (1)
20
A yellow amorphous powder. ½aꢀD þ 23 (c
1.0, MeOH). UV (MeOH)/nm: lmax (log 1)
218 (1.23), 256 (0.66), 280 (0.47), and 410
(0.18). IR (KBr) nmax: 3465, 1692, 1628,
1469, 1276, and 1081 cm21. 1H (400 MHz)
and 13C NMR (100 MHz) spectral data, see
Table 1. ESI-MS m/z: 583 [M 2 H]2, 431
[M 2 C7H5O4 (galloyl)]2. HR-ESI-MS
m/z: 583.1084 [M 2 H]2 (calcd for
C28H23O14, 583.1094).
3.3.2 Rhein 8-o-b-D-(60-
galloyl)glucopyranoside (2)
20
A yellow amorphous powder. ½aꢀD þ 10 (c
1.1, MeOH). UV (MeOH)/nm: lmax (log 1)
220 (0.59), 258 (0.32), 280 (0.21), and 410
(0.10). IR (KBr) nmax: 3471, 1700, 1631,
1359, 1270, and 1074 cm21. 1H (400 MHz)
and 13C NMR (100 MHz) spectral data, see
Table 1. ESI-MS m/z: 597 [M 2 H]2, 445
[M 2 C7H5O4 (galloyl)]2. HR-ESI-MS

Journal of Asian Natural Products Research
m/z: 597.0874 [M 2 H]2 (calcd for
Acknowledgements
C28H21O15, 597.0886).
1041
This work was financially supported by the
National Key Technology Research and
Development Program of China (No.
2007BAI37B05), the Western High-Tech
Project Action Plan of Chinese Academy of
Sciences (No. KG-CX2-YW-510), and the
National Natural Science Foundation of China
(No. 21075127).
3.4 Alkaline hydrolysis of 1
A solution of 1 (2 mg) in 1.0% NaOMeZ
MeOH (1.0 ml) was stirred at room
temperature for 1 h. The reaction mixture
was neutralized with CH3COOH. Com-
pound 4 was detected in the reaction
mixtures by co-TLC comparison with the
authentic sample (CHCl3–MeOH–H2O,
12:6:1; Rf ¼ 0.45).
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3.5 Scavenging activity of DPPH
radicals
The DPPH assay was carried out according
to the modified method of Chen et al. [11]:
aliquots of 30 ml of an ethanol solution
containing each sample were added to 3 ml
of 100 mM ethanol solution of DPPH.
Absorbance at 517 nm was determined
after 30 min at room temperature, and the
percent antioxidant activity (AA) was
calculated using the following formula:
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Thuong, I. Lee, B.S. Min, and K. Bae,
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AA% ¼ 100 2 {½ðAbssample 2 Absblank
£ 100ꢀ=Abscontrol}:
Þ
Ethanol (3 ml) was used as a blank.
100 mM of DPPH ethanol solution (3 ml)
was used as a negative control. The SC50
values denote the concentration of test
sample required to scavenge 50% DPPH
free radicals. Quercetin was used as a
positive control.