Preparation of novel antiproliferative emodin derivatives and studies on their cell cycle arrest, caspase dependent apoptosis and DNA binding interaction

Article abstract of DOI:10.1016/j.phymed.2013.03.015

Emodin (1) is the major bioactive compound of several herb species, which belongs to anthraquinone class of compound. As a part of our drug discovery program, large quantities of emodin (1) was isolated from the roots of Rheum emodi and a library of novel

Full text of DOI:10.1016/j.phymed.2013.03.015

Phytomedicine 20 (2013) 890–896
Contents lists available at SciVerse ScienceDirect
Phytomedicine
journal homepage: www.elsevier.de/phymed
Preparation of novel antiproliferative emodin derivatives and studies on their cell
cycle arrest, caspase dependent apoptosis and DNA binding interactionଝ
T. Narendera,∗, P. Sukanyaa, Komal Sharmab, Surendar Reddy Bathulab,∗
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226 001, UP, India
b Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226 001, UP, India
a r t i c l e i n f o
a b s t r a c t
Keywords:
Emodin (1) is the major bioactive compound of several herb species, which belongs to anthraquinone
class of compound. As a part of our drug discovery program, large quantities of emodin (1) was isolated
from the roots of Rheum emodi and a library of novel emodin derivatives 2–15 were prepared to evaluate
their antiproliferative activities against HepG2, MDA-MB-231 and NIH/3T3 cells lines. The derivatives
3 and 12 strongly inhibited the proliferation of HepG2 and MDA-MB-231 cancer cell line with an IC50
of 5.6, 13.03 and 10.44, 5.027, respectively, which is comparable to marketed drug epirubicin (III). The
compounds 3 and 12 were also capable of inducing cell cycle arrest and caspase dependent apoptosis
in HepG2 cell lines and exhibit DNA intercalating activity. These emodin derivatives hold promise for
developing safer alternatives to the marketed epirubicin.
Emodin derivatives
Rheum emodi
Anticancer agents
Anthraquinones
Apoptosis
Caspase-3
Cell cycle
© 2013 Elsevier GmbH. All rights reserved.
Introduction
cytotoxicity via Rad51 down regulation and ERK1/2 inactivation in
human breast cancer BCap-37 cells (Chen et al. 2009).
Anthraquinones are widely distributed secondary metabolites
of plants (aloe, cascara sagrada, senna and rhubarb), microbes,
lichens and insects, which possess various biological activities
(Zaprometnov 1993; Gorelik 1983; Fain 1999). Anthraquinones
isolated from the microbes such as daunorubicin (I), doxorubicin
(II), epirubicin (III) and several synthetic analogs such as mitox-
antrone (IV) and pixantrone (V) are in clinical use for the treatment
of various cancers (Fig. 1). Emodin (1) belongs to anthraquinone
class of compound is the major bioactive compounds of numerous
herb species such as Rheum, Polygonum (polygonaceae), Rhamnus
(Rhamnaceae) and Cassieae (senna) (Alves et al. 2004; Liang et al.
1993; Yang et al. 1999) and possesses immunosuppressive, anti-
cancer, anti-inflammatory, anti-atherosclerotic, and vasorelaxant
effects (Kuo et al. 2001; Huang et al. 1991; Zhou and Chen 1998;
Koyama et al. 1988). Several reports have shown that emodin (1)
has antiproliferative effects on many kinds of cancer cell lines such
as HER-2/neu over expressing breast cancer (Zhang et al. 1998,
1999), leukemia (Chun et al. 2010; Chen et al. 2002), hepatoma
(Shieh et al. 2004) and lung (Su et al. 2005) cancer cells. Also,
emodin-triggered apoptosis is mediated through the caspase and
mitochondria dependent pathways in proximal tubular epithelial
HK-2 cells (Wang et al. 2007). Emodin enhanced gefitinib induced
As a part of our drug discovery program on anticancer agents
from Indian medicinal plants, we isolated large quantities of
emodin (1) from the roots of Rheum emodi and planned to carry
out chemical transformation to improve its therapeutic applica-
tion. Chemical transformation of bioactive compounds of medicinal
herbs is one of the most common approaches in drug discovery
to improve the therapeutic properties. For example the anticancer
drugs teniposide and etoposide are derivatives of podophyllotoxin
and topotecan and irinotecan are analogs of camptothecin, which
have better therapeutic benefits than the parent natural products.
Materials and methods
General chemistry
IR spectra were recorded on Perkin-Elmer RX-1 spectrometer.
Using either KBr pellets (or) in neat. 1H NMR, 13C NMR, DEPT-90 and
DEPT-135 spectra were run on Bruker Advance DPX 300 MHz and
200 MHz in CDCl3.Chemical shifts are reported as values in ppm
relative to CHCl3 (7.26) in CDCl3 and TMS was used as internal
standard. ESI mass spectra were recorded on JEOL SX 102/DA-6000.
Chromatography was executed with silica gel (60–120 mesh) using
mixtures of chloroform, ethyl acetate and hexane as eluants.
Background of plant
ଝ
This is CDRI communication No. 8449.
Corresponding authors. Tel.: +91 522 2612411; fax: +91 522 2623405.
E-mail addresses: t narendra@cdri.res.in, tnarender@rediffmail.com
∗
R. emodi wall. (Family: Polygonaceae, commonly known as
(T. Narender), bsreddy@cdri.res.in (S.R. Bathula).
revand-chini and English name rhubarb) is a stout herb, distributed
0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.phymed.2013.03.015

T. Narender et al. / Phytomedicine 20 (2013) 890–896
891
and evaporated under reduced pressure. Then the crude product
was chromatographed on silica gel to afford the desired compound.
General method for esterification and acid formation (method C)
Compound 1 (100 mg, 0.00037 moles) magnetically stirred in a
solution of pyridine (2 ml) and acetic anhydride (0.00185 moles)
at 60–70 ◦C for 4 h. The reaction mixture was put into cold water
for crystallization, then filtered and dried. To the resultant crude
acetate was gradually added 10 ml of acetic anhydride and glacial
acetic acid mixture (1:1) and CrO3 at 45 ◦C and stirred for 10 h at
70 ◦C. Acetic anhydride and glacial acetic acid mixture was removed
by vacuum. It was then extracted with ethyl acetate (3 × 25 ml), the
organic layer was washed with water, dried over anhyd Na2SO4 and
evaporated under reduced pressure. Then the crude product was
chromatographed on silica gel to afford the desired compound.
Fig. 1. Anticancer drugs with anthraquinone skeleton: daunorubicin (I), doxoru-
bicin (II), epirubicin (III) mitoxantrone (IV), and pixantrone (V).
in the alpine and sub-alpine zones of the Himalayas (Agarwal et al.
2000; Singh et al. 2005). The roots of this species are used widely in
ayurvedic medicine. Roots of the Indian rhubarb is darker, inferior
in aroma, is a well known stomachic, bitter, cathartic and used all
over the world.
Biological study
Materials
MDA-MB-231, HepG2, and NIH/3T3 cell lines used in the present
study was obtained from ATCC (American Type Culture Collec-
tion, USA). Annexin V FITC Apoptosis Detection Kit, MTT dye
and Caspase-3 Activation Kit was procured from Sigma–Aldrich.
Absorbance was recorded using Eliza Plate Reader. Flow cytome-
try was performed using a FACScan (Becton Dickinson, Mountain
View, CA) flowcytometer.
Collection of medicinal plant
R. emodi wall. (Bark) was collected from India and the authen-
tification was done by Botany Division of Central Drug Research
Institute, Lucknow and kept in the herbarium for future reference.
Cell culture
Extraction
MDA-MB-231 cells were routinely maintained in Dulbecco’s
Modified Eagle Medium DMEM (Sigma–Aldrich) while HepG2,
NIH/3T3 cells were maintained in Roswell Park Memorial Insti-
tute (RPMI 1640, Merck) supplemented with 10% FBS (Merck) and
1% Antibiotic-Antimycotic Solution (Merck) at 37 ◦C in a humidi-
fied incubator with 5% CO2. All stock solution of solutions of the
compound were prepared in cell culture grade DMSO and stored in
−20 ◦C. Compounds were diluted in culture media prior to use in
experiments.
Powdered R. emodi wall. (Bark) (3 kg) were placed in glass per-
colator with 95% ethanol (10.l) and allowed to stand for 24 h at
room temperature. The percolate was collected and these processes
were repeated for four times. The combined percolate was evapo-
rated under reduced pressure at 50 ◦C to afford ethanol extract. The
weight of extract was found to be 200 g.
Isolation and purification of anthraquinone
Cell viability assay
The alcoholic extract was (200 g) chromatographed on a column
of silica gel (60–120 mesh), eluted with Hexane and chloroform
(70:30); recrystallization from methanol afford emodin (3 g). The
compound visualization was obtained under UV light, also shown
orange spot by spraying with 10% sulphuric acid in methanol.
Antiproliferative activity of the compounds was tested by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT) assay. Briefly, cells (4 × 103 cells/well) were cultured
in 96 well tissue culture plates and treated with different concen-
trations of compounds for 48 h. At the end of incubations, 10 ␮l
of MTT (10 mg/ml) was added to the wells and incubated for 3 h.
Absorbance was recorded at 540 nm using Eliza Plate Reader. All the
experiments were repeated at least thrice independently. Values
are expressed as mean SEM.
Preparation of emodin derivatives
General method for O-alkylation (method A)
To
a magnetically stirred solution of compound (100 mg,
0.00037 moles) in DMF (5 ml) at room temperature was treated
with the respective aliphatic halo long chain (RX) (0.00033 moles)
and K2CO3 (0.00092 moles). The whole reaction mixture was
stirred for 4 h at 60–70 ◦C. It was then extracted with ethyl acetate
(3 × 25 ml), the organic layer was washed with water, dried over
anhyd Na2SO4 and evaporated under reduced pressure. Then the
crude product was chromatographed on silica gel to afford the
desired compound.
Apoptosis studies
Quantitation of apoptotic cells by Annexin V staining was car-
ried out according to the manufacturer’s instructions (Calbiochem).
Briefly, HepG2 cells (5 × 105 cells/well) were seeded in 6 well
plates and treated with 5, 10 and 20 ␮M of compound 3 and 12
and staurosporine (1 ␮g/ml) as positive control for 24 h. After the
incubations, cells prepared as a suspension in 500 ␮l of cold PBS,
centrifuged for 5 min at 1000 × g then resuspended in 500 ␮l cold
1× binding buffer and added 1.25 ␮l of Annexin V FITC and incu-
bated for 15 min at RT in dark, centrifuge at 1000 × g for 5 min at
RT, remove supernatant, gently resuspend in 500 ␮l cold 1× bind-
ing buffer and added 10 ␮l PI. Flow cytometry was performed using
a FACScan (Becton Dickinson, Mountain View, CA) flowcytome-
ter, equipped with a single 488-nm argon laser. Annexin V-FITC
were analyzed using excitation and emission settings of 488 nm
and 535 nm (FL-1 channel); PI, 488 nm and 610 nm (FL-2 channel).
Debris and clumps were gated out using forward and orthogonal
General method for C-alkylation (method B)
To a magnetically stirred solution of amine (0.0022 moles)
cooled at 0 ◦C was slowly treated with formalin (0.00148 moles),
glacial acetic acid (0.0192 moles) and compound 1 (100 mg,
0.00037 moles). The whole reaction mixture was brought to RT
and stirred for 4 h, after pH was adjusted to 8 by addition of 20%
aq. NaOH. It was then extracted with ethyl acetate (3 × 25 ml), the
organic layer was washed with water, dried over anhyd. Na2SO4

892
T. Narender et al. / Phytomedicine 20 (2013) 890–896
light scatter. The experiment was repeated three times indepen-
dently.
Cell cycle analysis
For cell cycle distribution studies post-treatment, cells were
fixed in 70% ethanol, rehydrated in PBS with ribonuclease A
(100 ␮g/ml) and Triton-X (10 ␮g/ml) for 30 min at room temper-
ature then added PI (50 ␮g/ml) and incubated in dark for 30 min
and analyzed with FACScan flow cytometer (Becton Dickinson,
Mountain View, CA) equipped with an argon laser to give 488 nm
light.
Fig. 2. Preparation of O-alkylated derivatives 2–6 of emodin (1).
Caspase-3 activation assay
The activity of caspase-3 was determined using caspase-3
colorimetric assay kit (Sigma–Aldrich) according to the man-
ufacturer’s protocol. Briefly, HepG2 cells and NIH/3T3 cells (1
x 106cells/well) were treated with 0 ␮M, 10 ␮M and 20 ␮M
compounds 3 and 12 for 24 h. The cells were harvested and
lysed by addition of lysis buffer. Samples of the cell lysates
were mixed with colorimetric substrate (Ac-DEVD-pNA) and
incubated at 37 ◦C in the dark for 4 h. The absorbance was mea-
sured at 405 nm in an ELISA reader. Caspase-3 activity was
expressed as the change of the activity compared to the con-
trol.
Fig. 3. Preparation of C-alkylated derivatives 7–12 of emodin (1).
DNA intercalation assay
Calf thymus DNA (8.8 ␮M) was loaded with Tris buffer con-
taining ethidium bromide (4.4 ␮M). To each well were added
compounds 3, 12, and DOX at 5, 10 and 20 ␮M. After incuba-
tion for 30 min at 25 ◦C fluorescence was measured by using
fluorimeter in duplicate experiments with two control wells (no
compound = 100% fluorescence and no DNA = 0% fluorescence).
Fluorescence readings are reported as % fluorescence readings com-
pared to controls.
Statistical analysis
Fig. 4. Preparation of ester derivatives 13, 14 and acid (15) from emodin (1).
Each experiment was observed in triplicate. The data are pre-
sented as mean SD and compared using Student’s t-test. P < 0.05
or less was considered to be statistically significant.
acid in presence of triethylamine at 0 ◦C and acetic anhydride
in presence of pyridine respectively (Fig. 4). To transform the
methyl group in to acid functionality, emodin triacetate (14) was
oxidized with chromium trioxide in presence of glacial acetic
acid and acetic anhydride to provide acid (15) (Rahimipour et al.
2001).
Results and discussion
Chemistry
We here report the synthesis of novel O-alkylated derivatives
2–6 (Fig. 2), C-alkylated derivatives 7–12 (Fig. 3), acyl derivatives
13–14 (Fig. 4) and acid derivative 15 (Fig. 4) from 1 and their anti-
cancer profile against human hepatocellular carcinoma (HepG2),
metastatic human breast cancer (MDA-MB-231) and embryonic
fibroblast (NIH/3T3) cells lines. 3 and 12 showed comparable
in vitro anticancer activity with marketed drug epirubicin III (Fig. 1
and Table 1), therefore 3 and 12 were selected for apoptosis induc-
ing ability by flow cytometry, caspase-3 activity in HepG2 cells,
HepG2 cells on cell cycle distribution studies and DNA binding
interaction.
In order to prepare O-alkylated derivatives (2–6), hydroxyl
group at meta position of emodin (1) was alkylated with various
amine side chains under basic conditions using K2CO3 and DMF as
a solvent.
To study the substituent effect on anthraquinone nucleus
C-alkylated derivates 7–12 were prepared using Mannich reac-
tion protocol (Kemertelidze and Gotsiridze 1971a,b; Blicke 1942;
Brewster and Eliel 1953; Kintsurashvili et al. 1999).
Biological evaluation
Antiproliferative activity
All the compounds were screened against two cancer cell lines
(HepG2 and MDA-MB-231) and a non-cancer cell line (NIH/3T3).
The parent compound, emodin (1) has an IC50 of 291 ␮M, 119 ␮M
and 212 ␮M against MDA-MB-231, HepG2 and NIH/3T3 respec-
tively (Table 1). All the derivatives 2–15 prepared from emodin
(1) have exhibited improved in vitro anticancer activity against
MDA-MB-231 cell lines with better therapeutic index (Table 1).
Except 7, 8, and 13–15 all other derivatives have also exhib-
ited improved activity against HepG2 cell lines. Derivatives 2
(IC50 = 7.7 ␮M), 6 (IC50 = 4.2 ␮M), and 12 (IC50 = 5.0 ␮M) have IC50
below 10 ␮M against MDA-MB-231 cell lines, where as deriva-
tives 3 (IC50 = 5.64 ␮M) and 12 (IC50 = 10.4 ␮M) also have IC50 of
10 ␮M or below 10 ␮M against HepG2 cell line. Our structure
activity relationships indicated that O-alkylation and C-alkylation
of emodin (1) improves the activity in both cell lines, where
as esterification only improves the activity in MDA-MB-231 cell
line. O-alkylated derivative 3 found to be 21-fold more potent
in HepG2 cell line and 22-fold more potent in MDA-MB-231 cell
Since the emodin (1) contain free hydroxyl group we planned
to prepare few esters to study their anticancer activity. To prepare
ester derivative 13 and 14, emodin (1) was reacted with tiglic

T. Narender et al. / Phytomedicine 20 (2013) 890–896
893
Table 1
Chemical structures and in vitro anticancer activity (IC50 in ␮M) of emodin (1) and its derivatives (2–15).
Comp. no
Chemical structure
IC50(␮M)
HepG2
MDA-MB-231
291
NIH/3T3
1
2
3
4
5
6
119
212
54.49
5.64
63.19
23.15
27
7.72
13.03
22.14
35
17.86
11.61
145
29.98
40.99
4.24
7
8
9
348
73.3
340
122
12.29
29.16
122
17.88
17.82
10
11
63
41.16
10.43
112
217
62.54
12
10.44
5.027
23.42
13
14
216
200
35.95
2.142
570
26.47
15
III
218
4.6
8.103
7.7
245
Epirubicin (marketed drug)
3.2
line to its parent compound 1 and C-alkylated derivative 12 found
to be 11-fold more potent in HepG2 cell line and 58-fold more
potent in MDA-MB-231 cell lines to its parent compound 1. It
is noteworthy to mention here that 3 and 12 showed compara-
ble in vitro anticancer activity with marketed drug epirubicin III
(Fig. 1 and Table 1), therefore 3 and 12 were selected for cell cycle
arrest, caspase dependent apoptosis and DNA binding interaction
studies.

894
T. Narender et al. / Phytomedicine 20 (2013) 890–896
Fig. 5. Determination of apoptosis inducing ability of 3 and 12 by flow cytometry: HepG2 cells (5 × 105 cells) with 70–80% confluence were incubated in RPMI supplemented
with 10% FBS without (control) and staurosporine (1 ␮g/ml) as positive control with 5, 10 and 20 ␮M of derivatives 3 and 12. After 24 h, the cells were harvested, stained
with Annexin-V-FITC and PI, and analyzed by flow cytometry.
Apoptosis inducing ability studies by flow cytometry
staurosporine (1 ␮g/ml) as positive control in HepG2 cells. When
HepG2 cells were stained with annexin-V/PI and analyzed with
flow cytometer, early and late apoptosis (annexin V-stained) cells
were found to be increased in a dose-dependent manner (Fig. 5),
which indicates the induction of apoptosis by 3 and 12.
In order to characterize the cellular basis of antiproliferative
effects of selected derivatives 3 and 12, we initially investi-
gated the apoptosis inducing ability by flow cytometry in HepG2
cells (Muktapuram et al. 2012). Annexin-V specially binds to
phosphatidylserine and has been employed for determination of
apoptotic cells. Annexin-V/PI staining was performed to deter-
mine early, late apoptotic and necrotic cells followed by treatment
with 5, 10 and 20 ␮M concentration of derivatives 3 and 12 and
Cell cycle arrest studies
To determine the possible effect of these compounds on cell
cycle progression, HepG2 cells were treated with 5, 10 and 20 ␮M
Fig. 6. Effect of derivatives 3 and 12 treatment to HepG2 cells on cell cycle distribution. The cells treated with derivatives 3 and 12 for 24 h were collected and stained with
PI and analyzed by flow cytometry. Following FACS analysis, cellular DNA histograms were further analyzed by Modfit LT V3.2.1. The data are representative examples for
duplicate tests. The details are described in supporting information.

T. Narender et al. / Phytomedicine 20 (2013) 890–896
895
Fig. 8. DNA interacting ability of antiproliferative emodin derivatives 3, 12 and
doxorubicin (II); error bars represent the standard deviation of the mean, n = 3.
Fig. 7. Effect of 3 and 12 on caspase-3 activity in HepG2 cells and NIH/3T3 cells.
After 24 h treatment with or without derivatives 3 and 12, caspase-3 activities were
determined by mixing the cell lysates with colorimetric substrate (Ac-DEVD-pNA).
The absorbance was measured at 405 nm in an ELISA reader. Caspase-3 activity
was expressed as the change of the activity compared to the control. *P < 0.05 and
**P < 0.01 vs control.
preincubated with ethidium bromide for 30 min in buffer before
newly synthesized compound addition. Derivatives 3 and 12 were
incubated with ethidium bromide DNA complex for 30 min and
measured fluorescence. As shown in Fig. 8, the derivatives 3 and
12 have good DNA binding interaction similar to Doxorubicin (II).
The compound 12 has better DNA interaction than 3. These results
suggested that potent antiproliferative effect of the derivative 3 and
12 might be due to DNA intercalation.
of 3 and 12 then stained with propidium iodide (Fig. 6). To evaluate
the distribution of actively dividing cells in the cell cycle before the
induction of extensive apoptosis, cells were treated for 24 h, and
the percentage of apoptotic and nonapoptotic cells in each phase
of the cell cycle was determined by DNA flow cytometry (Sinha
et al. 2011). As shown in Fig. 6 cells in the G1/S phase increased
from 23.48% in controls to 24.66%, 49.73%, and 50.05% after treat-
ment with 3 and cells in the G1/S phase increased from 23.48% in
controls to 28.37%, 35.63%, and 46.29%, after treatment with deriva-
tive 12, indicating that derivatives 3 and 12 arrest cell cycle at G1/S
phase. Along with G1/S phase arrest, the sub-G0 population was
slightly increased in cells exposed to 10 and 20 ␮M of 3 and 12. The
results obtained in cell proliferation studies, and apoptosis induc-
tion assay also correlates the compound 3 and 12 effects on cell
cycle progression.
Conclusions
In conclusion we have isolated large quantities of emodin (1)
from the roots of R. emodi and prepared novel derivatives 2–15 and
evaluated their in vitro anticancer activity in HepG2 and MDA-MB-
231 cell lines. Some of the derivatives showed better therapeutic
effect than the parent compound. Among these derivatives 3 and
12 showed comparable in vitro anticancer activity to that of the
marketed drug epirubicin (III). The Compounds 3 and 12 were capa-
ble of inducing cell cycle arrest and caspase dependent apoptosis
in HepG2 cell lines and exhibit DNA intercalating activity. These
emodin derivatives hold a promise for developing safer alternatives
to the marketed Eupirubicin. Further work is progress in our lab-
oratory to evaluate their in vivo anticancer potential and in-depth
mechanistic aspects.
Caspase-3 activity in HepG2 cells
To find out whether the induction of apoptosis by 3 and 12 via
caspase mediated or not, we examined the caspase-3 activation in
HepG2 cells and NIH/3T3 cells. Caspases belongs to cysteine pro-
teases class, are the important proteins that regulate the apoptotic
response (Fig. 7). Among these, caspase-3 is a key protein in the
apoptosis mechanism since it executes both extrinsic and intrin-
sic pathways (Kamal et al. 2011; Wang et al. 2007). Our studies
indicated that a concentration dependent increase in caspase-3
activity with derivatives 3 and 12 treatment as compared with
the untreated control. Caspase-3 activation was enhanced 6.88
and 10.03-folds by 3 and 5.33and 8.66-folds by 12 in cells treated
with 10 and 20 ␮M respectively, when compared with control
group (Fig. 7) while in normal cells (NIH/3T3) there is no induction
of caspase-3 activity. These results confirmed that the apoptosis
induction is due to caspase-3 activation by 3 and 12 in HepG2
cells.
Conflict of interest
The authors have no conflict of interest.
Disclosure statement
The authors have nothing to disclose.
Acknowledgments
DNA binding interaction
In this study we evaluated the DNA interacting ability of antipro-
liferative emodin derivatives 3 and 12, using an ethidium bromide
displacement assay (Fig. 8). The fluorescence intensity of ethidium
bromide increases upon binding of DNA and subsequent displace-
ment of ethidium is apparent by a decrease in emission intensity
upon addition of competitive inhibitor (Bair et al. 2010; Dai et al.
2012). We have included doxorubicin II (Fig. 1), a known DNA
interacting anticancer drug for comparison. Calf thymus DNA was
Authors are thankful to Dr. T.K. Chakraborty, Director, CSIR-CDRI
for constant encouragement for the program on natural products of
biological importance. We also thank the Ministry of Earth Sciences
(MoES) and EMPOWER project from CSIR, New Delhi for providing
research grant and fellowship. Komal Sharma is thankful to DST
for INSPIRE fellowship. Mr. J.P. Chaturvedi, Miss. Astha Shukla and
Miss. Manali Agrawal for technical support, SAIF, CDRI for spectral
data.

896
T. Narender et al. / Phytomedicine 20 (2013) 890–896
Appendix A. Supplementary data
Kintsurashvili, L.A., Sikharulidze, M.I., Buyanov, V.N., Turabelidze, D.G., 1999. Syn-
thesis of amino derivatives of 1,6 8-trihydroxy-3-methyl-9,10-anthraquinone.
Chemistry of Natural Compounds 35, 6.
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.phymed.2013.03.015.
Koyama, M., Kelly, T.R., Watanabe, K.A., 1988. Novel type of potential anticancer
agents derived from chrysophanol and emodin. Some structure–activity rela-
tionship studies. Journal of Medicinal Chemistry 31, 283–284.
Kuo, Y.C., Meng, H.C., Tsai, W.J., 2001. Regulation of cell proliferation, inflammatory
cytokine production and calcium mobilization in primary human T lympho-
cytes by emodin from Polygonum hypoleucum Ohwi. Inflammation Research 50,
73–82.
References
Liang, J.W., Hsiu, S.L., Huang, H.C., Lee-Chao, P.D., 1993. HPLC analysis of emodin in
serum, herbs and Chinese herbal prescriptions. Journal of Food and Drug Analysis
1, 251–257.
Muktapuram, P.R., Gara, R.K., Sharma, K., Rohit, C., Srinivas, K., Mishra, D.P., Bathula,
S.R., 2012. Anticancer siRNA delivery by new anticancer molecule: a novel com-
bination strategy for cancer cell killing. European Journal of Medicinal Chemistry
56, 400–408.
Rahimipour, S., Bilkis, I., Péron, V., Gescheidt, G., Barbosa, F., Mazur, Y., Koch,
Y., Weiner, L., Fridkin, M., 2001. Generation of free radicals by emodic
acid and its [d-Lys6]GnRH-conjugate. Photochemistry and Photobiology. 74,
226–236.
Shieh, D.E., Chen, Y.Y., Yen, M.H., Chiang, L.C., Lin, C.C., 2004. Emodin-induced apo-
ptosis through p53-dependent pathway in human hepatoma cells. Life Sciences
74, 2279–2290.
Singh, S.S., Pandey, C.S., Singh, R., Agarwal, K.S., 2005. 1,8-Dihydroxyanthraquinone
derivatives from rhizomes of Rheum emodi. Indian Journal of Chemistry 43B,
1494–1496.
Sinha, S., Roy, S., Reddy, B.S., Pal, K., Sudhakar, G., Iyer, S., Dutta, S., Wang, E., Vohra,
P.K., Roy, K.R., Reddanna, P., Mukhopadhyay, D., Banerjee, R., 2011. Molecular
Cancer Research 9, 364.
Su, Y.T., Chang, H.L., Shyue, S.K., Hsu, S.L., 2005. Emodin induces apoptosis in human
lung adenocarcinoma cells through a reactive oxygen species-dependent mito-
chondrial signaling pathway. Biochemical Pharmacology 70, 229–241.
Wang, C., Wu, X., Chen, M., Duan, W., Sun, L., Yan, M., Zhang, L., 2007. Emodin induces
apoptosis through caspase 3-dependent pathway in HK-2 cells. Toxicology 231,
120–128.
Yang, F., Zhang, T., Tian, G., Cao, H., Liu, Q., Ito, Y., 1999. Preparative isolation
and purification of hydroxyanthraquinones from Rheum officinale Baill by high-
speed counter-current chromatography using pH-modulated stepwise elution.
Journal of Chromatography A 858, 103–107.
Agarwal, S.K., Singh, S.S., Verma, S., Kumar, S., 2000. Antifungal activity of
anthraquinone derivatives from Rheum emodi. Journal of Ethnopharmacology
72, 43–46.
Alves, D.S., Perez, F.L., Estepa, A., Micol, V., 2004. Membrane-related effects under-
lying the biological activity of the anthraquinones emodin and barbaloin.
Biochemical Pharmacology 68, 549.
Bair, J.S., Palchaudhuri, R., Hergenrother, P.J., 2010. Chemistry and biology of
deoxynyboquinone, a potent inducer of cancer cell death. Journal of the Ameri-
can Chemical Society 132, 5469–5478.
Blicke, F.F., 1942. Mannich reaction. In: Adams, R. (Ed.), Organic Reaction, vol. 1.
Wiley, New York, p. 303.
Brewster, J.H., Eliel, E.L., 1953. Carbon–carbon alkylation of amines and ammonium
salts. In: Adams, R. (Ed.), Organic Reactions. John Wiley and sons, New York, p.
8.
Chen, R.S., Jhan, J.Y., Su, Y.J., Lee, W.T., Cheng, C.M., Ciou, S.C., Lin, S.T., Chuang, S.M.,
Ko, J.C., Lin, Y.W., 2009. Emodin enhances gefitinib-induced cytotoxicity via
Rad51 down regulation and ERK1/2 inactivation. Experimental Cell Research
315, 2658–2672.
Chen, Y.C., Shen, S.C., Lee, W.R., Hsu, F.L., Lin, H.Y., Ko, C.H., Tseng, S.W., 2002. Emodin
induces apoptosis in human promyeloleukemic HL-60 cells accompanied by
activation of caspase 3 cascade but independent of reactive oxygen species
production. Biochemical Pharmacology 64, 1713–1724.
Chun, G.W., Jun, Q.Y., Bei, Z.L., Dan, T.J., Chong, W., Liang, Z., Dan, Z., Yan, W., 2010.
Anti-tumor activity of emodin against human chronic myelocytic leukemia K562
cell lines in vitro and in vivo. European Journal of Pharmacology 627, 33–41.
Dai, X.Y., Zeng, X.X., Peng, F., Han, Y.Y., Lin, H.J., Xu, Y.Z., Zhou, T., Xie, G., Deng, Y.,
Mao, Y.Q., Yu, L.T., Yang, L., Zhao, Y.L., 2012. A novel anticancer agent. SKLB70359,
inhibits human hepatic carcinoma cells proliferation via G0/G1 cell cycle arrest
and apoptosis induction. Cellular Physiology and Biochemistry 29, 281–290.
Fain, V.Ya., 1999. 9,10-Anthraquinones and their Use. USSR Academy of sciences,
Moscow (in Russian).
Zaprometnov, M.N., 1993. Phenolic Compounds. Distribution, Metabolism, and
Functions in Plants. USSR Academy of sciences (Nauka), Moscow (in Russian).
Zhang, L., Lau, Y.K., Kim, R.L.D.S., Chen, C.F., Hortobagyi, G.N., Chang, C., Hung,
M.C., 1998. Tyrosine kinase inhibitors, emodin and its derivative repress HER-
2/neu-induced cellular transformation and metastasis-associated properties.
Oncogene 16, 2855–2863.
Zhang, L., Lau, Y.K., Xia, W., Hortobagyi, G.N., Hung, M.C., 1999. Tyrosine kinase
inhibitor emodin suppresses growth of HER-2/neu-over expressing breast can-
cer cells in athymic mice and sensitizes these cells to the inhibitory effect of
paclitaxel. Clinical Cancer Research 5, 343–353.
Gorelik, M.V., 1983. Chemistry of Anthraquinone and Its Derivatives. USSR Academy
of sciences, Moscow (in Russian).
Huang, H.C., Chu, S.H., Chao, P.D., 1991. Vasorelaxants from Chinese herbs, emodin
and scoparone, possess immunosuppressive properties. European Journal of
Pharmacology 198, 211–213.
Kamal, A., Suresh, P., Mallareddy, A., Kumar, B.A., Reddy, P.V., Rajua, P., Tamboli,
J.R., Shaik, T.B., Jain, N., Kalivendi, S.V., 2011. Synthesis of a new 4-aza-2,
3-didehydropodophyllotoxin analogues as potent cytotoxic and antimitotic
agents. Bioorganic and Medicinal Chemistry 19, 2349–2358.
Kemertelidze, E.P., Gotsiridze, A.V., 1971a. Khimiya Prirodnykh Soedinenii, 519.
Kemertelidze, E.P., Gotsiridze, A.V., 1971b. Soobshcheniya Akademii Nauk Gruzii.
SSR 61, 93.
Zhou, X.M., Chen, Q.H., 1998. Biochemical study of Chinese rhubarb. XXII. Inhibitory
effect of anthraquinone derivatives on Na+-K+-ATPase of the rabbit renal
medulla and their diuretic action. Acta Pharmaceutica Sinica 23, 17–20.