Synthesis of novel anticancer iridoid derivatives and their cell cycle arrest and caspase dependent apoptosis

Article abstract of DOI:10.1016/j.phymed.2013.08.023

Nyctanthes arbortristis Linn (Oleaceae) is widely distributed in sub-Himalayan regions and southwards to Godavari, India commonly known as Harsingar and Night Jasmine. In continuation of our drug discovery programme on Indian medicinal plants, we isolated arbortristoside-A (1) and 7-O-trans-cinnamoyl 6β-hydroxyloganin (2) from the seeds of N. Arbortristis, which exhibited moderate in vitro anticancer activity. Chemical transformation of 2 led to significant improvement in the activity in derivative 8 and 15 against HepG2 (human hepatocellular carcinoma), MCF-7 (breast adenocarcinoma) cell lines. The compounds 8 and 15 were also capable of cell cycle arrest and caspase dependent apoptosis in HepG2 cell lines. These iridoid derivatives hold promise for developing safer alternatives to the marketed drugs.

Full text of DOI:10.1016/j.phymed.2013.08.023

Phytomedicine 21 (2014) 333–339
Contents lists available at ScienceDirect
Phytomedicine
journal homepage: www.elsevier.de/phymed
Synthesis of novel anticancer iridoid derivatives and their cell cycle
arrest and caspase dependent apoptosis
Sukanya Pandetia, Komal Sharmab, Surendar Reddy Bathulab,∗, Narender Tadigoppulaa,∗
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
Article history:
Received 31 May 2013
Accepted 22 August 2013
Nyctanthes arbortristis Linn (Oleaceae) is widely distributed in sub-Himalayan regions and southwards to
Godavari, India commonly known as Harsingar and Night Jasmine. In continuation of our drug discovery
programme on Indian medicinal plants, we isolated arbortristoside-A (1) and 7-O-trans-cinnamoyl 6␤-
hydroxyloganin (2) from the seeds of N. Arbortristis, which exhibited moderate in vitro anticancer activity.
Chemical transformation of 2 led to significant improvement in the activity in derivative 8 and 15 against
HepG2 (human hepatocellular carcinoma), MCF-7 (breast adenocarcinoma) cell lines. The compounds 8
and 15 were also capable of cell cycle arrest and caspase dependent apoptosis in HepG2 cell lines. These
iridoid derivatives hold promise for developing safer alternatives to the marketed drugs.
© 2013 Elsevier GmbH. All rights reserved.
Keywords:
7-O-trans-cinnamoyl 6␤-hydroxyloganin
derivatives
Arbortristoside-A
Anticancer agents
Apoptosis
Nyctanthes arbortristis
Introduction
1998; Saxena et al. 1984; Sanjita Das et al. 2008), antinociceptive
(Sanjita Das et al. 2008) and analgesic activity (Saxena et al. 1987).
Iridoids represent the group of cyclopentan – (c) – pyran
monoterpenoids. They are found as natural constituents in a large
number of plant families. Iridoids were first isolated in the lat-
ter part of the nineteenth century, but it was not until 1958 that
Halpern and Schmid (Halpern et al. 1958) proposed the basic
skeleton of the iridoids in their investigation of the structure of
plumieride, which possess various biological activities (Chopra
et al. 1956). The name iridoid is a generic term derived from the
names iridomyrmecin, iridolactone and iridodial compounds iso-
lated from some species of Iridomyrmex, a genus of ants, in which
they occur as defensive secretions (Roth and Eisner 1962). These
compounds have been referred to as pseudoindicans. They have
also been referred to as aucubin glucosides.
In continuation of our drug discovery programme on anti-
cancer agents from Indian medicinal plants, we isolated large
quantities of 7-O-trans-cinnamoyl-6␤-hydroxyloganin (2) from
the seeds of Nyctanthes arbortristis Linn 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 analogues of camptothecin, which
have better therapeutic benefits than the parent natural prod-
ucts. Towards this goal, we have synthesized novel derivatives
of compound 2 and evaluated their anticancer activity against
HepG2 (human hepatocellular carcinoma), MCF-7 (breast adeno-
carcinoma), MDAMB-231 and NIH/3T3 cell lines (Table 1). Further
we have studied the apoptosis inducing ability, effect on cell cycle
and caspase-3 activation studies of most active compounds 8 and
15.
Arbortristoside-A
(1)
and
7-O-trans-cinnamoyl-6␤-
hydroxyloganin (2) belongs to iridoid class of compounds are
the major bioactive compounds of numerous herb species such
as Nyctanthes arbortristis Linn (Division: Magnoliophyta; Class:
Magnoliopsida; Order: Lamiales; Family: Oleaceae), commonly
known as Harsingar and Night Jasmine and possesses leishmanici-
dal (Tandon et al. 1991), antiplasmodial (Tuntiwahwuttikul et al.
2003), antispermatogenic (Gupta et al. 2006), antiallergic (Gupta
et al. 1995), anti-inflammatory (Amrite et al. 2006; Patel et al.
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
∗
Corresponding authors. Tel.: +91 522 2612411; fax: +91 522 2623405.
E-mail addresses: bsreddy@cdri.res.in (S.R. Bathula), t narendra@cdri.res.in,
tnarender@rediffmail.com (N. Tadigoppula).
0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.phymed.2013.08.023

334
S. Pandeti et al. / Phytomedicine 21 (2014) 333–339
Table 1
120 g). n-Butanol was added to chloroform insoluble portion, the
n-butanol soluble fraction was evaporated under reduced pressure
at 60 ◦C afford n-butanol fraction (F003, 80 g).
Chemical structure and in vitro anticancer activity (IC50 in ␮M) of compounds 1, 2
and its derivatives (3–15).
Comp. No.
HepG2
MDAMB-231
MCF-7
NIH/3T3
1
2
89
78
80
76
87
79
78
84
Isolation and purification of Iridoids
3
4
5
6
7
8
9
10
11
12
13
14
469
134
113
42
56
12
145
79
74
23
78
29
14
4.6
2.1
1.24
270
125
97
49
45
10
156
83
310
147
98
45
44
14
151
87
76
32
76
37
16
3.7
4.6
3.84
280
140
120
55
59
19
167
92
89
34
93
45
31
3.2
3.6
4.57
n-Butanol fraction (80 g) was chromatographed on a column of
silica gel (60–120 mesh) and eluted with chloroform and methanol
in increasing polarity. Fractions were collected and then com-
bined on the basis of TLC pattern to get two subfractions (A
and B). Fraction A was rechromatographed on silica gel, eluting
with chloroform–methanol (96:4); recrystallization from methanol
afforded compound 1 (80 mg). Fraction B was rechromatographed
on silicagel (60–120 mesh), eluting with chloroform–methanol
(90:10); recrystallization from methanol afforded compound 2
(1.5 g). The compounds visualization was done under UV light, also
shown brown spot by spraying with 10% sulphuric acid in methanol.
82
16.8
82.6
32
15
18
Epirubicin
Paclitaxel (PTX)
Doxorubicin
7.7
1.24
3.26
Preparation of iridoid derivatives
General procedure for the dihydro compound
ppm relative to CHCl3/DMSO with TMS as internal standard. ESI
mass spectra were recorded on JEOL SX 102/DA-6000. Plates for
thin layer chromatography (TLC) were prepared from silica gel 60
GF254 (Merck) and activated by drying at 100 ◦C for 2 h. Chromatog-
raphy was executed with silica gel (60–120 mesh) using mixtures
of chloroform, methanol and hexane as eluants. Visualization was
obtained under UV light and spraying with 10% sulphuric acid in
methanol.
To a magnetically stirred solution of compound 2 (100 mg,
0.00018 mol) in methanol (10 ml) was added gradually
NiCl2 × 6H2O (0.00009 mol) at rt. When the clear solution acquired
a greenish colour, the whole reaction mixture was brought to 0 ◦C
and NaBH4 (0.00036 mol) was added portion wise. After addition
of NaBH4, the whole solution was stirred for 30 min at 0 ◦C to rt.
Methanol was removed by vacuum, and 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).
Background of plant
Nyctanthes arbortristis Linn (Oleaceae), commonly known as
Harsingar and Night Jasmine. It is a shrub growing to 10 m tall,
with flaky grey bark. The fruit is a flat brown heart-shaped to
round capsule 2 cm diameter, with two sections each containing
a single seed. Which have been claimed to possess multiple phar-
macological activities like antibacterial, antifungal, anti-influenza,
anti-inflammatory, analgesic, antipyretic, antihistaminic, antiulcer,
hypnotic, tranquilizing, hepatoprotective, antidiabetic, antianemic,
immunobioactivities, antioxidant, antispermatogenic, etc. (Rachna
and Mridula 2011).
General procedure for the amino hydroxylation
K2[OsO2(OH)2] (1.5 mol%) was dissolved with stirring in 10 ml
of aqueous solution of LiOH·H2O. After the addition of t-BuOH,
(DHQ)2PHAL was added and the mixture immersed in a cooling
bath set 0 ◦C. After the addition of compound 2, N-bromo acetamide
was added in one portion, which resulted in an immediate colour
change to green and the mixture vigorously stirred at the same
temperature. The reaction was monitored by TLC, and pH (full con-
version is indicated when the reaction mixture attains pH 7). After
4 h, the reaction mixture was treated with Na2SO3 and stirred at rt
Collection of medicinal plant
N. arbortristis Linn seeds were purchased from the local mar-
ket of Lucknow, India and the authentification was done by Botany
Division of Central Drug Research Institute, Lucknow and is kept in
the herbarium for future reference.
Extraction
Powdered N. arbortristis Linn seeds (4 kg) were placed in glass
percolator 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 300 g.
Fractionation
The ethanol extract was macerated with hexane. The hexane
soluble fraction was separated and evaporated under reduced pres-
sure to afford hexane fraction (F001, 80 g). Chloroform was added
to hexane insoluble portion, and the resultant solution was evapo-
rated under reduced pressure to afford chloroform fraction (F002,
Fig. 1. Regioselective hydrogenation of compounds
NaBH4/NiCl2·6H2O.
2
and
3
by using

S. Pandeti et al. / Phytomedicine 21 (2014) 333–339
335
(0.00027 mol).The whole reaction mixture was stirred for 4 h at
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
(Fig. 4).
General procedure for the esterification
To
a magnetically stirred solution compound 2 (100 mg,
0.00018 mol) in dry DCM (5 ml) maintained 0 ◦C after it added
Z-2-methyl but-2-enoyl chloride (0.00018 mol) and triethylamine
(0.00027 mol) at 0 ◦C for 1 h. DCM 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. 4).
Fig. 2. Amino hydroxylation of compounds 2 and 3 by using K2[OsO2(OH)2].
for 30 min. 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. 2).
General procedure for the decinnamylation
General procedure for the acetylation
To a magnetically stirred solution of compound 2 (100 mg,
0.00018 mol) in a mixture of solvents (H2O:THF:EtOH (1:1:1),
10 ml) was added hydroxylamine. Sulphuric acid salt (0.0009 mol)
and triethylamine (0.012 mol) at rt. The whole solution was stirred
for 6 h at 100–110 ◦C. Solvents were removed by vacuum, and
then extracted with ethyl acetate (3 × 25 ml), the organic layer
was washed with water, dried over anhyd Na2SO4 and evap-
orated under reduced pressure. Then the crude product was
chromatographed on silica gel to afford the desired compound 8
(Fig. 3).
Compound 2 (100 mg, 0.00018 mol) magnetically stirred in a
solution of pyridine (2 ml) and acetic anhydride (0.00163 mol) at
60–70 ◦C for 4 h. The reaction mixture was put into cold water for
crystallization, then filtered and dried to get desired compound
(Fig. 4).
General procedure for the oxidation
To a magnetically stirred solution of compound 8 (100 mg,
0.00018 mol) in dry acetone (3 ml) maintained 0 ◦C after it added
Jones reagent at 0 ◦C for 1 h. Acetone 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 10
(Fig. 3).
General procedure for the O-methylation of aromatic hyroxy
group
To a magnetically stirred solution of compound 2 (100 mg,
0.00018 mol) in DMF (5 ml) at room temperature was treated
with the dimethyl sulphate (0.00027 mol), TBAB (0.00018 mol) and
K2CO3 (0.00027 mol). The whole reaction mixture was stirred for
4 h at 100 ◦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
3 (Fig. 1).
Biological study
Materials
HepG2, MDA-MB-231, MCF-7, and NIH/3T3 cell lines used in
the present study was obtained from ATCC (American Type Cul-
ture Collection, USA). Annexin V FITC Apoptosis Detection Kit was
purchased from Calbiochem. MTT dye and Caspase-3 Activation Kit
was procured from Sigma–Aldrich. Absorbance was recorded using
Eliza Plate Reader. Flow cytometry was performed using a FACScan
(Becton Dickinson, Mountain View, CA) flowcytometer.
General procedure for the O-alkylation
To a magnetically stirred solution of compound 2 (100 mg,
0.00018 mol) in DMF (5 ml) at room temperature was treated
with the ethyl ␣-bromo isobutyrate (0.00021 mol) and K2CO3
Fig. 3. Decinnamoylation of compound 2 by NH2OH·H2SO4 followed by using oxidation and acetylation.

336
S. Pandeti et al. / Phytomedicine 21 (2014) 333–339
Fig. 4. Synthesis of ester analogues of compound 2.
Cell culture
analyzed with FACScan flow cytometer (Becton Dickinson, Moun-
tain View, CA) equipped with an argon laser to give 488 nm light.
MDA-MB-231 and MCF-7 cells were routinely maintained in
Dulbecco’s Modified Eagle Medium DMEM (Sigma–Aldrich) while
HepG2, NIH/3T3 cells were maintained in Roswell Park Memorial
Institute (RPMI 1640, Merck) supplemented with 10% FBS (Merck)
and 1% Antibiotic–antimycotic solution (Merck) at 37 ◦C in a humid-
ified incubator with 5% CO2. All stock solutions of compounds were
prepared in cell culture grade DMSO and stored in −20 ◦C. Com-
pounds were diluted in culture media prior to use in experiments.
Caspase-3 activation assay
The activity of caspase-3 was determined using caspase-3
colorimetric assay kit (Sigma–Aldrich) according to the manu-
facturer’s protocol. Briefly, HepG2 cells (1 × 106 cells/well) were
treated with 0 ␮M, 10 ␮M and 20 ␮M compounds 8 and 15 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 measured at 405 nm in an ELISA reader. Caspase-3
activity was expressed as the change of the activity compared to
the control.
Cell viability assay
Antiproliferative activity of the compounds were 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 + SD.
Statistical analysis
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.
Apoptosis studies
Results and discussion
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 compounds 8 and 15 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 incubated 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× binding buffer and added 10 ␮l PI. Flow cytometry was
performed using a FACScan (Becton Dickinson, Mountain View,
CA) flowcytometer, equipped with a single 488-nm argon laser.
Annexin V-FITC were analyzed using excitation and emission sett-
ings 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 light scatter. The experiment was repeated three
times independently.
Chemistry
Chemical transformation of the active compound was carried
out to study the structure–activity relationship. Initially the selec-
tive hydrogenation of olefinic bond (Khurana and Sharma 2004)
of cinnamoyl group was carried out on compound 2 using NaBH4,
NiCl2·6H2O to obtain the dihydro derivatives 4 and 5 (Fig. 1).
To increase the hydrophilic character of the compound asym-
metric amino hydroxylation was carried out on 2 and 3 using
K2[OsO2(OH)2], which provided aminohydroxylated (Subba Rao
et al. 2005) derivatives 6 and 7 (Fig. 2).
To find out the role of the cinnamoyl moiety in compound 2
we attempted to remove the cinnamoyl group from compound 2.
Hydrolysis of compound 2 under basic conditions such as NaOH
or LiOH or KOH desired compound was not formed. Therefore
NH2OH·H2SO4 (Fenoglio et al. 1996; Sun et al. 2001) was used to
selectively provide decinnamoylated derivative 8 (Fig. 3). The resul-
tant compound was acetylated to give compound 9. Compound 8
was also subjected to oxidation reaction, which led to generate a
keto compound 10 and subsequent acetylation gave 11.
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 tempera-
ture then added PI (50 ␮g/ml) and incubated in dark for 30 min and
Since the compound 2 contain free aromatic hydroxyl group we
prepared few ester derivatives (12–15) to study their anticancer
activity (Fig. 4).

S. Pandeti et al. / Phytomedicine 21 (2014) 333–339
337
Fig. 5. Determination of apoptosis inducing ability of 8 and 15 by flow cytometry: HepG2 cells (5 × 105 cells) with 70–80% confluence were incubated in RPMI supplemented
with 10% FBS without (control) with 5, 10 and 20 ␮M of derivatives 8 and 15. After 24 h, the cells were harvested, stained with Annexin-VFITC and PI, and analyzed by flow
cytometry.
Biological evaluation
manner (Fig. 5), which indicates the induction of apoptosis by
8 and 15.
Antiproliferative activity
All the compounds were screened against three cancer cell
lines (HepG2, MCF-7, MDA-MB-231) and a non-cancer cell line
(NIH/3T3). Except compounds 3, 4, 5 and 9 most of the deriva-
tives prepared from iridoid exhibited improved in vitro anticancer
activity against three cancer cell lines with better therapeutic
index (Table 1). Out of these active derivatives, 8 and 15 exhibited
improved activity against all cancer cell lines. Derivatives 8 showed
IC50 of 12, 10, 14 ␮M and derivative 15 showed IC50 of 14, 16, 18 ␮M
against HepG2, MCF-7, MDA-MB-231 cell lines respectively.
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
of 8 and 15 then stained with propidium iodide (Fig. 6) (Narender
et al. 2013). 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).
Apoptosis inducing ability studies by flow cytometry
Caspase-3 activity in HepG2 cells
In order to characterize the cellular basis of antiproliferative
effects of selected derivatives 8 and 15, we initially investigated
the apoptosis inducing ability by flow cytometry in HepG2 cells.
Annexin-V specially binds to phosphatidylserine and has been
employed for determination of apoptotic cells. Annexin-V/PI stain-
ing was performed to determine early, late apoptotic and necrotic
cells followed by treatment with 5, 10 and 20 ␮M concentra-
tion of derivatives 8 and 15 HepG2 cells (Muktapuram et al.
2012). 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
To find out whether the induction of apoptosis by 8 and 15 via
caspase mediated or not, we examined the caspase-3 activation in
HepG2 cells. Caspases belongs to cysteine proteases class, are the
important proteins that regulate the apoptotic response (Fig. 7).
Among these, caspase-3 is a key protein in the apoptosis mecha-
nism since it executes both extrinsic and intrinsic pathways (Kamal
et al. 2011; Wang et al. 2007). Our studies indicated that a concen-
tration dependent increase in caspase-3 activity with derivatives 8
and 15 treatment as compared with the untreated control. Caspase-
3 activation was enhanced 7.66 and 11.83 folds by 8 and 1.86, 4.66
folds by 15 in cells treated with 10 and 20 ␮M respectively, when

338
S. Pandeti et al. / Phytomedicine 21 (2014) 333–339
compared with control group (Fig. 7). These results confirmed that
the apoptosis induction is due to caspase-3 activation by 8 and 15
in HepG2 cells.
Conclusions
In conclusion we have discovered in vitro anticancer
activity in arbortristoside-A (1) and 7-O-trans-cinnamoyl-6␤-
hydroxyloganin (2) for the first time. Thirteen derivatives of
compound 2 prepared and evaluated their anticancer activity.
Derivative 8 and 15 turned out to be potent anticancer compounds
against three cancer cell lines. Our mechanistic studies indicated
that both derivatives 8 and 15 induce apoptosis and arrests cell
cycle. The induction of apoptosis appears to be due to caspase-3
activation. Further work is in progress in our laboratory develop
potent anticancer agent of iridoid class.
Supporting information
Spectral data of compounds 1–15 are available at
http://www.sciencedirect.com.
Conflict of interest
The authors have no conflict of interest.
Disclosure statement
The authors have nothing to disclose.
Acknowledgments
Authors are thankful to Dr. T.K. Chakraborty, Director, CSIR-CDRI
for constant encouragement for the programme on natural prod-
ucts of biological importance. Komal Sharma is thankful to DST
for INSPIRE fellowship. Dr. B.S. Reddy thanks BSC0104i project for
financial support. We also thank the ICMR, New Delhi for providing
research grant and fellowship. SAIF, CDRI for spectral data. This is
CDRI communication no. 8517.
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