SUPPLEMENTARY MATERIAL
Antiproliferative effect of isolated isoquinoline alkaloid from
Mucuna pruriens seeds in hepatic carcinoma cells
Pranesh Kumara, Atul Rawatb, Amit K Kesharia, Ashok K Singha, Siddhartha Maityc, Arnab Dec,
Amalesh Samantac and Sudipta Sahaa*
a
Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya
Vihar, Raebareli Road, Lucknow 226025, India; bCentre of Biomedical Research, Sanjay Gandhi
Post-Graduate Institute of Medical Sciences Campus, Raebareli Road, Lucknow 226014, Uttar
Pradesh, India; cDepartment of Pharmaceutical Technology, Jadavpur University, Kolkata
700032, India
*
Corresponding author email address: sudiptapharm@gmail.com
The present study was undertaken to investigate the antiproliferative action of isolated M1 (6,7Dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) from Mucuna pruriens seeds using human
hepatic carcinoma cell line (Huh-7 cells). Initially, docking studies was performed to find out the binding
affinities of M1 to caspase-3 and 8 enzymes. Later, cytotoxic action of M1 was measured by cell growth
inhibition (MTT), followed by caspase-3 & 8 enzymes assay colorimetrically. Our results collectively
suggested that M1 had strong binding affinity to caspase-8 in molecular modeling. M1 possessed
antiproliferative activity on Huh-7 cells (EC50 = 13.97 µM) and also inhibited the action of caspase-8
enzyme, signified process of apoptosis. M1 was active against Huh-7 cells that may be useful for future
hepatic cancer treatment.
Keywords: Isoquinoline alkaloid; Molecular docking; Huh-7 cells; Caspase 3 & 8
Experimental
General Experimental Procedures
UV spectra were recorded using Aquamate 8000 UV/VIS spectrophotometer (ThermoFisher Scientific,
MA, USA). IR spectra were obtained on a IRTracer-100 FTIR spectrophotometer (Shimatzu, Koyto,
Japan). NMR spectra were measured on Bruker 400 MHz NMR spectrometer (1H 400 MHz, 13C 100
MHz) NMR spectrometers (Rheinstetten, Germany). Direct-infusion mass spectroscopy (MS) data were
acquired using a hybrid triple quadrupole linear ion trap MS (QTRAP MS) equipped with an
electrospray ionization (ESI) source (2000 QTRAP, Applied Biosystems, Foster City, CA, USA).
Chemicals
Dulbecco’s Modified Eagle Medium (DMEM), caspase-3 and caspase-8 colorimetric assay kits were
purchased from Invitrogen Bioservices, India. Petroleum ether, ethyl acetate and methanol were
purchased from Merck (Merck, India). Dimethyl sulfoxide (DMSO, ACS grade), trypsin
ethylenediaminetetraacetic acid (trypsin-EDTA), Tris-HCl buffer, EDTA, sodium dodecyl sulfate and
protenase-K were obtained from S.D. Fine Chemicals, India. Huh-7 and RAW 264.7 cell lines were
purchased from ATCC (Manassas, VA, USA). Water was purified using a Milli-Q water purification
system (Millipore, Bedford, MA, USA). All other reagents were purchased from Sigma Aldrich, India.
Plant Materials, Extraction and Isolation
Seeds of MP were collected from eastern part of Uttar Pradesh, India and authenticated by Department of
Applied Plant Science, Babasaheb Bhimrao Ambedkar University, Lucknow. A voucher specimen of this
sample was deposited in the Department for future use (42/DAPS/BBAU/2014). The air dried seeds were
crushed and powered with were crushed by using a mixer grinder (Bajaj Appliances Pvt. Ltd., India).
About 750 g of powder was defatted with petroleum ether (60-80°C) and further extracted with methanol
(Tadesse et al., 2012). The pooled methanol extract was evaporated in vacuo using rotary evaporator (RV10, IKA, Germany) to give a residue of (21 g). Then, this extract was further chromatographed with
neutral alumina column chromatography with gradient elution of petroleum ether (60-80°C) and ethyl
acetate in a ratio of 90: 10, 50:10 and 20:10. The last fraction gave one white power which was
recrystalised with ethyl acetate (white solid, yield 105 mg, M1, Figure 1A).
Docking studies
Docking studies of M1 was performed with antiproliferative molecular targets namely caspase-3 (PDB:
1QXE) and caspase-8 (PDB: 1IBC) by using Autodock Vina. Before the docking study, we have
identified the active site domain with the help of DogP acitive Site recognizer. Grid box was set according
to best configuration of active site of amino acid sequence. Binding affinities (kcal/mol) and count of
probable hydrogen bonds were evaluated in this experiment.
Antiproliferative activity using Huh-7 cells
MTT [3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole] viability assay was
performed as described previously (Saha et al. 2010). Briefly, Huh-7 cells were seeded at 5.0×103
cells/well in 96-well plates. After 24 h, varying concentrations of MTX and M1 and (0.5 to 100 µM)
(n=6) in media was added to each well and the samples were incubated in a humidified condition 5% CO2
at 37°C for 72 h. After that, MTT solution (1 mg/ml, 50 µl) was added to each well and incubated for
another 4 h. Later, MTT and media were removed and 150 µl of DMSO was added. The absorbance was
measured at 570 nm wavelength using Multiskan™ GO Microplate Spectrophotometer (ThermoFisher
Scientific, MA, USA). Control and Blank sample was prepared using with and without cells containing
media. The final organic solvent concentration was 0.3% (v/v). Cell survival (% of control) was
calculated relative to untreated control cells. Data were analyzed by Graph Pad Prism 5 and represented
as the mean ± S.D. Statistical differences were analyzed using paired T–test and P<0.05 represented the
statistically significant data.
Caspase 3 and 8 colorimetric assay
Apoptotic activity in Huh-7 cells was determined using caspase-3 and caspase-8 colorimetric
assay kits which published previously (Saha et al. 2012). Briefly, 2x104 cells/well were seeded in 96-well
microplates and incubated for 24 h. Next day, varying concentrations of test compounds (0.5 to 10 µM)
were added to each well and incubated for another 24 h at 37°C (n=4). After 24 h treatment, 100 ml of
caspase-3 or caspase-8 reagents was added and the mixture further incubated for 2 h at room temperature.
Relative luminescence (RLU) was recorded using Multiskan™ GO Microplate Spectrophotometer at 405
nm wavelength and results expressed as fold change vsersus control. Statistical differences were analyzed
using one way-ANOVA and P<0.05 represented the statistically significant data.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 5.0 (San Diago, CA, USA). All results were
expressed as mean ± standard deviation (SD). The data was analyzed by one-way ANOVA (analysis of
variances) followed by Bonferroni multiple comparison test.
References
Saha S, Chan DSZ, Lee CY, Wong W, New LS, Chui WK, Yap CW, Chan ECY, Ho HK. 2012.
Pyrrolidinediones reduce the toxicity of thiazolidinediones and modify their anti-diabetic and anticancer properties. Eur J Pharmacol. 697:13–23.
Saha S, New LS, Ho HK, Chui WK, Chan ECY. 2010. Investigation of the role of the thiazolidinedione
ring of troglitazone in inducing hepatotoxicity. Toxicol Lett. 192:141–149.
Getahun T, Reneela P, Aman D. 2012. Isolation and characterization of natural products from Helinus
mystachnus (Rhamnaceae). J Chem Pharm Res. 4:1756-1762.
Figure S1. Docking images of M1 with caspase-3 (A) and caspase-8 (B) ligands. The mesh round circular
showed the binding affinity of ligand with amino acids involved in binding poses.
(A)
(B)
Figure S2. Viability of Huh-7 cells after treatment with M1 at varying concentrations for 72 h (n=6).
MTT was used as detecting reagents. Viability is expressed as a percentage of vehicle-treated control and
data was expressed as mean ± S.D. The calculated EC50 values for MTX and M1 were 2.99±0.11 and
13.97±0.13 µM, respectively. Statistically significant differences were observed between MTX and M1
(paired t-test, P<0.05) at and above 1.0 µM concentration.
% cell viability
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Figure S3. Characterizing the antiapoptotic properties of (A) and (C) MTX, (B) and (D) M1. Huh-7 cells
were treated with at concentrations of 0.5, 1, 5, 10, µM for 24 h and subjected to caspase-3 and capase-8
activity determination (n=3). Activity was expressed as relative luminescence units (RLU) and compared
against a DMSO-containing vehicle control. For, caspase-3, no statistical differences were demonstrated
between control and test groups (one-way ANOVA, P<0.001) at all concentrations. For caspase-8,
statistically significant differences were also observed between control and test groups at and above 1.0
µM (one-way ANOVA, P<0.001).
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