SUPPLEMENTARY MATERIAL
Cytotoxic constituents of Abutilon indicum leaves against U87MG human
glioblastoma cells
Rukaiyya Sirajuddin Khana, Mahibalan Senthia, Poorna Chandra Raoa, Ameer Bashab,
Mallika Alvalac, Dinesh Tummuric, Hironori Masubutid, Yoshinori Fujimotod and Ahil Sajeli
Beguma*
a
Department of Pharmacy, BITS-Pilani Hyderabad Campus, Jawahar Nagar, Shamirpet
Mandal, Hyderabad-500078, Telangana State, India;
b
Regional Agricultural Research
Station (ANGRAU), Palem, Mahabub Nagar District, Telangana State, India;
c
National
Institute of Pharmaceutical Education and Research-Hyderabad, Balanagar, Hyderabad500037, Telanagana, India;
d
Department of Chemistry and Materials Science, Tokyo
Institute of Technology, Meguro, Tokyo 152-8551, Japan.
* Corresponding author. Email: sajeli@hyderabad.bits-pilani.ac.in
The study was aimed to identify cytotoxic leads from Abutilon indicum leaves for treating
glioblastoma. The petroleum ether extract (AIP), methanol extract (AIM), chloroform and ethyl
acetate sub-fractions (AIM-C and AIM-E, respectively) prepared from AIM were tested for
cytotoxicity on U87MG human glioblastoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. These extracts exhibited considerable activity (IC50
values of 42.6–64.5 μg/mL). The most active AIM-C fraction was repeatedly chromatographed
to yield four known compounds, methyl trans-p-coumarate (1), methyl caffeate (2), syringic acid
(3) and pinellic acid (4). Cell viability assay of 1–4 against U87MG cells indicated 2 as most
active (IC50 value of 8.2 μg/mL), whereas the other three compounds were much less active.
Interestingly, compounds 1–4 were non-toxic towards normal human cells (HEK-293). The
content of 2 in the AIM-C was estimated as 3% by HPLC. Hence, presence of some more active
substances besides methyl caffeate (2) in AIM-C is anticipated.
Key Words: Abutilon indicum; MTT; U87MG; methyl caffeate; cytotoxicity; Malvaceae
1. Experimental Part
1.1. General
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay: MTT (Sigma–
Aldrich, Bangalore, India), PBS, RPMI medium and FBS (Gibco BRL, CA, USA). All the
other chemicals and reagents used were of analytical and molecular biology grade. Cell
viability: Multi-well plate reader (Victor3TM, Perkin Elmer). Lyophilization (CoolsafeTM,
Scanvac) Column chromatography (CC): silica gel (SiO2; spherical neutral (60-120 μm);
Merck chemicals). Rotary evaporation: Buchi Rotavapor R-210 (Switzerland). 1H- and 13CNMR spectra: Bruker DRX-500 spectrometer (500 and 125 MHz for 1H and
13
C, resp.) in
CDCl3 or CD3OD soln.; δ in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS:
Shimadzu LCMS-2020 Single beam Cod; in m/z. HPLC: Shimadzu class LC-20AD HPLC
system equipped with a Photo Diode Array (PDA) detector and a reverse phase (RP) column
(Phenomenex Kinetex 5 C-18 (100A); 250 mm × 4.6 mm).
1.2. Extraction of Plant Material
A. indicum leaves collected during the month of December from Jawahar Nagar village,
Shameerpet Mandal, Ranga Reddy District, Telangana, India was authenticated by Dr.
Chelladurai, Research Officer, C.C.R.A.S, Government of India, India. A specimen sample
(AI/2011/03) of raw material used for the investigation has been preserved in Department of
Pharmacy, BITS-Pilani Hyderabad Campus, Telangana, India. The leaves were shade dried
and pulverized by a mechanical grinder. Around 5.0 kg of powder was extracted with
petroleum ether (40-60 C, 3  1.5 L, 8 h) using Soxhlet apparatus. The petroleum ether
extract was then filtered and the filtrate was evaporated under reduced pressure. The dry
residue (127 g) was designated AIP. The marc was air-dried and extracted using methanol (3
 1.5 L, 50-55 C, 8 h). The methanolic extract was concentrated to one-eighth of the original
volume under reduced pressure using a rotary evaporator and lyophilized, which yielded 548
g of dry residue designated as AIM. Around 1.0 g of AIM was preserved for bioassay and the
remaining extract was fractionated against water using solvents of increasing polarities i.e.
hexane, chloroform (AIM-C), and ethyl acetate (AIM-E) and dried under reduced pressure
followed by lyophilization, the respective yields were found to be 68.7 g, 36.7 g, and 6.7 g.
1.3. Cell Culture and Treatment
U87MG (Malignant Glioma), HEK-293 (Human Embryonic Kidney) cells were grown in
RPMI-1640 supplemented with 10% heat inactivated FBS, 100 IU/mL penicillin, 100 mg/ml
streptomycin and 2 mM L-glutamine. Cultures were maintained in a humidified atmosphere
with 5% CO2 at 37C. The cultured cells were sub-cultured twice a week, seeding at a
density of about 2 × 103 cells/mL.
1.4. Cell Viability Assay
Cell viability was determined following the standard MTT assay. U87MG and HEK-293
cells (5 × 103 cells/well) were seeded to 96-well culture plate and cultured with and without
extract. The cell lines were treated with 10, 20, 30, 40 and 50 g/mL concentrations of AIP,
AIM, AIM-C and AIM-E in DMSO vehicle. Treatment was done for 72 h. At the end of
treatment, 20 L of MTT (5 mg/ml in PBS) was added to each well and incubated for an
additional 3 h at 37 ◦ C. The formazan crystals were dissolved in 100 L of DMSO and the
absorbance was read at 590 nm on a multi-well plate reader. Percent inhibition of
proliferation was calculated as a fraction of control.
1.5. Isolation of Chemical Constituents of AIM-C
Dried AIM-C (35.5 g) was chromatographed on SiO2 and eluted using solvent of increasing
polarity. Toluene: Ethyl acetate (0 – 10% ethyl acetate) eluate yielded fatty esters. The
Toluene: Ethyl acetate (15-25% ethyl acetate, 2 g) eluate showing positive color change with
FeCl3 reagent were pooled, which on rechromatography yielded four compounds, 4 (15.0
mg), 1 (4.8 mg), 2 (10.5 mg) and 3 (6.8 mg) in the order of elution. All compounds were
subjected for cell viability assay against U87MG and HEK-293 cells.
Methyl trans-p-coumarate (1)
Amorphous solid. M.p. 138-140 °C. 1H- and
13
C-NMR (CDCl3): in good agreement with
those reported data (Guzman et al. 2014). ESI-MS: 179 ([M+H]+).
Methyl caffeate (2)
Colourless crystals. M.p. 158-161 °C. 1H- and
13
C-NMR (CDCl3): in good agreement with
those reported data (Touafek et al. 2011). ESI-MS: 193 ([M-H]+).
Gallic acid 3,5-dimethyl ether (3)
Amorphous solid. M.p. 204-206 °C. 1H- and
13
C-NMR (CDCl3): in good agreement with
those reported values (Lee et al. 2010).
Pinellic acid (4)
Amorphous solid. M.p. 99-102 °C. 1H- and
13
C-NMR (CD3OD): in good agreement with
those reported values (Sunnam & Prasad, 2013). ESI-MS: 329 ([M-H]+).
1.6. HPLC quantification of 2 in AIM-C
The quantity of compound 2 present in AIM-C was analyzed by RP-HPLC with PDA
detector set at 250–350 nm under room temperature with a flow rate of 0.8 ml/min. The
mobile phase constituted a linear gradient from 60 to 40 % water in acetonitrile for 15 min. A
serial dilution of isolated compound 2 resulting to 100 ppm, 200 ppm, 300 ppm, 400 ppm
and 500 ppm solutions were used for preparing calibration curve (Concentration Vs Area
Under Curve). The amount of methyl caffeate (2) present in AIM-C was quantified from the
standard graph.
References
Guzman JD, Mortazavi PN, Munshi T, Evangelopoulos D, McHugh TD, Gibbons S,
Malkinson J, Bhakta S. 2014. 2-Hydroxy-substituted cinnamic acids and acetanilides
are selective growth inhibitors of Mycobacterium tuberculosis. Medicinal Chemistry
Communications. 5: 47–50.
Lee HJ, Khan M, Kang HY, Choi DH, Parkm MJ, Lee HJ. 2010. Rare natural products
from the wood of Magnolia grandiflora. Chemistry of Natural Compounds. 46:
289–290.
Sunnam SK, Prasad KR. 2013. Total synthesis of (+)-pinellic acid. Synthesis. 45: 1991–
1996.
Touafek O, Kabouche Z, Brouard I, Barrera Bermejo J. 2011. Flavonoids of Campanula
alata and their antioxidant activity. Chemistry of Natural Compounds. 46: 968–970.
Table S1: Cytotoxic effect of isolated compounds (50 M) against U87MG cells
Compound
1
2
3
4
Concentration in
g/mL
8.9
9.7
9.9
16.5
Percent growth
inhibition
6.00
51.13
9.24
12.36