SUPPLEMENTARY MATERIAL
Chemical composition, antibacterial and anticancer activities of volatile oil of Melicope
denhamii leaves
Sony Georgea, S Ajikumaran Naira, Ramaswamy Venkataramanb and Sabulal Babya*
a
Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic
Garden and Research Institute, Pacha-Palode, Thiruvananthapuram 695 562, Kerala, India;
b
Department of Chemistry, Sri Paramakalyani College (Manonmaniam Sundaranar
University, Tirunelveli), Alwarkurichi 627 412, Tamil Nadu, India
Abstract
Melicope denhamii leaf volatile oil was isolated by hydrodistillation, and twenty six
constituents comprising 95.95% of the leaf oil were characterized by gas chromatographic
techniques. Sesquiterpenes, zierone (22.49%) and -gurjunene (19.96%), were identified as
the major components. M. denhamii leaf oil tested against Gram +ve and Gram -ve bacteria
showed significant activity against Bacillus subtilis and Escherichia coli. Anticancer activity
of M. denhamii leaf oil against Dalton’s Lymphoma Ascites cells was assessed by trypan blue
exclusion and MTT assays, and the oil showed significant cytotoxicity at CD50 of 12.2
µg/mL. Induction of apoptosis on DLA cells by M. denhamii leaf oil was confirmed by
morphological observation, nuclear damage and comet assays.
*Corresponding author
Dr. Sabulal Baby
E-mail: sabulal@gmail.com
Tel: +91-472-2869226 ext. 214
Fax: +91-472-2869646
3. Experimental
3.1. General
Column chromatography: silica gel (SiO2; 100-200 mesh; SD Fine-Chem., Mumbai, India).
Refractive index: J257 Digital Refractometer, Rudolph Research Analytical, USA. Specific
rotation: Autopol IV Polarimeter, Rudolph Research Analytical, USA. 1H-NMR: 400 MHz
FT-NMR spectrometer, Bruker, Germany, in CDCl3, in ppm rel. to TMS. RPMI-1640
medium, phosphate buffered saline (PBS), trypan blue, streptomycin, penicillin, low melting
and high melting agar were purchased from Himedia, Mumbai, India and DMSO from SRL,
Mumbai, India. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT),
agarose, acridine orange, ethidium bromide, α-gurjunene and vincristine were purchased
from Sigma Aldrich, USA. Fetal bovine serum was purchased from Invitrogen, USA. All the
chemicals and reagents used were analytical grade.
3.2. Plant collection
Mature M. denhamii leaves were collected from Jawaharlal Nehru Tropical Botanic Garden
and Research Institute (JNTBGRI), Palode, Thiruvananthapuram, Kerala, India in February,
2011. Plant specimen was identified by Dr. E.S. Santhosh Kumar and a voucher specimen
No. 79305 was deposited in the Herbarium of JNTBGRI.
3.3. Essential oil isolation
M. denhamii fresh leaves (250 g) were hydrodistilled for 4 h using a Clevenger apparatus.
Isolated leaf oil was collected in diethyl ether, dried over anhydrous sodium sulfate and
refrigerated until analyzed.
3.4. Gas Chromatography-Mass Spectroscopy
GC-MS analysis was performed by split less injection of 1.0 μL of M. denhamii leaf oil on a
Hewlett Packard 6890 Gas Chromatograph fitted with an HP-5 (5% phenyl 95% dimethyl
polysiloxane, non-polar, 30 m x 0.32 mm, i.d., 0.25 m film thickness) capillary column,
coupled with a Model 5973 mass detector. GC-MS operation conditions: injector
temperature, 220oC; transfer line, 240oC; oven temperature programme, 60-246oC (3oC/min);
carrier gas, He at 1.4 mL/min. Mass spectra: Electron Impact (EI+) mode, 70 eV with a mass
range of 40 to 450 m/z; ion source temperature, 240oC. Linear retention indices (LRIs) of
constituents of M. denhamii leaf oil were determined in the HP-5 column using standard C5-
C30 hydrocarbons (Aldrich Chemical Company, USA). The oil constituents were identified
by mass spectral database match, comparison of spectra with literature and comparison of
linear retention indices (LRI) (Adams 2007; Dool and Kratz 1963).
3.5. Gas Chromatography-Flame Ionization Detection
M. denhamii leaf oil (100 L) was diluted to 3 mL using acetone and GC-FID analysis was
carried out by repeatedly (n = 4) injecting 1 L each of this solution onto a Shimadzu GC2010 Plus Gas Chromatograph with AOC-20i autoinjector and FID (Shimadzu, Japan), fitted
with an Rxi-5Sil MS capillary column (5% phenyl and 95% dimethyl polysiloxane, nonpolar, 30 m x 0.25 mm i.d., 0.25 m film thickness; Restek, USA). GC operation conditions:
injection mode, split; split ratio, 50; injector temperature, 270oC; oven temperature
programme, 60-250oC (3oC/min); hold time 2 min. at 250oC; carrier gas, N2 at 3 mL/min and
detector temperature 270oC. Relative percentages of individual components were calculated
from the peak area-percent report of volatiles from GC-FID data (n = 4).
3.6. Isolation of major constituent, identification and Co-GC
M. denhamii leaf oil (200 L) was subjected to column chromatography using silica gel (100200 mesh) and eluting with 100% petroleum ether (40-60) to 2% ethyl acetate in 10 mL
fractions. Fractions 29-32 on concentration yielded compound 1 (5 mg). Compound 1 was
identified as zierone using 1H-NMR. Co-injection of M. denhamii leaf oil with the authentic
standards of zierone and -gurjunene confirmed them as its two major components (Table 1).
3.7. Antibacterial assay
M. denhamii leaf oil was tested for antibacterial activity using the disc agar diffusion assay
(Sabulal et al. 2006). Gram (+)ve bacteria: Bacillus subtilis (two strains), Bacillus cereus,
Streptococcus mutans (two strains) and Gram (-)ve bacteria: Salmonella typhi, Pseudomonas
aeruginosa, Pseudomonas fluorescens, Proteus vulgaris, Serratia marcescens, Klebsiella
pneumoniae, Escherichia coli were obtained from the Institute of Microbial Technology
(IMTECH), Chandigarh, India as Microbial Type Culture Collection (MTCC) (Table 2). M.
denhamii leaf oil at 1:1 and 1:2 dilutions in dimethyl sulfoxide were tested against these
bacteria. Control discs impregnated with 10 μL of DMSO (inert solvent) and streptomycin
(reference for bacteria) at 2 μg/disc were used alongside the test discs in each experiment.
3.8. Cell lines
Dalton’s Lymphoma Ascites (DLA) cells, obtained from Amala Cancer Research Centre,
Thrissur, India were maintained as transplantable tumors in the peritoneal cavity of mice.
3.8.1. Collection of thymocytes
Swiss albino mice were sacrificed by cervical dislocation and thymus glands were carefully
separated without adjoining lymph nodes. The separated thymus glands were transferred to
RPMI-1640 and single cell suspension of thymocytes was prepared. Viability was assessed
by trypan blue exclusion method using a Neubauer counting chamber.
3.8.2. Collection of peritoneal macrophages
Swiss albino mice were sacrificed by cervical dislocation and immediately injected with 5
mL chilled RPMI-1640 medium to the peritoneal cavity and peritoneal exudate cells (PEC)
were collected. The glass adherent cell population (macrophages) was separated and viability
was assessed by trypan blue exclusion method using a Neubauer counting chamber.
3.8.3. Collection of bone marrow cells
Swiss albino mice were sacrificed by cervical dislocation, femur bones were separated
immediately and flushed with 5 mL chilled RPMI-1640 medium using a syringe fitted with
23 gauge (0.6 x 25 mm) needle. The flushed medium containing bone marrow cells was
collected and cell viability was assessed by trypan blue exclusion method using a Neubauer
counting chamber.
3.9. Preliminary cytotoxicity evaluation of M. denhamii leaf oil on DLA cells in vitro
The cytotoxicity of M. denhamii leaf oil on DLA cells was assessed by incubating 106 DLA
cells in 1 mL PBS containing vehicle (DMSO 0.01%) or different concentrations of leaf oil
(5, 10, 25, 50, 100 µg/mL) in 24 well plates for 3 h at 37oC in a water bath incubator. After 3
h of incubation, the cell viability was assessed by trypan blue exclusion method using a
Neubauer counting chamber (Shylesh et al. 2005). The cells appeared in blue colour were
counted as dead, while cell unstained were counted as live.
3.10. Determination of in vitro cytotoxicity of M. denhamii leaf oil on various cell types
Cytotoxicity of M. denhamii leaf oil on various cell types were assessed by incubating 1 mL
of RPMI medium supplemented with 10% FBS, streptomycin (100 µg/mL) and penicillin
(100 units/mL) seeded with 106 cells/mL of
DLA cells or peritoneal macrophages or
thymocytes or bone marrow cells with vehicle (DMSO 0.01%) or different concentrations of
leaf oil (10, 25 µg/mL) or vincristine (10, 25 µg/mL) in 24 well plates for 48 h in a CO 2
incubator at 37oC, 5% CO2, 95% air and 95% relative humidity. After 48 h of incubation the
cell viability was assessed by trypan blue exclusion method using a Neubauer counting
chamber. The cells appeared in blue colour were counted as dead, while cell unstained were
counted as live (Shylesh et al. 2005).
3.11. Determination of in vitro cytotoxicity of M. denhamii leaf oil on DLA cells by MTT
assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed
as described elsewhere (Shylesh et al. 2005). Briefly, each well of 24 well plates containing
1 mL of RPMI medium supplemented with 10% FBS, streptomycin (100 µg/mL) and
penicillin (100 units/mL) was seeded with 106 DLA cells and incubated with vehicle (DMSO
0.01%) or different concentrations of
leaf oil (5, 10, 25, 50, 100 µg/mL) or standard
anticancer drug, vincristine (5, 10, 25, 50, 100 µg/mL) for 48 h in a CO2 incubator at 37oC,
5% CO2, 95% air and 95% relative humidity. After incubation, 100 µL of 5 mg/mL MTT
solution was added to each well and cells were incubated for an additional 4 h. After final
incubation, the spent media was removed from the wells and the MTT formazan product was
dissolved in DMSO and optical density was measured at 570 nm using an ELISA plate
reader.
3.12. Apoptosis
DLA cells (106 cells/mL) incubated in a 24 well plate containing RPMI medium (1 mL/well)
supplemented with 10% FBS, streptomycin (100 µg/mL) and penicillin (100 units/mL) were
treated with vehicle (DMSO 0.01%) or M. denhamii leaf oil (25 µg/mL) or standard
anticancer drug, vincristine (25 µg/mL) in a CO2 incubator for 48 h at 37oC, 5% CO2, 95% air
and 95% relative humidity. After incubation, the DLA cells were observed under phase
contrast microscope to assess nuclear condensation and morphological changes of the cells.
Membrane blebbing and cell death on DLA cells were evaluated by fluorescent microscope.
Briefly, the treated cells were washed with PBS and mixed with acridine orange/ethidium
bromide stain (50 µg/mL). After staining, the cells were observed under an inverted
fluorescent phase contrast microscope (Olympus, USA) using blue filter and photographed
with digital camera for morphological changes and cell death (Shylesh et al. 2005). The
apoptotic DLA cells appeared in yellowish red colour whereas normal live DLA cells in
green colour.
3.13. Comet assay
Comet assay was carried out as described elsewhere (Arunkumar et al. 2012). Briefly, DLA
cells (106 cells/mL) were treated with vehicle (DMSO 0.01%) or M. denhamii leaf oil (25
µg/mL) or standard anticancer drug, vincristine (25 µg/mL), in 24 well plate for 48 h in
RPMI (1 mL/well) medium supplemented with 10% FBS, streptomycin (100 µg/mL) and
penicillin (100 units/mL) in a CO2 incubator with 5% CO2 at 37oC. After incubation DLA
cells were harvested, washed and suspended in PBS (pH 7.3). Cell suspension (10 µL, 10,000
cells) were added to 75 µL of low melting point agar, mixed thoroughly and spread uniformly
over the normal melting agar in a frosted slide. Over the cell paved layer, a third layer of low
melting point agar was added and a cover slip was placed over it after solidification. The
prepared slides were dipped in lysis buffer for 2 h and kept in electrophoresis buffer (pH
13.0) for 20 min. The treated slides were electrophoresed in a horizontal electrophoresis unit.
The slides were neutralized by drop wise addition of neutralization buffer (pH 7.5) and
stained with ethidium bromide (20 µg/mL). The stained slides were observed under
fluorescent microscope using green filter to asses DNA damage.
References
Adams
RP.
2007.
Identification
of
essential
oil
components
by
gas
chromatography/quadrupole mass spectroscopy. Carol Stream (IL): 4th ed. Allured
Publishing Corporation.
Arunkumar R, Ajikumaran SN, Subramoniam A. 2012. Induction of cell-specific apoptosis
and protection of mice from cancer challenge by a steroid positive compound
from Zornia diphylla (L.) Pers. J Pharmacol Pharmacother. 3:233-241.
Dool HV, Kratz PD. 1963. A generalization of the retention index system including linear
temperature programmed gas-liquid partition chromatography. J Chromatogr A. 11:463471.
Sabulal B, George V, Shiburaj S. 2006. Volatile constituents and antibacterial activity of the
flower oil of Evodia lunu-ankenda (Gaertn) Merr. J Essent Oil Res. 18:462-464.
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Table S1. Chemical composition of the leaf oil of Melicope denhamii.
Compound
FID (%)a
LRI
LRI
(Cal)
(Lit)
α-Copaene
1370
1374
2.50 ± 0.04
A, B, C
α-Gurjunene
1386
1409
19.96 ± 0.11
A, B, C, D
β-Elemene
1391
1389
8.18 ± 0.05
A, B, C
Cyperene
1394
1398
0.57 ± 0.01
A, B, C
β-Longipinene
1401
1400
1.31 ± 0.02
A, B, C
E-Caryophyllene
1417
1417
3.35 ± 0.09
A, B, C
β-Duprezianene
1421
1421
1.54 ± 0.14
A, B, C
Cumacrene
1472
1470
1.34 ± 0.05
A, B, C
γ-Gurjunene
1478
1475
0.67 ± 0.02
A, B, C
β-Vetispirene
1494
1493
0.68 ± 0.03
A, B, C
trans-Cycloisolongifol-5-ol
1511
1513
1.82 ± 0.02
A, B, C
Zierone
1576
1574
22.49 ± 0.16
A, B, C, D, E
Spathulenol
1584
1577
2.08 ± 0.06
A, B, C
Isoaromadendrene oxide
1590
-
4.28 ± 0.04
A
β-Biotone*
1599
1607
0.84 ± 0.02
A, B
trans-isolongifolanone*
1622
1625
1.26 ± 0.06
A, B
allo-Aromadendrene epoxide
1640
1639
0.57 ± 0.34
A, B, C
epi-Zizanone
1662
1668
0.65 ± 0.03
A, B, C
(Z)-α-Santalol
1668
1674
2.76 ± 0.05
A, B, C
Cyperotundone
1697
1695
1.58 ± 0.12
A, B, C
Nootkatol
1703
1714
3.93 ± 0.02
A, B, C
Curcumenol
1721
1733
7.69 ± 0.09
A, B, C
Cyclocolorenone
1760
1759
2.02 ± 0.09
A, B, C
2,4,6-Tris-tert-butylphenol*
1872
-
1.16 ± 0.03
A
Alloevodionol
1924
-
1.02 ± 0.04
A
Phytol
2083
-
1.70 ± 0.08
A
Number, % of constituents identified
Identification
techniquesb
26, 95.95%
Sesquiterpene hydrocarbons (%)
40.10
Oxygenated sesquiterpenes (%)
51.97
Others (diterpene, chromenes, phenolics %)
3.88
a
) n = 4;
b
) A - GC-MS mass spectral database match, B - LRI calculation, C -
Adams Index, D - Co-GC with authentic samples, E - NMR; * - tentatively
identified.
Table S2. Antibacterial activity of the leaf oil of Melicope denhamii.
Test organisms
MTCC
Leaf oil
Leaf oil
Streptomycin
No
(1:1 dilution)
(1:2 dilution)
(2 g/disc)
Zone of inhibition (mm)a
Gram +ve bacteria
Bacillus subtilis
96
8
7
15
Bacillus subtilis
441
18
16
21
Bacillus cereus
430
8
7
17
Streptococcus mutans
890
9
8
24
Streptococcus mutans
497
11
10
26
Salmonella typhi
733
6
No zone
16
Salmonella typhi
734
8
No zone
22
Pseudomonas fluorescens
103
10
8
17
Pseudomonas aeruginosa
741
No zone
No zone
15
Proteus vulgaris
426
9
7
20
Serratia marcescens
97
10
8
17
Klebsiella pneumoniae
109
No zone
No zone
16
Escherichia coli
443
9
9
10
Gram -ve bacteria
a
) Diameter of zone of inhibition in mm excluding the diameter of the disc.
Table S3. Cytotoxicity of Melicope denhamii leaf oil on DLA cells by trypan blue exclusion
assay.
Test material
M. denhamii
% cell death
leaf oil (µg/mL)
Control
0
0
5
23 ± 1.3
10
53 ± 2.0
25
100
50
100
100
100
(0.01% DMSO)
Leaf oil
Values are mean ± S.D. of three separate determinations. DLA cells were incubated at 37 oC
in phosphate buffered saline (pH 7.3) for 3 h.
Table S4. Cytotoxicity of Melicope denhamii leaf oil on different cell types.
Test material
M. denhamii
leaf oil
% Cell death
DLA
(µg/mL)
Leaf oil
Vincristine
Macrophage
Thymocytes
s
Bone marrow
cells
10
51 ± 2.0
9 ± 1.0
11 ± 1.3
9 ± 2.0
25
100
14 ± 2.0
18 ± 1.0
20 ± 2.3
10
100
18 ± 3.0
15 ± 2.0
19 ± 3.3
25
100
26 ± 2.0
21 ± 2.3
31 ± 3.0
Values are mean ± S.D. of three separate determinations. Cells were incubated at 37oC for 48
h in RPMI media in CO2 incubator. Percentage cell death was zero for control (0.01%
DMSO).
Zierone
α-Gurjunene
Figure S1. Major components in M. denhamii leaf oil, zierone and α-gurjunene.
Figure S2. [i] DLA cells stained with acridine orange-ethidium bromide under fluorescent
microscope: (a) DLA cells treated with DMSO (0.01%) appeared in green color (live), (b)
DLA cells treated with M. denhamii leaf oil (25 μg/mL) appeared in yellowish red (dead
cells), (c) DLA cells treated with vincristine (25 μg/mL) appeared in yellowish red (dead
cells); [ii] Morphological changes under phase contrast microscopy: (d) DLA cells treated
with DMSO (0.01%) showed no membrane blebbing and nuclear condensation, (e) DLA cells
treated with M. denhamii leaf oil (25 μg/mL) showed membrane blebbing and nuclear
condensation, (f) DLA cells treated with vincristine (25 μg/mL) showed membrane blebbing
and nuclear condensation; [iii] DLA cells in comet assay (single cell gel electrophoresis)
viewed under fluorescent microscopy: (g) DLA cells treated with DMSO (0.01%) showed
nuclear integrity, (h) DLA cells treated with M. denhamii leaf oil (25 μg/mL) showed nuclear
DNA damage and comet formation, (i) DLA cells treated with vincristine (25 μg/mL) showed
nuclear DNA damage and comet formation.