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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*


aPhytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram 695 562, Kerala, India;

bDepartment 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 GC-2010 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, non-polar, 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 (100-200 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 CO2 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. 11:463-471.

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.

Shylesh BS, Ajikumaran SN, Subramoniam A. 2005. Induction of cell specific apoptosis and protection from Dalton’s lymphoma challenge in mice by an active fraction from Emilia Sonchifolia. Indian J Pharmacol. 37:232-237.

Table S1. Chemical composition of the leaf oil of Melicope denhamii.

Compound

LRI

(Cal)


LRI

(Lit)


FID (%)a

Identification techniquesb

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

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

No


Leaf oil

(1:1 dilution)



Leaf oil

(1:2 dilution)



Streptomycin

(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

Gram -ve bacteria















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


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

leaf oil (µg/mL)



% cell death

Control

(0.01% DMSO)



0

0


Leaf oil

5

23 ± 1.3

10

53 ± 2.0

25

100

50

100

100

100

Values are mean ± S.D. of three separate determinations. DLA cells were incubated at 37oC 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 (µg/mL)

% Cell death




DLA

Macrophages

Thymocytes

Bone marrow cells

Leaf oil

10

51 ± 2.0

9 ± 1.0

11 ± 1.3

9 ± 2.0

25

100

14 ± 2.0

18 ± 1.0

20 ± 2.3

Vincristine

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.


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