Professional Documents
Culture Documents
0250-7005/2007 $2.00+.40
3293
Family
Place of Collection
Voucher No.
% w/w
Labiatae
Labiatae
Labiatae
Labiatae
Lauraceae
Lauraceae
Ayoun-kourkoush
Tarshich
Ain-Saad
Ain-Saad
Nahr-Ibrahim
Nahr-Ibrahim
AS2
AS4
AS10
AS13
AS14
AS14
1.5
1.8
2
2.25
2.9
3
3294
Cancer cell lines. The human amelanotic melanoma cell line C32,
(American Type Culture Collection, ATCC, Rockville, MD, USA)
(ATCC No: CRL 1585), renal cell adenocarcinoma ACHN (ATCC
No: CRL-1611) and hormone-dependent prostate carcinoma
LNCaP (ATCC No: CRL-1740) were cultured in RPMI-1640
medium while the human breast cancer cell line MCF-7 (ATCC
No: HTB-22D) was cultured in DMEM medium. Both media were
supplemented with 10% foetal bovine serum, 1% L-glutamine and
1% penicillin/streptomycin (Sigma-Aldrich, Milan, Italy). The cell
lines were maintained at 37C in a 5% CO2 atmosphere and 95%
humidity. The cultures were passed once a week by trypsinization
using a 1:30 dilution of standard trypsin-EDTA solution (Gibco,
Milan, Italy).
Assessment of cytotoxicity. The cytotoxic assay was performed as
previously described (22). The protein-staining sulforodamine B
(SRB) assay was used for measurement of cell proliferation. The
test is based on the estimation of cell number indirectly by
providing a sensitive index of total cellular protein content which
is linear to cell density. The cells were trypsinized, counted and
placed in 96-well plates at optimal plating density of each cell line
was determined over a range from 5x104 to 15x104 ensuring
exponential growth throughout the experimental period and a
linear relationship between absorbance at 490 nm and cell number
where analysed by the SRB assay, and incubated to allow for cell
attachment. After 24 h, the cells were treated with serial dilutions
of the samples. Each sample was initially dissolved in DMSO and
further diluted in medium to produce different concentrations.
One hundred microliters/well of each dilution were added to the
plates in six replicates to obtain the final concentrations ranging
from 5 to 400 g/ml for the essential oils, and from 2 to 50 g/ml
for the commercially available identified constituents (linalool,
limonene, 1,8-cineole, trans-caryophyllene, and -humulene). The
final mixture used for treating the cells contained not more than
0.5% of the solvent (DMSO), the same as in the solvent-control
wells. After 48 h of exposure, 100 l of ice-cold 40%
trichloroacetic acid (TCA) was added to each well, left for 1 h at
4C, and washed with distilled water. The TCA-fixed cells were
stained for 30 min with 50 l of 0.4 (w/v)% SRB in 1% acetic acid.
The plates were washed with 1% HOAc and air dried overnight.
For reading of the plate, the bound dye was solubilised with 100 l
of 10 mM tris base (tris[hydroxymethyl]aminomethane). All
products were purchased from Sigma-Aldrich. The absorbance of
each well was read on a Molecular Devices SpectraMax Plus Plate
Results
3295
Compound
-Thujene
0.890.11
-Pinene
10.150.32
Camphene
1.080.09
Sabinene
8.640.15
-Pinene
2.900.18
-Myrcene
0.680.03
-Phellandrene
tr
-3-Carene
-Terpinene
1.100.12
p-Cymene
10.760.53
-Phellandrene
Limonene*
0.570.09
1,8 Cineole*
0.280.06
-Ocimene
-Terpinene
7.560.11
-Terpinolene
0.620.15
Fenchone
Linalool*
2.810.11
-Thujone
0.080.11
-Isothujone
cis-p-2-Menthen-1-ol
-Campholene aldehyde
0.250.11
Camphor
Terpinen-4-ol
-Terpineol
1.530.16
Isoborneol
cis-Piperitol
0.100.06
Isopulegone
0.100.02
Methyl thymylether
Pulegone
tr
Neryl acetate
0.260.03
Bornyl acetate
-Terpinyl acetate
Thymol
9.920.07
-Fenchyl acetate
Carvacrol
4.980.08
1-p-Menthen-8-yl acetate
0.400.08
-Cubebene
Eugenol
-Ylangene
-Copaene
1.670.13
-Bergamotene
-Bourbonene
0.240.05
-Elemene
0.210.01
Methyl eugenol
Zingiberene
-Gurjunene
0.510.06
trans-Caryophyllene*
3.670.11
Aromadendrene
-Farnesene
-Humulene*
0.340.03
trans--Farnesene
Epi-bicyclosesquiphellandrene1.680.14
Ar-curcumene
-Cubebene
-Zingibirene
-Guaiene
-
0.500.06
8.660.14
0.110.08
12.760.34
8.900.23
1.480.09
0.720.07
0.440.06
32.850.64
0.180.09
1.110.11
0.220.05
0.720.13
0.270.11
0.140.04
-
0.100.01
3. 670.03
1.690.04
1.640.03
2.140.01
0.560.01
0.110.07
0.150.01
0.120.05
0.100.01
9.430.07
21.830.18
0.100.01
0.120.02
0.350.04
0.400.02
0.310.01
0.230.01
0.230.08
0.170.01
0.100.04
1.00.06
0.320.04
0.100.01
0.130.01
-
0.440.09
5.720.12
0.140.06
6.170.14
3.460.08
0.450.10
0.120.03
0.860.05
2.230.11
1.100.08
35.150.36
0.080.005
1.500.09
0.490.07
7.080.14
4.420.07
2.420.04
4.430.10
13.520.25
3.730.16
0.170.06
0.100.08
2.520.04
0.380.08
0.220.03
0.920.05
6.810.12
0.390.01
17.080.25
6.480.09
1.130.03
tr
3.600.06
6.010.09
8.560.11
6.330.12
2.860.06
0.320.05
2.430.08
0.380.02
2.050.05
0.520.03
0.220.01
0.200.01
0.100.01
0.480.01
0.630.01
3.990.01
0.290.01
2.400.03
-
0.160.01
4.720.11
2.550.08
6.970.21
3.010.14
tr
0.170.01
1.080.04
0.120.07
0.670.07
0.920.06
4.110.22
0.300.05
0.490.12
1.150.18
0.470.09
1.760.13
0.350.06
-
1.200.05
43.620.44
0.390.08
tr
12.990.13
1.480.02
5.710.12
3.180.32
0.240.06
1.050.07
0.990.04
3.410.45
tr
Table II continued
3296
Table II continued
Essential oils
tR
Compound
14.89
15.01
15.09
15.15
15.21
15.54
15.67
15.76
15.88
15.90
16.14
16.27
16.44
16.61
17.37
17.76
18.64
19.31
19.39
20.82
Germacrene D
-Selinene
-Muurolene
-Cadinene
-Cadinene
-Guaiene
Palustrol
Spathulenol
-Gurjunene
Viridiflorol
-Guaiene
Junipene
-Maaliene
-Bisabolol
Oxo--Ylangene
Neophytadiene
Biformene
Eremanthin
Rimuene
Dehydrocostus lactone
Identification
1
0.110.02
0.370.04
3.110.12
0.990.08
0.610.07
0.780.04
0.150.07
0.100.05
0.710.24
80.91
2
2.160.32
0.570.14
0.570.13
0.880.11
1.170.15
0.810.11
1.010.35
0.100.08
86.67
3
0.360.02
0.140.01
3.650.09
7.570.12
56.82
0.310.02
-
1.510.07
0.330.11
0.160.03
3.080.04
-
97.21
79.26
tr
0.10.01
1.160.05
0.110.37
94.29
(1) Satureia thymbra, (2) Sideritis perfoliata, Laurus nobilis (3) fruits and (4) leaves (5) Pistacia palestina, and (6) Salvia officinalis. tR: Retention
time (as min). *Identification confirmed with authentic standard. % identification: peak area identified relative to total peak area %). tr: <0.05%.
Satureja thymbra L.
Sideritis perfoliata L.
Laurus nobilis L. (fruits)
Laurus nobilis L. (leaves)
Pistacia palestina Boiss.
Salvia officinalis L.
Cell line
C32
ACHN
154.301.2**
100.901.1**
75.451.2**
209.691.4**
356.981.3**
367.431.5**
155.881.1**
98.580.5**
78.241.5**
202.621.7**
204.701.8**
108.701.2**
MCF-7 LNCaP
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
Vinblastine (for ACHN, C32, and LNCaP,) and Taxol (for MCF-7)
were used as positive control. Data are given as the mean of at least
three independent experimentsS.D. **p<0.01 vs. control.
Discussion
L. nobilis fruit oil exhibited the most interesting biological
activity on the ACHN and C32 cell growth inhibition, while
the activity of leaf oil was considerably less, which can be
attributed to the different composition with regards to the
predominant compounds of the oils. This result is of some
importance, since data on the cytotoxic activity of L. nobilis
are scarce. Costunolide and zaluzanin D, two of the
3297
3298
Conclusion
The six essential oils and some of their mono- and
sesquiterpenes had the ability to inhibit tumor cell growth.
Further evaluation is warranted to clarify the mechanism of
action and their potential use.
In Memoriam
This work is dedicated to the memory of Professor Dr. C.F. Pollera,
who was an inspiration to scientists and played a key role in Clinical
Oncology, not only in Italy, but also in the international arena.
References
1 Heinrich M and Bremner P: Ethnobotany and ethnopharmacytheir role for anti-cancer drug development. Curr Drug Targets
7: 239-245, 2006.
2 Cordell GA, Beecher CW and Pezzuto JM: Can ethnopharmacology contribute to the development of new anticancer
drugs? J Ethnopharmacol 32: 117-33, 1991.
3 Heinrich M and Gibbons S: Ethnopharmacology in drug
discovery: an analysis of its role and potential contribution. J
Pharm Pharmacol 53: 425-432, 2001.
4 Cavalieri E, Mariotto S, Fabrizi C, de Prati AC, Gottardo R,
Leone S, Berra LV, Lauro GM, Ciampa AR and Suzuki H:
alpha-Bisabolol, a nontoxic natural compound, strongly induces
apoptosis in glioma cells. Biochem Biophys Res Commun 315:
589-594, 2004.
5 de Sousa AC, Alviano DS, Blank AF, Alves PB, Alviano CS and
Gattass CR: Melissa officinalis L. essential oil: antitumoral and
antioxidant activities. J Pharm Pharmacol 56: 677-681, 2004.
6 Prashar A, Locke IC and Evans CS: Cytotoxicity of lavender oil
and its major components to human skin cells. Cell Prolif 37:
221-229, 2004.
7 Yoo CB, Han KT, Cho KS, Ha J, Park HJ, Nam JH, Kil UH
and Lee KT: Eugenol isolated from the essential oil of Eugenia
caryophyllata induces a reactive oxygen species-mediated
apoptosis in HL-60 human promyelocytic leukaemia cells.
Cancer Lett 225: 41-52, 2005.
8 Sigurdsson S, Ogmundsdottir HM and Gudbjarnason S: The
cytotoxic effect of two chemotypes of essential oils from the fruits
of Angelica archangelica L. Anticancer Res 25: 1877-1880, 2005.
9 Prashar A, Locke IC and Evans CS: Cytotoxicity of clove
(Syzygium aromaticum) oil and its major components to human
skin cells. Cell Prolif 39: 241-248, 2006.
10 Lampronti I, Saab AM and Gambari R: Antiproliferative
activity of essential oils derived from plants belonging to the
Magnoliophyta division. Int J Oncol 29: 989-995, 2006.
11 Gomez-Serranillos MP, El-Naggar T, Villar AM and Carretero
ME: Analysis and retention behaviour in high-performance
liquid chromatography of terpenic plant constituents (Sideritis
spp.) with pharmacological interest. J Chromatogr B Analyt
Technol Biomed Life Sci 812: 379-383, 2004.
3299