Phytochemical profile of Juniperus oxycedrus ssp. oxycedrus berries: A new monoterpene glucoside and evaluation of the effects on cancer cell lines

Article abstract of DOI:10.1016/j.phytol.2014.08.015

Phytochemical investigation of polar extract from Juniperus oxycedrus spp. oxycedrus berries leds to the isolation of one new monoterpene glucoside (3R,6E)-3,7 dimethyl 8-hydroxy-6-octenoic acid 8-O-β-d-glucopyranoside along with seven known components, some of them were initially isolated from Juniperus communis L. berries. Their structures were established on the basis of extensive 1D and 2D NMR (¹H, ¹³C, COSY, HMBC, HSQC, ROESY) and ESI-MS studies. The n-butanol fraction and isolated components, shikimic acid (2), compound 3, 4 and 5 were evaluated, in vitro, for their effect on cell viability against human malignant melanoma (A375), breast (MCF-7) and lung (H460) cancer cell lines. Shikimic acid exhibited selective effect on cell viability only against breast MCF-7 cell lines reaching IC₅₀ value at dose of 30 μM and also induced the level decrease of vascular endothelial growth factor (VEGF) and five pro-inflammatory cytokines suggesting its potential anti-inflammatory effect.

Full text of DOI:10.1016/j.phytol.2014.08.015

Phytochemistry Letters 10 (2014) 152–159
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Phytochemical profile of Juniperus oxycedrus ssp. oxycedrus berries:
A new monoterpene glucoside and evaluation of the effects on
cancer cell lines
Simona De Marino a, Carmen Festa a, Franco Zollo a, Fabiola Rusolo b, Francesca Capone b,
Eliana Guerriero b, Susan Costantini b, Vincenzo De Felice c, Maria Iorizzi c,
*
a
`
Dipartimento di Chimica delle Sostanze Naturali, Universita degli Studi di Napoli ‘‘Federico II’’, Via D. Montesano 49, I-80131 Napoli, Italy
b Centro Ricerche Oncologiche di Mercogliano, Istituto Nazionale per lo Studio e la Cura dei Tumori ‘‘Fondazione Giovanni Pascale’’, IRCCS, Italy
c
`
Dipartimento di Bioscienze e Territorio, Universita degli Studi del Molise, Contrada Fonte Lappone, I-86090 Pesche, Isernia, Italy
A R T I C L E I N F O
A B S T R A C T
Article history:
Phytochemical investigation of polar extract from Juniperus oxycedrus spp. oxycedrus berries leds to the
Received 14 July 2014
isolation of one new monoterpene glucoside (3R,6E)-3,7 dimethyl 8-hydroxy-6-octenoic acid 8-O-b-D-
Received in revised form 20 August 2014
Accepted 24 August 2014
Available online 4 September 2014
glucopyranoside along with seven known components, some of them were initially isolated from
Juniperus communis L. berries. Their structures were established on the basis of extensive 1D and 2D NMR
(1H, 13C, COSY, HMBC, HSQC, ROESY) and ESI-MS studies. The n-butanol fraction and isolated
components, shikimic acid (2), compound 3, 4 and 5 were evaluated, in vitro, for their effect on cell
viability against human malignant melanoma (A375), breast (MCF-7) and lung (H460) cancer cell lines.
Shikimic acid exhibited selective effect on cell viability only against breast MCF-7 cell lines reaching IC50
Keywords:
Juniperus oxycedrus ssp. oxycedrus L.
Monoterpene glucoside
NMR
value at dose of 30
mM and also induced the level decrease of vascular endothelial growth factor (VEGF)
and five pro-inflammatory cytokines suggesting its potential anti-inflammatory effect.
Viability evaluation
Cytokinome profile
Anti-inflammatory capacity
ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
in pharmacy as ‘‘juniper tar’’, is an empyreumatic oil widely
employed in human and veterinary dermatology to treat chronic
Juniperus oxycedrus L. (Cupressaceae) is one of 10 species in the
genus Juniperus throughout the world (Adams, 1998). This shrubs
or small tree grows wild in stony places of the Mediterranean and
Near East countries and is one of the most appreciate plants for its
essential oil richness and for its biologically active components
extensively used in folk medicine. Phytochemical studies carried
out on Juniperus taxa demonstrated the presence of a wide array of
secondary metabolites with a variety of pharmacological effects
(Moreno et al., 1998). Juniper berries have long been used as flavors
to aromatize alcoholic beverages such as the famous gin or
aromatic grappa and, in northern Europeae, particularly in
Scandinavian cuisine are used to ‘‘impart a sharp, clear flavour’’
to meat dishes, especially wild birds and game meats (Montagne,
1999). J. oxycedrus was utilized since the antiquity to produce a
sesquiterpene-rich oil named cade oil obtained by destructive
distillation of branches and wood of the plant. Cade oil, also known
eczema and other skin diseases (Chalchat et al., 1990). Cade oil has
also keratolytic and antipruritic properties (Leung and Foster,
1996) and rectified ‘‘juniper tar’’ was used as fragrance component
in soaps, detergents, creams and parfumes (Leung and Foster,
1996). J. oxycedrus has an extensively history of use in global folk
medicine for various disorder, such as hyperglycaemia, bronchitis
and pneumonia (Sanchez de Medina et al., 1994), decoction of the
plant is also used in traditional medicine in Spain as mouth
analgesic and for stomach disorders (Fernandez et al., 1996), in
Turkish folk medicine the boiled fruit extract has widely been used
in the treatment of gastrointestinal disorders, common colds, and
as diuretic (Akkol et al., 2009), while the resin was used for wound
healing (Yesilada et al., 1993).
Aqueous and methanol extracts of J. oxycedrus leaves showed in
vitro antimicrobial activity (Karaman et al., 2003) while methanol
and dichloromethane extracts of leaves and stems have been found
to reduce the blood pressure of normotensive rats (Bello et al.,
1997), to inhibit the response to histamine, serotonin and
acetylcholine (Moreno et al., 1998) and possess analgesic
properties (Moreno et al., 1998). The methanolic extracts of fruits
*
Corresponding author. Tel.: +39 0874 404100; fax: +39 0874 404123.
E-mail address: iorizzi@unimol.it (M. Iorizzi).
http://dx.doi.org/10.1016/j.phytol.2014.08.015
1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

S. De Marino et al. / Phytochemistry Letters 10 (2014) 152–159
153
and leaves exhibited antinociceptive and anti-inflammatory
activities (Akkol et al., 2009), antioxidant (Lizzo et al., 2007) and
antifungal activities have been also reported (Amri et al., 2013).
Recently, polyphenolic fraction from J. oxycedrus badia exhib-
ited acethylcholinesterase inhibitory activity and protected
neurons from injury in the neurodegeneration cell model (Tavares
et al., 2012).
coumpound 2, (iv) a slowdown in cell cycle G0/G1 phase respect
in MCF-7 cells after treatment with compound 2 to untreated cells,
and (v) the compound 2 induced the level decrease of VEGF and
other five pro-inflammatory cytokines suggesting its potential
anti-inflammatory effect.
2. Results and discussion
There are many reports on the chemical composition of the oils
from leaves (Chalchat et al., 1990; Adams et al. 1999), berries (Boti
et al., 2006) and less frequently wood (Barrero et al., 1993) of
J. oxycedrus, however intraspecific variation in the chemical compo-
nents of these essential oil was observed according to harvesting time,
geographical origin and starting material. Concerning leaf oil of
J. oxycedrus, chemical studies have reported in varying details the
composition from different geographic areas as from Corsica (Boti
et al., 2006), Croatia (Milos and Radonic, 2000), Spain (Adams, 1998),
Italy (Valentini et al., 2003), Tunisia (Medini et al., 2010), Greece (Stassi
et al., 1995), Lebanon (Lizzo et al., 2007), Turkey (Sezik et al., 2005).
Most of the study reported in literature up to now on J.
oxycedrus are focused on the chemical composition of the berry’s
essential oil and limited data are reported on non-volatile
components. The methanol extract from ripe berries of J. oxycedrus
from Turkey was recently tested on cancer cell viability by using a
human hepatocellular liver carcinoma (HepG2) cell line (Taviano
et al., 2013). In previous works we have analyzed the seasonal
variations in the metabolic fingerprint of the J. communis berries
(Falasca et al., 2014) and the ability of imbricatolic acid to prevent
cell cycle progression in human lung tumor cells (CaLu-6) (De
Marino et al., 2011).
Natural products obtained mainly from plants have been used
as prominent source of phytochemicals that have been shown to
exert a wide range of biological activity.
The methanolic crude extracts of J. oxycedrus ssp. oxycedrus
berries were submitted to extractive and chromatographic
analyses. Four extracts were obtained using n-hexane, CHCl3,
n-BuOH and water following the modified Kupchan’s partitioning
procedure (Kupchan et al., 1973).
Purification of the n-buOH fractions let to the isolation of one
new monoterpene glucoside (1), with four known ones 2–5
identified as shikimic acid (2) (Ali et al., 2009), the monoterpene
glucoside (3) previously isolated from J. communis var. depressa
(Nakanishi et al., 2005), the n-butyl
b-D-fructofuranoside (4) (Hu
and Chen, 1997) and the phenyl propanoid rhamnoside (5) isolated
from Pinus massoniana Lamb. (Lundgren et al., 1985) and from
Chamaecyparies obtusa Sieb. et Zucc. (Matsubara et al., 1990)
(Fig. 1). Their structures were elucidated using chemical and
spectroscopic methods including 1D and 2D NMR experiments and
spectrometric techniques.
2.1. Structural elucidation
As a part of our ongoing investigations toward the discovery of
bioactive secondary metabolites from plants origin, one new
monoterpene glucoside (1) and seven known components were
isolated.
Herein, we also report the evaluation of effects of crude
n-butanol extract and its pure compounds 2, 3, 4 and 5 on three
human cancer cell lines, A375 (malignant melanoma), MCF-7,
(breast), H460 (lung). In overall the experiments performed on
cells showed that: (i) the n-butanol extract decreased the cell
viability only in MCF-7 cells, (ii) the compound 2 had effect on
Compound 1 was obtained as an amorphous powder and
25
exhibited a negative optical rotation ([
a
]
ꢀ5.7, c. 0.12, MeOH).
D
In the positive and negative ESI-MS quasimolecular ion peaks were
observed at m/z 371 [M+Na]+ and m/z 347 [M]ꢀ respectively. Its
molecular formula was determined as C18H28O8 based on the
positive-mode HRESI-MS analysis (m/z 371.1679 [M+Na]+; calc. for
C
18H28O8Na 371.1676) and 13C NMR data. The 1H NMR spectrum
(Table 1) exhibited signals due to two methyl groups ( H 0.95 and
1.70), one olefinic proton at H 5.50, one methine, three methylene
and one primary alcoholic function resonating at H 4.05 and 4.20.
Furthermore, one anomeric signal at dH 4.26 (d, J = 7.8 Hz) was
d
d
MCF-7 viability at the concentration of 30
mM, (iii) no significant
d
increase of apoptosis in MCF-7 cells after treatment with
Table 1
1H and 13C NMR data (CD3OD, 500 and 125 MHz) of compound 1 and selected signals in the (R)- and (S)-PGME amide of 1.
Position
dH (J in Hz)
dC
HMBC
(R)-PGME amide
(S)-PGME amide
1
2
–
181.7
46.9
1.96 m
C-1, C-3, C-9
2.19 m
3
4
1.95 m
31.6
37.9
1.24 m
1.41 m
5
2.09 m
26.0
C-4, C-6, C-7
C-8
2.07 m
6
7
8
5.50 br t (6.6)
–
130.5
132.4
75.5
5.475 (br t)
5.487 (br t)
4.05 d (11.7)
4.20 d (11.7)
0.95 d (6.1)
1.70 s
C-6, C-7, C-10, C-10
9
19.9
13.8
C-2, C-3, C-4
C-6, C-7, C-8
0.934 (6.2)
1.731 s
0.893 (6.2)
1.746 s
10
Glucose
10
20
30
40
50
60
4.26 d (7.8)
3.19 dd (7.8, 8.3)
3.34a
102.1
74.8
77.8
71.5
77.5
62.6
C-8
4.272 (7.7)
4.271 (7.7)
3.26a
3.24a
3.66 dd (11.9, 5.4)
3.86 dd (11.9, 2.3)
a
Signals overlapped.

154
S. De Marino et al. / Phytochemistry Letters 10 (2014) 152–159
recognized with
a
large vicinal coupling constant (3JH1,H2
)
(Demetzos and Dimas, 2001) including the inhibition of cell
indicating the trans-diaxial orientation (
b
-configuration). The
b-glucose unit and
proliferation in several cell lines (Dimas et al., 2006).
13C NMR spectrum of 1 showed signals due to
one signal for a C–O group (dC 181.7). The 1H–1H-COSY implied
connectivity in the alkylic chain, from H2-2 to H-6, an isolated spin
system for H2-8 and CH3-10, together with signals of a D-
glucopyranose. The HMBC experiment showed correlations
reported in Table 1 and indicated the glycosidic linkage at C-8
2.2. Colorimetric assay with sulforhodamine B, analysis of apoptosis
and cell cycle
Cancer chemotherapy regimen makes use of cytotoxic com-
pounds that target proliferating cell populations and most of
cytotoxic drugs are from plants origin.
position, clarified with a cross peak between H-10 (
C 75.5). The double bond between C-6 and C-7 was E-configured
based on a NOESY cross-peak observed between H-6 ( H 5.50) and
H2-8 at H 4.20 and H 4.05. NOESY was also observed for CH3-10
dH 1.70) with H-5 at dH 2.07 (Fig. 1).
Acidic hydrolysis of 1 afforded -glucose which was confirmed
dH 4.26) and C-8
(
d
The cell viability of several cancer lines of melanoma (A375),
breast (MCF-7) and lung (H460) was determined after 24 h
stimulation with crude n-butanol extract by colorimetric assay
(sulforhodamine B) to identify the IC50 concentration, correspond-
ing to the chemical extract amount that causes 50% inhibition of
cell growth (Fig. 3). However, it is important to underline that
firstly we performed the tests after 24, 36, 48 and 60 h and the
results were perfectly conserved. Hence we reported in this paper
the results obtained after 24 h in according to other our recent
papers (Rusolo et al., 2013; Costantini et al., 2014). Moreover, since
the solvent can be not healthy for the cells, we tested the effect of
only n-butanol on three cancer cell lines and verified that it had not
biological effect on cells and the related cell viability remained
equal to 100%. Therefore the cellular viability of untreated cells
was used as control.
d
d
d
(
D
by GC–MS analysis (Hara et al., 1987). The absolute configuration
at C-3 in 1 was determined by NMR methodology (PGME method);
1 was converted into its (R)- and (S)-phenylglicine methyl ester
(PGME) amides which enabled the determination of the absolute
configuration of
b
,b
disubstituted carboxylic acids (Yabuuchi and
Kusumi, 2000).
The Dd [d (R-PGME amide)-d (S-PGME amide)] values obtained
from the 1H NMR spectra of the PGME amides (Table 1) suggested
that the absolute configuration at C-3 in 1 was R and was
elucidated as shown in Fig. 2. These data were in accordance with
the presence of (3R,6E)-3,7-dimethyl-8-hydroxy-6 octenoic acid 8-
The decrease in cell viability became significant only for breast
cancer cell line, MCF-7, reaching IC50 at the concentrations of
O-
b
-D-glucopyranoside.
In the present study we also characterized four known labdane
200
mg. Then, we tested on MCF-7 cells the pure compounds 2, 3, 4
diterpenoids from n-hexane and chloroform extracts. It is
especially noteworthy that the isolated metabolites of the J.
oxycedrus, are similar to those identified in the corresponding
extracts from berries of J. communis, a plant which grows in the
same geographical area in the central Italy. The n-hexane soluble
fraction was purified by chromatographic separation yielding the
known labdane diterpenoids cis-communic acid, myrcecommunic
acid, sandarocopimaric acid (De Marino et al., 2011). The
purification of CHCl3 soluble fraction gave the natural imbricatolic
acid (De Marino et al., 2011). Labdane diterpenes, isolated from
several plants families, show a variety of biological activities
and 5. Only compound 2, shikimic acid, as evidenced in Fig. 4,
showed a significant decrease in cell viability of reaching IC50 value
at dose of 30
mM. Compound 1 has not been tested because its
amount was not enough for the test.
In the next step we evaluated the ability of compound 2 to
induce apoptosis in MCF-7 and has been calculated the rate of
apoptosis by identification of four populations:
1) live cells which are not undergoing to apoptosis: annexin V (ꢀ)
and cells dead marker (ꢀ);
2) cells in early apoptosis: annexin V (+) and cells dead marker (ꢀ);
Fig. 1. Natural compound isolated from J. oxycedrus ssp. oxycedrus L.

S. De Marino et al. / Phytochemistry Letters 10 (2014) 152–159
155
Table 2
Rate of apoptosis (A) and cell percentages in G0/G1, S and G2/M phases (B)
evaluated in MCF-7 cells, non treated and treated for 24 h, with 30 mM of compound
2A.
Cells
Live
Cells in
early
Cells in late
apoptosis
Dead
cells
cells
apoptosis
Fig. 2. Dd values [Dd (in ppm) dR
of 1.
ꢀ
dS] obtained for the (R)- and (S)-PGME amides
A
MCF-7 non treated
MCF-7 treated
95.40%
92.31%
0.48%
3.40%
1.89%
2.78%
2.23%
1.51%
Cells
G0/G1
S
G2/M
B
MCF-7 non treated
MCF-7 treated
48.5%
56.5%
32.5%
21.3%
17.4%
19.2%
Human Cytokine 27-Plex Panel. The results obtained were
compared with untreated cells used as control. These experiments
showed that the cytokine levels in the supernatants after
treatment with compounds 3, 4 and 5 were similar to those in
control cells whereas the levels of VEGF and five pro-inflammatory
cytokines, like IL-8, IL-12, IP-10 (CXCL10), RANTES (CCL5) and
MCP-1 (CCL2) decreased in dose dependent way at increasing
levels of compound 2 (Table 3 and Fig. 5).
The cytokines IL-8 and VEGF promote tumor angiogenesis,
growth, and metastasis and are coexpressed in a variety of cancers.
The promoters of their genes contain different recognition sites for
transcription factors nuclear factor (NF)-kappaB (Bancroft et al.,
2001).
Fig. 3. Cell viability determined after 24 h stimulation with J. oxycedrus n-butanol
extract on A375, MCF-7 and H460 cells and estimated as % compared to untreated
cells.
MCP-1 and RANTES are potent chemoattractants belonging to
the CC subfamily of chemokines that are involved in immunoreg-
ulatory and inflammatory processes with ability to recruit,
activate, co-stimulate T cells and monocytes and attract above
mentioned cells into the place of destruction/inflammation (Conti
and Di Gioacchino, 2001). In literature it is reported that MCP-1
and RANTES expression is regulated from NF-kB and the blocking
of MCP-1 activity in immunodeficient mice bearing human breast
carcinomas resulted in significant prolongation of the survival of
tumor-bearing mice (Salcedo et al., 2000).
Therefore, the reduction of IL-8, VEGF, MCP-1 and RANTES could
Fig. 4. Cell viability determined after 24 h stimulation with different concentrations
of the compounds 2, 3, 4 and 5 on MCF-7 cells and estimated as % compared to
untreated cells.
indicate that they inhibit the activation of NF-kB by blocking
tumor growth.
On the other hand IL-12 is an interleukin, naturally produced by
dendritic cells, macrophages and human B-lymphoblastoid cells in
response to antigenic stimulation. It stimulates, from T and natural
3) cells in late apoptosis: annexin V (+) and cells dead marker (+);
4) dead cells which do not cross the apoptotic process: annexin V
(ꢀ) and cells dead marker (+).
killer (NK) cells, the production of interferon-gamma (IFN-g),
which in turn increases the production of a chemokine called
inducible protein-10 (CXCL10) that mediates this anti-angiogenic
effect (Coma et al., 2006). This can explain because both IL-12 and
IP-10 decreased after treatment with compound 2 in MCF-7 cells.
Taking into consideration these data, it is suggest a possible
anti-inflammatory effect of compound 2 on MCF-7 cells and its
In details, the cells dead marker used is 7-Aminoactinomycin D
(7-AAD).
The treatment with compound 2 did not show significant
increase of apoptosis in MCF-7 (Table 2A). This was shown by a
density increase of cells in the plot of live cells and the presence of
few cells in early apoptosis/dead and, even further, in late
apoptosis.
We also evaluated the cell cycle in MCF-7 cells treated with
compound 2. The treatment showed a slowdown in cell cycle
G0/G1 phase respect to untreated cells (Table 2B) with a slight
lengthening of G0/G1 phase peak and a shortening of S phase
peaks. In fact, the cell percentages in G0/G1, S and G2/M phases
were 48.5%, 32.5% and 17.4% in untreated cells and 56.5%, 21.3%
and 19.2% in treated cells.
capacity of reducing NF-kB activation.
Recent data have evidenced that inflammation is a critical
component of tumor development. A growing body of epidemio-
logic evidence has emerged showing that long-standing inflam-
mation promotes tumor development, growth, and progression
(Multhoff et al., 2012). For this reason, many research groups are
working to find new possible anti-inflammatory molecules.
Therefore, concerning breast cancer is a complex and hetero-
geneous disease, we have evaluated the cytokine florescence
intensities after treatment with compounds 2 by Human Cytokine
27-Plex Panel also in supernatants of another breast cancer cell
line, MDA-MB231, and in this case the cytokine levels resulted
similar to those in control cells. It could depend from the intrinsic
difference between MDA-MB231 and MCF7 human breast cancer
2.3. Cytokine level evaluation on MCF-7 supernatants
We also evaluated the cytokine florescence intensities in MCF-7
supernatants after treatment with compounds 2, 3, 4 and 5 by

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S. De Marino et al. / Phytochemistry Letters 10 (2014) 152–159
as internal standard. The Heteronuclear Single-Quantum Coher-
1
ence (HSQC) spectra were optimized for an average JCH of
140 Hz; the gradient-enhanced Heteronuclear Multiple Bond
3
Correlation (HMBC) experiment were optimized for a JCH of
8 Hz. Nuclear Overhauser Effect (NOE) measurements were
performed by 2D ROESY experiment. Through-space 1H connec-
tivities were evidenced using a ROESY experiment with mixing
times of 300 ms. HPLC was performed using a Waters 510 pump
equipped with a Waters Rheodine injector and a Waters 401
differential refractometer as detector, using a 30 cm ꢁ 3.9 mm;
i.d., C18
m-Bondapak (Waters, Milford, MA, USA) columns; flow
rate was 1 mL minꢀ1, and HPLC on a JASCO PU-2089 Plus
equipped with a UV/vis detector (UV-2075 Plus JASCO) at
270 nm using a Luna C18 column (150 mm ꢁ 4.60 mm i.d., 3
mm,
Phenomenex).
The GC/MS analysis was carried out with an Agilent Technolo-
gies 6890N Network gas chromatograph coupled to an Agilent
Technologies 5973 Network quadrupole mass selective spectrom-
eter and provided with a split/splitless injection port. Helium was
used as carrier gas at a linear velocity of 40 cm/s. Droplet counter-
current chromatography (DCCC) was performed on a DCC-A
apparatus (Tokyo Rikakikai Co., Tokyo, Japan) equipped with 250
glass-columns. The purity of compounds was determined to be
greater than 95% by HPLC, MS and NMR.
3.2. Plant material
Selected ripe berries of wild growing plants of J. oxycedrus L.
(Cupressaceae) were collected in the mountain areas of Isernia
(central Italy) in October 2010 and identified by Dr. Paola Fortini. A
voucher specimen is deposited (JO-301-10) at the Herbarium of
Dipartimento di Bioscienze e Territorio, University of Molise
(Pesche, Isernia). Berries were kept frozen at ꢀ20 8C until analyzed.
3.3. Extraction and isolation
Fresh ripe berries (100 g) were crushed and extracted with
MeOH (3ꢁ 700 mL) at room temperature. The combined extracts
(15 g) were concentrated and subjected to a modified Kupchan’s
(Kupchan et al., 1973) partitioning procedure, as reported in De
Marino et al. (2011), obtaining three fractions: n-hexane extract
(3.2 g), CHCl3 extract (2.5 g), n-butanol extract (2.0 g) and an
aqueous residue. The n-hexane extract was separated by column
chromatography on silica gel and then purified by HPLC in the
same conditions reported in De Marino et al. (2011), to give mainly
cis-communic acid and myrcecommunic acid. The CHCl3 extract
(1.5 g) was fractionated by DCCC using CHCl3/MeOH/H2O (7:13:8)
in the ascending mode (the lower phase was the stationary phase)
to give three main fractions. Fractions were combined and
monitored by TLC on silica gel with CHCl3/MeOH/H2O (80:18:2)
Fig. 5. Cytokine levels evaluated in MCF-7 cells after treatment with compound 2.
We indicate with * the comparisons between the levels in the untreated and treated
cells resulted statistically significant with p < 0.05 by T-test. The labels show the
concentrations corresponding to the different colors.
cell lines. Indeed, MCF7 cells are estrogen receptor alpha
(ERalpha)-positive, weakly invasive and luminal epithelial-like,
whereas MDA-MB231 cells are ERalpha-negative, highly invasive
and fibroblast-like. Hence, further studies will be performed to
evaluate the cytokine levels on other breast cancer cell lines
because many different cell lines are known for this cancer
(Holliday and Speirs, 2011).
as eluent and then separated by HPLC (C18
m-Bondapak column;
30 cm ꢁ 3.9 mm i.d.). Fraction 1 purified by HPLC with MeOH/H2O
(7:3) contained mainly imbricatolic acid; fraction 2 processed with
MeOH/H2O 8:2 yielded myrcecommunic acid and sandarocopi-
maric acid; fraction 3 contained several aldehydes and were
purified by HPLC (MeOH/H2O 75:25) as eluent.
3. Experimental
3.1. General experiment procedure
The n-BuOH extract (2 g) was submitted to DCCC with n-BuOH/
Me2CO/H2O (3:1:5) in the descending mode (the upper phase was
the stationary phase), to give three main fractions A-C. The
obtained fractions were monitored by TLC on Silica gel plates with
n-BuOH/OHAc/H2O (12:3:5) and CHCl3/MeOH/H2O (80:18:2).
Fraction A (180 mg) mainly contained free sugars and compound
High-resolution ESI mass spectrometry (HR-ESI-MS) was
recorded on a Micromass QTOF spectrometer and electrospray
ionization mass spectrometry (ESI-MS) experiments were per-
formed on an Applied Biosystem API 2000 triple-quadrupole mass
spectrometer. Optical rotations were determined on a Jasco P-2000
polarimeter. NMR spectra were obtained on Varian Inova 500 NMR
spectrometer (1H at 500 MHz and 13C at 125 MHz) equipped with a
2 (6.3 mg) purified by HPLC on a C18
m-Bondapak column (MeOH/
H2O 15:85) as eluent. Fraction B (550 mg) contained the new
monoterpene glucoside 1 (1.2 mg), the glucoside 3 (1.8 mg) and
the fructofuranoside derivative 4 (mg 1.5) and they were purified
Sun hardware, d(ppm), J in Hz, spectra referred to CD3OH (dH = 3.31)

S. De Marino et al. / Phytochemistry Letters 10 (2014) 152–159
157
Table 3
Cytokine levels evaluated in MCF-7 cells after treatment with compound 2 by Human Cytokine 27-Plex Panel.
CTRL
52.8
5
m
M
10
m
M
20
m
M
30
m
M
40
m
M
50 mM
PDGF-bb
IL-1b
52.2
32.6
50.0
31.9
34.7
51.9
32.4
34.2
49.6
32.8
34.8
49.1
32.9
34.5
49.3
32.3
34.4
33.8
35.2
IL-1ra
IL-2
35.7
116.5
28.3
115.5
26.0
114.0
27.2
115.0
24.7
112.5
23.8
113.6
23.6
113.1
23.3
IL-4
IL-5
16.0
14.7
14.2
13.0
12.7
12.5
12.6
IL-6
97.7
93.8
97.8
95.5
94.5
93.6
93.2
IL-7
46.2
39.8
37.5
41.5
41.5
39.9
40.1
IL-8
612.2
140.7
359.0
1082.2
81.3
590.3
142.7
344.7
915.2
80.0
169.0
137.3
347.7
846.0
77.8
42.8
24.7
23.6
22.9
IL-9
137.8
356.9
732.7
79.7
139.0
358.8
372.3
77.2
136.9
349.6
372.1
76.5
138.7
348.9
371.3
77.9
IL-10
IL-12
IL-13
IL-15
216.5
99.3
214.8
109.0
29.5
219.0
104.5
28.5
219.8
108.3
28.0
207.5
104.5
28.2
211.5
103.5
28.1
213.9
103.9
28.5
IL-17
Eotaxin
FGF basic
G-CSF
GM-CSF
IFN-g
IP-10
MCP-1
MIP-1a
MIP-1b
RANTES
TNF-a
VEGF
29.7
93.7
95.7
93.8
92.2
92.3
91.7
90.3
42.3
37.7
34.7
35.6
37.3
37.5
36.7
1732.2
39.2
1738.2
31.8
1725.0
32.3
1729.7
29.3
1713.5
34.3
1723.9
34.8
1698.7
34.6
26.0
16.3
15.7
15.3
14.3
13.9
13.7
3028.7
33.5
2508.8
32.0
920.5
31.8
235.8
28.8
163.2
26.7
163.2
29.6
163.2
28.5
65.3
69.8
69.0
66.0
65.2
67.6
66.4
159.8
25.7
65.0
66.7
60.3
59.8
59.3
58.9
24.3
23.0
22.0
20.0
21.3
20.9
4923.7
4395.0
3985.5
3539.3
2136.0
2136.0
2136.0
by HPLC with MeOH/H2O 15:85, 25:75 and 20:80 respectively, as
eluent; fraction C was separated by HPLC (MeOH/H2O 45:55) to
(R)-PGME amide of compound 1: 1H NMR (500 MHz in CD3OD)
d
7.38 (5H, m, phenyl), 5.57 (1H, d, CHNH), 3.75 (3H, s, OCH3). Other
signals are in Table 1. ESI-MS m/z 496 [M+H]+.
give compound
components.
5 (2.2 mg) and a mixture of polyphenolic
(S)-PGME amide of compound 1: 1H NMR (500 MHz in CD3OD)
d
7.40 (5H, m, phenyl), 5.58 (1H, d, CHNH), 3.77 (3H, s, OCH3). Other
signals are in Table 1. ESI-MS m/z 496 [M+H]+. See supplementary
material for 1H NMR, COSY, HSQC, HMBC spectra.
3.3.1. Acid hydrolysis of 1
Compound 1 (0.3 mg) was hydrolyzed with 2 N CF3CO2H (2 mL)
at 110 8C in a sealed tube for 8 h. After cooling, the solution was
diluted with H2O (5 mL) and extracted with AcOEt (3ꢁ 2 mL). The
aqueous layer was evaporated to dryness under reduced pressure
and the residue was reacted with 0.1 M
hydrochloride in anhydrous pyridine (200
3.3.3. Shikimic acid (2)
White amorphous powder, ([
positive ESI-MS m/z 175 [M+H]+.
25
a
]
D
ꢀ148.7, c. 0.20, MeOH);
ꢀ35.0, c. 0.18, MeOH);
L
-cysteine methyl ester
m
L) for 1 h at 60 8C
(Hara, Okabe, & Mihashi, 1987). 1-(Trimethylsilyl)imidazole in
pyridine was added and the thiazolidine derivatives analyzed by
3.3.4. Monoterpene glucoside (3)
25
White amorphous powder, ([a]
D
GC–MS.
retention times of their derivatives with those of
(Rt = 17.88 min) and -glucose (Rt = 19.20 min). The hydrolysate
was subjected to GC–MS for identification of the sugar moiety on a
m film thick-
D-Glucose were confirmed in 1 by comparison of the
positive ESI-MS m/z 369 [M+Na]+.
D
-glucose
L
3.3.5. n-Butyl
Colorless oil, ([
237 [M+H]+.
b-D-fructofuranoside (4)
25
a
]
D
ꢀ28.8, c. 0.15, MeOH); positive ESI-MS m/z
HP-5MS capillary column (30 m ꢁ 0.25 mm, 0.25
m
ness, Agilent USA). GC oven temperature was kept constant at
180 8C. The injector temperature was 230 8C. The temperature of
the ion source and the transfer line was 250 and 280 8C,
respectively. Mass spectra were taken at 70 eV and the mass
range was from 40 to 350 a.m.u.
3.3.6. Phenyl propanoid rhamnoside (5)
25
White amorphous powder, ([
a
]
D
+33.9, c. 0.22, MeOH);
positive ESI-MS m/z 515 [M+Na]+.
3.4. Cell culture
3.3.2. (R)- and (S)-PGME amides of compound 1
To an ice cooled DMF solution (500
PGME (0.5 mg) were added PyBOP (1.0 mg), HOBt (0.5 mg) and N-
methylmorpholine (25 L) and stirring was continued at room
temperature for 5 h. After addition of 5% HCl (1 mL), the mixture
was extracted with EtOAc (2.5 mL). The extract was washed with
saturated NaHCO3 aq (1 mL) and brine (1 mL), and then
m
L) of 1 (0.4 mg) and (R)-
Cell cultures were kept in growth conditions identical or as
similar as possible. A375 and MCF-7, malignant melanoma and
breast cancer cell lines, were kept in culture and expanded at 37 8C
in a humidified atmosphere of 5% CO2 in culture medium DMEM
(Dulbecco’s Modified Eagle’s Medium, Lonza, Verviers, Belgium),
supplemented with 10% FBS (Invitrogen, Camarillo, CA, USA) at 1%
penicillin/streptomycin 100ꢁ (Euroclone, Devon, UK), 1% Gluta-
max 100ꢁ (Invitrogen). The other human lung cancer cell line,
H460, was kept in culture and expanded in humidified atmosphere
m
concentrated in vacuo to afford the (R)-PGME amide of
1
(0.5 mg). The (S)-PGME amide of 1 was prepared according to
the same procedure as described above. Each diasteroisomer was
subjected to HPLC (Luna C18
3
m
m) eluting with MeOH/H2O (from
at 37 8C and 5% CO2 incubator in culture medium RPMI1640 w/o L-
20% to 100% MeOH) gradient 20% to 100% MeOH in 15 min, then
hold 100% MeOH for 10 min, by UV detector at 270 nm.
Glutamine (Lonza, Verviers, Belgium), supplemented with 10% FBS
(Invitrogen, Camarillo, CA, USA) at 1%, penicillin/streptomycin

158
S. De Marino et al. / Phytochemistry Letters 10 (2014) 152–159
100ꢁ (Euroclone, Devon, UK), 1% Glutamax 100ꢁ (Invitrogen).
Phosphate buffer 10ꢁ (PBS phosphate buffered saline Ca2+ and
Mg2+ free) and trypsin (Ca2+ and Mg2+ free) were supplied by
Euroclone.
removing of the supernatant, 1 mL of ice cold 70% ethanol was
added to the resuspending cell pellet in the residual PBS. The
tubes were capped and freezed at ꢀ20 8C for at least 3 h prior to
staining. Ethanol-fixed cells were centrifuged at 300 ꢁ g for
5 min at room temperature and pellet was resuspended in PBS.
The cells were centrifuged again at 300 ꢁ g for 5 min at room
temperature, the supernatant was removed and discarded and
3.5. Colorimetric assay with sulforhodamine B
First of all, cell proliferation was assessed in presence of
n-butanolic extract, by colorimetric assay with sulforhodamine
B (SRB, Sigma Aldrich) on the three cell lines and compared to
untreated cells. The cells (4.5 ꢁ 104–5 ꢁ 104) were seeded in
cell pellet was resuspended in 200 m
L of MuseTM Cell Cycle
Reagent and incubated for 30 min at room temperature,
protected from light. Cell suspension samples were transferred
to a 1.5-mL microcentrifuge tube prior to analysis on MuseTM
Cell Analyzer. The MuseTM Cell Cycle Assay uses a premixed
reagent which includes the nuclear DNA intercalating stain
propidium iodide (PI) and RNAse A in a proprietary formulation.
PI discriminates cells at different stages of the cell cycle, based
on differential DNA content in the presence of RNAse to increase
the specificity of DNA staining. Resting cells (G0/G1) contain two
copies of each chromosome. As cells begin cycling, they
synthesize chromosomal DNA (S phase). Fluorescence intensity
from PI increases until all chromosomal DNA has doubled (G2/M
phase). At this stage, the G2/M cells fluoresce with twice the
intensity of the G0/G1 population. The G2/M cells eventually
divide into two cells.
96-multiwell plates in 200
mL of culture medium, and left to grow
for 24 h at 37 8C for allowing adhesion. Then, the cells were treated
with J. oxycedrus extract using different concentrations: 50 g,
80 g, 200 g, 300 g and 400 g and then incubated for 24 h.
In particular, the extract was dissolved in dimethyl sulfoxide
m
m
m
m
m
(DMSO, Sigma–Aldrich) at a concentration of 100 mg/mL. In cell
cultures the DMSO concentration remained always below 0.1%, a
dose that did not exert toxic effects. Then, the cells were fixed by
the addiction of 10% trichloroacetic acid (TCA, Sigma–Aldrich) per
well for at least 1 h at 4 8C. Subsequently, the cells were washed
with distilled water and were dried to air.
100
mL of SRB was added to each well and the plate was
incubated for 30 min at RT, protected from light. To remove the dye
excess the cells were washed using 1% acetic acid. The number of
viable cells was directly proportional to the protein bound-dye
The assay utilizes PI-based staining of DNA content to
discriminate and measure the percentage of cells in each cell
cycle phase (G0/G1, S, and G2/M). The Muse Cell Cycle Software
Module performs calculations automatically.
formation which was then solubilized with 100
mL of 10 mM Tris
base solution pH 10.5 per well, shaking the plates for at least
15 min on a gyratory shaker to homogenize the dye solution.
Measure of OD was performed by using an automated 96-well
plate reader (Bio-Rad, Hercules, CA, USA; Microplate Reader) at a
wavelength of 540 nm. All experiments were performed in
triplicate and were repeated for three times. The cellular viability
was estimated as % compared to untreated cells.
Data is displayed in two plots:
ꢂ
ꢂ
DNA content index and cell size index dot plot.
DNA content index histogram with markers available to analyze
the cell populations in each phase of the cycle.
Based on the reduction in cell viability observed in MCF-7 cell
line after 24 h of treatment, we also treated the MCF-7 with the
3.8. Bio-Plex assay
compounds 2, 3, 4 and 5 at different concentrations 5
20 M, 30 M, 40 M and 50 M.
m
M, 10
m
M,
The levels of a panel of numerous cytokines, chemokines, and
growth factors were evaluated at the same time by BioPlex assay.
The simultaneous quantitative determination of a large panel of
cytokines, able to report the correct ratios and dynamics between
highly and poorly represented molecules, has emerged as an
accurate, simple, specific, noninvasive, reproducible and less
expensive method (Rusolo et al., 2013; Costantini et al., 2014).
The multiplex biometric ELISA-based immunoassay, containing
m
m
m
m
3.6. Apoptosis assay
The cells (1 ꢁ 106) have been harvested and washed two times
with cold PBS. Subsequently, the cells have been labeled by
Annexin V & Dead Cell Assay kit in according to instructions of
producer (Merck Millipore, Darmstadt, Germany). This assay is
based on the detection of phosphatidylserine (PS) on the surface of
apoptotic cells using Annexin V conjugated FITC in combination
with cell death marker, 7-actinomycin D (7-AAD). This staining
allows quantitative analysis of live cells, cells in early and later
apoptosis and dead cells. The samples have been tested on MuseTM
Analyzer (Merck Millipore) and were analyzed by a software
supplied by the same company.
dyed microspheres conjugated with
a monoclonal antibody
specific for a target protein was used, according to the manufac-
turer’s instructions (Bio-Plex Bio-Rad), to evaluate the levels of
different cytokines by Human Cytokine 27-Plex Panel after
treatment with compounds 2, 3, 4 and 5 in MCF-7 supernatants.
In particular, the following cytokines were evaluated: fourteen
interleukins (IL), IL-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-12 (p70), IL-13, IL-15, IL-17, and eotaxin (CCL11), basic
fibroblast growth factor (FGF), granulocyte colony-stimulating
factor (G-CSF), granulocyte macrophage colony-stimulating factor
3.7. Cell Cycle Assay
(GM-CSF), interferon-gamma (IFN-
ligand 10 (CXCL10), monocyte chemo-attractant protein-1 (MCP-1),
two macrophage inflammatory protein–1s, MIP-1 and MIP-1
platelet-derived growth factor- (PDGF- ), RANTES (regulated on
activation, normal T cell expressed and secreted), tumor necrosis
factor- (TNF- ) and VEGF. Each experiment was performed in
duplicate as previously described (Rusolo et al., 2013). Protein levels
were determined using a Bio-Plex array reader (Luminex, Austin, TX,
USA) that quantitates multiplex immunoassays in a 96-well format
withverysmallfluidvolumes. Theanalytelevelwascalculatedusing
a standard curve, with software provided by the manufacturer (Bio-
Plex Manager Software).
g), chemokine (C-X-C motif)
The MuseTM Cell Cycle Assay uses a premixed reagent which
includes the nuclear DNA intercalating stain propidium iodide (PI)
and RNAse A in a proprietary formulation. PI discriminates cells at
different stages of the cell cycle, based on differential DNA content
in the presence of RNAse to increase the specificity of DNA staining.
It is important to have a single cell suspension prior to ethanol
fixation and the minimum recommended number of cells for
fixation in a tube is 1 ꢁ 106 cells. The samples were centrifuged at
300 ꢁ g for 5 min and after removing and discarding the
supernatant, an appropriate volume of PBS was added to each
tube (1 mL of PBS per 1 ꢁ 106 cells). After centrifugation and
a
b,
b
b
a
a

S. De Marino et al. / Phytochemistry Letters 10 (2014) 152–159
159
Fernandez, A., Ortuno, I., Martos, A., Fernandez, C., 1996. Saber Y utilizacion de
The cytokines levels evaluated in MCF-7 supernatants were
compared by T-test. Values of p < 0.05 were considered to be
statistically significant.
`
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`
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`
Fantasma (Universita del Molise) for the technical support.
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