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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Dipartimento di Chimica delle Sostanze Naturali, Universita` degli Studi di Napoli Federico II, Via D. Montesano 49, I-80131 Napoli, Italy
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
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 14 July 2014
Received in revised form 20 August 2014
Accepted 24 August 2014
Available online 4 September 2014
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-b-Dglucopyranoside 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 mM and also induced the level decrease of vascular endothelial growth factor (VEGF)
and ve pro-inammatory cytokines suggesting its potential anti-inammatory effect.
2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Juniperus oxycedrus ssp. oxycedrus L.
Monoterpene glucoside
NMR
Viability evaluation
Cytokinome prole
Anti-inammatory capacity
1. Introduction
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 avors
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 avour
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
* 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/ 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
153
Table 1
1
H 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
1
2
3
4
5
6
7
8
9
10
Glucose
10
20
30
40
50
60
a
Signals overlapped.
dH (J in Hz)
dC
HMBC
1.96 m
2.19 m
1.95 m
1.24 m
1.41 m
2.09 m
2.07 m
5.50 br t (6.6)
4.05 d (11.7)
4.20 d (11.7)
0.95 d (6.1)
1.70 s
181.7
46.9
4.26 d (7.8)
3.19 dd (7.8, 8.3)
3.34a
3.26a
3.24a
3.66 dd (11.9, 5.4)
3.86 dd (11.9, 2.3)
102.1
74.8
77.8
71.5
77.5
62.6
(R)-PGME amide
(S)-PGME amide
5.475 (br t)
5.487 (br t)
0.934 (6.2)
1.731 s
0.893 (6.2)
1.746 s
C-8
4.272 (7.7)
4.271 (7.7)
31.6
37.9
26.0
130.5
132.4
75.5
19.9
13.8
154
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
Fig. 2. Dd values [Dd (in ppm) dR dS] obtained for the (R)- and (S)-PGME amides
of 1.
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 (+).
In details, the cells dead marker used is 7-Aminoactinomycin D
(7-AAD).
The treatment with compound 2 did not show signicant
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.
2.3. Cytokine level evaluation on MCF-7 supernatants
We also evaluated the cytokine orescence intensities in MCF-7
supernatants after treatment with compounds 2, 3, 4 and 5 by
A
MCF-7 non treated
MCF-7 treated
Live
cells
Cells in
early
apoptosis
Cells in late
apoptosis
Dead
cells
95.40%
92.31%
0.48%
3.40%
1.89%
2.78%
2.23%
1.51%
Cells
G0/G1
G2/M
B
MCF-7 non treated
MCF-7 treated
48.5%
56.5%
32.5%
21.3%
17.4%
19.2%
156
as internal standard. The Heteronuclear Single-Quantum Coherence (HSQC) spectra were optimized for an average 1JCH of
140 Hz; the gradient-enhanced Heteronuclear Multiple Bond
Correlation (HMBC) experiment were optimized for a 3JCH of
8 Hz. Nuclear Overhauser Effect (NOE) measurements were
performed by 2D ROESY experiment. Through-space 1H connectivities 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; ow
rate was 1 mL min1, 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 Technologies 6890N Network gas chromatograph coupled to an Agilent
Technologies 5973 Network quadrupole mass selective spectrometer and provided with a split/splitless injection port. Helium was
used as carrier gas at a linear velocity of 40 cm/s. Droplet countercurrent 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 identied 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
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 signicant with p < 0.05 by T-test. The labels show the
concentrations corresponding to the different colors.
157
Table 3
Cytokine levels evaluated in MCF-7 cells after treatment with compound 2 by Human Cytokine 27-Plex Panel.
PDGF-bb
IL-1b
IL-1ra
IL-2
IL-4
IL-5
IL-6
IL-7
IL-8
IL-9
IL-10
IL-12
IL-13
IL-15
IL-17
Eotaxin
FGF basic
G-CSF
GM-CSF
IFN-g
IP-10
MCP-1
MIP-1a
MIP-1b
RANTES
TNF-a
VEGF
CTRL
5 mM
10 mM
20 mM
30 mM
40 mM
50 mM
52.8
33.8
35.2
116.5
28.3
16.0
97.7
46.2
612.2
140.7
359.0
1082.2
81.3
216.5
99.3
29.7
93.7
42.3
1732.2
39.2
26.0
3028.7
33.5
65.3
159.8
25.7
4923.7
52.2
32.6
35.7
115.5
26.0
14.7
93.8
39.8
590.3
142.7
344.7
915.2
80.0
214.8
109.0
29.5
95.7
37.7
1738.2
31.8
16.3
2508.8
32.0
69.8
65.0
24.3
4395.0
50.0
31.9
34.7
114.0
27.2
14.2
97.8
37.5
169.0
137.3
347.7
846.0
77.8
219.0
104.5
28.5
93.8
34.7
1725.0
32.3
15.7
920.5
31.8
69.0
66.7
23.0
3985.5
51.9
32.4
34.2
115.0
24.7
13.0
95.5
41.5
42.8
137.8
356.9
732.7
79.7
219.8
108.3
28.0
92.2
35.6
1729.7
29.3
15.3
235.8
28.8
66.0
60.3
22.0
3539.3
49.6
32.8
34.8
112.5
23.8
12.7
94.5
41.5
24.7
139.0
358.8
372.3
77.2
207.5
104.5
28.2
92.3
37.3
1713.5
34.3
14.3
163.2
26.7
65.2
59.8
20.0
2136.0
49.1
32.9
34.5
113.6
23.6
12.5
93.6
39.9
23.6
136.9
349.6
372.1
76.5
211.5
103.5
28.1
91.7
37.5
1723.9
34.8
13.9
163.2
29.6
67.6
59.3
21.3
2136.0
49.3
32.3
34.4
113.1
23.3
12.6
93.2
40.1
22.9
138.7
348.9
371.3
77.9
213.9
103.9
28.5
90.3
36.7
1698.7
34.6
13.7
163.2
28.5
66.4
58.9
20.9
2136.0
158
159
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