390 B.

MARONGIU
FLAVOUR ET AL.
AND FRAGRANCE JOURNAL
Flavour Fragr. J. 2003; 18: 390397
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1224

Extraction of Juniperus oxycedrus ssp. oxycedrus

essential oil by supercritical carbon dioxide: influence
of some process parameters and biological activity
Bruno Marongiu,1* Silvia Porcedda,1 Alessandra Caredda,1 Barbara De Gioannis,1 Laura Vargiu2 and
Paolo La Colla3
1
Dipartimento di Scienze Chimiche, Universit degli Studi di Cagliari, Cittadella Universitaria, St. Prov. Monserrato, Sestu Km
0.770, I-09042 Monserrato, Italy
2
Idenix, University of Cagliari Collaborative Laboratory
3
Dipartimento di Biologia Sperimentale, Sez. Microbiologia, Cittadella Universitaria, St. Prov. Monserrato, Sestu Km 0.770, I-
09042 Monserrato, Italy
Received 2 August 2002
Revised 8 January 2003
Accepted 14 January 2003

ABSTRACT: Supercritical carbon dioxide extraction of the leaves and berries of Juniperus oxycedrus L. ssp.
oxycedrus, coupled with a two-stage separation, allowed the entrapment of cuticular waxes in the rst separator
(90 bar, 10 C), thereby allowing pure essential oils to be collected in the second separator (15 bar, 10 C). An
extraction carried out on a charge of leaves, at 90 bar and 50 C, gave a yield of 0.8% (w/w) with respect to the
charged material, of an oil whose major constituents were germacrene D (15.9%), manoyl oxide (10.2%) and 1-epi-
cubenol (5.4%). The berry oil obtained, at the same pressure and temperature as those for the leaves, gave a yield
of 0.45% (w/w) and was composed chiey of germacrene D (13.8%), -pinene (10.5%) and -myrcene (8.1%).
At a constant temperature of 50 C, different extraction pressures (80, 90 and 100 bar) were tested in order to
obtain the juniper wood essential oil. The extraction yield depended strongly on the extraction pressure, reaching
a maximum of 14.7% (w/w) at 100 bar. The main constituents in the extract were -cadinene, calamenene, cubenol
and 1-epi-cubenol. Hydrodistillation of the wood of J. oxycedrus gave a yield of 11.0% (w/w) of essential oil, with
a reduced level of sesquiterpene hydrocarbons and an enhanced amount of oxygenated sesquiterpenes, with respect
to the oil obtained by supercritical carbon dioxide extraction. The extracts obtained at different pressures were tested
for cytotoxicity, antiviral and antimicrobial activities. The results showed that the extracts of leaves and berries
obtained at 200 bar were cytotoxic against different cell lines used to support virus growth. As far as antiviral activiy
is concerned, some of the extracts were active against a single-stranded RNA+ virus (Poliovirus-1). When tested for
antimicrobial activity, none of the samples were shown to be active. Copyright 2003 John Wiley & Sons, Ltd.
KEY WORDS: extraction; essential oil; Juniperus oxycedrus L. ssp. oxycedrus; supercritical carbon dioxide;
cytotoxicity; antiviral; antimicrobial

Introduction products.5 The oil of juniper wood is obtained from the

The genus Juniperus consists of approximately 60 spe-
cies.1 Juniperus oxycedrus L., juniper (also known as
cade tree2), is a tree that grows up to 8 m in height and
is native to the Mediterranean region.3 J. oxycedrus L.
ssp. oxycedrus also grows wild in Sardinia, especially in
the mountainous central region.4 Cade oil (also called
juniper tar oil) has been obtained since antiquity by dry
(empyreumatic) distillation of the wood.2 This oil, used in
human and veterinary dermatology and cosmetology,2
is composed of both the essential oil and decomposed
* Correspondence to: B. Marongiu, Dipartimento di Scienze Chimiche,
Universit degli Studi di Cagliari, Cittadella Universitaria, St. Prov.
Monserrato, Sestu Km 0.770, I-09042 Monserrato, Italy.
E-mail: maronb@unica.it
wood or branches of juniper by water or steam distilla-
tion.6 The process, even when optimized to avoid hydro-
lysis and thermal degradation of the oil, always furnishes
a product with characteristic odour notes.7 Supercritical
uid extraction (SFE) is a valid alternative for the pro-
duction of avours and fragrances from natural materials.
Compressed CO2 is able to solubilize hydrocarbon and
oxygenated mono- and sesquiterpenes,8 the main constitu-
ents of essential oils. The separation of the extractant is
easy and the oil is devoid of residues that pose a risk for
human use. In recent years, the composition of steam-
distilled oils of leaves of J. oxycedrus L. ssp. oxycedrus
from Greece and Spain have been reported by Adams
et al.9 Some data on the essential of this subspecies has
been presented also by Picci et al.4 No data on the berry
and wood essential oils composition of ssp. oxycedrus

Copyright 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 390397
 SUPERCRITICAL CO2 EXTRACTION OF JUNIPERUS OXYCEDRUS OXYCEDRUS
was found. The present study was undertaken to verify
the possibility of obtaining, in a single extraction stage,
the volatile content from the leaves, berries and wood of
J. oxycedrus L. ssp. oxycedrus by means of supercritical
carbon dioxide extraction. The biological activity of all of
the derived extracts was also investigated.
Materials and Methods
Materials
Leaves and berries of J. oxycedrus ssp. oxycedrus
(Cupressaceae) were gathered in May 2000 from trees
growing wild on Monte Arcosu (Capoterra) and Ardasai
(Seui) in the southern and central regions of Sardinia
(Italy), respectively. The plant was identied by Pro-
fessor. Mauro Ballero, Department of Botanic Sciences,
University of Cagliari, and a voucher specimen was
deposited at the Universitys Herbarium (CAG No. 1652).
After picking, the leaves and berries were air-dried at
room temperature in the shade for 3 weeks. They had a
nal moisture content of 8.2% and 11.9% (w/w), respect-
ively, on a dry basis. Seasoned wood, from Seui, was
20 years old and had a water content of 5.6% (w/w).
Before SFE, the vegetable matter was ground and the
resultant particles treated by mechanical sieving (300
800 m). Carbon dioxide (CO2, purity 99%) was supplied
by Societ Italiana Ossigeno (SIO), Cagliari, Italy.
SFE Apparatus
Supercritical CO2 extractions were performed in a laborat-
ory apparatus10 equipped with a 400 ml extraction vessel,
which operated in a single-pass mode by passing CO2
through the xed bed of vegetable particles. Two fractions
of the extract were recovered in two separator vessels
(300 and 200 ml) which were connected in series. The
cooling of the rst separator was achieved by using a
thermostated bath (Neslab, Model CC-100II; accuracy of
0.1 C). The use of the second separator allowed the dis-
charge of the liquid product at desired time intervals. In
this section, the temperature was maintained at the desired
value using two methods, rst, by the utilization of a heat-
ing ribbon wrapped around the piping dividing the two
separators, and second, by means of a water-thermostated
system connected to the second separator. A high-pressure
diaphragm pump (Lewa, Model EL 1) with a maximum
capacity of 6 kg/h, pumped liquid CO2 at the desired ow
rate. The CO2 was then heated to the extraction tempera-
ture in a thermostated oven (accurate to 0.02 C). Extrac-
tion was carried out in a semibatch mode: batch charging
of vegetable matter and continuous ow of solvent. The
ow of CO2 was monitored by a calibrated rotameter
(Sho-rate, Model 1355) positioned after the last separator.
Copyright 2003 John Wiley & Sons, Ltd.
391
The total CO2 delivered during an extraction was mea-
sured by a dry test meter. Temperatures and pressures
along the extraction apparatus were measured by thermo-
couple and Bourdon-tube test gauges, respectively. Pres-
sure was regulated by high-pressure valves under manual
control, located on different points of the apparatus.
Hydrodistillation
Hydrodistillations (HD) were performed in a circulat-
ory Clevenger-type apparatus for 5 h using 100 g of plant
material from the same samples employed in the SFE
experiments.
GCMS Analysis
A Hewlett-Packard 5890 Series II gas chromatograph (GC)
was used for analysing the extracts. It was equipped with a
splitsplitless injector and a DB5-MS fused silica column
of 5% phenyl-methylpolysiloxane (30 m 0.25 mm i.d.,
lm thickness 0.25 m). The GC conditions used were:
programmed heating from 60 to 280 C at 3 C/min,
followed by 30 min under isothermal conditions; the in-
jector was maintained at 250 C; helium, with a ow rate
of 1.0 ml/min, was the GC carrier gas; the sample (1 l)
was injected in split mode (1:20). The GC was tted with
a quadrupole mass spectrometer (MS), Model HP 5989
A. MS conditions were as follows: ionization energy,
70 eV; electronic impact ion source temperature, 200 C;
quadrupole temperature, 100 C; scan rate, 1.6 scan/s;
mass range, m/z 40500. Software to handle mass spe-
ctra and to record chromatograms was MS ChemStation
(Hewlett-Packard), using NIST98 and LIBR(TP)11 mass
spectra libraries. Run samples were diluted in chloro-
form at a dilution ratio of 1:100 (w/w). Chromatographic
results were expressed as area-percentages, calculated
without applying any response factor, and are reported
as a function of retention times (Rt) and Kovts indices
(KI).10 No duplicate analyses were performed. Identica-
tions were made by matching both their mass spectra and
(KI) values with those reported in the literature and those
of pure compounds, whenever possible.
Biological Analysis
Extracts were dissolved in DMSO at 100 mg/ml and then
diluted in culture medium.
Cytotoxicity
Exponentially growing human CD4+ lymphocytes (MT-4)
and baby hamster kidney (BHK-21) cells were resus-
pended in growth medium containing serial dilutions of
Flavour Fragr. J. 2003; 18: 390397
392 B. MARONGIU ET AL.

the extracts. Cell viability was determined after 96 h at cuticular waxes are washed off with mild conditions of
37 C by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- extraction.14 Waxes, when present, were entrapped in the
tetrazolium bromide (MTT) method.13 Green monkey rst separator set at the same pressure of the extractor
kidney (Vero) cells were seeded into 24-well plates in and at a temperature of 10 C. The oil was recovered in
growth medium and incubated overnight at 37 C, in a the second separator at 15 bar and 10 C. The juniper
humidied CO2 (5%) atmosphere. New growth medium wood essential oil was obtained, essentially, by a single-
containing serial dilutions of the extracts was then added. stage separation since preliminary tests proved that the
After incubation for 3 days, cultures were harvested, matrix was devoid of cuticular waxes.
trypsinized, resuspended in maintenance medium and
counted.
Leaves
MTT Assay
The leaf essential oil of J. oxycedrus ssp. oxycedrus was
The activity of the extracts against HIV-1 was based obtained by SFE under the following conditions: P =
on the inhibition of virus-induced cytopathogenicity in 90 bar, T = 50 C and CO2 = 1.0 kg/h. A 4 h run gave a
acutely infected MT-4 cells, at a multiplicity of infection yield, with respect to the charged material, of 0.8%
(m.o.i.) of 0.01. Briey, 50 l culture medium containing (w/w) of an oil whose major constituents (Table 1) were
1 104 cells were added to each well of at-bottomed germacrene D (15.9%), manoyl oxide (10.2%) and 1-epi-
microtitre trays containing 50 l culture medium with cubenol (5.4%). Adams et al.9 studied this subspecies,
or without test compounds. 20 l of an HIV suspension originating from Greece and Spain, and noted that the
containing 100 (HIV-1) CCID50, was then added. After steam-distilled oils were dominated by -pinene and
a 4 day incubation, cell viability was determined by the limonene and that the yield varied from 0.20 to 0.42%
MTT method. Activity against Reovirus was based on the (calculated as oil weight/weight of oven dried, extracted
inhibition of virus-induced cytopathogenicity in acutely leaves). Germacrene D and manoyl oxide were present as
infected BHK-21 cells. The cells were seeded overnight at minor constituents. Picci et al.4 reported the qualitative
a rate of 5 104 well into 96-well plates, and incubated in
growth medium at 37 C in a humidied CO2 (5%) atmo- Table 1. Retention times (Rt), Kovts indices (KI) and
sphere. Cell monolayers were infected with 50 l proper chromatographic area percentages of compounds
found in the SFE extract of leaves of J. oxycedrus
virus dilution, to give a m.o.i of 0.01. Serial dilutions of obtained at 90 bar, 50 C and fCO2 = 1.0 kg/h
test compounds in Dulbeccos modied Eagles medium,
supplemented with 2% inactivated fetal calf serum, were Rt KI Compounds* SFE
then added. After a 3 day incubation at 37 C, the num- 4.57 929 -Pinene 1.4
ber of viable cells was determined by the MTT method. 11.44 1125 trans-Verbenol 3.1
13.99 1191 Verbenone 3.0
20.03 1340 Terpinyl acetate 1.6
Plaque Reduction Assays 23.04 1406 (E)-Caryophyllene 4.2
24.57 1444 -Humulene 3.5
For each compound the 50% effective concentration 25.45 1464 -Muurolene 2.9
25.67 1469 Germacrene D 15.9
(EC50) was determined in duplicate 24-well plates by 26.30 1484 epi-Cubebol 1.8
plaque reduction assays. Cell monolayers (Vero) were 26.45 1487 -Muurolene 2.3
infected with 100 PFU/well of virus (HSV-1, Sb-1 and 27.02 1500 -Cadinene 2.1
27.12 1502 Cubebol 4.0
VSV). Serial dilutions of the test compounds in 27.25 1505 -Cadinene 3.0
Dulbeccos modied Eagles medium, supplemented with 27.52 1512 NI 2.2
2% inactivated serum and 0.75% methyl cellulose, were 28.79 1541 cis-Muurol-5-en-4- -ol 2.2
29.75 1563 Caryophyllene oxide 4.0
then added to the monolayers. The cultures were further 30.03 1569 Globulol 0.4
incubated at 37 C for 3 days, then xed with 50% 30.86 1587 Humulene epoxide II 2.8
ethanol and 0.8% crystal violet, and then washed and air- 31.18 1594 trans-Isolongifolene 2.7
31.62 1605 1-epi-Cubenol 5.4
dried. The plaques formed were counted. 32.69 1636 -Cadinol 1.9
33.39 1656 NI 3.2
33.80 1668 (Z)-Nerolidol acetate 2.7
34.12 1676 NI 2.4
Results and Discussion 44.63 1971 Manoyl oxide 10.2
46.62 2030 Abietatriene 4.2
Extraction 47.62 2061 Abietadiene 2.3
53.94 2268 4-epi-Abietal 4.5

The apparatus employed, owing to a two-stage separa- Total oil 99.9

Total identied 92.1
tion, allowed leaf and berry oil free of cuticular waxes
to be obtained. Indeed, being located on the surface, the * NI, unidentied.

Copyright 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 390397
 SUPERCRITICAL CO2 EXTRACTION OF JUNIPERUS OXYCEDRUS OXYCEDRUS 393

composition of two samples of Sardinian leaf essential oil Table 2. Retention times (Rt), Kovts indices (KI) and
obtained by water distillation at a yield of 0.20% (w/w). chromatographic area percentages of compounds
found in the SFE extract of berries of J. oxycedrus
They identied the following constituents: -pinene (main obtained at 90 bar, 50 C and fCO2 = 1.5 kg/h
constituent), camphene, -pinene, 3-carene, myrcene,
limonene, -terpinene, p-cymene, caryophyllene and Rt KI Compounds* SFE
humulene. Milos and Radonic15 studied a HD oil of 4.68 933 -Pinene 10.5
J. oxycedrus from Croatia and also found -pinene as 5.05 948 Camphene tr
5.60 968 Sabinene tr
the main constituent, followed by manoyl oxide, present 5.75 973 -Pinene 0.9
in percentages very close to our values. The yield was 6.06 983 -Myrcene 8.1
0.05% (w/w). The oils reported in the literature are richer 7.12 1016 p-Cymene tr
in -pinene than our sample. This may be due to vari- 7.27
7.33
1020
1022
Limonene
-Phellandrene
1.5
0.2
ability of composition and partly to the fact that, usually, 9.21 1070 Terpinolene 0.4
water or steam distillation is performed on fresh intact 9.79 1083 Perillene 0.4
10.82 1106 -Campholenal tr
material, whereas carrying out a SFE is more favourable 11.39 1123 trans-Pinocarveol 0.5
for treating a dried and ground matrix. These treatments 11.63 1130 Camphor 0.4
may cause an unwanted loss of part of the volatile con- 12.02 1141 Camphene hydrate 0.2
13.03 1168 Terpin-4-ol 0.4
stituents. From the extracted matrix, a further extraction 13.67 1184 -Terpineol tr
was performed using a single separator for 3 h at 200 bar 14.15 1195 Verbenone tr
and at the previously quoted values of T and . This SFE 15.55 1232 Neral 2.9
16.88 1266 Geranial 5.1
produced a greenish semi-solid mass at a yield of 1.6% 17.47 1280 Bornyl acetate tr
(w/w). No residues of essential oil were detected in this 20.16 1343 -Cubebene 1.2
extract from the GC trace. With the exception of linear 21.37 1370 -Copaene 0.5
22.00 1383 -Elemene tr
alkanes and aliphatic acids, all the compounds identied 23.24 1411 (E)-Caryophyllene 2.7
were either hydrocarbon or oxygenated diterpenes. Pro- 23.68 1422 -Gurjunene tr
minent components were the alcohols manool, incensole, 23.88 1427 trans- -Bergamotene tr
24.77 1449 -Caryophyllene 4.0
sclareol, larixol, totarol and ferruginol. 25.51 1466 Germacrene D isomer 1.2
25.68 1470 -Muurolene 1.0
25.96 1476 Germacrene D 13.8
26.19 1481 Valencene 0.4
Berries 26.47 1487 -Selinene tr
26.63 1491 -Muurolene 1.7
Extractions were carried out at the same pressure and 27.08 1501 -Bisabolene tr
-Cadinene
temperature as with the leaves, using a CO2 = 1.5 kg/h. 27.23
27.53
1505
1512 -Cadinene
3.0
7.5
It gave a berry oil with a pale yellow colour containing 27.67 1515 trans-Calamenene 5.2
60 constituents (Table 2). The extraction yield was 0.45% 28.01 1523 Cadina-1,4-diene 0.3
-Cadinene
(w/w). Germacrene D (13.8%), -pinene (10.5%) and 28.12
28.35
1526
1531 -Calacorene
0.6
0.7
-myrcene (8.1%) were prominent constituents. Menut 28.58 1537 NI 0.4
et al.,16 Milos and Radonic15 and Guerra et al.17 reported 29.28 1552 (E)-Nerolidol 1.6
29.77 1563 Caryophyllene alcohol tr
the composition of the oil of berries of J. oxycedrus 29.94 1567 Caryophyllene oxide 0.9
obtained by hydro- or steam distillation. These invest- 30.25 1574 Gleenol 1.8
igators found as main constituents -pinene (percentages 30.38 1577 NI 0.3
31.06 1591 Humulene epoxide II 0.6
higher than 50%) and myrcene. The yields were 0.7% 31.39 1598 NI 0.2
(w/w),16 0.04% (w/w)15 and 0.50% (v/w).17 Also in this 31.85 1611 1-epi-Cubenol 4.5
case our sample was found to be poorer in -pinene than 32.42 1628 Cubenol 4.5
32.57 1633 -Muurolol 1.4
the oil described in the literature. 32.89 1642 -Cadinol 1.0
33.25 1652 NI 0.4
33.55 1661 Cadalene 0.9
36.93 1668 14-Oxy- -muurolene 0.4
Wood 37.43 1754 1-Pentadecanol 2.6
44.41 1965 1-Eicosene 1.1
Table 3 reports the percentage composition of the 44.81 1976 NI 0.4
61.35 2627 NI 0.5
hydrodistilled oil and that of the oil obtained by SFE at 67.05 2879 NI 0.9
50 C, CO2 = 1.5 kg/h, and at three different pressures.
Total oil 99.7
The oil yields were 5.2, 11.4 and 14.7% (w/w) at pres- Total identied 96.6
sures of 80, 90 and 100 bar, respectively. These results
are summarized in Figure 1, where Y%, the percentage * NI, unidentied.

tr, trace < 0.1%.
ratios of moil/mmatrix (mass of oil/mass of the charged
material) of samples collected at xed intervals of time,

Copyright 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 390397
394 B. MARONGIU ET AL.

Table 3. Retention times (Rt), Kovts indices (KI) and chromatographic area percentages of compounds found in
the SFE extract of wood of J. oxycedrus obtained at 50 C, fCO2 = 1.5 kg/h, and at different pressures: 80, 90 and
100 bar (E[80], E[90] and E[100], respectively) and of the hydrodistilled oil (HD)

(Rt) (KI) Compounds* E[80] E[90] E[100] HD

7.05 1013 p-Cymene tr tr tr
7.18 1017 Limonene tr tr tr
15.93 1242 Thymoquinone tr tr tr
20.03 1340 -Cubebene 0.2 0.3 0.1 0.1
21.25 1367 -Copaene 0.9 1.0 0.7 0.5
21.91 1382 -Elemene 2.2 2.4 1.7 1.3
22.38 1391 (Z)-Caryophyllene tr tr tr tr
23.18 1410 (E)-Caryophyllene 6.0 6.4 5.0 3.7
23.59 1420 -Elemene tr 0.2 0.1 0.1
23.81 1425 -Guaiene 0.5 0.5 0.4 0.2
24.33 1438 cis-Muurola-3,5-diene 0.5 0.3 0.4 0.3
24.70 1447 -Humulene 5.2 5.2 4.1 3.2
24.82 1450 cis-Muurola-4(14),5-diene 0.3 tr tr tr
25.21 1459 allo-Aromadendrene tr 0.3 0.2 0.2
25.44 1464 Germacrene D isomer 4.9 6.1 4.9 3.2
25.52 1466 -Cadinene tr tr 0.1 0.3
25.61 1468 -Muurolene 0.7 tr 1.0 tr
26.06 1478 -Selinene 1.2 1.0 0.2 0.6
26.16 1481 cis- -Guaiene 0.2 0.3 tr 0.2
26.35 1485 -Selinene 1.0 0.9 0.9 0.6
26.55 1489 -Muurolene 4.2 3.4 3.3 2.1
27.11 1502 -Cadinene 0.5 0.4 0.3 0.2
27.61 1514 -Cadinene 23.9 22.8 21.4 14.0
27.74 1517 trans-Calamene 11.7 8.2 7.8 6.0
27.96 1522 Cubenene 1.0 1.0 1.0 0.7
28.06 1524 -Cadinene 1.9 1.1 1.5 0.8
28.31 1530 -Calacorene 2.6 1.9 2.1 1.5
28.44 1533 NI 1.1 tr 0.1 0.1
28.54 1536 NI tr 1.0
28.80 1542 Elemol tr 0.8 1.0 tr
28.89 1544 Germacrene B tr tr tr tr
29.09 1548 -Calacorene 0.7 0.6 0.6 0.6
29.41 1555 NI tr 0.2 0.2 0.2
29.67 1561 Caryophyllene alcohol 0.3 0.2 0.3 0.8
29.70 1562 NI 0.4 0.3 0.4
29.86 1565 Caryophyllene oxide 1.1 1.3 0.9 1.5
29.97 1568 Globulol tr tr tr
30.18 1572 Gleenol 2.6 2.6 2.9 3.3
30.52 1580 cis-Arteannuic alcohol tr 0.2 0.2 0.3
30.84 1587 NI 0.4 0.4 0.5 0.8
30.98 1590 Humulene epoxide II 0.9 1.0 0.6 1.1
31.31 1597 NI tr 0.5 tr 0.8
31.92 1613 1-epi-Cubenol 7.0 8.6 8.5 12.5
32.19 1621 -Acorenol 0.4 tr 0.5 0.2
32.48 1630 Cubenol 6.6 7.2 8.5 10.5
32.63 1634 -Muurolol 2.1 2.8 3.2 4.8
32.92 1643 -Cadinol 1.3 1.9 2.1 3.6
33.29 1653 NI 0.6 1.7 1.2 3.0
33.51 1659 Cadalene 1.8 1.2 1.8 1.5
33.74 1666 NI 0.4
33.80 1668 NI tr 0.5 0.7 1.2
34.38 1684 Juniper camphor tr 0.2 0.3 0.5
34.53 1688 NI tr tr 0.2 0.3
34.78 1695 NI tr tr 0.1 0.3
35.04 1702 14-Hydroxy- -humulene 0.3 0.9 1.0 2.2
35.17 1705 NI tr tr 0.2
35.51 1715 NI tr 0.2 tr 0.6
36.02 1729 NI tr tr 0.1 0.2
36.12 1732 NI tr tr tr 0.2
36.23 1735 NI tr tr 0.2 0.2
36.46 1741 -Acoradienol tr tr 0.2 0.2
36.86 1752 14-Oxy- -muurolene 0.4 0.7 0.8 1.1
37.00 1756 NI 0.2
37.10 1759 2-Hexenyl cynnamaldehyde tr 0.3 0.3 0.3
37.17 1760 NI 0.1
37.31 1764 14-Hydroxy- -muurolene 0.5 0.9 1.4 2.3
37.55 1771 NI tr tr 0.2
37.74 1776 NI tr 0.3 0.4 0.2

Copyright 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 390397
 SUPERCRITICAL CO2 EXTRACTION OF JUNIPERUS OXYCEDRUS OXYCEDRUS 395

Table 3. (Continued)
(Rt) (KI) Compounds* E[80] E[90] E[100] HD

38.10 1785 14-Hydroxy- -cadinene 0.47 0.7 1.2 2.2

38.19 1787 NI tr 0.2 0.3
38.61 1798 -Vetivone tr tr 0.2
38.92 1807 NI tr tr 0.2 0.1
40.45 1852 NI tr tr 0.1 0.1
43.70 1944 epi-13-Manool tr tr
51.23 2176 NI 0.1 tr
54.8 2297 trans-Ferruginol 0.3 0.2
55.04 2306 NI tr 0.3 0.1
Total oil 99.0 99.9 98.9 100.0
Total identied 96.1 95.8 93.9 89.7

* NI, unidentied compound.

tr, trace < 0.1%.

compounds (NI) for each column extract. Despite the age

Figure 1. Percentage yield (w/w), Y%, of J. oxycedrus
wood essential oil against extraction time, obtained at
50 C, fCO2 = 1.5 kg/h and at three different pressures:
80, 90 and 100 bar
have been reported against the time of extraction. It
is evident that the higher extraction pressure allows a
reduction in the processing time and gives a higher yield.
Indeed, under these conditions the compositions of the
oils obtained at 80, 90 and 100 bar do not differ sub-
stantially from each other, and all the extracts possessed
good aromatic qualities. Only traces of monoterpenes
were found in each extracted oil. The major constitu-
ents in the supercritical uid extracts were -cadinene,
(21.423.9%), trans-calamenene (7.811.7%), cubenol
(6.68.5%) and 1-epi-cubenol (7.08.6%). The hydro-
distilled oil was obtained at a yield of 11.0% (w/w)
and had reduced sesquiterpene hydrocarbons (SH) con-
tents, and higher amounts of oxygenated sesquiterpenes
(OS), with respect to the supercritical uid extracts. The
percentages of the major constituents of the supercrit-
ical extracts in the hydrodistilled oil were: -cadinene
(14.0%), trans-calamenene (6.0%), cubenol (10.5%), and
1-epi-cubenol (12.5%). Unfortunately, a large number of
OS in the HD oil have not been identied. At the bottom
of Table 3 are the reported percentages of the unidentied
of the starting material, our results for the chemical com-
position of the extracts are in good agreement with those
reported by Chalchat et al.2 and by Barrero et al.,6 both
concerning oils of J. oxycedrus. In contrast, the yields are
not comparable, e.g. the yield of the steam-distilled oil
obtained by Barrero et al.6 was 2.6% (w/w) against our
value, relative to water distillation, of 11.0% (w/w). The
chemical composition of the samples collected after every
hour is deducible from Table 4, which reports the results
concerning the more important constituents obtained dur-
ing a run performed at the following conditions: P =
90 bar, T = 50 C, CO2 = 1.5 kg/h, time of extraction, t =
10 hours. Approximately, all compounds (SH) with a re-
tention time up to 30.0 min share a decreasing trend in
the percentages, while the others (OS) change in opposite
direction. This effect, concerning hydrocarbon and
oxygenated monoterpenes as light compounds and hydro-
carbon and oxygenated sesquiterpenes as heavy com-
pounds, has been observed, e.g. by Reverchon et al.14 in
the extraction of volatiles from leaves of Salvia ofcinalis.
Shorter diffusion times of lighter molecules cause a rough
selective extraction. Extraction runs of longer duration are
necessary to obtain an oil of reproducible composition.
Some experiments were also performed at different values
of CO2 ow. Figure 2(a,b) show graphs representing the
percentage yield (w/w) of extraction against extraction
time and ms/m0 (the specic amount of solvent; ms is the
mass of CO2 and m0 is the mass of the charged matrix).
From the curves, one can deduce that in the SFE of
juniper wood, the rate of extraction is inuenced by
external diffusion. The highest tested value of solvent
ow is adequate to shorten the extraction time.
Biological Activity
The extracts were assayed for cytotoxicity, antiviral
[Herpes simplex type 1 (HSV-1), Poliovirus 1 (Sb-1),
Vesicular stomatitis virus (VSV), Reovirus (Reo) and
Yellow fever virus (YFV)], antimicrobial (Staphylococcus

Copyright 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 390397
396 B. MARONGIU ET AL.

Table 4. Chromatographic area percentages of the main constituents found in the SFE extract of wood of
J. oxycedrus collected after each hour of extraction: 1 h, 2 h, etc. Extraction conditions were as follows: 90 bar,
50 C and fCO2 = 1.5 kg/h

Compounds 1h 2h 3h 4h 5h 6h 7h 8h 9h 10 h
-Copaene 1.4 1.4 0.7 0.4 0.4 0.3 0.2 0.2
-Elemene 3.7 3.3 1.9 1.6 1.0 1.0 1.0 0.9 0.6 0.5
(E)-Caryophyllene 9.4 8.8 5.6 5.1 4.0 3.2 3.1 3.0 2.2 1.8
-Humulene 6.9 6.5 4.6 4.7 3.6 3.1 3.0 2.8 2.2 1.8
Germacrene D isomer 6.4 7.2 5.4 4.5 4.0 3.7 3.4 3.2 2.5 2.1
-Muurolene 3.6 4.0 3.5 2.8 2.6 2.7 2.6 2.4 2.0 1.8
-Cadinene 25.8 26.6 25.8 22.0 20.0 19.6 19.1 18.1 15.1 12.7
trans-Calamenene 8.9 4.4 6.6 13.4 13.3 7.6 7.3 6.6 6.6 6.2
-Calacorene 1.7 1.8 1.8 1.9 1.9 1.7 1.7 1.6 1.4 1.3
Gleenol 2.1 2.2 3.0 3.0 3.4 3.0 3.3 3.1 3.4 3.4
1-epi-Cubenol 7.0 6.6 9.9 12.1 12.0 11.3 11.4 11.6 12.5 12.5
Cubenol 5.7 5.7 8.5 10.0 11.4 9.8 9.9 10.2 10.7 11.1
-Muurolol 1.9 1.8 2.6 3.8 5.5 4.6 4.5 4.3 5.0 5.5
-Cadinol 1.2 1.3 1.9 2.4 3.2 3.2 3.2 3.2 3.7 4.0
14-Hydroxy- -humulene 0.4 0.6 0.7 0.7 1.4 1.9 2.1 2.1 2.5 2.7
14-Oxy- -muurolene 0.4 0.4 0.6 1.2 1.2 1.2 1.3 1.4 1.5 1.6
14-Hydroxy- -muurolene 0.4 0.5 0.6 1.9 2.8 2.9 3.0 3.7 4.1
14-Hydroxy- -cadinene 0.4 0.3 0.4 1.8 2.5 2.6 2.9 3.5 4.1

aureus, Salmonella spp.) and antifungal (Candida

albicans, Aspergillus fumigatus) activities, as shown in
Tables 5 and 6.
The extracts from the leaves and berries of J.
oxycedrus, obtained at 200 bar, were cytotoxic for MT-
4 cells (Table 5). On the other hand, the extracts from the
wood, obtained at 200 bar, were active against Poliovirus
(Sb-1) at non-cytotoxic concentrations. Interestingly, the
extract of berries obtained at 90 bar, but not that obtained
at 200 bar, was active against Poliovirus (Table 6).
Poliovirus is the representative member of the Family
Picornaviridae usually used in antiviral screening assays.
This family also includes Coxsackieviruses, Echoviruses
and Hepatitis A, which are the most common and import-
ant human pathogens.18,19 Since Coxsackieviruses share
many characteristics with Poliovirus, we are now assay-
ing the active extracts against Coxsackievirus serotype B2.

Table 5.

Extracts of J. oxycedrus CC50a EC50b

(MT-4) (HIV-1)
Wood 200 atm > 100 > 100.011
Leaves 200 atm 5.6 > 5.6
Berries 200 atm 17 > 17
Wood 90 bar > 100 > 100
Berries 90 atm 100 > 100
*Azidothymidine (AZT) 100 0.011
a
Compound concentration (g/ml) required to reduce the viability of mock-
infected MT-4 cells by 50%, as determined by the MTT method.
b
Figure 2. Percentage yield (w/w), Y%, of J. oxycedrus Compound concentration (g/ml) required to achieve 50% protection of
wood essential oil obtained at 90 bar, 50 C, and at MT-4 cells from the HIV-1 induced cytopathogenicity, as determined by the
MTT method.
three different values of solvent flow: fCO2 = 0.9, 1.2 (* Azidothymidine has been reported as a reference compound. Data repres-
and 1.5 kg/h; (a) against extraction time; (b) against ent mean values for two separate experiments. Variation among duplicate
the specific amount of solvent (ms /m0) samples was less than 15%.)

Copyright 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 390397
 SUPERCRITICAL CO2 EXTRACTION OF JUNIPERUS OXYCEDRUS OXYCEDRUS 397

Table 6.
Extracts of CC50 EC50
J. oxycedrus
VEROa BHK-21a,b Herpes Poliovirus-1c Vesicular Reovirusd Yellow fever
simplex-1c (Sb-1) stomatitis virusc (Reo) viruse
(HSV-1) (VSV) (YFV)

Wood 200 atm > 100 > 100 > 100 20 > 100 > 100 > 100
Leaves 200 atm 60 60 > 60 > 60 > 60 > 60 > 60
Berries 200 atm 60 60 > 60 > 60 > 60 > 60 > 60
Wood 90 bar > 100 > 100 > 100 > 100 > 100 > 100 > 100
Berries 90 atm > 100 > 100 > 100 60 > 100 > 100 > 100

a
Compound concentration (g/ml) required to reduce the number of mock-infected VERO and BHK-21 monolayers by 50%.
b
Compound concentration (g/ml) required to reduce the viability of mock-infected BHK-21 monolayers by 50%, as determined by the MTT method.
c
Compound concentration (g/ml) required to reduce the HSV-1/Sb1/VSV plaque number by 50% in VERO monolayers.
d
Compound concentration (g/ml) required to achieve 50% protection of BHK-21 cells from the Reovirus-induced cytopathogenicity, as determined by the
MTT method.
e
Compound concentration (g/ml) required to reduce the YFV plaque number by 50% in BHK-21 monolayers. (Data represent mean values for
two separate experiments. Variation among duplicate samples was less than 15%.)

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