Eur. J. Wood Prod. (2012) 70:615–620
DOI 10.1007/s00107-012-0596-9
O R I G I NA L S O R I G I NA L A R B E I T E N
Chemical composition and biological activity of the essential oil
from the wood of Pinus heldreichii Christ. var. leucodermis
K. Graikou · O. Gortzi · G. Mantanis · I. Chinou
Received: 2 March 2011 / Published online: 19 January 2012
© Springer-Verlag 2012
Abstract The chemical composition of the essential oil obtained by steam distillation from the wood of P. heldreichii,
collected from north Greece area was determined by GC and
GC/MS for the first time. Forty constituents (corresponding
to 96.3% of the total weight) were identified. The main components were: limonene, cembrene, longifolene, α-pinene,
methyl chavicol, kaurene and cembrene A. The antimicrobial activity of the oil was evaluated against six Gram positive and Gram negative bacteria and three human pathogenic
fungi, using the agar dilution technique. Strong activities
against most of the tested microorganisms were exhibited.
Moreover, the oil showed a very promising antioxidant activity through Rancimat method.
mit GC und GC/MS analysiert. Vierzig verschiedene Inhaltsstoffe (entsprechend bis 96.3 % des Gesamtgewichts)
konnten identifiziert werden. Die Hauptinhaltsstoffe waren:
Limonen, Cembren, Longifolen, α-Pinen, Methylchavicol,
Kauren und Cembren A. Die antimikrobielle Aktivität des
ätherischen Öls wurde an sechs Gram positiven und negativen Bakterien sowie drei pathogenen Pilzen mit der Agardiffusionsmethode bestimmt. Das ätherische Öl zeigte eine
deutliche antimikrobielle Aktivität gegen die meisten geprüften Mikroorganismen. Die antioxidative Wirkung des
ätherischen Öls, die mit der Rancimat Methode getestet wurde, war vielversprechend.
Chemische Zusammensetzung und biologische
Aktivitäten des ätherischen Öls aus dem Holz
von Pinus leucodermis
1 Introduction
Zusammenfassung Das ätherische Öl aus Holz von P. heldreichii, (Nordgriechenland) wurde mittels Wasserdampfdestillation gewonnen und die chemische Zusammensetzung
K. Graikou · I. Chinou ()
Dept. of Pharmacy, Div. of Pharmacognosy & Chemistry
of Natural Products, University of Athens, Panepistimiopolis
Zografou, 15771, Athens, Greece
e-mail: ichinou@pharm.uoa.gr
O. Gortzi
Dept. of Food Technology, Technological Educational Institution
(TEI) of Larissa, Terma Temponera Str., 43100, Karditsa, Greece
G. Mantanis
Lab. of Wood Technology, Department of Wood & Furniture
Design and Technology, Technological Educational Institute
(TEI) of Larissa, 43100, Karditsa, Greece
The genus Pinus belongs to the family Pinaceae and the
monotypic subfamily Pinoideae. There are about 115 species
of Pinus and their natural distribution ranges from arctic
and subarctic regions of Eurasia and North America south
to subtropical and tropical regions of Central America and
Asia (Farjon 1984; Silba 1986; Rushforth 1987).
The species was first described as Pinus heldreichii by the
Swiss botanist K. Hermann Christ in 1863 from specimens
collected on Mount Olympus, and then described a second
time as P. leucodermis in 1864; the author of the second description was the Austrian botanist F. Antoin. Some minor
morphological differences have been claimed between the
two descriptions (leading to the maintenance of both as separate taxa by a few botanists), but this is not supported by
modern studies of the species, which show that both names
refer to the same taxon and are synonyms.
Pinus heldreichii Christ. var. leucodermis is a forest
species endemic to the Balkan peninsula and one of the least
616
studied species, often called white bark pine. It is encountered in the mountains of southeastern Europe, in southwestern Bulgaria, Bosnia, Albania, the former Yugoslav Republic of Macedonia, Serbia, northern Greece and locally in
southern Italy, growing at 900–2,500 m altitude (Tsoumis
1983). It is an evergreen tree, usually up to 25–35 m high,
that has up to 2 m trunk diameter.
The tree is called Bosnian pine [English], Panzerkiefer
[German], pin d’ecorce blanche [French], il pino loricato
[Italian], robolo [Greek], cherna mura [Bulgarian, Serbian],
whitebark pine, or Heldreich pine.
P. heldreichii wood is commonly used in Greece as a
valuable material for making wine barrels. It is a rare, but
excellent species for the timber industry, which is known
to have a remarkable natural durability (Petrovic and Miric
1981).
Lange et al. (1994) investigated the xylem oleoresin
which was tapped from several P. heldreichii trees in
Kosovo and thunbergol and cembrol (84.8%) were identified as its main components. In addition, this work revealed
that limonene (79.4%), α-pinene (11.2%) and longifolene
(5.6%) were the main components of gum turpentine oil.
Obst (1998) also reported that 83% of the Bosnian pine turpentine is composed of limonene.
Additionally, several studies have been carried out on the
chemical analyses of the essential oil from the needles of P.
heldreichii. Maric et al. (2007) found that Bosnian pine needles material from Herzegovina is rich in limonene (52.8%),
germacrene D (15.8), α-pinene (10.2%) and caryoplyllene
(7.7%). Quite similar results were reported by Nikolic et
al. (2007) in a research study carried out on Bosnian pine
needle volatiles from different populations in Serbia and
Montenegro, as well as by Petrakis et al. (2001) in a study
on the needles’ oils from trees growing in Greece, where
they concluded the following chemical profile: limonene ≫
α-pinene > germacrene-D > β-caryophyllene > β-pinene.
Since the 16th century, a high interest has arisen in P.
heldreichii wood, apart from wine barrel production, also as
the main resource for housing, dairying and storage of goods
(Todaro et al. 2007).
However, information describing its chemical properties
is very limited in the literature. In this study here, the chemical composition as well as the antioxidant and the antimicrobial profile of the essential oil from the wood of P. heldreichii is reported for the first time.
2 Material and methods
2.1 Plant material and isolation of the essential oil
Straight-grained mature heartwood from ten trees of P. heldreichii, with an average number of 6–8 annual rings cm−2 ,
Eur. J. Wood Prod. (2012) 70:615–620
was collected from a high-altitude forest area of Pindos
in north-west Greece. After a slow air-drying process of
six months, small, clear wood specimens were prepared.
Voucher specimen is kept in the Laboratory of Pharmacognosy & Chemistry of Natural Products, University of
Athens. The dried wood was subjected to hydro-distillation
for 4 h, in 0.4 l of water, in a Clevenger-type apparatus,
with a water-cooled oil receiver to reduce formation of artifacts due to overheating during hydro-distillation (British
Pharmacopoeia 1993). The wood essential oil was collected
over water and dried over anhydrous sodium sulfate (Panreac Quimica S.A. Barcelona, Spain) and it was stored at
4–6 ◦ C until analyzed.
2.2 Essential oil analysis
The oil was analyzed by GC on a Perkin-Elmer 8500
gas chromatograph with FID, fitted with a Supelcowax-10
fused silica capillary column (30 m x 0.32; film thickness,
0.25 µm). The column temperature was programmed from
75 to 200 ◦ C at a rate of 2.5 ◦ C/min. The injector and detector temperatures were programmed at 230 ◦ C and 300 ◦ C,
respectively. Helium was used as carrier gas at a flow rate
of 0.6 ml/min. The GC-MS analysis was carried out using a
Hewlett Packard 5973-6890 GC-MS operating on EI mode
(equipped with a HP 5MS 30 m × 0.25 mm × 0.25 µm film
thickness capillary column). Helium (1 ml/min) was used
as carrier gas. Temperature program: the initial temperature of the column was 60 ◦ C (for 5 min), then raised to
280 ◦ C within 3 ◦ C/min, and held there for 30 min (total
time: 93.33 min).The compounds were identified by comparison of their retention indexes (RI) (Massada 1976), retention times (RT) and mass spectra with those of authentic
samples and/or the NIST/NBS, NIST02, Wiley 575 libraries
spectra and the literature (Adams 2007). The percentage
composition of the essential oil is based on peak areas obtained without FID factor correction.
2.3 Antimicrobial activity
Antimicrobial activity of the essential oils against bacteria and fungi was determined by using the agar dilution
technique. The microorganisms included two Gram-positive
bacteria: Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 12228); four Gram-negative
bacteria: Escherichia coli (ATCC 25922), Enterobacter
cloacae (ATCC 13047), Klebsiella pneumoniae (ATCC
13883) and Pseudomonas aeruginosa (ATCC 227853); and
the pathogenic fungi Candida albicans (10231), C. tropicalis (13801) and C. glabrata (28838). Standard antibiotics
(netilmicin, amoxicillin and clavulanic acid) were used in
order to control the sensitivity of the tested bacteria and 5flucytocine, amphotericin B and itraconazole were used in
Eur. J. Wood Prod. (2012) 70:615–620
order to control the tested fungi. Pure limonene, α-pinene
and cembrene were also tested to compare their antimicrobial activities with the ones of the assayed oil. All technical data have been described previously (Runyoro et al.
2010). Minimum inhibitory concentrations (MICs) were determined for the oil sample and the standard pure compounds under identical conditions, for comparison purposes.
The experiments in all cases were repeated three times.
617
Table 1 Main components of the essential oil from P. heldreichii wood
Tab. 1 Hauptinhaltsstoffe des ätherischen Öls aus dem Holz von
P. heldreichii
Compound*
2.5 Statistical analysis
The results were expressed as average values. Data are expressed as means ± S.D.
3 Results and discussion
Method of
identification
1
α-pinene
6.43
931
a, b, c
2
camphene
0.37
942
a, b, c
3
β-pinene
0.25
966
a, b, c
a, b, c
myrcene
1.09
975
5
limonene
28.70
1040
a, b, c
6
α-terpinolene
0.34
1085
a, b, c
7
exo-fenchol
0.63
1121
a, b, c
8
β-terpineol
0.11
1150
a, b, c
9
camphene hydrate
0.20
1154
a, b, c
10
borneol
0.83
1170
a, b, c
11
terpinen-4-ol
0.26
1177
a, b, c
12
methyl chavicol
5.94
1195
a, b, c
13
γ -terpineol
2.48
1199
a, b, c
14
trans-carveol
0.15
1217
a, b, c
15
cis-carveol
0.06
1231
a, b, c
16
carvacrol methyl ether
0.05
1236
a, b, c
17
carvone
0.16
1242
a, b, c
18
iso-bornyl acetate
0.05
1282
a, b, c
19
α-longipinene
0.86
1350
a, b, c
20
α-ylangene
0.13
1367
a, b, c
21
longicyclene
0.27
1372
a, b, c
22
sativene
0.35
1389
a, b, c
23
longifolene
6.89
1412
a, b, c
24
trans-β-farnesene
0.25
1453
a, b, c
25
β-chamigrene
0.12
1472
a, b, c
26
β-selinene
1.05
1489
a, b, c
27
α-selinene
0.42
1495
a, b, c
28
β-himachalene
0.07
1498
a, b, c
29
β-bisabolene
0.06
1506
a, b, c
30
juniperol
0.51
1601
a,c
31
cembrene
23.82
1936
a, b, c
32
cembrene A
5.58
1946
a, b, c
33
kaurene type
0.24
1957
c
34
phenanthrene type
0.22
1974
c
35
kaur-15-ene
0.16
1997
a, b, c
36
kaurene
5.88
2043
a, b, c
37
abietadiene
0.39
2083
a, b, c
38
sandaracopimarinal
0.37
2182
a, b, c
39
dehydro abietal
0.06
2270
a, b, c
40
4-epi-abietal
0.47
2307
a, b, c
Total
* Compounds
The results obtained from the chemical composition are
summarized in Table 1. The identified components represent
96.30% of the total oil. The percentages of the constituents
HP-5§
4
2.4 Antioxidant activity
The method used was a modification of the Rancimat
method reported by Lalas and Tsaknis (2002). About three
grams of oil consisting of purified sunflower oil and pinus
oil (0%-Control, 1%, and 2%) were accurately weighed into
the reaction vessel of the Rancimat 743 (Metrhom LTD,
Herisau, CH 9101, Switzerland). The reaction vessels were
placed in the apparatus. The conditions were set at 70 °C
and 7 L/h. The protection factor (P.F.) was calculated as
P.F. = (induction period with antioxidant)/(induction period
without antioxidant). A protection factor greater than 1 indicates inhibition of the lipid oxidation. The higher the value,
the better is the antioxidant activity (Lalas and Dourtoglou
2003).
Preparation of oil for Rancimat method: Sunflower oil
(Sol, Elais S.A., Athens, Greece) was purified from trace
metals and other prooxidants via adsorption chromatography to yield purified sunflower oil triacylglycerol fractions
according to the method described by Fuster et al. (1998).
A glass column (40 × 2.5 cm i.d.) (wrapped with aluminum
foil to prevent light-induced oxidations during the purification process), plugged with glass wool, was packed with
250 g of alumina (activated at 100 ◦ C for 8 h and then
at 200 ◦ C for 12 h) suspended in n-hexane, capped with
sea sand, and conditioned by prewashing with 200 ml of
n-hexane. The oil (100 ml) was dissolved in an equal volume
of hexane and passed through the column, which was then
washed with 200 ml of n-hexane. The hexane (total volume
300 ml) was evaporated using a rotary evaporator and the triacylglycerols were collected in an aluminium foil-wrapped
flask.
% in ess.oil
§
96.3%
listed in order of elution from a HP-5 MS column
Retention indices (KI) on HP-5 MS capillary column
a = Retention time; b = Retention Index; c = mass spectra
618
Eur. J. Wood Prod. (2012) 70:615–620
are based on normalization of peak areas without application
of the response correction factor. The major components are:
limonene 28.70%, cembrene 23.82%, longifolene 6.89%, αpinene 6.43%, methyl chavicol 5.94%, kaurene 5.88% and
cembrene A 5.58%.
It has to be noted, the presence in the oil of a total
37.18% of monoterpene hydrocarbons and 36.07% of diterpenes followed by oxygenated monoterpenes 10.92% and
sesquiterpenes 10.47% (Table 2). High level of monoterpenes (∼ 60%) was also detected in the essential oil from
the needles of P. heldreichii growing in Greece (Petrakis et
al. 2001).
Comparing the results of this study with previously reported ones on essential oils from different parts of the tree,
both wood and needles oil have limonene and α-pinene as
the most abundant components, while the rest major constituents are not comparable. So, in the wood oil, cembrene
Table 2 Composition of different classes of terpenes in the essential
oil of P. heldreichii wood
Tab. 2 Zusammensetzung der verschiedenen Terpenklassen im ätherischen Öl von P. heldreichii Holz
Compounds
% in essential oil
Monoterpene
37.18
Sesquiterpene
10.47
Diterpenes
36.07
83.72
Hydrocarbons
Oxygenated monoterpenes
10.92
Oxygenated Sesquiterpene
0.51
Oxygenated Diterpenes
0.90
12.33
Oxygenated compounds
Others
0.22
Total
96.27
Table 3 Main components (%
in essential oil) from different
parts of P. heldreichii
Tab. 3 Hauptinhaltsstoffe
(Anteil im ätherischen Öl in %)
verschiedener Pflanzenteile von
P. heldreichii
and longifolene (23.82% and 6.89% respectively) are among
its main constituents, while they are almost absent in the
needle oil (Table 3).
On the other hand, the composition of the wood oil in
comparison with the xylem oleoresin (Lange et al. 1994)
shows a completely different chemical profile. Wood oil has
only some similarities with gum turpentine oil (Lange et al.
1994) regarding the presence of its main constituents (αpinene, β-pinene, limonene, methyl chavicol longifolene)
but with totally different percentages (limonene ∼ 80% in
gum oil but only 29% in wood oil) while cembrene, cembrene A and kaurene appeared only in wood oil (Table 3).
The wood oil was tested against Gram (±) bacteria and
fungi and exhibited a wide profile of antimicrobial activity (Table 4) against all tested microorganisms (MIC values 2.00–3.74 mg/ml) while pure limonene was almost inactive and α-pinene showed a moderate antimicrobial profile (MIC values 2–15 mg/ml) in accordance with previously reported results (Runyoro et al. 2010). The expressed
quite strong activity of the wood oil of P. heldreichii could
be mainly attributed to the content in cembrene (23.82%)
which shows a strong activity against Gram-positive bacteria and pathogenic fungi (Table 4) as it has been also previously reported (Chen et al. 2009). Cembrene which is the
main constituent only of the wood oil of the tree seems to
be recognized as the most valuable compound for its antimicrobial activity.
The protection of P. heldreichii wood oil on purified sunflower oil was also studied (Table 5). In all ratios used, wood
oil improved significantly the resistance of sunflower oil to
oxidation. The results obtained, using the Rancimat method
to evaluate the antioxidant activity, showed that P. heldreichii wood oil can be considered as a good natural source
with significant antioxidant activity. This activity can be attributed to the main constituents and/or to synergy among
Compounds
Wood Xylem oleoresin Gum turpentine Needles
Needles
(Lange et al.
oil (Lange et al. (Petrakis et al. (Maric et al.
1994)
1994)
2001)
2007)
α-pinene
6.43
–
11.16
13.8
10.2
17.51
β-pinene
0.25
–
0.47
4.2
3.0
5.66
limonene
28.70 –
Needles
(Nikolic et al.
2007)
79.44
34.3
52.8
26.30
methyl chavicol 5.94
–
0.18
–
–
–
longifolene
1.1
5.62
–
–
–
β-caryophyllene –
6.89
–
0.09
8.4
7.7
10.41
germacrene D
–
–
–
12.8
15.8
13.53
cembrene
23.82 5.6 or 4.1
–
0.2
0.1
–
cembrene A
5.58
–
–
–
–
–
kaurene
5.88
–
–
0.3
–
–
thunbergol/
cembrol (1:2)
–
84.8
–
–
–
–
Eur. J. Wood Prod. (2012) 70:615–620
619
Table 4 Antimicrobial activities (MIC mg/ml) of the studied P. heldreichii essential oil and its main components
Tab. 4 Antimikrobielle Aktivität (MIC mg/ml) des ätherischen Öls von P. heldreichii und seiner Hauptinhaltsstoffe
Speciesessential oils
S. aureus
S. epidermidis P. aeruginosa E. cloacae
K. pneumoniae E. coli
C. albicans C. tropicalis C. glabrata
P. heldreichii
2.20
2.45
3.00
3.25
3.74
3.12
2.75
2.20
Limonene
>20
>20
>25
>25
>25
>20
>25
>25
>25
α-Pinene
7.50
9.50
6.00
15.00
8.00
2.00
4.00
4.00
2.00
Cembrene
1.40
1.54
4.72
>25
5.26
6.70
4.84
3.71
3.27
Itraconazole
NT
NT
NT
NT
NT
NT
1 × 10−3
0.1 × 10−3
1 × 10−3
NT
0.1 × 10−3
1 × 10−3
10 × 10−3
1 × 10−3
0.5 × 10−3
0.4 × 10−3
5-Flucytocine
NT
NT
Amphotericin B NT
NT
NT
NT
2.00
NT
NT
NT
NT
NT
Netilmicin
4 × 10−3
4 × 10−3
8.8 × 10−3
8 × 10−3
8 × 10−3
10 × 10−3
NT
NT
NT
Amoxicillin
2 × 10−3
2 × 10−3
2.4 × 10−3
2.8 × 10−3 2.2 × 10−3
2 × 10−3
NT
NT
NT
Clavulanic acid
0.5 × 10−3 0.5 × 10−3
1 × 10−3
1.6 × 10−3 1 × 10−3
1.2 × 10−3 NT
NT
NT
NT = not tested
Table 5 Protection factor of various concentrations of P. heldreichii
wood oil on purified sunflower oil
Tab. 5 Antioxidativer Effekt verschiedener Konzentrationen des ätherischen Öls aus dem Holz von P. heldreichii auf Sonnenblumenöl
Sample
Oil in sunflower oil (%)
Protection factor
P. heldreichii oil
1
2.17 (±0.06)
P. heldreichii oil
2
2.08 (±0.02)
Values are means of triplicate determinations and standard deviation
(SD) is given in parenthesis
the different oil components. The measured antioxidant
power depends on the chosen method, the concentration and
the nature and physicochemical properties of the studied antioxidant (Martos-Viuda et al. 2010). However, the increase
in the quantity of wood oil added to the mixture did not increase the induction period. Studies indicate that some dietary compounds may have concentration-dependent antioxidant or prooxidant activities. It has been observed that the
activity of some antioxidants does not increase linearly with
increasing concentration. At sufficiently high levels of addition, it may even become a pro-oxidant (Schuler 1990). It is
well known that phenolic compounds are strong antioxidant
agents, but recently it has been published that essential oils
rich in nonphenolic compounds may also exhibit antioxidant
properties (Kordali et al. 2005). It has been published in recent literature, that compounds like limonene, α-pinene, βpinene and kaurene type diterpenoids, which are among the
most abundant in the studied wood oil, have shown strong
to moderate antioxidant activity (Ruberto and Baratta 2000;
Thirugnanasampandan et al. 2008), so the exhibited antioxidant activity could suggest to be attributed mainly to them.
4 Conclusion
The GC and GC/MS analyses of the essential oil from P. heldreichii wood led to the identification of forty constituents
(corresponding to 96.30% of the total weight) among which
limonene, cembrene, longifolene and α-pinene have been
found as the most abundant ones.
Besides, the oil exhibited a broad spectrum of strong antimicrobial activities especially against Gram positive bacteria (MIC values 2.20–2.45 mg/ml) and human pathogenic
fungi (MIC values 2.0–2.5 mg/ml). This activity could be
mainly attributed to the high content of cembrene identified in significant amounts (∼ 24%) only in the wood oil
of P. heldreichii, among the previous studied volatiles, from
different parts of the tree. However, further investigation
should be carried out on new series of pathogenic microorganisms, in order to validate a potent antiseptic use in the
field of cosmetics.
Moreover, the antioxidant activity of P. heldreichii oil
could find further potential application in the area of food
protection mostly in the process of food storage.
Finally, except for the well known extensive use of P. heldreichii wood in the timber industry, this study contributes
to the further exploitation of the wood oil in the above mentioned different commercial areas of high interest.
References
Adams RP (2007) Identification of essential oil components by gas
chromatography/mass spectroscopy. Allured, Carol Stream
British Pharmacopoeia (1993) vol I, international ed. HMSO, London
Chen S, Liu J, Gong H, Yang D (2009) Identification and antibacterial
activity of secondary metabolites from Taxus endophytic fungus.
Chin J Biotechnol 25:368–374
Farjon A (1984) Pines: drawings and descriptions of the genus Pinus.
Brill & Backhuys, Leiden
620
Fuster MD, Lampi AM, Hopia A, Kamal-Eldin A (1998) Effects of
α- and γ -tocopherols on the autoxidation of purified sunflower
triacylglycerols. Lipids 33:715–722
Kordali S, Cakir A, Mavi A, Kilic H, Yildirim A (2005) Screening of
chemical composition and antifungal and antioxidant activities of
the essential oils from three Turkish Artemisia species. J Agric
Food Chem 53:1408–1416
Lalas S, Tsaknis J (2002) Extraction and identification of natural
antioxidant from the seeds of Moringa oleifera tree variety of
Malawi. J Am Oil Chem Soc 79:677–683
Lalas S, Dourtoglou V (2003) Use of rosemary extract in preventing
oxidation during deep fat frying of potato chips. J Am Oil Chem
Soc 80:579–583
Lange W, Janežic TS, Spanoudaki M (1994) Cembratrienols and other
components of white bark pine (Pinus heldreichii) oleoresin. Phytochemistry 36:1277–1279
Maric S, Jukic M, Katalinic V, Milos M (2007) Comparison of chemical composition and free radical scavenging ability of glycosidically bound and free volatiles from Bosnian pine (Pinus heldreichii Christ. var. leucodermis). Molecules 12:283–289
Martos-Viuda M, Navajas YR, Zapata ES, Fernandez-Lopez J, PérezÁlvarez J (2010) Antioxidant activity of essential oils of five spice
plants widely used in a Mediterranean diet. Flavour Frag J 25:13–
19
Massada Y (1976) Analysis of essential oil by gas chromatography and
spectrometry. Wiley, New York
Nikolic B, Ristic M, Bojovic S, Marin PD (2007) Variability of the needle essential oils of Pinus heldreichii from different populations in
Montenegro and Serbia. Chem Biodivers 4:905–911
Obst JR (1998) Special (secondary) metabolites from wood. In: Forest
products biotechnology. Taylor & Francis, New York, p 153
Eur. J. Wood Prod. (2012) 70:615–620
Petrovic M, Miric M (1981) Resistance to decay fungi of the wood of
munica (Pinus heldreichii), silver fir and Norway spruce in comparison with Scots pine. J Sumarstvo 34:27–34 (in Serbo-Croat)
Petrakis PV, Tsitsimpikou C, Tzakou O, Couladis M, Vagias C, Roussis V (2001) Needle volatiles from five Pinus species growing in
Greece. Flavour Frag J 16:249–252
Runyoro D, Ngassapa O, Vagionas K, Aligiannis N, Graikou K, Chinou I (2010) Chemical composition and antimicrobial activity of
the essential oils of four Ocimum species growing in Tanzania.
Food Chem 119:311–316
Ruberto G, Baratta MT (2000) Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem
69:167–174
Rushforth KD (1987) Conifers: facts on file publications. New York,
pp 232
Schuler P (1990) Natural antioxidants exploited commercially. In:
Hudson BJF (ed) Food antioxidants. Elsevier, London, pp 99–170
Silba J (1986) An international census of the Coniferae. In: Moldenke
HN, Moldenke AL (eds) Phytologia memoirs no. 8, Corvallis,
OR, USA
Todaro L, Andreu L, D’Alessandro CM, Gutierrez E, Cherubini P,
Saracino A (2007) Response of Pinus leucodermis to climate and
anthropogenic activity in the National Park of Pollino (Basilicata,
Southern Italy). Biol Conserv 137:507–519
Thirugnanasampandan R, Jayakumar R, Narmatha Bai V, Martin E,
Rajendra Prasad KJ (2008) Antiacetylcholinesterase and antioxidant ent-Kaurene diterpenoid, melissoidesin from Isodon wightii
(Bentham) H. Hara. Nat Prod Res 22:681–688
Tsoumis G (1983) Science and technology of wood. Van Nostrand
Reinhold, New York, pp 456