FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fra`r[ J[ 1999^ 04] 323Ð326 "1999#

Analysis of the essential oil volatiles of Douglas fir

(Pseudotsuga menziesii) from Bulgaria
Leopold Jirovetz,1* Christiane Puschmann,1 Albena Stojanova,2 Stojan Metodiev2 and Gerhard Buchbauer1
1
Institute of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
2
Higher Institute of Food and Flavour Industries, 26 Maritza, Plovdiv 4002, Bulgaria

Received 16 March 1999

Revised 5 July 1999
Accepted 07 July 1999

ABSTRACT] The essential oils of the bark\ fresh needles with twigs\ air!dried needles and twigs and fresh young
needles of Douglas _r "Pseudotsu`a menziesii# from Bulgaria were analysed by GC\ GCÐMS and olfactometry[
More than 49 compounds could be identi_ed by mass spectra and retention!time correlations\ as well as olfactory
evaluations[ As main compounds\ the monoterpenes b!pinene\ sabinene\ "Z#!b!ocimene\ "E#!b!ocimene\ a!
terpinolene\ a!terpineol\ citronellyl acetate\ a!terpinene\ limonene and g!terpinene were found[ An odour descrip!
tion is given\ as well as an assignment of the characterized odour to identi_ed active oil constituents[ Additionally\
_rst data on biological tests of these natural products are discussed[ Copyright Þ 1999 John Wiley + Sons\ Ltd[

KEY WORDS] Douglas _r^ essential oil^ GC^ GCÐMS^ olfactory evaluation^ biological e}ects

Introduction Experimental
Douglas _r "Pseudotsu`a menziesii# Mirb[ Franco\ fam! Sample Preparation
ily Pinaceae# is a wide!spread conifer species in America
The raw material was gathered in May 0888 in the
and Europe\ with di}erent compositions of volatiles in
region of Kazanlik in the Balkan Mountains[ The bark
the essential oils\ depending on the various plant parts[
"sample 0#\ fresh needles with twigs "sample 1#\ air!dried
Besides the pleasant _r odour\ used in various _elds of
needles with twigs "sample 2# and fresh young needles
cosmetics and perfumery\ biological activities were also
reported on this valuable natural product[ Some useful "sample 3# were cut to a size of 0 cm[ The essential oils
were obtained by steam distillation of about 499 g cut
e}ects on bears\ wild boar\ deer and sheep\ in cor!
raw material from each sample in a tinned copper lab!
relation with the composition of odour compounds\
have been studied extensively in the past[0\5\7\8 Also\ anti! oratory apparatus of 4 dm2 for 1 h[ The steam dis!
tillation was carried out at the rate of 09) and
microbial and vermicidal activities against\ for example\
atmospheric pressure of the steam "9[0 MPa#[ The yields
bacteria\ fungi and worms are well known from essential
of essential oils were as follows] 9[43) from the bark\
oils of Douglas _r[1\04\05 The composition of the volatiles\
9[45) from fresh needles with twigs\ 9[55) from air!
depending on seasonal and geographic conditions\ is
dried needles with twigs\ and 9[49) from the fresh
responsible for these e}ects[ Therefore\ the aim of this
young needles[ The samples were stored in a refrigerator
work was to give detailed information of the com!
until analysis[
position of essential oils of "0# the bark\ "1# the fresh
needles with twigs\ "2# the air!dried needles with twigs\
and "3# the fresh young needles\ of Bulgarian Douglas
_r\ which has been investigated very little in the past\02
and to correlate the identi_ed components with the Olfactory Evaluations
odour descriptions of the single samples[ Additionally\
_rst data from microbial tests on these essential oils The essential oils were diluted with dichloromethane
from Pseudotsu`a menziesii from Bulgaria are discussed[ "0 ] 09# and 09 ml of each sample was placed on a
commercial odour strip "Dragoco\ Germany#[ The
 Correspondence to] L[ Jirovetz\ Institute of Pharmaceutical Chemistry\ Uni!
odour of the single oils was evaluated by professional
versity of Vienna\ Althanstrasse 03\ A!0989 Vienna\ Austria[ perfumers[

Copyright Þ 1999 John Wiley + Sons\ Ltd[


Analysis
Gas Chromato`raphyÐFlame Ionization Detection
"GCÐFID#
A GC!03A with FID and C!R5A!Chromatopac inte!
grator "Shimadzu\ Japan# and a GC!2699 with FID
"Varian\ Germany# and C!R0B!Chromatopac inte!
grator "Shimadzu\ Japan# were used for GC analyses[
Parameters were as follows] carrier gas\ hydrogen^ injec!
tor temperature\ 149>C^ detector temperature\ 179>C^
temperature programme\ 39Ð149>C\ with a heating rate
of 5>C:min^ columns\ 59 m×9[21 mm bonded FSOT!
RSL!199 fused silica "_lm thickness 9[14 mm^ Bio!Rad\
Germany# and 59 m×9[21 mm bonded DB!WAX fused
silica "_lm thickness 9[49 mm^ Restek\ USA#[ Quanti!
_cation was carried out by percentage of peak area
calculations[ The identi_cation of single aroma com!
pounds was partly performed by the comparison of
retention times with reference data2\3\6\00\01 as well as
private retention index libraries[
Gas Chromato`raphyÐMass Spectrometry "GCÐMS#
For GCÐMS analyses\ the following were used] GC!
06A with a QP4999 Mass Spectrometer "class4k!sof!
tware^ Shimadzu\ Japan#\ GC!HP4789 with HP4869!
MSD "ChemStation software^ Hewlett!Packard\ USA#
and a GCQ "GCQ!software^ Finnigan!Spectronex\
USA:Germany#[ Parameters were as follows] carrier
gas\ helium^ injector!temperature\ 149>C^ interface!
heating\ 179>C^ ion source heating\ 199>C^ EI mode\ 69
eV^ mass range\ 30Ð449 amu^ for other parameters\ see
section on GCÐFID[ Mass spectra correlations were
with Wiley\ NIST\ NBS and private libraries on!line\ as
well as published data3\4 o}!line[
Microbiolo`ical tests
The following microorganisms were used] Staphy!
lococcus aureus\ Serratia marcescens\ Bacillus subtilis\
Proteus vul`aris\ Saccharomyces cerevisiae and Asper!
`illus ni`er[ The antibacterial activity was studied using
the agar!di}usion!cup method09\03 cups 9[5 cm and vol!
ume 9[90 ml for each product[ After cultivation at 26>C
for 13 h "bacteria#\ 29>C for 13 h "yeast#\ and 29>C for
13 h "mould fungi#\ the diameter of the zones obtained
was measured[
Results and Discussion
Using gas chromatographicÐspectroscopic "GC and
GCÐMS# and olfactory "odour evaluations# methods\
nearly 59 constituents of the essential oils of bark\ fresh
Copyright Þ 1999 John Wiley + Sons\ Ltd[
ESSENTIAL OIL OF PSEUDOTSUGA MENZIESII 324
needles with twigs\ air!dried needles with twigs and fresh
young needles of Douglas _r were identi_ed "see Table
0#[ Main compounds "concentration higher than 2)\
calculated as percentage of peak area from GC analyses#
of the essential oil of the Douglas _r bark "sample 0# are]
"Z#!b! "08[2)# and "E#!b!ocimene "04[3)#\ b!pinene
"04[0)#\ sabinene "03[8)#\ a!terpinolene "00[2)#\ a!
terpineol "2[7)# and citronellyl acetate "2[0)#[ From
the fresh needles with twigs "sample 1#\ b!pinene
"13[3)#\ sabinene "11[1)#\ a!terpinolene "07[7)#\
citronellyl acetate "4[8)#\ "Z#!b!ocimene "4[5)#\ a!ter!
pineol "4[3)#\ g!terpinene "2[8)# and geranyl acetate
"2[4)# were determined as principal constituents[ As
main compounds of the essential oil of the air!dried
needles with twigs "sample 2#\ b!pinene "20[1)#\ sabi!
nene "18[6)#\ a!terpinolene "06[2)#\ "Z#!b!ocimene
"3[8)# and a!terpineol "2[7)# were obtained^ and those
from the fresh young needles "sample 3# essential oil
were b!pinene "28[4)#\ sabinene "10[9)#\ a!terpinolene
"19[1)# and "Z#!b!ocimene "6[0)#[ A signi_cant cor!
relation of these data with olfactory evaluations "see
Table 1# of the samples can be given as follows[ The
combination of the main compounds b!pinene\ sabinene
and a!terpinolene with "Z#! and "E#!b!ocimene in a com!
parable concentration is responsible for the typical _r
odour[ The dominating citronella note of sample 1 can
be correlated to higher concentrations of citronellyl
acetate and geranyl acetate "together 8[3)^ both odour!
active constituents in citronella oils#[ The teatree!oil!
odour impression of sample 2 oil is the result of a higher
amount of a!terpinolene and a!terpineol\ while the tur!
pentine oil side!notes are known from b!pinene and
partly a!terpinolene[
Using various microorganisms "S[ aureus\ S[ marce!
scens\ B[ subtilis\ P[ vul`aris\ S[ cerevisiae and A[ ni`er#
the antimicrobial activity "MIC  minimum inhibiting
concentration in accordance to a reference1# of the four
essential oil samples was tested[ The _rst results are as
follows "see Table 2#[ All four samples show an anti!
bacterial activity against Gram!negative and Gram!
positive bacteria[ The bacteriostatic action is most
clearly expressed for the essential oil of fresh needles
with twigs "sample 1#[ These e}ects are\ however\
weaker against yeast and mould fungi[
In conclusion\ we can report that Bulgarian Douglas
_r essential oils "bark\ needles and twigs# are rich in
b!pinene\ sabinene\ a!terpinolene\ cis! and trans!b!
ocimene\ citronellyl acetate\ a!terpineol\ g!terpinene
and geranyl acetate[ These compounds are also respon!
sible for the typical pleasant odour of the di}erent
samples[ All of the four investigated essential oils
show antimicrobial activity and will therefore be used
for extensive tests in future studies[ To the best of
our knowledge\ these detailed analyses on essential
oils of Pseudotsu`a menziesii from Bulgaria were made
for the _rst time[
Flavour Fra`r[ J[ 1999^ 04] 323Ð326
325 L[ JIROVETZ ET AL[

Table 1. Composition of the essential oils of the bark (sample 1), fresh needles with
twigs (sample 2), air-dried needles with twigs (sample 3) and fresh young needles
(sample 4)
Compounda 0 1 2 3 Identi_cation RIb
2Z!Hexen!0!ol ndc nd 9[0 9[0 RI\ MSd 730
Hexanol nd nd 9[0 9[0 RI\ MS 738
a!Thujene 9[0 nd nd nd RI\ MS 824
a!Pinene 0[2 9[0 9[0 nd RI\ MS\ Oe 833
Heptanol 9[0 nd nd nd RI\ MS 841
Camphene 9[1 9[1 9[0 9[0 RI\ MS 843
2Z!Hexenyl acetate nd nd 9[0 9[0 RI\ MS 861
Sabinene 03[8 11[1 18[6 10[9 RI\ MS 863
b!Pinene 04[0 13[3 20[1 28[4 RI\ MS\ O 867
b!Myrcene 1[0 0[2 9[6 9[5 RI\ MS 880
a!Phellandrene 9[0 nd 9[0 9[0 RI\ MS 0996
0\3!Cineole 9[1 nd 9[0 nd RI\ MS 0998
a!Terpinene 9[8 0[7 9[5 9[8 RI\ MS 0905
p!Cymene 1[9 9[5 9[0 9[0 RI\ MS 0908
0\7!Cineole nd nd nd 9[0 RI\ MS 0910
Limonene 1[7 0[9 9[3 9[3 RI\ MS\ O 0914
"Z#!b!Ocimene 08[2 4[5 3[8 6[0 RI\ MS 0917
b!Phellandrene 9[6 0[9 9[4 9[4 RI\ MS 0920
"E#!b!Ocimene 04[3 0[7 9[7 9[2 RI\ MS 0939
g!Terpinene 0[7 2[8 0[3 0[7 RI\ MS 0946
Dihydro!myrcenol 9[1 nd 9[0 9[0 RI\ MS 0953
a!Terpinolene 00[2 07[7 06[2 19[1 RI\ MS\ O 0966
Linalool nd nd 9[0 9[0 RI\ MS 0985
Myrcenol nd 9[4 9[0 9[1 RI\ MS 0092
b!Phenylethanol 9[0 nd nd 9[0 RI\ MS 0094
b!Pinene oxide 9[3 nd 9[0 9[0 RI\ MS 0098
Fenchol 9[2 nd 9[0 9[1 RI\ MS 0005
Isopulegol nd nd 9[0 nd RI\ MS 0018
Camphor nd nd 9[0 9[0 RI\ MS 0021
trans!Pinocarveol 9[1 nd nd 9[0 RI\ MS 0024
Citronellal nd nd 9[2 9[0 RI\ MS 0028
trans!Verbenol nd nd nd 9[0 RI\ MS 0030
Borneol 9[0 nd 9[0 9[0 RI\ MS 0052
p!Cymen!7!ol 9[2 nd 9[0 9[0 RI\ MS 0057
a!Terpineol 2[7 4[3 2[7 0[8 RI\ MS\ O 0075
Dihydrocarveol nd nd 9[0 9[0 RI\ MS 0089
Octyl acetate 9[0 nd 9[0 nd RI\ MS 0083
b!Cyclocitral 9[2 nd 9[0 9[0 RI\ MS 0190
Carvone nd nd 9[1 9[0 RI\ MS 0116
Geraniol 9[2 nd 9[0 9[0 RI\ MS\ O 0127
Isopulegyl acetate nd nd 9[0 nd RI\ MS 0148
Thymol 9[1 nd 9[0 nd RI\ MS 0160
Bornyl acetate nd nd 9[0 9[2 RI\ MS 0167
Citronellyl acetate 2[0 4[8 1[1 9[4 RI\ MS\ O 0223
a!Longipinene nd nd 9[0 9[0 RI\ MS 0259
Geranyl acetate 9[6 2[4 0[0 9[2 RI\ MS\ O 0253
a!Cubebene nd nd nd 9[0 RI\ MS 0269
a!Copaene 9[0 nd 9[0 nd RI\ MS 0263
Decyl acetate 9[0 nd 9[0 9[0 RI\ MS 0281
b!Elemene 9[0 nd 9[0 9[0 RI\ MS 0399
b!Caryophyllene nd nd 9[0 9[0 RI\ MS 0329
"E#!b!Farnesene nd nd 9[1 nd RI\ MS 0325
a!Humulene 9[0 9[4 9[3 9[2 RI\ MS 0335
Germacrene D 9[1 9[6 9[2 9[5 RI\ MS 0360
a!Muurolene 9[0 nd 9[0 9[0 RI\ MS 0384
d!Cadinene nd nd 9[1 9[1 RI\ MS 0405
Caryophyllene oxide I 9[2 9[5 9[3 9[0 RI\ MS 0466
d!Cadinol nd nd 9[0 nd RI\ MS 0564
Unknown 9[5 9[1 9[2 9[3
a
In order of their retention index "non!polar column#[
b
Identi_ed by retention index correlations using reference data "non!polar column#[
c
Not identi_ed[
d
Identi_ed by mass spectra correlations using reference data "see section on GCÐMS#[
e
Identi_ed by olfactory evaluations[

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 323Ð326
 ESSENTIAL OIL OF PSEUDOTSUGA MENZIESII 326

Table 2. Olfactory evaluations of the four essential oil References

samples from Bulgarian Douglas fir
0[ Buchbauer G\ Jirovetz L\ Wasicky M\ Nikiforov A[ J[ A`ric[
Sample 0[ Essential oil of the bark] typical _r oil odour Food Chem[ 0883^ 31] 1741[
Sample 1[ Essential oil of the fresh leaves with twigs] citronella 1[ Chalchat JC\ Garry R[Ph\ Bastide P\ Fabre F\ Malhuret R[ Plant
oil top!note\ later _r needle odour Med[ Phytother[ 0880^ 14] 073[
Sample 2[ Essential oil of the air!dried leaves with twigs] teatree 2[ Davies NW[ J[ Chromato`r[ 0889^ 492] 0[
oil odour notes\ later palmarosa oil and _r oil notes 3[ Jennings W\ Shibamoto T[ Qualitative Analysis of Flavor and
Sample 3[ Essential oil of the fresh young leaves] typical _r nee! Fra`rance Volatiles by Glass Capillary Gas Chromato`raphy[ Aca!
dle odour with turpentine oil side!notes demic Press] New York\ 0879[
4[ Joulain D\ Konig WA[ The Atlas of Spectral Data of Sesquiterpene
Hydrocarbons[ E[B[!Verlag] Hamburg\ 0887[
5[ Kimball BA\ Craver RK\ Johnston JJ\ Nolte DL[ J[ Hi`h Resol[
Table 3. Antimicrobial activities of Douglas fir essential Chromato`r[ 0884^ 07] 110[
oils 6[ Kondjoyan N\ Berdaque J!L[ A Compilation of Relative Retention
Indices for the Analysis of Aromatic Compounds[ Edition du
Microorganism Sample Sample Sample Sample Laboratoire Flaveur] Saint Genes Champelle\ 0885[
0 1 2 3 7[ Oh HK\ Sakai T\ Jones MB\ Longhurst WM[ Appl[ Microbiol[
0856^ 04] 666[
Staphyllococcus aureus 08 13 19 6 8[ Sakai T\ Maarse H\ Kepner RE\ Jennings WG\ Longhurst WG\
Serratia marcescens 03 19 05 19 Longhurst WM[ J[ A`ric[ Food Chem[ 0856^ 04] 0969[
Bacillus subtilis 07 07 07 11 09[ Sojanova A\ Metodiev S\ Karova E[ Antimicrobial activity of
Proteus vul`aris 03 10 03 11 aromatic products[ 2[ Essential oil of Pseudotsu`a menziesii
Saccharomyces cerevisiae 07 03 03 03 "Mirb[# Franco[ Poster presentation "No[ MM 56# at the First
Asper`illus ni`er 6 05 03 03 Balkan Conference of Microbiology\ {Microbiologia Balkania
|88|\ Plovdiv\ Bulgaria\ October 4Ð8\ 0888[
 mm Values of inhibition zones[
00[ Tudor E[ Rev[ Roumaine Chim[ 0887^ 32] 476[
01[ Tudor E[ J[ Chromato`r[ A 0886^ 668] 176[
02[ Yonov N[ Sci[ Woods 0881^ 3] 58[
Acknowled`ements * The authors acknowledge the olfactory evalu! 03[ Zaika L[ J[ Food Safety 0877^ 8] 86[
ations by Mr Victor Hausmann and Mr Wolfgan Hoppner\ Chief 04[ Zou J\ Cates RG[ J[ Chem[ Ecol[ 0884^ 10] 276[
Perfumers of Dragoco Co[\ Vienna\ Austria# as well as help in bio! 05[ Zou J\ Cates RG[ J[ Chem[ Ecol[ 0886^ 12] 1202[
logical tests by Professor Dr E[ Karova\ Higher Institute of Food and
Flavours\ Plovdiv\ Bulgaria[

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 323Ð326