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FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fra`r[ J[ 1999^ 04] 089Ð083

Essential oil and glycosidically bound volatiles of


Origanum vulgare L. ssp. hirtum (Link) Ietswaart
J. Mastelić, M. Milos̆ and I. Jerković
Department of Organic Chemistry, Faculty of Chemical Technology, N. Tesle 10/V, 21000 Split, Croatia

Received 12 June 0888


Revised 19 December 0888
Accepted 00 January 1999

ABSTRACT] The glycosidically bound volatiles were isolated from dried plant material by percolation with
ethyl acetate and by extraction with water during hydrodistillation of essential oil[ Fifteen volatile aglycones
were identi_ed by gas chromatography!mass spectrometry "GC!MS# on two columns with di}erent polarity of
the stationary phases[ The main aglycones were] thymoquinone\ benzyl alcohol\ thymol\ 1!phenylethanol\
carvacrol and 0!octen!2!ol[ The content of aglycones was 08 and 10 mg kg−0 with respect to the method of
isolation[ The chemical composition of aglycones was compared with the chemical composition of essential oil\
which consisted mainly of thymol and carvacrol[
After the hydrolysis of glycosides\ D!"¦#!glucose and unknown disaccharide were identi_ed[ The hydrolysis
of disaccharide\ D!"¦#!glucose and D!"¦#!galactose were detected by TLC and GC!MS as trimethylsilylethers[
Copyright Þ 1999 John Wiley + Sons\ Ltd[

KEY WORDS] Ori`anum vul`are ssp[ hirtum^ Lamiaceae^ glycosidically bound volatiles^ sugar moiety^ essential
oil\ gas chromatography!mass spectrometry

Introduction antioxidant activity of di}erent Ori`anum species have


been intensively investigated[15Ð20 Glycosidically bound
Ori`anum vul`are L[ ssp[ hirtum "Link# Ietswaart is a volatiles were partially investigated in Ori`anum vul`are
famous aromatic and medicinal herb[ Due to its high by Merkx and B[ Svendsen[21 They identi_ed eugenol
content of thymol and carvacrol\ the essential oil of and an isomer of methoxy!vinylphenol as the main agly!
oregano is an interesting product in pharmacology and cones[
food industry[ The herb\ its extracts and its essential oil The aim of this study was to determine the com!
serve as folk remedies[ The plant itself is widely used in position of aglycones and sugar moiety of gly!
the Mediterranean and in the Latin American cuisines cosidically!bound volatile compounds from Ori`anum
"chilli and pizza#[ vul`are L[ ssp[ hirtum "Link# Ietswaart[ The percolation
Volatile agylcones were detected in many aromatic with ethyl acetate at room temperature and the extrac!
and nonaromatic plants[ Among volatile aglycones tion with water during hydrodistillation of essential oil
from plants of Lamiaceae family aliphatic alcohols\ ter! were compared as methods for the isolation of glyco!
penes\ coumarin and phenylpropane compounds were sides[ The composition of volatile aglycones and of
identi_ed[0 The sugar moiety of these glycosides can be essential oil was also compared[
monosaccharidic and disaccharidic[ b!D!glucopyranose
is the most common monosaccharide found in many
plants of the Lamiaceae1Ð7 family\ and in numerous
other plants[8Ð07 b!D!galatopyranose is present in Thy! Experimental
mus vul`aris08 and b!L!arabinopyranose in Gymnocladus
chinensis[19 The disaccharidic sugar moiety of these gly! Plant Material
cosides can be divided into several groups] rhamnosyl!
glucoside^10\11 arabinosyl!glucoside^10\12 apiosyl!glu! The plant material was collected in the Mediterranean
coside11\13 and galactosyl!glucoside sugar moiety[14 region of Croatia\ near Split\ after ~owering in Sep!
The essential oils\ the geographic variations and the tember 0887[ The dried plant material "leaves and apical
stalks# was used for investigation[ The voucher speci!
 Correspondence to] J[ Mastelic\ Department of Organic Chemistry\ Faculty
men is deposited at Department of Organic Chemistry\
of Chemical Technology\ N[ Tesle 09:V\ 10999 split\ Croatia[ Faculty of Chemical Technology\ University of Split[

Copyright Þ 1999 John Wiley + Sons\ Ltd[


ESSENTIAL OIL OF ORIGANUM VULGARE 080

Isolation of Glycosides Isolation of Carbohydrates


The glycosides were isolated by two methods] After removing the aglycones\ the aqueous extracts
"Method A and Method B# were concentrated to 9[4 ml
Method A in a rotating evaporator under reduced pressure\ and
One hundred grams of plant material was extracted heated in a boiling water bath for 09 min[ The salts and
by exhaustive percolation with ethyl acetate at room the denatured enzyme were removed as precipitate by
temperature[ Octyl!b!D!glucoside\ 499 mg\ was added as addition of 09 ml of ethanol[ The ethanol extract was
internal standard22 in ethyl acetate[ After percolation\ concentrated to 9[4 ml and this solution was used for
the extract was concentrated to dryness in a rotating thin layer chromatography "TLC# of carbohydrates[
evaporator under reduced pressure up to 39>C[ The
residue was dissolved in ethanol and puri_ed by selective Isolation and Acidic Hydrolysis of a Disaccharide
precipitation of the ballast compounds using water[ The
precipitation of acidic balast compounds was performed Glucose and disaccharide\ which were detected by TLC
in ethanol with conc[ ammonia[23 Finally\ the puri! analyses of ethanolic extract\ were separated by ~ash
_cation was performed by ~ash chromatography on a chromatography on silica gel column "09 g silica gel 59\
silica gel column applying ethyl acetate ] ethanol ] i[d[ 1[4 cm#[ Elution was performed with 199 ml mixture
ammonia 5 ] 2 ] 0 v:v:v[ TLC analyses showed the of ethyl acetate ] ethanol 5 ] 1 v:v and 199 ml ethanol[
absence of free carbohydrates in the glycosidic fraction[ The fraction of disaccharide was hydrolyzed with HC0
The obtained glycosidic fraction was concentrated to "pH 0# for 29 min at 099>C[ After the hydrolysis\ neu!
dryness\ dissolved in a citrate bu}er "pH 4[4^ 4 ml# and tralization and drying\ the residue was dissolved in 9[4
the remaining free terpenes and hydrophobic com! ml methanol[ The precipitate of NaCl was removed and
pounds were removed with penthane!dicloromethane the methanolic solution was subjected to TLC and GC!
as described in a previous paper[22 MS analyses[

Method B Acidic Hydrolysis of Glycosides


One hundred grams of plant material was submitted\
for 2 h\ to a simultaneous extraction and hydro! The acidic hydrolysis of isolated glycosides was per!
distillation in Clevenger type apparatus[ Octyl!b!D!glu! formed with HC0 "pH 0# for 0 hr at 099>C[ The obtained
coside\ 499 mg\ was added to water intended for aglycones were not isolated and investigated[ The iso!
hydrodistillation as internal standard[ The essential oil lation of liberated saccharides was performed as given
was separated\ successfully dried over Na1SO3 and in above paragraph "neutralization\ drying and dis!
stored[ The aqueous extract was separated and the solving of residue#[
residual plant material was extracted once more with
299 ml of boiling water[ The pooled aqueous extracts
Identification of Carbohydrates by TLC
were concentrated to 29 ml in a rotating evaporator
under reduced pressure\ up to 49>C[ Balast components TLC was employed to separate and identify the carbo!
were removed by precipitation with ethanol[ Further hydrates from the hydrolysates[ The carbohydrates
puri_cation\ ~ash chromatography\ dissolving in bu}er were chromatographed on silica gel with di}erent
and removing the hydrophobic compounds were per! mobile phases[24Ð26 The authentic samples of carbo!
formed as described in Method A[ hydrates as 9[1) methanolic solutions were used for
references[ Detection] aniline!diphenylamine!phos!
phoric acid as reagent[ The individual spots were ident!
Enzymatic Hydrolysis of Glycosides and Separation
i_ed by comparison of their RF!values with those of
of Aglycones
authentic samples[
b!Glucosidase from almonds "{Fluka|\ 19 mg# was
added to the glycosidic solutions "from Method A and Preparation of Trimethylsilylethers
Method B# along with 2 ml pentane for the trapping of
liberated aglycones[ The hydrolysis was carried out for The solution of carbohydrates was concentrated to dry!
61 h\ at 29>C with the mixture being shaken occasion! ness and dissolved in 9[4 ml pyridine[ The mixture of
ally[ After the hydrolysis\ the pentane layer was separ! hexamethyldisylazane and trimethylchlorsilane "2 ml
ated[ The remaining aglycones were extracted from the 1 ]0 v:v# was added to the solution[ After a 2 h reaction
aqueous layer with pentane "09×1 ml#[ The combined at room temperature\ the solvent and the remaining
pentane extracts were dried over an anhydrous sodium reagents were removed by evaporation under reduced
sulphate\ concentrated to _nal volume of 9[4 ml\ and 0 pressure[ The residue was dissolved in 9[4 ml pentane
ml was used for GC!MS analysis[ and concentrated once more to dryness[ Finally\ the

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 089Ð083
081 J[ MASTELIC AND I[ JERKOVIC
ł \ M[ MILOS ł

residue was dissolved in 9[4 ml pentane ] ether 0 ]0 v:v Table 1. Constituents and percentage composition of
and this solution was used for GC!MS analyses of essential oil from Origanum vulgare L. ssp. hirtum (Link)
carbohydrates[ Ietswaart isolated by hydrodistillation
Peak Compound I0 Peak area Methods of
No[ "HP!19M# ) identi_cation
Gas Chromatography-Mass Spectrometry (GC-MS)
Hydrocarbons
The volatile aglycones and the essential oil were 0[ a!Thujene 0923 9[4 I0\ I1\ MS
1[ b!Pinene 0986 9[2 I0\ I1\ MS
analyzed by gas chromatography!mass spectrometry 2[ Sabinene 0001 9[1 I0\ I1\ MS
"Hewlett!Packard\ model 4789\ with a mass selective 3[ d!2!Carene 0020 9[2 I0\ I1\ MS
detector\ model 4860A#[ Two columns with di}erent 4[ b!Phelandrene 0101 9[0 I0\ I1\ MS
5[ g!Terpinene 0120 1[9 I0\ I1\ MS
polarity of stationary phases were used[ GC operating 6[ Terpinolene 0159 9[2 I0\ Ð\ MS
conditions were]22\23 column HP!19M "Carbowax 19M#\ 7[ p!Cymene 0162 00[7 I0\ Ð\ MS
49 m×9[1 mm i[d[\ _lm thickness 9[1 mm\ column tem! 8[ Caryophyllene 0467 9[6 I0\ I1\ MS
09[ a!Humulene 0526 9[0 I0\ I1\ MS
perature programmed from 69>C isothermal for 3 min\ 01[ a!Murolene 0582 9[2 I0\ I1\ MS
then increased to 079>C at a rate of 3>C min−0^ column 02[ b!Farnesene 0588 9[2 I0\ I1\ MS
HP!090 "Methylsilicone#\ 14 m×9[1 mm i[d[\ _lm thick! 03[ b!Bisabolene 0690 9[6 I0\ I1\ MS
04[ d!Cadinene 0618 9[1 I0\ I1\ MS
ness 9[1 mm\ column temperature programmed from
69>C isothermal for 1 min\ then increased to 199>C at Oxygene containing compounds
a rate of 2>C mm−0^ carrier gas helium\ ~ow rate 0 ml 05[ 0!Octen!2!ol 0304 1[9 I0\ I1\ MS
06[ Bornyl acetate 0431 9[0 I0\ I1\ MS
min−0\ injector temperature 149>C\ volume injected 0 07[ Terpinen!3!ol 0448 0[9 I0\ I1\ MS
m\ split ratio 0 ] 49[ MS conditions] ionization voltage 08[ Methylthymyl ether 0454 9[4 I0\ Ð\ MS
69 eV\ ion source temperature 179>C\ mass range 29Ð 19[ 1!Isopropyl!0!methoxy!
3!methyl benzene 0465 9[3 I0\ I1\ MS
299 mass units[ The analysis of carbohydrates as tri! 10[ Borneol 0544 0[0 I0\ I1\ MS
methylsilylethers was performed on column HP!090 11[ p!Cymen!7!ol 0676 9[0 I0\ I1\ MS
with the same GC!MS conditions[ In this case mass 12[ Thymol 1012 38[2 I0\ I1\ MS
13[ Carvacrol 1038 13[5 I0\ I1\ MS
range was 29Ð599 mass units[
Total 85[2)

Identification and Quantitative Determination I0  retention indices on HP!19M^ I1  retention indices on HP!090^
MS  mass spectra^ Ð  not detected[
Individual peaks were identi_ed by comparison of their
retention indices with those of authentic samples\ as
well as by comparison of their mass spectra with those Glycosidically Bound Volatiles
stored in data base "Wiley library#[ The percentage com!
position of the samples was computed from the GC The content of glycosidically bound volatile compounds
peak areas without using correction factors[ The con! in dried plant material was 08 mg kg−0 "Method A#
tents of aglycones were calculated from the GC!peak and 10 mg kg−0 "Method B#[ Fifteen aglycones were
areas related to the GC!peak area of 0!octanol "lib! identi_ed[ Among the aglycones were identi_ed ali!
erated from octyl!b!D!glucoside#[ Preliminary GC!MS phatic alcohols\ terpene compounds and derivatives of
analysis showed the absence of 0!octanol as potential phenylpropanes[ The results are shown in Table 1[ The
aglycone[ main aglycones for both methods were] thymoquinone
"36[9 and 39[1)#\ benzyl alcohol "1[9 and 7[8)#\ thy!
mol "5[7 and 6[4)#\ 1!phenylethanol "9[7 and 4[5)#\ 0!
octen!2!ol "4[3 and 0[2)# and carvacrol "4[4 and 0[8)#[
Results and Discussion Minor di}erences among the content and the com!
Free Volatile Compounds position of aglycones depend on the isolation methods[
By comparison of the chemical composition of the
The yield of essential oil was 1[8)[ The content and essential oil "Table 0# and aglycones "Table 1# _ve com!
chemical composition of the essential oil is given in pounds were established to be identical] 0!octen!2!ol\
Table 0[ Sixteen compounds were identi_ed rep! terpinen!3!ol\ p!cymen!7!ol\ thymol and carvacrol[ Our
resenting 85[2) of the total oil[ The main components results show only moderate correlation in the chemical
were thymol "38[2)#\ carvacrol "13[5)# and p!cymene composition of free and glycosidically bound volatiles[
"00[7)#[ The essential oil also contained smaller quan! The aglycones\ such as aliphatic alcohols\ 1!phenyl!
tities of g!terpinene "1[9)#\ 0!octen!2!ol "1[9)#\ bor! ethanol\ benzyl alcohol\ eugenol\ linalool\ geraniol\
neol "0[0)# and terpinen!3!ol "0[9)#[ Similar results neral\ a!terpineol and terpinen!3!ol can\ more or less\
were reported for the Green origin of Ori`anum vul`are be considered as ubiquitous in aglycone fractions of
ssp[ hirtum[20 Lamiaceae0 family[ From our results it can be seen that

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 089Ð083
ESSENTIAL OIL OF ORIGANUM VULGARE 082

Table 2. Identified constituents and percentage com- 080"39#\ 036"13#\ 017"7#\ 092"5#\ 62"78#[ b!anomer of
position of volatile aglycones from Origanum vulgare L. glucopyranose] M¦ 324"1#\ 294"1#\ 180"1#\ 106"06#\
ssp. hirtum (Link) Ietswaart isolated by two different 193"099#\ 080"27#\ 078"3#\ 036"10#\ 017"5#\ 092"5#\
methodsa 62"66#\ a!anomer of galactopyranose] M¦ 324"0#\
Peak Compound Peak area ")# Methods of 294"2#\ 154"1#\ 106"16#\ 194"10#\ 193"099#\ 080"37#\
No[ identi_cation 036"11#\ 017"00#\ 092"5#\ 62"67#\ b!anomer of gal!
A B
actopyranose] M¦ 324"2#\ 250"1#\ 208"3#\ 107"00#\
0[ 2!Hexen!0!ol tr 2[3 I0\ I1\ MS 106"49#\ 194"11#\ 193"099#\ 081"07#\ 080"82#\ 036"23#[ In
1[ 0!Octen!2!ol 4[3 0[2 I0\ I1\ MS
2[ Benzaldehyde 9[7 9[8 I0\ I1\ MS
general\ galactose is rarely found as the glycone moiety
3[ Terpinen!3!ol 9[5 Ð I0\ I1\ MS of glycosidically bound volatiles[ This monosaccharide
4[ Thymoquinone 36[9 39[1 I0\ I1\ MS unity was identi_ed as glycone part in glycosides of
5[ Methyl salicylate Ð 9[5 I0\ I1\ MS
Thymus vul`aris "Lamiaceae#\08 as well as the disac!
6[ Benzyl alcohol 1[9 7[8 I0\ I1\ MS
7[ 1!Phenyl ethanol 9[7 4[5 I0\ I1\ MS charide part in glycosides of b!D!glucopyranosyl!"0!1#!
8[ p!Cymen!7!ol 9[7 0[1 I0\ I1\ MS b!D!galactopyranose in Coleus forskohlii "Lamiaceae#[14
09[ Eugenol 9[3 Ð I0\ I1\ MS
00[ Thymol 5[7 6[4 I0\ I1\ MS Acknowled`ements * This work was supported by Croatian National
01[ Carvacrol 4[4 0[8 I0\ I1\ MS Grant\ Project 900!992[
02[ 0!H!Indole 1[1 9[7 I0\ I1\ MS
03[ 1!"p!Metoxyphenyl# ethanol Ð 0[2 Ð\ Ð\ MS
04[ Butyl phtalate monoester 2[9 tr Ð\ Ð\ MS

Total 64[2) 63[9) References


a
Methods used] A!Percolation with ethyl acetate and enzymatic hydrolysis^ 0[ Stahl!Biskup E\ Intert F\ Holthuijzen J\ Stengele M\ Schulz G[
B!Extraction with water during hydrodistillation and enzymatic hydrolysis[
I0  retention indices on HP!19M^ I1  retention indices on HP!090^
Flavour Fra`r[ J[ 0882^ 7] 50[
MS  mass spectra^ Ð  Not detected^ tr  trace ³ 9[0)[ 1[ Sakata I\ Mitsui T[ A`ric[ Biol[ Chem[ 0864^ 28] 0218[
2[ Sakata I\ Koshimizu K[ A`ric[ Biol[ Chem[ 0867^ 31] 0848[
3[ Martinkus C\ Croteau R[ Plant Physiol[ 0870^ 57] 88[
4[ Lang E\ Horster H[ Planta Med[ 0866^ 20] 001[
most of them were not identi_ed among the oregano 5[ Skopp K\ Horster H[ Planta Med[ 0865^ 18] 197[
aglycone compounds[ 6[ Sasaki H\ Tagushi H\ Endo T\ Yoshioka I\ Itaka Y[ Chem[ Pharm[
Bull[ 0870^ 18] 0525[
7[ Cubo M\ Sasaki H\ Endo T\ Tagushi H\ Yoshioka I[ Chem[
Pharm[ Bull[ 0875^ 230] 2986[
Carbohydrates 8[ Francis MJO\ Allcock C[ Phytochem[ 0858^ 7] 0228[
09[ Willis RDH\ Scriven FM[ Phytochem[ 0868^ 07] 674[
After removing the aglycones from aqueous solutions 00[ Tackeda Y\ Fukomoto K\ Tachibana M\ Shingu T\ Tetsuro F\
the released carbohydrates were identi_ed by TLC on a Ichihara T[ Phytochem[ 0889^ 18] 0480[
silica gel with solvents of di}erent polarity[ After the 01[ Takeo T[ Phytochem[ 0870^ 19] 1034[
02[ Banthorpe DV\ Mann J[ Phytochem[ 0860^ 00] 1478[
enzymatic hydrolysis of the glycosides D!"¦#!glucose as 03[ Myakado M\ Ohno N\ Kirai H\ Yoshioka H[ Phytochem[ 0863^
the main glycone and one unknown disaccharide was 02] 1770[
found\ RF!values of disaccharide in di}erent solvents 04[ Tschesche R\ Ciper F\ Breimaier E[ Chem[ Ber[ 0866^ 009] 2000[
05[ Huiying L\ Shoushen L\ McCabe T\ Clardy J[ Planta Med[ 0873^
were similar to those of "¦#!lactose[ The most e}ective 49] 490[
mobile phase was n!buthanol ] acetic acid ] ether ] water 06[ Krauss R\ Spiteler G[ Phytochem[ 0880^ 29] 0192[
8 ] 5 ] 2 ] 0 v:v:v with the following RF values] D"¦#! 07[ Inoshiri S\ Saiki M\ Kohda H\ Otsuka H\ Yamasaki K[ Phyto!
chem[ 0877^ 16] 1758[
glucose 9[33\ D"¦#!galactose 9[28\ "¦#!lactose 9[19[ The 08[ Mulkens A[ Pharm[ Acta Helv[ 0876^ 51] 118[
acidic hydrolysis of isolated disaccharide produced\ 19[ Konoshima T\ Sawada T[ Chem[ Pharm[ Bull[ 0873^ 21] 1506[
after enzymatic hydrolysis\ D!"¦#!glucose and D!"¦#! 10[ Williams PJ\ Strauss CR\ Wilson B\ Massy!Westropp R[ Phyto!
chem[ 0871^ 10] 1902[
galactose "in 0 ] 0 ratio#[ The same monosaccharides 11[ Nagao T\ Okabe H\ Yamagauchi T[ Pharm[ Bull[ 0877^ 25] 460[
were identi_ed after a direct acidic hydrolysis of gly! 12[ Huiying L\ Shoushen L\ McCabe T\ Clardy J[ Planta Med[ 0873^
cosides "without previous enzymatic hydrolysis#[ The 49] 490[
13[ Bredenkamp MW\ Drewes SE\ Wenther GL[ Phytochem[ 0878^
acidic hydrolisate contained D!"¦#!glucose "77)# and 17] 152[
D!"¦#!galactose "01)#[ The agylcones obtained by 14[ Ahmed B\ Vishwakarma RA[ Phytochem[ 0877^ 16] 2298[
acidic hydrolysis of glycosides contained many artefacts 15[ Kokkini S\ Karousou A\ Dardioti A\ Krigas N\ Lanaras T[ Phy!
tochem[ 0886^ 33] 772[
"due to reactions of elimination and:or rearrangement# 16[ Russo M\ Galletti GC\ Bocchini P\ Carnacini A[ J[ A`ric[ Food
and were therefore not further investigated[ Chem[ 0887^ 35] 2630[
Finally\ D!"¦#!glucose and D!"¦#!galactose were 17[ Lagouri V\ Blekas G\ Tsimidou M\ Kokkini S\ Boskou D[ Z[
Lebensm Unters Forsch[ 0882^ 086] 19[
identi_ed by GC!MS as trimethylsilylethers on HP!090 18[ Kokkini S\ Vokou D[ Biochem[ Syst[ Ecol[ 0882^ 10] 286[
column by coinjection with authentic compounds[ The 29[ Kokkini S\ Karousou R\ Vokou D[ Biochem[ Syst[ Ecol[ 0883^
anomers of glucose and galactose gave the following 11] 406[
20[ Vokou D\ Kokkini S\ Bessiere JM[ Biochem[ Syst[ Ecol[ 0882^ 10]
ion fragments\ m:z ")# a!anomer of glucopyranose] 176[
M¦ 324"1#\ 282"1#\ 294"2#\ 180"1#\ 106"06#\ 193"099#\ 21[ Merkx YM\ Baerheim Svendsen A[ Planta Med[ 0878^ 44] 77[

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 089Ð083
083 J[ MASTELIC AND I[ JERKOVIC
ł \ M[ MILOS ł

22[ Mastelic J\ Milos² M\ Kus²trak D\ Radonic A[ Croat[ Chem[ Acta[ 25[ Soczewinski E\ Wojciah M\ Pachowicz K[ Chem[ Anal[ "Warsaw#
0887^ 60] 036[ 0887^ 32] 712[
23[ Mastelic J\ Kus²trak D[ Acta Pharm[ 0886^ 36] 022[ 26[ Franken!Luykx JMM\ Klopper WJ[ Brauwissenschaft 0856^ 19]
24[ Bieganowska ML\ Petruzynik A[ Chem[ Anal[ "Warsaw# 0886^ 062[
31] 235[

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 089Ð083

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