129
FARMACIA, 2008, Vol.LVI, 2
ANALYSIS OF VOLATILE COMPOUNDS OF
OFFICINAL TILIAE FLOS BY GAS –
CHROMATOGRAPHY COUPLED WITH MASS
SPECTROMETRY
VALERIA RĂDULESCU1*, ELIZA OPREA2
University of Medicine and Pharmacy “Carol Davila”, Faculty of
Pharmacy, 6 Traian Vuia, 020956, Bucharest, Romania
2
University of Bucharest, Faculty of Chemistry, 4-12 Regina Elisabeta,
030018, Bucharest, Romania
*corresponding author: valeriaradulescu@netscape.net
1
Abstract
The hydro-distilled and the infusion obtained from Tilia platyphyllos (Tiliaceae)
flowers were extracted on 500mg octadecylsilane (C18) cartridges. The volatile compounds
retained on C18 cartridge were eluted with dichloromethane. The hydro-distilled and
infusion extracts were analyzed by gas chromatography coupled with mass spectrometry
(GC/MS). In hydro-distilled and infusion extracts were identified 36 and 20 compounds,
respectively.
The main constituents of hydro-distilled were 2-phenylethanol with 26.07%, six
monoterpenic hydrocarbons, eight monoterpenic alcohols, four phenol-ethers, eight
carbonylic compounds, four esters and four alkanes.
In the infusion extract, 2-phenylethanol represented 29.48% of the total area;
other important compounds were: six monoterpenic hydrocarbons (totalling 12.23%) and
six alkanes (22.17%). A few other major compounds were also identified: 2-phenylethyl
butanoate (12.11%), 4-methyl-2.6-ditertbuthylphenol (5.01%), vomifoliol (4.44%).
Abstract
Hidrodistilatul şi infuzia obţinute din flori de Tilia platyphyllos (Tiliaceae) au
fost supuse extracţiei pe cartuş cu octadecilsilan (C18) de 500mg. Compuşii volatili reţinuţi
pe cartuşul C18 au fost eluaţi cu diclorometan. Extractele (hidrodistilatul şi infuzia) au fost
analizate prin gaz –cromatografie cuplată cu spectrometrie de masă, fiind identificaţi 36 şi,
respectiv, 20 de compuşi.
Componentele principale ale hidrodistilatului au fost: 2-feniletanol cu 26,07%,
şase hidrocarburi monoterpenice, opt alcooli monoterpenici, patru fenol-eteri, opt compuşi
carbonilici, patru esteri şi patru alcani. În infuzie, 2-feniletanolul a reprezentat 29,48% din
aria totală; alţi compuşi importanţi au fost: şase hidrocarburi monoterpenice (totalizând
12,23%) şi şase alcani (22,17%). Au mai fost identificaţi: butanoat de 2-feniletil (12,11%),
4-metil-2,6-diterţbutilfenol (5,01%), vomifoliol (4,44%).


Tilia platyphyllos
solid Phase Extraction


infusion
GC/MS
130
FARMACIA, 2008, Vol.LVI, 2
INTRODUCTION
In the European Pharmacopoeia (EP), the inflorescence of Tilia
platyphyllos Scop, Tilia cordata Miller and Tilia vulgaris Heyne are
accepted as officinal species [1]. The German Commission E Monographs
has approved linden flower, with or without bracts, for the treatment of
colds and cold related coughs [2].
In spite of the fact that in Romania there are many species, only
flowers with bracts from three species are used for medicinal purpose: Tilia
platyphyllos Scop., Tilia cordata Miller and Tilia tomentosa Moench.
Linden flowers, Tiliae flos, have been used in phytotherapy, as they have
antispasmodic, sudorific, expectorant, diuretic and sedative effects.
The pharmaceutical properties of the linden flower are attributed to
its chemical compounds: flavonoids, volatile oil, tannins and mucilage
polysaccharide [3- 8].
The flavonoid composition of the flowers, bracts and leaves of
different officinal species of Tilia were studied using high performance
liquid chromatography [9- 13].
The sedative activity of the linden flower has been attributed to the
volatile oil components [7] although the volatile oil content is low (0.020.1%).
Gas chromatographic studies concerning the chemical composition
of essential oil showed very different results for different species, depending
on the method used for the sample preparation for chromatographic analysis
[14- 16].
The aim of the present study is to elucidate the chemical
composition in volatile compounds of flowers of Tilia platyphyllos Scop.
MATERIALS AND METHODS
Reagents and solvents
All solvents and reagents were purchased from Merck, Darmstadt,
Germany:
- dichloromethane, methanol were SupraSolv for gas chromatography;
- cartridges for solid phase extraction were LiChrolut RP-18 columns,
500 mg;
- anhydrous Na2SO4 granulated for organic trace analysis
The n-alkanes C8 – C20 used for the determination of the retention
Kovats index were from Fluka.
FARMACIA, 2008, Vol.LVI, 2
131
Plant material
The plant material of Tilia platyphyllos was harvested during
flowering stage from Bucharest Botanical Garden. The flowers and bracts
were separated from leaves and manually grounded.
Solid phase extraction of flowers hydro-distilled
45g of Tilia platyphyllos flowers were hydro-distilled in a
Clevenger-type apparatus for 4 h [17].
In order to determine the contents in volatile oil of the linden
flower, in the process of hydro-distillation, xylene was used. On the other
hand, in order to be able to analyze very light (highly volatile) compounds,
the hydro-distillation was made without organic solvent.
The volatile compounds from hydro-distilled were isolated by
solid-phase extraction (SPE) [18, 19].
A 500 mg C18 solid phase extraction cartridge was conditioned by
eluting it with 2x4mL dichloromethane, followed by 2x4mL of methanol.
The cartridge was allowed to dry after each flush. Then, 2x4mL of distilled
water were passed through the cartridge (the cartridge should not be allowed
to dry before sample application).
By applying a slight vacuum, the obtained hydro-distilled (diluted
at 200mL) were allowed to pass through the cartridge at a flow of 12mL/min. The cartridge was eluted with 5mL water, and then dried for 5-10
minutes with nitrogen. The compounds retained on C18 cartridge were eluted
with 2x2.5mL of dichloromethane and 2μL from this solution were injected
for GC analysis.
Infusion sample preparation
100mL of boiled water were poured over samples of 2g of Tilia
platyphyllos flowers. After approximately 30 minutes, the cold extracts were
filtered on glass fibre filter. The residue from the filter was rinsed 3-4 times
with distilled water, and the final filtrated infusion was adjusted to 100mL.
Solid phase extraction of flowers infusion
A 500 mg C18 solid phase extraction cartridge was conditioned by
the same method as solid phase extraction of flower hydro-distilled. By
applying a slight vacuum, the obtained infusions were allowed to pass
through the cartridge at a flow of 1-2mL/min. The cartridge was then dried
for 5-10 minutes with nitrogen. The compounds retained on C18 cartridge
were eluted with 2 x 1,5mL of dichloromethane.
132
FARMACIA, 2008, Vol.LVI, 2
Gas chromatography – mass spectrometry
GC–MS analyses were carried out with a Fisons Instruments GC
8000, equipped with an electron impact quadrupole, MD 800 mass
spectrometer detector. The electron ionization energy was 70eV, ion-source
temperature 200C and the interface temperature 280C. A split–splitless
injection (split ratio 1:30) at 280C was employed.
A fused silica column 5% phenylpoly (dimethylsiloxane) (DB-5MS
30m x 0.32mm i.d. and 0.25m film thickness, J&W Scientific) was used.
The oven temperature was programmed as follows: from 40C (3 min hold)
raised with 5C/min to 200C (2 min hold), then with 15C/min to 250C and
finally hold at 250C for 10 min. The carrier gas (helium) flow rate was
2mL/min. 2µL of sample were injected. These conditions were applied for
hydro-distilled and infusion samples. Data acquisition was performed with
MassLab software for the mass range 30–600u with a scan speed of 1 scan/s.
The identification of compounds was performed by comparing their
mass spectra with data from Adams [20] US National Institute of Standards
and Technology (NIST, USA), WILEY 1996 Ed. mass spectra library and a
personal library of 600 spectra. The identification of compounds was also
based on the Kovats retention indices (KRI).
The Kovats retention indices were calculated using n-alkanes C8 –
C20 and the experimental values were compared to those reported in
literature [21, 22].
RESULTS AND DISCUSSION
The average content in essential oil of Tilia platyphyllos samples
was 0.08% (two determinations).
In figure 1 is shown the chromatogram of hydro-distilled extract.
Table I shows the relative content of volatile compounds from
hydro-distilled extract expressed as percentage from total area.
In hydro-distilled, 36 compounds with content between 0.15% and
26.07% were identified, adding up to 90.65% of the total area. The main
constituent of the flowers hydro-distilled was 2-phenylethanol with a
content of 26.07%, known for its antimicrobial, antiseptic properties and
also used in the formulation of pharmaceutical and perfume products, as
fragrance and preservative.
The percentage of monoterpenic hydrocarbons varies between 0.15%
for -thujene and 2.19 % for -carene, with a total of 6.79 % of the area. The
content of monoterpenic alcohols varies from 1.34% for cis-carveol to 5.60% for
p-cymene-8-ol, the total percentage of monoterpenic alcohols being 18.91%.
133
FARMACIA, 2008, Vol.LVI, 2
13.609
30.902
Sc an EI+
TIC
2.89e7
RT
19.533
15.828
15.425
13.133
26.592
12.986
26.904
19.349
15.223
14.159
18.689
24.997
20.688
24.575
21.788
21.201
24.502
31.525
22.650
29.178
17.259
17.919
22.870
11.482
27.711
23.897
10.932
rt
10.000
12.000
14.000
16.000
18.000
20.000
22.000
24.000
26.000
Figure 1
The Chromatogram of hydro-distilled extract
28.000
30.000
tR (min)
134
FARMACIA, 2008, Vol.LVI, 2
Table I
The chemical composition of hydro-distilled extract
No.
Compounds
tR (min.)
KRI
Area %
1 α-thujene
6.674
908
0.15
2 α -pinene
6.861
913
1.30
3 sabinene
8.312
951
1.40
4 β -pinene
8.383
952
1.20
5 decane
9.418
1000
1.60
6 δ-carene
9.557
1004
2.19
7 benzaldehyde
10.382
1030
0.55
8 phenylacetaldehyde
11.354
1057
0.18
9 linalool
12.986
1105
1.22
10 hotrienol
13.133
1109
1.55
11 2-phenylethanol
13.609
1124
26.07
12 cis-p-mentha-2,8-dien-1-ol
14.159
1141
2.51
13 borneol
15.223
1175
1.54
14 4-menth-1-en-4-ol
15.425
1181
2.86
15 anethofurane (dill ether)
15.572
1186
0.85
16 p-cymen-8-ol
15.828
1194
5.60
17 α -terpineol
15.975
1198
2.29
18 cis-carveol
16.177
1205
1.34
19 cuminaldehyde
17.497
1249
0.31
20 5-pentyl-3H-furan-2-one
18.139
1271
0.70
21 safranal
18.689
1289
1.25
22 2-methoxy-4-vinyl-phenol
19.533
1319
8.35
23 eugenol
20.688
1360
2.43
24 β -damascenone
21.201
1378
1.55
25 tetradecane
21.788
1400
1.01
26 methyleugenol
22.006
1408
1.27
27 isoamylbenzoate
22.870
1441
0.71
28 cis-geranyl acetone
23.145
1451
0.76
29 β -ionone
23.897
1480
0.18
30 benzyl tiglate
24.502
1503
0.93
31 1.2-dimethoxy-4-(1-propenyl)benzene
24.575
1506
1.88
32 2-allyl-5,5-dimethyl-1,3-ciclohexanedione
24.997
1523
3.17
33 phenylethyl tiglate
26.592
1587
2.50
34 hexadecane
26.904
1600
1.30
35 benzyl benzoate
30.902
1772
7.33
36 octadecane
31.525
1800
0.62
Total
90.65
The hydro-distilled is also rich in phenolic and phenolic-ethers compounds:
8.35% 2-methoxy-4-vinyl-phenol, 2.43% eugenol and 1.27% methyleugenol.
FARMACIA, 2008, Vol.LVI, 2
135
There are many carbonyl compounds varying from 0.18% for
phenylacetaldehyde to 3.17% for 2-allyl-5.5-dimethyl-1.3-cyclohexanedione.
Four esters were found: 7.33% benzyl benzoate, 2.5% phenylethyl tiglate,
0.93% benzyl tiglate and 0.71% isoamyl benzoate. The total content of
alkanes (C14, C16, C18) in hydro-distilled extract was 4.53%.
Table II shows the relative content of volatile compounds from infusion
extract expressed as percentage from the total area. 20 compounds ranging
between 0.2% and 29.48% were identified adding up to 91.69% of the total area.
The main component of the infusion extract was, just like in the
hydro-distilled, 2-phenylethanol with a content of 29.48%. The ester of this
alcohol with butyric acid is also found in a significant quantity (12.11%).
The relative content of monoterpenic hydrocarbons is 12.23%,
varying from 0.20% for -thujene to 5.95% for -carene. The alkanes are also
present in high quantities in the infusion extract, with a total of 22.17%. The
alkyl-phenol, 4-methyl-2.6-ditertbutylphenol, a compound with potential
antioxidant activity, is present in a quantity of 5.01% from the total area.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Table II
Chemical composition of infusion extract
Compounds
tR (min.)
KRI
Area %
6.678
908
0.20
α-thujene
6.861
913
1.99
α -pinene
8.310
951
2.13
sabinene
8.383
952
1.96
β-pinene
9.410
1000
0.84
decane
9.538
1004
5.95
δ-carene
10.345
1027
1.41
limonene
14.031
1137
29.48
2-phenylethanol
16.048
1200
4.83
dodecane
21.824
1400
6.38
tetradecane
22.998
1445
12.11
2-phenylethyl butanoate
24.208
1492
1.76
β -ionone
24.483
1502
5.01
4-methyl-2.6-ditertbuthylphenol
24.832
1516
0.61
β -methyl-ionone
26.941
1600
5.31
hexadecane
28.756
1678
0.98
3-oxo- α -ionol
28.958
1687
1.49
3-oxo-7,8-dihydro- α -ionone
29.215
1700
2.03
heptadecane
31.562
1800
2.78
octadecane
32.130
1824
4.44
vomifoliol
91.69
Total
136
FARMACIA, 2008, Vol.LVI, 2
The vomifoliol, a keto-alcohol, with a content of 4.44% from the
total area in infusion extract, has been reported to play an important role as
an endogenous regulator of the stomata aperture [23, 24]. This compound
was also identified in other plants [25, 26], being reported the predominant
volatile compound in eucalyptus honey [27].
CONCLUSIONS
Although the flowers of Tilia platyphyllos studied in this paper
have a small quantity of essential oil, by solid-phase extraction and GC/MS
analysis there were identified 36 volatile organic compounds in hydrodistilled extract and 20 compounds in the infusion extract.
The study could be continued on other officinal species of linden,
Tilia cordata and Tilia tomentosa. An interesting direction of study would
be the analysis of volatile compounds from Tilia honey, in order to
determine linden specific honey markers.
REFERENCES
1. ***European Pharmacopoeia 5th Edition, Tiliae flos, Strasbourg,
2004, 1914-1915
2. Blumenthal M., Busse W.R., Goldberg A., et al. (eds.), The
Complete German Commission E Monographs: Therapeutic Guide
to Herbal Medicines, Austin: American Botanical Council and
Boston: Integrative Medicine Communications, 1998, 163
3. Bisset N., Herbal Drugs and Phytopharmaceuticals, Stuttgart,
Germany, CRC Press, 1994, 496
4. Buchbauer G., Jirovetz L., Jaeger W., Passiflora and lime-blossoms:
motility effects after inhalation of the essential oils and of some of
the main constituents in animal experiment, Arch. Pharm., 1992,
325, 247-248
5. Chevallier A., Encyclopedia of Medicinal Plants, New York, DK
Publishing Inc., 1996, 275
6. Duke J.A., CRC Handbook of Medicinal Herbs. Boca Raton, FL,
CRC Press, 1985, 485
7. Newall C.A., Anderson L.A., Phillipson J.D., Herbal Medicines: A
Guide for Health-Care Professionals, London: The Pharmaceutical
Press, 1996, 181
8. Viola H., Wolfman C., Levi de Stein M., Wasowski C., Peña C.,
Medina J. H., Paladini A. C., Isolation of pharmacologically active
FARMACIA, 2008, Vol.LVI, 2
137
benzodiazepine receptor ligands from Tilia tomentosa (Tiliaceae) J.
Ethnopharmacol., 1994, 44, 47-53
9. Jenke D.R., Chromatographic method validation: a review of current
practices and procedures. II. Guidelines for primary validation
parameters, J. Liq. Chrom. Rel. Technol., 1996, 19, 737-757
10. Pietta P., Mauri P., Bruno A., Zini L., High-performance liquid
chromatography and micellar electrokinetic chromatography of
flavonol glycosides from Tilia, J. Chromatogr. A, 1993, 638, 357361
11. Pietta P., Facino R.M., Carini M., Mauri P., Thermospray liquid
chromatography - mass spectrometry of flavonol glycosides from
medicinal plants, J. Chromatogr. A, 1994, 661, 121-126
12. Toker G., Aslan M., Yesilada E., Memisoglu M., ITO S.,
Comparative evaluation of the flavonoid content in officinal Tiliae
flos and Turkish lime species for quality assessment, J. Pharm.
Biomed. Anal. 2001, 26, 111-121
13. Toker G, Memisoglu M., Yesilada E., Aslan M., Main Flavonoids of
Tilia argentea DESF. ex DC. Leaves, Turk. J. Chem. 2004, 28, 745750
14. Bernasconi R., Gebistorf J., Essential oils of linden blossoms and
chemotaxonomy of the Tilia genus, Pharm. Acta Helv., 1968, 43,
677-688
15. Buchbauer G., Remberg B., Jirovetz L., Nikiforov A., Comparative
headspace analysis of living and fresh cut lime tree flowers (Tiliae
flores), Flavour Fragr. J., 2006, 10, 221-224
16. Toker G., Baser K.H.L., Kurkcuoglu M., Ozek T., Composition of
Essential Oils from Tilia L. species growing in Turkey, J. Essent. Oil
Res., 1999, 11, 369-374
17. *** Farmacopeea Română, ediţia a-X a, Editura Medicală,
Bucureşti, 1993, 1064
18. Oprea E., Rădulescu V., Chiliment S., Cercetări privind compoziţia
chimică a uleiului volatil şi a infuziei din flori de Sambucus nigra L.,
Rev. Chim., 2004, 55, 410-412
19. Rădulescu V., Chiliment S., Oprea E., Capillary gas
chromatography–mass spectrometry of volatile and semi-volatile
compounds of Salvia officinalis, J. Chromatogr. A, 2004, 1027, 121126
20. Adams R. P., Identification of Essential Oil Components by Gas
Chromatography / Quadrupole Mass Spectroscopy, Allured Publ.
Corp., Carol Stream, Illinois, 2001
138
FARMACIA, 2008, Vol.LVI, 2
21. www.flavornet.org
22. www.pherobase.com
23. Andersson R., Lundgren R., Monoaryl and cyclohexenone
glycosides from needles of Pinus sylvestris, Phytochem., 1988, 27,
559-562
24. Stuart K.L., Roberts E.V., Whittle Y.G., A general method for
vomifoliol detection, Phytochem., 1976, 15, 332-333
25. Hammami S., Ben Jannet H., Bergaoui A., Ciavatta L., Cimino G.,
Mighri Z., Isolation and Structure Elucidation of a Flavanone, a
Flavanone Glycoside and Vomifoliol from Echiochilon fruticosum
Growing in Tunisia, Molecules 2004, 9, 602-608
26. Siddiqui B.S., Kardar M.N., Ali S.T., Khan S., Two New and a
Known Compound from Lawsonia inermis, Helv. Chim. Acta, 2003,
86, 2164 - 2169
27. Vasquez, L., Díaz-Maroto, M., Guchu, E., Pérez-Coello, M.,
Analysis of volatile compounds of eucalyptus honey by solid phase
extraction followed by gas chromatography coupled to mass
spectrometry, Eur. Food Res. Technol., 2006, 224, 27-31
28. Camelia Ioana Peev, Diana-Simona Antal, L. Vlase, Cristina
Dehelean, Z. Szabadai, Conţinutul în polifenoli ai mugurilor foliari
de tei argintiu (Tilia tomentosa Moench), Farmacia, 2006, LIV, 2,
79- 86