FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2001; 16: 209–211

DOI: 10.1002/ffj.981

Volatile constituents from the leaves of four

Libanothamus species from the Venezuelan Andes

A. Usubillaga,∗ R. Aparicio, M. Romero, L. B. Rojas and N. Khouri

Instituto de Investigaciones de la Facultad de Farmacia, Universidad de Los Andes, Merida, Venezuela

Received 8 August 2000

Revised 6 December 2000
Accepted 6 December 2000

ABSTRACT: The essential oil from the leaves of Libanothamus humbertii Cuatr., Libanothamus lucidus (Aristeg)
Cuatr., Libanothamus nerifolia (B. ex H) Ernst, and Libanothamus occultus (Blake) Cuatr, collected in the
Andes near Merida, were obtained by hydrodistillation. The oil yield varied between 0.05% and 0.13% and
monoterpene hydrocarbons constituted 83.6–90% of the oils. Limonene (19.4%) is the most abundant component
in L. humbertii, ˛-thujene (30.2%) in L. lucidus, sabinene (25.6%) in L. nerifolia and ˛-pinene (31.3%) in L.
occultus. Copyright  2001 John Wiley & Sons, Ltd.

KEY WORDS: Libanothamus humbertii; Libanothamus lucidus; Libanothamus nerifolia; Libanothamus occultus;
Asteraceae; essential oil; limonene; ˛-thujene; sabinene; ˛-pinene

Introduction
The genus Libanothamus (Asteraceae) belongs to the
subtribe Espeletiinae (tribe Helianthae). It is formed
by 14 species2 of small resinous trees that grow in
the Andes of Venezuela and Colombia at altitudes of
2200–3500 m above sea level. Humboldt collected one
of these trees, locally known as incienso, at Silla de
Caracas and named it Trixis nerifolia.7 Ernst6 created
the genus Libanothamus for this species, unaware it had
been previously transferred to the genus Espeletia by
Schutz. After an exhaustive work on anatomy, pollen
morphology and chromosome counting on all available
specimens of Espeletia, Cuatrecasas in 1976 created the
subtribe Espeletiinae,4 which was divided in to seven
genera, one of them the genus Libanothamus.
A previous study of L. nerifolia 3 revealed the pres-
ence of (
)-kaur-16-ene diterpenes. Since no reports
have been published on the composition of the volatile
constituents of these plants, the present study was under-
taken in order to obtain knowledge of their chemical
composition.
Ł Correspondence to: A. Usubillaga, Facultad de Farmacia, Instituto
de Investigaciones, Universidad de Los Andes, Merida, Venezuela.
E-mail: usubillaga@cantv.net
Contract/grant sponsor: Consejo Nacional de Ciencia y Tecnologia
(CONICIT), Venezuela.
Contract/grant number: S1-97000048.
Experimental
Plant Material
Leaves of these trees were collected at full flowering
stage. Sites, dates and altitudes of collection are given
in Table 1. Voucher specimens have been deposited at
the MERF Herbarium (Herbarium of the Faculty of
Pharmacy in Merida, Venezuela). The botanical identifi-
cation was made by Professor Pablo Melendez, MERF
Herbarium.
Isolation of the Volatile Oil
Fresh leaves were cut into small pieces and the volatile
fraction was isolated by hydrodistillation for 3 h using
a Clevenger-type trap. The yield of oil was 0.05–
0.13% (v/w).
Gas Chromatography
GC analyses were performed using a Perkin-Elmer Auto-
System gas chromatograph equipped with FID detec-
tor and data-handling system. Two capillary columns
of different polarities were used: a 5% phenylmethyl
polysiloxane fused-silica column (30 m ð 0.25 mm i.d.,
film thickness 0.25 µm; HP-5, Hewlett-Packard, CA,
USA) and a polyethylene glycol fused-silica column

Copyright  2001 John Wiley & Sons, Ltd.

210 A. USUBILLAGA ET AL.

Table 1. Sites of collection and oil yields of Libanothamus species

Altitude Oil yield

Species Date Place (m) Voucher (v/w%)
L. humbertii 12-02-99 Laguna Negra 3200 A. Usubillaga 10 0.05
L. lucidus 11-24-99 La Aguada 3400 A. Usubillaga 08 0.10
L. nerifolia 11-06-98 El Batallon 2800 R. Aparicio 01 0.13
L. occultus 11-06-98 El Batallon 2800 R. Aparicio 02 0.11

(60 m ð 0.25 mm i.d., film thickness 0.25 µm; HP-
WAX, Hewlett Packard, CA, USA). For the HP-5 col-
umn, oven temperature was programmed from 50 ° C to
200 ° C at 3 ° C/min, then at 8 ° C/min up to 280 ° C. For
the HP-WAX column, the temperature was programmed
from 50 ° C to 230 ° C at 3 ° C/min and held isothermal
at this temperature for 20 min. In both cases injec-
tor and detector temperatures were 200 ° C and 280 ° C,
respectively. The carrier gas was helium at 0.8 ml/min.
The samples (1.0 µl) were injected using a split ratio
of 1 : 100. Retention indices were calculated relative to
C8 –C24 alkanes. The percentage composition of the oils
was calculated by the normalization method from the
GC peak areas.
Gas Chromatography–Mass Spectrometry
GC–MS analyses were carried out on a Model 5973
Hewlett-Packard GC–MS system fitted with a HP-5MS
fused-silica column (30 m ð 0.25 mm i.d., film thick-
ness 0.25 µm, Hewlett-Packard). The oven temperature
programme was the same as that used for the HP-5
column for GC analysis; the transfer line temperature
was programmed from 150 ° C to 280 ° C; source temper-
ature, 230 ° C; quadrupole temperature, 150 ° C; carrier
gas, helium, adjusted to a linear velocity of 34 cm/s;
ionization energy, 70 eV; scan range, 10–500 amu;
2.2 scans/s. Samples (1.0 µl) were injected using a
Hewlett-Packard ALS injector with split ratio 100 : 1.
The identity of the oil components was established from
their GC retention indices,1,5,8 by comparison of their
MS spectra with those of standard compounds available
in the laboratory, and by library search (Wiley, 6th edn).
Results and Discussion
The essential oils isolated from the four Libanothamus
species were obtained in yields of 0.05–0.13%. The
identified oil components represent 95.5–98.4% of the
total oils. They are listed in Table 2 in order of elution
on the HP-5 column.
Limonene (19.4%) was the most abundant component
in the oil of L. humbertii but ˛-thujene (18.1%) and ˛-
phellandrene (16.5%) were also abundant. In the oil of L.
lucidus, ˛-thujene (30.2%) and ˛-pinene (22.2%) were
the two dominant components. In L. nerifolia, sabinene

Table 2. Percentage composition of the essential oils isolated from the leaves of L. humbertii, L. lucidus, L. nerifolia
and L. occultus

Component RI1 RI2 L. humbertii L. lucidus L. nerifolia L. occultus Identification methods

1 ˛-Thujenea,b 920 1020 18.1 30.2 18.1 – GC–MS, RI1 , RI2
2 ˛-Pinenea,b 926 1029 11.4 22.2 4.2 31.3 GC–MS, RI1 , RI2
3 Camphenea,b 940 1069 – 1.2 0.3 0.1 GC–MS, RI1 , RI2
4 Sabinenea,b 962 1125 3.3 2.5 25.6 0.5 GC–MS, RI1 , RI2
5 ˇ-Pinenea,b 968 1115 3.4 5.7 1.8 13.0 GC–MS, RI1 , RI2
6 Myrcenea,b 981 1152 6.7 1.5 2.3 17.1 GC–MS, RI1 , RI2
7 ˛-Phellandrenea,b 997 1170 16.5 10.2 11.0 6.7 GC–MS, RI1 , RI2
8 3-Carenea,b 1000 1135 0.3 0.3 0.2 1.2 GC–MS, RI1 , RI2
9 ˛-Terpinenea,b 1008 1183 0.4 0.5 0.8 0.4 GC–MS, RI1 , RI2
10 p-Cymenea,b 1020 1261 6.5 5.5 3.5 11.0 GC–MS, RI1 , RI2
11 Limonenea,b 1028 1205 19.4 1.2 18.6 1.3 GC–MS, RI1 , RI2
12 (Z)-ˇ-Ocimenea,b 1030 1230 0.3 0.2 0.2 0.3 GC–MS, RI1 , RI2
13 (E)-ˇ-Ocimenea,b 1047 1252 0.6 0.4 0.3 0.5 GC–MS, RI1 , RI2
14
-Terpinenea,b 1055 1256 0.5 0.8 1.3 0.4 GC–MS, RI1 , RI2
15 ˛-Terpinolenea,b 1080 1280 1.7 0.6 0.7 0.8 GC–MS, RI1 , RI2
16 Linalola,b 1091 1511 0.1 0.1 0.4 0.4 GC–MS, RI1 , RI2
17 Nonanala,c 1095 1394 0.2 – – – GC–MS, RI1 , RI2
18 ˇ-Thujonea 1106 – 0.2 0.3 0.1 – GC–MS, RI1
19 Menth-2-en-1-ola,b 1117 1630 0.2 – 0.3 – GC–MS, RI1 , RI2
20 ˛-Campholene aldehydea,b 1121 1428 t 0.6 0.3 0.5 GC–MS, RI1 , RI2
21 trans-Verbenola 1138 – – 0.4 0.3 2.3 GC–MS, RI1 , RI2
22 Pinocarvonea 1153 – – 0.2 0.2 0.4 GC–MS, RI1 , RI2

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 209–211
 VOLATILE CONSTITUENTS OF LIBANOTHAMUS SPP. LEAVES 211

Table 2. (continued)
Component RI1 RI2 L. humbertii L. lucidus L. nerifolia L. occultus Identification methods
23 p-Mentha-1,5-dien-8-ola 1159 – – 0.4 – t GC–MS, RI1 , RI2
24 Terpin-4-ola,b 1170 1637 0.8 1.8 0.4 0.2 GC–MS, RI1 , RI2
25 ˛-Terpineola,b 1181 1729 – 0.3 – 0.3 GC–MS, RI1 , RI2
26 Myrtenala 1184 – – 0.3 – – GC–MS, RI1
27 Verbenonea,b 1196 1735 – 0.1 – – GC–MS, RI1 , RI2
28 Thymol methyl ethera 1225 – 0.6 1.7 – 0.2 GC–MS, RI1
29 ˛-Copaenea,b 1366 1510 0.4 – 0.3 0.3 GC–MS, RI1 , RI2
30 ˇ-Cubebenea,b 1379 1550 – – – 0.5 GC–MS, RI1 , RI2
31 ˇ-Caryophyllenea,b 1408 1615 0.6 3.5 2.4 0.8 GC–MS, RI1 , RI2
32 ˛-Humulenea,b 1446 1672 – – – 0.3 GC–MS, RI1 , RI2
33 trans-ˇ-Farnesenea,b 1450 1685 – – 0.2 – GC–MS, RI1 , RI2
34 Aromadendranea 1452 – – 0.2 – – GC–MS, RI1
35 Germacrene-Da,b 1473 1719 2.9 – 0.6 3.8 GC–MS, RI1 , RI2
36 Isovaleric acid phenethyl estera 1482 – 1.2 – – – GC–MS, RI1
37 Bicyclogermacrenea,b 1487 1747 1.0 – 1.5 0.4 GC–MS, RI1 , RI2
38 ˛-Muurolenea,b 1490 1758 – – – 0.3 GC–MS, RI1
39 υ-Cadinenea,b 1515 1773 1.0 – – 0.6 GC–MS, RI1 , RI2
40 Nerolidol hEia,b 1558 2010 – – – 0.4 GC–MS, RI1 , RI2
41 Spathulenola,b 1570 2155 – 1.3 – – GC–MS, RI1 , RI2
42 Caryophyllene oxidea,b 1576 1990 – 1.0 – – GC–MS, RI1 , RI2
43 Manoyl oxidea 1977Ł – – 0.2 – – GC–MS, RI1 , S
44 Kaur-16-en-19-ald,e 2330Ł – 0.2 0.1 t 0.2 GC–MS, RI1 , S
Identified compounds (%) 99.4 95.5 95.9 96.4
Grouped components
Monoterpene hydrocarbons 90.0 83.6 88.9 84.6
Oxygen-containing monoterpenes 1.9 6.2 2.0 4.3
Sesquiterpene hydrocarbons 7.1 3.7 5.0 7.0
Oxygen-containing sesquiterpenes – 2.3 – 0.4
Diterpenes 0.2 0.3 t 0.2
GC–MS = compounds identified by computer comparison with Wiley Mass Spectra Library (6th edn).
RI1 D retention index on HP-5 capillary column.
RI2 D retention index on HP-Wax column.
S D standard compound.
Ł Measured isothermally.
a
Adams, 1995.
b
Davies, 1990.
c
Jennings and Shibamoto, 1980.
d
Khouri et al., 2000.
e Usubillaga et al., 1999.

(25.6%) was the most abundant constituent, followed References

closely by limonene (18.6%) and ˛-thujene (18.1%). On
the other hand, in L. occultus ˛-pinene (31.3%) was most
abundant, followed by myrcene (17.1%).
The monoterpene hydrocarbons constituted the main
oil fraction of the Libanothamus species studied, rang-
ing from 83.6% to 90% of the oil, whereas oxygen-
containing monoterpenes (2.0–6.3%) and sesquiterpenes
(5.0–7.1%) occurred in relatively smaller proportions.
(
)-Kaur-16-ene-19-al, previously reported in other
Espeletiinae,9,10 is also present in the four Libanothamus
species studied.
Acknowledgements—The Authors would like to thank the Consejo
Nacional de Ciencia y Tecnologia (CONICIT, Grant S1-97000048)
for financial support.
1. Adams RP. Identification of Essential Oil Components by Gas
Chromatography/Mass Spectroscopy. Allured: Carol Stream, II,
1995.
2. Badillo V. Alcance 1994; 45: 1.
3. Bohlmann F, Suding H, Cuatrecasas J, King RM, Robinson H.
Phytochemistry 1980; 19: 267.
4. Cuatrecasas. J. Phytologia 1976; 35: 43.
5. Davies NW. J. Chromatogr. 1990; 503: 1.
6. Ernst A. Vargasia 1870; 572.
7. Humboldt A. Rel. Hist. 1814; 1: 605.
8. Jennings W, Shibamoto T. Quantitative Analysis of Flavor and
Fragrance Volatiles by Glass Capillary Gas Chromatography. Aca-
demic Press: New York, 1980.
9. Khouri N, Usubillaga A, Rojas LB, Galarraga F. Flavour Fragr. J.
2000; 15: 263.
10. Usubillaga A, Khouri N, Visbal T. J. Essent. Oil Res. 1999; 11:
757.

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 209–211