The essential oil of Bupleurum fruticosum L. from Corsica: A comprehensive study

Article abstract of DOI:10.1002/cbdv.200800310

A detailed analysis of Bupleurum fruticosum oil was carried out by combination of GC (RI), GC/ MS, and 13C-NMR analyses. After fractionation by column chromatography, 34 components accounting for 97.8% of the oils were identified. The main component was β

Full text of DOI:10.1002/cbdv.200800310

2244
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
The Essential Oil of Bupleurum fruticosum L. from Corsica:
A Comprehensive Study
by Kai Liu, Marie Laure Lota, Joseph Casanova, and Fe´lix Tomi*
´
Universite de Corse-CNRS, UMR 6134 SPE, Equipe Chimie et Biomasse, Route des Sanguinaires,
F-20000 Ajaccio (phone: þ33-495-524122; fax: þ33-495-524142; e-mail: felix.tomi@univ-corse.fr)
A detailed analysis of Bupleurum fruticosum oil was carried out by combination of GC (RI), GC/
MS, and 13C-NMR analyses. After fractionation by column chromatography, 34 components accounting
for 97.8% of the oils were identified. The main component was b-phellandrene (67.7%), followed by
sabinene (9.3%), and limonene (5.6%). The evolution of the chemical composition according to the
stages of development of the plant was investigated as well as the composition of leaf, twig, and flower
oils. A solvent-free microwave extraction (SFME) of aerial parts was carried out and the composition of
the extract compared with that of the essential oil. Finally, 57 oil samples isolated from aerial parts of
individual plants, collected all around Corsica, were analyzed, and the data were submitted to statistical
analysis. Although the contents of the main components varied, only one group emerged, accompanied
with some atypical compositions.
Introduction. – Bupleurum is a genus of the Apiaceae family comprising ca. 200
species and primarily located in the Northern hemisphere, Eurasia, and North Africa.
In the Mediterranean basin and North Africa, ca. 25 species are represented with some
restricted endemism (B. dianthifolium, Italy; B. barceloi and B. bourgaei, Balearic
Islands, Spain). About 50 species from this genus (e.g., B. falcatum, B. chinense, B.
fruticosum, etc.) have been studied chemically, and more than 100 compounds have
been isolated (saikosaponins, phenylpropanoids, lignans, coumarins, flavonoids, sterols,
etc.) [1].
Bupleurum fruticosum L. is an evergreen, glaucous, perennial, bushy shrub, 1-to-2-
m high, with simple elliptic-oblong leaves, which is represented all around the
Mediterranean basin and in the islands [1–3]. B. fruticosum emits a strong odor and is
repulsive for animals. On the contrary, the fragrant and tasty fruits have been used as
spices [4].
Only a few studies were concerned with the chemical composition of the essential
oil from Bupleurum fruticosum. The first study dates back to 1911 and reported the
occurrence, in oil from Sardinia (Italy), of a-pinene, b-phellandrene, and the so-called
buplerol, a monoterpene alcohol never identified later in the B. fruticosum oils [5]. The
next study was reported in 1970 by Peyron and Roubeau [2]. They identified various
monocyclic and bicyclic monoterpenes in an oil sample isolated from the flower heads
of plants harvested in the French Alpilles.
Since 1987, various investigations on the chemical composition of B. fruticosum
essential oil were conducted using modern analytical techniques (e.g., GC/MS in
combination with GC retention indices (RI)). Although they related to wild growing
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
2245
and cultivated plants as well as different parts of the plant (aerial parts, leaves, stems,
flowers, or fruits), the results may be summarized by the occurrence of four chemical
compositions, all dominated by monoterpene hydrocarbons.
The composition dominated by b-phellandrene was observed in oil samples of
various origins: i) wild plants from Sardinia, Italy (64.5%; sabinene, 20.7%; aerial parts,
leaves, stems, and flowers) [6], ii) in vitro multiplied plants in the Botanical Garden of
the University of Urbino, Italy, (60.9%; sabinene, 12.8%; leaves) [7], and iii) wild
plants of Eastern Libya (49.3%; a-pinene, 15.3%; epigean parts) [8]. Otherwise, a-
pinene and b-pinene (41.2 and 35.9%, resp.) were the major components of fruit oil
from Spain [9].
Finally, it should be mentioned that oils with different compositions were isolated
from leaves and stems of the same plant cultivated at the Istituto Botanico di Urbino,
Italy. Leaf oil was dominated by b-phellandrene and sabinene (41.7 and 35.8%, resp.
[7], or 38.7 and 39.7%, resp. [4]), while stem oil contained mostly g-terpinene and a-
phellandrene (49.8 and 18.3%, resp. [7], or 48.8 and 12.2%, resp. [4]).
An oil sample from Sicily, Italy, isolated by hydrodistillation/simultaneous solvent
extraction, contained a-pinene (21.7%), b-phellandrene (21.3%), and b-pinene
(13.2%) as main components [10].
The aim of the present study was to give an overview of the essential oil from
Bupleurum fruticosum L. growing wild in Corsica. First, we reported on the detailed
analysis of a bulk sample, by combination of chromatographic and spectroscopic
techniques. Then, we compared the compositions of leaf, stem, and flower oils. We
followed the evolution of the chemical composition of the oil from aerial parts
according to the stage of development of the shrub (before, during, and after
flowering). Finally, we examined the chemical variability. An interesting point, in our
opinion, is that sampling, preparation of the oils, and analyses were carried out at two
periods separated by ten years, i.e., in 1997 and 2007. Individual components were
identified by combination of GC (RI), GC/MS, and 13C-NMR.
Results and Discussion. – Detailed Analysis of an Oil Sample of Bupleurum
fruticosum. The essential oil was isolated by vapor distillation of the aerial parts of
Bupleurum fruticosum by the society U Mandriolu. It was analyzed by combination of
GC (RI), GC/MS, and 13C-NMR. To identify more components and to confirm the
identification of some compounds, the bulk sample was submitted to column
chromatography (CC) and the fractions were analyzed by GC (RI) and 13C-NMR.
Moreover, two minor esters, cinnamyl isovalerate and cinnamyl 2-methylbutyrate were
identified by comparison of their spectral NMR data with those of authentic samples
prepared by esterification of cinnamyl alcohol.
Individual components of the essential oil, their retention indices on apolar and
polar columns, as well as their relative percentages and the mode of identification are
reported in Table 1. In total, 34 components, which represented 97.8% of the total
amount of the oil, were identified, most of them (25) by GC (RI), GC/MS, and
13C-NMR. Among the identified compounds, 18 were hydrocarbons, and the oil was
characterized by a high content of b-phellandrene (67.7%). Two monoterpenes,
sabinene (9.3%) and limonene (5.6%), were present in an appreciable amount. A few
acyclic, non-terpenic compounds (decan-1-ol, hexyl acetate, octyl acetate, and hexyl

2246
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Table 1. Components of the Essential Oil Isolated from Aerial Parts of Bupleurum fruticosum L. from
Corsica
Componentsa)
Retention index (RI)
Composition [%]
Identification mode
BP-1
BP-20
a-Thujene
a-Pinene
Camphene
Sabinene
b-Pinene
Myrcene
Hexyl acetate
a-Phellandrene
d-Car-3-ene
a-Terpinene
p-Cymene
923
931
944
967
972
982
994
999
1027
1027
1073
1129
1116
1166
1275
1171
1153
1176
1276
1207
1222
1250
1288
1550
1683
1605
1678
1478
1448
1768
1583
1666
1762
1669
1713
1738
1730
2237
2093
2037
2310
2334
0.1
1.6
0.1
9.3
0.3
2.3
0.1
2.4
0.4
0.2
0.3
5.6
67.7
1.2
0.2
0.1
0.1
0.3
0.5
0.5
0.2
0.8
trc)
0.3
1.9
trc)
trc)
0.2
trc)
0.1
0.3
0.1
0.3
0.4
RI, MS
RI, MS, 13C-NMR
RI, MS
RI, MS, 13C-NMR
RI, MS
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
RI, MS, 13C-NMR
1006
1011
1013
1026
1026
1050
1080
1084
1157
1163
1176
1193
1226
1255
1271
1335
1361
1448
1479
1495
1503
1517
1587
1598
1646
1655
Limoneneb)
b-Phellandreneb)
g-Terpinene
Terpinolene
Linalool
Cryptone
Terpinen-4-ol
Estragole
Octyl acetate
Hexyl isovalerate
Decanol
Bornyl acetate
Citronellyl acetate
Geranyl acetate
(E)-b-Farnesene
Germacrene D
Bicyclogermacrene
b-Bisabolene
Elemicine
Viridiflorol
Ledol
Cinnamyl 2-methylbutyrate
Cinnamyl isovalerate
Total
97.8
a) Order of elution and percentages of components are given for the apolar column (BP-1), except for
compounds with b). b) Percentages are given for the polar column (BP-20). c) tr: traces.
isovalerate) were also identified. Phenyl propanoids were represented by cinnamyl
esters, estragole, and elemicine (Fig. 1).
The actual content of b-phellandrene was determined using quantitative 13C-NMR
spectroscopy. Waiting a period of 5 T1 of the longest T1 value before applying another
pulse, combined with a 908 pulse angle, allows both maximal steady-state magnetization
and complete relaxation of each C-atom [11]. Applying the aforementioned

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
2247
Fig. 1. Structures of some compounds present in the essential oil of Bupleurum fruticosum
parameters with the gated-decoupling technique, which provides the suppression of the
nuclear Overhauser enhancement (NOE), is well-known as the standard sequence for
quantitative NMR measurements [12]. The mean value of the integrals of the signals of
the protonated C-atoms was compared to those of the CH2 groups of diglyme, used as
internal standard (see Exper. Part). The percentage of b-phellandrene determined by
GC (FID) is in agreement with that evaluated from NMR data (67.7 and 65.8%, resp.).
Essential Oils from Various Parts of the Plant. It could happen that essential oils
isolated from different parts of the plant have different compositions. To verify this
point, we first compared the composition of leaf and stem oils isolated from two
individual plants (Samples A and B), and we observed, in both cases, that the oils
exhibited a very similar composition, the yield of the leaf oil (2.1 and 3.6%) being
higher than that of the stem oil (1.6 and 2.1%). More interesting is the comparison of
leaf and stem oils with flower oil. The yield of oil isolated from leaves and stems (1.6
and 2.2%) is higher than that isolated from flowers (1.1 and 1.3%). The nine major
components of the corresponding oils, identified by GC (RI) and 13C-NMR, accounted
for 95.9–97.9% of the total amount (Table 2). Although the composition is quite
similar with respect to the monoterpene hydrocarbons and oxygenated monoterpenes,
Table 2. Chemical Composition (major components) [%] of the Flower and the Leaf and Stem Essential
Oils of Bupleurum fruticosum L. from Corsica
Componentsa)
Sample A
Sample B
Flower
Leaf and stem
Flower
Leaf and stem
a-Pinene
Sabinene
Myrcene
a-Phellandrene
Limoneneb)
b-Phellandreneb)
Estragole
Decanol
Geranyl acetate
1.5
2.5
2.0
3.0
5.5
71.3
4.8
1.1
2.0
1.5
2.3
3.4
5.7
77.3
–
1.6
1.0
1.9
1.1
2.3
3.3
6.0
79.1
trc)
0.7
2.0
3.0
5.5
71.8
6.6
1.3
1.2
2.4
2.3
1.7
1.2
Yield [% (w/w)]
1.1
1.6
1.3
2.2
a) Order of elution and percentages of components are given for the apolar column (BP-1), except for
compounds with b). b) Percentages are given for the polar column (BP-20). c) tr: traces.

2248
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
some differences are observed for phenyl propanoids. Indeed, it could be pointed out
that the flower oil contained appreciable amounts of estragole (6.6 and 4.8%), not
present in leaf and stem oils.
Solvent-Free Microwave Extraction. Solvent-free microwave extraction (SFME) is
an original combination of microwaves and dry distillation [13]. The essential oils from
two individual samples (Samples C and D), extracted by SFME for 30 min, exhibited a
chemical composition qualitatively and quantitatively similar to those obtained by
conventional hydrodistillation with a Clevenger apparatus for 3–4 h (Table 3). We just
noted that the content of the major component, b-phellandrene, was slightly lower, and
that higher percentages of oxygenated compounds were observed by SFME in
comparison with conventional hydrodistillation. In terms of time and energy, SFME
allows, in general, substantial savings of costs and may be considered as a green
technology. However, due to the very low yields (0.21 and 0.25%) obtained by this
technique in comparison to those attained with conventional hydrodistillation (1.71 and
1.86%), SFME seems not appropriate for the industrial isolation of the volatiles of
Bupleurum fruticosum.
Table 3. Chemical Composition (major components) [%] of the Essential Oil Isolated from Aerial Parts
of Bupleurum fruticosum by SFME and by Conventional Hydrodistillation (HD)
Componentsa)
Sample C
Sample D
HD
SFME
HD
SFME
a-Pinene
Sabinene
b-Pinene
Myrcene
a-Phellandrene
para-Cymene
Limoneneb)
b-Phellandreneb)
Octyl acetate
Decanol
Geranyl acetate
Elemicin
Cinnamyl isovalerate
2.1
1.0
0.4
2.4
3.4
0.2
6.1
79.7
0.3
0.4
0.8
0.2
0.1
1.1
0.7
0.2
2.0
3.5
0.2
6.1
76.8
0.5
1.3
1.2
0.9
0.6
2.1
1.0
0.3
2.3
3.3
0.3
6.1
79.1
0.3
0.4
1.6
0.1
0.2
1.3
0.9
0.3
2.0
2.8
0.4
5.9
77.5
0.5
1.0
2.5
0.2
0.5
Yield [% (w/w)]
1.71
0.21
1.86
0.25
a) Order of elution and percentages of components are given for the apolar column (BP-1), except for
compounds with b). b) Percentages are given for the polar column (BP-20).
Evolution of the Composition before, during, and after Flowering. The composition
of essential oils isolated from aerial parts of plants collected before (May 2005), during
(July or September 2005), and after (November 2005) the flowering period are
reported in Table 4. The 13 major components, which represented from 92.6 to 96.6%
of the total amount of the oils, were identified by GC (RI) and 13C-NMR. b-
Phellandrene is strongly predominant all along the physiological cycle of the plant. The
percentage of this major component (70.4–78.3%) varied slightly between samples,
although the two samples varied in their composition by the content of sabinene (6.0

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
2249
and 8.2% in Sample E vs. 1.2 and 1.5% in Sample F). There was no significant variation
in the composition of the essential oils collected before, during, and after the flowering
period. Conversely, the yield of essential oils varied drastically, being lower (0.8 and
1.2%) before and higher during and after the flowering period (2.4–2.9%) (Table 4).
Table 4. Evolution of the Chemical Composition (major components) in [%] of the Essential Oils Isolated
from Aerial Parts of Bupleurum fruticosum before (BF), during (DF), and after (AF) Flowering
Componentsa)
RI
Sample E
Sample F
BP-1
BP-20
BF
DF
AF
BF
DF
AF
a-Pinene
Sabinene
Myrcene
Hexyl acetate
a-Phellandrene
p-Cymene
931
967
982
993
999
1013
1025
1026
1050
1157
1163
1255
1361
1027
1129
1166
1273
1171
1276
1207
1222
1250
1683
1605
1768
1762
1.9
7.3
2.3
0.2
2.6
0.3
5.6
72.8
0.3
0.3
0.6
0.3
1.2
1.8
6.0
2.1
0.1
3.0
0.2
5.4
73.6
0.3
–
1.8
8.2
2.2
0.2
2.5
0.5
5.4
70.4
1.4
0.2
0.9
0.8
2.1
2.0
1.5
2.1
0.1
3.0
0.3
5.8
75.7
0.1
0.8
0.2
1.2
1.8
2.0
1.2
2.3
0.5
3.5
0.1
5.9
78.3
0.1
–
1.9
1.2
2.0
1.2
2.3
0.6
5.9
71.6
0.2
2.1
0.1
1.6
1.9
Limoneneb)
b-Phellandreneb)
g-Terpinene
Cryptone
Terpinen-4-ol
Decanol
Geranyl acetate
0.4
1.2
1.6
–
0.2
1.5
Yield [% (w/w)]
1.2
2.9
2.6
0.8
2.4
2.4
a) Order of elution and percentages of components are given for the apolar column (BP-1), except for
compounds with b). b) Percentages are given for the polar column (BP-20).
Chemical Variability. Aerial parts of 57 individual plants of Bupleurum fruticosum
were collected in various places in Corsica (Fig. 2) from July to September 1997 (20
samples) and from April (or July) to November 2007 (37 samples). The fresh material
was hydrodistilled and the oil samples were analyzed by GC (RI). Some samples,
selected on the basis of their chromatographic profile, were submitted to GC/MS and/
or 13C-NMR analysis. The 57 compositions were submitted to principal component
analysis (PCA; Fig. 3), and k-means partitioning was performed on all the terpene data
with individual compounds expressed as a percentage. In PCA, the first two axes
accounted for 86.56 and 8.36%, respectively. Only one group emerged, although a few
samples, characterized by a lower content of b-phellandrene and a higher content of
sabinene, were present (Table 5).
As expected, b-phellandrene was by far the major component. Indeed, its content
varied between 59.0 and 80.3% for all samples, except one (49.1%). Moreover, its
content was higher than 70.0% for 72% of the samples. Among other monoterpenes,
sabinene accounted for more than 10% in 10 samples out of 57 and reached 29.2% in
one sample. Limonene (4.0–9.2%) was always present at appreciable content, while a-
pinene and b-pinene reached punctually 8.8 and 6.6%, respectively.
Conclusions. – It appears from that study on a large group of samples (57),
harvested in various locations of a well-defined area, that Bupleurum fruticosum

2250
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
&
Fig. 2. Sampling locations of Bupleurum fruticosum. .: Main city; : Sampling location: 1, Barchetta; 2,
Caporalino; 3, Cardo; 4, Casanova de Venaco; 5, Casella; 6, Cateraggio; 7, Erbalunga; 8, Luri; 9,
Marinca; 10, Meria; 11, Morsiglia; 12, Nonza; 13, Patrimonio; 14, Pino; 15, Ponte Leccia; 16, Ponte
Novo; 17, Saint-Pierre de Venaco; 18, San Nicolau; 19, Sisco; 20, Tolare; 21, Tolla.
growing wild in the island of Corsica produces a b-phellandrene-rich oil (more than
70% for 72% of the samples). For comparison purposes, it could be noted that the
highest percentage of b-phellandrene reported so far in the literature reached 64.5%
(Italy). No sample dominated by g-terpinene, a-pinene, b-pinene, or sabinene was
detected. Leaves and stems produced oils with similar composition. The composition of
flower oil, although dominated by b-phellandrene, exhibited a small content of the

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
2251
Fig. 3. Principal component analysis scatter plot of 57 samples of Bupleurum fruticosum essential oil from
Corsica
Table 5. Chemical Variability (major components) [%] of 57 Samples of Bupleurum fruticosum Essential
Oil. Results are given as mean value (Mean) with standard deviation (SD), minimum content (Min),
and maximum content (Max).
Componentsa)
Mean
SD
Min
Max
a-Pinene
Sabinene
b-Pinene
Myrcene
a-Phellandrene
p-Cymene
2.1
5.3
0.5
1.5
2.6
1.1
5.6
72.5
0.6
0.4
0.6
0.4
1.0
1.6
1.18
5.67
1.03
0.88
0.49
1.09
0.63
6.09
0.73
0.48
0.57
0.32
0.50
0.45
1.6
0.6
0.3
1.8
1.7
0.1
4.0
49.1
trc)
–
8.8
29.2
6.6
2.5
3.6
1.1
9.2
80.3
3.1
3.1
2.1
1.7
Limoneneb)
b-Phellandreneb)
g-Terpinene
Cryptone
Terpinen-4-ol
Estragole
Decanol
trc)
0
0.2
0.6
2.5
2.5
Geranyl acetate
a) Order of elution and percentages of components are given for the apolar column (BP-1), except for
compounds with b). b) Percentages are given for the polar column (BP-20). c) tr: traces.
phenylpropanoid estragole (Fig. 1). The composition of oils extracted from aerial parts
varied only slightly, qualitatively and quantitatively, along the physiological cycle of the
plant. However, the yield was higher during and after the flowering stage. Finally, oil
produced by SFME had a similar composition than that isolated by conventional
hydrodistillation, but the yield was drastically lower.

2252
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
´
The authors are indebted to the Collectivite Territoriale de Corse (ARP Ressources Naturelles) and to
the European Community Program INTERREG 3 Corsica-Sardinia-Tuscany for partial financial
support. We also thank Mr. Alessandri from the society U Mandriolu, Corsica, for the gift of an oil sample
and Mrs. Hugot for the identification of the plant material.
Experimental Part
Plant Material. To differentiate oils isolated from various parts of the plant, leaves, stems, and flowers
of Bupleurum fruticosum L. were collected from two plants growing wild near Corte, in the centre of
Corsica, in September 2005, and they were submitted independently to hydrodistillation. Aerial parts of
two individual plants were collected in May, in July or September, and in November 2005 to follow the
evolution of the composition before, during, and after flowering. Finally, aerial parts of individual plants
were collected from July to September 1997 (20 samples) and 2007 (37 samples), in the following
locations: Barchetta, Caporalino, Cardo, Casanova de Venaco, Casella, Cateraggio, Erbalunga, Luri,
Marinca, Meria, Morsiglia, Nonza, Patrimonio, Pino, Ponte Leccia, Ponte Novo, Saint-Pierre de Venaco,
San Nicolau, Sisco, Tolare, and Tolla (Fig. 2). It could be noted that B. fruticosum is abundant in the so-
called ꢂAlpine Corsicaꢃ and it is scarce in the other part, called ꢂCrystalline Corsicaꢃ [14]. Identification of
the species was confirmed by Mrs. Laetitia Hugot, Office de lꢀEnvironnement de la Corse.
Essential-Oil Isolation and Fractionation. The oil sample used for detailed analysis was supplied by
the society U Mandriolu, Corsica. It was obtained by vapor distillation in an industrial apparatus, with
plants harvested during July 2005, near Corte. The bulk sample (1.030 g) was submitted to column
chromatography (CC) on silica gel (SiO2). Elution with a solvent gradient of increasing polarity
(pentane/Et2O 100 :0–0 :100) led to four fractions: Fr. 1 (812 mg) and Fr. 2 (18 mg) eluted with pentane,
Fr. 3 (115 mg) eluted with pentane/Et2O 97:3, and Fr. 4 (86 mg) eluted with Et2O.
Otherwise, the fresh material (aerial parts or various organs of the plant) was submitted to
hydrodistillation for 3 h, using a Clevenger-type apparatus. The yield of oil was expressed as % (w/w) vs.
fresh material. Solvent-free microwave extraction (SFME) was carried out using a Milestone DryDist
microwave apparatus. The temp. was monitored and controlled by feedback to the microwave power
regulator. Extraction time, 30 min; power, 500 W.
Analytical GC. GC Analysis was carried out with a Perkin-Elmer Autosystem GC apparatus
equipped with FID and two fused-silica cap. columns (50 mꢀ0.22 mm i.d., film thickness 0.25 mm), BP-1
(poly(dimethylsiloxane)) and BP-20 (poly(ethyleneglycol)). The oven temp. was programmed from 60
to 2208 at 28/min and then held isothermal at 2208 for 20 min; injector temp., 2508; detector temp., 2508;
carrier gas, He (0.8 ml/min); split, 1:60. The relative proportions of the oil constituents were expressed as
percentage obtained by peak area normalization, without using correcting factors. Retention indices (RI)
were determined rel. to the retention times of a series of n-alkanes with linear interpolation (Target
Compounds software from Perkin-Elmer).
GC/MS Analysis. The essential oils were analyzed with a Perkin-Elmer TurboMass detector
(quadrupole), directly coupled to a Perkin-Elmer Autosystem XL, equipped with a fused-silica cap.
column (60 mꢀ0.22 mm i.d., film thickness 0.25 mm), Rtx-1 (polydimethyl siloxane). Carrier gas, He
(1 ml/min); split, 1:80; injection vol., 0.2 ml; injector temp., 2508; oven temp. programmed from 608 to
2308 at 28/min and then held isothermal at 2308 for 45 min; ion-source temp., 1508; energy ionization,
70 eV; electron ionization mass spectra were acquired over the mass range 35–350 Da.
NMR Analysis. All 13C-NMR spectra were recorded in CDCl3, with chemical shifts referred to int.
Me4Si. Spectra of mixtures (essential oil or CC fractions) isolated in 2007 were recorded on a Bruker
AVANCE 400 Fourier Transform spectrometer, operating at 100.63 MHz, equipped with a 5-mm probe,
with the following parameters: pulse width, 4 ms (flip angle 458); acquisition time, 2.7 s for 128 K data
table with a spectral width of 24000 Hz (240 ppm); CPD mode decoupling; digital resolution, 0.183 Hz/
pt. The number of accumulated scans was 3000 for each sample (ca. 40 mg of the sample in 0.5 ml of
CDCl3). Spectra of oil samples isolated in 1997 were recorded on a Bruker AC 200 Fourier Transform
spectrometer operating at 50.323 MHz, equipped with a 10-mm probe, with the following parameters:
pulse width, 5.0 ms (flip angle 458); spectral width, 12500 Hz (250 ppm); CPD mode decoupling. The

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
2253
number of accumulated scans was 3000–5000 for each sample (200 mg of the oil in 2 ml of CDCl3). An
exponential multiplication of the free induction decay with line broadening of 1.0 Hz was applied before
Fourier transformation.
1H-, 13C-, and 2D-NMR Spectra (DEPT, HSQC, HMBC) of pure compounds were recorded using
Bruker microprograms (Bruker AVANCE 400). For T1 measurements, the longitudinal relaxation delays
of the 13C-nuclei (T1 values) were determined for each protonated C-atom of b-phellandrene (Fig. 1) and
for the two CH2 groups of diglyme (bis(2-methoxyethyl) ether) by the inversion-recovery method, using
the standard sequence 1808-t-908-D1, with a relaxation delay D1 of 10–50 s (Table 6). Each delay of
inversion (t) was thus taken into account for the computation of the corresponding T1 using Eqn. 1.
IP ¼ I0 þ p ꢁ eꢂt=T
(1)
1
Table 6. 1H-, 13C-, and 2D-NMR Data of b-Phellandrene. At 400/100 MHz, respectively, in CDCl3; d in
ppm, J in Hz.
Position
d(C)
T1 [s]
DEPT
d(H)
1
2
3
4
5
6
7
8
9
143.72
129.62
134.15
42.16
25.81
30.25
109.95
31.99
n.m.a)
7.9
7.9
7.4
4.0
4.2
4.6
7.5
3.5
C
–
CH
CH
CH
CH2
CH2
CH2
CH
CH3
CH3
6.16 (dd, J¼9.8, 2.7)
5.75 (br. d, J¼9.8)
2.00–2.10 (m)
1.77 (dq, J¼12.7, 4.0), 1.43 (tdd, J ¼ 12.7, 10.0, 3.8)
2.44 (dt, J ¼ 14.9, 3.8), 2.29 (dd, J ¼ 14.9, 12.7)
4.76–4.78 (m), 4.73–4.75 (m)
1.60–1.70 (m)
19.54
19.73
0.94 (d, J¼6.7)
10
3.5
0.91 (d, J¼6.7)
a) n.m.: Not measured
For the quant. determination of b-phellandrene in the oil sample of the society U Mandriolu, a
quantitative spectrum was acquired using the inverse gated decoupling sequence with the following
parameters: pulse width, 8.5 ms (flip angle 908); acquisition time, 2.7 s for 128 K data table with a spectral
width of 24000 Hz (240 ppm); repetition time 40 s (5ꢀlongest T1 of protonated C-atoms, Table 6); digital
resolution, 0.183 Hz/pt. The number of accumulated scans was 256 (52 mg of the oil sample in 0.5 ml of
CDCl3). Diglyme was used as int. standard (T1 of CH2 group C-atoms, 3.8 s). The amount, mP in mg, of b-
phellandrene was determined using Eqn. 2:
2AP ꢁ MP ꢁ mD
mP
¼
(2)
AD ꢁ MD
AP, mean values of the integrals of the protonated C-atoms of b-phellandrene; AD, mean values of the
integrals of the two CH2 groups of diglyme used as int. standard; MP and MD, molecular weight [g/mol] of
b-phellandrene and diglyme, resp.; mD, weight of diglyme [mg]. Experimental data: AP, 1.711; AD, 1.006;
MP, 136.23 g/mol; MD, 134.17 g/mol; mD, 10.0 mg; mEO, 52.0 mg; mP, 34.2; mP%¼65.8.
Identification of Components. Identification of the individual components was based: i) on
comparison of their GC retention indices (RI) on polar and apolar columns with those of authentic
compounds and literature data [15][16], ii) on computer matching with commercial mass spectral
libraries [17–19] and comparison of spectra with literature data [15][20][21], and iii) on comparison of
the signals in the 13C-NMR spectra of the mixtures with those of reference spectra compiled in the
laboratory spectral library, with the help of a laboratory-made software [22–24]. In the investigated
samples, individual components were identified by NMR at contents as low as 0.5%.
Preparation of Cinnamyl Esters. To a soln. of cinnamyl alcohol (276 mg, 2.05 mmol or 519 mg,
3.87 mmol, resp.) in 30 ml CH2Cl2 and Et3N (326 mg, 3.22 mmol or 780 mg, 7.74 mmol, resp.) cooled to

2254
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
08, a soln. of 2-methylbutyryl chloride (518 mg, 4.29 mmol) or isovaleryl chloride (978 mg, 7.74 mmol) in
CH2Cl2 (30 ml) was added dropwise. The mixture was stirred until r.t. was reached and then refluxed for
3 h. The mixture was poured into 200 ml of cold H2O. After decantation, the org. phase was separated,
washed twice with H2O, dried (Na2SO4), and concentrated in vacuum. The crude esters were purified by
CC (SiO2). Yields (not optimized) were 80 and 79%, resp.
Cinnamyl 2-Methylbutyrate. 1H-NMR: 7.37–7.40 (m, 2 H); 7.29–7.33 (m, 2 H); 7.23–7.26 (m, 1 H);
6.65 (d, J¼15.9, 1 H); 6.28 (dt, J¼15.9, 6.4, 1 H); 4.74 (dd, J¼6.4, 1.2, 2 H); 2.43 (sext., J¼7.0, 1 H);
1.66–1.77 (m, 1 H); 1.44–1.55 (m, 1 H); 1.17 (d, J¼7.0, 3 H); 0.92 (t, J¼7.4, 3 H). 13C-NMR: 176.56;
136.32; 133.96; 128.61; 128.02; 126.61; 123.48; 64.79; 41.12; 26.83; 16.64; 11.65.
Cinnamyl Isovalerate. 1H-NMR: 7.37–7.40 (m, 2 H); 7.30–7.35 (m, 2 H); 7.25–7.28 (m, 1 H); 6.65 (d,
J¼15.8, 1 H); 6.28 (dt, J¼15.8, 6.4, 1 H); 4.74 (dd, J¼6.4, 1.4, 2 H); 2.24 (d, J¼7.3, 2 H); 2.08–2.19 (m,
1 H); 0.97 (d, J¼6.7, 6 H). 13C-NMR: 172.96; 136.28; 134.08; 128.61; 128.04; 126.61; 123.37; 64.79; 43.44;
25.75; 22.44.
REFERENCES
[1] S. L. Pan, ꢂTraditional Herbal Medicines for Modern Times. Bupleurum Species – Scientific
Evaluation and Clinical Applicationꢃ, Taylor & Francis, New York, 2006.
[2] L. Peyron, M. Roubeau, Plant. Med. Phytother. 1970, 4, 172.
[3] V. Picci, A. Atzei, A. Manunta, Rivista Ital. EPPOS, 1974, 56, 239.
[4] A. Manunta, B. Tirillini, D. Fraternale, J. Essent. Oil Res. 1992, 4, 461.
[5] L. Francesconi, E. Sernagiotto, Atti R. Acc. Lincei 1913, 22, 34.
[6] A. Manunta, I. Morelli, V. Pizzi, Plant. Med. Phytother. 1987, 21, 20.
[7] A. Bertoli, L. Pistelli, I. Morelli, D. Fraternale, L. Giamperi, D. Ricci, Plant Sci. 2004, 167, 807.
[8] L. Giamperi, D. Ricci, D. Fraternale, A. Manunta, R. Tabacchi, J. Essent. Oil Res. 1998, 10, 369.
[9] I. Lorente, M. A. Ocete, A. Zarzuelo, M. M. Cabo, J. Jimenez, J. Nat. Prod. 1989, 52, 267.
[10] G. Dugo, A. Trozzi, A.Verzera, A. Rapisarda, Essenze Derivati Agrumari 2002, 70, 201.
[11] H. Gꢁnther, in ꢂLa Spectroscopie de RMNꢃ, Masson, Paris, 1994, p. 246.
[12] J. R. Mooney, in ꢂAnalytical NMRꢃ, Eds. L. D. Field, S. Sternhell, John Wiley & Sons, Chichester,
1989, p. 45.
[13] M. E. Lucchesi, F. Chemat, J. Smadja, J. Microwave Power Electromagn. Energ. 2004, 39, 135.
´
´
[14] J. Gamisans, D. Jeanmonod, ꢂLa Vegetation de la Corseꢃ, Edisud, Aix-en-Provence, 1999.
[15] D. Joulain, W. A. Kçnig, ꢂThe Atlas of Spectral Data of Sesquiterpene Hydrocarbonsꢃ, EB-Verlag,
Hamburg, 1998.
[16] N. W. Davies, J. Chromatogr., A 1990, 503, 1.
[17] F. W. McLafferty, D. B. Stauffer, ꢂWiley Registry of Mass Spectral Data: Mass Spectrometry Library
Search System Bench-Top/PBM, version 3.10dꢃ, 6th edn., Palisade, Newfield, 1994.
[18] National Institute of Standards and Technology, ꢂThe NIST/EPA/NIH Mass Spectral Libraryꢃ, PC
Version 1.7, Perkin-Elmer Corporation, Norwalk, CT, 1999.
[19] W. A. Kçnig, D. H, Hochmuth, D. Joulain, ꢂTerpenoids and Related Constituents of Essential Oils.
Library of MassFinder 2.1ꢃ, Institute of Organic Chemistry, Hamburg, 2001.
[20] F. W. McLafferty, D. B. Stauffer, ꢂThe Wiley/NBS Registry of Mass Spectral Dataꢃ, 4th edn., Wiley-
Interscience, New York, 1988.
[21] R. P. Adams, ꢂIdentification of Essential Oil Components by Gas Chromatography/Mass Spectro-
scopyꢃ, 4th edn., Allured, Carol Stream, Illinois, 2007.
[22] F. Tomi, P. Bradesi, A. Bighelli, J. Casanova, J. Magn. Reson. Anal. 1995, 1, 25.
[23] S. Rezzi, A. Bighelli, V. Castola, J. Casanova, Appl. Spectrosc. 2002, 56, 312.
[24] F. Tomi, J. Casanova, Acta Horticult. 2006, 723, 185.
Received October 17, 2008