BiochemicalSystematicsand Ecology, Vol.25, No. 4, pp.

353-358, 1997
) Pergamon © 1997 ElsevierScience Ltd
All rights reserved.Printedin Great Britain
0305-1978/97 $17.00+0.00
PII: S0305-1978(96)00106-8

Phytochemical and Chemotaxonomic Studies of Foeniculum

vulgare

B. MUCKENSTURM,*$ D. FOECHTERLEN,* J.-P. REDURON,T P. DANTONT

and M. HILDENBRANDt
*Universit~ de Haute Alsace, E.N.S.C.Mu., 3, rue Alfred Werner, F-68093 Mulhouse, France;
tConservatoire botanique, Ville de Mulhouse, 68062 Mulhouse, France

Key W o r d Index--Umbelliferae; phenylpropanoids; monoterpenoids; n-butylanisole; rotundifolone; 10-

nonacosanone.
Abstract--Different populations of Foeniculum vulgate contain 10-nonacosanone as a specific chemical
marker. Foeniculurn vulgate subsp, piperitum is characterized by the presence of rotundifolone, p-Butylanisole is
present in traces in fennel which contains a large amount of trans-anethole. A chemical classification based on
the amount of estragole, trans-anethole iimonene and fenchone is proposed for the different varieties and
chemotypes of F. vulgare subsp, vulgate. © 1997 Elsevier Science Ltd

Introduction
Fennel is a plant belonging to the Umbelliferae (Apiaceae) family, known and used by
humans since antiquity. Because of its flavor, it was cultivated in every country sur-
rounding the Mediterranean sea. Its therapeutic (Puelo, 1980) and culinary utilization
was so large that fennel was exported from country to country for centuries. Today, all
these cultures have given rise to a great complexity, and simple observation of the
plant's morphological characteristics is not sufficient to classify the different fennel
species and subspecies (Jansen, 1981 ).
The present classification of fennel plants is based mainly on their utilization. There is
only one species of fennel: Foeniculum vulgare Miller. This species is divided in two
subspecies: subsp, vulgare and subsp, piperitum (Ucria) Coutinho.
Foeniculum vulgare subsp, vulgare regroups all the cultivated and naturalized fennels
with a more or less aniseed taste, and it is subdivided in four varieties. Variety azoricurn
Miller is an annual fennel in which the hypertrophied sheaf of the basal leaves consti-
tutes a bulb that is used as a vegetable. Variety dulce Miller (sweet fennel) is an annual
or biannual fennel with sweet-tasting fruits used as a condiment. Cultivar 'Bronze" Miller
(sweet fennel bronze) is a horticultural variety of fennel. Variety vulgare brings together
all the cultivated fennels, or those which have escaped from culture, with a bitter after-
taste. It is used in the food industry to extract trans-anethole and so give an aniseed taste
to various products.
Foeniculum vulgare subsp, piperitum is an uncultivated perennial plant with short, rigid
lobed leaves and narrow umbels that produces small fruits. This subspecies is devoid of
aniseed fragrance, contains no anethole (Gildemeister and Hoffmann, 1916) and is

~tCorresponding author.

(Received 2 April 1 996; accepted 1 8 October 1996)

353
354 B, MUCKENSTURM ETAL.

often confused with a chemotype of F. vulgate var. vulgate, which also presents a bitter,
but distinct, taste and is cultivated for anethole production (Desmarest, 1978).
As a part of a general chemotaxonomic survey of the Apiaceae, we tried to find out
whether the several fennel taxa are also chemically different. To determine whether it is
possible to characterize the different species and subspecies of fennel with the help of
chemistry, we analyzed by G C - M S the volatile portion of every part of the plant of dif-
ferent fennel populations.

M a t e r i a l s and M e t h o d s
BiologicalmateriaL The fennel plants were collected directly from nature or cultivated in the greenhouses of the
Plant Conservatory of Mulhouse (CBM) (Haut-Rhin, France) or bought in the local market. We studied the
same species and subspecies cultivated at CBM and harvested in autumn 1995.
Foeniculum vulgate subsp, piperitum was collected in autumn 1993 in Valbonne (Alpes-maritimes, France).
Foeniculum vulgate var. vulgate was collected during the same period in Valbonne (Alpes-maritimes, France),
in Belvezet (Gard, France) and a population cultivated at Milly-la-For~t originated from the Durance valley.
The sweet fennel plants (F. vulgate var. dulce) came from SuCre (lie-de-France, France) and from the local
market. Variety azoricurnwas bought in a local market and in Alberobello (Italy). Cultivar "Bronze' was bought
in a seed shop (Chiltern Seeds, U.K.) and grown in the CBM. Voucher specimens are deposited in the her-
barium of the Botanic Institute of Strasbourg (France).
Extraction. The different parts of the plants (1 g of roots, stems, leaves or seeds) were minced separately and
extracted by crushing with diethylether (20 ml). The supernatent solution was then decanted and concentrated
to 1 ml before injection.
Methods. The samples were analyzed without further purification by gas chromatography (HP 5890 series
II) using a 2 5 m × 0 . 1 5 m m i.d. BPX5 (non-polar) capillary column (film thickness 0.25~m) with He as carrier
gas and split/splitless injector, coupled with a mass spectrometer (quadripolar mass selective detector, HP
5971 ). Temperature program: 80°C (3 rain), then 80-310°C at 3.5°C/min and 310°C (15 min); injector tem-
perature: 230°C; transfer line temperature: 310°C. Area percentages were obtained using the HP G1034B
Software for MS station. Identification of known compounds involved comparison of spectral data with an
internal spectral library and retention times.
The unknown products were isolated with successive chromatography on silica gel and silica gel/10% silver
nitrate by using petroleum ether-ethyl acetate mixtures as eluent and identified by 1H-N M R spectra (250 M Hz,
CDCI3), 13C-NMR spectra and MS.
10-Nonacosanone (1) could be isolated from commercial fennel powder in the pure state, without chro-
matography. By continuous extraction for 24h with hot petroleum ether of 4 5 g of fennel powder in a
Kumagawa apparatus, concentration and recrystallization from petroleum ether, we obtained 593 mg (1.3%) of
pure 10-nonacosanone (1). MS:m/z422 (M +, 2), 337 (2), 311 (8), 310 (8), 295 (M-CgH19, 54), 171 (67),
170 (21), 155 (M-C19H39, 100), 110 (23), 85 (36), 71 (77), 57 (53), 43 (48). 1H-NMR (250 MHz, CDCI3):
5 0.87, t (J1-2 =J28-29 = 7.4 Hz, 6H, H-l, H-29); 1.25, m (44H, H-2 to 7 and H-13 to 28); 1.56, m (4H, H-8 H-
12),2.41t (Js_9=J~1_12=7.5Hz, 4H, H - 9 H - 1 1 ) . 13 C - N M R ( C D C I 3 ) : 5 1 4 . 2 ( C - 1 , C - 2 9 ) , 2 2 . 7 ( C - 2 , C-28),
23.9 (C-8, C-12), 29.2 (C-7, C-13), 29.4 (C-4, C-6, C-14, C-26), 29,6 (C-5, C-15 to 25), 31.9 (C-3, C-27),
42.8 (C-9, C-11), 211.7 (C-10).
Rotundifolone (2) was isolated from the ether extract of F. vulgare subsp, piperitum seeds on an SiO2 chro-
matography column, eluted with petroleum ether-ethyl acetate (AcOEt) mixture (8:2). MS: m/z 166 (M +, 28),
151 ( M - 1 5 , 16), 138 ( M - 2 8 , 100), 137 (30), 123 (28), 109 (22), 95 (17), 81 (18), 79 (27), 68 (58), 67
(84), 57 (18), 53 (28), 43 (27), 41 (35). ~H-NMR (250MHz,CDCI3): 5 1.45, s (3H, H-7); 1.78, d
(J9-1 o = 1.5 Hz, 3 H, H-9); 1.86, ddd (Jsax-6eq= 14.6 Hz, JB,x-Sax = 6.2 Hz, Jsax-seq = 12.3 HZ, 1 H, H -6ax); 2.09,
dd (J9-1o = l . 5 H z , Jlo-5eq =2.5Hz, 3H, H-10); 2.1, dddd (J6ax-6~ =14.6Hz, J6~-seq =2.3Hz,
J6eq-S~ = 5.5 Hz, J6a×-2 = 0.2 Hz, 1 H, H-6eq); 2.37, m (J6ax-seq-- 15.6 Hz, J5ax~ax = 6.2 Hz, 1 H, H-5ax); 2.48,
m (Jsax-seq = 15.6 Hz, 1 H, H - 5eq); 3.22, d (Jz~ax -- 0.2 Hz, 1 H, H -2). 13C_N M R (CDCI3): 8 21.8 (C- 7), 23.1
(C-5, C-9, C-10), 27.9 (C-6), 63.4 (C-1), 127.6 (C-8), 149.5 (C-4), 198.4 (C-3).
The mass spectra of trans-anethole, cis-anethole and estragole are quite similar. Identification can be
achieved by considering retention times. Injection of reference compounds shows that the retention times are
in the order allyl, cis and trans isomers, as is generally the case for phenylpropanoids.
Cis-anethole, needed as a reference compound, was synthesized by dibromination of commercial trans-
anethole, elimination of HBr and hydrogenation, in a way similar to a known method (Naves, 1960).
Careful examination of the mass spectra of minor products led us to suspect the presence of para-butyla-
nisole as a small peak close to and just after the peak of trans-anethole. To verify that hypothesis, we synthe-
sized para-butylanisole by a non-ambiguous method. Comparison of the mass spectra and retention times
confirmed the identity of the natural and synthetic products.
STUDIESOF FOENICULUMVULGARE 355

Para-butyrylanisole was synthesized by FriedeI-Craft acylation of anisole by butyryl chloride in the presence
of AICI3. MS: 178 (M ÷, 18), 163 ( M - 1 5 , 2), 150 ( M - 2 8 , 8), 147 (4), 135 (M-C3H7, 100), 107 (8), 104
(2), 92 (8), 77 (11), 64 (4), 50 (2), 41 (2). 1H-NMR (250 MHz, CDCI3): 5 0.98, t (J=7.5 Hz, 3H, H-IO);
1.74, sextuplet (J=7.5Hz, 2H, H-9); 2.89, t (J=7.5Hz, 2H, H-8); 3.85, s (3H, H-11); 6.91, dd (J6_5=
J2_3=7.5 Hz, Js_2= 2 Hz, 2H, H-6, H-2); 7.93, dd (Js_s=J2_3=7.5Hz, J3_5=2Hz, 2H, H-5, H-3). 13C-NMR
(CDCI3): ~ 13.9 (C-10), 18.0 (C-9), 40.2 (C-8), 55.4 (C-11), 113.6 (C-3 C-5), 130.2 (C-2 C-6), 130.3 (C-
1), 163.3 (C-4), 199.0 (C-7).
Para-butylanisole (3) was obtained via the Clemensen reduction of para-butyrylanisole. MS: 164 (M, 18),
134 (2), 121 (M-C3H7, 100), 105 (2), 103 (2), 91 (4), 77 (4), 65 (2), 51 (1).IH-NMR (250MHz, CDCI3):
5 0.95, t (J= 7.3 Hz, 3H, H-10); 1.36, sextuplet (J= 7.3 Hz, 2H, H-9); 1.58, sextuplet (J = 7.3 Hz, 2H, H-8);
3.81, s (3H, H-11); 2.57, t (J=7.4Hz, 2H, H-7); 6.91, dd (Js_5=J2_3=6.5Hz, Js_2=2.1 Hz, 2H, H-6 H-2);
7.93, dd (J6_s=J2_3=6.5 Hz, J3_5=2.1 Hz, 2H, H-5 H-3). 13C-NMR (CDCI3): ~ 13.9 (C-10), 22.3 (C-9),
33.9 (C-8), 34.7 (C-7), 55.2 (C-11), 113.6 (C-3 C-5), 129.2 (C-2 C-6), 135.0 (C-1), 157.6 (C-4).

Results and Discussion

The composition given in previous literature reports generally concerns essential oils
(Ravid et al., 1983; Nigam et al., 1987; Sur et al., 1991; Zhao et al., 1991). As our
samples are obtained by solvent extraction, without any purification, we observe addi-
tional compounds which cannot be steam-distilled. This necessitates frequent cleaning
of the injector, as non-volatile compounds remain inside. However, this enabled us to
discover the presence of 10-nonacosanone (1) in the aerial parts of all fennel analyzed
so far. The amount given in the following tables is largely minimized as the column
temperature needed (310°C) is higher than the injection port temperature used (240°C).
It is possible to isolate large quantities of isomer-free lO-nonacosanone without chro-
matography. This is the first report of lO-nonacosanone in Foeniculum vulgare. It was
known as a natural product from Bupleurum species (Cauwet and Carbonnier, 1977)
and from a tree fern (Soeder and Hodgin, 1975). We also found it as a constituent of
Anethum graveolens L. (Muckensturm, unpublished work), which was considered in the
past as belonging to the same genus as Foeniculum vulgate (Linnaeus, 1753). We con-
sider it as a chemical marker of these three genus of Umbelliferae.
We note that the subspecies piperitum is chemically different from the subspecies
vulgare: subsp, piperitum does not contain any estragole (methylchavicol) or trans-
anethole but shows the presence of rotundifolone (2) (2-5% peak area in the leaves and
up to 25% in the seeds). Rotundifolone is a monoterpenoid with a characteristic bitter
taste, first found as a major component of Mentha rotundifolia L. essential oil (Shimizu,
1956). It can be considered as a specific chemical marker of the subsp, piperitum. A

CgHI9 C C19H39

0
I
o~

1 2 3
356 B. M U C K E N S T U R M ETAL.

trace amount of piperitenone is also present. In the course of our work, a publication
appeared (Badoc et al., 1994) confirming this finding.
Para-butylanisole had not previously been known as a natural product. We found it in
several fennel seeds as a small peak (~0,1% peak area or less) in F. vulgare var. azoricum
and var. vulgare. The presence of para-butylanisole is also to be noticed in some com-
mercial trans-anethole in an amount of ~0.1%.
A small peak of cis-anethole is present in every fennel where trans-anethole is present
in a large amount, with an intensity of approximately 0.5% of the trans-anethole peak.
The results obtained within each population are very homogeneous. Foeniculum
vulgare var. vulgare presents great composition differences with varying populations.
This is a result of the definition of the variety vulgare, which merges all the plants that
have escaped from culture with an aniseed bitter after-taste. With the aim of clarifying
the status of var. vulgare, we propose to subdivide it into three chemotypes according to
their relative compositions. We will call them chtyp, estragole, chtyp, estragole/anethole
and chtyp, anethole in the following tables.

L ea ves
The study of the green part of the plant is sufficient for the characterization of the
different chemotypes and subspecies. The subsp, piperitum, as noted before, is clearly
distinguished by the presence of rotundifolone. For the subsp, vulgare we conven-
tionally divide the var. vulgare into three chemotypes:

chtyp, estragole estragole is the major compound

chtyp, estragole/anethole estragole and anethole are present in the same amount
chtyp, anethole trans-anethole is the major compound

Representative specimens of these three chemotypes were harvested respectively in

Belvezet, Milly and Valbonne. The experimental values are reported in Table 1.
To establish the difference between var. dulce and cultivar "Bronze" we have to con-
sider the peak of fenchone (~5% peak area in var. dulce against 20% in cultivar
"Bronze").

TABLE 1. M A I N VOLATILE CONSTITUENTS" OF THE GREEN PARTS OF FENNEL

Subsp. vulgare

Subsp. vat cuJtivar vat var. vulgare var. vulgare var

Compound piperitum dulce "Bronze" azoricum c h t y p estragole chtyp, estragole/anethole chtyp, anethole

~- Pinene 1 8 2 1 10 20 traces
Sabinene 2 2 2 traces 3 5 1
~x-Phellandrene 60 10 1 traces 15 10 10
Limonene 5 3 2 15 1 2 4
I$- Phellandrene 10 1 2 traces ~ 2 1
Fenchone -- 5 20 1 5 1 10
Estragole -- 2 4 2 47 21 10
trans-Anethole -- 60 58 65 2 22 55
Rotundifolone 5 . . . . . .
10-Nonaeosanone 10 2 1 2 2 3 5

"Amount of compounds in % area. 100% is the total area of all peaks integrated on the chromatograms. Peaks with an area < 1% are
not considered in this table unless they are present in another variety, in which case they are noted as "traces".
STUDIES OF FOENICULUM VULGARE 357

To establish the difference between the three chemotypes of var. vulgare we have to
consider the peaks of estragole and trans-anethole, as follows. Chtyp. estragole: % peak
area of estragole >40%; chtyp, anethole: % peak area of trans-anethole >40%; chtyp.
estragole/anethole: any intermediate composition.
For the var, azoricum we notice the % peak area of limonene ~1 5%, compared with 2-
5% in the other varieties.

Seeds
On the contrary, the chemical composition of the seeds is similar for the three che-
motypes of var. vulgate, but makes the difference between subspecies and varieties.
Rotundifolone (2), which is characteristic for subsp, piperitum, is present in an
amount of ~25% peak area of the volatile constituents in the seeds. It is the second
major product of these seeds after limonene (peak area ~30%).
The presence of trans-anethole and estragole confirmed the appurtenance of all the
varieties to the subsp, vulgate, but in the case of the seeds, the chemical composition of
the varieties is different enough to characterize them. Foeniculum vulgare var. vulgare is
characterized by the fact that its major compound is estragole (~-50-60% peak area)
whereas in the other varieties the estragole only reaches 3-4% peak area and the major
product is trans-anethole (~60% peak area). Foeniculum vulgare var. dulce (sweet
fennel) is characterized by the presence of trans-anethole (~60% peak area) and ---20%
peak area of fenchone. As can be seen in Table 2, cultivar "Bronze" seeds show the
same chemical composition as var. dulce seeds. This cultivar is also called sweet fennel
bronze; it is included in var. dulce. Foeniculum vulgare var. azoricum is characterized by
its major compound, trans-anethole (~60% peak area), by the amount of limonene
(~10% peak area compared with 2-3% in the other varieties) and by the amount of
fenchone (only ~6% peak area compared with 20% in the other varieties).

Roots
The root compositions of the different fennels are very similar. No root contains
estragole or anethole (responsible for the aniseed aromaticity of fennel). The compo-

TABLE 2. M A I N VOLATILE CONSTITUENTS" OF THE SEEDS OF FENNEL

Subsp. vulgate

Subsp. var. cultivar var. var. vulgate var. vulgam var.

Compound piperitum dulce "Bronze" azoricum chtyp, estragole chtyp, estragole/anethole chtyp, anethole

¢- Pinene traces 6 6 2 1 6 4
Sabinene 1 2 2 1 1 2 7
~- Phellandrene 2 traces traces traces 1 traces 1
Limonene 30 2 2 10 7 3 3
~- Phellandrene traces 1 2 traces traces traces traces
y-Terpinene 15 1 traces traces 2 1 2
Terpinolene 12 . . . . . .
Fenchone -- 22 20 6 25 21 20
Estragole -- 3 4 4 60 50 61
trans-Anethole -- 60 60 65 1 10 4
Rotundifolone 25 . . . . . .
10-Nonacosanone 1 1 1 1 1 1 3

*Amount of compounds in % area. 100% is the total area of all peaks integrated on the chromatograms. Peaks with an area < 1% are
not considered in this table unless they are present in another variety, in which case they are noted as "traces".
358 B. MUCKENSTURMETAL.

sition of the roots of the subsp, vulgate is h o m o g e n e o u s w h a t e v e r the variety c o n -

sidered. These roots s h o w s the presence of d i l l a p i o l e ( ~ 9 0 % peak area) and t e r p i n o l e n e
( ~ 2 - 5 % peak area). For the subsp, piperitum, the same p r o d u c t s are f o u n d in the roots
but w i t h less d i l l a p i o l e ( ~ 3 0 % peak area) and more t e r p i n o l e n e ( ~ 5 0 % peak area). R o o t
c o m p o s i t i o n c a n n o t be used for c h e m o t a x o n o m y .

References
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