PHYTOCHEMICAL ANALYSIS

Phytochem. Anal. 15, 249–256 (2004)CARBON DIOXIDE EXTRACTION OF CHAMOMILE FLOWERS

SUPERCRITICAL 249
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002.pca.775

Supercritical Carbon Dioxide Extraction of

Chamomile Flowers: Extraction Efficiency,
Stability, and In-line Inclusion of Chamomile–
Carbon Dioxide Extract in β -cyclodextrin

C. S. Kaiser, H. Römpp and P. C. Schmidt*

Department of Pharmaceutical Technology, Auf der Morgenstelle 8, D-72076 Tübingen, Germany

The extraction of chamomile flowers using supercritical carbon dioxide was investigated with respect to extrac-
tion efficiency and compared with solvent extraction. The stability of matricine, a sensitive constituent of the
essential oil of chamomile, in these extracts was studied during storage at different temperatures over 6 months.
Matricine was stable at −30°C. A slight decrease (80–90% recovery) occurred at +5°C, whereas complete de-
composition of matricine took place within 3–4 months at room temperature and at +30°C, respectively. An
in-line inclusion of chamomile constituents in β -cyclodextrin ( β -CD) during the extraction process was assessed
and inclusion rates between 40 and 95% were obtained depending on the amount of β -CD and the type of chamo-
mile constituent. No further stabilization of matricine in the carbon dioxide extract/β -CD complexes was achieved.
High residual water contents in the complexes even after freeze-drying were identified as accelerating the
decomposition. In addition, the extractability of flavonoids, such as apigenin and apigenin-7-glucoside, was
determined. Apigenin-7-glucoside, the more hydrophilic substance, was not extractable with pure carbon
dioxide and showed a recovery of 11% using methanol modified carbon dioxide (18%, w/w) at 60°C and 380 bar.
Extraction conditions in the two-phase region of the binary mixture carbon dioxide–methanol (70°C, 100 bar)
led to a drastic change in fluid polarity and hence extractability increased to 92–95%. Copyright © 2004 John
Wiley & Sons, Ltd.
Keywords: supercritical fluid extraction; carbon dioxide; β-cyclodextrin; modifier matricine; chamomile.

et al., 1983). Therefore, the production of matricine-

INTRODUCTION
Chamomilla recutita L. Rauschert is one of the oldest and
best known herbal remedies. Extracts of chamomile
flowers are used in various pharmaceutical prepara-
tions such as liquids, inhalations or ointments in
the treatment of stomatitis, bronchitis, peptic ulcer,
gastrointestinal spasms, diarrhoea, indigestion and
dermal inflammations.
The active components can be divided into a lipo-
philic group, consisting mainly of the essential oil com-
pounds, and a hydrophilic group containing particularly
flavonoids and derivatives. The most important constitu-
ents of these groups are (−)-α-bisabolol and bisabolol
oxides, matricine, spiroether and β -farnesene on the one
hand and apigenin and apigenin-7-glucoside on the other
(see selected structures in Fig. 1).
Various pharmacological activities, mainly the anti-
spasmodic and anti-inflammatory properties, of chamo-
mile components have been reviewed by Carle and
Gomaa (1992). Matricine, a 6,7-guaianolide primarily
existing in chamomile flowers, contributes essentially
to the anti-inflammatory effect. The blue coloured
chamazulene is a decomposition product of matricine,
generated during steam distillation and shows signific-
antly less activity compared with matricine (Jakovlev
* Correspondence to: P. C. Schmidt, Department of Pharmaceutical Technology,
Auf der Morgenstelle 8, D-72076 Tübingen, Germany.
Email: peter-christian.schmidt@uni-tuebingen.de
containing extracts is desirable, although this compound
is fairly unstable.
Supercritical fluids have been established as solvents
for a great variety of applications (Kaiser et al., 2001),
mainly for the production of lipophilic extracts under
mild conditions. Supercritical fluid extraction (SFE) of
chamomile flowers has been studied by several authors
with different emphases (Stahl and Schilz, 1976; Stahl
and Schütz, 1978; Reverchon and Senatore, 1994; Pekic
et al., 1995; Scalia et al., 1999; Zekovic, 2000; Povh et al.,
2001).
β -Cyclodextrins (β -CD) are cyclic oligosaccharides,
made up of seven glucose rings linked by α-1,4 glycosidic
bonds. They are capable of including in their cavity
slightly polar molecules by non-covalent interaction
forces like Van der Waals forces and hydrogen bonds.
Processing of extract components as guest molecules by
inclusion in β -cyclodextrin as a host molecule is a known
method for the protection and stabilization of volatile
or unstable substances (Szente et al., 1981; Szente and
Szejtli, 1988). Furthermore, liquid or paste-like sub-
stances, such as carbon dioxide extracts, are converted
into solid, free-flowing powders and inclusion in β -CD
reduces the intense flavour of these extracts.
The present work provides a comprehensive study
on the extraction of chamomile flowers by supercritical
carbon dioxide, focusing on extraction efficiency, mass
balances and stability of matricine in these extracts. The
possibility of an in-line inclusion of chamomile carbon
dioxide extracts in β -CD during the extraction process

Copyright © 2004 John Wiley & Sons, Ltd. Received

Phytochem. Anal. 22 April
15: 249–256 2003
(2004)
Copyright © 2004 John Wiley & Sons, Ltd. Revised 10 July 2003
Accepted 5 August 2003
250 C. S. KAISER ET AL.
Figure 1. Selected active components of chamomile flowers.
has been investigated. The benefits and limitations of
using methanol-modified carbon dioxide in pilot-scale
extraction have been evaluated for the processing of
polar solutes like flavonoids and flavonoid glycosides.
EXPERIMENTAL
Materials
Chamomile flowers (type Manzana, harvested in
2001) of the (−)-α-bisabolol chemotype with negligible
contents of (−)-α-bisabolol oxides were provided by
Robugen (Esslingen, Germany). The raw material was
employed as a crude drug or ground at two levels using
a Pulverisette® cutting mill (Fritsch, Idar-Oberstein,
Germany). (−)-α-Bisabolol, apigenin and apigenin-7-
glucoside reference substances were purchased from
Roth (Karlsruhe, Germany) and cis- and trans-spiroether
were a gift from Robugen (Esslingen, Germany). Matric-
ine standard substance was isolated by Ness (Schmidt
and Ness, 1993). β -CD was supplied by Wacker-Chemie
(Munich, Germany), carbon dioxide (technical grade)
and gases for GC (type 5.0) by Messer Griesheim
(Darmstadt, Germany) and solvents (gradient grade) as
well as n-hexadecane by Merck (Darmstadt, Germany).
Water for HPLC was produced using reverse-osmosis
followed by distillation.
SFE apparatus
All extractions were performed on a pilot-scale batch
extraction plant (Sitec, Maur, Switzerland). Liquid
carbon dioxide was stored in a carbon dioxide tank and
compressed by a high pressure pump to the desired ex-
traction pressure. Before passing the extraction vessel, a
heat exchanger provided the chosen extraction tempera-
ture. The extracted material was separated from carbon
dioxide by pressure reduction into the liquid state in a
separator. Therein liquid carbon dioxide was continu-
ously evaporated by adequate heating and left the
separation vessel in the gaseous state. It was liquefied
again by a condenser and recycled into the carbon
Copyright © 2004 John Wiley & Sons, Ltd.
dioxide tank. A modifier could be added to the super-
critical carbon dioxide by a separate modifier supplying
system.
Supercritical fluid extraction
Extractability and stability of chamomile extract
constituents using pure carbon dioxide. Extractions
were carried out with 200 g of crude drug, 210 g coarsely
ground and 260 g finely ground chamomile flowers. The
comminution was carried out directly before extraction.
The resulting particle sizes were >3.15 mm for the major
fraction of crude drug, between 0.5 and 1.4 mm for
coarsely and <0.5 mm for finely ground drug. The extrac-
tion conditions were 40°C, 90 bar, according to a carbon
dioxide density of 540 kg/m3, and 8 kg/h carbon dioxide
for 3 h. Separation took place in liquid carbon dioxide
at 8°C and 40 bar. After rapid decompression of the
separation vessel, extracts were taken off which were
included in the solidified carbon dioxide. After evapora-
tion of carbon dioxide, the extracts were frozen at −30°C
and subjected to a freeze-drying programme for 24 h
(Lyovac GT 2, Finn-Aqua, Hürth, Germany). Three
extracts of each comminution level were produced and
divided into four parts for storage at −30°C, +5°C, room
temperature or +30°C.
Extraction efficiency of flavonoids. Three consecutive
extractions (trials 1a, 1b, 1c) with the same raw material
were carried out with 260 g of finely ground crude
drug. The first two were conducted at 60°C and 380 bar
(carbon dioxide density, 880 kg/m3) with 2 L methanol as
modifier (molar fraction 23% and mass fraction 18%).
A third extraction with the same raw material was per-
formed at 70°C and 100 bar (carbon dioxide density,
350 kg/m3) in the two-phase region of the binary mixture
carbon dioxide:methanol with 2 L methanol (molar frac-
tion of 24%, mass fraction of 19%) as modifier.
For comparison, a single-stage extraction (trial 2)
at 70°C and 100 bar was performed (2 L methanol;
molar fraction 28%; mass fraction 22%). The extraction
time was 3 h for all experiments. After the withdrawal
of the extract, a sample of 10 g of the extracted plant
material was taken out of the extraction vessel for the
Phytochem. Anal. 15: 249–256 (2004)
 SUPERCRITICAL CARBON DIOXIDE EXTRACTION OF CHAMOMILE FLOWERS
determination of the residual amount of components
remaining in the plant material after SFE.
Extracts containing methanol were concentrated using
a vacuum rotary evaporator (Büchi, Flawil, Switzerland)
and then dried at 35°C for 2 days in a vacuum drying
oven (Binder, Tuttlingen, Germany).
In-line inclusion of carbon dioxide extracts in β -CD. The
extractions were carried out with 210 g of coarsely
ground chamomile flowers at 40°C, 90 bar and 8 kg/h
carbon dioxide for 3 h. Aliquots of 5, 10, 15 or 20 g β -
CD, according to mass ratios of 2–8:1 (β -CD: carbon
dioxide extract) were placed in the separation vessel
prior to the extraction. Withdrawal of the extract:β -CD
mixture and freeze-drying were carried out as described
above. The extractions were conducted in triplicate.
Sample preparation for HPLC and GC analysis
Carbon dioxide extract (50–100 mg), accurately weighed,
was dissolved in methanol:dichloromethane (1:1, v/v) for
pure carbon dioxide extracts and in methanol for modi-
fied carbon dioxide extracts; 2.0 mL of internal standard
solution (275 mg n-hexadecane:500 mL n-hexane) were
added and the solution made up to 50.0 mL in a volu-
metric flask.
For the determination of the total content of lipophilic
components in the crude drug, 5 g of chamomile flowers
were macerated twice with 100 mL of methanol:
dichloromethane (1:1, v/v) on a magnetic stirrer for
60 min. The drug:solvent ratio was 1:40, which ensured
complete extraction of the lipophilic chamomile carbon
dioxide constituents (Ness and Schmidt, 1995). The
sample was filtered, 4.0 mL of internal standard solu-
tion were added and made up to 100.0 mL.
For the determination of the residual content
of lipophilic components in the crude drug after SFE,
maceration was conducted once with 100 mL methanol:
dichloromethane. The residual content represents the
chamomile components that are not removed by SFE.
For the determination of hydrophilic components,
the same macerations were adopted using methanol
as extracting solvent. All samples were filtered
through a 0.45 µm regenerated cellulose filter (Sartorius,
Göttingen, Germany; type 18406-25) prior to analysis by
HPLC and GC.
Analysis of carbon dioxide extract:β -CD complexes
and determination of the inclusion rates
After freeze-drying, carbon dioxide extract:β -CD
complexes were gently homogenized using a pestle
and mortar. For the determination of the inclusion and
free fraction of the extract components, a hexane wash
procedure described by Waleczek et al. (2002) was per-
formed. A sample of the carbon dioxide extract:β -CD
complex (1.5 g) was accurately weighed and washed with
5.0 mL n-hexane to remove adhered components from
the outer surface of the β -CD particles. The n-hexane
phase was decanted, 1.0 mL internal standard solution
was added and the solution was made up to 25.0 mL in a
volumetric flask, before analysis by GC and HPLC. The
remaining slurry from the washing procedure was dis-
solved in 10.0 mL dimethylsulphoxide, 30 mL methanol:
Copyright © 2004 John Wiley & Sons, Ltd.
251
dichloromethane (1:1, v/v) were added and the sample
was equilibrated for 12 h. After filtration, the resulting
solution, representing the inclusion part of chamomile
constituents, was analysed by GC and HPLC.
HPLC analysis
A Waters (Millipore, Milford, MA, USA) model
616 pump with a 600S controller equipped with a
SIL-9A auto-injector and a SPD-6A UV-detector (both
from Shimadzu, Duisburg, Germany) was employed.
Data analysis was carried out using LC10 software
(Shimadzu). Separations were performed on a Nucleosil
100-5 RP18 (250 × 4.6 mm i.d.) column (Macherey
and Nagel, Düren, Germany) protected by a Nucleosil
100-5 RP18 (8 × 4 mm i.d.) guard column. The injection
volume was 20 µL.
Analysis of matricine and dicycloethers was carried
out as described by Schmidt and Ness (1993). The elu-
tion was conducted using a gradient programme with
mobile phase A containing water:methanol:acetonitrile
(50:38:12, v/v) and methanol as mobile phase B at a flow
rate of 1.0 mL/min. The gradient programme was as
follows: 0–15 min 100% A, 15–18 min 100–70% A, 18–
30 min 70–30% A, 30–33 min 30–100% A, 33–40 min
100% A. The UV detection wavelength was 244 nm.
Calibration was performed with matricine in the range
1.527–45.81 µg/mL (r 2 = 0.9984), with cis-dicycloether in
the range 65.674–326.35 µg/mL (r 2 = 0.9980) and with
trans-dicycloether in the range 9.787–48.94 µg/mL (r 2 =
0.9979).
Flavonoids (apigenin and apigenin-7-glucoside)
were determined according to Schmidt and Vogel
(1992) at a UV detection wavelength of 355 nm.
Acetonitrile:phosphoric acid (1000:2, v/v) was used as
mobile phase A and water:phosphoric acid (1000:2, v/v)
as mobile phase B at a flow rate of 1.2 mL/min. The
gradient programme was as follows: 0–15 min 22% A,
15–20 min 50% A, 20–25 min 50–75% A, 25–30 min
75% A, 30–35 min 75–22% A, 35–45 min 22% A. The
quantity was calculated from an external standard
calibration in the range 7.012–140.24 µg/mL (r 2 = 0.9986)
for apigenin-7-glucoside and in the range 2.616–26.16 µg/
mL (r 2 = 0.9989) for apigenin.
GC analysis
A Hewlett-Packard (Böblingen, Germany) 5890 II gas
chromatograph with a HP 7673A auto-injector, a flame
ionisation detector and a HP 3392A integrator was used.
Extracts were injected in the split mode (1:10) and separ-
ated on a 30 m × 0.25 mm i.d. capillary column (Optima
δ -3, Macherey-Nagel, Düren, Germany) using hydrogen
as a carrier gas. The separation was performed follow-
ing the methods of Ness and Schmidt (1995). The
temperature programme was 120°C rising to 170°C at
7°C/min, 170–194°C at 3°C/min, 194–300°C at 5°C/min
and finally held at 300°C for 5 min. The injector and
detector temperatures were 280 and 250°C, respectively.
The quantity of (−)-α-bisabolol was calculated from a
calibration with n-hexadecane as internal standard in
the range of 24.196–241.96 µg/mL. The C25-n-alkane,
which represents the lipophilic, non-volatile fraction
of cuticular waxes, was calculated as (−)-α-bisabolol.
Phytochem. Anal. 15: 249–256 (2004)
252 C. S. KAISER ET AL.

The substance was identified by comparison of GC
chromatogram pattern with the literature (Reverchon
and Senatore, 1994).
Determination of water content
Water contents of the extracts were determined
with Karl–Fischer titration using SM-Titrino 702 and
Titration Stand 703 (Metrohm, Filderstadt, Germany)
with Hydranal Composite 2 (Riedel-de-Haën, Seelze,
Germany) as a one-component reagent in methanol as
solvent. Sodium tartrate dihydrate was used for deter-
mination of the titre.
RESULTS AND DISCUSSION
Extractability and stability of chamomile constituents
using pure carbon dioxide
The extraction conditions with a relatively low carbon
dioxide density of 540 kg/m3 were optimised with respect
to yield and selectivity for components of the essential
oil of chamomile. As noted by Reverchon et al. (1995),
sesquiterpene hydrocarbons and oxygenated derivatives
which represent the major fraction of the essential oil
are readily soluble in supercritical carbon dioxide and
hence high carbon dioxide densities are not required
because they would lead to co-extraction of undesired
by-products. A mass balance of the extraction process
with pure carbon dioxide for selected chamomile con-
stituents is given in Table 1. The total content was deter-
mined by solvent maceration and was set as 100%. For
all substances, an increase in extractability with a higher
comminution level was obvious; this is in accordance
with studies on extraction kinetics by Pekic et al. (1994).
Essential oil compounds are located inside vegetable
structures and therefore the reduction of particle sizes
reduces the mass transfer resistance and promotes
extractability. With respect to extractability and sta-
bility, three different classes of components could be
observed.
(−)-α-Bisabolol, a sesquiterpene alcohol and the C25-
n-alkane showed high recoveries in the supercritical ex-
tract (58–92%). After addition of the residual content,
determined by solvent maceration in the extracted plant
material, a total recovery of 80–100% was found. These
substances were characterized by good extractability and

Table 1. Recovery of chamomile constituents in the carbon dioxide extract and residual con-
tent of the extracted plant materiala

Recovery, % (mean ± standard deviation)b

Extract Residual Total Contentc (mg/100 g)

β -Farnesene
crude drug 16.2 ± 6.2 31.0 ± 2.3 47.1 ± 8.3
coarsely ground 22.4 ± 2.2 18.2 ± 1.2 40.7 ± 3.0 162.8 ± 1.6
finely ground 38.7 ± 8.4 7.0 ± 0.5 45.7 ± 8.1
α-Bisabolol
crude drug 57.7 ± 4.3 23.1 ± 0.6 80.9 ± 4.0
coarsely ground 71.7 ± 2.0 11.4 ± 0.5 83.1 ± 2.1 355.1 ± 12.5
finely ground 91.7 ± 2.2 5.5 ± 0.5 97.2 ± 2.4
C25-n-alkane
crude drug 61.0 ± 3.4 15.3 ± 1.8 76.4 ± 1.8
coarsely ground 75.5 ± 2.1 17.5 ± 1.4 93.0 ± 1.4 120.8 ± 5.4
finely ground 91.9 ± 1.2 7.4 ± 0.6 99.2 ± 1.8
Apigenin-7-gluc.
crude drug ndd 76.6 ± 2.6 76.6 ± 2.6
coarsely ground nd 89.7 ± 19.4 89.7 ± 19.4 384.8 ± 8.5
finely ground nd 94.2 ± 4.8 94.2 ± 4.8
Apigenin
crude drug 20.9 ± 0.8 73.2 ± 0.2 94.1 ± 1.0
coarsely ground 27.3 ± 1.0 54.7 ± 9.0 82.0 ± 8.2 8.78 ± 1.4
finely ground 33.4 ± 0.7 67.4 ± 0.3 100.8 ± 0.5
Matricine
crude drug 34.2 ± 4.3 35.1 ± 0.5 69.3 ± 4.8
coarsely ground 45.0 ± 5.1 18.1 ± 0.9 63.1 ± 5.3 197.5 ± 13.8
finely ground 65.2 ± 6.7 9.9 ± 2.5 75.0 ± 4.2
trans-Spiroether
crude drug 34.9 ± 4.6 37.9 ± 1.7 72.7 ± 4.5
coarsely ground 55.8 ± 8.8 34.6 ± 2.0 90.5 ± 10.8 135.2 ± 4.8
finely ground 71.7 ± 2.8 22.8 ± 1.4 94.4 ± 3.6
cis-Spiroether
crude drug 31.1 ± 3.2 34.1 ± 2.4 65.1 ± 5.5
coarsely ground 55.2 ± 2.0 22.0 ± 4.7 77.2 ± 4.6 526.8 ± 17.4
finely ground 72.7 ± 4.6 9.7 ± 0.9 82.4 ± 4.0
a
Experiment to conditions: 40°C; 90 bar; 8 kg/h carbon dioxide; 3 h.
b
n = 3.
c
mean ± standard deviation (n = 3), determined by double maceration.
d
nd = not detectable.

Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 249–256 (2004)
 SUPERCRITICAL CARBON DIOXIDE EXTRACTION OF CHAMOMILE FLOWERS
stability. Since solvent maceration of the crude drug was
performed for the determination of the residual content,
poor diffusivity of chamomile constituents through the
barrier of entire cells resulted in lower recoveries and
hence a higher deficit in the mass balance. If comminu-
tion had an influence on the stability of the extracted
substances, a higher deficit in the recovery would have
been observed for components of finely ground drug due
to degradation processes. Thus, a negative effect of com-
minution on the stability of extracted components was
ruled out.
Apigenin and particularly the more hydrophilic
apigenin-7-glucoside were stable but pure carbon diox-
ide was not an adequate solvent for extraction. Apigenin
showed a yield of 21–34% in the carbon dioxide extract,
whereas apigenin-7-glucoside was not detected at all.
With solvent maceration the missing amount was found
in the extracted plant material.
A third group of substances, β -farnesene, matricine
and the spiroethers, revealed a higher deficit in the total
recovery and this was attributed to poor stability. β -
Farnesene, an non-substituted sesquiterpene, was the
most volatile substance, and its loss took place during
freeze-drying of the extract. This was confirmed by the
investigation of extracts, dried at room temperature and
ambient pressure, in which the recovery of β -farnesene
in the extract was 61.6 ± 1.8% (mean ± standard devia-
tion, n = 3) for coarsely ground drug, which was three
times higher than after freeze-drying. Cis-spiroether may
have undergone isomerisation to trans-spiroether, and
both isomers are prone to rapid light-induced degrada-
tion (Grünhagen, 1994).
The degradation mechanism of matricine was investi-
gated by Ness (1995), and the first step was found to be
a hydrolytic cleavage of the acetic acid ester (Ness, 1995).
Therefore the stability of matricine is strongly influenced
by the water content of the extract. During storage
at room temperature and at +30°C, matricine as a com-
ponent of a carbon dioxide extract was rapidly decom-
posed and was no longer detectable after 3 and 4 months,
respectively (Fig. 2). If ground plant material was
extracted, the decomposition seemed to accelerate, but
Figure 2. Stability of matricine gained by SFE depending on storage conditions
and level of comminution. (- - -) finely ground; (– –) coarsely ground; (—) crude
drug; (䊏) −30°C; (䉬) +5°C; (䊊) room temperature; (×) +30°C (error bars: mean ±
standard deviation, n = 3).
Copyright © 2004 John Wiley & Sons, Ltd.
253
the differences were not significant and levelled off at the
end of the investigation. After storage at +5°C, the
matricine content ranged between 80 and 90% based on
the starting material. At reference conditions of −30°C,
matricine was stable (99–101%). For comparison, in an
aqueous solution, particularly at pH values below 5, the
degradation of matricine into chamazulene-carboxylic
acid and finally into chamazulene took place within 2
days (Ness et al., 1996). Although the solubility of water
in supercritical carbon dioxide was only ca. 0.16% (w/w)
according to King et al. (1992), the large amount of
carbon dioxide (24 kg) used in one experiment allowed
the extraction of 8–10 g of water. Water, which was
partially separated from the lipophilic matrix of the
extract, showed a pH value of 3–4. During the extrac-
tion process co-extracted water was saturated with
carbon dioxide, thus a low pH value resulted due to the
formation of carbonic acid. Moreover, the degrada-
tion of matricine into chamazulene carboxylic acid
influenced the pH value and could lead to an auto-
catalytic degradation. However, even after freeze-drying,
the extracts contained a residual amount of water, which
accelerated the degradation of matricine. A comparison
of single extracts with respect to their water content and
their matricine stability revealed a strong correlation
(Table 2).
Extraction efficiency of flavonoids
The extraction of chamomile flavonoids by means of
supercritical carbon dioxide was investigated by Scalia
et al. (1999). They obtained recoveries of 0.5 and 56.1%
for apigenin-7-glucoside and apigenin, respectively, at
202 bar, 40°C, 17 kg/h carbon dioxide for 2 h. The addi-
tion of 5% (v/v) methanol as a modifier was performed
only on analytical scale and this enhanced the recoveries
to 14.6 for apigenin-7-glucoside and 143.3% for apigenin
based on the amount extracted by maceration. Thus, the
above result of no detectable apigenin-7-glucoside in an
unmodified carbon dioxide extract (40°C, 90 bar) was not
unexpected. Zekovic (2000) reported “poor” solubility
Phytochem. Anal. 15: 249–256 (2004)
254 C. S. KAISER ET AL.

Table 2. Water content of single carbon dioxide extracts after phase on the other, containing between 50 and 60 mol%
freeze-drying and correlation with matricine stability methanol (Semenova et al., 1979). Thus, a liquid meth-
anol phase, saturated with carbon dioxide, actually
Matricine content (%) represented the extraction fluid. The dielectric constant,
Level of Water
which was correlated with polarity and thus solvent
comminution contenta (%) RTb, 2 months
power for polar solutes, was approximately 2.0 for pure
Finely ground 0.55 ± 0.03 39.9 carbon dioxide (35°C, 100 bar) and was increased to 4.5
Finely ground 1.97 ± 0.04 5.1 by the addition of 26.7 mol% methanol (65°C, 303.5 bar).
Finely ground 3.42 ± 0.06 2.3 The splitting in the two-phase region (65°C, 100.3 bar,
Coarsely ground 0.58 ± 0.05 41.7 57.5 mol% methanol) resulted in a dielectric constant of
Coarsely ground 0.50 ± 0.06 42.6 12.4 (Roskar et al., 1992). The percentage of methanol
Coarsely ground 1.40 ± 0.16 5.2 was found to play the key role for the solvent power
Crude drug 0.61 ± 0.01 52.5 of the modified fluid, whereas the dependency on fluid
Crude drug 0.66 ± 0.01 48.1 density (pressure and temperature) was rather small.
Crude drug 0.89 ± 0.03 51.1
A fractionated extraction with pure and modified
a
Mean ± standard deviation (n = 3). supercritical carbon dioxide allowed the selective enrich-
b
Room temperature. ment of either the essential oil components or the more
hydrophilic substances like flavonoids. Thus, an initial
for apigenin and no solubility for apigenin-7-glucoside in extraction step with a low carbon dioxide density (40°C,
carbon dioxide at 40°C and 200 bar. 90 bar) removed 80–100% of essential oil components.
To improve the extractability, further extractions with In a second extraction step methanol-modified carbon
modified carbon dioxide were assessed. Yield, content of dioxide was used for the production of an extract en-
apigenin-7-glucoside in the extract and total recovery riched in flavonoids. A supercritical fluid extract contain-
compared with solvent maceration are given in Table 3. ing the whole polarity range of chamomile constituents
A high extraction temperature (60°C) at a carbon diox- was obtained when the extraction was carried out in the
ide density of 880 kg/m3 (extraction pressure 380 bar) two-phase-region of the binary mixture methanol:carbon
caused a rising vapour pressure and therefore a higher dioxide (experiment 2). The recoveries compared with
solubility of solutes in a supercritical fluid. The extract solvent maceration are listed in Table 4. Matricine was
yield was 7.2% (1a) and hence much higher than the not detected because of thermal degradation during
yield of approximately 1.2% achieved by pure SFE at extraction (60°C) and drying procedures. The loss of
40°C and 90 bar. However, the recovery of apigenin- β -farnesene was lower than after freeze-drying. Isomer-
7-glucoside in these extracts was comparatively low isation from cis-spiroether to trans-spiroether is the
(8.8%). Experiment 1b was performed with the pre- reason for significantly higher recoveries of trans-
extracted drug of experiment 1a. A total recovery of spiroether. Starting from the crude drug, such an extract
11.2% apigenin-7-glucoside was achieved. Because of included all constituents of pharmacological interest
a low extract yield (2.3%) the content of apigenin-7- enriched by a factor of 6–7. Considering the solvent
glucoside in the extract was comparatively high (19.5 mg/ consumption of supercritical fluid extraction with
g in 1b vs. 4.66 mg/g in 1a). The almost correspond- methanol-modified carbon dioxide, a reduction of 80%
ing yields of apigenin-7-glucoside in extract 1a and 1b compared with solvent maceration was achieved and
indicated that the extraction of apigenin-7-glucoside only one extraction step with modified carbon dioxide
was limited by a poor solubility and was not a problem was necessary to obtain extracts containing lipophilic
of saturation of the fluid. The subsequent extraction and hydrophilic components.
in the two-phase region of the binary mixture carbon
dioxide:methanol resulted in an almost complete extrac-
tion of apigenin-7-glucoside (total recovery of 95%, In-line-inclusion of chamomile:carbon dioxide extracts
experiment 2c) and thus was regarded as proof of in β -CD
the solubility-limited extraction of apigenin-7-glucoside.
Derived from the phase diagrams of the carbon dioxide: The formation of β -CD complexes can be achieved by
methanol system, it was apparent that the extracting different methods (Hedges, 1998; Loftsson, 1999). Using
solvent splits up into a gaseous and carbon dioxide-rich the co-precipitation method, an aqueous solution of
phase on the one hand and a liquid and methanol-rich β -cyclodextrin was prepared at 75°C, and the guest
Table 3. Experimental conditions for the determination of the extractability of apigenin-7-
glucoside

Experimental conditions Apigenin-7-glucoside

Yield of
Experimenta Pressure Temperature extract Contentb Total recoveryb
number (bar) (°C) (% crude drug)b (mg/g extract) (%)

1ac 380 60 7.24 ± 0.03 4.66 ± 0.55 8.78 ± 1.08

1bc 380 60 2.29 ± 0.02 19.5 ± 3.87 11.2 ± 2.35
1cc 100 70 9.65 ± 1.32 43.2 ± 0.76 95.2 ± 1.68
2 100 70 16.3 ± 1.45 21.9 ± 2.36 92.6 ± 10.5
a
Each experiment was conducted twice.
b
Mean ± range (n = 2).
c
Experiments were carried out consecutively with the same starting plant material.

Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 249–256 (2004)
 SUPERCRITICAL CARBON DIOXIDE EXTRACTION OF CHAMOMILE FLOWERS 255

Table 4. Recovery of a total carbon dioxide extract (100 bar, 70°C) compared to repeated solvent maceration (= 100%)

Recovery, % (mean ± range, n = 2)

β -Farnesene (−)-α-Bisabolol C25-n-alkane Apigenin Apigenin-7-glucoside Matricine trans-Spiroether cis-Spiroether

82.1 100.5 111.1 134.2 92.6 nda 126.0 96.0

± 6.7 ± 0.7 ± 10.7 ± 16.8 ±10.5 ± 6.4 ± 3.2
a
Not detectable.

compound was added to the solution while stirring. Cool- Table 5. Molar ratios n( β -CD):n(extract component) as
ing of the solution to room temperature induced precipi- related to mass ratio m( β -CD):m(extract)
tation of the complex. The slurry method as well as the
paste or kneading method used less solvent and thus CDs Mass ratio
were employed as suspensions. The use of supercritical
2:1 4:1 6:1 8:1
carbon dioxide in complex formation has been reported
by several authors. Van Hees et al. (1999) studied the Extract Molar ratios
formation of a piroxicam:β -CD inclusion complex in component β -CD:extract component
supercritical carbon dioxide by pressurizing a physical
mixture of both components in a static mode and ob- β -Farnesene 7.7 15.5 23.2 31.0
tained high inclusion rates (>90%) at 150°C and 300–450 (−)-α-Bisabolol 2.4 4.8 7.2 9.6
bar. An ibuprofen:methyl-β -CD complex was success- C25-n-alkane 8.7 17.4 26.1 34.8
fully prepared by passing ibuprofen-loaded carbon diox- Matricine 9.6 19.2 28.8 38.4
ide (35°C, 130–220 bar) through a methyl-β -CD packed trans-Spiroether 8.2 16.3 24.5 32.6
cis-Spiroether 1.8 3.6 5.5 7.3
bed (Charoenchaitrakool et al., 2002). A similar set-up
was used by Kamihira et al. (1990) for the formation of
inclusion complexes between cyclodextrins and aromatic
compounds at 10–40°C and 20–60 bar. Adda and Lorne bonds. Besides van der Waals forces, which play the
(1989) improved the recovery of volatile compounds by key role for penetration and binding of a guest-molecule
the use of β -CD as trapping medium in the separator of in the hydrophobic cavity of a β -CD molecule, hydro-
a supercritical extraction plant. gen bonds between guest and hydroxyls were formed
In this work, inclusion during the SFE process was (Saenger, 1980). The matricine molecule is capable
investigated. The complex formation was supposed to of forming hydrogen bonds, which is not possible for
occur in liquid carbon dioxide in the separation vessel β -farnesene or the C25-n-alkane. Dicycloethers have a
(40 bar), where extract components were still partially stretched, rigid structure fixed by two triple-bonds and
soluble and β -CD was suspended. Molar ratios n(β - therefore it was assumed that parts of these molecules
CD):n(compound) for the experiments depended on the remain outside the cavity and thus are more easily re-
content of a component in the extract and on the mo- moved by washing with n-hexane.
lecular weight of the component. The calculated values Although high inclusion rates for matricine were
are given in Table 5. The mass ratios were chosen accord- attained, no further stabilization was achieved. The re-
ing to Waleczek et al. (2002). Considering the extract covery of matricine after a storage period of 1 month at
as a pseudo pure compound to be included with an different temperatures in comparison to the data of a
estimated average molecular weight of 350, the total β - crude carbon dioxide extract revealed that the average
CD:compound ratios ranged between 0.6 and 2.5. values were even below those of the crude extract. A
Initially the content of extract components in a carbon possible reason may be the non-negligible water content
dioxide extract:β -CD complex was compared with a of β -CD (13.7 ± 0.2%) before freeze-drying and a
pure carbon dioxide extract. They were in the same residual content of 6.7 ± 0.4% thereafter. Furthermore,
range for (−)-α-bisabolol, C25-n-alkane, matricine and co-extracted water could not be separated as easily as
dicycloethers. However, β -farnesene as the most volatile from a pure lipophilic carbon dioxide extract, but was
component could be partially protected from volatiliza- absorbed and retained in the carbon dioxide extract:β -
tion during freeze-drying. The extent of the protection CD complex. In the presence of 10 g co-extracted water
depended on the applied mass of β-CD. (50–200% of the β -CD amount), an equilibrium between
Inclusion rates as ratios of included fraction to the the insoluble and soluble complex existed, resulting in
total content depended on the mass ratio of β - free guest molecules that were prone to decomposition.
CD:extract. For the highest mass ratio (8:1), the inclu- Another explanation may be that β -CDs act as artificial
sion rates ranged between 74% (C25-n-alkane) and 96% enzymes catalysing the hydrolysis of esters if they are
(β -farnesene). Except for matricine, a reduction of the included in the β -CD-cavity (Fernandez et al., 2001);
β -CD amount led to a decline in the inclusion rates. hydrolysis of the cyclic ester structure of matricine is also
For the C25-n-alkane, inclusion rates of 40–50%, for the the first step in a possible degradation mechanism.
dicycloether 50–60% and for β -farnesene and (−)-α-
bisabolol 50–70% were obtained. Matricine showed in-
clusion rates of 85–93%, independent of the amount of Acknowledgements
β -CD. The different affinities of chamomile constituents
The authors gratefully acknowledge Robugen (Esslingen, Germany)
included in the cavity of a β -CD molecule were ascribed for the supply of chamomile flowers and dicycloether reference sub-
to different molar ratios as well as to molecular shape, stances. In addition we would like to thank Professor Tsige Gebre-
size and the ability to form intermolecular, non-covalent Mariam, University of Addis Ababa for reading the manuscript.

Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 249–256 (2004)
256 C. S. KAISER ET AL.
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