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Composition profile of horsetail (Equisetum

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organic solvents extraction

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DOI: 10.1016/j.supflu.2004.07.004

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 J. of Supercritical Fluids 33 (2005) 131–138

Composition profile of horsetail (Equisetum giganteum L.) oleoresin:

comparing SFE and organic solvents extraction
Eliane M.Z. Michielina , Louisiane F.V. Brescianib , Leandro Danielskic ,
Rosendo A. Yunesb , Sandra R.S. Ferreiraa,∗
a LATESC/EQA – CTC/UFSC, Laboratório de Termodinâmica e Extração Supercrı́tica – Departamento de Engenharia Quı́mica e Engenharia
de Alimentos – Centro Tecnológico/Universidade Federal de Santa Catarina, Caixa Postal 476, CEP 88040-900, Florianópolis, SC, Brazil
b QMC – CFM/UFSC – Departamento de Quı́mica – Centro de Ciências Fı́sicas e Matemáticas/Universidade

Federal de Santa Catarina, Florianópolis, SC, Brazil

c Technische Universitaet Hamburg – Harburg – AB 6-03, D-21071 Hamburg, Germany

Received in revised form 12 July 2004; accepted 21 July 2004

Abstract

The composition profile of horsetail (Equisetum giganteum L.) oleoresin obtained by supercritical fluid extraction (SFE) and organic
solvents such as hexane and dichloromethane (DCM) was investigated and the results were compared to evaluate the processes efficiency. To
observe the selectivity characteristic of the supercritical fluid process, carbon dioxide was used as solvent with density varying from 719.1 to
923.0 kg/m3 . The operating conditions were 303 and 313 K and 12, 15, 17, 20, 25 and 30 MPa and the SFE yield was up to 1.44% (w/w). In
order to investigate the extraction process of the horsetail oleoresin, the main subject of this work was the qualitative evaluation of the extracts
by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) analysis, due to the non-polar characteristic conferred
by the supercritical solvent. The compounds identification of the horsetail extracts was based on a database for natural products. The influence
of the solvent density and the extraction time in the oleoresin composition was also investigated. The evaluation of the composition profile
indicates that the supercritical CO2 extracts are remarkably different than the organic solvent extracts.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Horsetail (Equisetum giganteum L.); Supercritical CO2 ; Composition

1. Introduction Nowadays, besides the constant improvement of alterna-

Supercritical fluid extraction (SFE) is becoming an ef-
ficient and worldwide spread technique to obtain valuable
natural substances, like essential oils and oleoresins, from
complex materials. This operation has been considered as
an attractive process to replace conventional ones, such as
steam distillation and organic solvent extraction, specially
concerned to product quality and the environmental aspects
involved [1,2]. The SFE can also offers the possibility for
process selectivity, extract fractionation and essential oil pu-
rification, as a result of the solvent density control [3].
∗ Corresponding author. Tel.: +55 48 331 9448; fax: +55 48 331 9687.
E-mail address: sandra@enq.ufsc.br (S.R.S. Ferreira).
tive techniques, sort of SFE, phytochemical studies involv-
ing medicinal plants are in constant development, specially
concerned to the isolation of active principles and the char-
acterization of new components with therapeutic activity.
In the endless field of natural products, the Equisetum
giganteum L., also known as horsetail or giant horsetail, is
commonly used in herbal medicine and as an easy substitute
of the Equisetum arvense (field horsetail). The giant horse-
tail is a lower vascular plant used medicinally as diuretic,
hemostatic and astringent. The plant extract is also suitable
for diarrhea, gonorrhea and kidney stone cure, among other
applications [4–6].
The current techniques to obtain plant extracts, such as
steam distillation and organic solvent extraction, usually re-
quire several hours or even days, spending large solvent

0896-8446/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2004.07.004
132 E.M.Z. Michielin et al. / J. of Supercritical Fluids 33 (2005) 131–138

volumes that must be recovered. Besides difficulties in the

extraction step, the solute/solvent separation may result in
degradation of the thermolabile components and the product
may present traces of the solvent used, reducing its quality
[7–10].
Therefore, due to the so called inconveniences of the or-
ganic solvent processes, the objective of this work was to
evaluate the SFE of horsetail (E. giganteum L.) oleoresin at
different operational conditions and discuss the temperature,
pressure and time dependence in the extract composition pro-
file. The oleoresin composition profile and the process yield
were compared to organic solvent extractions performed with
n-hexane and dichloromethane (DCM).

Fig. 1. SFE unit: CO2 reservoir; S (surge tank); P (pump); E (extractor); TC

(thermostatic baths); MV (micrometering valve); C (sample collector); BL
2. Material and methods (wet test meter), PI1, PI2 and PI3 (pressure).

2.1. Plant material

Dried horsetail aerial parts purchased from Chamel Ind.
e Com. (Campo Largo, PR, Brazil) were stored at 263 K
in a domestic freezer. The plant authenticity was evaluated
and the voucher specimen (E. giganteum L.) was deposited
at the HASU herbarium under no. 15378 (UNISINOS, São
Leopoldo, RS, Brazil). The plant material was grounded im-
mediately before the extraction with supercritical CO2 . The
fixed bed of particles was formed by grounded horsetail aerial
parts, obtained using a coffee grinder (Melitta, SP, Brazil).
The particle size was classified with sieves and the interme-
diary fraction (mesh −20 + 32 and mean particle diameter
0.92 mm) was used to settle the bed of E. giganteum L. inside
the extractor. The fixed bed was formed with 40.0 ± 0.5 ×
10−3 kg of triturated horsetail, placed slowly inside the ex-
tractor to obtain a uniform bed and avoid wall effects and
channeling. After the fixed bed formation, the extractor was
connected downstream the pump and the experiments were
conducted using a static period of 3 h. This period consist of a
contact between solvent and solid matrix, before the effective
extraction, in order to increase the solute solubilization.
2.2. Supercritical fluid extraction (SFE)
A schematic design of the SFE unit used in this work is
shown in Fig. 1. The process used CO2 99.9% pure, deliv-
ered at pressure up to 6 MPa (White Martins, Brazil). The
extraction line, formed by a 0.2 L stainless steel surge tank
(Suprilab, SP, Brazil), was connected to the pump (Thermo
Separation Products, Model 3200P/F, CA, USA). The ex-
traction pressure was monitored in an analogical manome-
ter (Header, 400 ± 2.5 bar). The surge tank was pressurized
and the temperature was controlled at 276 K by a thermo-
static bath (Microquı́mica, Model MQBTZ99-20, SC, Brazil,
±0.1 ◦ C) to guarantee liquid CO2 at the pump head suction.
The solvent flows through a temperature-controlled coil (2 m,
1/8 stainless steel tube) to establish the operational tem-
perature and follows to the extractor, a jacket stainless steel
column (40 cm length × 2.1 cm inner diameter – Suprilab,
SP, Brazil), where the fixed bed (grounded horsetail parti-
cles) was formed. Downstream the extractor a micrometering
valve (MV) (Autoclave Engineers, Model 30VRMM4812,
PA, USA) was connected for the solvent expansion. The so-
lute (horsetail oleoresin) was collected in amber flasks placed
at the end of the process line and connected to a calibrated
wet test meter (LATESC/EQA-UFSC) where the flow rate
was monitored and the amount of CO2 measured. Samples
were collected at pre-established time intervals, up to ap-
proximately 5 h process and weighed in an analytical balance
(OHAUS, Model AS200S ±0.0001 g, USA). The overall ex-
traction curves were obtained with mass of solute versus ex-
traction time and the experiments were carried out at 303 and
313 K and from 12 to 30 MPa.
2.3. Organic solvent extraction
The conventional method, used to obtain the horsetail ole-
oresin, consists in a cold maceration of the plant, to avoid ther-
mal degradation. The extraction was performed with dried
plant powder (0.33 kg), placed in methanol (3.5 L) for 5 days.
The resulting extract was evaporated at reduced pressure, up
to 10–20% of the initial volume, to obtain the crude extract
(CE). The CE was then partionated with n-hexane or DCM
(0.45 L for each organic solvent) to allow the separation of
the extract components according to their polarity.
2.4. Extract components identification
The main components of the horsetail oleoresin were sepa-
rated and quantified by gas chromatography. A gas chromato-
graph (Shimadzu, Model CGS-14A, Kyoto, Japan) equipped
with a FID detector was used to analyze the extracts. The sam-
ples investigated were obtained from SFE at different oper-
ational conditions (solvent density) and different extraction
time. Horsetail oleoresins produced by conventional meth-
ods, with n-hexane and DCM, were also analyzed.
 E.M.Z. Michielin et al. / J. of Supercritical Fluids 33 (2005) 131–138
The flow rate for the carrier gas (hydrogen) and the make-
up gas (nitrogen) was 2 mL/min, chromatograms were run
with on-column 1 ␮L sample injection diluted with DCM
at 1 mL. The GC analysis was performed using split-mode
injection with split rate at 1/20.
For the chromatographic analysis a non-polar LM1 col-
umn with 0.25 mm inner diameter and 0.3 ␮m film thickness
was used. The temperature program (based on the compo-
nents retention time) started at 313 K with 6 K/min heating
rate up to 443 K, followed by 8 K/min up to 575 K. The de-
tector and the injector temperatures were 593 and 553 K, re-
spectively.
The area of the chromatogram peaks, correspondent to
the sample components were integrated using the program
Cromatografia (Microquı́mica, Brazil). The main compo-
nents of the horsetail oleoresin were identified by gas
chromatography–mass spectrometry (GC–MS) investigation
(Shimadzu, Model GCMS-QP2000A, Kyoto, Japan) and us-
ing a database for natural products (NIST Mass-Spectral Li-
brary with Windows search program – Version 2), where MS
results were compared. The temperature program used in the
GC–MS analysis was the same for the GC analysis.
3. Results and discussion
3.1. Overall extraction curve
Typical overall extraction curves (OEC) that represent the
supercritical fluid extraction of horsetail oleoresin are shown
in Fig. 2. The curves were obtained from extractions at 303
and 313 K, pressures from 12 to 25 MPa and flow rate from
3.88 × 10−5 to 7.65 × 10−5 kg CO2 /s. The OEC in Fig. 2
show the constant extraction rate period (initial portion of
the curves) and the decreasing extraction rate period, repre-
senting the influence of the mass transfer mechanisms. In the
beginning of the curves (up to 70 min extraction) the convec-
tion is the dominant mass transfer mechanism, while in the
end the diffusion mechanism controls the mass transfer. The
Fig. 2. Temperature and pressure effects in the OEC: (
) 3.88 × 10−5 kg/s and ρ CO2 923.0 kg/m3 , () 3.93 × 10−5 kg/s and ρ CO2 880.2 kg/m3 , () 6.18
× 10−5 kg/s and ρ CO2 891.2 kg/m3 , (×) 6.85 × 10−5 kg/s and ρ CO2 840.6 kg/m3 , ( ) 6.5 × 10−5 kg/s and ρ CO2 847.6 kg/m3 , (•) 7.55 × 10−5 kg/s and ρ
CO2 781.2 kg/m3 , (+) 7.65 × 10−5 kg/s and ρ CO2 809.7 kg/m3 , (−) 6.53 × 10−5 kg/s and ρ CO2 719.1 kg/m3 .
133
Fig. 3. Effect of the CO2 flow rate in the OEC at 20 MPa and 313 K.
curves shown in Fig. 2 indicate an increase in the constant ex-
traction rate with increasing pressure at constant temperature.
The effect of the CO2 flow rate in the OEC is observed in
Fig. 3 for 20 MPa and 313 K and flow rates 1.85 × 10−5 and
4.73 × 10−5 kg CO2 /s. The curves show an increase in the
extraction rate with increasing the solvent flow rate. At 1.85
× 10−5 kg CO2 /s the extraction yield obtained at 720 min
process was 0.75% (w/w), otherwise, using 4.73 × 10−5 kg
CO2 /s the same extraction yield was obtained at 288 min.
Reverchon et al. [12] observed similar effect for the extraction
of hiprose seed oil with supercritical CO2 , reducing 50% of
the extraction time due to an increase in the flow rate from 1
to 2 gCO2 /min.
Important process characteristics such as solubility and
selectivity, as well as process yield, are pressure- and
temperature-dependent, i.e., solvent density-dependent. The
values of supercritical CO2 density ρ (kg/m3 ), for the range
of temperature and pressure used in this work, are presented
in Table 1 [11]. The extraction yields obtained with supercrit-
ical CO2 at different operating conditions (303 and 313 K and
12–30 MPa) and with n-hexane and dichloromethane (DCM),
for the organic solvent extraction, are compared in Table 1.
The highest yield was obtained with SFE at 313 K and 30 MPa
(1.44%, w/w). The yield obtained with hexane extraction was
compared, in terms of order of magnitude, with SFE at densi-
ties up to 810 kgCO2 /m3 . Otherwise, the yield obtained with
134 E.M.Z. Michielin et al. / J. of Supercritical Fluids 33 (2005) 131–138

Table 1 in CO2 , where the crossover was at 30 MPa for the isotherms
Yield obtained in different extraction processes and CO2 properties 313 and 333 K and at 35 MPa for temperatures from 333 to
Organic solvent extraction Supercritical fluid extraction 353 K. For ␤-carotene, the crossover pressure lays between
Solvent Yield (%, w/w) MPa/K ρCO2 (kg/m3 ) Yield (%) 15 and 17.5 MPa for the temperature range of 313–333 K.
Hexane 0.31 12/303 809.7 0.35
Vasapoloo et al. [14] observed the same behavior for the ex-
DCM 0.78 12/313 719.1 0.34 traction of lycopene from tomato with supercritical CO2 and
15/303 847.6 0.37 reported an increase in the extraction yield with temperature,
15/313 781.2 0.39 from 45 to 60 ◦ C, at constant pressure.
17/303 867.1 0.47 Moreover, the enhancement in the extraction yield indi-
17/313 808.7 0.51
20/303 891.2 0.76
cates the decrease in the process selectivity for complex mix-
20/313 840.6 0.81 tures, observed through the composition profile of the oleo-
25/303 923.0 0.91 resin (next section).
25/313 880.2 0.94
30/313 910.6 1.44 3.2. Horsetail extract composition

DCM, a low polarity solvent, is comparable with the one The horsetail oleoresin is a greenish viscous product,
obtained with SC-CO2 at 20 MPa and 303 K. formed basically by high molecular weight compounds, from
The results for the supercritical extraction yields are com- around 160–430 kg/kg mol. Results of the GC–MS analy-
pared in Fig. 4 through the yield isotherms. The results indi- sis from horsetail extracts obtained with supercritical fluid
cate an increase in the process yield with pressure at constant extraction and with n-hexane extraction are presented in
temperature for 303 and 313 K. This behavior is justified by Fig. 5a and b, respectively. The high-pressure extract (Fig. 5a)
the increase in the solvent density with pressure. was obtained at 313 K and 20 MPa (840.6 kg/m3 ) and col-
The effect of temperature on the extraction rate, at con- lected in the first portion of the OEC (up to 100 min ex-
stant pressure, is due to two mechanisms: an increase in the traction time). Table 2 shows the identified components of
process temperature increases the solubility due to solute va- the horsetail oleoresin, with the respective retention time in
por pressure enhancement and reduces the solubility due to the chromatographic column and the compounds molecular
the decrease in the solvent density. These opposing effects weight and formula. The substances listed in Table 2 indi-
result in the crossover of solubility isotherms [14]. cate the presence of a series of components with non-polar
In Fig. 4, the horsetail oleoresin yield decreases with aspects, a SC-CO2 extraction characteristic. Also, from the
temperature at 12 MPa but increases for pressures above
15 MPa and up to 25 MPa. The isotherms show an inver-
sion pattern between 12 and 15 MPa, possibly representing
the crossover region. Therefore, according to the results pre-
sented in Table 1 and Fig. 4, the density effect in the oleoresin
solubilization is dominant at 12 MPa. Otherwise, the vapor
pressure is the main effect in the solubilization process from
15 to 25 MPa, for the range of temperatures and pressures
studied in this work.
Güçlü-Üstündag and Temelli [13] reported several sys-
tems with isotherm inversion, such as tocopherol solubility

Fig. 5. (a) GC–MS analysis for horsetail oleoresin obtained with supercrit-
Fig. 4. Pressure effect in the process yield at constant temperature: yield ical CO2 at 20 MPa, 313 K and 5.45 × 10−5 kg CO2 /s. (b) GC–MS analysis
isotherms at 303 and 323 K. for horsetail oleoresin obtained with n-hexane.
 E.M.Z. Michielin et al. / J. of Supercritical Fluids 33 (2005) 131–138 135

Table 2
Components identified in the horsetail oleoresin
Peak Compound RT (min) Mol. (kg/kgmol) Molecular formula
1 Dodecanoic acid 26.36 200 C12 H24 O2
2 3-Nonynoic acid methyl ester 28.73 168 C10 H16 O2
3 3,6-Dimethyl decane 38.06 170 C12 H26
4 n-Heneicosane 40.26 296 C21 H44
5 26-Hydroxicholesterol 41.56 402 C27 H46 O2
6 Ergosta-4,7,22-trien-3-one 42.60 394 C28 H42 O
7 8,12-Dimethyl-4Z,8E,12E-octadecatriene 43.93 276 C20 H36
8 Methenolone 45.26 302 C20 H30 O2
9 Gorgost-5-en-3-ol 46.13 426 C30 H50 O
10 2,6,10,14-Hexadecatetraen-1-ol,3,7,11,15-tetramethyl-acetat (E,E,E) 46.36 332 C22 H36 O2
11 Z-13-Octadecenal 47.90 266 C18 H34 O
12 Bufa-20,22-dienolide,3,14-dihydroxy 52.93 386 C24 H34 O4

chromatograms in Fig. 5a and b and the results in Table 2, for
a set of components analyzed in this work, the CO2 extraction
present lower selectivity for non-polar components related to
the n-hexane extraction. Among the identified components
were found alkanes like n-heneicosane, a cuticular hydro-
carbon; fatty acids like dodecanoic acid, a glyceride; methyl
esters such as 3-nonynoic acid methyl ester; steroid triter-
penes such as ergosta-4,7,22-trien-3-one and gorgost-5-en-3-
ol; a naturally-occurring anabolic steroids like methenolone
[15,16].
The influence of the process temperature and pressure on
the solute composition is presented in Table 3. The results are
shown in molar fraction and % peak area for samples obtained
in the initial part of the OEC (up to 100 min) and at different
extracting conditions. The molar fraction values of the horse-
tail components were obtained considering only the identified
substances present at the oleoresin. Table 3 shows an increase
in the number of identified components with temperature and
pressure, indicating a more complex composition profile for
the extract obtained at 313 K and 20 MPa. The results listed
in Table 3 are also evaluated though Fig. 6 shows the be-
havior of components 1, 2, 3, 6 and 8 with temperature and
pressure. Dodecanoic acid (compound 1) was not detected at
12 MPa and its concentration increases with temperature for
20 MPa. For the aliphatic hydrocarbon 3,6-dimethyl decane
(compound 3) the solute vapor pressure effect is also observed
by the concentration decrease with the solvent density and in-
creases with temperature for 12 and 20 MPa. The behavior of
the extraction rate for the steroids ergosta-4,7,22-trien-3-one
(compound 6) and methenolone (compound 8) represent the
complexity of the solvent density effect in the extract com-
position. For methenolone the concentration increases with
temperature at 12 MPa and decreases at 20 MPa. Otherwise,
increasing temperature, the concentration of ergosta-4,7,22-
trien-3-one decreases at 12 MPa and increases at 20 MPa.
This complex behavior can be explained by the intermolecu-
lar interactions and the vapor pressure of the oleoresin com-
ponents.
The effect of the extraction time in the solute composi-
tion was also investigated and the results are presented in
Table 4 in terms of components molar fraction. The mo-
lar fraction values were again calculated considering only
the identified components of the horsetail oleoresin. The re-
sults in Table 4 represent samples obtained at 20 MPa, 313 K
and 5.33 × 10−5 kg CO2 /s, and at three different parts of
the OEC, the beginning (up to 100 min extraction time), the

Table 3
Effect of the CO2 density in the horsetail oleoresin composition (molar fraction and % area of the peaks)
Peak Compound Molar fraction: T (K)/P (MPa) % Peak area: T (K)/P (MPa)

313/12 303/12 313/20 303/20 313/12 303/12 313/20 303/20

1 Dodecanoic acid – – 0.2077 0.0953 – – 14.917 6.163
2 3-Nonynoic acid methyl ester – – 0.2893 0.0118 – – 24.717 0.917
3 3,6-Dimethyl decane 0.0625 0.0688 0.0389 0.0164 5.943 4.206 3.288 1.253
4 n-Heneicosane 0.1632 0.1551 0.0927 0.1353 8.225 5.451 4.492 5.910
5 26-Hydroxicholesterol 0.0405 – 0.0276 0.0164 1.505 – 0.988 0.527
6 Ergosta-4,7,22-trien-3-one 0.1322 0.2029 0.0634 0.0302 5.008 5.357 2.308 0.989
7 8,12-Dimethyl-4Z,8E,12E-octadecatriene 0.0652 – 0.0267 0.0249 3.524 – 1.390 1.175
8 Methenolone 0.4838 0.4671 0.1859 0.5295 23.895 16.099 8.840 22.661
9 Gorgost-5-en-3-ol – – 0.0133 – – – 0.446 –
10 2,6,10,14-Hexadecatetraen-1-ol,3,7,11,15- 0.0274 0.038 0.0148 0.0622 1.233 1.196 0.639 2.422
tetramethyl-acetato (E,E,E)
11 Z-13-Octadecenal 0.0252 0.0264 0.0158 0.0261 1.406 1.032 0.845 1.264
12 Bufa-20,22-dienolide,3,14-dihydroxy – 0.0417 0.0239 0.0518 – 1.131 0.892 1.737
NI Non-identified components – – – – 49.261 65.528 36.238 54.982
136 E.M.Z. Michielin et al. / J. of Supercritical Fluids 33 (2005) 131–138

Fig. 6. Effect of solvent density in the oleoresin composition (1) dodecanoic acid; (3) 3,6-dimethyl decane; (6) ergosta-4,7,22-trien-3-one; (8) methenolone.

Table 4
Effect of the extraction time in the oleoresin composition
Peak Compound Molar fraction

0–100 (min) 100–270 (min) 270–380 (min)

1 Dodecanoic acid 0.2077 0.1543 0.2174
2 3-Nonynoic acid methyl ester 0.2893 0.0162 0.0916
3 3,6-Dimethyl decane 0.0389 0.0486 0.0443
4 n-Heneicosane 0.0927 0.0795 0.0704
5 26-Hydroxicholesterol 0.0276 0.0362 0.0318
6 Ergosta-4,7,22-trien-3-one 0.0634 0.1432 0.1048
7 8,12-Dimethyl-4Z,8E,12E-octadecatriene 0.0267 0.0902 0.0643
8 Methenolone 0.1859 0.3476 0.2766
9 Gorgost-5-en-3-ol 0.0133 0.0196 0.0254
10 2,6,10,14-Hexadecatetraen-1-ol,3,7,11,15-tetramethyl-acetato (E,E,E) 0.0148 0.0194 0.0229
11 Z-13-Octadecenal 0.0158 0.0184 0.0142
12 Bufa-20,22-dienolide,3,14-dihydroxy 0.0239 0.0268 0.0363
Composition expressed in molar fraction for samples obtained at 313 K and 20 MPa.

intermediate fraction (from 100 to 270 min) and the final por-
tion of the OEC (from 270 to 380 min). Fig. 7 shows the be-
havior of the molar fraction of the components (1) dodecanoic
acid, (2) 3-nonynoic acid methyl ester, (4) n-heneicosane, (8)
methenolone and (12) bufa-20,22-dienolide,3,14-dihydroxy
with the extraction time. For components (1) and (2) the com-
position decreases from the beginning of the extraction to
the intermediate fraction and increases at the final portion of
the OEC. Component 4 decreases with extraction time while
component 12 increases, although its concentration is low
and practically constant. Component 8 has opposite extrac-
tion behavior compared to component 1, i.e., its concentra-
tion increases up to 270 min then decreases, probably due
to its exhaustion or the synergetic effect of the remaining
components of the oleoresin. Therefore, for the operational
conditions studied in this work, the intermediate fraction of

Fig. 7. Effect of the extraction time in the horsetail oleoresin composition: (1) dodecanoic acid; (2) 3-nonynoic acid methyl ester; (4) n-heneicosane; (8)
methenolone; (12) bufa-20,22-dienolide,3,14-dihydroxy.
 E.M.Z. Michielin et al. / J. of Supercritical Fluids 33 (2005) 131–138 137

Table 5
Comparing results from SFE and organic solvent extractions (hexane, dichloromethane) (% peak area)
Compounds RT (min)a % Peak area
SFE (MPa, K) n-Hexane DCM

20,313 12,303
2-Nonadecanone 19.76 – – 9.35 8.86
Heptadecanoic acid 21.43 – – 37.56 39.92
12,15-Octadecadienoic acid, methyl ester 23.33 – – 5.31 3.08
Dodecanoic acid 26.36 14.917 – – –
3-Nonynoic acid methyl ester 28.73 24.717 – – –
Ergosta-7,22-dien-3-ol 33.96 – – 6.67 8.44
Stigmasta-4,22-diene 34.96 – – 11.23 12.75
3,6-Dimethyl decane 38.06 3.288 4.206 – –
Cholest-7-en-3-ol,14-methyl 38.43 – – 5.84 4.73
n-Heneicosane 40.26 4.492 5.451 – –
26-Hydroxicholesterol 41.56 0.988 – – –
Ergosta-4,7,22-trien-3-one 42.60 2.308 5.357 – –
8,12-Dimethyl-4Z,8E,12E-octadecatriene 43.93 1.390 – – –
Methenolone 45.26 8.840 16.099 – –
Gorgost-5-en-3-ol 46.13 0.446 – – –
2,6,10,14-Hexadecatetraen-1-ol,3,7,11,15-tetramethyl-acetato (E,E,E) 46.36 0.639 1.196 – –
Z-13-Octadecenal 47.90 0.845 1.032 – –
Bufa-20,22-dienolide,3,14-dihydroxy 52.93 0.892 1.131 – –
Non-identified components – 36.238 65.528 24.04 22.22
a RT: retention time.

the OEC (from 100 to 270 min) presented the highest concen-
tration of target components such as steroid triterpenes (peaks
5, 6, 8) and also triterpene hydrocarbons (peaks 3 and 7).
The influence of the extraction process on the oleoresin
composition was investigated and the results are compared
in Table 5, in terms of % peak area, for extracts obtained
with SC-CO2 (313 K/20 MPa and 303 K/12 MPa), n-hexane
and DCM.
The results indicate a remarkable difference in the com-
position profile of the horsetail oleoresin between the super-
critical extracts and the conventional ones (organic solvents).
Considering the characteristic of the analytical method used
in this work (gas chromatography), the conventional oleo-
resin show mainly components with retention time lower than
38 min and the CO2 extracts were rich in components with
retention time from 26 to 53 min. Besides the difference in the
extract yield using n-hexane and DCM (Table 1), the com-
position profile of both extracts was comparable in terms
of identified components (Table 5). Those extracts, although
present different components than the SC extracts, indicate
the same class of components: alkanes, fatty acids, methyl
esters and steroids triterpenes, present in all samples evalu-
ated. This behavior is caused mostly by the differences at the
solvents polarity and affinity to the solute.
4. Conclusions
0.78% (w/w). The pressure effect in the SFE yield was also
observed and the results indicate that, increasing pressure
from 12 to 30 MPa at constant temperature, the extraction
yield increases. The influence of the extraction temperature
indicates an increase in the extraction rate by increasing tem-
perature at 12 MPa and decreases the extraction rate with
temperature from 15 to 25 MPa. This behavior shows the
isotherms crossing between 12 and 15 MPa. It was also ob-
served the influence of CO2 density and extraction time in the
solute composition profile. The components polarity in the
high-pressure extracts increased with the extraction time. The
CO2 extracts shown the presence of important components
such as methenolone, phyto-steroids (ergosta-4,7,22-trien-3-
one) and hidrocarbons (3,6-dimethyl decane; n-heneicosane
and 8,12-dimethyl-4Z,8E,12E-octadecatriene), mostly ab-
sent in the conventional oleoresin. The study of horsetail
extraction using supercritical CO2 confirms the ability of the
high-pressure process to obtain complex mixtures formed ba-
sically by high molecular weight components (426 g/mol).
Acknowledgements
The authors wish to acknowledge CAPES and CNPq for
the financial support and Professor Paulo G. Windish from
UNISINOS, São Leopoldo, RS, Brazil, for the plant identifi-
cation.

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