Industrial Crops and Products 36 (2012) 401–404

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Industrial Crops and Products

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Fatty acid composition and oxidation stability of hemp (Cannabis sativa L.)
seed oil extracted by supercritical carbon dioxide
C. Da Porto ∗ , D. Decorti, F. Tubaro
Department of Food Science, University of Udine, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Supercritical carbon dioxide (SC-CO2 ) was employed to extract oil from hemp (Cannabis sativa L.) seeds.
Received 1 July 2011 For ground seeds, the supercritical extraction was carried out at temperatures of 40, 60 and 80 ◦ C and
Received in revised form pressures of 300 and 400 bar. Different solvent-ratios were applied. Supercritical CO2 extractions were
15 September 2011
compared with a conventional technique, n-hexane in Soxhlet. The extraction yields, fatty acid compo-
Accepted 25 September 2011
sition of the oil and oxidation stability were determined. The seed samples used in this work contained
Available online 29 November 2011
81% PUFAs, of which 59.6% was linoleic acid (␻-6), 3.4% ␥-linolenic (␻-3), and 18% ␣-linolenic (␻-6). The
highest oil yield from seeds was 22%, corresponding to 72% recovery, at 300 bar and 40 ◦ C and at 400 bar
Keywords:
Supercritical CO2
and 80 ◦ C. The highest oxidation stability corresponding to 2.16 mM Eq Vit E was obtained at 300 bar and
Cannabis sativa L. 80 ◦ C.
Hempseed oil © 2011 Elsevier B.V. All rights reserved.
FAME analysis
PUFA’s
Oxidation stability

1. Introduction Within hemp seed oil, ␥-tocopherol is present in significantly

Hemp cultivation (Cannabis sativa L.) of cultivar Felina charac-
terized by a low concentration of ␦-9-tetrahydrocannabinol (≤0.2%
THC) (Lercker et al., 1992) as recommended by the Regulation
EC no. 1124/2008 (12 November 2008) has been reintroduced in
Friuli-Venezia Giulia region (North East Italy) both for fibre and oil
applications.
Hemp seed oil contains linoleic acid (LA, 18:2 n-6) and ␣-
linolenic acid (ALA, 18:3 n-3) as its major omega-6 and omega-3
polyunsaturated fatty acids (PUFA), respectively. Linolenic and
linoleic fatty acids are known as essential fatty acids because
humans cannot produce them themselves, and must obtain them
in their diet. These fatty acids comprise the most desirable con-
tents of the oil, especially due to the ratios in which they exist. The
3:1 ratio of LA to ALA is alleged to be optimal for nutrition (Deferne
and Pate, 1996; Calloway and Laakkonen, 1996) the lowering of LDL
cholesterol and high blood pressure for cardiovascular disease pre-
vention (Jones, 1995). The additional presence of ␥-linolenic acid
(GLA, 18:3 n-3) in hemp seed oil ultimately makes its also ideal as
an ingredient for cosmetics (light body oils, lipid-enriched creams,
etc.) (Rausch, 1995).
∗ Corresponding author. Fax: +39 0432 558120.
E-mail address: carla.daporto@uniud.it (C. Da Porto).
higher quantities than ␣-tocopherol; the Fedora cultivar resulted
to contain about 0.5 g/L of ␥-tocopherol with only trace amounts
of ␣-tocopherol (Jones, 1995). Antioxidant property of cold-pressed
hemp seed oil has been investigated and its effectiveness in inhibit-
ing free radicals resulted higher than olive oil (extra-virgin) (Yu
et al., 2005; Ramadan and Moersel, 2006). Antioxidants are well rec-
ognized for their potential in health promotion and prevention of
aging-related diseases, including cancer and heart disease (Cadenas
and Davies, 2000). The versatility of the hempseed lends itself to the
development of numerous products for food, cosmetic, therapeutic,
functional food and nutraceutical industries.
Extraction of oil from hemp seed is usually carried out by cold-
pressing methods or employing organic solvents. Approximately
35% of the available oil remains in the seed cake using the pressing
process and best quality oil is obtained only from the first fractions
recovered.
Supercritical carbon dioxide fluid extraction (SC-CO2 ) has
become an important separation technique in the field of food and
nutraceutical applications (Ravento et al., 2002; Corrêa et al., 2008).
With this technology it is possible to extract heat sensitive, eas-
ily oxidized compounds such as PUFAs, and to avoid using any
toxic solvent like n-hexane, leaving residues in the product, since
CO2 is non-toxic, non-flammable, inexpensive, separable from the
extracted solutes and reaches supercritical conditions easily (32 ◦ C
and 74 bar). Supercritical carbon dioxide (SC-CO2 ) has been estab-

0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2011.09.015
402 C. Da Porto et al. / Industrial Crops and Products 36 (2012) 401–404
Table 1
Supercritical CO2 experimental conditions tested for hempseed oil extraction.
Sample T (◦ C) P (bar) Flow rate (kg/h) QCO2 (kg CO2 /kg feed)
A 40 300 10 45
B 60 300 10 60
C 80 300 10 30
D 40 400 10 60
E 60 400 10 30
F 80 400 10 45
lished as a good alternative solvent for lipid extraction, such as in
the extraction of PUFAs from fish lipids (Corrêa et al., 2008; Rubio-
Rodríguez et al., 2008) and the extraction of vegetable oils (Gomez
et al., 1996; Nodar et al., 2002; Westerman et al., 2005; Özkal et al.,
2005). Despite the extensive works carried out on supercritical CO2
extraction of oil from various seeds, the information on the SC-
CO2 extraction of oil from hemp (C. sativa L.) seed carried out by
a pilot scale apparatus is limited. The objectives of this work was
to establish a preliminary set of supercritical CO2 extraction condi-
tions (pressure, temperature, and flow rate) to obtain high quality
hempseed oil. The quality of the oil extracted used SC-CO2 in terms
of its fatty acid composition oxidation stability, was compared with
that obtained using n-hexane.
2. Materials and methods
2.1. Characterization of the seeds
Hemp (C. sativa L.) seed Felina cultivar, grown in Prato Car-
nico (Udine) in 2010, was obtained from experimental cultivation.
Seed samples were natural dried in a cool, windy and dry location.
Oilseeds were finely ground in a grinder and subsequently garbled
to 1.50 mm particle size (Dp). The extraction process (8 h at 70 ◦ C)
of oil from ground hempseed samples were performed in a Soxhlet
extractor using 30 g of seed and 240 mL of n-hexane followed by
solvent removal under vacuum at 60 ◦ C.
2.2. SC-CO2 apparatus and procedure
The experiments were carried out using a high pressure pilot
plant (SCF 100 serie 2MP PLC, SEPARECO, Italy) equipped with 1 L
volume extractor vessel (E1) and two separators, the first one is a
gravimetric separator (S1), the second one is an high performance
cyclonic separator (S2). The high pressure vessel contains an extrac-
tion basket of 700 mL, closed with porous stainless steel disks. The
CO2 is pressurized by a high pressure diaphragm pump (400 bar)
with jacketed heads for cooling. Flow rate can be regulated between
2 and 10 kg/h. The flow sheet of this plant, which encloses also the
fractionation column not used in the experiments, is given in Fig. 1.
About 3 × 10−1 kg of ground and garbled seeds (d = 0.44 g/mL)
were loaded into the extraction vessel for each experimental
extraction. Flow rate was regulated at 10 kg/h. After extraction,
the samples were centrifuged and the oil fraction separated. The
operating conditions of each experiment are reported in Table 1.
2.3. GC analysis of fatty acids
The fatty acid methyl esters (FAME) were prepared by trans-
esterification of oil with 2 N KOH in methanol and n-hexane. Gas
chromatographic (GC) analysis of FAME were performed in a Var-
ian 3400 gas chromatograph equipped with a SP-2380 fused-silica
column (Supelco, Bellafonte, PA) (30 m × 0.32 mm i.d., film thick-
ness 0.20 ␮m), a split injector at 250 ◦ C; flame ionization detector
at 260 ◦ C. Helium was used as carrier gas and the split ratio was used
1:50. The programmed temperature was: 2 min at 50 ◦ C, 50–250 ◦ C
at 4 ◦ C/min. The identification of FAME was based on external
standards using commercial reference compounds (Sigma). Each
FAME sample was analysed three times.
2.4. Oxidation stability assay
The oxidation stability of hempseed oil samples was evaluated
by crocin kinetic competition test as described by Tubaro et al.
(1996). The results were expressed as equivalent millimolar con-
centration of Trolox.
2.5. Statistical analysis
Each sample was analysed in triplicate. The tests for statistical
significance of difference between treatments for volatile com-
pounds data was performed by analysis of variance (ANOVA) at
significance level of ˛ = 0.05. The effect of treatments on means
of samples between treatments were compared using Tukey’s test
(˛ = 0.05).
3. Results and discussion
Moisture content of the hempseed sample was 7.8 ± 0.2%.
Oil content of the seeds determined by Soxhlet extraction was
30.6 ± 0.04% lower than the values (≈36%) reported for cultivar
FIN-314 grown in Finland (Calloway and Laakkonen, 1996).
The effects of pressure and temperature on the SC-CO2 extrac-
tion of hempseeds were investigated at pressures of 300 and
400 bar and temperatures of 40, 60 and 80 ◦ C. The SC-CO2 flow rate
was maintained constant at 10 kg/h and the mean particle size was
1.50 mm. Table 2 shows the fatty acid composition (%) obtained by
the supercritical extractions and the conventional Soxhlet extrac-
tion with n-hexane. No significant differences were found when the
hempseed oil was extracted by n-hexane in Soxhlet or by SC-CO2
under different experimental conditions were analysed by FAME
analysis. The main components were linoleic, ␣-linolenic, and oleic
acid (88.63% for the oil extracted by n-hexane and a mean value of
88.76% for the oil extracted under different SC-CO2 operating con-
ditions), followed by the palmitic acid (5.37% for the oil extracted
by n-hexane and a mean value of 5.17% for the oil extracted under
different SC-CO2 operating conditions), and ␥-linolenic acid (3.48%
for the oil extracted by n-hexane and a mean value of 3.47% for
the oil extracted under different SC-CO2 operating conditions). This
composition agrees with results reported by Oomah et al. (2002)
for oil extracted from the hempseeds cultivar Fasamo subjected
to microwave treatments. The linoleic acid content of hampseed
oil was similar to that of soybean and maize oils (54–59%). The
stearic, oleic, and linoleic acid contents of hempseed oil, compris-
ing about 72% of total fatty acids, resembled those of cotton-seed
oil (Van Niekerk and Burger, 1985). The polyunsaturated fatty acids
(PUFA) of the oil amounted to 81% of total fatty acids, while the
monounsaturated and saturated fatty acids amounted to 11.5 and
7.5%, respectively. Hence, the ratio of polyunsaturated to monoun-
saturated to saturated was 81:11:7. The oil was characterized by
high polyunsaturated/saturated (P/S) ratio of 10.5. A high ratio of
P/S is regarded favorably for the reduction of serum cholesterol
and arteriosclerosis and prevention of heart diseases (Rudel et al.,
1998). Similarly, the ratio of ␻-6 to ␻-3 ratio fatty acids was 3.3,
much higher than most vegetable oils.
Table 3 shows the results of the supercritical carbon dioxide
extractions and the conventional Soxhlet extraction as regard oil
yield (% w/w), recovery (% total oil) and oxidation stability (mM
Eq Vit E). The highest oil yield was 22%, which corresponds to 72%
recovery. It was obtained at 300 bar and 40 ◦ C and at 400 bar and
80 ◦ C with a solvent-ratio of 40 kg CO2 per kg of feed. Similar result
has been found by Nodar et al. (2002) for the oil contained in soy-
bean seeds at 300 bar and 40 ◦ C. The highest oxidation stability,
 C. Da Porto et al. / Industrial Crops and Products 36 (2012) 401–404 403

Fig. 1. Flowsheet of the pilot scale apparatus (SCF 100 serie 2MP PLC, SEPARECO, Italy). B1: storage tank; E1: extraction vessel; S#: separators; H#: heat exchangers; C1:
condenser; HV#: hand valves; MV1: membrane valve; NRV#: no return valves; P: diaphragm pumps; F2: flow meter; T: fractionation tower; M#: manometers; k: safety
devices; FL1: Coriolis mass flow meter; L: liquid storage tank.

Table 2
Fatty acid composition of hempseed oil, cultivar Felina, extracted under different SC-CO2 experimental conditions (A: 40 ◦ C, 300 bar, 45 kg CO2 /kg feed; B: 60 ◦ C, 300 bar,
60 kg CO2 /kg feed; C: 80 ◦ C, 300 bar, 30 kg CO2 /kg feed; D: 40 ◦ C, 400 bar, 60 kg CO2 /kg feed; E: 60 ◦ C, 400 bar, 30 kg CO2 /kg feed; F: 80 ◦ C, 400 bar, 45 kg CO2 /kg feed) and by
Soxhlet extraction with n-hexane.

SC-CO2 extraction Soxhlet

A B C D E F

Fatty acid composition (%)

Palmitic acid (C16:0) 5.19 ± 0.03a 5.13 ± 0.04 5.36 ± 0.03 5.23 ± 0.05 5.07 ± 0.05 5.05 ± 0.03 5.37 ± 0.13
Stearic acid (C18:0) 1.57 ± 0.03 1.56 ± 0.01 1.45 ± 0.05 1.74 ± 0.04 1.75 ± 0.01 1.70 ± 0.04 1.56 ± 0.05
Oleic acid (C18:l) 10.99 ± 0.35 11.30 ± 0.20 11.31 ± 0.26 11.11 ± 0.38 11.33 ± 0.25 11.46 ± 0.23 11.51 ± 1.05
Linoleic acid (C18:2␻6) 59.77 ± 0.74 59.23 ± 0.81 59.49 ± 0.75 59.30 ± 0.73 59.33 ± 0.63 59.45 ± 0.53 59.16 ± 0.85
␥-Linolenic acid (C18:3␻6) 3.42 ± 0.16 3.58 ± 0.14 3.46 ± 0.12 3.50 ± 0.18 3.49 ± 0.21 3.39 ± 0.19 3.48 ± 0.15
␣-Linolenic acid (C18:3␻3) 18.15 ± 0.31 18.20 ± 0.31 17.95 ± 0.35 18.12 ± 0.41 18.04 ± 0.37 18.01 ± 0.43 17.96 ± 0.23
Eicosenoic acid (C20:l) 0.78 ± 0.03 0.81 ± 0.01 0.81 ± 0.04 0.79 ± 0.04 0.79 ± 0.05 0.78 ± 0.07 0.80 ± 0.01
Behenic acid (C22:0) 0.13 ± 0.02 0.20 ± 0.05 0.16 ± 0.02 0.18 ± 0.03 0.18 ± 0.03 0.17 ± 0.02 0.18 ± 0.03

EFAs sum 77.92 77.43 77.45 77.43 77.38 77.40 77.12

␻-6/␻-3 ratio 3.29 3.25 3.31 3.29 3.28 3.25 3.29

PUFAs sum 81.35 81.00 80.91 80.93 80.87 80.85 80.60

Monounsaturated 11.12 11.49 11.47 11.30 11.51 11.63 11.66
Saturated 7.54 7.51 7.62 7.77 7.61 7.52 7.74
Polyunsaturated/saturated ratio 10.79 10.79 10.62 10.42 10.62 10.76 10.42
a
Each data represents the mean of three replicates ± standard deviation.

Table 3
Oil yield, recovery and oxidation stability of hempseed oil, cultivar Felina, extracted under different SC-CO2 experimental conditions and by Soxhlet extraction with n-hexane.

Pressure (bar) Temperature (◦ C) QCO2 (kg CO2 /kg feed) Oil yield (g oil/100 g seeds) Recovery (% total oil) Oxidation stability (mM Eq Vit E)

300 40 40 22.1 ± 0.7 aa 72.2 ± 0.5 aa 0.40 ± 0.04 b

60 50 17.2 ± 0.5 c 56.2 ± 1.2 c 0.17 ± 0.03 c
80 30 16.8 ± 0.7 d 55.0 ± 0.6 d 2.18 ± 0.04 a

400 40 50 20.5 ± 0.8 b 66.9 ± 1.1 b 0.26 ± 0.08 b

60 30 17.3 ± 0.7 c 56.6 ± 0.7 c 0.16 ± 0.08 c
80 40 22.1 ± 0.3 a 72.1 ± 1.0 a 0.03 ± 0.02 d

Soxhlet (n-hexane)b 30.6 ± 0.4 100 0.17 ± 0.07

Vergin olive oil 0.53 ± 0.1
a
Values with different letter within columns indicate significant differences (p < 0.05).
b
It was considering that the hexane-extracted 100% of the oil.
404 C. Da Porto et al. / Industrial Crops and Products 36 (2012) 401–404
corresponding to 2.16 mM Eq Vit E, about 4 times that of vergin
olive oil, was obtained by SC-CO2 extraction carried out at 300 bar
and 80 ◦ C. Under these operating conditions, the SC-CO2 -extracted
oil resulted more protected against oxidation than the other SC-CO2
samples (A, B, D, E, and F) and the n-hexane-extracted oil. Schmidt
and Pokorny (2005) reported that the most active antioxidants in
lipids are ␥-tocopherol and ␦-tocopherol, decreased in the order:
␥ > ␦ > ␤ > ␣-tocopherol. ␥-Tocopherol was reported to be the pre-
dominant tocopherol in hempseed oil (Jones, 1995; Yu et al., 2005;
Ramadan and Moersel, 2006). It is interesting to note that at 300 bar
and 80 ◦ C the highest oxidation stability (2.16 mM Eq Vit E) and
lowest oil yield (16.8%, w/w) were obtained. Such behavior could
be explained by the greater selectivity of the SC-CO2 in extract-
ing the tocopherols than the oil. Authors such as Leo et al. (2005)
explain that this greater selectivity of the CO2 for the tocopherols
means that the tocopherol concentration in the oil is higher during
the initial extraction phase, then decreasing during the subsequent
extraction phases.
4. Conclusion
Hemp seed oil is a non-traditional oil which may serve as dietary
source of EFA’s and natural antioxidants for health promotion and
disease prevention or may be used as ingredient for functional food.
The results indicated that supercritical extractions at 300 bar and
40 ◦ C and at 400 bar and 80 ◦ C with a solvent-ratio of 40 kg CO2 per
kg of feed were more effective on the oil yield, whereas at 300 bar,
80 ◦ C and a solvent-ratio of 30 kg CO2 per kg of feed the highest
oxidation stability was obtained.
Acknowledgement
The authors wish to thank Friuli-Venezia Giulia Region (art. 17,
LR 26/2005) for its financial support.
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